To Expedite Resilient Solar in the Caribbean, Collaboration is Key – Benzinga

Posted on by

A new Rocky Mountain Institute report entails how policymakers and regulators can collaborate to quicken solar photovoltaic installation across the Caribbeanimproving communities' livelihoods, enabling cost savings, building resilience and reducing dependency on foreign oil.

NEW YORK (PRWEB) July 14, 2020

Rocky Mountain Institute (RMI), the Clinton Foundation and the United Nations Office of the High Representative for the Least Developed Countries, Landlocked Developing Countries, and Small Island Developing States released a joint report that details how island policymakers and regulators can work together to enhance the resilience of solar photovoltaic (PV) systems across the Caribbean.

The Caribbean and Small Island Developing States (SIDS) continue to face more frequent and intense weather events. When critical infrastructure, like hospitals and schools, are without power, communities suffer deeply. Solar PV systems are a way to increase the resilience of the grid and greatly improve people's access to reliable electricity, but they are useless if they fail.

The report underscores the need to ensure resilient solar PV systems are installed in the most cost-effective and collaborative way. Other key topics include incentivizing the use of the correct equipment and ensuring building codes incorporate resilient solar PV design and construction standards.

"I cannot stress enough the value of stakeholder collaboration. Collaboration ensures that the correct equipment is available, known best practices are enforced, and that systems are built to the highest standards. These components ensure that islands build the most resilient solar projects and ultimately more resilient communities," said Jules Kortenhorst, Chief Executive Officer, RMI.

Key recommendations for collaboration presented in the report include:

"In the Sustainable Development Goals, the world committed to ensuring access to affordable, reliable and modern energy for all. Many Small Island Developing States are emerging as frontrunners in the pursuit of renewable energy, with solar power leading the way. International support in the form of access to finance, investments and technology will be critical to accelerate their transitionand their resilience," said Fekitamoeloa Katoa Utoikamanu, High Representative for the UN-OHRLLS.

"President Clinton's commitment to the environment continues to drive our work across the Caribbean and island states, finding scalable, replicable solutions for clean energy. We are proud to partner with RMI and UN OHRLLS to share these best practices with the global community. Today, it is even more important to find reliable, cost-effective energy solutions to keep essential services up and running in the face of extreme weather events and the current pandemic," explained Kevin Thurm, chief executive officer, Clinton Foundation.

Solar Under Storm for Policymakers is the third in a series of reports by RMI and partners that focus on installing hurricane-resilient solar systems. The first Solar Under Storm guide discussed the root causes of past solar failures focused on ground-mounted systems. Solar Under Storm Part II explored best practices for equipment and procedures for rooftop systems.

To download the Solar Under Storm for Policymakers report,visit Solar Under Storm for Policymakers: Select Best Practices for Resilient Photovoltaic Systems for Small Island Developing States.

Media Inquiries please contact:

Nick Steel, Media Relations Manager, T: +1 347-574-0887, E: nsteel@rmi.org

Conor O'Loughlin, Head of Advocacy, E: conoroloughlin@un.org

Notes to Editors

About Rocky Mountain Institute

Rocky Mountain Institute (RMI)an independent nonprofit founded in 1982transforms global energy use to create a clean, prosperous, and secure low-carbon future. It engages businesses, communities, institutions, and entrepreneurs to accelerate the adoption of market-based solutions that cost-effectively shift from fossil fuels to efficiency and renewables. RMI has offices in Basalt and Boulder, Colorado; New York City; the San Francisco Bay Area; Washington, D.C.; and Beijing.

More information on RMI can be found at http://www.rmi.org or follow us on Twitter @RockyMtnInst.

About the Clinton Foundation

Building on a lifetime of public service, President Clinton established the Clinton Foundation on the simple belief that everyone deserves a chance to succeed, everyone has a responsibility to act, and we all do better when we work together.

For nearly two decades, that belief has energized the work of the Foundation in overcoming complex challenges and improving the lives of people across the United States and around the world. As an operating foundation, we work on issues directly or with strategic partners from the business, government, and nonprofit sectors to create economic opportunity, improve public health, and inspire civic engagement and service.

Our programs are designed to make a real difference today while serving as proven models for tomorrow. The goal of every effort is to use available resources to get better results fasterat the lowest possible cost. We firmly believe that when diverse groups of people bring resources together in the spirit of true cooperation, transformative ideas will emerge to drive life-changing action. Learn more at https://www.clintonfoundation.org/, on Facebook at Facebook.com/ClintonFoundation, and on Twitter @ClintonFdn.

About the United Nations Office of the High Representative for the Least Developed Countries, Land Locked Countries, and Small Island Developing States

The United Nations Office of the High Representative for the Least Developed Countries, Landlocked Developing Countries, and Small Island Developing States (UN-OHRLLS) assists vulnerable countries in areas including economic growth, poverty reduction, and meeting targets laid out in the Sustainable Development Goals.

For the original version on PRWeb visit: https://www.prweb.com/releases/to_expedite_resilient_solar_in_the_caribbean_collaboration_is_key/prweb17254640.htm

See the article here:

To Expedite Resilient Solar in the Caribbean, Collaboration is Key - Benzinga

Everybody Profits From Nonprofit Caribbean Tourism Bodies – South Florida Caribbean News

Posted on by

A joint editorial by Frank J. Comito, CEO and Director General, Caribbean Hotel and Tourism Association (CHTA); and Brian D. Frontin, President, Caribbean Society of Hotel Association Executives (CSHAE) and CEO, Trinidad Hotels, Restaurants & Tourism Association (THRTA)

MIAMI The nonprofit tourism associations of the Caribbean are integral to the regions recovery and to the sustained profitability of the tourism and hospitality sector. And, during these difficult Coronavirus Disease (COVID-19) times it is even more important to support these vital development-driver institutions if we are to emerge from the pandemic more quickly and in the best economic shape.

Moving beyond this pandemic well-prepared for the new realities of global tourism requires governments to collaborate closely with the private sector, speaking with one voice through their national associations and the Caribbean Hotel and Tourism Association (CHTA) on hurdling the barriers lining the path back to profitability.

We know from experience with hurricanes and health crisis situations that when the business community and governments work in a unified way they are able to accelerate the return to full employment, restoring tax revenue, rebuilding dynamic business, and enhancing education, health and other government services.

The most important thing the business community can do is to support its local and regional hotel and tourism associations by being as collaborative and engaged as possible on a variety of issues which affect their bottom line and the well-being of the communities where they operate.

Nonprofit organizations are societys biggest (little-known) change agents, especially the business-led NGOs like ours, marshaling and leveraging resources at the local, regional and international levels.

Their historic contributions to the economies and development of the Caribbean region are considerable, working tirelessly for decades on advocacy, training and education, research, marketing, beautification, environmental protection, energy efficiency, product development and community enhancements.

One can easily state that through the dedicated volunteer business leadership of these nonprofit organizations, businesses and governments throughout the Caribbean have flourished.

Today, however, the very survival and future viability of many of these organizations is in question.

Leadership, beacons of guidance, providing answers, support and advice through crises are provided by national and regional associations to help businesses make it through the storms and create a path towards recovery.

We have received plaudits for the important benefits accruing from membership of CHTA and national associations which shone brightly over the past 100 days as tourism stakeholders, in many instances working in step with governments, have worked on response, mitigation and recovery.

The rapid Caribbean-wide response of CHTA to the crisis was made possible by national tourism associations assembling data from countries and territories, which were fed to member groups directly to support local efforts or through such avenues as the resilience series of webinars launched by CHTA.

The value of our membership associations has been proven so often that we may be suffering from our own success as destinations and resorts appear to be taking our collective efforts for granted. Our biggest challenge as nonprofit organizations is to get many more in the business community to realize that we are their insurance policy. But we need them to pay the correct premium for this insurance coverage.

We are supported by voluntary contributions and only about half of the private sector in most jurisdictions actually come to the table to help make the difference in their own industry often because they dont know the extent of what the organizations do for them, but also unfortunately because some businesses simply dont see supporting the collective as part of their responsibility, but are willing to freely accept the benefits to their businesses which these associations bring about, thanks to the dedicated leadership and support of some of their fellow businesses. This simply is not fair play.

Those who are disengaged dont seem to realize theyre hurting themselves and that its absolutely essential, especially during crises like the one we have now, to come to the table and lend talent and resources for the benefit not only of their own properties, but for all the Caribbean enterprises, because as we know all too well, in small island states, a rising tide raises all boats. At the minimum, a modest financial dues investment should be committed.

Every association in the Caribbean is financially strapped, but we in regional and national organizations are being asked on a daily basis to do far more with fewer resources during the toughest of times. Without the urgent support of industry stakeholders, some of these long-standing national hotel and tourism associations may soon be forced to close their doors.

Such closures would be lamentable because we are all in this together, not just those in the major breadwinner of the region, tourism, but also the majority of private sector concerns linked to the sector.

Banks, insurance companies, telecommunications firms, wholesalers, shipping firms and service providers are among the many industry players whose success has been built through tourism.

Decades of development work by the regions 33 national hotel and tourism associations and CHTA have contributed to their collective success. Now, in this time of incredible vulnerability, as best they can, we need these businesses, along with all tourism-related businesses, to support our nonprofit organizations and associations.

After all, this would be a business imperative not an act of charity because they will be investing in the recovery of the most important revenue producer of the region. The return on their investment in tourism associations will help to reignite tourism faster, which will more readily accrue benefits to these businesses in the medium to long term.

The pandemic lockdowns have allowed our associations to make our industry smarter and stronger by intensifying training in the sector with heightened hygiene and health and safety guidelines. Our training modules are now being carefully studied by the private sector another benefit produced by nonprofits that helps corporations.

CHTA has focused a lot of effort on improving and honing health, safety and environmental standards collaboratively with the Caribbean Public Health Agency (CARPHA), the Caribbean Tourism Organization (CTO), the Organization of Eastern Caribbean States Commission (OECSC), and the Jamaica-based Global Tourism Resiliency and Crisis Management Centre (GTRCMC) during the COVID-19 pandemic.

Such initiatives will help countries and territories rebound and recover faster, while building consumer confidence, and confidence in our own employees as they adhere to protocols that can keep them safe as well.

Importantly, this coordinated skills boosting will build confidence in travelers as word gets out that the Caribbean is really helping to mitigate or remove altogether health and safety risks.

The point is, tourism is everybodys business and for everybodys benefit: governments benefit because they generate tax revenue from our generation of business for the economy and many jobs and spin-off businesses are created, broadening tourisms impact even further.

Those businesses which have benefited so richly from tourism can look at ways of supporting their local national hotel and tourism associations and CHTA, because tourism generates a tremendous value that enhances the health, wealth, environment and well-being of all our people.

We applaud those businesses and their dedicated owners, operators, managers and supervisors in the region who have invested leadership, time and money into making these institutions possible.

We challenge those who have not stepped up to the plate, at a minimum, with the remittance of a small annual dues investment, to do so. Time and time again, whether theyve realized it or not, our nonprofit organizations have been there for them and it is not only long overdue, it is fair play and simply good business for them to step up in this time of tremendous need for their own benefit, if nothing else.

Standing by and watching nonprofit tourism associations struggle or fail is just bad business.

See the rest here:

Everybody Profits From Nonprofit Caribbean Tourism Bodies - South Florida Caribbean News

You could move to Barbados and work there remotely for a year thanks to a new incentive – Insider – INSIDER

Posted on by

If you can work from anywhere, would you stay where you are?

As much of the world has taken to working remotely because of the coronavirus pandemic, many employees are no longer tied to where their offices are.

Barbados is banking on the idea that many people will choose a tropical paradise over their current digs.

Imagine working from here. LU LIN/Shuttershock

Barbados Prime Minister Mia Amor Mottley said last week that the Caribbean country was planning to introduce a "12-month Barbados Welcome Stamp" that would allow visitors to stay for up to a year and work remotely.

Mottley said the stamp would allow "persons to come and work from here overseas, digitally so, so that persons don't need to remain in the countries in which they are" but beyond that, information is scarce. A representative for Barbados Tourism Marketing told Insider that "details on the visa are still being finalized by the Government of Barbados."

The idea is that as short-term travel becomes increasingly difficult, a 12-month stamp would make the trip more worthwhile. It would also bring much-needed tourism dollars to the island.

Mottley said on June 26 that Barbados planned to reopen to tourists on July 12, this Sunday, when some air travel is expected to resume.

"We will continue to take a risk-based approach to the protection of our country, our people and our visitors," Mottley said.

It's important to note, however, that nonessential travel is still discouraged in many places, including the US, where the Centers for Disease Control and Prevention has warned against it.

The island is a British Commonwealth nation. Styve Reineck/Shutterstock

A recent press release from Barbados Tourism Marketing said visitors from high-risk countries (more than 10,000 new cases in the past seven days) were "strongly encouraged" to take a COVID-19 test within 72 hours of their departure. Travelers from low-risk spots (with fewer than 100 cases in the past week) could take the test up to a week before departure.

Travelers who didn't take a test before their arrival will have to take one when they land, then quarantine at their expense until they receive the results, which could take about two days. Should they test positive, "they will be placed in isolation where they will receive care from the Ministry of Health and Wellness," the press release said.

Visitors will also have to fill out an online embarkation/disembarkation form related to their health and face temperature checks at the airport, the press release said.

According to data from Johns Hopkins University, Barbados had 98 confirmed COVID-19 cases and seven deaths as of Wednesday.

While it's still unclear when this welcome stamp might launch, Mottley said: "The government is committed to working with you on the promotion of new concepts like the 12-month Barbados Welcome Stamp, being able to open our borders to persons traveling and making it as hospitable as ever for all of us."

Representatives for the Barbados Government Information Service did not immediately respond to Insider's request for comment.

See the article here:

You could move to Barbados and work there remotely for a year thanks to a new incentive - Insider - INSIDER

Two Caribbean destinations tap the brakes on reopening as US numbers continue to climb – Travelweek

Posted on by

The CTO's Johnson JohnRose (left) with St. Maarten's Minister of Tourism, Ludmila de Weever (right) take part in the CTO's 'Plain Speaking' Facebook Live event this morning

TORONTO The Dutch Caribbean destination of St. Maarten has pushed back its reopening date for U.S. travellers from July 15 to Aug. 1.

As for Canadian travellers, St. Maartens Minister of Tourism Ludmila de Weever says Canada is in St. Maartens lower-risk bracket, and its really about when [Canadians] are ready to fly. Were ready to accept them.

De Weever took part in a CTO Facebook Live session this morning, updating the trade on St. Maartens reopening plans.

Meanwhile, Grenada has announced a new reopening date, one month later than originally planned.

Here are all the latest updates

ST. MAARTEN

On this mornings Facebook Live session, part of the CTOs Plain Speaking series, de Weever said that while St. Maarten originally planned to welcome back U.S. flights starting tomorrow, July 15, that date has now been pushed back to Aug. 1.

The one constant is that things are always changing, she said.

Everybody got a bit nervous when they heard we were reopening to the U.S., she added. However, Aug. 1 is now a hard deadline. August 1 is in the books now. What you dont want to do is keep pushing dates with airlines. You have to give them a date and stick to it.

