Monthly Archives: January 2022

RedChIP identifies noncoding RNAs associated with genomic sites occupied by Polycomb and CTCF proteins – pnas.org

Posted: January 3, 2022 at 1:28 am

Abstract

Nuclear noncoding RNAs (ncRNAs) are key regulators of gene expression and chromatin organization. The progress in studying nuclear ncRNAs depends on the ability to identify the genome-wide spectrum of contacts of ncRNAs with chromatin. To address this question, a panel of RNADNA proximity ligation techniques has been developed. However, neither of these techniques examines proteins involved in RNAchromatin interactions. Here, we introduce RedChIP, a technique combining RNADNA proximity ligation and chromatin immunoprecipitation for identifying RNAchromatin interactions mediated by a particular protein. Using antibodies against architectural protein CTCF and the EZH2 subunit of the Polycomb repressive complex 2, we identify a spectrum of cis- and trans-acting ncRNAs enriched at Polycomb- and CTCF-binding sites in human cells, which may be involved in Polycomb-mediated gene repression and CTCF-dependent chromatin looping. By providing a protein-centric view of RNADNA interactions, RedChIP represents an important tool for studies of nuclear ncRNAs.

The majority of the eukaryotic genome is transcribed into coding and noncoding RNAs (ncRNAs). ncRNAs fulfill various functions both in the cytoplasm and the cell nucleus. Nuclear ncRNAs are attracted to different genomic regions and mediate the activation or repression of genes located in these regions and are also implicated in the genome three-dimensional organization (1, 2). In particular, recent studies indicate that RNA is essential for the chromatin targeting of Polycomb repressive complexes (3) and the organization of CTCF-dependent chromatin loops (4). However, these studies do not show which particular RNAs are involved.

Helpful in studying nuclear functions of ncRNAs are methods that map the sites of ncRNA associations with the genome. Initially developed for probing genomic interactions of one particular RNA, these methods are now available in an all-vs.-all version allowing simultaneous detection of the sites of chromosomal locations for all RNA molecules present in the nucleus (reviewed in ref. 1). An important drawback of these techniques, however, is that they do not disclose the proteins involved in RNADNA interactions.

To identify RNAs that could be involved in the functioning of DNA-bound proteins, we developed a hybrid approachRedChIPcombining an RNADNA proximity ligation technique [Red-C (5)] with chromatin immunoprecipitation (ChIP). Using antibodies against CTCF and EZH2, we identified various ncRNAs interacting with DNA at the sites of deposition of the above-mentioned proteins.

The RedChIP experimental procedure is analogous to HiChIP used for protein-centric mapping of DNADNA interactions (6), with the difference being that RNADNA interactions are analyzed instead of DNADNA interactions. Briefly, RNAproteinDNA complexes are cross-linked in living cells, and RNA and DNA fragments are ligated in situ using a bridge adapter. Ligated complexes are solubilized by sonication and subjected to IP using antibodies against a protein of interest. RNADNA chimeric molecules are then purified and sequenced, thus reporting RNADNA interactions that may be mediated by a particular protein and proteinDNA interactions that may be mediated by various RNAs (Fig. 1A). An aliquot of the material (input fraction) is processed without an IP step to record the total set of RNADNA interactions mediated by any proteins, as in a regular Red-C experiment.

RedChIP technique. (A) Outline of the experimental procedure. (B) A region of Chr17 encompassing Hoxb genes showing distribution of DNA and RNA portions in IP (RedChIP) and input (RedC) fractions from experiments with EZH2 and CTCF antibodies. Shown alongside are ChIP-seq peaks of EZH2 in H1-hESCs (human embryonic stem cells) and ChIP-seq peaks of CTCF in K562 cells as well as total RNA-seq profiles for H1-hESCs and K562 cells (from ENCODE). (C) Distribution of DNA portions around EZH2 peaks in hESCs and CTCF peaks in K562. (D) Ratio of the number of RNA contacts detected in different chromatin types in IP fraction to the number of RNA contacts detected in the same chromatin types in input fraction.

We used antibodies against EZH2, a catalytic subunit of the PRC2 complex, to study the Polycomb-dependent RNADNA interactome in human embryonic stem cells. We also used antibodies against CTCF to study the CTCF-dependent RNADNA interactome in human K562 cells. DNA portions of the chimeric molecules showed a clear preference for the binding sites of corresponding proteins in the IP fraction (Fig.1 B and C), indicating successful IP. Accordingly, we observed an enrichment of DNA portions in chromatin types typical for poised promoters and Polycomb-repressed regions in the IP fraction from the EZH2 experiment and an enrichment of DNA portions in chromatin types typical for insulators and promoters in the IP fraction from the CTCF experiment (Fig. 1D). Meanwhile, RNA portions of the chimeric molecules showed correlation with RNA-sequencing (RNA-seq) profiles both in IP and input fractions (Fig. 1B), reflecting the origination of RNA portions from various transcripts. We combined the contacts of RNA portions originating from a single gene, thus obtaining a whole-genome contact profile for each annotated RNA.

