How to use precision medicine to personalize COVID-19 treatment according to the patients genes – TheStreet

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Courtesy of Colin Allen, University of Pittsburgh and David Finegold, University of Pittsburgh

Tom Hanks and his wife, Rita Wilson, were among the earliest celebrities to catch the novel coronavirus. In an interview at the beginning of July, Hanks described how differently COVID-19 had affected each of them in March.

My wife lost her sense of taste and smell, she had severe nausea, she had a much higher fever than I did. I just had crippling body aches, he said. I was very fatigued all the time and I couldnt concentrate on anything for more than about 12 minutes.

Why does COVID-19 present such different symptoms or none at all in different people?

Preexisting conditions can only be part of the story. Hanks is over 60 and is a Type 2 diabetic, putting him in a high-risk group. Nevertheless, he survived his brush with the virus with no pneumonia and apparently without any long-lasting effects. Knowing what causes variation in different patients could help physicians tailor their treatments to individual patients an approach known as precision medicine.

In recent years, a gene-centric approach to precision medicine has been promoted as the future of medicine. It underlies the massive effort funded by the U.S. National Institutes of Health to collect over a million DNA samples under the All of Us initiative that began in 2015.

But the imagined future did not include COVID-19. In the rush to find a COVID-19 vaccine and effective therapies, precision medicine has been insignificant. Why is this? And what are its potential contributions?

We are a physician geneticist and a philosopher of science who began a discussion about the promise and potential pitfalls of precision medicine before the arrival of COVID-19. If precision medicine is the future of medicine, then its application to pandemics generally, and COVID-19 in particular, may yet prove to be highly significant. But its role so far has been limited. Precision medicine must consider more than just genetics. It requires an integrative omic approach that must collect information from multiple sources beyond just genes and at scales ranging from molecules to society.

Inherited diseases such as sickle cell anemia and Tay-Sachs disease follow a predictable pattern. But such direct genetic causes are perhaps the exception rather than the rule when it comes to health outcomes. Some heritable conditions for instance, psoriasis or the many forms of cancer depend on complex combinations of genes, environmental and social factors whose individual contributions to the disease are difficult to isolate. At best, the presence of certain genes constitutes a risk factor in a population but does not fully determine the outcome for an individual person carrying those genes.

The situation becomes yet more complicated for infectious diseases.

Viruses and bacteria have their own genomes that interact in complex ways with the cells in the people they infect. The genome of SARS-CoV-2 underlying COVID-19 has been extensively sequenced. Its mutations are identified and traced worldwide, helping epidemiologists understand the spread of the virus. However, the interactions between SARS-CoV-2 RNA and human DNA, and the effect on people of the viruss mutations, remain unknown.

Tom Hanks and his wife caught the virus and recovered in a matter of weeks. Presumably each was infected over the course of a few minutes of exposure to another infected person, involving cellular mechanisms that operate on a timescale of milliseconds.

But the drama of their illness, and that of the many victims with far worse outcomes, is taking place in the context of a global pandemic that has already lasted months and may continue for years. People will need to adopt changes in their behavior for weeks or months at a time.

What should a precision medicine approach be in a pandemic? The gene-centric vision of precision medicine encourages people to expect individualized gene-targeted fixes. But, genes, behavior and social groups interact over multiple timescales.

To capture all the data needed for such an approach is beyond possibility in the current crisis. A nuanced approach to the COVID-19 pandemic will depend heavily on imprecise population level public health interventions: mask-wearing, social distancing and working from home. Nevertheless, there is an opportunity to begin gathering the kinds of data that would allow for a more comprehensive precision medicine approach one that is fully aware of the complex interactions between genomes and social behavior.

With unlimited resources, a precision medicine approach would begin by analyzing the genomes of a large group of people already known to be exposed to SARS-CoV-2 yet asymptomatic, along with a similar-sized group with identified risk factors who are dying from the disease or are severely ill.

An early study of this kind by Precisionlife Ltd data mined genetic samples of 976 known COVID-19 cases. Of these, 68 high-risk genes were identified as risk factors for poor COVID-19 outcomes, with 17 of them deemed likely to be good targets for drug developments. But, as with all such statistical approaches, the full spectrum of causes underlying their association with the disease is not something the analysis provides. Other studies of this kind are appearing with increasing frequency, but there is no certainty in such fast-moving areas of science. Disentangling all the relevant factors is a process that will take months to years.

To date, precision medicine has proven better suited to inherited diseases and to diseases such as cancer, involving mutations acquired during a persons lifetime, than to infectious diseases. There are examples where susceptibility to infection can be caused by malfunction of unique genes such as the family of inherited immune disorders known as agammaglobulinemia, but these are few and far between.

Many physicians assume that most diseases involve multiple genes and are thus not amenable to a precision approach. In the absence of the kind of information needed for a multi-omic approach, there is a clear challenge and opportunity for precision medicine here: If it is to be the future of medicine, in order to complement and expand our existing knowledge and approaches, it needs to shift from its gene-centric origins toward a broader view that includes variables like proteins and metabolites. It must consider the relationships between genes and their physical manifestations on scales that range from days to decades, and from molecules to the global society.

Colin Allen, Distinguished Professor of History & Philosophy of Science, University of Pittsburgh and David Finegold, Professor, Department of Human Genetics, Pitt Public Health, University of Pittsburgh

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How to use precision medicine to personalize COVID-19 treatment according to the patients genes - TheStreet

Meiotic chromosome synapsis depends on multivalent SYCE1-SIX6OS1 interactions that are disrupted in cases of human infertility – Science Advances

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Abstract

Meiotic reductional division depends on the synaptonemal complex (SC), a supramolecular protein assembly that mediates homologous chromosomes synapsis and promotes crossover formation. The mammalian SC has eight structural components, including SYCE1, the only central element protein with known causative mutations in human infertility. We combine mouse genetics, cellular, and biochemical studies to reveal that SYCE1 undergoes multivalent interactions with SC component SIX6OS1. The N terminus of SIX6OS1 binds and disrupts SYCE1s core dimeric structure to form a 1:1 complex, while their downstream sequences provide a distinct second interface. These interfaces are separately disrupted by SYCE1 mutations associated with nonobstructive azoospermia and premature ovarian failure (POF), respectively. Mice harboring SYCE1s POF mutation and a targeted deletion within SIX6OS1s N terminus are infertile with failure of chromosome synapsis. We conclude that both SYCE1-SIX6OS1 binding interfaces are essential for SC assembly, thus explaining how SYCE1s reported clinical mutations give rise to human infertility.

Meiotic cell division is defined by a unique and highly dynamic program of events that result in homologous chromosome synapsis, crossover (CO) formation, and subsequent homolog segregation into haploid germ cells (13). Homologous chromosome pairs are established through interhomolog recombination searches from up to 400 induced double-strand breaks (DSBs) per cell (4). Once established, local recombination-mediated alignments are converted into the single continuous synapsis of aligned homologous chromosomes through the zipper-like assembly of the synaptonemal complex (SC) (5). The SCs supramolecular protein structure mediates continuous 100-nm tethering between homologous chromosome axes and provides the necessary three-dimensional framework for crossover formation (2). Following SC disassembly, crossovers provide the sole physical links between homologs at metaphase I, so are essential for ensuring correct homolog segregation in addition to providing genetic diversity (2).

The SC has an iconic and highly conserved tripartite structure that has been observed across meiotically reproducing eukaryotes (6). This consists of lateral elements (LEs) that coat the two homologous chromosome axes and a midline central element (CE), with a series of transverse filaments that bind together these longitudinal electron-dense structures (Fig. 1A) (7). The protein components of the mammalian SC have been identified as transverse filaments protein SYCP1 (Synaptonemal complex protein 1) (8), CE proteins SYCE1, SYCE2, and SYCE3 (Synaptonemal complex central element proteins 1 to 3), SIX6OS1, and TEX12 (Testis-expressed protein 12) (912), and LE proteins SYCP2 and SYCP3 (13, 14). All transverse filament and CE components are essential for SC assembly, and their individual disruption leads to infertility owing to meiotic arrest with failure of DSB repair (10, 11, 1518). In contrast, disruption of LE components produces a sexual dimorphism of male infertility and female subfertility (19, 20), with SYCP3 deficiency in females promoting germ cell aneuploidy and embryonic death (21).

(A) Schematic of the SC demonstrating its tripartite structure of two chromosome-bound LEs and a midline CE. Synapsis is achieved through N-terminal head-to-head assembly of SYCP1 molecules, which are bound via their C termini to meiotic chromosomes. SYCP1 head-to-head assembly is structurally supported within the CE by SYCE3 (red), an SYCE1-SIX6OS1 complex (yellow), and SYCE2-TEX12 fibrous assemblies (green). (B) Human SYCE1 (top) and SIX6OS1 (bottom) sequence schematics indicating the location and consequence of infertility-associated mutations of SYCE1 and 1021 internal deletion of SIX6OS1, alongside the principal constructs used in this study. (C) SDSpolyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified recombinant proteins used in this study. The dominant degradation product of SYCE1POF is indicated by an asterisk; its identity was confirmed by the observed cleavage of degraded MBP- and His-SYCE1POF fusion proteins upon treatment with TEV protease (fig. S1, A and B), consistent with it representing C-terminal degradation down to SYCE1s structural core. Mw, weight-average molecular weight. (D) SEC-MALS analysis. SYCE1core (yellow), SYCE1POF (green), and full-length SYCE1 (violet) are dimeric species of 36, 48 (39 kDa for the degradation product), and 86 kDa, respectively (theoretical dimers: 37, 55, and 80 kDa). dRI, differential refractive index. Data for SYCE1core and full-length SYCE1 are reproduced from (28).

In recent years, a variety of cellular imaging, biochemical and structural biology approaches have begun to uncover the molecular structures, interactions, and mechanisms responsible for mammalian SC assembly. SYCP1 self-assembles into a supramolecular lattice that provides the underlying 100-nm synapsis between chromosome axes (22, 23), while SYCP3 assembles into regularly repeating filaments that support chromosomal looping (24, 25). The five CE proteins provide essential structural supports for the SYCP1 lattice that enable its continuous and cooperative extension along the entire chromosome length. In this capacity, CE proteins have been categorized as synaptic initiation factors (SYCE3, SYCE1, and SIX6OS1) and elongation factors (SYCE2 and TEX12), of which their disruption leads to complete loss of tripartite SC structure and failure of extension of short SC-like stretches, respectively (10, 11, 1618). Of synaptic initiation factors, SYCE3 forms dimers that undergo potentially limitless self-assembly (26, 27), SYCE1 forms antiparallel dimeric assemblies (28), and SIX6OS1 is an SYCE1-interacting protein of unknown structure (11). These likely act as short-range structural supports between SYCP1 molecules, possibly in transverse, longitudinal, and vertical orientations to stabilize a local three-dimensional SYCP1 lattice (22). In contrast, SYCE2 and TEX12 exist as a seemingly constitutive complex that undergoes self-assembly into fibers of many micrometers in length (29), which likely provide the long-range structural supports that stabilize continuous growth of the SYCP1 lattice along the entire chromosome axis (22).