She added that the airlines have been so understanding. In our discussions, one said to us, Any decision you make, we respect that. You have to keep your country safe. That kind of understanding, coming from these major airline companies, that means a lot.

People may get frustrated and lose patience but at the end of the day we are looking out for our visitors as well as our residents.

Presenting todays Facebook Live session along with de Weever was the CTOs Communications Specialist, Johnson JohnRose. If youre going to open to the U.S., you have to find that balance between lives and livelihoods, he said.

De Weever agreed, noting we are so grateful people want to come here, but were a small location and our capacities are limited.

The destination reopened to Europe and several Caribbean islands on July 1. So far that stage of the reopening process is going well, says de Weever. Anyone coming to St. Maarten must upload the results of their negative PCR test (taken within 72 hours of arrival) and complete a health questionnaire.

St. Maartens Health Declaration Platform, with all these details and more, is at stmaartenentry.com.

De Weever said St. Maarten is looking at reducing the number of people allowed into the destination. Not all airlines are doing reduced capacity [onboard]. Even with the Europe flights weve had already, were getting about 1,000 passengers per week. Thats already a lot and the flights and getting fuller and fuller. So we are considering [limiting the number of arrivals]. But we havent come to a final decision. Its one thing if airlines are reducing onboard capacity. But for airlines who arent doing that, its a different story.

St. Maarten will be watching the COVID-19 numbers in the U.S. very carefully in the next couple of weeks. Eighty percent of our market is North America, i.e. the U.S., said de Weever.

Two helpful sites for anyone looking to travel to St. Maarten, or agents with clients looking to travel to the destination, are stmaartenentry.com and stmaartenupdates.com.

GRENADA

Grenada has a new reopening date for international visitors, including the Canadian market.

Initially scheduled to reopen July 1, Grenada has remained closed following a June 28 address from Grenadas Prime Minister Dr. Keith Mitchell, who called for a more cautious approach to reopening the countrys borders.

Now Grenada will reopen to international travellers on Aug. 1. The reopening date for regional visitors is July 15.

Canada is on Grenadas list of medium-risk countries, along with the UK and other EU countries.

Protocols include a mandatory certified copy of a negative PCR test dated no more than 7 days prior to departure. All passengers must also undergo a rapid test on arrival at the airport. If the rapid test is positive, the traveller will then be tested using swab/PCR and placed in quarantine at an approved accommodation at their expense, for 2 4 days, pending the PCR result. If that subsequent test is positive they will remain in quarantine for up to 14 days, or until they have tested negative on PCR.

All travellers must also complete, sign and submit a Health Declaration Form, and download and register on Grenadas contact tracing app. Both can be found at covid19.gov.gd.

Non-national must also have travel insurance covering COVID-19, or declare that they will bear the cost for treatment and isolation, according to a statement from Grenadas Ministry of Tourism and Civil Aviation.

Editor at Travelweek

Kathryn is Editor at Travelweek and has worked for the company since 1995. She has travelled to more than 50 countries and counts Hong Kong, Jerusalem, the Swiss Alps and the Galapagos Islands among her favourite destinations.

Read the original here:

Two Caribbean destinations tap the brakes on reopening as US numbers continue to climb - Travelweek

All the Pirates of the Caribbean Movies Ranked – Collider.com

Posted on by

Pirates of the Caribbean was never supposed to work.

The film was born out of an initiative, started by former Disney CEO Michael Eisner, to mine the companys many theme park attractions for potential movie franchises. It was already off to a wobbly start with Brian De Palmas Mission to Mars and, er, The Country Bears. And pirate movies, of any kind, were seen as box office kryptonite, especially since the last big budget endeavor, 1995s Cutthroat Island, bankrupted its studio and made the Guinness Book of World Records for the biggest flop. While in production, Disney executives were nervous about Johnny Depps fey portrayal of the lead pirate, Captain Jack Sparrow, and the dark tone being conjured by director Gore Verbinski. When the film was released in 2003, it was the first Disney film to carry a PG-13 rating.

And yet somehow, Pirates of the Caribbean has become one of the most dependably bankable franchises in Hollywood. Even the last film, 2017s Pirates of the Caribbean: Dead Men Tell No Tales, which was already mired in Depps controversial private life, grossed $800 million worldwide. And as it was inspired by a theme park attraction, so has it inspired theme park attractions, with Jack Sparrow being added to the classic attraction and a brand new, state-of-the-art ride based on the franchise, Pirates of the Caribbean: Battle for the Sunken Treasure, developed for Shanghai Disneyland (it debuted with the rest of the park in 2016). And recent rumors have suggest that the franchise will be resurrected soon enough, with one possibility being a female-led entry starring Margot Robbie and written by Christina Hodson.

But which Pirates of the Caribbean entry is the most swashbuckling, supernatural creature-filled joy? And which entry should be stranded ashore. Youll have to read on to find out, ya lousy landlubber.

Read the original post:

All the Pirates of the Caribbean Movies Ranked - Collider.com

Airlines Are Working Toward A Sustainable Caribbean Solution – St. John Source

Posted on by

Basil Springer, Ph.D.

Do not be conformed to this world, but be transformed by the renewal of your mind, that by testing you may discern what is the will of God, what is good and acceptable and perfect. Romans 12:2

LIAT (1974) Ltd. (Leeward Islands Air Transport) has been hemorrhaging for a long time, primarily because of weaknesses in governance structure, political interference, undercapitalization, unimaginative marketing, high cost, low productivity and myopic partnership practices.

The advent of COVID-19 forced the already debt-ridden airline to suspend commercial services in early April 2020. The borders of most of its 15 destinations are still closed to commercial traffic and the exclusive government shareholders are seriously considering airline liquidation.

Last week, LIAT announced that no further information about its future will be available until after its next annual general meeting, which has not yet been scheduled.

Let us hope that good sense will prevail and that the minds of the leaders of this region will be renewed to find an efficient solution that strengthens the current weaknesses of LIAT and exploits the uniqueness of the Caribbean region as an attractive, warm weather destination.

The efficient Caribbean airline industry is essential for regional unity, without which some countries will struggle to survive.

The role of government in open market economies is to set policy regarding the legal and social framework, create a user-friendly enabling environment for the private sector to do business, provide public goods and services, stabilize the economy and negotiate global public-private sector partnerships. The general rules of government are not structured to manage commercial activities.

Over the last 20 years, I have had the opportunity to establish more than one trust under the trust laws of the Caribbean. As they search for a sustainable Caribbean airline solution, I would respectfully suggest that the governments of the Caribbean destinations served by LIAT should establish a Trust for the benefit of the people of the region.

Experienced private sector trustees, approved by the governments, would be appointed to govern the trust. The trustees would report to the governments, say, once every six months, to give an account of their stewardship. The governments would be responsible for capitalizing a Fund for the Trust and the management of the trust would be entrusted to the private sector. The management would report to the trustees monthly. The trust would be structured to permit private sector investment in the fund through a specific class of shares.

A knowledgeable marketing communications firm with expertise in public relations and digital marketing would be selected to promote the Caribbean globally as a diverse multicultural, multiethnic, multireligious, multi-culinary, multi-musical genre and multilingual region.

Based on the economic growth philosophy of tax the outputs, not the inputs, governments should remove the tax component from the regional airfares and hence increase the turnover of passenger traffic. The management would introduce a performance optimization system aimed at achieving high productivity yields from human resources, technology and innovative processes.

COVID-19 has had a devastating impact on the global airline industry. Nevertheless, the Caribbean governments and private sector must work together in smart partnerships with established foreign airlines and financial partners to benefit from their superior investment resources, experience, and airline industry expertise towards their mutual benefit.

*The above is a column written by Basil Springer, who isa director of the New Jersey-based Caribbean Media Exchange on Sustainable Tourism (CMEx), which has hosted many international events as well as Marketplace Excellence (MPE) a Public Relations, Marketing and Media Company. He is the Chairman of Global Business Innovation Corporation, which launched the Caribbean Food Business Innovation Revolution initiative in Trinidad in January 2015.

View post:

Airlines Are Working Toward A Sustainable Caribbean Solution - St. John Source

Healthcare Nanotechnology Market Size By Product Analysis, Application, End-Users, Regional Outlook, Competitive Strategies And Forecast Up To 2026 -…

Posted on by

New Jersey, United States,- Latest update on Healthcare Nanotechnology Market Analysis report published with extensive market research, Healthcare Nanotechnology Market growth analysis, and forecast by 2026. this report is highly predictive as it holds the overall market analysis of topmost companies into the Healthcare Nanotechnology industry. With the classified Healthcare Nanotechnology market research based on various growing regions, this report provides leading players portfolio along with sales, growth, market share, and so on.

The research report of the Healthcare Nanotechnology market is predicted to accrue a significant remuneration portfolio by the end of the predicted time period. It includes parameters with respect to the Healthcare Nanotechnology market dynamics incorporating varied driving forces affecting the commercialization graph of this business vertical and risks prevailing in the sphere. In addition, it also speaks about the Healthcare Nanotechnology Market growth opportunities in the industry.

Healthcare Nanotechnology Market Report covers the manufacturers data, including shipment, price, revenue, gross profit, interview record, business distribution etc., these data help the consumer know about the competitors better. This report also covers all the regions and countries of the world, which shows a regional development status, including Healthcare Nanotechnology market size, volume and value, as well as price data.

Healthcare Nanotechnology Market competition by top Manufacturers:

Healthcare Nanotechnology Market Classification by Types:

Healthcare Nanotechnology Market Size by End-user Application:

Listing a few pointers from the report:

The objective of the Healthcare Nanotechnology Market Report:

Cataloging the competitive terrain of the Healthcare Nanotechnology market:

Unveiling the geographical penetration of the Healthcare Nanotechnology market:

The report of the Healthcare Nanotechnology market is an in-depth analysis of the business vertical projected to record a commendable annual growth rate over the estimated time period. It also comprises of a precise evaluation of the dynamics related to this marketplace. The purpose of the Healthcare Nanotechnology Market report is to provide important information related to the industry deliverables such as market size, valuation forecast, sales volume, etc.

Major Highlights from Table of contents are listed below for quick lookup into Healthcare Nanotechnology Market report

About Us:

Market Research Intellect provides syndicated and customized research reports to clients from various industries and organizations with the aim of delivering functional expertise. We provide reports for all industries including Energy, Technology, Manufacturing and Construction, Chemicals and Materials, Food and Beverage, and more. These reports deliver an in-depth study of the market with industry analysis, the market value for regions and countries, and trends that are pertinent to the industry.

Contact Us:

Mr. Steven Fernandes

Market Research Intellect

New Jersey ( USA )

Tel: +1-650-781-4080

See more here:

Healthcare Nanotechnology Market Size By Product Analysis, Application, End-Users, Regional Outlook, Competitive Strategies And Forecast Up To 2026 -...

New Comprehensive Report on Nanomedicine Market to Witness an Outstanding Growth during 2020 2025 with Top Players Like – Jewish Life News

Posted on by

Nanomedicine Market Overview 2020 2025

This has brought along several changes in This report also covers the impact of COVID-19 on the global market.

The risingtechnology in Nanomedicine Marketis also depicted in thisresearchreport. Factors that are boosting the growth of the market, and giving a positive push to thrive in the global market is explained in detail.

Get a Sample PDF copy of the report @ https://reportsinsights.com/sample/91246

Key Competitors of the Global Nanomedicine Market are: , GE Healthcare, Johnson & Johnson, Mallinckrodt plc, Merck & Co. Inc., Nanosphere Inc., Pfizer Inc., Sigma-Tau Pharmaceuticals Inc., Smith & Nephew PLC, Stryker Corp, Teva Pharmaceutical Industries Ltd., UCB (Union chimique belge) S.A,

Historical data available in the report elaborates on the development of the Nanomedicine on national, regional and international levels. Nanomedicine Market Research Report presents a detailed analysis based on the thorough research of the overall market, particularly on questions that border on the market size, growth scenario, potential opportunities, operation landscape, trend analysis, and competitive analysis.

Major Product Types covered are:Regenerative MedicineIn-vitro & In-vivo DiagnosticsVaccinesDrug Delivery

Major Applications of Nanomedicine covered are:Clinical CardiologyUrologyGeneticsOrthopedicsOphthalmology

This study report on global Nanomedicine market throws light on the crucial trends and dynamics impacting the development of the market, including the restraints, drivers, and opportunities.

To get this report at a profitable rate.: https://reportsinsights.com/discount/91246

The fundamental purpose of Nanomedicine Market report is to provide a correct and strategic analysis of the Nanomedicine industry. The report scrutinizes each segment and sub-segments presents before you a 360-degree view of the said market.

Market Scenario:

The report further highlights the development trends in the global Nanomedicine market. Factors that are driving the market growth and fueling its segments are also analyzed in the report. The report also highlights on its applications, types, deployments, components, developments of this market.

Highlights following key factors:

:-Business descriptionA detailed description of the companys operations and business divisions.:-Corporate strategyAnalysts summarization of the companys business strategy.:-SWOT AnalysisA detailed analysis of the companys strengths, weakness, opportunities and threats.:-Company historyProgression of key events associated with the company.:-Major products and servicesA list of major products, services and brands of the company.:-Key competitorsA list of key competitors to the company.:-Important locations and subsidiariesA list and contact details of key locations and subsidiaries of the company.:-Detailed financial ratios for the past five yearsThe latest financial ratios derived from the annual financial statements published by the company with 5 years history.

Our report offers:

Market share assessments for the regional and country level segments. Market share analysis of the top industry players. Strategic recommendations for the new entrants. Market forecasts for a minimum of 9 years of all the mentioned segments, sub segments and the regional markets. Market Trends (Drivers, Constraints, Opportunities, Threats, Challenges, Investment Opportunities, and recommendations). Strategic recommendations in key business segments based on the market estimations. Competitive landscaping mapping the key common trends. Company profiling with detailed strategies, financials, and recent developments. Supply chain trends mapping the latest technological advancements.

Access full Report Description, TOC, Table of Figure, Chart, etc. @ https://reportsinsights.com/industry-forecast/Nanomedicine-Market-91246

About US:

Reports Insights is the leading research industry that offers contextual and data-centric research services to its customers across the globe. The firm assists its clients to strategize business policies and accomplish sustainable growth in their respective market domain. The industry provides consulting services, syndicated research reports, and customized research reports.

Contact US:

:(US) +1-214-272-0234

:(APAC) +91-7972263819

Email:[emailprotected]

Sales:[emailprotected]

Link:

New Comprehensive Report on Nanomedicine Market to Witness an Outstanding Growth during 2020 2025 with Top Players Like - Jewish Life News

Engineering a far-red lightactivated split-Cas9 system for remote-controlled genome editing of internal organs and tumors – Science Advances

Posted on by

INTRODUCTION

Many studies have shown that the CRISPR-Cas9 system is a revolutionary technology (1, 2). This relatively easy-to-use technology has provided unprecedented opportunities for scientific research and disease treatments, including applications in high-throughput screening and functional genomics research and treatment of virus infections (3), genetic diseases (4), and cancer (5). Nevertheless, there are now several well-known disadvantages with the CRISPR-Cas9 system, including the fact that single guide RNAs (sgRNAs) can sometimes lead to off-target effects such as double-strand breaks in untargeted genome regions, which can cause unintended adverse consequences such as gene mutations, insertions, deletions, and even tumorigenic events (6). Seeking to overcome these challenges, several strategies have been developed to improve the precision of CRISPR-Cas9 gene editing, including Cas9 modifications (e.g., Cas9 nickase and high-fidelity variants), prime editors, base editors, and selecting sgRNAs with minimal off-target capacity (7, 8). Recently, some inducible Cas9 expression systems have been developed to limit the activity or lifetime of Cas9, thereby lowering the probability of off-target effects by reducing the exposure time of a cells genome to the Cas9 nuclease (9).