We then focused on the analysis of contacts of individual RNAs in the experiment with EZH2 antibodies. We first aimed to identify cis-acting RNAs that fulfill their functions in the vicinity of an encoding gene. For each RNA, we selected a fraction of cis contacts established with DNA regions surrounding the gene (1 Mb of gene boundaries including the gene) and compared the number of cis contacts between EZH2-precipitated and input fractions. We found that the degree of enrichment in the IP fraction correlated with the percentage of contacts detected in Polycomb-specific and poised promoter-specific chromatin types but not any other chromatin types in the area under study (Fig. 2A). We identified 10 long intergenic ncRNAs (lincRNAs) with a fold enrichment of >1.3 in both replicates (Fig. 2C). Among the identified RNAs is Kcnq1ot1 (fold change = 1.5), a well-known example of antisense lincRNA involved in the Polycomb-mediated silencing of several genes in the same locus (7). High fold enrichment was also observed for AC078778, antisense lincRNAs from the HOXC locus. Notably, antisense RNAs, on average, demonstrate higher fold enrichment than other lincRNAs and mRNAs of protein-coding genes (Fig. 2G), indicating the enrichment of the group of antisense RNAs with RNAs that may mediate Polycomb targeting.

Identification of ncRNAs associated with genomic regions occupied by EZH2 in hESCs and by CTCF in K562 cells. (A) Ratio of the number of cis contacts of individual RNAs between EZH2-precipitated and input fractions (x axis) vs. the percentage of cis contacts detected in different chromatin types in EZH2-precipitated fraction (y axis). (B) The same as A for CTCF-precipitated fraction. (C and D) Ratio of the number of cis (C) or trans (D) contacts of individual RNAs between EZH2-precipitated and input fractions for rep1 and rep2. (E and F) Ratio of the number of cis (E) or trans (F) contacts of individual RNAs between CTCF-precipitated and input fractions for rep1 and rep2. (G) Distribution of fold changes from C for different RNA biotypes (antisense, n = 44; linc, n = 162; protein coding, n = 4,184). *P < 0.05, ***P < 0.001. (H) Intersection of RNAs enriched in RedChIP and fRIP-seq.

At the final step of the analysis, we searched for trans-acting RNAs that could participate in Polycomb functioning genome-wide. We compared the number of trans contacts (contacts with nonparental chromosomes) for each RNA between EZH2-precipitated and input fractions and looked for RNAs showing an elevated number of contacts in the IP fraction. The highest enrichment was observed for antisense RNA KIF5C-AS1, snRNA RNU5B-1, and SNORD3a RNA (Fig. 2D). These RNAs are good candidates to act as global mediators of Polycomb activity.

The above types of analysis were then performed for the data from the experiment with CTCF antibodies. In the analysis of cis-acting RNAs, we observed a correlation of RNA enrichment in the CTCF-precipitated fraction with the percentage of contacts detected in promoter-, enhancer-, and insulator-specific chromatin type (Fig. 2B). We identified seven lincRNAs with a fold enrichment of >1.3 in both replicates (Fig. 2E). These lincRNAs might participate in loading CTCF to its DNA sites and organization of promoter-enhancer specific and other chromatin loops within genomic loci from where lincRNAs are produced. In the analysis of trans-acting RNAs, the highest fold enrichment was observed for snRNA RNU12 (Fig. 2F). Notably, U12 RNA is the second top by the total number of contacts among all RNAs in K562 cells (1.1% of all contacts). The potential involvement of RNU12 RNA in the functions of CTCF requires further experimental evidence.

Remarkably, the set of RNAs enriched in RedChIP significantly intersects the set of RNAs enriched in RNA IP (formaldehyde RNA IP-sequencing, fRIP-seq) experiments (Fig. 2H). Importantly, 18 of 22 ncRNAs overrepresented in CTCF- and EZH2-RedChIP samples are fRIP-positive, indicating these ncRNAs indeed interact with the studied proteins.

Collectively, the present study results demonstrate the utility of the RedChIP protocol for identifying RNAs that may target nonhistone proteins to various locations on chromosomes or mediate interactions of these proteins with DNA. The identification of RNAs that are known to target Polycomb complexes to repressed genomic domains strongly supports the validity of the experimental approach, whereas identifying a set of RNAs possessing similar characteristics will stimulate studies of their possible role in Polycomb and CTCF functioning. The RedChIP technique can be used for identifying RNAs associated with genomic regions occupied by any protein of interest.