Owing to the essential roles of meiotic recombination, synapsis, and chromosome dynamics in mammalian meiosis (15, 3034), their defects are associated with human infertility, recurrent miscarriage, and aneuploidies (35, 36). As genetic causes of infertility, they typically fall within the category of idiopathic cases, having no readily diagnosable and clinically resolvable cause. Within the 10 to 15% of couples who suffer from infertility, approximately 25% are idiopathic and of likely genetic origin, comprising 50 to 80% of cases of nonobstructive azoospermia (NOA) and premature ovarian failure (POF) (36, 37). While individual infertility mutations are inherently unlikely to become widespread in a population, they can be found within families, especially when consanguineous (38), and provide crucial insights into their common targets and the molecular mechanisms that they disrupt.

Within the SC, familial infertility mutations have been identified for SYCP3 and SYCE1 (36). All identified SYCP3 mutations are autosomal dominant and alter or delete its structural cores C terminus that mediates filamentous assembly, so likely sequester wild-type (WT) molecules into inactive complexes (24, 36). In contrast, the three identified SYCE1 mutations are autosomal recessive and were found in two familial cases of NOA and one of POF (36). The two NOA cases are splice-site mutations, c.197-2A>G and c.375-2A>G, which are predicted to result in a truncated product of amino acids 1 to 65 and an internal deletion of amino acids 126 to 155, respectively (39, 40). These remove or delete part of human SYCE1s structural core that is encoded by amino acids 25 to 179, so can be explained by disruption of its dimeric structure (Fig. 1B) (28, 36). The POF mutation c.613C>T generates a premature stop codon (p.Gln241*) to give a truncated product of amino acids 1 to 240, relative to the canonical 351amino acid isoform (Fig. 1B) (41). However, as this truncation lies outside SYCE1s structural core, the molecular mechanism that is disrupted, and thereby responsible for infertility, remains unknown.

Here, we combine mouse genetics and cellular and biochemical studies to reveal a multivalent interaction mode between SYCE1 and SIX6OS1 that is disrupted by infertility-associated mutations of SYCE1. We find that the SIX6OS1 N terminus binds and disrupts the core dimeric structure of SYCE1 (amino acids 25 to 179) to form a 1:1 complex as the first interface, and its downstream sequence binds to SYCE1 amino acids 177 to 305 as the second interface. SYCE1s infertility-associated mutations c.375-2A>G (NOA) and c.613C>T (POF) specifically disrupt the first and second interfaces, respectively. Mice harboring the SYCE1 POF mutation and a targeted deletion within SIX6OS1 (which disrupts the first interface) are infertile, with failure of SC assembly. We conclude that both SYCE1-SIX6OS1 binding interfaces are essential for SC assembly and meiotic division, thus explaining how human infertility results from the differential targeting of binding interfaces by SYCE1s reported clinical mutations.

The SYCE1 POF mutation c.613C>T encodes a premature stop codon (p.Gln241*) that is predicted to generate a truncated protein product of amino acids 1 to 240, relative to SYCE1s canonical 351amino acid isoform (Fig. 1B) (41). We previously demonstrated that an N-terminal structural core encoded by amino acids 25 to 179 (SYCE1core) forms an -helical antiparallel coiled-coil structure that mediates head-of-head dimerization of SYCE1 (28). As this core region is retained (Fig. 1B), we predicted that SYCE1s antiparallel dimeric structure would be maintained within the 1- to 240-amino acid truncated product of the POF mutation (SYCE1pof). To test this, we purified recombinant SYCE1pof, generating purified material that contained approximately equal quantities of the full protein and a degradation product of apparent size consistent with degradation to the C-terminal boundary of its structural core (Fig. 1C and fig. S1, A and B). Circular dichroism (CD) spectroscopy confirmed that SYCE1pof contains a proportion of -helical structure consistent with retention of the 25179 core structure (fig. S1C), and SYCE1pof and SYCE1core demonstrated identical melting temperatures (Tm) of 39C (fig. S1D). Furthermore, analysis by size exclusion chromatography multiangle light scattering (SEC-MALS) confirmed that the full and degraded proteins are homodimers of 48 and 39 kDa, respectively (Fig. 1D). We conclude that SYCE1pof retains the dimeric structure imposed by its core 25179 region, so its SC and meiotic defects must result from additional structural or functional roles of its deleted C terminus.

Having established its retention of core dimeric structure, we next sought to determine the structural and functional consequence of the SYCE1 POF mutation on the SC and meiotic division in vivo. We thus generated mice harboring mutations of Syce1 alleles to introduce stop codons at amino acid position 243, equivalent to the human p.Gln241* mutation (figs. S2 and S3). While heterozygotes (designated Syce1POF/WT) were fertile, both male and female homozygotes (designated Syce1POF/POF) were infertile, replicating the autosomal recessive pattern of the POF mutation in humans (41). In male mutant mice, we observed reduced testis size (63% smaller, n = 3 mice at 2 months of age; fig. S4A) and a zygotene-like arrest similar to that observed in the SYCE1 knockout (16). There was defective SC assembly, with reduced staining for SYCP1 (Fig. 2A) and SYCE3 (Fig. 2B) and no staining for SYCE1 (Fig. 2C), SIX6OS1 (Fig. 2D), and SYCE2-TEX12 (fig. S4, B and C). Analysis of SYCE1 expression in the testis of Syce1POF/POF mice confirmed the presence of Syce1 transcript and a protein product of the correct molecular weight, albeit at reduced levels in comparison with WT (fig. S4, D and E, and table S1A). The Syce1POF open reading frame achieved WT levels of protein expression in a heterologous 293T cellular system (fig. S4F). We next studied the kinetics of DSB repair. Meiotic DSBs are generated by the nuclease SPO11 and are then resected to form single-stranded DNA ends that invade into the homologous chromosome by the recombinases RAD51 (DNA repair protein RAD51 homolog 1) and DMC1 (Meiotic recombination protein DMC1/LIM15 homolog) (42). DSBs are labeled by the presence of phosphorylated H2AX (-H2AX) (43). The distribution of -H2AX in mutant spermatocytes was similar to that found in WT cells at early prophase I but show increased staining at zygotene-like arrest (Fig. 2E). The distributions of RAD51 and DMC1 were detected on aligned LEs (Fig. 2, F and G) but in absence of mismatch repair protein MLH1 (DNA mismatch repair protein Mlh1) (marker of crossing-overs) (Fig. 2H). Together, these data indicate generation of DSBs but with failure of their repair and CO formation in Syce1POF/POF. In female mutant mice, we observed no follicles in adult ovaries (fig. S5A), and embryonic oocytes demonstrated zygotene arrest with mostly unaligned chromosome axes, recapitulating the human POF syndrome. Analysis of the SC revealed similar defects, with reduction in SYCP1 and SYCE3 (Fig. 3, A and B) staining (though to a lesser extent than males), and absence of SYCE1, SIX6OS1 (Fig. 3, C and D), and SYCE2-TEX12 (fig. S5, B and C). The distribution of -H2AX, RAD51, and DMC1 labeling in zygotene-like mutant oocytes was also increased and lacked MLH1 foci (Fig. 3, E to H). Thus, the SYCE1 POF mutation leads to male and female infertility with phenotypes of failed DSB repair, synapsis, and lastly SC assembly, similar to those previously observed upon disruption of structural components of the SC CE (10, 11, 1618).

(A) Double immunolabeling of WT pachytene and Syce1POF/POF zygotene-like spermatocytes with SYCP3 (red) and SYCP1 (green). In Syce1POF/POF spermatocytes, AEs fail to synapse and show a weak staining of SYCP1 along the axial elements (AEs). a.u., arbitrary units. (B to D) Double immunolabeling of spermatocyte spreads with SYCP3 (red) and the CE proteins (green). Syce1POF/POF zygotene-like spermatocytes showed a highly reduced signal of SYCE3 (B) and the absence of (C) SYCE1 and (D) SIX6OS1 from the AEs. (E) Double immunolabeling of -H2AX (green) and SYCP3 (red) in spermatocyte spreads from WT and Syce1POF/POF mice. -H2AX staining was persistent in Syce1POF/POF zygotene-like spermatocytes, but was restricted to the sex body in WT pachytene cells. (F and G) Double immunofluorescence of (F) RAD51 or (G) DMC1 (green) and SYCP3 (red). Syce1POF/POF zygotene-like spermatocytes showed increased numbers of foci of RAD51 and DMC1 along the AEs in comparison with WT, indicating unrepaired DSBs. (H) Double immunolabeling of MLH1 (green) and SYCP3 (red) showing the absence of COs (MLH1) in arrested Syce1POF/POF spermatocytes. Fluorescence intensity levels (A, B, and E) and number of foci (F and G) from WT and zygotene-like arrested spermatocytes are quantified in the right-hand plots. Welchs t test analysis: ***P < 0.0001. Scale bars, 10 m.

(A) Double immunolabeling of oocyte spreads from WT and Syce1POF/POF mice with SYCP3 (red) and SYCP1 (green). Syce1POF/POF oocytes became arrested in a zygotene-like stage where AEs remain unsynapsed and unaligned, with reduced levels of SYCP1. (B to D) Double immunolabeling of oocyte spreads with SYCP3 (red) and the CE proteins (green). Syce1POF/POF zygotene-like oocytes showed reduced SYCE3 signal (B) and a complete absence of (C) SYCE1 and (D) SIX6OS1 from the AEs. IP, immunoprecipitation. (E) Double immunostaining of spread preparations of WT pachytene and Syce1POF/POF zygotene-like oocytes with -H2AX (green) and SYCP3 (red). In Syce1POF/POF oocytes, the levels of -H2AX increased and were more restricted to AEs in comparison with WT pachytene cells. (F to G) Double immunolabeling of (F) RAD51 or (G) DMC1 (green) and SYCP3 (red), showing higher numbers of foci in AEs from mutant oocytes. (H) Labeling of MLH1 (green) and SYCP3 (red). MLH1 foci are absent from the AEs of Syce1POF/POF oocytes. Fluorescence intensity levels (A, B, and E) and number of foci (F and G) from WT and Syce1POF/POF zygotene-like oocytes are quantified in the right-hand plots. Welchs t test analysis: ***P < 0.0001. Scale bars, 10 m.

As the Syce1POF/POF mouse strain indicated a clear structural defect in the SC, we wondered whether the POF mutation may disrupt the known interaction between SYCE1 and fellow SC CE components SIX6OS1 and SYCE3 (11). The expression of SYCE1 and SIX6OS1 in COS7 cells produced cytoplasmic signals that became colocalized in foci upon coexpression (95% cells; Fig. 4A and fig. S6), in keeping with our previous findings (11). SYCE1pof formed similar or slightly reduced numbers of foci that equally colocalized with SIX6OS1, indicating a retention of SIX6OS1 binding (89% cells; Fig. 4A). We further demonstrated a similar coimmunoprecipitation of SIX6OS1 by WT SYCE1 and SYCE1pof upon coexpression in human embryonic kidney (HEK) 293 cells (Fig. 4B). Thus, the SYCE1-SIX6OS1 interaction is retained in the SYCE1 POF mutation. Could other disrupted functions contribute to the effect of the POF mutation? The only other known SYCE1 interactor is SYCE3, which undergoes low-affinity binding, as determined by its dissociation during purification (fig. S7, A and B). In contrast with the WT protein, the expression of SYCE1pof (cytoplasmic foci) in COS7 cells failed to recruit SYCE3 (preferentially nuclear) to their cytoplasmic foci (colocalization between SYCE3 and SYCE1 was observed for 95% of cells expressing WT SYCE1 and 21% of cells expressing SYCE1pof; Fig. 4C and fig. S6). Similarly, SYCE1pof failed to coimmunoprecipitate SYCE3 upon coexpression in HEK293 cells (Fig. 4D). Thus, while the SYCE1-SIX6OS1 complex is retained, the low-affinity SYCE1-SYCE3 complex is largely abolished in the SYCE1 POF mutation.