There are a variety of chemically induced CRISPR-Cas9 systems, including doxycycline-regulated Cas9 (10), trimethoprim (TMP) (11) and 4-hydroxytamoxifen (4-OHT)controlled Cas9 (12), rapamycin-inducible split-Cas9 (13), 4-OHTresponsive inteindependent Cas9 (14), and 4-OHTresponsive nuclear receptors split-Cas9 (15), among others. However, a notable adverse effect of these systems is the potential for cytotoxicity from the chemical inducers: Doxycycline can negatively affect cell numbers and colony formation (16), TMP can inhibit uptake of folic acid by the cells (17), 4-OHT can increase cytosolic levels of autophagosomes and cause irregularly clumped chromatin in the nuclei (18), and rapamycin can perturb the endogenous mammalian target of rapamycin pathway (19). Moreover, once these agents are inside the cells or present in an in vivo context, these inducer chemicals can diffuse freely, limiting the spatial resolution of editing induction. In addition, it is difficult to rapidly remove the inducer compounds, so they can persist for a long time, making it difficult to turn Cas9 activity on and off quickly and precisely.

These limitations have helped motivate the development of multiple systems based on the optical control of Cas9 activity because light is a reversible and noninvasive inducer modality that potentially offers fine precise spatiotemporal resolution. The first reported example of a photoactivatable Cas9 system was paCas9 system based on blue light (20). In the paCas9 system, Cas9 nucleases are fragmented into two nonfunctional fragments that can be reconstituted as an active nuclease under blue light illumination based on dimerization of their respective fusion domains, the positive Magnet (pMag) or negative Magnet (nMag) proteins from the filamentous fungus Neurospora crassa (21). Later studies reported the ultraviolet (UV) lightmediated cleavage of a synthesized complementary oligonucleotide element that normally inactivates the editing-guiding function of sgRNAs (22).

There is also a recently reported blue lightbased anti-CRISPR system comprising AcrIIA4 (23) (a potent Cas9 inhibitor) and the LOV2 blue-light photosensor (24). Without illumination, the AcrIIA4-LOV2 complex remains bound to Cas9, inhibiting its nuclease activity. Under blue light illumination, the AcrIIA4-LOV2 complex is separated from Cas9 and its editing activity can be restored (25). However, neither UV nor blue light is able to penetrate deeply into the body, owing to the strong absorption and scattering of these light energies by biological tissues (26). UV light hardly penetrates the skin and blue light does merely by 1 mm (27, 28). This substantial limitation, viewed alongside the fact that UV and prolonged blue light exposure can cause cytotoxicity (29, 30), highlights the difficulty of applying these light-induced Cas9 systems for in vivo research applications and clinical translation.

We have, for some time, been investigating far-red light (FRL)inducible genetic systems due to the deep tissue penetration of FRL with above 5 mm beneath the surface of skin (27, 28). We here report our development of an FRL-activated split-Cas9 (FAST) system that can be used to noninvasively induce gene editing activity in cells located deep inside animal tissues. The FAST system relies on two split-Cas9 fusion proteins with high-affinity binding domains: One half of Cas9 is constitutively expressed, while the other is under the FRL-inducible control of the bacterial phytochrome BphS optical controllable system previously established by our group (31). We initially assembled the FAST system components in human embryonic kidney (HEK)293 cells and used light-emitting diode (LED)based FRL illumination to demonstrate successful activation of targeted genome editing. Next, after achieving FRL-inducible editing in diverse human cell lines, experiments with implants confirmed that FAST was able to robustly activate editing in cells positioned in subdermal animal tissues. Experiments with the transgenic tdTomato reporter mouse line established FRL-induced FASTmediated editing of mouse somatic cells (hepatocytes in the liver), and work with cell cycleinactivating gene edits of cancer cells in xenograft tumor mice demonstrate how FAST can be deployed against disease. Thus, beyond extending the optogenetic toolbox for gene editing of mammalian cells to include induction by the highly in vivocompatible and deep tissuepenetrating energies of FRL, our study extends this initial technology to demonstrate applications relevant for basic biological and biomedical research.

To develop an optogenetically controlled device for genome editing with deep tissuepenetrative capacity and with negligible phototoxicity in vivo, first, we constructed an FRL-controlled full-length Cas9 system based on our previously reported orthogonal FRL-triggered optogenetic system (FRL-v2) (31). However, there was serious background leakage in dark state with low-induction performance under illumination. Therefore, we focused on building a FAST system based on split-Cas9 (13) and FRL-v2, which comprises the bacterial FRL-activated cyclic diguanylate monophosphate (c-di-GMP) synthase (BphS) and a c-di-GMPresponsive hybrid transactivator, p65-VP64-BldD. For the FAST system, we then fused the N-terminal Cas9 fragment [Cas9(N)] to the Coh2 domain from Clostridium thermocellum (32) and fused the C-terminal Cas9 fragment [Cas9(C)] to the DocS domain from the same bacterium. Expression of the NLS-Cas9(N)-Coh2 fusion protein is driven by the FRL-v2specific chimeric promoter (PFRL), while expression of the DocS-Cas9(C)-NES fusion protein is driven by a constitutive promoter (PhCMV). A complete Cas9 protein can be reconstituted upon FRL illumination because of the high-affinity interaction of the Coh2 and DocS domains (Fig. 1). Confirming the editing activity of the reconstituted Cas9, we found that HEK-293 cells cotransfected with pXY137 (PhCMV-p65-VP64-BldD-pA::PhCMV-BphS-P2A-YhjH-pA, 100 ng), pYH20 [PFRL-NLS-Cas9(N)-Linker-Coh2-pA, 50 ng], pYH102 [PhCMV-DocS-Linker-Cas9(C)-NES-pA, 100 ng], and pYW57 [PU6-sgRNA (CCR5)-pA, 50 ng] successfully edited the targeted human CCR5 locus (11.9% indel frequency) upon FRL illumination (1 mW/cm2; from an LED source, 730 nm); no editing was detected for dark control cells (Fig. 2, A and B). These detected edits were analyzed by the mismatch-sensitive T7 endonuclease I (T7E1) assay. We further used Sanger sequencing to confirm that the FRL-induced, FAST-mediated edits (indel mutations) occurred in the targeted region of the human CCR5 locus at a frequency of ~20% using the tracking of indels by decomposition (TIDE) analysis (fig. S1).

(A) Schematic of the split-Cas9 fusion protein components of the FAST system. Coh2 and DocS are two C. thermocellum proteins that interact with high affinity. Cas9 is formed from two separate (N- and C-terminal) Cas9 fragments that individually lack nuclease activity. When Cas9s two fragments Cas9(N) and Cas9(C) are respectively fused with Coh2 and DocS, they readily combine to reconstitute a nuclease-active form of Cas9. (B) Schematic of the FAST system, as deployed in mammalian cells, based on the fragments detailed in (A). FRL (~730 nm) activates the engineered bacterial photoreceptor BphS, which converts guanosine triposphate (GTP) into c-di-GMP. c-di-GMP can bind to BldD (derived from sporulating actinomycete bacteria) and be translocated into the nucleus. This induces dimerization of the synthetic transcriptional activators p65-VP64-BldD [BldD fused with p65 (the nuclear factor Btransactivating domain) and VP64 (a tetramer of the herpes simplex virusderived VP16 activation domain)], after which they bind to PFRL to activate expression of the N-terminal fusion fragment of split-Cas9. The other (C-terminal) fusion fragment is constitutively expressed, as driven by the human cytomegalovirus promoter (PhCMV). DNA double-strand breaks are formed by Cas9 after the Coh2-DocS heterodimerizationmediated reconstitution of the two fusion fragments.

(A) Time schedule of FRL-controlled gene editing in HEK-293 cells. Cells were illuminated (1 mW/cm2; 730 nm) for 4 hours once a day for 2 days and were collected at 48 hours after the first illumination for further analysis. (B) A mismatch-sensitive T7 endonuclease I (T7E1) assay to test HEK-293 cells (6 104) transfected with full-length Cas9 (pHP1) or the FAST system (pXY137, pYH20, and pYH102), together with the sgRNA targeting to CCR5 locus (pYW57). FRL-mediated editing (indel deletions) of the human EMX1, CXCR4, and VEGFA loci by FAST was performed using the same experimental procedure as that used when targeting the CCR5 gene. (C) FRL-mediated multiplex editing of the human CCR5 and CXCR4 loci. (D) FAST-mediated DNA insertion via homology-directed repair (HDR), achieved by adding a single-stranded oligodeoxynucleotide (ssODN) template (10 M), bearing a HindIII restriction endonuclease site. Homologous arms are indicated in red. The target sites of sgRNA (EMX1) are marked in blue. HEK-293 cells (6 104) were cotransfected with full-length Cas9 (pHP1) or the FAST system (pXY137, pYH20, and pYH102) and the sgRNA targeting to EMX1 locus (pYH227) via a nucleofection method. In (B) to (D), n = 2 from two independent experiments. Red arrows indicate the expected cleavage bands. Detailed description of genetic components and transfection mixtures are provided in tables S1 and S5. N.D., not detectable.

We next confirmed that the FAST system can cleave different targeted endogenous genomic loci and induce indel mutations via nonhomologous end joining (NHEJ) in an FRL-dependent manner by designing sgRNAs targeting three additional human genes (EMX1, CXCR4, and VEGFA), and these induced indel mutations were detected by T7E1 assay. With each of these sgRNAs, FRL-induced but not dark-induced indel mutations were observed (Fig. 2B). We also confirmed that the FAST system can cleave targeted exogenous d2EYFP reporter efficiently (fig. S2). In addition to single gene targeting, we also tested whether our FAST system can simultaneously edit multiple target sites. Using one sgRNA targeting CCR5 and another sgRNA targeting CXCR4, the FAST system was capable of inducing the desired indel mutations at the two target sites upon FRL illumination (Fig. 2C), demonstrating optogenetic multiplexed control of NHEJ-mediated indel mutations in mammalian cells.

We further investigated whether FAST can be used for homology-directed repair (HDR)mediated genome editing. The FAST system components and a donor template (single-stranded oligodeoxynucleotide containing a HindIII site) were electroporated into HEK-293 cells. Assessment of HDR events at the EMX1 locus using restriction endonuclease assays showed that the FAST system induced HindIII site integration at the EMX1 locus at a frequency of 5.7% under FRL illumination; no HDR events were detected in dark controls (Fig. 2D). Together, these results establish that the FAST system can be deployed for optogenetic control of NHEJ-/HDR-mediated indel mutations.

To demonstrate photoactivatable regulation of gene editing in diverse mammalian cell lines, we introduced the FAST system into four different human cell lines, and it achieved successful FRL-induced gene editing (CCR5 locus) in each of them (Fig. 3A). Next, experiments testing the FRL illumination intensity and duration-dependent activity of the FAST system showed that the frequency of edits (indel mutations at CCR5) increased along with illumination intensity and with illumination time (Fig. 3, B and C), indicating the tunability of the FAST system. We also used a photomask to establish proof of principle for spatially controlled gene editing with the FAST system (Fig. 3, D and E). We also conducted an experiment with two rounds of FRL illumination to verify repeated induction cycles of the FAST system wherein the first round of illumination achieved indel mutations guided by an sgRNA targeting CXCR4 locus, followed by transfection of a second sgRNA targeting the CCR5 locus, which guided successful indel mutations after the second FRL illumination. However, engineered cells shifted to the dark did not have indel mutations in CCR5 locus (fig. S3, A and B). This result indicates that the FAST system is reusable and reversible.

(A) FAST-mediated gene editing in four human cell lines. (B) Illumination intensitydependent FAST gene editing. In (A) and (B), cells were collected for mismatch-sensitive T7E1 assays, as indicated in the time schedule of Fig. 2A. (C) Evaluation of exposure timedependent FAST system gene editing performance. Cells were collected for T7E1 assays at 24 hours after the start of the second illumination. (D) Schematic of the photomask device used to demonstrate the spatial regulation of FAST-mediated gene editing. Cells were illuminated through a photomask containing a 7-mm line pattern. (E) Spatial control of FRL-dependent gene editing mediated by the FAST system. HEK-293 cells (3 106) were cotransfected with the FAST system, sgRNA (pYW57), and a frameshift enhanced green fluorescent protein (EGFP) reporter containing a CCR5 locus (pYH244) and were illuminated with FRL (0.5 mW/cm2; 730 nm; 2-min on, 2-min off) for 48 hours. EGFP is not expressed without Cas9 activity because the EGFP sequence is out of frame. Upon double-strand cleavage by Cas9, the frameshifts caused via DNA repair by NHEJ enable EGFP expression. The fluorescence of EGFP was assessed via fluorescence meter ChemiScope 4300 Pro imaging equipment (Clinx) at 48 hours. In (A) to (C), n = 2 from two independent experiments. Red arrows indicate the expected cleavage bands. Detailed description of genetic components and transfection mixtures are provided in tables S1 and S5. SEAP, human placental secreted alkaline phosphatase.

We then evaluated the photocytotoxicity of FRL (730 nm) or blue light (470 nm) illumination on mammalian cells. When HEK-293cells were transfected with human placental secreted alkaline phosphatase (pSEAP2)-control-and then exposed to FRL or blue light for different intensity, the SEAP expression demonstrated that the FRL exposure resulted in negligible cytotoxicity. However, a marked difference was observed from the blue light illumination, which substantially reduced cell viability (fig. S4, A and B). Moreover, we did not observe substantially increased cytotoxicity with FRL illumination of cells engineered with the FAST system (fig. S4, C and D), indicating the inertness and noncytotoxicity of the system constituents. In short, neither FRL illumination nor the ectopic presence of FAST system constituents was verified to influence the gene expression capacity of the engineered cells. In addition, we also compared the controllable gene editing performance of our FAST system with the rapamycin-responsive split-Cas9 system (13) and the blue lightcontrolled paCas9 system (20) that have been reported. The results showed that the genome editing efficiency of rapamycin-responsive split-Cas9 system was lower than the FAST system (fig. S5, A and B), and the paCas9 system had relative higher background leakage in the dark. Our FAST system showed notable induction of indel mutations under FRL illumination but with negligible background in the dark (fig. S5, C and D). Off-target activity of the FAST system was also assessed simply. We checked a potential off-target site of human BMP1 locus, as reported previously (33). The indel frequencies were determined through T7E1 assay at the on-target and potential off-target sites of BMP1. As a result, no mutations were detected at the potential off-target site after editing by our FAST system (fig. S6, A and B). This is probably due to the FAST-mediated transient expression of split-Cas9 that lowered the probability of off-target effects by reducing the exposure time of a cells genome to the Cas9 nuclease (79). However, there might be off-target effects that can still occur in illuminated cells.