Cells are fixed with formaldehyde, DNA is fragmented with NlaIII restriction enzyme, and the ends are blunted and A-tailed. RNA 3 ends are ligated to a biotinylated bridge adapter followed by ligation of the opposite ends of the bridges with DNA ends in spatial proximity. Ligated complexes are solubilized by sonication, immunoprecipitated, and washed. RNADNA chimeras are purified, and DNA is digested with MmeI restriction enzyme. After biotin pull-down, reverse transcription is initiated from the bridge with template switching at the RNA 5 end, allowing for the incorporation of an Illumina adapter. Another Illumina adapter is ligated to DNA ends, and the chimeras are amplified and paired-end sequenced. The detailed protocol and sequencing data processing are described in SI Appendix. For sample processing statistics, refer to Dataset S1. For read processing statistics, refer to Dataset S2.

Raw fastq reads and processed TSV files with contacts are available at Gene Expression Omnibus (accession no. GSE174474). The code for read processing is available as RedClib on GitHub: https://github.com/agalitsyna/RedClib.

This work was supported by the Russian Science Foundation (21-64-00001) and by the Russian Ministry of Science and Higher Education (075-15-2021-1062).

Author contributions: A.A. Gavrilov, E.L.A., and S.V.R. designed research; A.A. Gavrilov and E.B.D. performed research; A.A. Gavrilov, R.I.S., M.D.M., and A.A. Galitsyna analyzed data; and A.A. Gavrilov and S.V.R. wrote the paper.

The authors declare no competing interest.

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2116222119/-/DCSupplemental.

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LifeBank Chain (LBC) Focuses on Research and Development in the Field of Genetics and Cell Science – Markets Herald

Posted: at 1:28 am

GENE & CELL MEDICINE LTD located in Israel and Singapore started a new project:LifeBank Chain (LBC). The project LBC plans to build a genetic and cell data collaboration platform.

Genetic research seeks to understand the process of trait inheritance from parents to offspring.Human genetic research is revealing the nature of human bioinformatics and giving scientists a powerful approach to study various health issues of human life.

Cell research focuses on stem cell and immune cell therapies, which are an extremely promising approach for the treatment of many diseases with an immune component including cancer, autoimmune disease, and chronic inflammation.

The wide applications of these new biological technologies in the medical field greatly reshaped the traditional pharmaceutical industry, whose focus was not only put on the treatment of disease as before but also on gene diagnosis and prevention, which opened the door to the world of a personalized and precise medicine.

Blockchain is an emerging technology that has attracted increasing attention from both researchers and practitioners. The functionalities of blockchain technology and smart contracts provide an opportunity over the large gene and cell data to support genetic and cell data integrity and security while giving patients control over their own data.

LBC plans to build a genetic and cell data collaboration platform incorporating an extensible cross-chain service system based on individual and institutional nodes. The platform product service layer abstracts all typical kinds of gene and cell blockchain applications and provides the full functions and implementation framework of typical applications.

The goal ofLifeBank Chain (LBC) is to establish a global-level service platform for sharing and utilizing human genetic and cell data through secured blockchain technologies.The LBC blockchain is designed to provide genetic and cell research industry partners with enterprise-level blockchain infrastructure, industry solutions, and secure, reliable, and flexible blockchain services. LBC will work together with medical practitioners to provide full-solution ancillary reagent services and provide flexible and pioneering tools to simplify therapy workflow at every step of the medical process.

LBC will form a professional and shared social organization LBC Life Alliance inviting life technology companies, scientific research institutes, medical institutions, etc. to jointly solve medical, health, disease, and public health problems, and jointly build the application standards of gene and stem cell medical technology on the blockchain, and contribute to the cause of human health.

LifeBank Chain enables healthcare professionals to manage the medical data and do research in an auditable, transparent, and secure way on LBCs distributed network. LBC continues to closely monitor the evolution of genetics and cell therapy in different medical subspecialties around the world.

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Letters: Freedom demands the responsibility of sacrifice in COVID fight – Charleston Post Courier

Posted: at 1:26 am

A sure sign that a nation is losing its soul is when its people are no longer willing to sacrifice for the common good. Some people seem to have forgotten what it means to be a patriot.

Casualties from COVID-19 have surpassed 800,000 deaths and 50 million wounded. This war against the virus has lasted less than two years.

If an enemy invaded our country causing similar casualties, would you be willing to sacrifice to defeat it?

Freedom is not about what we want to do, but rather what we should do.

Unfortunately it is no longer about we but more and more about me.

As a result, the responsibility that freedom demands is becoming ignored. Freedom boils down to one thing: choice.

Do you want to fly on an airplane? You have a choice to either wear a mask or not fly.

Your employer requires you to be vaccinated. You have a choice to either get vaccinated or find another job.

Most of our laws are the result of people choosing to be irresponsible. Freedom permits them to choose to be responsible or irresponsible. If they choose to be irresponsible, they pay a price for their freedom of choice, yet some believe they should be able to choose to be irresponsible without paying any price.

The basic tenets of responsibility in a free democratic society are simple: Keep your word, and respect others and their property.

Freedom demands little more and nothing less.

DOUGLAS DEVLAMING

Charleston

Here is an example of an internet scam.