(A) Mouse SIX6OS1 colocalized with mouse SYCE1 and SYCE1POF in a cytoplasmatic punctate pattern upon coexpression in COS7 cells; the percentage of cells exhibiting colocalization is shown in the right-hand plot (n = 100 cells). DAPI, 4,6-diamidino-2-phenylindole. (B) HEK293T cells were cotransfected with the indicated expression vectors. Protein complexes were immunoprecipitated with anti-Flag or antienhanced green fluorescent protein (EGFP) antibodies, or mouse immunoglobulin G (IgG) as a negative control, and were analyzed by immunoblotting with the indicated antibody. GFP-mSIX6OS1 coimmunoprecipitated with Flag-mSYCE1 and Flag-mSYCE1POF, suggesting that the POF mutation of SYCE1 alone is insufficient to block the interaction. (C) COS7 cells were transfected with mouse Syce3 in combination with mouse Syce1 or Syce1pof as indicated. SYCE1 colocalized with SYCE3 in its own cytoplasmatic punctate pattern, and colocalization was substantially diminished for SYCE1POF (n = 100 cells). (D) Immunoprecipitation of protein complexes from HEK293T-cotransfected cells with an anti-Myc or anti-EGFP antibody or mouse IgG. SYCE1 coimmunoprecipitated with SYCE3, and the interaction was disrupted for SYCE1 POF, suggesting that the C-terminal region of SYCE1 is required for its interaction with SYCE3. The untransfected lanes in (B) and (D) show the absence of all the proteins in total protein extracts from untransfected 293T cells. Scale bars, 20 m.

What is the molecular basis of SIX6OS1 binding by SYCE1? As this is retained in SYCE1pof, we reasoned that SIX6OS1 binding must be mediated by SYCE1s structural core. We screened SYCE1core against a library of SIX6OS1 constructs through bacterial coexpression and identified a robust interaction with amino acids 1 to 67 of SIX6OS1, herein referred to as SIX6OS1N (Figs. 1B and 5A). We were able to purify the SYCE1core-SIX6OS1N complex by reciprocal affinity chromatography, ion exchange, and size exclusion chromatography (Fig. 5B) and found it to be stable under all experimental conditions tested. We were further able to purify similar complexes for SYCE1pof (with the same degradation product as upon isolated expression) and full-length SYCE1 (Fig. 1C and fig. S1B), confirming that SIX6OS1 binding is retained by all constructs containing the 25179 core. CD analysis revealed similar -helical content for SYCE1-SIX6OS1N complexes as for their isolated SYCE1 proteins (fig. S1C). CD thermal denaturation revealed slightly increased cooperativity of unfolding and melting temperatures for SYCE1-SIX6OS1N complexes relative to their isolated SYCE1 proteins (increasing from 39 to 43C, 39 to 41C, and 38 to 40C for SYCE1core, SYCE1pof, and full length, respectively; Fig. 5C and fig. S1D). SEC-MALS analysis revealed that all three SYCE1-SIX6OS1N complexes are 1:1, with molecular weights of 27, 37, and 46 kDa, respectively (Fig. 5D and fig. S7C). Thus, the SYCE1core undergoes conformation change from an antiparallel homodimer to a 1:1 complex upon binding to SIX6OS1N (Fig. 5E).

(A) Amylose pulldown following coexpression of MBP-SIX6OS1 175, 167, 175 1021, and free MBP with His-SYCE1core. (B) SDS-PAGE of the copurification of the SYCE1core-SIX6OS1n complex. Ni-NTA, Ninitrilotriacetic acid. (C) CD thermal denaturation recording the CD helical signature at 222 nm between 5 and 95C, as % unfolded; estimated melting temperatures (Tm) are indicated. (D) SEC-MALS analysis. SYCE1core-SIX6OS1n (blue), SYCE1POF-SIX6OS1n (red) and full-length SYCE1-SIX6OS1n (black) are 1:1 complexes of 27, 37 (29 kDa for the degradation product complex), and 46 kDa, respectively (theoretical 1:1 to 27, 36, and 48 kDa), while MBP-SIX6OS1n (gray) is a 57-kDa monomer (theoretical, 53 kDa). SDS-PAGE of the SYCE1POF-SIX6OS1n sample is shown in Fig. 1C. (E) Schematic of the conformational change of the SYCE1core antiparallel dimer (yellow) into a 1:1 SYCE1core-SIX6OS1n complex (yellow-blue). (F and G) SEC-SAXS analysis. (F) SEC-SAXS P(r) interatomic distance distributions of SYCE1core-SIX6OS1n (blue), SYCE1POF-SIX6OS1n (red), and SYCE1core (yellow), revealing maximum dimensions (Dmax) of 138, 180, and 186 , respectively. Their cross-sectional radii (Rc) are indicated (fig. S7D). (G) SAXS ab initio models of SYCE1core-SIX6OS1n (blue) and SYCE1core (yellow); averaged models were generated from 20 independent DAMMIF runs. Data for SYCE1core and full-length SYCE1 are reproduced from (28).

We analyzed the conformation of the SYCE1core-SIX6OS1N complex by size exclusion chromatography small-angle x-ray scattering (SEC-SAXS; fig. S7, D and E). The SAXS real-space pair-distance P(r) distribution (the distribution of interatomic distances within a protein structure) demonstrates positive skew, indicating that SYCE1core-SIX6OS1N retains the rod-like structure of SYCE1core, but with a reduction in its molecular length from 186 to 138 (Fig. 5F). Furthermore, its cross-sectional radius is slightly increased from 9 to 11 (fig. S7F), suggesting an increase from a two- to four-helical coiled coil. These geometric changes are consistent with the SYCE1core-SIX6OS1N 1:1 complex forming a shorter but wider coiled coil than the isolated SYCE1core dimer, as indicated by their SAXS ab initio models (Fig. 5G). Furthermore, the SAXS P(r) distribution of SYCE1pof indicates a similar elongated structure but with an increased tail to a maximum dimension of 180 (Fig. 5F), consistent with it containing the same SYCE1core-SIX6OS1N structure with an extended and potentially unstructured C terminus to amino acid 240. We conclude that SYCE1core mediates a direct interaction with SIX6OS1N that imposes a conformational change to a 1:1 complex that adopts a shorter and wider coiled-coil conformation than the isolated SYCE1core antiparallel homodimer.

Does the SYCE1core-SIX6OS1N complex represent the sole means by which SYCE1 interacts with SIX6OS1? We were unable to obtain soluble biochemical complexes containing SIX6OS1 sequences beyond its N terminus and so used yeast two-hybrid (Y2H) to test SYCE1 binding by full-length SIX6OS1. Having confirmed direct binding of SYCE1core to full-length SIX6OS1, we used C-terminal truncation to dissect its minimal binding site to amino acids 1 to 75, in keeping with our biochemical findings, and identified an additional interaction between SYCE1 177305 and full-length SIX6OS1 (Fig. 6A).

(A) Y2H analysis of interactions between SYCE1 and SIX6OS1 in which positive reactions are indicated by the growth of blue colonies. These data are representative of three repeats. (B) Schematic of the SYCE1-SIX6OS1 interaction based on the Y2H data in (A), with the two binding sites highlighted in red and green. The SYCE1 POF mutation blocks the second binding interface between SYCE1 177305 and SIX6OS1 downstream sequence within region 1262, whereas the SIX6OS1 1021 deletion blocks the first binding interface between SYCE1core (25179) and SIX6OS1n (167). (C) COS7 cells were transfected with mouse Six6os1 1021 alone or in combination with mouse Syce1. SIX6OS1 1021 showed nuclear localization with some cytoplasmatic signal and colocalized in cytoplasmic foci with SYCE1; the percentage of cells exhibiting colocalization is shown. Scale bars, 20 m. (D) Coimmunoprecipitation of SIX6OS1 1021 and Flag-SYCE1 from cotransfected HEK293T cells using anti-Myc or anti-EGFP antibodies, or mouse IgG as a negative control. SIX6OS1 1021 coimmunoprecipitated SYCE1, indicating that the second SYCE1 binding interface is retained. The untransfected lanes confirm the absence of SIX6OS1 1021 and SYCE1 in total protein extracts of untransfected 293T cells.

To establish whether SYCE1core and 177305 bind to the same or distinct sites within SIX6OS1, we established an internal deletion of SIX6OS1 amino acids 10 to 21 (1021) that blocks formation of the SYCE1core-SIX6OS1N biochemical complex (Fig. 5A). SIX6OS1 122 did not interact with any SYCE1 construct (Fig. 6A), indicating that amino acids 10 to 21 are necessary but not sufficient for SYCE1core binding. While 1021 completely abrogated the Y2H interaction of full-length SIX6OS1 with SYCE1core (25179), it retained a robust interaction with SYCE1 177305, suggesting distinct SIX6OS1-binding sites (Fig. 6A). Furthermore, 1021 blocked the ability of SIX6OS1 1262 to interact with SYCE1core and SYCE1pof (amino acids 25 to 240) while retaining its binding to full-length and 25315 SYCE1 (Fig. 6A). Thus, SYCE1 undergoes multivalent interactions with SIX6OS1, with the first binding interface mediated by SYCE1core and SIX6OS1N (167), and the second interface mediated by SYCE1 177305 and downstream sequence within SIX6OS1 1262. Furthermore, the first and second binding interfaces are specifically disrupted by SIX6OS1 deletion 1021 and the SYCE1 POF mutation, respectively, and in both cases, an SYCE1-SIX6OS1 complex is retained through the unaffected alternative site (Fig. 6B).

Our biochemical and Y2H analyses concluded that SIX6OS1 1021 would disrupt the first SYCE1-SIX6OS1 binding interface while retaining complex formation through the second interface. In support of this, we found that SIX6OS1 1021 retained its ability to form intense colocalized foci with SYCE1 upon coexpression in COS7 cells (98% of the cells; Fig. 6C), similar to our previous observations for the SYCE1 POF mutation (Fig. 4A). Similarly, SIX6OS1 1021 retained its ability to coimmunoprecipitate SYCE1 upon coexpression in HEK293 cells (Fig. 6D). Thus, localization and coimmunoprecipitation data from heterologous systems support our Y2H findings that the second SYCE1-SIX6OS1 binding interface is retained in SIX6OS1 1021, mirroring the retention of only the second binding interface that is predicted for the 126155 deletion of the SYCE1 c.375-2A>G NOA mutation (40).