Having established the basic performance characteristics of the FAST system in human cells, we next conducted experiments with mice to verify the systems capacity to induce gene editing based on the tissue-penetrating capacity of FRL. Specifically, we conducted an experiment using hollow fiber implantation of HEK-293 cells equipped with the FAST system into the dorsum of mice and exposed to FRL illumination (10 mW/cm2; alternating 2-min on/off for 4 hours) (Fig. 4A). Notably, the FRL illumination of the FAST cell-bearing mice induced notable activation of gene editing (~11.4% of the cells retrieved from the implant fibers was edited at the CCR5 locus versus not detectable for dark control cells) (Fig. 4B). These results demonstrate that the FAST system can be used to activate gene editing inside animal tissues, exploiting the physical properties of FRL as an inducer modality.

(A) Schematic for the time schedule and experimental procedure for FRL-controlled gene editing in mice harboring hollow fiber implants with HEK-293 cells. Pairs of 2.5-cm hollow fibers containing a total of 5 106 transgenic HEK-293 cells (engineered with FAST system) were subcutaneously implanted on the dorsum of wild-type mice and illuminated with FRL (10 mW/cm2; 730 nm; 2-min on, 2-min off) for 4 hours each day for 2 days. Cells were collected from the hollow fiber implants at 48 hours after the first illumination and assessed with mismatch-sensitive T7E1 assay to assess targeted gene editing efficiency (CCR5 locus). (B) Representative T7E1 assay for FAST-mediated indel mutations. n = 3 mice. The red arrow indicates the expected cleavage bands. Detailed description of genetic components and transfection mixtures are provided in table S1 and S5.

We obtained transgenic mice harboring a homozygous Rosa26 CAG [cytomegalovirus (CMV) enhancer fused to the chicken beta-actin] promoter loxP-STOP-loxP-tdTomato cassette present in all cells. In this model, tdTomato is silent because of the stop signal [three repeats of the simian virus 40 (SV40) polyadenylate (polyA) sequence], but the deletion of the stop cassette allows transcription of the tdTomato gene, resulting in fluorescence expression. The Cas9-mediated DNA cleavage of the stop sequence guided by sgRNAs can initiate CAG promoter to drive tdTomato expression (34). Therefore, we used this mouse model to examine the in vivo genome editing performance of the FAST system in mice somatic cells (Fig. 5A). We used hydrodynamic injection to introduce the FAST system components, along with an sgRNA designed to target the deletion of the SV40 polyA stop cassette, which should activate tdTomato reporter protein expression upon successful editing. Note that it is difficult to activate tdTomato expression by Cas9 system as the desired edit requires two cuts on the same allele; we eventually achieved the desired edit, but it required optimization of the delivery mode for the FAST components. Briefly, we chose hydrodynamic injection because it is known to result in enrichment of plasmids (and thus, transgene expression) in liver cells (35). We reduced the overall number of plasmids by combining some constructs (fig. S7, A and B) and explored a number of different injection time and illumination schedules (Fig. 5A), but we only detected weak tdTomato signals in the FRL-illuminated FAST mice (fig. S8).

(A) Schematic showing the time schedule and experimental procedure for assessing in vivo gene editing. The minicircle iteration of the FAST system pYH412, pYH413, and pYH414 at a 7:15:4 (w/w/w) ratio were injected hydrodynamically via tail vein. Twenty-four hours after injection, mice were illuminated with FRL (10 mW/cm2; 730 nm; 2-min on, 2-min off) for 4 hours per day for 3 days. A second injection of the minicircle-based FAST system components was performed on the fifth day, followed by 4 hours daily illumination for three additional days. In our design, the tdTomato reporter protein was expressed after a stop cassette was destroyed by Cas9 editing. (B) Fluorescence IVIS image of mouse livers. (C) The frequency of edits (targeting the aforementioned stop cassette) by monitoring fluorescence intensity of the tdTomato reporter in Gt(ROSA)26Sortm14(CAG-tdTomato)Hze mice. (D) Representative fluorescence microscopy images of tdTomato and tdTomato+ hepatocytes present in frozen liver sections from FRL-illuminated mice. Blue indicates 4,6-diamidino-2-phenylindole (DAPI) staining nuclei; red indicates endogenous tdTomato expression. The images represent typical results from three independent measurements. Scale bar, 100 m. Data in (C) are means SEM; n = 3 mice. P values were calculated by Students t test. ****P < 0.0001 versus control.

We speculated that this apparently weak induction of editing activity may result from rapid degradation of the plasmids, so we constructed minicircle (36) iterations of our FAST system. Minicircle DNA vectors without the bacterial backbone of the plasmid, markedly reducing the possibility of random integration of bacterial DNA sequences into the genome, have been shown to maintain gene expression in cells for long durations because these molecules are resistant to degradation (37). We delivered the minicircle iterations of the FAST via hydrodynamic injection and used FRL illumination schedules as follows: alternating 2-min on/off for 4 hours, once each day for 3 days; we then monitored the fluorescence signal intensity in livers. FRL illumination of the mice bearing the FAST system resulted in strong editing and thus, tdTomato reporter expression (Fig. 5, B and C). We also detected strong tdTomato expression in liver sections prepared from the FRL-illuminated FAST mice (Fig. 5D), and Sanger sequencing of genomic DNA extracted from the livers verified the success of the targeted excision of the SV40 polyA stop cassette in the FRL-induced FAST mice (fig. S9). Collectively, these results demonstrate that the FAST system can be used for in vivo editing of the genomes of somatic cells located in the internal organs of mice.

We further investigated the optogenetic activation of the FAST system in tumor models as proof-of-concept examples for therapeutic genome editing. The polo-like kinase (PLK1) protein is a highly conserved serine-threonine kinase that promotes cell division, and strong PLK1 expression is a marker in various types of tumor (38). Extensive work has established that inhibition or depletion of PLK1 leads to cell-cycle arrest, apoptosis, and a so-called mitotic catastrophe in cancer cells, which provides a promising modality for anticancer therapy (39, 40). After initially confirming that the FAST system can edit the PLK1 locus (indel mutations and extensive apoptosis) in the FRL-illuminated human lung cancer A549 cells in vitro (fig. S10, A to D), we then evaluated the tumor therapy application of our FAST system by testing the in-tumor editing performance of the FAST system for the disruption of the PLK1 locus in mice bearing A549 xenograft tumors.

We first delivered the minicircle iterations of the FAST system alongside a PLK1-targeting sgRNA minicircle vector when the tumors had reached 80 to 100 mm3; note that we also injected transfection reagent, a cationic polymer-coated nanoparticle (APC), (41) to facilitate the transfection of tumor cells in situ. Subsequently, FRL illumination was delivered to the xenograft-bearing mice via LED for 4 hours each day for 7 days (Fig. 6A), and tumor development was monitored by measuring the sizes of the tumors every 2 days. Notable inhibition of tumor growth was observed for the FAST mice that received FRL illumination; no such inhibition was observed for the dark control FAST or FRL-illuminated vehicle control mice (Fig. 6, B to D). Mismatch-sensitive T7E1 assays confirmed that the FRL-induced FAST system achieved the desired genome disruption of PLK1 gene in the tumor tissue (Fig. 6E) at a frequency of ~21.5% detected by TIDE analysis (Fig. 6F). Moreover, quantitative real-time polymerase chain reaction (qRT-PCR) verified the expected reductions in tumor PLK1 mRNA expression upon FRL illumination (Fig. 6G). Consistent with the observed antitumor efficacy, subsequent histologic analysis of tumor sections revealed extensive cancer cell necrosis (Fig. 6H) and very extensive cell apoptosis [via both terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate nick end labeling (TUNEL) and caspase-3labeling assays; Fig. 6, I and J]. Thus, FRL-triggered FAST-mediated gene editing can inhibit cancer cell growth in xenograft tumors in mice. These results further indicate that our FAST system could be deployed for deep tissue gene editing.

(A) Schematic showing the time schedule and experimental procedure for the in-tumor FAST-mediated gene editing. The minicircle iteration of the FAST system targeting to PLK1 locus pYH412, pYH420, and pYH414 at a 7:15:4 (w/w/w) ratio were injected intratumorally. Twenty-four hours after per injection, mice were illuminated with FRL (10 mW/cm2; 730 nm; 2-min on, 2-min off) for 4 hours per day totally for 7 days. (B) Images of tumor tissues from the different treatments. (C) Tumor growth curves for the different treatments. (D) The weight of tumor tissues after the different treatments. (E) Indel mutations in the tumor tissues detected via mismatch-sensitive T7E1 assays. Red arrows indicate the expected cleavage bands. (F) The gene editing efficacy quantified by the TIDE analysis. (G) Relative mRNA expression levels of the PLK1 gene quantified by quantitative real-time polymerase chain reaction (qRT-PCR). The data are means SEM; n = 5 mice. P values were calculated by Students t test. ****P < 0.0001 versus control. (H) Representative fluorescence microscopy images of hematoxylin and eosin (H&E) staining of tumor tissues. The images represent typical results from three independent measurements. Scale bar, 100 m. Representative fluorescence microscopy images of TUNEL staining (I) and caspase-3 (J) staining of tumor tissues. The images represent typical results from three independent measurements. Scale bars, 100 m. Photo credit: Yuanhuan Yu, East China Normal University.

CRISPR-Cas9 is an undeniably revolutionary technology that is changing biological and medical research (4, 5, 42), and several innovative extensions of the basic CRISPR-Cas9 concept have enabled a new era of conditional genome editing activation iterations with chemical (1015) and UV/blue light inducers (20, 22, 25). Nevertheless, limitations with these systems warrant the development of alternatives that exploit different induction sources. The FAST system we developed in the present study opens the door for spatiotemporally selective induction of Cas9 genome editing deep inside animal tissues. It bears emphasis that our induction uses LED lights rather than lasers or optical fibers, highlighting that FAST should be very easy to deploy in a wide range of experimental contexts. Although we did face initial hurdles with induction efficiency for in vivo applications, our development of a minicircle-based iteration of the FAST system easily overcame this and permitted robust editing in mouse livers. The deep tissuepenetrating utility of the FAST system was applied to achieve anticancer therapy by disrupting PLK1 gene in mice bearing A549 xenograft tumors. In this way, we could greatly reduce side effects of the anticancer drugs and promote the precision treatment of cancers. We also envision that the FAST system can be used to study the function of cancer-associated genes during tumor development process by controlling gene knockout or interference in specific tissues at different time nodes.

While we do demonstrate FAST system applications for biological research and the treatment of disease, the present paper merely reports the initial proof-of-principle study. Given that FAST is a fully genetically encoded system, a variety of vectors, alternative plasmids, and tissue-specific promoters could be used to selectively deliver FAST system components to diverse tissues, and we fully anticipate that adeno-associated virus vectors will become a popular modality for this task. Moreover, there is no obvious factor to prevent the deployment of FAST as a genome-integrated stable system, which should enable researchers to selectively activate targeted editing anywhere that they are able to supply sgRNAs and FRL illumination from an LED.

We anticipate that the combination of precise temporal control and deep tissue penetration will enable rapid-uptake FAST in a variety of research communities. Chemical inducers can cause adverse effects in cells and can diffuse freely, and the complexity of cellular and organismal metabolism makes it exceedingly difficult to precisely control the spatiotemporal dynamics of inducible gene editing systems (1619). In this light, perhaps researchers can deploy FAST and FRL induction strategies to explore the development, basic biology, or etiopathological basis of diverse processes that occur in animal internal organs such as the heart, lungs, liver, kidneys, etc., and in tissues, including muscles and bone marrow. In theory, the FAST system should give researchers previously unattainable precise control of conditional genetic knockout and knock-in experiments. A huge variety of temporal illumination schemes should be feasible with FAST because FRL has low phototoxicity, representing a clear advantage over UV- and blue lightbased Cas9 induction systems. Moreover, FAST may offer neuroscientists an alternative to the presently popular optical fiber implantationbased approaches for optogenetic-based gene editing research.

In summary, we have developed a FAST system that is apparently safe (negligible phototoxicity to mammalian cells, high tissue permeability, and noninvasiveness). With FRL as its fundamental basis, the FAST system offers excellent tunability (robust induction of gene editing and almost negligible background activity) and precise controllability (illumination intensity dependent, exposure time dependent, and strong spatiotemporal specificity), making it suitable and practical for the many biological and biomedical applications that require gene editing in vivo, especially for processes that occur within animal tissues.

The FAST system consists of the following main components: the FRL sensors (BphS and p65-VP64-BldD) (31), interacting proteins (cohesion Coh2 and dockerin DocS from C. thermocellum) (32), and the N- and C-terminal fragments of Streptococcus pyogenes Cas9 [Cas9(N) (residues 2 to 713) and Cas9(C) (residues 714 to 1368)] (13). Complementary DNAs (cDNAs) encoding BphS and p65-VP64-BldD were prepared, as previously described (31). cDNAs encoding Coh2 and DocS were chemically synthesized by the company Genewiz Inc. cDNAs encoding the N- and C-terminal fragments of Cas9 fused with a nuclear localization signal from SV40 T antigen were amplified from the Addgene plasmid 42230. The inducible Cas9 was constructed on the basis of the Cas9(N) and Cas9(C) fragments fused with Coh2 and DocS, respectively, which were cloned through Gibson assembly according to the manufacturers instructions [Seamless Assembly Cloning Kit; catalog no. BACR(C) 20144001; OBiO Technology Inc.]. All genetic components have been validated by sequencing (Genewiz Inc.). Plasmids constructed and used in this study are provided in table S1.

The sgRNAs targeting CCR5, EMX1, CXCR4, VEGFA, BMP1, tdTomato stop cassette, and PLK1 were generated by annealed oligos and cloned into the BbsI site of a constitutive mammalian PU6-driven sgRNA expression vector (pYH49). The PU6-sgRNA fragment was PCR amplified from the Addgene plasmid 58767 and then cloned into the corresponding sites (MluI/XbaI) of pcDNA3.1(+) to obtain the pYH49 expression vector. The target sequences and oligonucleotides used for sgRNA construction are listed in table S2.

All cell types {HEK-293 [CRL-1573; American Type Culture Collection (ATCC)], HeLa (CCL-2; ATCC), telomerase-immortalized human mesenchymal stem cells (43), and HEK-293derived Hana3A cells engineered for constitutive expression of RTP1, RTP2, REEP1, and Go} were cultured at 37C in a humidified atmosphere, containing 5% CO2 in Dulbeccos modified Eagles medium (DMEM; catalog no. C11995500BT; Gibco) supplemented with 10% fetal bovine serum (FBS; catalog no. 16000-044; Gibco) and 1% (v/v) penicillin/streptomycin solution (catalog no. ST488-1/ST488-2; Beyotime Inc.). All cell lines were regularly tested for the absence of mycoplasma and bacterial contamination. Cells were transfected with an optimized polyethyleneimine (PEI)based protocol (44). Briefly, cells were seeded in a 24-well cell culture plate (6 104 cells per well) 18 hours before transfection and were subsequently cotransfected with corresponding plasmid mixtures for 6 hours with 50 l of PEI and DNA mixture [PEI and DNA at a ratio of 3:1 or 5:1 (w/w)] (PEI molecular weight, 40,000; stock solution of 1 mg/ml in ddH2O; catalog no. 24765; Polysciences Inc.). At 12 hours after transfection, the culture plate was placed below a custom-designed 4 6 LED array (1 mW/cm2; 730 nm) for illumination.