First, you fill out a short survey sent to your phone. There are no questions about personal information, so OK.

And its all done via text message.

Then, you receive a message that youve won an iPhone 13.

You are told that the phone is in the warehouse.

To receive it, you only need to pay a $1.64 shipping charge.

Once the payment is made, the phone will be loaded onto a truck for delivery and sent to you. You just have to enter your credit card or bank account information.

Then, when you dont pay the money or give them your financial information, you start receiving messages about delivery requests and late payment notices from the delivery company.

Dont fall prey to these schemes trying to get your money.

STEPHEN BRINKMAN

Charleston

Make a difference in the New Year.

More than 60% of residents of long-term care facilities in South Carolina have no visitors.

They have no family or friends who are available to visit them or spend time with them. The lack of social contact and a support system often results in depression and decline.

Residents of our community can do something to help these friends and neighbors.

They can join the Volunteer Ombudsman Program, which is sponsored by the South Carolina Department on Aging, Office of the State Long Term Care Ombudsman.

The function of the volunteer in residential facilities is to provide encouragement and meet an essential need.

The efforts, presence and commitment of this program make a significant difference in the lives of many.

Volunteers help to diminish the sense of isolation that residents can experience, and help facilitate a sense of self-determination and achievement.

I encourage you to connect with residents who live in long-term care facilities, participate in conversations, and encourage self-advocacy.

Volunteers help to ensure that the voices of long-term care residents do not go unheard and show residents they have not been forgotten.

Working as a volunteer ombudsman will help to ensure that the voices of long-term care residents do not go unheard.

Contact the Volunteer Long-Term Care Ombudsman Program online at volunteerombudsman@tridentaaa.org or by phone at 843-554-2276.

LINDA NAERT

Long Term Care Ombudsman Volunteer Coordinator

North Charleston

Years ago, my mother gave me a Dec. 26 deadline to write thank-you notes for gifts received for Christmas and get them mailed by New Years.

So Charleston, here is my thank-you note to the Charleston Music Hall, Charleston Symphony Orchestra and Charleston School of the Arts for their recent holiday program of music.

I have never liked the song The Twelve Days of Christmas. But the rendition or I should say the spoofing of it by four gentlemen at the Charleston Music Hall is something all should experience.

The usual order of gifts was mixed up. By the second verse, the audience was writhing with laughter.

Not to be outdone, CSO conductor Yuri Bekker got the audience involved.

Two elves went through the audience and encouraged several people to sing the five golden rings part of the song.

After initially showing looks of panic, a man and a woman sang the phrase great. Then the rest of the audience was eager to join in as well.

The best part was saved for last.

Charleston County School of the Arts musicians and choral group lit up the theater.

About a dozen deaf students signed the words to Silent Night, which left many breathless.

Thank you, Charleston, for once again making this season very merry and bright in spite of COVID.

CHRISTINE EBEL

Charleston

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Finding freedom as an LGBTQ person in a city after escaping a small town – LGBTQ Nation

Posted: at 1:26 am

Living in a small town isnt always easy as an LGBTQ person. It can be isolating, make people feel trapped and unable to be their true selves. However, that doesnt mean the future is destined to be miserable.

Read these stories from people who have moved out of their small towns and found freedom in big cities.

Related: This group offers stand-in parents for LGBTQ weddings. It all started with a viral video.

Dinesh grew up in a relatively conservative Gujarati Hindu household. He was raised in a tight-knit community where everyone knows everyone and no one wants to be seen to go against the norm. Any different aspects of his personality were far from celebrated, and any aspirations for him to just be him fell on deaf ears.

His family, most notably his grandmother, really pushed home to him the elements of culture, tradition and religion. As a child these elements seemed jovial and lively to him, but as his understanding grew of his faith and culture, he found his own relationship with his heritage.

It may seem odd in a way that I kind of never had a coming out moment. I explored myself and learnt more about myself through my experiences. I lived as my true self in every parameter of my life, and at home I was just me too. My identity wasnt discussed, but in a way it didnt matter to me, not out of a dismissive place or malice, but because I did as I pleased everywhere else and I believed nobody had the power to judge me or stop me from doing that, Dinesh shared.

However, this element of judgment became an issue as both his personal and professional life evolved. All of a sudden, he was judged for his lifestyle and looked down upon. Many thought that his lifestyle did not or could not coincide with his faith or culture and professionalism, simply thinking his viewpoints were naive and uninformed because he was young.

So, Dinesh moved to London and started his own business at just 15.

I am a very proud Brit but Im also a very proud Indian man and growing up in the UK, experiencing the culture and heritage that seemed diluted with judgment and unnecessary conservatism, left me longing to experience a missing part of myself.

So, he started traveling India, absorbing everything he could from the food culture to the heritage, learning a great deal more about his faith and, in turn, discovering a missing part of himself.