Having established that the severe phenotype of the SYCE1 POF mutation likely results from the disruption of the second SYCE1-SIX6OS1 binding interface and its interaction with SYCE3, we wondered whether a similar phenotype would result from the sole disruption of the first SYCE1-SIX6OS1 binding interface. To test this, we generated mice harboring mutations of Six6os1 alleles encoding internal in-frame deletions of amino acids 10 to 21 (equivalent numbering to the human protein) (fig. S8, A and B). While heterozygotes (designated Six6os11021/WT) were fertile, both male and female homozygotes (designated Six6os11021/1021) were infertile, similar to the SYCE1 POF mutation. In males, we observed reduced testis size (Fig. 7A) and a zygotene-like arrest similar to that observed in the Six6os1 and Syce1 knockouts (11, 16). The mutant spermatocytes were defective in synapsis and SC assembly, with reduced staining for SC proteins SYCP1 (Fig. 7B) and SYCE3 (Fig. 7C) and no staining for SYCE2-TEX12 (Fig. 7, F and G). In contrast with their complete absence in the SYCE1 POF mutation, we observed some residual staining for SYCE1 (Fig. 7D) and SIX6OS1 (Fig. 7E) even though the levels of transcription of Six6os11021 appeared to be increased in the mutant testis (fig. S9 and table S1B). We detected -H2AX (fig. S10A) and DMC1/RAD51 foci (fig. S10, B and C) on aligned axial elements but no MLH1 foci (fig. S10D), indicating the proper induction of DSBs with their failed repair and absence of COs. Thus, SIX6OS1 1021 leads to infertility with a phenotype of failed DSB repair and SC assembly, similar to the SYCE1 POF mutation and those reported for disruption of structural components of the CE (10, 11, 1618).

(A) Genetic deletion of amino acids 10 to 21 of SIX6OS1 led to a reduction of the testis size compared to the WT (mice of 3 months of age). (B) Double immunolabeling of WT pachytene and Six6os1/ zygotene-like spermatocytes with SYCP3 (red) and SYCP1 (green). AEs failed to synapse in Six6os1/ spermatocytes despite partial alignment, with reduced loading of SYCP1 along the AEs. (C to G) Double immunolabeling of spermatocyte spreads with SYCP3 (red) and all CE components (green). Six6os1/ zygotene-like spermatocytes showed reduced signals of (C) SYCE3, (D) SYCE1, and (E) SIX6OS1, and the absence of (F) SYCE2 and (G) TEX12 from the AEs. Scale bars, 10 m. Plots represent the quantification of fluorescence intensity levels in Six6os1/ zygotene-like and WT pachytene spermatocytes (B to E). Welchs t test analysis: ***P < 0.0001. (H) Schematic of how the SYCE1 antiparallel dimer (yellow) undergoes conformational change upon interaction with SIX6OS1 (blue) to form a possible 1:1 complex through consecutive binding interfaces mediated by SYCE1core-SIX6OS1n (site 1) and SYCE1 177305 and downstream sequence within SIX6OS1 1262 (site 2). The consequence of SYCE1 mutations associated with POF (c.613C>T) and NOA (c.375-2A>G) and SIX6OS1 1021 on the integrity, predicted stoichiometry, and conformation of resultant SYCE1-SIX6OS1 complexes is illustrated. Photo credit (A): Laura Gmez-H, Instituto de Biologa Celular y Molecular del Cncer.

Thus, we conclude that both first and second SYCE1-SIX6OS1 binding interfaces are essential for SC assembly and meiotic progression. Furthermore, these findings explain how the sole disruption of individual SYCE1-SIX6OS1 binding interfaces by SYCE1 NOA (c.375-2A>G) and POF (c.613C>T) mutations result in the reported familial cases of human infertility.

The structural and functional integrity of the SC is contingent on the structure and assembly of is constituent protein components. Here, we report that SC assembly depends on multivalent interactions between CE components SYCE1 and SIX6OS1 that are disrupted by infertility-associated mutations of SYCE1. The first binding interface is formed by the structural core of SYCE1 (SYCE1core; amino acids 25 to 179), which undergoes conformational change from an antiparallel homodimer to a 1:1 complex upon interaction with SIX6OS1s N terminus (SIX6OS1N; amino acids 1 to 67). The second binding interface is formed by downstream sequence within SIX6OS1 1262 interacting directly with SYCE1 177305. Through the generation of mice harboring an internal deletion of SIX6OS1s N terminus (1021) and the SYCE1 POF mutation (murine p.Gln243*), which specifically block the first and second binding interfaces, respectively, we find that integrity of both SYCE1-SIX6OS1 binding interfaces is essential for SC assembly and meiotic progression in vivo.

What is the structure of the SYCE1-SIX6OS1 complex? SEC-SAXS analysis revealed that the SYCE1core-SIX6OS1N 1:1 complex formed by the first binding interface has a length and cross-sectional radius of 138 and 11 , in comparison with 186 and 9 for the SYCE1core dimer. We previously reported a model for SYCE1core in which amino acids 52 to 179 form an antiparallel dimeric coiled coil containing a midline kink, with helices of amino acids 25 to 50 packing against this structural core (fig. S11A) (28). A maximum dimension of 138 for SYCE1core-SIX6OS1N suggests a coiled-coil length of approximately 92 amino acids, given a helical rise of 1.5 per amino acid (44). This could be explained by the 52179 region forming a helix-turn-helix structure through exaggeration of the kink to a full turn, which may combine with the helix formed by amino acids 25 to 50 and an helix from SIX6OS1N to form a four-helical coiled coil, consistent with its 11- cross-sectional radius (fig. S11B). The second binding interface between SYCE1 177305 and downstream sequence within SIX6OS1 1262 suggests that SYCE1core-SIX6OS1N likely adopts a parallel configuration to form a single SYCE1-SIX6OS1 1:1 complex of consecutive first and second binding interfaces (Fig. 7H).

Our analysis of the SYCE1-SIX6OS1 complex reveals how the three reported clinical mutations of SYCE1 differentially affect its interaction with SIX6OS1. The SYCE1 NOA mutation c.197-2A>G is predicted to result in a truncated product of amino acids 1 to 65 (39), which would disrupt both binding sites and so likely abrogates SYCE1-SIX6OS1 complex formation and thus works as a null mutation. The SYCE1 NOA mutation c.375-2A>G is predicted to result in internal deletion of amino acids 126 to 155 (40), which would disrupt the first binding interface while retaining the second binding interface, and so is likely to result in a conformationally altered 1:1 complex (Fig. 7H). In contrast, while 1021 SIX6OS1 similarly disrupts the first binding interface and retains the second binding interface, the SYCE1core remains unaffected and so is predicted to enable formation of a head-to-head 2:2 complex (Fig. 7H). The SYCE1 POF mutation c.613C>T generates a premature stop codon (p.Gln241*) that gives a truncated product of amino acids 1 to 240 (41), which we have demonstrated disrupts the second binding interface while retaining the first binding interface (Fig. 7H). Thus, the latter two infertility-associated mutations of SYCE1 specifically disrupt one SYCE1-SIX6OS1 interface while retaining the other, which combine with our mouse genetic studies to confirm that both interfaces are essential for the structural assembly of the SC and its function in meiosis.

What are the structural roles of SYCE1 and SYCE1-SIX6OS1 within the SC? Our analyses of Syce1POF/POF and Six6os11021/ 1021 mouse strains revealed similar phenotypes with retention of some SYCP1 and SYCE3 recruitment to chromosome axes, with absence or substantial reduction of SYCE1 and SIX6OS1, and lack of recruitment of SYCE2-TEX12. This pattern suggests a hierarchical model of SC assembly in which SYCE1 and SYCE1-SIX6OS1 lie downstream of SYCP1 and SYCE3, and upstream of SYCE2-TEX12 (Fig. 1A), which is consistent with existing knockout data (10, 11, 1518). The disruption of SYCE3 binding by the POF mutation suggests that its SYCE1-SIX6OS1 complex would be defective for SC recruitment, whereas the SYCE1-SYCE3 interaction, and hence SC recruitment, should be retained for the SYCE1-SIX6OS1 complex of the SIX6OS1 1021 internal deletion. This explains the greater severity of the CE loading defect in Syce1POF/POF than Six6os11021/ 1021, in which SYCE1 and SIX6OS1 staining was substantially reduced in the latter (83.77% of SYCE1 reduction, 0.12 0.02 in the Six6os11021/ 1021 versus 0.73 0.21 in the WT; 68.27% of SIX6OS1 reduction, 0.24 0.02 in the Six6os11021/ 1021 versus 0.76 0.15 in the WT) but completely absent in the former. Thus, we conclude that the first and second SYCE1-SIX6OS1 interfaces are essential for initiation of SC CE formation and likely function by stabilizing a local three-dimensional SC structure that mediates recruitment and self-assembly of SYCE2-TEX12 into fibers that mediate SC elongation along the chromosome axis. Furthermore, the SYCE1 POF mutation is likely worsened by its additional disruption of SYCE3 binding that removes the residual SYCE1-SIX6OS1 SC recruitment observed for the SIX6OS1 1021 internal deletion.

The existence of SYCE1core as an isolated antiparallel homodimer and in a 1:1 complex with SIX6OS1N raises the question of which is the biologically relevant conformation. It is important to highlight that the CD melting temperatures of SYCE1-SIX6OS1N complexes and isolated SYCE1 dimers are very similar, ranging between 38 and 41C. In contrast, highly stable SC components SYCE2-TEX12 and SYCP3 have melting temperatures of approximately 65C (24, 29). Thus, the relatively low melting temperatures of SYCE1-SIX6OS1N complexes and SYCE1 suggest that they may undergo conformational change in vivo, with each conformation functioning at different stages of meiosis and/or at different locations within the SC. Furthermore, our analysis of SYCE1 infertility-associated mutations and a targeted internal deletion of SIX6OS1 revealed at least four possible conformations of SYCE1 and SYCE1-SIX6OS1 complexes (Fig. 7H). Owing to the direct competition between SIX6OS1N binding and SYCE1core dimerization, these conformations could be achieved in the absence of mutations, through alterations of protein levels, local concentrations, allosteric changes, and posttranslational modifications. Hence, alterative conformations of SYCE1 and SYCE1-SIX6OS1 are intriguing candidates for local structural heterogeneity and the propagation of signals along the length of the SC, which could function in roles such as crossover enforcement and interference. Thus, as we progress toward a full molecular understanding of the mammalian SC, the multivalent SYCE1-SIX6OS1 interactions described herein provide tantalizing possibilities for a dynamic role of SC structure in its enigmatic functions in the mechanics of meiosis.

Human SYCE1 sequences were cloned into pHAT4 and pMAT11 vectors (45) for bacterial expression as His- and His-MBP (Maltose-Binding Protein) fusions with TEV (Tobacco Etch Virus) cleavage sites for fusion protein removal. Human SIX6OS1 was cloned into pRSF-Duet1 vectors with a TEV-cleavable N-terminal MBP fusion for coexpression with SYCE1. Proteins were expressed in BL21(DE3) Escherichia coli cells (Novagen), in 2xYT (Yeast Extract Tryptone) media. Expression was induced with addition of 0.5 mM isopropyl--d-thiogalactopyranoside with the cells incubated at 25C for 16 hours. Cells were lysed via sonication in 20 mM tris (pH 8.0) and 500 mM KCl, followed by centrifugation. Supernatant was applied to an amylose (New England Biolabs) affinity chromatography column, followed by HiTrap Q HP (GE Healthcare) anion exchange chromatography. His- and His-MBP/MBP tags were removed by incubation with TEV protease at 4C for 16 hours. The cleaved proteins were further purified by HiTrap Q HP (GE Healthcare) anion exchange chromatography followed by size exclusion chromatography (HiLoad 16/600 Superdex 200, GE Healthcare). The purified proteins/complexes were concentrated using Microsep Advance 3 kDa (PALL) centrifugal filter units and stored at 80C. Protein samples were analyzed for purity using Coomassie-stained SDSpolyacrylamide gel electrophoresis. Protein molecular weights and extinction coefficients were calculated using ExPASY ProtParam (http://web.expasy.org/protparam/) with protein concentrations determined using a Cary 60 ultraviolet (UV) spectrophotometer (Agilent).