For HDR-mediated genome editing experiments, 6 105 HEK-293 cells were nucleofected with the FAST system plasmids (pXY137, 200 ng; pYH20, 100 ng; and pYH102, 200 ng), sgRNA expression vector (pYH227, 100 ng; targeting EMX1), and 10 M single-stranded oligonucleotide donor using the SF Cell Line 4D-Nucleofector X Kit L (catalog no. V4XC-2024; Lonza) and the CM-130 program (4D-Nucleofector System; Lonza). At 24 hours after nucleofection, cells were illuminated by FRL (1 mW/cm2; 730 nm) for 4 hours once a day for 2 days, and then cells were collected at 48 hours after the first illumination for analysis. Genomic DNA was isolated using a TIANamp Genomic DNA Extraction Kit (catalog no. DP304; TIANGEN Biotech Inc.) according to the manufacturers instructions.

Genomic DNA was extracted from cells or tissues using the TIANamp Genomic DNA Extraction Kit (catalog no. DP304; TIANGEN Biotech Inc.) according to the manufacturers instructions. The genomic region containing the target sites was PCR amplified using the 2 Taq Plus Master Mix II (Dye Plus) DNA polymerase (catalog no. P213; Vazyme Inc.). The primers used for PCR amplification are listed in table S3. The PCR amplicons were purified using HiPure Gel Pure Micro Kits (catalog no. D2111-03; Magen Inc.) according to the manufacturers protocol. Purified PCR products (300 ng) were mixed with 1.5 l of 10 M buffer for restriction enzyme (catalog no.1093A; Takara Bio) and ultrapure water to a final volume of 15 l and reannealed (95C, 5 min; 94C, 2 s, 0.1C per cycle, 200 times; 75C, 1 s, 0.1C per cycle, 600 times; and 16C, 5 min) to form heteroduplex DNA. After reannealing, the heteroduplexed DNA was treated with 5 U of T7E1 (catalog no. M0302; New England BioLabs) for 1 hour at 37C and then analyzed by 1.5% agarose gel electrophoresis. Gels were stained with GelRed (catalog no. 41003; Biotium) and imaged with Tanon 3500 gel imaging system (Tanon Science & Technology Inc.). Relative band intensities were calculated by ImageJ software. Indel percentage was determined by the formula 100% [1 (1 (b + c)/(a + b + c))1/2], in which a is the integrated intensity of the undigested PCR product, and b and c are the integrated intensities of each cleavage product.

Sequence of the gene region containing the target sequence was amplified by PCR. Purified PCR amplicons from the nuclease target site were cloned into the T-vector pMD19 (catalog no. 3271; Takara Bio). Thirty clones were randomly selected and sequenced using each genes PCR forward primers by the Sanger method (45). Primers used for PCR amplification are listed in table S3.

Target regions were amplified by PCR. Purified PCR samples were analyzed by Sanger sequencing. The sequencing data files (.ab1 format) were imported into the TIDE Web tool (https://tide.nki.nl/) (46) to quantify nature and frequency of generated indels.

The genomic PCR and purification were performed, as described above. Purified PCR products were mixed with 15 U of HindIII (catalog no. 1060B; Takara Bio), 2 l of 10 M buffer for restriction enzyme, and ultrapure water to a final volume of 20 l and then incubated at 37C for 3 hours. The digested products were analyzed by agarose gel electrophoresis. Gel staining and imaging were performed, as described above. Quantification was calculated on the basis of relative band intensities. The HDR percentage was determined by the formula 100% (b + c)/(a + b + c), in which a is the intensity of the undigested PCR product, and b and c are the intensities of each HindIII-digested product.

HEK-293 cells (6 104) were cotransfected with the FAST system (pXY137, 100 ng; pYH20, 50 ng; and pYH102, 100 ng), the sgRNA targeting d2EYFP (pYH410, 50 ng), and the d2EYFP reporter plasmid (pYW110, 200 ng). At 12 hours after transfection, cells were illuminated (1 mW/cm2; 730 nm) for 4 hours once a day for 2 days and were harvested after trypsinization and washed in phosphate-buffered saline (PBS) for three times. About 10,000 events were collected per sample and analyzed with a BD LSRFortessa cell analyzer (BD Biosciences) equipped for d2EYFP [488-nm laser, 513-nm longpass filter, and 520/30 nm emission filter (passband centered on 530 nm; passband width of 30 nm)] detection. Data were analyzed using the FlowJo V10 software.

The production of human placental SEAP in cell culture medium was quantified using a p-nitrophenylphosphatebased light absorbance time course assay, as previously reported (31). Briefly, 120 l of substrate solution [100 l of 2 SEAP buffer containing 20 mM homoarginine, 1 mM MgCl2, and 21% (v/v) diethanolamine (pH 9.8) and 20 l of substrate solution containing 120 mM p-nitrophenylphosphate] were added to 80 l of heat-inactivated (65C, 30 min) cell culture supernatant. The time course of absorbance at 405 nm was measured by using a Synergy H1 hybrid multimode microplate reader (BioTek Instruments Inc.) installed with the Gen5 software (version 2.04).

Cell viability was assayed using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cytotoxicity assay kit (catalog no. E606334-0250; Sangon Biotech Inc.) according to the manufacturers instructions. Briefly, 10 l of MTT reagent (5 mg/ml) was added to each well of 96-well plates. The samples were mixed gently and incubated for 4 hours in a CO2 incubator. Formazan solubilization solution (100 l) was added into each well. The plate was put on a shaker to mix gently for 10 min to dissolve the formazan crystals, and then the plate was read with a Synergy H1 microplate reader (BioTek Instruments Inc.) at 570 nm.

The off-target sites of the BMP1 gene were examined according to the previously reported procedure (33). Genomic DNA was extracted, as described above, and the region of genome containing the possible nuclease off-target sites was PCR amplified using appropriate primers (table S3). The following procedures were similar to those of on-target examination by T7E1 assay, as described above.

Minicircles are episomal DNA vectors that allow sustained transgene expression in quiescent cells and tissues. Minicircle DNA vectors were prepared, as previously described (36). Minicircle-producing system contains the Escherichia coli strain ZYCY10P3S2T (a genetically modified minicircle-producing bacterial strain) and the empty minicircle-producing plasmid pMC.BESPX (gene of interest would be cloned into this plasmid). Briefly, ZYCY10P3S2T competent cells prepared with standard protocol, as previously described (36), were transformed with the minicircle-producing plasmid pMC.BESPX carrying the gene of interest. The transformed cells were cultured and induced by 0.01% l-arabinose to produce minicircle DNA vectors that were devoid of the bacterial plasmid DNA backbone and contain only genes of interest.

The in vivo DNA delivery reagent APC is a cationic polymer-coated nanoparticle composed of biocompatible polystyrene sulfonate and -cyclodextrinPEI (Mw, 25 kDa) and prepared, as previously reported (41). First, the seed solution was prepared by adding freshly prepared 600 l of NaBH4 (10 mM) into 5-ml mixture of HAuCl43H2O (0.5 mM) and cetyltrimethylammonium bromide (CTAB; 0.1 M) and incubated at 30C for 30 min. Ten milliliters of HAuCl43H2O (1 mM), 10 ml of CTAB (0.2 M), 120 l of AgNO3 (0.1 M), and 600 l of hydroquinone (0.1 M) were mixed together as growth solution. When the color of the growth solution turned from yellow to colorless, 320 l of seed solution was added. The desired longitudinal surface plasmon resonance peak was obtained after keeping the reaction mixture undisturbed in dark at 30C for 12 hours. The products were then gathered by centrifugation at 7000 RCF (relative centrifugal force) for 10 min at 30C. The supernatant was removed, and the precipitate was resuspended in 2 ml of 30C ultrapure water. Furthermore, 1 ml of the products from last step [Au (0.2 mg/ml)] was added to 10 ml of polysodium 4-styrenesulfonate (2 mg/ml) dissolved in NaCl (1 mM) solution and stirred for 1 hour at 30C. The solution was centrifuged at 7000 RCF for 10 min, and the residue was resuspended to obtain 2 ml of biocompatible polystyrene sulfonatecoated nanoparticle solution. Last, 1 ml of biocompatible polystyrene sulfonatecoated nanoparticles was added to 10 ml of -cyclodextrinPEI (2 mg/ml) dispersed in NaCl (1 mM) solution and stirred for 1 hour at 30C to obtain APC.

Apoptosis analysis at the cellular level was assessed using the Annexin Vfluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Detection Kit (catalog no. E606336; Sangon Biotech Inc.). Briefly, A549 cells (3 104) cotransfected with the minicircle iterations of the FAST system and the sgRNA targeting PLK1 {pYH412 (PhCMV-p65-VP64-BldD-pA::PhCMV-BphS-P2A-YhjH-pA, 135 ng), pYH414 [PFRL-NLS-Cas9(N)-Linker-Coh2-pA, 77 ng], and pYH420 [PU6-sgRNA (PLK1)::PhCMV-DocS-Linker-Cas9(C)-NES-pA, 288 ng]} were illuminated by FRL (1 mW/cm2; 730 nm) for 4 hours once a day for 2 days and were then collected at 48 hours after the first illumination for analysis. The subsequent procedures were performed according to the manufacturers instructions and analyzed by flow cytometry (BD LSRFortessa cell analyzer; BD Biosciences). The LSRFortessa was equipped with green fluorescence channel (488-nm laser, 530/30 nm emission filter, 505 nm longpass dichroic mirror) and red fluorescence channel (561-nm laser, 610/20 nm emission filter, 595 nm longpass dichroic mirror). A gate was applied on forward scatter and side scatter to remove debris from cell populations. Data were analyzed using the FlowJo V10 software.

Total RNA of cells or tissues was extracted using the RNAiso Plus kit (catalog no. 9109; Takara Bio). A total of 500 ng of RNA was reverse transcribed into cDNA using a PrimeScript RT Reagent Kit with the genomic DNA Eraser (catalog no. RR047; Takara Bio). Quantitative PCR (qPCR) reactions were performed on the LightCycler 96 real-time PCR instrument (Roche Life Science) using the SYBR Premix Ex Taq (catalog no. RR420; Takara Bio). Program for qPCR amplifications were as follows: 95C for 10 min, followed by 40 cycles at 95C for 10 s, 60C for 15 s, and 72C for 10 s, and then 95C for 10 s, 60C for 60 s, 97C for 1 s, and last, 37C for 30 s. The qPCR primers used in this study are listed in table S4. Samples were normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the endogenous control. Standard Ct method was used to obtain relative mRNA expression level.

Wild-type mice [8 week old, male, C57BL/6J, East China Normal University (ECNU) Laboratory Animal Center] were randomly divided into two groups. The semipermeable KrosFlo polyvinylidene fluoride hollow fiber membrane (Spectrum Laboratories Inc.; notably, the light-absorption properties of this material to lights of 300 to 1000 nm are almost the same) implants containing optogenetically engineered HEK-293 cells (pairs of 2.5-cm hollow fibers containing a total of 5 106 engineered cells) were subcutaneously implanted beneath the dorsal skin of the mice under anesthesia (two 2.5-cm hollow fibers in each mouse). At 1 hour after implantation, the mice were illuminated by FRL (10 mW/cm2; 730 nm; 2-min on, 2-min off, alternating, to avoid the thermal discomfort in mice caused by continuous illumination) for 4 hours once a day for 2 days. The control mice were kept in dark. Cells were then collected from the implanted hollow fibers at 48 hours after the first illumination, and the genomic DNA was extracted for mismatch-sensitive T7E1 assay to quantify the indel mutations of the endogenous gene CCR5.

The transgenetic Ai14 tdTomato reporter mice [6 week old, female, Gt(ROSA)26Sortm14(CAG-tdTomato)Hze, from the Jackson laboratory; Ai14 is a Cre reporter allele designed to have a loxP-flanked stop cassette, preventing the transcription of a CAG promoterdriven red fluorescent tdTomato, all inserted into the Gt(ROSA)26Sor locus] were randomly divided into three groups (vehicle, FAST without illumination, and FAST with FRL). The minicircle DNA vectors encoding the FAST system {pYH412 (PhCMV-p65-VP64-BldD-pA::PhCMV-BphS-P2A-YhjH-pA, 81 g), pYH413 [PU6-sgRNA (tdtomato stop cassette)::PhCMV-DocS-Linker-Cas9(C)-NES-pA, 173 g], and pYH414 [PFRL-NLS-Cas9(N)-Linker-Coh2-pA, 46 g]} were dissolved in Ringers solution [NaCl (8.6 g/liter), KCl (0.3 g/liter), and CaCl2 (0.28 g/liter)] and injected into mices tail vein by hydrodynamic injection. The injection volume of the DNA mixture solution was 100 l per mouse weight (gram). Twenty-four hours after injection, mice were illuminated with FRL (10 mW/cm2; 730 nm; 2-min on, 2-min off, alternating, to avoid the thermal discomfort in mice caused by continuous illumination) for 4 hours per day for 3 days (according to the time schedule in Fig. 5A). A second-round injection of the minicircle-based FAST system was performed on the fifth day, followed by 4 hours of daily illumination for three additional days. On the 15th day after the first hydrodynamic injection, mice were euthanized, and the livers were isolated for fluorescence imaging or histological analysis. The tdTomato signal from isolated liver was detected using IVIS Lumina II in vivo imaging system (PerkinElmer, USA) and frozen tissue section histological analysis.

First, dissected liver tissue blocks were soaked in 4% paraformaldehyde for 2 hours. Subsequently, the tissue blocks were stepwise dehydrated with 15% sucrose solution overnight and then soaked in 30% sucrose solution for another 3 hours. After being washed three times with PBS, freshly dissected tissue blocks (<5 mm thick) were placed on to a prelabeled tissue base mold and embedded in Tissue-Tek optimal cutting temperature (O.C.T.) compound (catalog no. 4583; Sakura). These tissue blocks were stored at 80C for freezing until ready for sectioning. The tissues were sliced into frozen sections with 5-m thickness using Cryostat Microtome (Clinical Cryostat; CM1950; Leica) for further processing or stored at 80C ultralow-temperature freezer.