Experiencing gay culture in India was interesting. Being bombarded by Asian communities in the UK with judgment made me feel as though I would be experiencing the same within India. But to my surprise, it seemed like gay culture was thriving, as so many individuals were expressing their true selves. I was used to things being underground with conservatism prevailing to a degree, but in Mumbai there did seem to be an air of acceptance and change.

Dinesh was astonished by the resilience of queer-identifying people in India, and their acceptance of anyone into their community seemed far more advanced anything hed experienced previously.

He has since found many south Asians within both India and the UK reaching out for guidance and advice on navigating the often tumultuous road between culture and their queer identity. He said its been eye-opening, but it made him want to be there for more people.

Dinesh now feels more comfortable in his identity than ever, as a gay man, a practicing Hindu and Gujarati Indian, who is also British and loves to cook. These aspects of his personal identity arent ones he feels he needs to prove to anyone.

It makes me really happy to see change, and how so many south Asians are now reaching out to guide and support others. Ive had quite a rollercoaster ride of a life over the last six years, but I feel very fortunate for all of that. I really encourage anyone struggling with their identity to reach out and connect with others. The melding of south Asian culture and our faiths and heritage with a queer identity at times seems daunting, but it doesnt have to be. I believe that you can live life as your true self, and no one has the power to take that away from you.

Sion grew up in a small village and was quite literally the only gay in the village.

However, he didnt fully realize his sexuality until he grew older, as growing up in a small village meant he didnt think being gay was a thing. He didnt think it could be a part of his life.

Quite simply, the concept of being gay did not exist for me. It was hidden in the media for the most part, and was not apparent in day-to-day life. I had never knowingly met or spoken to another gay person living in my small village or indeed in the neighbouring towns, perhaps if I had I might have had a moment of realization sooner, he said.

Given that he did not have an awareness of people being gay or that gay relationships existed, Sion did not follow the common story of coming out in a small town and escaping to the big city. In fact it was quite the opposite.

He left his small village and headed to the big city for university, while very much still in the closet.

It was during this time he met people from all walks of life, as his eyes were finally opened. Sion found himself in the right environment with the right people, and felt safe to start exploring his sexuality.

It wasnt until a year later when he properly came out, after going to a gay club with his housemates and waking up the next morning with a shifted mindset and sense of clarify the next morning.

A month later I came out to my friend in strictest confidence and he subsequently made it his mission to get me out for our final night out of our first year of university that night I met my now husband.

Fast forward seven months of dating and coming out to friends and family one by one, Sion had finally made it fully out of the closet with a new boyfriend in hand.

It scares me to think how things would have been different and much more difficult had I not had the chance to change my environment from small village to big city, he admitted.

Fast forward again 13 years, and hes now living his best life alongside his husband. He gets to travel the world and share his experiences with other LGBTQ people in a bid to make the world safer and coming out easier than it was for him he wouldnt have it any other way.

Growing up in a small town can often make you feel far more visible as an LGBTQ person. I didnt realize this would be the case until I tried to come out at the age of 15, shared Emilie.

Her coming out didnt go as well as she had hoped and she felt under a lot of scrutiny, that people saw her as deviant and exotic and, in some cases, threatening. It was attention she hadnt counted on and certainly didnt enjoy it.

The sort of books and films I was into growing up featured positive responses to characters being their authentic selves. And these characters were often rewarded with love, excitement and adventure. But life just went on for me. Nothing remarkable happened, and sometimes it felt the big queer life I should be leading was happening somewhere without me.

This prompted her to move away from the sleepy countryside and start afresh in a big city.

Once she had settled, her anxiety over being judged, outed or feeling like her authentic self wasnt welcome began to decrease.

She no longer felt like she was standing out, because the city hosted such a mix of people, and she could see herself everywhere. She says it was joyous to develop self confidence and, a decade later, that confidence sticks with her no matter where in the world she is.

All the queer spaces and events I got to immerse myself in gave me the courage to come out for a second time a few years ago. I had the privilege of marching at London Pride, I was openly dating, my family and friends and everyone at work knew who I was, and no one had any issues. It was great.

Emilie stresses that she has to continuously come out to new people in new situation, but thanks to the incredible reactions shes had to bring who she is, that gets easier every time, something she believes her move to the city is responsible for.

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Prince Charles praises ‘brave individuals’ who are ‘standing up for freedom’ in heartfelt New Year message – Woman & Home

Posted: at 1:26 am

Prince Charles has written a powerful New Year message to those fighting for freedom in war torn countries such as Afghanistan, Syria and Myanmar. His human rights message said we should take a moment to 'remember the many people around the world who are standing up for freedom'.

The New Year's Instagram post, which the Duke posted yesterday, and has since amassed thousands of likes started with, "As we start a New Year, we might take a moment to remember the many people around the world who are standing up for freedom and human rights."

The message was accompanied by a picture of him visiting an International Rescue Committee centre, an organisation that works with communities displaced by war and conflict, in Jordan at the end of last year.