Far-UV CD spectra were collected using a Jasco J-810 spectropolarimeter (Institute for Cell and Molecular Biosciences, Newcastle University). Wavelength scans were recorded at 4C from 260 to 185 nm at 0.2-nm intervals using a 0.2-mm path length quartz cuvette (Hellma). Protein samples were measured at 0.2 to 0.4 mg/ml in 10 mM Na2HPO4 (pH 7.5) and 150 mM NaF. Nine measurements were taken for each sample, averaged, buffer-corrected and converted to mean residue ellipticity (MRE) ([]) (1000 degcm2dmol1 per residue). Spectral deconvolutions were carried out using the Dichroweb CDSSTR algorithm (http://dichroweb.cryst.bbk.ac.uk). CD thermal melts were recorded at 222 nm between 5 and 95C, at intervals of 0.5C with a 1C/min ramping rate. Protein samples were measured at 0.1 mg/ml in 20 mM tris (pH 8.0), 150 mM KCl, and 2 mM dithiothreitol (DTT), using a 1-mm path length quartz cuvette (Hellma). The data were plotted as % unfolded after conversion to MRE ([]222,x-[]222,5)/([]222,95-[]222,5). The melting temperature was determined as the temperature at which the proteins are 50% unfolded.

SEC-MALS analysis of protein samples was carried out at concentrations of 5 to 20 mg/ml in 20 mM tris (pH 8.0), 150 mM KCl, and 2 mM DTT. Samples were loaded onto a Superdex 200 Increase 10/300 GL (GE Healthcare) column at 0.5 ml/min using an KTA Pure (GE Healthcare) system. The eluate was fed into a DAWN HELEOS II MALS detector (Wyatt Technology), followed by an Optilab T-rEX differential refractometer (Wyatt Technology). SEC-MALS data were collected and analyzed using ASTRA 6 software (Wyatt Technology), using Zimm plot extrapolation with a 0.185 ml/g dn/dc value to determine absolute protein molecular weights.

SEC-SAXS experiments were carried out on beamline B21 at the Diamond Light Source synchrotron facility (Oxfordshire, UK). Protein samples at concentrations 6 to 20 mg/ml were loaded onto a Superdex 200 Increase 10/300 GL size exclusion chromatography column (GE Healthcare) in 20 mM tris (pH 8.0) and 150 mM KCl at 0.5 ml/min using an Agilent 1200 high-performance liquid chromatography system. The eluate was fed through the experimental cell, with SAXS data recorded at 12.4 keV, in 3.0-s frames with a detector distance of 4.014 m. Sctter 3.0 (www.bioisis.net) was used to subtract and average the frames and carry out the Guinier analysis for the Rg and cross-sectional Rg (Rc). P(r) distributions were fitted using PRIMUS. Ab initio modeling was performed using DAMMIF (46) imposing P1 symmetry. Twenty independent runs were averaged. The PyMOL Molecular Graphics System, Version 2.0 Schrdinger, LLC was used to generate images of the SAXS ab initio models.

Constructs of human SYCE1 and SIX6OS1 were cloned into pGBKT7 and pGADT7 vectors (Clontech). Y2H experiments were carried out using the Matchmaker Gold system (Clontech) according to the manufacturers guidelines. Y187 yeast strain was transformed with pGBKT7 vectors, while the Y2H gold strain was transformed with pGADT7 vectors. Yeast transformations were carried out using standard lithium acetate methods. Mating of the two strains was carried out in 0.5 ml 2 YPDA (Yeast Peptone Dextrose Adenine) at 30C, 40 rpm, by mixing respective colonies. After 24 hours, the cultures were centrifuged and pellets were resuspended in 0.5xYPDA. These were then plated onto SD/Trp/Leu to select for mated colonies and onto SD/Trp/Leu/Ade/His with X--gal to detect mated colonies through ADE1, HIS3, and MEL1 reporter gene activation. Plates were then incubated for 5 days at 30C.

For developing the Syce1POF/POF model, Syce1single-guide RNA (sgRNA) 5-TGACTTCTTTCCACACTATC-3 targeting the intron 10 was predicted at https://eu.idtdna.com/site/order/designtool/index/CRISPR_SEQUENCE. This crRNA (CRISPR RNA), the tracrRNA (trans-activating CRISPR RNA), and the ssODN (single-stranded donor oligonucleotides) (5-GGGACTCTTCCTCCGAAGCCATGAGGCAGCTGCAGCAATGTAAGATGCAGGGTGGGGCAGGAGGAGGAAATGTCTAGCACTGACTTCTTTCCACACCCCCAGGTAGATCTTCAAGGATGAGAACAAGAAAGCTGAGG

AGTTCCTAGAGGCTGCAGCTCAGCAGCACGAGCAGCTGCAGCAGAGGTGCCACCAGCTACAG-3) were produced by chemical synthesis at IDT. The ssODN contains the mutated base (C>T, p.Gln241*) and the peptidyl-glycine -amidating monooxygenase (PAM) was mutated by substituting it by the human intron sequence (ACTATCAG > CCCCCAG). The crRNA and tracrRNA were annealed to obtain the mature sgRNA. A mixture containing the sgRNAs, recombinant Cas9 protein (IDT), and the ssODN [Cas9 (30 ng/l), annealed sgRNA (20 ng/l each), and ssODN (10 ng/l)] were microinjected into B6/CBA F2 zygotes (hybrids between strains C57BL/6 J and CBA/J) (47) at the Transgenic Facility of the University of Salamanca. Edited founders were identified by polymerase chain reaction (PCR) amplification (Taq polymerase, NZYTech) with primers flanking the exon 11 (primer F 5-CTGTAGAGAAACTGATGAAAGT-3 and R 5-CAAGAAAATATGAAGAGACATAC-3) producing an amplicon of 398 base pairs (bp) for both edited and WT alleles, and either direct sequenced or subcloned into pBlueScript (Stratagene) followed by Sanger sequencing, selecting the point mutation in the targeted region of Syce1 (fig. S2). For generating the Six6os11021/ 1021 (named as Six6os1/), Six6os1-crRNA G68 5-ATCTGTTTGTCAGTTTGGAC-3 and Six6os1-crRNA G75 5-TACTTATGTCTTGCTCATAC-3 targeting exons 2 and 3 and the ssODN (5-GTTCTTACTTTATGTATGCTCTTTTATATATGGCTTCTGAAAGTTTTATTATTTATTTTACACAGTGTCCAAGATGAATGATAATCTGTTTGTCAGTTTGCAAGACATAAGTATTAAAGAAGATACGATTCAAAGAATTAATAGTAAGTAGTTTTGCATGAAATAAATATTTTAGTCTTTTGGTTTTATCTTATATAGCA-3) were predicted, produced, and microinjected, as previously described. Edited founders with the predicted deletion were identified through PCR using primers flanking this region (primer F 5-CACTTACATTTTCCTTTTAAGAATGC-3 and R 5-CCCCTCTCATACATACAAGTTGC-3). The 1021 allele was 285 bp long versus 413 bp of the WT allele (fig. S8, A and B). The founders were crossed with WT C57BL/6 J to eliminate possible unwanted off-targets. Heterozygous mice were resequenced and crossed to give rise to edited homozygous. Genotyping was performed by analysis of the PCR products of genomic DNA with primers F and R.

Histology. For histological analysis of ovaries, after the necropsy of the mice, their ovaries were removed and fixed in formol 10%. They were processed into serial paraffin sections and stained with hematoxylin and eosin. The samples were analyzed using a microscope OLYMPUS BX51, and images were taken with a digital camera OLYMPUS DP70.

Immunocytology. Testes were detunicated and processed for spreading using a conventional dry-down technique. Oocytes from fetal ovaries (E17.5 embryos) were digested with collagenase, incubated in hypotonic buffer, disaggregated, and fixed in paraformaldehyde. Both meiocyte preparations were incubated with the following primary antibodies for immunofluorescence (IF): rabbit SIX6OS1 R1 and R2 [1:100, Proteogenix (11)], rabbit SYCE1 17406-1-AP (1:50, Proteintech), guinea pig SYCE1 (1:100, provided by C. Hg), mouse SYCP3 immunoglobulin G (IgG) sc-74569 (1:1000, Santa Cruz Biotechnology), rabbit serum SYCP3 K921 (1:500), rabbit SYCP1 IgG ab15090 (1:200), guinea pig SYCE3(1:20, provided by R. Benavente), guinea pig SYCE2 (1:100, provided by C. Hg), rabbit TEX12 IgG (1:100, provided by R. Benavente), rabbit anti--H2AX (ser139) IgG #07-164 (1:200) (Millipore), mouse MLH1 51-1327GR (1:5, BD Biosciences), rabbit RAD51 PC130 (1:50, Calbiochem), and rabbit DMC1 R1 and R2 (1:500, Proteogenix). The secondary antibodies used were goat Alexa 555 -mouse A-32727, goat Alexa 488 -mouse A-11001, donkey Alexa 555 -rabbit A-31572 (1:200, Thermo Fisher Scientific), goat Alexa 488Fab -rabbit 111-547-003, and donkey fluorescein isothiocyanate guinea pig 706-095-148 (1:100, Jackson Immunoresearch). Slides were visualized at room temperature using a microscope (Axioplan 2; Carl Zeiss Inc.) with 63 objectives with an aperture of 1.4 (Carl Zeiss Inc.). Images were taken with a digital camera (ORCA-ER; Hamamatsu) and processed with OPENLAB 4.0.3 and Photoshop (Adobe). Quantification of fluorescence signals was performed using ImageJ software.

HEK293T and COS7 cell lines were and obtained from the American Type Culture Collection (ATCC). Cell lines were tested for mycoplasma contamination (Mycoplasma PCR ELISA, Sigma-Aldrich). They were transfected with Jetpei (PolyPlus) according to the manufacturers protocol.

Immunoprecipitation and Western blotting. HEK293T cells were transiently transfected, and whole-cell extracts were prepared and cleared with protein G Sepharose beads (GE Healthcare) for 1 hour. The antibody was added for 2 hours, and immunocomplexes were isolated by adsorption to protein G Sepharose beads overnight. After washing, the proteins were eluted from the beads with 2 SDS gel-loading buffer 100 mM tris-HCl (pH 7), 4% SDS, 0.2% bromophenol blue, 200 mM -mercaptoethanol, and 20% glycerol and loaded onto reducing polyacrylamide SDS gels. The proteins were detected by Western blotting with the indicated antibodies. Immunoprecipitations were performed using mouse -Flag IgG (5 g; F1804, Sigma-Aldrich), mouse green fluorescent protein (-GFP) IgG (4 g; CSB-MA000051M0m, Cusabio), mouse -Myc obtained from hybridoma cell myc-1-9E10.2 ATCC (4 g), and ChromPure mouse IgG (5 g/1 mg protein; 015-000-003). Primary antibodies used for Western blotting were rabbit -Flag IgG (1:2000; F7425 Sigma-Aldrich), goat -GFP IgG (sc-5385, Santa Cruz Biotechnology) (1:3000), and rabbit -Myc Tag IgG (1:3000; #06-549, Millipore). Secondary horseradish peroxidaseconjugated -mouse (715-035-150, Jackson ImmunoResearch), -rabbit (711-035-152, Jackson ImmunoResearch), or -goat (705-035-147, Jackson ImmunoResearch) antibodies were used at 1:5000 dilution. Antibodies were detected by using Immobilon Western Chemiluminescent HRP Substrate from Millipore. Both Syce1POF and Six6os1 1021 complementary DNAs (cDNAs) used for IF and coimmunoprecipitation experiments were reverse transcription PCRamplified (the primers used for it were Syce1 S 5-GAGCAGTATGGCCACCAGACC-3 and Syce AS 5-GAGGAGGGTATTAGGTCCTGC-3; Six6os1 S 5-AGTGTCCAAGATGAATGATAATCTG-3 and Six6os1 AS 5-GTTCAAAAATAATAACTCAAAAAAAC-3) from total RNA extracted from Syce1POF/POF and Six6os11021/ 1021 mice, respectively. PCR-amplified fragments were cloned in pcDNA3-based mammalian expression vectors with different tags (enhanced GFP or Flag) and verified by Sanger sequencing.