A total of 5 106 of A549 cells were suspended in 0.2 ml of sterile PBS and subcutaneously injected onto the back of the 6-week-old female BALB/c nude mice (ECNU Laboratory Animal Center). When the tumor size reached about 80 to 100 mm3, APC/FAST complex containing 20 l of APC and the minicircle iteration of the FAST system {pYH412 (PhCMV-p65-VP64-BldD-pA::PhCMV-BphS-P2A-YhjH-pA, 2.7 g), pYH414 [PFRL-NLS-Cas9(N)-Linker-Coh2-pA, 1.5 g], and pYH420 [PU6-sgRNA (PLK1)::PhCMV-DocS-Linker-Cas9(C)-NES-pA, 5.8 g]} were injected intratumorally. These injected mice were randomly divided into two groups (dark and illumination). Injections were conducted under anesthesia once every 2 days for five times. Twenty-four hours after every injection, mice were illuminated with FRL (10 mW/cm2; 730 nm; 2-min on, 2-min off, alternating, to avoid the thermal discomfort in mice caused by continuous illumination) according to the time schedule in Fig. 6A or kept in dark. Mice of the vehicle control group were intratumorally injected with 20 l of APC and 50 l of PBS and were then illuminated with FRL (10 mW/cm2; 730 nm; 2-min on, 2-min off), as indicated in Fig. 6A. The tumor sizes and the body weights of mice were measured every 2 days. On the 15th day after the first intratumor injection, all mice were sacrificed and tumor weights were recorded. The tumor volumes were measured using a digital caliper and calculated by the following formula: tumor volume = [length of tumor (width of tumor)2]/2. Then, tumors were isolated for indel mutation analysis and tumor apoptosis detection by hematoxylin and eosin (H&E) staining, TUNEL, and caspase-3labeling assays.

Glass slides that hold the frozen tissue sections were washed with PBS three times for 5 min each time, transferred to 0.5% Triton X-100 (dissolved in PBS; Sigma-Aldrich) for 10 min, and washed with PBS twice for 5 min each time. The slides were rinsed in running tap water at room temperature for 1 min. The samples were then stained in hematoxylin staining solution (catalog no. E607317; Sangon Biotech Inc.) for 8 min and washed in running tap water for 10 min. Next, the samples were differentiated in 1% acid alcohol for 10 s, washed in running tap water for 30 min, and were then counterstained in eosin staining solution (catalog no. E607321; Sangon Biotech Inc.) for 30 s to 1 min and washed in running tap water for 10 min. Last, the tissue sections were sealed by a drop of mounting medium over the tissue and then covered by a coverslip. The prepared slides were then observed by a microscope (DMI8; Leica) equipped with an Olympus digital camera (Olympus DP71; Olympus).

A TUNEL Apoptosis Assay Kit (catalog no. 30063; Beyotime Biotechnology Inc.) was used to evaluate tumor tissue apoptosis according to the manufacturers instructions. After washing three times with PBS, the slides were incubated with 4,6-diamidino-2-phenylindole (DAPI) solutions (5 g/ml; catalog no. C1002; Beyotime Inc.) for 2 to 5 min at room temperature. The slides were further washed three times with PBS and mounted with the antifade mounting media. Last, the slides were sealed and observed by a fluorescence microscope (DMI8; Leica) equipped with an Olympus digital camera (Olympus DP71; Olympus). TUNEL-positive nuclei were stained green, and all other nuclei were stained blue.

Isolated tumor frozen tissue sections were thawed at room temperature for 15 min and rehydrated in PBS for 10 min. The tissue samples were surrounded with a hydrophobic barrier using a barrier pen after draining the excess PBS. Then, the slides were soaked in 0.5% Triton X-100 (dissolved in PBS; catalog no. 9002-93-1; Sigma-Aldrich) for 20 min. Nonspecific staining between the primary antibodies and the tissue samples was blocked by incubating sections in the block buffer (1% FBS in PBS) for 1 hour at room temperature. After incubating with the anticaspase-3 antibody (1:100; catalog no. ab32351; Abcam) overnight at 4C, the slides were washed three times for 15 min each time in PBS and then incubated with the Alexa Fluor 555 goat anti-rabbit immunoglobulin G antibody (1:500; catalog no. ab150078; Abcam) for 1 hour at room temperature. After washing three times with PBS, the slides were incubated with DAPI solutions (5 g/ml; catalog no. C1002; Beyotime Inc.) for 2 to 5 min at room temperature. The slides were further washed three times with PBS and mounted with the antifade mounting media. Last, the slides were sealed and observed by a fluorescence microscope (DMI8; Leica) equipped with an Olympus digital camera (Olympus DP71; Olympus). Caspase-3positive cytoplasm was stained red, and all nuclei were stained blue.

All experiments involving animals were conducted in strict adherence to the guidelines of the ECNU Animal Care and Use Committee and in direct accordance with the Ministry of Science and Technology of the Peoples Republic of China on Animal Care. The protocols were approved by the ECNU Animal Care and Use Committee (protocol IDs, m20180105 and m20190607). All mice were euthanized after the termination of the experiments.

All in vitro data represent means SD and are described separately in the figure legends. For the animal experiments, each treatment group consisted of randomly selected mice (n = 3 to 5). Comparisons between groups were performed using Students t test, and the results are expressed as means SEM. GraphPad Prism software (version 6) was used for statistical analysis.

Acknowledgments: We are grateful to all the laboratory members for cooperation in this study, especially J. Jiang, S. Zhu, and X. Yang. Funding: This work was financially supported by the grants from the National Key R&D Program of China, Synthetic Biology Research (no. 2019YFA0904500), the National Natural Science Foundation of China (NSFC; no. 31971346 and no. 31861143016), the Science and Technology Commission of Shanghai Municipality (no. 18JC1411000), the Thousand Youth Talents Plan of China, and the Fundamental Research Funds for the Central Universities to H.Y. This work was also partially supported by NSFC no. 31901023 to N.G. We also thank the ECNU Multifunctional Platform for Innovation (011) for supporting the mouse experiments and the Instruments Sharing Platform of School of Life Sciences, ECNU. Author contributions: H.Y. conceived the project. H.Y. and Y.Y. designed the experiment, analyzed the results, and wrote the manuscript. Y.Y., X.W., J.S., H.L., and Y.C. performed the experimental work. Y.P., D.L., and N.G. analyzed the results and revised the manuscript. All authors edited and approved the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors. All genetic components related to this paper are available with a material transfer agreement and can be requested from H.Y. (hfye{at}bio.ecnu.edu.cn).

Read more:

Engineering a far-red lightactivated split-Cas9 system for remote-controlled genome editing of internal organs and tumors - Science Advances

COVID-19 Analysis | Humanized Mice Model Market by Trends, Dynamic Innovation in Technology and 2026 Forecasts – Cole of Duty

Posted on by

Persistence Market Research (PMR) has published a new research report on humanized mice model. The report has been titled,Humanized Mice Model Market: Global Industry Key Analysis, Size, Share, Growth, Trends and Forecast 2017 2026.In order to extemporize pre-clinical studies, humanized mice models surface as a better solution. With further development in genomic analysis, there is also a chance of refinement in the mouse models.

Humanized mice models have been in use mice for better understanding of the disease, in order to design effective therapies, create accurate models of drug metabolism and improve the understanding of mammalian and human genome function. Market players are incessantly competing for a significant value share and expansion of their global outreach by investing in technologies.

Get Sample Copy of Report @ https://www.persistencemarketresearch.com/samples/22714

Company Profiles

Get To Know Methodology of Report @ https://www.persistencemarketresearch.com/methodology/22714

With growing need to develop better varieties of humanized mice model and improve the quality and outcome of research studies, the demand for humanized mice models is increasing. However, various technical restrictions may pose as a challenge for the development of the market. Some of the leading companies operating in the market are The Jackson Laboratory, Crown Bioscience Inc. (JSR Life Science), Charles River Laboratories International, Inc., Taconic Biosciences, Inc., Ingenious Targeting Laboratory Inc., Trans Genic Inc., Ltd., GenOway, Creative Animodel, and Horizon Discovery Group plc.

According to the report, the globalhumanized mice model marketis projected to exhibit a CAGR of 6.0% from 2017 to 2026. In 2017, the market was worth US$ 67.5 Mn and it has been estimated that it will touch a valuation of US$ 113.5 Mn by the end of 2026.

Technological Development to Trigger Demand for Humanized Mice Models

Novel technologies are being developed with each passing day, which permit robust genetic modification of mice, which accelerates the development of new immune deficient mice models for capitalizing on the discovery of novel approaches to enhance the engraftment of human cells or tissues.

Moreover, speedy technological advancements have been witnessed in the last 20 years, which comprise the genetic engineering of mouse genome such as the development of knock-out mouse, knock-in mouse as well as the transgenic mouse.

Access Full Report @ https://www.persistencemarketresearch.com/checkout/22714

The aptitude to engraft mouse liver with human hepatocytes and mouse bone marrow with human hematopoietic stem cells is also foreseen to provide new opportunities, proliferating the market growth. These models also overcome the limitations of xenograft models as they elucidate disease etiology, tumor progression and metastasis.

Moreover, humanized mice strains provide better research models than working with mutant mouse protein. They are considered as more realistic models for tumor studies than the dish-grown cancer cells, and are also used for safety evaluation when neither normal mice nor rats can be used in particular biologics.

Availability of Substitutes to Hinder Market Growth

In addition to mice, other genetically modified species that can be used as models are being carefully measured for research purpose. Lately, knockout of CFTR gene has been stated in the pig as it is similar in size to humans and may be a superior model for the research on cardiovascular and metabolic diseases. Experimental advancements employing computer simulation, in silico, in vitro, and other non-animal approaches are also being taken up for the replacement of animal studies.

View original post here:

COVID-19 Analysis | Humanized Mice Model Market by Trends, Dynamic Innovation in Technology and 2026 Forecasts - Cole of Duty

A New Economies ETF to Invest in Technological Advances of Tomorrow – ETF Trends

Posted on by

As we witness the of new economies fueled by innovative companies disrupting traditional industries, investors can also consider targeted exchange traded fund strategies that can capitalize on the growth opportunities through leveraging advancements in exponential processing power, artificial intelligence, robotics, automation, and more.

In the recent webcast, Investing in the New Economy: Growth Opportunities through Technology Advances, Matthew Bartolini, Head of SPDR Americas Research, State Street Global Advisors, will delve, highlighted the rapid shifts in the way we interact with the world this year, with increased video conferencing, working from home and rising demand for telehealth after the coronavirus outbreak upended the normal economy.

Societal trends have been upended, with consumer and corporate behavior altered, leading to an increase in interest in how to stay connected, Bartolini said.

Consequently, as we move on and adapt, technological innovation will be at the forefront of reshaping our way of life. It will touch every industry and be the catalyst for new ones, he added.

For example, Bartolini andRic Edelman, Founder, Edelman Financial Engines, listed out potential opportunities in advanced medicine, telehealth services, remote access capabilities, document storage, videoconferencing, digital payments, interactive home entertainment, new forms of media consumption, cybersecurity and updated infrastructures in what may be described as a tech inflection point.

Investors are likely to seek out specific exposure tools to capture one or all of these next-generation (NextGen) trends, and those thematic funds are a diverse group of exposures and construction, Bartolini added.

As a way to help investors capture these new economies, John van Moyland, Managing Director, Global Head of S&P Kensho Indices, S&P Dow Jones Indices, highlighted the S&P Kensho New Economies Composite indices, a comprehensive framework with which to understand and precisely capture exponential innovation and growth. For example, the Intelligent Infrastructure segment captures industries involving smart buildings and smart grids; Smart Transportation covers drones, autonomous vehicles, and advanced transportation systems; and Future Security encompasses space, robotics, cybersecurity, smart borders, wearables, drones, and virtual reality.

You need to understand which companies are leading the innovation, van Moyland said. The current classification schemes, such as GICS, are not particularly well suited to identifying specific areas of innovation: as functional models, they arent sufficiently precise.

The Kensho indexing methodology starts with expert curation, combined with advanced topic modeling, to create a comprehensive model of the target industry. The indices incorporate A.I. selection to identify companies from SEC filings to capture both pure-play names and supporting ecosystems. The companies are then categorized according to their focus on the new economy.

The components are weighted by an algorithmic proxy where a holding is overweight as subsector demonstrates enormous potential and expectations are high; increasing underweight approaching the peak of inflated expectations as visibility is growing rapidly; underweight as the subsector corrects from over-inflated expectations with negative returns and high volatility; and lastly, increasing overweight as the subsector gains equilibrium between market expectation and reality.

By targeting firms within the sectors driving innovation within the new economy, the result is a high beta, multi-cap, high active share exposure to pursue long-term growth potential, Bartolini said.

Bartolini highlighted the SPDR Kensho New Economies Composite ETF (NYSEArca: KOMP) as a way to complement a core position with a broad-based exposure focused on companies driving innovation within the Fourth Industrial Revolution. KOMP includes New Economy industries, ranging from 3D printing to genetic engineering, which can provide investors access to a comprehensive and diversified set of companies propelling innovation.

Financial advisors who are interested in learning more about investment opportunities in technology advances can watch the webcast here on demand.

View original post here:

A New Economies ETF to Invest in Technological Advances of Tomorrow - ETF Trends

Explained: How Different Vaccines Work, Including COVAXIN and ZyCoV-D – The Wire

Posted on by

The world is waiting with bated breath for a functional COVID-19 vaccine. In the past century, vaccines have rescued humankind from many infectious diseases.

What makes designing a potential vaccine for COVID-19 challenging? To answer this question, it is important to understand how a vaccine elicits an appropriate immune response in the body.

Active and passive immunisation

The immune response towards any infection can be passive or active. Passive immunisation is when antibodies are directly transferred from one individual to another. Such passively transferred antibodies accord immediate protection but it wanes gradually and the individual eventually becomes susceptible to the disease again. It could be natural or artificial.

Placental transfer of antibodies from the mother to the foetus gives natural passive immunity. Convalescent plasma therapy, involving the transfer of plasma containing specific antibodies from recovered individuals to susceptible individuals, provides passive immunity as well.

Vaccines provide active immunisation: they deliberately introduce a foreign substance, called an antigen, into the body to induce the body to mount an immune response. Though the protection is not conferred immediately, the immunity lasts for a considerably longer period once established. Repeated doses of the same antigen could boost immunity further.

How does a vaccine work?

A vaccine works like a virus and initiates an immune response but without causing major illness.

The immune response is brought on by different types of cells. However, a specialised group of white blood cells, called the B and T lymphocytes, are important to sustain the immune response in the long run.

When an antigen enters the body, cells called dendritic cells get attracted to it, and then carry the antigen to T lymphocytes. The T lymphocytes identify these antigens and bind to them.

Meanwhile, B lymphocytes also pick up the antigens, process them and present them to the T lymphocytes. After this interaction, T lymphocytes release signalling molecules called cytokines, which stimulate the B lymphocytes. In response, the B lymphocytes rapidly turn into plasma and memory B cells. One B lymphocyte can produce thousands of such daughter cells in a few days.

The plasma cells are responsible for secreting antibodies that will tackle the antigen and eliminate the infection. And once the infection has been removed from the body, the plasma cells die while memory B cells rest in the bone marrow, and keep secreting low levels of antibodies.

When the body is exposed to the same antigen again, the circulating antibodies bind to the antigen. This is what they mean when they say the immune system becomes familiar with the antigen, and the immune response the second time is even more effective. This memory is known as immunological memory, and it forms the basis of vaccination.

Live attenuated and inactivated killed vaccines

Ideally, a vaccine should trigger an adequate immune response without harming the body. There are different types of vaccines to achieve this outcome. Conventional vaccines fall into two broad categories: live attenuated vaccines and inactivated killed vaccines.