Prince Charles' message of hope went on to say, "In places such as Afghanistan, Syria and Myanmar, among others, the threats and reality of political and religious persecution and insecurity are coupled with an increasingly dire humanitarian situation.

He went on to pay tribute to those who faced these situations last year, "In the face of such adversity, incredibly brave individuals, local communities and international organizations are responding to great needs by providing vital assistance."

While the Prince Of Wales, who spent four days on a royal tour of the Middle East with Camilla in November, also had a wish for 2022, "I pray for peaceful resolutions to these conflicts and that we might all be blessed with the courage to support those in need, wherever they may be."

Prince Charles has been a patron of the British Red Cross since 2003 and a patron of the International Rescue Committee since January 2020.

Founded in 1933 the International Rescue Committee is at work in over 40 countries to help people to survive, recover and gaincontrol of their future.

Charles and Camilla, like Kate and William, had a quiet festive periodand were said to have spent Christmas day with the Queen, after she cancelled a big Christmas celebration at Sandringham.

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Freedom Transit rings in New Year with new amenities – Observer-Reporter

Posted: at 1:26 am

Freedom Transit is leaning into the New year, new you mantra.

The transportation company, which connects greater Washington County to the Mon Valley and Pittsburgh, recently completed upgrades to five area bus stops and rolled out a mobile app that provides riders with real-time bus information.

Its something thats been in the works for about ... the past year, said Alexandra Sakalik, Freedom Transits outreach and marketing director. Dark hits at 4, 4:30, now. We wanted something that was safer for our riders.

To ensure rider safety and make waiting for the bus more comfortable, Freedom installed new shelters, complete with benches and lights, at five locations throughout Washington and Canonsburg.

The sites upgraded with shelters, seating and solar-powered lighting are stops at Beau Street Park and Ride, Crumrine Tower, Jefferson Avenue outside of Pathways, Valleyview Terrace and Southpointe Park and Ride.

These are just safer places for our riders to wait, especially now that we have the benches, Sakalik said. Weve already identified a couple locations for our next phase.

Sakalik said rider input and traffic patterns factored into which stops received upgrades, and the next phase of upgrades should be completed in fall 2022.

While some riders wait for seating and lighting to be installed at additional stops, the wait for a real-time bus ride app is over for all Freedom Transit commuters.

At the beginning of December, the company launched a mobile app that offers a variety of commuter data. Created in collaboration with developer Modeshift, the app uses Google Maps so riders can track their bus in real-time and locate the bus stop nearest them.

Lets just say youre closest to the Local A, said Sakalik. (The app) will tell you how many more minutes, give you directions in order to get to the bus stop.

Riders can also link their smartcards to the app, which means the app can be used in place of physical bus passes. Commuters can also pay for rides through the app.

As of Wednesday afternoon, Sakalik said more than 70 Freedom Transit riders had installed the app to their smartphones a number much higher than the company expected, given the app launched only two weeks ago.

We think thats a pretty good number, Sakalik said, noting the first pass sold through the app was a Prexie pass (Freedom Transit offers Washington School District students discounted rides).

Freedom Transit is, like several area transit companies, looking for ways to welcome commuters back on busses after the number of public transportation riders dipped during the global pandemic. The company hopes new amenities, including free on-board WiFi, which was rolled out over the summer, and upgraded stops will encourage folks to hop aboard.

This past year has really been an up-and-coming year for us, said Sakalik. I think a lot of transit agencies are pulling out all the stops to get riders back. We have a lot going on. Were just happy to be a part of that change and happy to get new riders and bring back old riders alike.

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Moving to Texas for more freedom? Not so fast. Study ranks the state 49th in personal liberties – San Antonio Current

Posted: at 1:26 am

For a second consecutive year, Texas posted the highest annual population growth among U.S. states, according to Census figures, and many of its new residentscite the state'slaissez-faire governance aspart of the draw.

But if youre searching for greater personal freedoms, a new study suggests you'd best look elsewhere.That analysis, published by self-described libertarian think tank the CATO Institute, found that the Lone Star State a place where you can open-carry without a mask on ranks 49th among U.S. states when it comes to personal freedoms.

CATO gave Texas high scores when it comes to economic and fiscal freedom it ranks No. 10 and No. 12 on those, respectively and the study ranked the state No. 21 overall when it comes to a variety of freedoms.

But that's not to say Texas is a beacon in the personal freedom category. The state's "generally aggressive" criminal justice record helps explainits finish near the bottom.

More Texas citizens are imprisoned per capita than those of any other democracy on the planet, for example. And although only 12% of the state is African American, that demographic makes up a majority of the states inmates. Drug-related arrests are around the national average, but possession of fewer than two ounces of weed under the state's "harsh" cannabis laws can land violators a 180-day sentence in the county jail.

The study also cited the Texas' tough stance on gambling as a drag on its personal freedom ranking. Even sports betting remains illegal here, even though the practice is permitted in 30 states and the District of Columbia.