Total RNA was isolated from testis of WT and mutant mice. To analyze the expression of Syce1 and Six6os1 mRNAs, equal amounts of cDNA were synthesized using SuperScript II Reverse Transcriptase (Invitrogen, Life Technologies) and Oligo (dT). Quantitative PCR (qPCR) was performed using FastStart Universal SYBR Green Master Mix (ROX) (Roche) and specific forward and reverse primers: qSYCE1_F 5-GGACATGGTGAAAAAGTTGCAG-3 and qSYCE1_R 5-CAGTTCCTTCTGCAGGTTGTC-3 for Syce1, and qSIX6OS1_F 5-GCTGAATGTGGAGATAAAGAG-3 and qSIX6OS1_R 5-AGGAGTTTCAGGAGTTTGAGG-3 for Six6os1. All qPCR reactions were performed at 95C for 10 min and then 40 cycles of 95C for 15 s and 62C for 1 min on the iQ5 Thermal Cycler (Bio-Rad). -Actin was amplified as a housekeeping gene with the primers q-actin_F 5-GGCACCACACCTTCTACAATG-3and q-actin_R 5-GTGGTGGTGAAGCTGTAGCC-3.

Statistics. To compare counts between genotypes, we used the Welchs t test (unequal variances t test), which was appropriate as the count data were not highly skewed (i.e., were reasonably approximated by a normal distribution) and, in most cases, showed unequal variance. We applied a two-sided test in all the cases. Asterisks denote statistical significance: *P < 0.01, **P < 0.001, and ***P < 0.0001.

Acknowledgments: We thank Diamond Light Source and the staff of beamline B21 (proposals sm15836, sm21777, and sm23510). We thank H. Waller for assistance with CD data collection. Funding: O.R.D. is a Sir Henry Dale Fellow jointly funded by the Wellcome Trust and Royal Society (grant number 104158/Z/14/Z). This work was supported by MINECO (BFU2017-89408-R) and by Junta de Castilla y Leon (CSI239P18). F.S.-S., L.G.-H., and N.F.-M. are supported by European Social Fund/JCyLe grants (EDU/556/2019, EDU/1083/2013, and EDU/310/2015). CIC-IBMCC is supported by the Programa de Apoyo a Planes Estratgicos de Investigacin de Estructuras de Investigacin de Excelencia cofunded by the CastillaLen autonomous government and the European Regional Development Fund (CLC201701). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Ethics statement: Mice were housed in a temperature-controlled facility (specific pathogen free) using individually ventilated cages, standard diet, and a 12-hour light/dark cycle, according to European Union laws at the Servicio de Experimentacion Animal, SEA. Mouse protocols were approved by the Ethics Committee for Animal Experimentation of the University of Salamanca (USAL). We made every effort to minimize suffering and to improve animal welfare. Blinded experiments were not possible since the phenotype was obvious between WT and mutant mice for all of the experimental procedures used. No randomization methods were applied since the animals were not divided in groups or treatments. The minimum size used for each analysis was two animals per genotype. Author contributions: F.S.-S., L.G.-H., O.M.D., N.F.-M., C.G.-P., M.S.-M., and O.R.D. performed experiments. O.R.D. and A.M.P. designed experiments, analyzed data, and wrote the manuscript. A.M.P., E.L., and O.R.D. supervised and designed the work. Competing financial 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.

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Meiotic chromosome synapsis depends on multivalent SYCE1-SIX6OS1 interactions that are disrupted in cases of human infertility - Science Advances

Thought to Be Extinct, New Guinea’s Singing Dogs Found Alive in the Wild – Smithsonian Magazine

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The New Guinea singing dog was thought to be extinct in the wild, but new genetic research suggests their distinctive howl still echoes in the highlands of the Oceanic islands, reports James Gorman for the New York Times.

Not seen in the wild by scientists since the 1970s, conservation biologists thought the only New Guinea singing dogs left on Earth were the 200 to 300 captive animals residing in zoos and sanctuaries, reports Michael Price for Science.

But anecdotal reports and a pair of photographs suggested a similarly tan-colored, medium-sized wild dog was roaming the mountainous terrain near a gold mine on Papua, the western, Indonesian half of the large island north of Australia.

The locals called them the highland wild dog, James McIntyre, president of the New Guinea Highland Wild Dog Foundation and co-author of the paper, tells the Times. The New Guinea singing dog was the name developed by caucasians. Because I didnt know what they were, I just called them the highland wild dogs.

To find out what these highland wild dogs really were, McIntyre trekked into the rugged terrain surrounding the Grasberg Mine, one of the worlds largest gold and copper mines, in 2016. The expedition produced 149 photographs of 15 individual dogs as well as an array of fecal samples. Per Science, if one were trying to cast a pooch for the role of the New Guinea singing dog, the wild dogs looked, acted and sounded the part.

However, the fecal samples didnt have enough genetic material for a proper analysis, so in 2018 the researchers returned and collected blood samples from three of the animals, according to the paper which was published this week in the journal Proceedings of the National Academy of Sciences.

These three samples were used to sequence the highland wild dogs genomes. The researchers then compared the dogs nuclear DNA with 16 captive New Guinea singing dogs, 25 dingoes as well as more than 1,000 individuals from 161 additional breeds.

The genetic analysis suggests that these highland wild dogs are in fact part of a wild population of New Guinea singing dogs. Crucially, the newly revealed wild population is much more genetically diverse than captive singing dogs, which descended from just eight individuals and are severely inbred, reports Katie Hunt for CNN.

Assuming these highland wild dogs are the original New Guinea singing dogs, so to speak, that really gives us a fantastic opportunity for conservation biology, Elaine Ostrander, a geneticist at the U.S. National Human Genome Research Institute and co-author of the study, tells Ed Cara of Gizmodo. Itll give us a chance to reintroduce the original genetics of these dogs into this conservation population.

Both the wild dogs and the captive singing dogs are close relatives of the Australian dingo, and relatively distant relatives of domestic dogs. The New Guinea singing dogs closest domesticated relatives are East Asian breeds including the chow chow, Akita and shiba inu, according to Science. This connection suggests that the singing dog may have split off from the ancestors of these Asian breeds some 3,500 years ago when humans and a few canine companions migrated to Oceania, per the Times.

Ostrander tells the Times that the genome of the wild singing dogs offers researchers a missing piece that we didnt really have before, that may help clarify the history of dog domestication.

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Online Roulette for Real Money – Best 3 Casinos to Win Big

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People love to gamble, and technology has made it possible to visit an online casino, where they can sign up for free. Playing American roulette is what a lot of people enjoy. Since they can play American roulette from wherever they are, as long as they have access to the Internet, they are happy.

When a person wants to play roulette online, it is a good idea for them to read up on the rules and ways that they can play it better in order to win more money. Here are the 3 best sites to play roulette online:

Whenever a person first goes on the site, they will need to open an account. This is free to do, and their information is protected. If they should have any difficulties, the customer service agents can assist them right away. They are trained and experienced at what they do. When they handle an issue, they are kind and courteous to their customers.

All of these casinos handle deposits and withdrawals with the utmost of care. They protect an individuals privacy at all times. Customers are given options for deposits and withdrawals so they can find one that works the best for their situation.

When people gamble online, they will want to pay attention to any promotions that may be offered to them by the casino. Since these promotions can help the gambler to earn more money, they should always be aware of what they are. They can benefit from them as they return to gamble on the sites over and over again.

People love the roulette wheel because it is a game of chance. Hitting big on the roulette wheel can mean winning some nice money. A gambler will want to know how the roulette wheel works so they have a better chance of winning. An online roulette game is thrilling and fun for many gamblers.

The atmosphere of the online casinos is a vibrant one. They have intense graphics and excellent, sound systems. People get excited when they visit their sites. The atmosphere leads the gambler to have an exciting experience as they try their luck at winning. For a gambler, online sites are fascinating to them, they enjoy coming back to try their luck again and again.

Playing the online roulette game can be something that people want to do on a regular basis. Now, they can play the online roulette game from wherever they need to go for business or pleasure. All they need is a laptop or cell phone to access the Internet, and they will be all set to have an exciting experience gambling online. It is something that people enjoy, and many of them win big money.

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Online Roulette for Real Money - Best 3 Casinos to Win Big

Roulette – Online Roulette games you can play for free, no …

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Get ready for the biggest thrill of your life once you get rolling with the exciting game of roulette. Before playing in a game it is important to understand some things about roulette odds. There are several versions of roulette that have slightly different rules involved. This makes the game accessible and entertaining for every type of player out there. Most importantly, there are two different types of roulette wheels, one called the American roulette and one is European roulette wheel.

While there are two major variations of roulette, there are more similarities than not. To play in a game of roulette, first locate the roulette table layout. This green table is situated next to the roulette wheel and has loads of spots for you to take your best bet. Of all the choices, there are two categories of bets called the "inside bets" or "outside bets". The inside bets are the higher payout because the odds are high. Inside bets include a bet on a single lucky number, splits, or small combinations of numbers on the table layout. Place an outside bet for a lower payout but better odds. Outside bets include a bet on red or black, even or odd, a certain dozen of numbers, or a column.

The roulette table is managed by a croupier and a dealer. The roulette wheel is always spinning and anyone in the action needs to place their uniquely colored chip on the table. Once the dealer calls an end to the bets, you better have your stake on the table. If you are a winner, then you are paid out according to the inside or outside payout.

With the different varieties of roulette, let's talk about how to play American roulette since it is the simpler version to play. The American roulette wheel has numbers 1-36 and includes a 0 and a 00 slot. It is played in North America, as its name suggests. When the ball drops into its final slot, let's hope that it doesn't land in the 0 or 00. The house has two chances to take the win and American roulette puts the house edge at 5.26%.

European roulette only has the one zero slot on the wheel cutting the house edge in half to 2.7%. European roulette is played in most European countries, of course. There are a couple of rules that may be instituted in the game that are great news to improve your odds against the house. In European roulette, you may find the "en prison" or "la partage rules". If the ball lands in the zero slot in the roulette spin, "la partage" rules allow the player to receive half of their bet back. If you are playing at a table using the "en prison" rule, that means the bet will be "imprisoned" when the ball lands on the zero for one more spin. If your bet hits on the next spin, then your bet is released from prison with no winnings.