Live attenuated vaccines contain whole virus particles. Inducing the virus to replicate under unnatural conditions reduces its virulence. For example, researchers could have injected the virus into an unnatural host, causing the virus to eventually lose its adaptation towards the actual host, and transform to a less virulent form. That is, it can no longer cause disease as well as it could before. This process is called attenuation.

The level of attenuation is critical to a vaccines success. Over-attenuation could render the vaccine ineffective, while under-attenuation could cause the vaccine itself to produce disease.

The chickenpox, measles, mumps and rubella vaccines are all live vaccines.

Inactivated vaccines contain a part of the virus instead of the whole. During preparation, researchers remove those parts of the virus required for viral replication, making these vaccines safer than the live attenuated type.

On the flip side, inactivated vaccines in general dont accord long-lasting protection, like live vaccines. Sometimes, a substance called an adjuvant is added to inactivated vaccines to boost the immune response and make them last considerably longer. However, including an adjuvant increases their overall cost.

The COVAXIN vaccine against COVID-19, developed by Hyderabad-based Bharat Biotech, is an inactivated vaccine with alum as adjuvant.

DNA vaccines

Apart from conventional vaccines, in the last few years, researchers have tried a new generation of vaccines. One of them is a DNA vaccine. The ZyCoV-D vaccine being developed by Zydus Cadila is of this type.

Cells have DNA in their chromosomes and also outside the chromosomes in a form called plasmids. First, researchers obtain plasmids from a bacterium. Then, they separate some genetic material from the virus and insert it into the plasmid, and inject this plasmid into the body.

The viral genes then integrate themselves into cells in the body and begin to express foreign proteins. This triggers an immune response.

Plasmid DNA can be easily constructed and manipulated by genetic engineering. So its not hard to produce DNA vaccines in large volumes which then means they are quite cost-effective.

However, although experiments with DNA vaccines on animal models have been successful, not one is currently available for human use. One reason is that DNA vaccines have been found to elicit a less pronounced immune response in humans than in animals. Scientists are working on several strategies to overcome this shortcoming.

In this context, if ZyCoV-D successfully completes human clinical trials and is found to be efficacious (and safe), the occasion will undoubtedly break new ground in the history of vaccines.

Niranjana Rajalakshmiis a veterinary microbiologist.

Read the original post:

Explained: How Different Vaccines Work, Including COVAXIN and ZyCoV-D - The Wire

Liver Protein Could Be Behind Exercises Brain Benefits – Technology Networks

Posted on by

Whether you opt for a stroll in the park, a few lengths of the swimming pool or perhaps an all-out HIIT session that makes you question how your legs will possibly carry you around for the rest of the day exercise is often either a passion or a chore.We know that it is good for us science says so. Exercise can dramatically reduce your risk of developing a major illness, such as Type 2 diabetes, heart disease or cancer, by up to 50%, in addition to helping you live longer. It's also good for the aging brain exercise is correlated with a reduced risk for mild cognitive impairment and also improves cognition in populations that are at-risk for Alzheimer's disease.

Alana Horowitz, a graduate student in the Villeda lab at the University of California San Francisco (UCSF), told Technology Networks: "In interventional studies, where participants will engage in supervised exercise, researchers can more specifically observe the state of memory before and after exercise. Theyve shown that improvements in fitness level from these exercise regimens correlate with improvements in cognitive tasks, such as delayed word recall."

What's remarkable about these data, Horowitz says, is that exercise is effective regardless of when it's implemented. You can start exercising at mid-life or later in life, in your 50s, 60s or 70s and still reap the cognitive benefits.

The health benefits associated with exercise are often sufficient to inspire individuals to exercise, even if it isn't their favourite task in the world. However, due to physical limitations such as age or fragility, not everyone is able to easily hop on a treadmill.

In a new study, published in the journal Science, Horowitz and colleagues at UCSF make some interesting discoveries surrounding a little-studied liver protein. Their findings suggest that it might be responsible for the neurological benefits associated with exercise in the aging brain. Their work is in mice, but it's possible that with extended research their findings could one day be helpful for individuals that are physically unable to exercise.

Firstly, Horowitz and colleagues assessed the brains of aged mice who had been exercising for several weeks: "Our mice underwent what we call a 'voluntary running' paradigm for six weeks. We added a running wheel to each of their cages, so they could run as much or as little as they wished," said Horowitz. Age-matched mice were provided with nesting material as a control measure.

The scientists found that exercise induced adult neurogenesis, increased expression of brain-derived neurotrophic factor and improved hippocampal-dependent learning and memory when compared to controls.

The blood from active mice was then transferred to sedentary mice over a treatment period of four weeks, which resulted in improvements in learning and memory tasks and increased neurogenesis that mirrored the earlier results found in the active mice. What components of the active mice's blood could have caused these changes in the brain?

"We first narrowed our candidate pool to 12 factors that changed in both mature and aged mice. This gave us confidence that we were looking at a robust phenomenon. From those 12 candidates, we did a deep dive in the literature to look for any hints that the proteins might be linked to cognition or neurogenesis. We chose to pursue Gpld1 because, although it had not been previously linked to cognition, it was implicated in the widest range of processes from hormonal responses to metabolic processes," said Horowitz.

"We figured that if the protein had already been investigated thoroughly, someone would have stumbled upon this effect," Saula Villeda, assistant professor in the Department of Anatomy at UCSF, added in a press release. "I like to say -- if you're going to take a risk by exploring something new, you might as well go big!"

The scientists discovered that, post-exercise, Gpld1 a protein produced in the liver was increasingly expressed in the blood circulation of the mice, and this correlated with improvements in the animal's cognitive performance assessed by a radial arm water maze test (RAWM) and contextual fear conditioning behavioral tests. Interestingly, elevated Gpld1 has also been found in the blood of active elderly adults when compared to sedentary adults in human research.

Once overproduction of Gpld1 had been triggered, the mice were subject to a multitude of tests including RAWM, forced alternation (a Y maze test) and novel object recognition tests (NOR). "Our cognitive tests are designed to be sensitive to age-related changes. They can detect cognitive deficits associated with age and if were able to reverse that decline with our interventions."

But can assessing the cognition of mice be compared to an assessment in humans? "Some of the tests are actually pretty similar to cognitive tests used in humans. The limitation to them is what motivates the mouse to participate in the task. For example, if you wanted to test my memory, you can just ask me 'Look at these two objects, which one have I shown you before?'. We cant ask that to a mouse, so we have to take advantage of their primal instincts, such as their natural tendency to explore an object they havent seen before, to measure their memory," said Horowitz.

Taken together, these results imply that Gpld1 could be a molecule implicated in the cognitive improvements associated with exercise. When asked for how long the improvements were observed in the mice overexpressing Gpld1, Horowitz said: "The short answer is, we dont know the limit of these effects yet. We measured as far out as 60 days after treatment and saw benefits at that time point. We havent tracked further out to see if or when the effects fade."

To be honest, I didnt expect to succeed in finding a single molecule that could account for so much of the benefits of exercise on the brain. It seemed more likely that exercise would exert many small, subtle effects that add up to a large benefit, but which would be hard to isolate. Villeda said. When I saw these data, I was completely floored.

But is Gpld1 the only molecule responsible for inducing these effects? Horowitz suspects not.

Whilst the scientists did test another candidate from their pool of 30 proteins Pon1, a liver-derived circulating factor it was not sufficient to significantly enhance cognition. "I fully expect there to be other candidates that can have similar effects or synergies with Gpld1 to benefit other aspects of brain function," Horowitz commented.

The physiological role of Gpld1 is somewhat elusive. Nevertheless, cleavages of its substrates are implicated in signaling cascades linked to blood coagulation and inflammation. The team therefore propose that, considering inflammation and blood coagulation are elevated in aging individuals and are linked to cognitive decline, Gpld1 might be eliciting some form of reversal effect on these processes.

Analyzing the protein's physiological role will be their next step, according to Horowitz: "While we tested changes in neurogenesis and cognition, effects on other cell types or brain processes are still unknown. We also need to figure out how the targets of Gpld1 mediate these effects. Were actively working to tease apart these complex signaling networks and are really excited to see where it leads."

Hypothetically speaking, might there one day be a pill that can produce the beneficial brain effects of exercise, without actually exercising? Whilst this could improve the lives of individuals that are physically limited, it's very early days, and there are several factors that need further exploration and clarification.

For example, neurogenesis was one of the measured variables in this research. However, debate over whether neurogenesis even occurs in the adult human brain is still ongoing, which could be a potential caveat to translating the findings from animal models to humans. But Horowitz believes that neurogenesis is just one piece of the puzzle, stating that she would expect changes in other cells and processes, such as mature neurons and neuroinflammation, that would more fully account for the cognitive effects that were seen in the study.

Reference: Horowitz et al. (2020). Blood factors transfer beneficial effects of exercise on neurogenesis and cognition to the aged brain. Science. DOI: 10.1126/science.aaw2622.

Alana Horowitz was speaking to Molly Campbell, Science Writer, Technology Networks.

Read more here:

Liver Protein Could Be Behind Exercises Brain Benefits - Technology Networks

Global CRISPR-Based Therapeutics Market 2020 Segmented by Product, Application, Key Players and Regional Analysis to 2025 – Cole of Duty

Posted on by

Recently updated and published a report titled Global CRISPR-Based Therapeutics Market 2020 by Company, Regions, Type and Application, Forecast to 2025, by Researchstore.biz highlights lubricants market growth potentials which can assist the stakeholders to understand key trends and prospects in the market as well as identify the growth opportunities and competitive scenarios. The report contains factors such as market size, market share, market segmentation, significant growth drivers, market competition, and diverse features affecting the global CRISPR-Based Therapeutics market. The report analyzes each segment in the global CRISPR-Based Therapeutics market. The report focuses on the markets growth potential, which can help investors identify scope and opportunities.

The report scrutinizes the market framework and based on that it presents a summary of policies and improvement plans. The report features premium quality data figures associated with financial figures of the industry like market size, sales data, and revenue figures through graphics, diagrams, charts, and numbers. These figures make the report more influential and easy to understand. The report recognizes the technical barriers, other issues, and cost-effectiveness affecting the market. It offers useful guidelines for players to secure a position of strength in the global CRISPR-Based Therapeutics market.

DOWNLOAD FREE SAMPLE REPORT: https://www.researchstore.biz/sample-request/41907

NOTE: This report takes into account the current and future impacts of COVID-19 on this industry and offers you an in-dept analysis of Multi Channel Flame Photometers market.

Company Profiles:

Every market participant needs to be familiar with the competitive scenario in the global CRISPR-Based Therapeutics industry. In order to fulfill the requirements, the industry analysts have examined the strategic activities of the competitors to help the key players increase their competitiveness. The report studies the progress of leading players based on crucial parameters, including market share, new developments, global reach, local competition, price, and production.

The major market players that are operating in the global CRISPR-Based Therapeutics market are Caribou Biosciences , Thermo Fisher Scientific , Merck KGaA , Addgene , Takara Bio USA , CRISPR THERAPEUTICS , Intellia Therapeutics , Editas Medicine , Mirus Bio LLC , Horizon Discovery Group , GE Healthcare Dharmacon . All these market players are profiled in the market report.

The study provides an in-depth overview of country-level break-up classified as potentially high growth rate territory, countries with the highest market share in past and current scenario. Some of the regional break-up classified in the study are: North America (United States, Canada and Mexico), Europe (Germany, France, UK, Russia and Italy), Asia-Pacific (China, Japan, Korea, India and Southeast Asia), South America (Brazil, Argentina, etc.), Middle East& Africa (Saudi Arabia, Egypt, Nigeria and South Africa)

Market segmentation by product: Genome Editing, Genetic Engineering, gRNA Database/Gene Librar, CRISPR Plasmid, Human Stem Cells, Genetically Modified Organisms/Crops, Cell Line Engineering

Market segmentation by application: Biotechnology Companies, Pharmaceutical Companies, Academic Institutes, Research and Development Institutes

ACCESS FULL REPORT: https://www.researchstore.biz/report/global-crispr-based-therapeutics-market-41907

The Following Doubts Are Addressed In The Market Report:

Customization of the Report:This report can be customized to meet the clients requirements. Please connect with our sales team ([emailprotected]), who will ensure that you get a report that suits your needs. You can also get in touch with our executives on +1-201-465-4211 to share your research requirements.

About Us

Researchstore.biz is a fully dedicated global market research agency providing thorough quantitative and qualitative analysis of extensive market research.Our corporate is identified by recognition and enthusiasm for what it offers, which unites its staff across the world.We are desired market researchers proving a reliable source of extensive market analysis on which readers can rely on. Our research team consist of some of the best market researchers, sector and analysis executives in the nation, because of which Researchstore.biz is considered as one of the most vigorous market research enterprises. Researchstore.biz finds perfect solutions according to the requirements of research with considerations of content and methods. Unique and out of the box technologies, techniques and solutions are implemented all through the research reports.

Contact UsMark StoneHead of Business DevelopmentPhone: +1-201-465-4211Email: [emailprotected]Web: http://www.researchstore.biz

See the rest here:

Global CRISPR-Based Therapeutics Market 2020 Segmented by Product, Application, Key Players and Regional Analysis to 2025 - Cole of Duty

David Seymour rejects that ACT’s paying lip service to anti-1080 activists with conservation policy – Newshub

Posted on by

Seymour said ACT has a "rational" view on 1080 and that it's a "necessary poison" - but he thinks the Department of Conservation should be more focused on finding different ways to protect unique flora and fauna.

Newshub revealed last year that the Department of Conservation has increased spending on finding alternatives to 1080 in recent years - but Seymour wants more resources put towards it.

"I think ultimately what comes out of the Department of Conservation is that they're not as serious about it as they could be. I think if given the opportunity they will just keep on using 1080 indiscriminately," he told Magic Talk.

"I think what the Government needs to do is say, 'What is your actual policy and your statement of position on minimising this?' They're not doing that and instead they're paying lip service and carrying on."

Magic Talk host Sean Plunket told Seymour the same could be said of ACT.

"In the absence of a specific policy or a request for more funding or a specific research project, I can't see it as anything but lip service."

Seymour pushed back. He said ACT has put forward a policy and that ahead of the September election the public needs to know where the party stands on the controversial poison.

"It's telling people exactly where you stand and it's the rational position... Our policy is to keep the pressure on, that we do need to find alternatives. I think it's useful for people to know that's where we stand."

ACT is proposing ring-fenced contestable science funding to prioritise research and development into viable alternatives to 1080.

ACT would also require the Department of Conservation to "use all measures and resources at their disposal to minimise the by-kill of game animals and other non-target species during 1080 operations".

New Zealand First is also opposed to the use of 1080.

Back in November it celebrated $3.5 million from the Provincial Growth Fund going towards developing predator control approaches to help reduce the need for repeated 1080 use.

Green MP and Conservation Minister Eugenie Sage has stopped work being done to use genetic engineering as a solution to dropping 1080, despite official advice suggesting it could be used to help rid New Zealand of predators.

Sage believes 1080 is the safest and most effective means of getting rid of pests.

"Aerial operations using biodegradable 1080 are the safest and most effective means of knocking down rats, stoats and possums over large areas of rugged terrain," she told Newshub in 2019.