What may be most surprising to some about the CATO analysis is where the Lone Star State is positioned when it comes to the rights of gun owners. Texas ranked No. 31, while Kansas topped the nation with the leniency of its gun laws.

Texas may be a cheaper place to live, its people friendlier and its climate balmier than most of the country. But if youre looking for more freedom, you may want to consider Nevada, the freest state in the country, according to the CATO report.

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Moving to Texas for more freedom? Not so fast. Study ranks the state 49th in personal liberties - San Antonio Current

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The Boston Celtics, my political activism, and a call for greater freedom – The Boston Globe

Posted: at 1:26 am

This holiday season has given me time to reflect on my return to Boston as a Celtics player and my longer journey to reclaim my freedom as an American citizen. In the past few months, Ive changed my last name to Freedom, become a US citizen, and continued my political activism, having most recently launched a campaign in which I wear custom shoes dedicated to human rights crises around the world. Ive doubled down on my advocacy, using every opportunity to keep world attention on human rights, whether on the court, as a regular guest in the media, or in meetings with politicians.

The journey to get here wasnt easy. The last time I spoke with my parents was in 2015. Any contact with me could get them arrested. Turkish authorities forced my family to publicly disown me, imprisoned my father on charges that he was a member of a terrorist organization (he has since been acquitted), revoked my passport, and issued 10 arrest warrants against me in four years. Fortunately, my teammates have been there every step of the way to get me where I am today, becoming family after mine was broken up.

When I was targeted by Turkey for speaking out against President Recep Tayyip Erdogan, my teammates consistently checked in on me and offered to help in any way they could. When Celtics games were pulled from Chinas Tencent streaming service this season in retaliation for my wearing shoes that called for greater freedom in Tibet, my teammates were the first to express their unconditional support.

They encourage me to stand up for whats right, give voice to innocent people, and always remind me they have my back no matter what. Theyre my No. 1 source of motivation and hope.

My activism has also brought the team closer together. Our locker room has become a forum for deeper discussions about how to effectively seek justice in the world. My shoes spark running conversations about the messages behind them. And my citizenship process gave us a chance to discuss American history, government, the Constitution, and even the current situation facing marginalized communities. America is far from a perfect union with its own set of appalling human rights abuses.

Most shockingly, the United States leads the world in jailing its own people, mostly due to crimes related to poverty, mental illness, or addiction social and health issues that need to be addressed. As a country that accounts for nearly 5 percent of the global population, the United States has 20 percent of the worlds prison population, with people of color suffering disproportionately. The consequences dont end with incarceration, as former inmates can go on to face a lifetime of barriers to finding work and re-entering society. Even a progressive state like Massachusetts locks up people at higher rates than many countries. According to a 2020 Harvard Law report on racial disparities in Massachusetts criminal system, Black and Latinx people receive longer prison sentences than similarly situated white people for similar offenses. The devastating impact of mass incarceration on our communities is a stark reminder of the work that needs to be done at home.

In the lead-up to my citizenship test, my teammates printed out the questions to ensure I was ready. Theyd test me over and over again, in the locker room, on the plane, and during our health treatments. When I finally passed, they were ecstatic and offered to throw me a party. But I said all I want are some American-themed cupcakes. To my surprise, the next day I found the perfect red and blue cupcakes in our dining hall, made by my Celtic brothers themselves.

Wherever I find myself, Boston sport fans similarly show gratitude and love for what I stand for. I get standing ovations whenever I check into games. I feel at home here in Massachusetts. And its because we all know this is bigger than basketball.

Enes Kanter Freedom is a center for the Boston Celtics.

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The Boston Celtics, my political activism, and a call for greater freedom - The Boston Globe

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Letter to the editor: Portland vaccine passport backers targeting others’ freedom – pressherald.com

Posted: at 1:26 am

Anyone notice the Portland restaurants calling for vaccine passports are all upscale establishments downtown? Id wager theyre not having quite the time making payroll as many of their hundred-plus counterparts all across the city.

This has been one of the hallmarks of the pandemic: people in positions of comfort speaking broadly for everyone. As was pointed out early on, we arent all in the same lifeboat. The failure to honestly acknowledge this has been a mighty example of the systemic inequality that this crowd loves to complain about. And yes, Im talking about the left.

What happened to the Democrats who thought the Patriot Act was a grotesque overreach of constitutional rights? How can they chant my body, my choice and then take away a childs right to an education if they dont submit to an experimental vaccine?

These same Democrats have stood back and watched (from home) while the largest transfer of wealth in history has migrated from the middle class to the 1 percent. Theyve happily hitched their wagons to Big Pharma and Big Tech and in doing so have become the same defenders of the ultra wealthy who they despised when George W. Bush was in office.

Freedom and privacy arent antiquated talking points of the alt-right. Theyre fundamentals of human decency and a civilized society. These restaurants are more than welcome to insulate themselves in a homogenous nest of zero-risk-takers if theyre that worried. Thats a right they actually do have.