There are endless ways for you to play and enjoy roulette. With choices from European and American roulette and now the online versions offer unrivaled convenience. If you are feeling hesitant, the free online roulette rooms are a great way to experience the rules and flow of play. After hundreds of years, roulette has no evidence of shrinking popularity. Give it a whirl and you can easily see why!

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From Japan to the world – IAG – Inside Asian Gaming

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Japans Sega Sammy Creation is getting in early by developing slot titles that incorporate some elements of popular Japanese pachislot games.

As Japan continues to ponder the prospect of an integrated resort industry, Sega Sammy is working to bring creative EGM content to the world, installing the first Virtua Fighter video slot machine at Vietnams Macau Gaming Club in June. IAG spoke with Masaki Yamamoto, Vice President, Executive Officer and Managing Director of the Research and Development Division at Sega Sammy Creation the casino sales company under Sega Sammy Holdings and Hayato Sasaki, who serves as Director of Sales.

Shintaro Kamimura: How has Sega Sammy Creation been affected by COVID-19?Hayato Sasaki: Due to so many casinos not accepting customers at this time, we havent been getting orders from casinos. Also, since the Japanese government has been moving slowly [due to COVID-19], it caused delays in various projects. However, I think the same is true for all manufacturers.

SK: What was the reason for going with the Virtua Fighter slot motif this time?Masaki Yamamoto: Virtua Fighter is the first 3D fighting game and has sold all over the world, so we thought it would be well-received all over the world. We started with about 3,000 title options, and this is one of the dozen or so that we narrowed it down to.The target demographic for Virtua Fighter and House of the Dead is people in their 40s to 50s who actually played the games back in the day, and since they still have new series coming out, we hope it will also resonate with the younger generations.

SK: It really has the game aspects that you find in Japanese pachislot.HS: Its a 5-line game, but is made with amusement elements like pachislot. The casinos have actually come to us saying the free game portion is too long, and theyre worried its hurting their income. The next problem is finding the balance between the casinos income and the pachislot-type amusement that even we want to play.MY: But we arent giving up on this at all. We believe that this sort of gaming element is necessary to allow customers to enjoy casinos for long periods of time. We plan to incorporate both operator and user opinions to continue making improvements.

SK: Do you think pachislot-type gaming aspects will hit big once there are IRs in Japan?HS: Yes. In that sense we really want to take the lead [in a Japanese IR]. Of course, other companies are developing products, but we are developing in Japan, so we dont want to lose in a Japanese IR. Naturally we are also developing products with existing pachislot themes. We should probably be able to make that announcement in six months or so.MY: There will be at most three IRs in Japan. Even if each location has 3,000 slot machines, thats a total of 9,000. Its a very small market. However, if certain machines become popular in Japan and customers leave with the impression that Japan has great game content, thats an opportunity to expand sales to overseas markets. I think it will help give Japanese manufacturers strength. Were adding aspects to meet the needs of the market and also things that we personally want to do.

SK: This is the idea of new CEO Scott Winzeler?MY: Thats right. He always says, Start with the core, then we can do what we want. He started out in Japanese pachislot at IGT, so I think he understands that side of things very well, and his way of thinking is a really good reference.

SK: Can you tell us about your electronic table games?HS: The Maximum Fortune series features baccarat, roulette and Sic Bo. You can even squeeze the baccarat cards with the squeeze button. In Sic Bo you can squeeze the dice with a button too. In roulette, there is a pachislot-type feature that gives prior notification of a win by the dragon ramp lighting up on the screen for certain hits.

SK: Most of your table games are released under the Genesis Slant cabinet and slots under the Genesis Star cabinet. Weve heard there is very little need for repairs?HS: The rate of breakdowns is extremely low. The maintenance company actually told us they need more to do. We are quite confident in our cabinet products after making tens of millions of pachinko, pachislot, amusement and consumer products. That is our biggest sales pitch.

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From Japan to the world - IAG - Inside Asian Gaming

Total and Macquarie to build 2GW floating offshore wind farm portfolio – Power Technology

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]]]]]]>]]]]>]]> Total and Macquarie aim to develop the largest floating wind projects to date. Credit: Matt Artz on Unsplash.

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Total and Macquaries Green Investment Group have teamed up to develop five floating offshore wind projects in South Korea with a capacity of up to 2GW.

As part of the 50/50 partnership, the two companies have agreed to develop floating offshore wind projects off the eastern and southern coasts near Ulsan and South Jeolla provinces of the country.

Total chairman and CEO Patrick Pouyann said: Our entry in the floating offshore wind segment in South Korea is in line with Totals strategy to profitably develop renewable energy worldwide and contribute to our net-zero ambition.

We strongly believe in the potential of floating offshore wind in South Korea, which will play a key role in achieving the countrys renewables objectives.

An on-site wind data collection campaign has already begun, and the two companies intend to begin construction of the first 500MW project by the end of 2023.

Last July, South Korea unveiled its Green New Deal plan through which it aims to develop renewable energy that reach at least 20% of the power mix by 2030, including 12GW of offshore wind capacities.

The Total/Macquarie partnership will become effective from autumn 2020, subject to regulatory approvals and satisfaction of other conditions precedent.

In June this year, Macquarie-owned Green Investment Group and Enso Energy formed a joint venture to develop a subsidy-free solar and battery portfolio in the UK.

In another development, a consortium consisting of Korea Hydro & Nuclear Power, Alpha Asset Management, Sprott Korea Investment, Hana Financial Investment, Korea Investment & Securities, and a group of Korean infrastructure investors have signed definitive agreements to acquire a 49.9% stake in a portfolio of wind generating assets in the US.

The 852MW wind portfolio includes four operating wind farms. These include the 218MW Bishop Hill wind farm and the 226MW California Ridge wind farm, both located in Illinois.

The remaining two facilities are the 201MW Prairie Breeze wind farm in Nebraska, and the 207MW Rattlesnake wind farm in Texas.

Brookfield Renewable and Invenergy has sold its interests in these projects for an enterprise value of approximately $1.5bn.

The deal, which is subject to satisfaction of customary closing conditions, is expected to close in the fourth quarter of 2020. Brookfield will retain 50.1% equity interest in the portfolio.

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Total and Macquarie to build 2GW floating offshore wind farm portfolio - Power Technology

Heat wave on the way: Potential for offshore wind event next week – The Union of Grass Valley

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A heat wave is headed this way and the National Weather Service has issued an excessive heat watch through the entire Sacramento and San Joaquin Valleys up to the Sierra crest.

Temperatures will be highest over the holiday weekend, with high to very high heat risk forecast for Sunday and Monday.

The reason for this unseasonable heat wave for this time of year is the level of high pressure in the upper ridge is forecast to strengthen, the sinking air associated with that will cause temps to rise 15 to 20 degrees above normal for this time of year, National Weather Service Meteorologist Scott Rowe said.

Today, the warming trend begins with highs around 90 degrees, 92 on Friday, 97 forecast for Saturday, 102 Sunday, and 99 Monday.

Its unexpected to see heat waves of this magnitude late in the season, Rowe said. We are in September now.

Looking ahead to the extended forecast, temps do tend to decline on Tuesday, when we start the new work week, Rowe said.

Wildfire smoke wafting over areas of Northern California will also play a part in high and low temperatures, potentially keeping daytime highs from reaching their forecasted extremes.

Smoke acts as a cloud, Rowe said in reference to the ability for smoke to reduce high temperatures. On the other hand, for the overnight lows, it would help keep temperatures in. It mutes the extremes a little in the end.

Overnight lows are expected to stay in the upper 70s and low 80s, providing little overnight moisture recovery and increased wildfire risk.

A potential offshore north wind event is being eyed for Tuesday or Wednesday, though confidence at this point is low.

We may be transferring from heat concerns to fire weather concerns rather quickly, Rowe said.

Its still a little bit early to tell, Rowe said of the potential north to northeast wind event. It may be a little too early to discern. Depends on how cool it gets in the great basin.

If the offshore wind event were to materialize, the weather service foresees anywhere from 15 to 20 mph winds on Tuesday or Wednesday.

Going to have to get through the heat wave to see what kind of models we will have, Rowe said.

With the holiday weekend coming up the National Weather Service wants to remind people to stay hydrated and take care of themselves in the heat.

If folks do decide to go out on a hike or any trails or area waterways, there may not be that much relief from the heat for those coming up from the valley.

To contact Multimedia Reporter Elias Funez, email efunez@theunion.com or call 530-477-4230.

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Clean Ocean Action Pushes for Environmentally Responsible Offshore Wind Projects – The SandPaper

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In an effort to ensure offshore wind energy near New Jersey is developed in the most environmentally responsible manner possible, nonprofit Clean Ocean Action recently submitted detailed comments to the state Board of Public Utilities regarding plans for offshore wind development. COA policy attorney Peter Blair prepared and submitted correspondence to Aida Camacho-Welch, board secretary, to provide recommendations for the BPUs Offshore Wind Strategic Plan as well as its draft solicitation guidance document for the next offshore wind project.

COA consists of a broad-based coalition of more than 125 active boating, business, community, conservation, diving, environmental, fishing, religious, service, student, surfing and womens groups in New Jersey. Alliance for a Living Ocean, on Long Beach Island, is one of the nonprofits partner organizations. The group has actively followed offshore wind development in the New York/New Jersey Bight for the past decade and, over the last several years, has actively engaged with the BPU, the state Department of Environmental Protection and other state and federal agencies concerning offshore wind.

We need to rapidly transition to a future powered by clean renewable energy, the organization noted last month, citing the acceleration of climate change, which leads to increased temperatures, stronger storms, ocean acidification and sea level rise. As states address and design pathways to achieving climate and clean energy goals, offshore wind has become a prominent option.

The turn to wind power the use of turbines to capture kinetic energy from the wind and generate electricity is an incredibly important issue, says COA, and the nonprofit aims to foster this sustainable industry while also protecting marine and coastal ecosystems.

The BPUs strategic plan, Blair remarked in his comments to Camacho-Welch, is a critical energy blueprint that will guide the necessary transition away from fossil fuels to a system powered by offshore wind energy. It is clear that the NJBPU and state agencies have been hard at work to assess the scope and magnitude of the OSW development.

While offshore wind is necessary to achieve the states climate goals, he added, there are significant potential impacts to the marine and coastal environment from the rapid development of this marine intensive and coastally-dependent industry.

COA urges the state to proactively balance the development of offshore wind with the serious concerns regarding its impacts, including the impacts that are currently under-evaluated. Therefore, it is imperative that the state develop a comprehensive blueprint designed to ensure the environmentally responsible development of New Jerseys goal of 7,500 megawatts by 2035. COA applauds the leadership of NJBPU in developing the strategic plan to act as that blueprint.

The letter also seeks clarity on how the recommendations contained in the strategic plan will be developed into meaningful community engagement and environmental protection for coastal and ocean areas and proposes several recommendations to help strengthen the plan.

In his comments about the draft solicitation guidance document, meanwhile, Blair writes, COA is encouraged by the level of detail outlined in the Draft Guidance Document and appreciates the requirements that are forward-looking in terms of ensuring a clean resilient energy grid. This includes the requirements that the applicants address whether the project will include energy storage capabilities, and information related to the developers intent to use technologies aimed at reducing peak demand electric generation. These are just some of the key aspects that illustrate N.J.s commitment to ensuring offshore wind development is done right.