You can read more about 1080 here.

Read the original post:

David Seymour rejects that ACT's paying lip service to anti-1080 activists with conservation policy - Newshub

The Trump supporters who changed their minds: ‘I’d rather vote for a tuna fish sandwich’ – The Guardian

Posted on by

The anti-Trumpers are at it again only this time, theyre Republicans.

Kevin, a lifelong Republican voter and pastor from Arizona, says he voted for Trump in 2016 with high hopes for the future. He knew that Trump didnt have the same political experience as the other contenders, but he was optimistic he could grow into his new role.

Now he says: Ive seen how he has tried to divide our country and that is not something I want, nor what our country should have This man is an absolute danger to our country.

Kevins experience of voting for Trump and then quickly realizing hed made a mistake is one of many being used by Republican Voters Against Trump (RVAT), which wants to boot Trump out of office later this year. The group is seeking testimony from former Trump voters through its website, which displays the best quotes so far with pride. (Id vote for a tuna fish sandwich before I vote for Donald Trump again, reads one.)

Sarah Longwell, a Republican consultant who co-founded RVAT, said they have received hundreds of testimonies in recent months.

The group is funded by millionaire neoconservative pundit Bill Kristol, who was formerly chief of staff to the vice-president under George HW Bush, but uses testimony from distinctly non-political voices to make an impact.

One of the reasons they are so compelling is because you can tell how authentic they are, how deeply they feel this a lot of them want to get something off their chest, says Longwell. The testimonies are not scripted or paid for, but they are the result of a lot of workshopping.

Jeffrey Farmer, from Massachussetts, certainly fits the bill of a non-polished but frustrated voice: he is immunocompromised and angered by Trumps response to the pandemic. And he is certainly the voice of a media-trained, focus-group prepped politico just a person who formerly backed Trump.

I dont even know why Im doing this stupid thing, because this is not what I do. I dont do social media or anything. But I cant take this any more, he says.

Farmer voted for Trump in 2016 because of how much he disliked Hillary Clinton, but describes him as being Like a Tasmanian devil, who spends all day complaining on Twitter instead of doing his job.

This guy couldnt lead his way out of a wet frickin paper bag, says Farmer.

Longwell, herself a disgruntled Republican, says she initially started looking for answers after Trump won the presidency.

I have been alarmed by him from the beginning, she says and so, around 2017, she began to search for answers. I wanted to know how the party got taken over by Donald Trump, she says. She ran focus groups with soft Trump voters who voted for him 2016 but rated him as doing somewhat badly or very badly and tried to understand how to persuade them against him.

The key thing, she found, was for them to hear from people like themselves.

One thing we found is that the cultural aspect played a big role in [the 2016 election], she says. Youd get women whod say I voted for him and I cried, or, I voted for him and then I had to take a shower afterwards. But they were surrounded by people who talk about how all Democrats are socialists or whatever, she says.

As a lifelong supporters of the Republican party though, does she really want Biden to win? Longwell says that she absolutely, unequivocally does.

Donald Trump has a negative impact on the future of the Republican party, says Longwell. He has sort of hijacked the party and really poisoned the country, and has turned it into a nationalist populist party. Theres a section of Republicans who do not find that attractive, and Im one of them.

The best thing for the party long term is for him to get defeated soundly, and for the party to rethink its direction.

See more here:

The Trump supporters who changed their minds: 'I'd rather vote for a tuna fish sandwich' - The Guardian

Transitioning From Live Poker To Online Poker | My Blog – Tight Poker

Posted on by

Tuesday, July 14th, 2020 | Written by Renee

Live poker is among various industries severely impacted by the coronavirus pandemic, with land-based casinos and poker rooms forced to close. While some have started to reopen, others have permanently shut down. Thousands of players who used to play in live settings are now contemplating on formally transitioning to online poker.

If you are one of those players, then playing cash games online would be a great way to restart your career and build your bankroll. While a lot of players might already know the nitty gritty of playing online cash games, some just lack the knowledge. If you are the latter and are considering to try your hand at the virtual felts, the tips below will come in handy as you move to the online poker tables.

Get Ready For Faster Game Speed

Both live and online poker follow essentially the same rules when it comes to cash games, however there are also differences in some aspects, including speed. In a live poker game, a full ring usually involves about 25-30 hands being dealt per hour, but online, that number increases to 60-80 hands each hour. But that is only if you play just one table. Expect that number to increase further the moment you multi-table, playing more than one game at the same time.

Also take note that if you play an online cash game, you will have less time to act. This is because the game involves a time bank that puts a limit to how long you can make a decision. This is so much different compared to a live setting where there is a lot of downtime between hands.

Dont Go Straight To Multi-tabling

If youre just starting to get to know how online cash games work, you better stick with just one table and then advance to the next level once youre ready. Take things one step at a time no need to rush; if you do, you wont be able to put enough focus on your game which will then lead to potential losses. You can add more tables if you have established a decent win rate.

Start Small

If you want to achieve something, its always practical to begin with tiny steps. It will allow you to properly gauge your skills and improve them along the way without putting too much of your bankroll on the line.

In a live game, a $1/$2 stake is the level preferred by most beginners. However in the online world, new players are encouraged to begin in much lower stakes, like $0.10/$0.25, or $0.25/$0.50. This is because you will most probably compete with fellow beginners at this level, compared to the $1/$2 games where you will likely take on the more experienced online crushers.

Manage your bankroll properly by not rushing to go to the higher stakes. Begin from the bottom and then slowly climb your way to the top.

Get Yourself A Reliable Gaming Set-Up

If you plan on taking your game online, one of the most important things to consider is whether or not your current set-up can withstand long hours of play without unnecessary interruptions.

A good set-up will enable you to play with much needed comfort and convenience, helping you to perform better. You can go for a two-monitor set-up, or use your laptop and install an external monitor. Get a comfy chair, too. Remember, you dont have to invest in the most high-tech, expensive equipment. Just make sure the set-up does not interfere with your play.

Get Acquainted With Online Poker Tools

One major difference in playing online is that you get to use third-party software to enhance your decision-making skills. While not all sites are allowing these tools, some do, and it would be great if you have proper knowledge on how to use them. Do your homework. There are a lot of online resources regarding poker tools like Holdem Manager, Poker Tracker, PIOSolver and Simple Postflop. All you have to do is to study, practice, and learn.

Read more:

Transitioning From Live Poker To Online Poker | My Blog - Tight Poker

Letter to the Editor – Wouldn’t following the Golden Rule call on us to wear a mask? – Crookston Daily Times

Posted on by

Shouldnt we all practice the Golden Rule by wearing masks? A new parable for these times may help reconsider how safe the COVID19 virus really is:

Out of the lands of the world rose the free marketers. And they lived high and mighty free of restraints. They believed they were entitled to take all the earths bounty. And those that were not of them, those others, those losers, had sinned and deserved misery. In this, free market hypocrisy denied equally both the lives of the living as well the unborn.

Then there arose a mighty plague in all the lands. A plague that smote the rich and the poor, the high and the low. And many died.

And yet, the high and mighty with their minions denied this fierce plague. They told all who would listen, Fear not. Keep the markets open. Let there be freedom in our lands.

But the wrath of the mighty plague increased. And still more passed through the doors of death. And many families were lost in grief.

Still, the high found sanctuary behind their walls of spiritual pride. As the powerful, they resisted most strongly, as the powerful can. They sold immunity in those churches to all who came in.

The warnings from the wise about such gatherings were not heard. A few could not hide, nor would too many even try to hide from the plague. The high said, We are protected in the blood of Jesus. No harm will come to us. We will be free to worship as we please.

Then again, the high were obsessed with reopening the markets without any care. Despite the now great and still growing death toll, they said, What is more important, free market or human life? What have you got to lose?"

And it came to pass, countless many were stricken dead. A great cry grew throughout all the lands, God, why have you forsaken us?

Finally, in the fever of those who remained, the voice of God was heard at last. You have forsaken me. I sent warnings with few deaths. But your shouts were of remaining free. When more died, your spiritual blindness would not even then let you see. Then after the countless many died, your markets were once more thrown open and again without restraint.

For all these sins, how shall each of you be left not to die?

Erwin R. RudFosston, Minnesota

Go here to read the rest:

Letter to the Editor - Wouldn't following the Golden Rule call on us to wear a mask? - Crookston Daily Times

Coronavirus in Wales: Face coverings mandatory on trains and buses – BBC News

Posted on by

Media playback is unsupported on your device

Three-layer face coverings will be mandatory on public transport in Wales from 27 July, the first minister has said.

At the daily Welsh Government coronavirus briefing, Mark Drakeford said this would also be the case for taxis and other situations where 2m social distancing was not possible.

Last month, Health Minister Vaughan Gething recommended their use.

But he stopped short of making them mandatory.

Transport for Wales said it had worked with the Welsh Government throughout the pandemic and Great Western Railway said it encouraged all customers to follow the new rule.

The Welsh Conservatives questioned why the measure was not being introduced immediately and Plaid Cymru called for masks to be made mandatory for all indoor spaces.

The union Unite said it warmly welcomed the decision.

Mr Drakeford said: "For the sake of simplicity and consistency, as well as being part of our plan tohelp reduce the risk of transmission while on public transport where it is not always possible to maintain a two metre physical distance, it will become mandatory for people to wear a three-layer face covering while travelling - this includes taxis."

Media playback is unsupported on your device

Face coverings are currently required on public transport in England, Scotland and Northern Ireland, while wearing a face covering in shops and supermarkets in England is to become mandatory from 24 July.

Having a different rule for wearing masks on public transport in Wales and England was "not sustainable in the long term", Mr Drakeford said.

"Our decision to make face coverings mandatory on public transport is a combination of the fact that we know as the economy gets back into operation more people will need to use public transport to go to work and for other purposes, and when more people need to use confined spaces then additional protections need to be introduced in order to overcome the fact that two-metre social distancing will not always be possible," he added.

Taxi driver Stephen Clifford, from Newport, does not believe it will work for customers

He said: "Most of people the customers wouldn't wear them.

"We'd lose an awful lot of money. If you've got to have it, you have got to have it. And what if we had to provide them? It's hard to say."

Media playback is unsupported on your device

Asked why coverings were not mandatory in other public spaces, Mr Drakeford said: "The advice of the Welsh Government is that if places are crowded then face coverings are advisory. Where places are not crowded it is a matter for the individual citizen to make that decision."

Coronavirus is now "at its lowest ebb" since the pandemic began, he added, saying the Welsh Government's response had to be "proportionate".

While Mr Drakeford said masks would not be mandatory for shoppers, businesses may ask people to wear them.

He said the retail sector had made "huge efforts" to introduce measures to maintain physical distancing, including putting up one-way systems, limiting the number people who can enter a shop and putting up screens at checkouts.

But, he added: "At this point in time, when the prevalence of coronavirus is low, we are not mandating the use of face coverings in other public places, but many people may choose to wear them - and there is nothing to stop that happening in Wales.

"Our advice may change if cases of coronavirus begin to increase."

The first minister said the Welsh Government had made changes to regulations which recognise there are some occasions when it is not always possible to maintain a distance of 2m.

"These include maintaining hygiene standards and limiting close face-to-face interaction, wherever reasonable," he added.

Mr Drakeford also said 300,000 coronavirus tests have been carried out in Wales, with 17,000 of them positive.

He urged people to carry on following the "golden rules" such as washing hands frequently.

He said there had been a "real change in working patterns, with more people working from home", adding: "We need to see flexible working become a permanent feature of working life in Wales and the Welsh government will lead the way in this."

Reacting to the announcement, Conservative Covid recovery spokesman Darren Millar said: "We must still take every precaution to avoid a second wave of cases, and making wearing face masks mandatory from today may go some way to achieving this - but only if brought in now."

The British Medical Association (BMA) also called for the new rules to be "implemented without delay". It said face coverings should be used whenever people could not keep a safe distance.

Plaid Cymru leader Adam Price welcomed the move but said: "In acknowledging that face coverings make a crucial difference on trains, buses and in taxis, the question must be begged of Welsh Government - why not in shops also?

"The latest guidance, while a step in the right direction, still doesn't go far enough."

Unite Wales regional secretary Peter Hughes said: "This decision will improve safety on our buses, trains and taxis it will also greatly increase the confidence of the general public to travel on public transport as lockdown measures are eased."

Read the original:

Coronavirus in Wales: Face coverings mandatory on trains and buses - BBC News

On Grand Ole Opry, Margo Price Asks Show to Book the Real Lady A – Variety

Posted on by

In a televised live appearance on The Grand Ole Opry Saturday night, Nashville-based singer-songwriter Margo Price offered an unexpected bit of advocacy between songs, name-checking the group formerly known as Lady Antebellum before suggesting that the show should book Anita White, whom she called the real Lady A.

Price made the suggestion immediately after performing a cover of Skip a Rope, a No. 1 country hit from the 1960s that dealt with racism and other social ills.

Before going into the final number of her set, the title track from her new album Thats How Rumors Get Started, Price said, I would just like to commend the Opry for coming out and saying black lives matter. I think its so important at this time. And I hope that we can continue to go one step further in so many of these Nashville institutions and support the voices of our Black brothers and sisters when they need it most.

She continued, You know, Lady Antebellum has had a platform here. I think it would be really wonderful if yall invited Anita White the real Lady A here to come and sing. You know, country music owes such a great deal of what we have to Black artists and Black music, and there (is) just no place for sexism, racism in this music.

On Twitter Sunday, Price referred to some discord over her remarks. Some people are disappointed by my words about ending racism in this country, she wrote, but I will never be ashamed to stand up for what is right.

Price has not pulled any punches recently in her feelings about the former Lady Antebellum wanting to share the name with the Seattle blues singer who has long used it. Three days before playing the Opry, Price forwarded a news story about the band suing Price to establish that both artists could use the name Lady A, tweeting: Sooooo they changed their name but does the A stand for antebellum or ahole.

Skip a Rope was recorded by Henson Cargill in 1967 and spent five weeks at the top of the country chart in 1968, going on to be nominated for a CMA Award for song of the year. Sometimes cited by country music historians as an example of a golden age of social commentary in the genre, the song included lines like, Cheat on your taxes, dont be a fool / Now what was that they said about a Golden Rule? / Never mind the rules, just play to win / And hate your neighbor for the shade of his skin.

Preceding Price on the Opry Saturday was one of the newest Black country stars in a genre that hasnt always had a surplus of them, Jimmie Allen. Also appearing on the bill (filmed live without an audience) were the Gatlin Brothers; given their avowed support for Donald Trump, that could have made for some interesting conversation backstage, if Price and the Gatlins werent too socially distanced.

Price has not been shy to speak up on other topics recently. Earlier in the month, she tweeted, Would be cool if, instead of arresting peaceful protesters, the police would go downtown on Broadway and arrest bar owners like Steve Smith and all the other asshat(s) who are walking around without masks on and spreading Covid.

Prices TV tour behind her new album continues with two more appearances today. After performing on PBS/CNN Internationals Amanpour, shell be seen in a pre-recorded full-band video on Jimmy Kimmel Live!

More:

On Grand Ole Opry, Margo Price Asks Show to Book the Real Lady A - Variety