William HiltonPortland

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Letter to the editor: Portland vaccine passport backers targeting others' freedom - pressherald.com

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The bill on social media incitement is a danger to freedom of expression – Haaretz

Posted: at 1:25 am

The bill to prevent incitement on social media is supposed to address a real problem that ought to be addressed. But despite its good intentions, the bills provisions are excessive and completely disconnected from the requirements of proportionality. It is extremely dangerous to freedom of expression, in both the short term and the long, and will also be of dubious effectiveness. Moreover, its far from clear that alternatives were properly considered.

The bill would turn district court judges into censors at the request of prosecutors. Granted, judges wouldnt be able to prevent publication a priori, but they could order content removed from a website. It fails to draw the vital distinction between social media and local or foreign news sites, which obey the rules of journalistic ethics and editorial responsibility. Its provisions would apply to both alike.

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Two conditions are listed for issuing a content removal order. First, the contents publication must be a criminal offense. Second, theres a real chance that letting it remain online would undermine personal safety, public safety or national security.

The first condition is extremely broad, which in and of itself makes the bill disproportionate. Were the criminal offenses that restrict free speech here only the ones suited to a democratic country, like incitement to violence, that would be one thing. But they arent.

We inherited outdated offenses that contradict democracy from the British Mandate, which wasnt a democratic regime. These include insulting a public servant, contempt of court, offending religious sensibilities and, worst of all, incitement to sedition. This alone would be enough to spark contempt for the government and public dissatisfaction. But Israels legislature also added poorly worded criminal provisions on issues like revealing official secrets and slander.

Despite repeated urging by courts and academics that the definitions of these crimes be adapted to a democratic system of government, nothing has been done. And now, the government seeks to turn them into a platform for serious harm to freedom of expression. It would be no exaggeration to say that massive use of these offenses could even endanger freedom of expression.

Granted, the prosecution generally exercises restraint in their use, but thats in criminal proceedings against individuals. Its not clear similar restraint would be exercised about content removal. Moreover, theres no guarantee that current prosecution practices wont change tomorrow.

Nor is the second condition reassuring. While harm to personal safety is clear and defined, national security and public safety are extremely vague terms that can be infinitely expanded.

Court rulings allow publication to be barred only to avoid grave harm to an important public interest, and only when such harm is a near certainty. But the bill turns its back on these essential limitations, thereby showing contempt for freedom of expression.

Sometimes, the harm that publishing something does to a significant public interest is offset by the very public value of publishing it, such as its contribution to freedom of political, religious or artistic expression. But the bill makes no mention of this idea.

Moreover, in the event of a criminal conviction, the court will be authorized to order content removed even if the second condition isnt met. But if removing content is a preventive measure, what justifies doing so if the conditions justifying prevention havent been met?

This broad willingness to undermine freedom of expression ought to worry us. Israelis seem to be greatly concerned about their own freedom of expression and that of people who think like them, but they waive this concern with regard to conflicting opinions. The understanding that our commitment to freedom of expression is actually judged by our treatment of offensive, outrageous, stomach-churning statements hasnt taken root. Without full freedom of expression, democracy cant exist even in the thin, procedural sense of the term. Without full freedom of expression, self-actualization, cultural flourishing and progress are all impossible. A bill like this contributes to eroding the principle of freedom of expression.

If district court judges are authorized to serve as a kind of censor, the day is not far off when proposals will be made to expand their powers by letting them prevent publication a priori, even in the classic, non-internet media. The day is also not far off when proposals will be made to turn the content removal order into a basis for conviction of the very crime that the order deemed the content to commit.

The bill also reflects the Knessets fondness for legal proceedings that rely on classified information that the party injured by the order isnt allowed to see, even if this material is inadmissible as evidence. Nor does the bill insist that the party liable to be harmed by the order be present during the proceedings. In other words, these proceedings will be far from due process.

In addition, the bill reflects contempt for the criminal process, since a judicial ruling that a law was violated will essentially replace a criminal conviction obtained with all the procedural safeguards that criminal trials are supposed to include. If its possible to determine that a crime was committed through such an abbreviated process, why even hold criminal trials, with all their complications?

One also has to ask whether this system which is based on the states employment of force via the judicial branch will even be effective in achieving its goals. After all, quite a lot of time will pass before the prosecution decides to go to court and the court holds a hearing and rules.

But the nature of the internet is that offensive, juicy content spreads like wildfire in all directions, to other online platforms and the mainstream media. Thus, the question is whether this isnt a vain pursuit, because it will respond too late.

Its highly unlikely that the government thoroughly explored alternatives that would empower the individual who was harmed and assign liability to the social media companies. These companies cant be trusted to keep their platforms from being turned into a no mans land where justified, proportionate prohibitions on truly dangerous speech are violated. But they could be made to do so through professional intermediaries free of economic or political interests.

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The bill on social media incitement is a danger to freedom of expression - Haaretz

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