However, COA does encourage the BPU to include additional requirements, outlined in the comments, to further ensure that the development of offshore wind does not come at the expense of N.J.s marine and coastal ecosystems. These include added obligations for developers in the Environmental Protection Plan, Fisheries Protection Plan, Economic Development Plan, Interconnection Plan, and Operation and Maintenance Plan.

As Blair emphasized, COA, overall, advocates for a balanced approach to wind power that recognizes the urgency of developing affordable and reliable renewable energy in the context of the numerous potential negative impacts offshore wind development may have.

Juliet Kaszas-Hoch

juliet@thesandpaper.net

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Offshore energy technology developers offered free access to test facilities in Europe – Energy Live News – Energy Made Easy

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EMEC Fall of Warness tidal test site. Image: Colin Keldie/Ocean_2G/EMEC

Developers of offshore energy technologies are being invited to apply for free access to test facilities in Europe.

TheMaRINET2 (Marine Renewables Infrastructure Network) project is open to developers of offshore wind, wave and tidal energy at full system, subsystem, component and sensor level.

The programme is a network of 39 partners in 13 European countries, involving research centres and organisations that work together to progress offshore renewable energy technologies.

Successful applicants will be able to access the test facilities, including the European Marine Energy Centre (EMEC) in Scotland, for free between 18th January and 17th July 2021.

MaRINET2 has awarded almost 5 million (4.4m) in free testing access across a European network of 57 research facilities so far, with the aim of accelerating the progress of the offshore renewables sector towards full-scale industrialisation.

Danielle Moodie, Project Manager at EMEC said: As a world-leading test site in marine energy testing, EMEC experiences a range of wave and tidal conditions across our sites in Orkney, suitable for testing a variety of offshore technologies. With flexible test infrastructure, we are seeing increasing interest from floating offshore wind and supply chain companies looking to put foundations, components and data acquisition technologies to the test as well.

MaRINET2 is a good opportunity to reduce the cost of open-water testing for energy generators or subsystems so in this last call for applications, we are looking to encourage applications from across the offshore renewables market.

The call closes on 16th October 2020.

If you enjoyed this story you can sign up to our weekly email forEnergy Live News and if youre interested in hearing more about the journey to net zero by 2050, you can also sign up to thefuture Net Zeronewsletter.

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Offshore energy technology developers offered free access to test facilities in Europe - Energy Live News - Energy Made Easy

EU money can be used to offshore migrants on boats – EUobserver

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EU purse strings are open for member states to hold migrants on boats offshore - under certain legal conditions.

The statement by the Brussels-executive on Tuesday (1 September) follows Maltese media reports that the government in Valletta intends to spend over 1m a month to detain migrants and refugees on a large Cypriot-flagged passenger ship.

The commission's comments are a general statement - not linked to the specifics of Malta's decision - noting that people on board must have access to asylum procedures in order to secure EU funds.

"This is also related to the place where the activity would take place, as EU asylum law does not apply in international waters," said a commission spokesperson, in an email.

The commission further noted that the money can only be used to finance food, medical assistance, and boat personnel.

The Maltese government did not respond as of writing on whether it intends to use EU funding to pay for the million-euro a month plan.

But earlier this week The Shift news, an independent media outlet in Malta, reported the government was trying to finance the project from the EU budget.

Similar ideas first broached in 2016 by the Italian government, to create so-called floating hotspots, where migrants are screened on boats, failed to gain much support.

Rome at the time proposed health, security and identity checks on the boats before bringing rescued people on shore for further asylum evaluation.

But case law at the European Court of Strasbourg complicated the proposals, given wider issues over detention and human rights.

It is not immediately clear if the Maltese government intends to carry out screenings, or offer those it detains on the boat access to asylum procedures.

Both Malta and Italy had earlier this year declared their ports unsafe given the pandemic.

But the scheme points to a wider a problem of how to parcel out people, initially rescued at sea, among EU states.

For over 10 days, some 350 people were left stranded on the charity rescue boat, Sea-Watch 4 until granted permission to disembark in Palermo, Sicily.

Such incidents have been occurring on and off since 2018.

The European Commission is often called to help coordinate the subsequent relocations.

It says that over 2,000 people rescued at sea were transferred from Malta and Italy to other EU states and Norway between 27 June 2018 and 3 August 2020.

Of those, some 1,090 were transferred from Malta and 967 from Italy. Another 849 remain to be relocated from both.

Some 1,251 were transferred after disembarkations that took place in 2019, and 68 were transferred after disembarkations that took place in 2020.

"These applicants were transferred to Germany, Spain, Finland, France, Ireland, Lithuania, Luxembourg, Portugal and Romania," said the commission.

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Second MHI Vestas SOV Hits the Water in Turkey – Offshore WIND

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Turkeys Cemre Shipyard has launched the second of the three service operation vessels (SOVs) being built for MHI Vestas and Esvagt.

The vessels are of the Havyard 831 L SOV design andwill servicethe Borssele III and IV offshore wind farm in the Netherlands, the Triton Knoll wind farm offshore England, and theMoray Eastwind farm off Scotland.

Cemre Shipyard launched the first SOV in April 2020. All three vessels were initially scheduled to be delivered by early 2021.

Esvagt has since accepted a delay in the deliveries of the vessels due to what was described as Havyard going through financial whirlwinds. The new delivery dates have since not been disclosed.

All three vessels are under 15-year charter contracts with MHI Vestas.

The 70.5-metre-long SOVs will be equipped with a compensated walk-to-work gangway and daughter craft, as well as multiple other features, and will have the capacity to accommodate up to 60 persons.

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Australia offers exploration acreage across five offshore basins – Offshore Oil and Gas Magazine

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(Courtesy Department of Industry, Science, Energy and Resources)

Offshore staff

CANBERRA, Australia The coalition government of Australia has issued the countrys 2020 Offshore Petroleum Exploration Acreage Release.

This covers 42 areas in waters offshore Western Australia, Victoria, Northern Territory and the territory of Ashmore and Cartier Islands off northwest Australia.

A total of 100,000 sq km (38,610 sq mi) of acreage is available for exploration across the Bonaparte, Browse, Northern Carnarvon, Otway, and Gippsland basins.

Federal Minister for Resources, Water and Northern Australia, Keith Pitt said: The government strongly believes exploration will play a key role in helping our economy recover from the pandemic.

This years release is dominated by areas in established oil and gas provinces with existing infrastructure. This is consistent with the drive to ensure major projects have and maintain steady supply into the future.

Most importantly, I want to make sure Australia remains an attractive destination for exploration investment and that we can continue to deliver long-term energy security for all Australians.

All areas offered are based on industry nominations and were subject to a public consultation process. Bidding will close on June 1, 2021.

09/02/2020

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Australia offers exploration acreage across five offshore basins - Offshore Oil and Gas Magazine

CWind Phantom to Support Yunlin OWF Construction – Offshore WIND

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Wpd, the developer of the Taiwanese Yunlin offshore wind farm, has awarded CWind Taiwan with a contract for a crew transfer vessel (CTV) which will support the projects two-year construction phase.

The vessel operator will deploy CWind Phantom for crew transfer during construction works.

The 27-metre CTV, built in 2015 and officially named in 2017, was imported to Taiwan and re-flagged in 2019, after which the vessel started its contract for the Formosa 1 offshore wind farm.

The vessel is currently carrying out an UXO survey at an offshore wind farm site in Taiwan, CWind said in a press release from 1 September.

Weve been supporting wpd in their GuanYin and Yunlin projects since 2018 and were pleased to continue to provide critical services to the customer with this additional scope of work. It is both a great honour that our fleet and crew are widely recognised by our clients and a ringing endorsement of our decision to provide market-leading, fully localised solutions to the Taiwanese offshore wind industry, said Ethan Wang, Commercial Director at CWind Taiwan.

The Yunlin offshore wind farm, scheduled for commissioning in 2021, will also see a locally-built CTV being used during its operations and maintenance (O&M) phase. The Prosperous 1(1), launched at the beginning of August, was ordered by the developer wpd and the service provider Deutsche Windtechnik.

The 640 MW project will comprise 80 Siemens Gamesa 8 MW turbines installed some 8 kilometres off the coast of Taiwan.

For its 350 MW Guanyin project in Taiwan, wpd recently signed several supply agreements, with the project expected to officially kick off after receiving the remaining permits.

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Benefits of NOMA versus ad hoc arbitration in shipping and offshore contracts – Lexology

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Introduction

Arbitration is the most commonly used dispute resolution mechanism in shipping and offshore contracts. However, parties often tend to spend little or no effort reflecting on the type of arbitration solution chosen (ie, ad hoc versus institutional arbitration). This article highlights the benefits of agreeing to arbitration under the rules of the Nordic Offshore and Maritime Arbitration Association (NOMA) versus ad hoc arbitration.

Arbitration in Norway has traditionally been ad hoc, based on the rules of the Norwegian Arbitration Act 2004 (NAA) and similar preceding legislation. Ad hoc arbitration is not administered by an institution, leaving the parties to agree how the proceedings will be conducted to determine the particular dispute. This generally provides greater flexibility to the parties as the NAA is somewhat limited in scope and contains only limited regulations. However, where the parties are unable to agree on procedural aspects of the arbitration, the tribunal has broad discretion as to how the arbitration will be conducted. Therefore, unless the parties agree otherwise, the NAA states that the arbitration will be conducted in such manner (procedurally) as the tribunal considers appropriate. As these characteristics of ad hoc arbitration can sometimes create a lack of transparency and predictability, ad hoc arbitration is often perceived as something of a black box.

The alternative to ad hoc arbitration is institutional arbitration, where the arbitration is administered by and conducted pursuant to pre-established rules and procedures of arbitration institutions such as the International Chamber of Commerce (ICC), the London Maritime Arbitrators Association (LMAA) or NOMA, considered further below. Institutional arbitration generally ensures a high degree of foreseeability in respect of how the arbitration will be conducted, particularly as regards the procedural rules. However, pre-established arbitration rules will limit flexibility, although parties are usually free to vary those rules by agreement, even after a dispute has arisen.

NOMA arbitration Nordic best practice

NOMA was established on 28 November 2017 as a common Nordic alternative to both the traditional ad hoc and established institutional arbitrations. The lack of transparency and foreseeability in ad hoc arbitration was seen as making international parties reluctant to agree to refer their disputes to arbitration in Norway and the Nordics. Conversely, the rules of traditional arbitration institutions such as ICC and LMAA were perceived to be too rigid and incompatible with the more flexible Nordic legal tradition. Thus, NOMA's ambition was to preserve and codify a Nordic best practice for the conduct of arbitration and to limit as much as possible the institutionalisation of those rules and best practices. The result is the NOMA Rules and NOMA Best Practice Guidelines.

Unlike traditional arbitration institutions, NOMA does not charge administrative fees, offering the use of the NOMA Rules and Best Practice Guidelines free of charge. Further, NOMA does not administer the arbitration proceedings, only stepping in for certain limited matters upon the parties' request. NOMA therefore ensures a certain degree of flexibility and party autonomy, while at the same time promoting transparency and foreseeability in the arbitration process.

Benefits of NOMA versus ad hoc arbitration

It is a general and overarching feature of the NOMA Rules and Best Practice Guidelines that they promote efficiency and simplicity in the arbitration process.

Efficiency (both in cost and time) is achieved in particular through detailed provisions in the NOMA Guidelines on case preparations and procedural rules. For instance, NOMA requires a case management conference (CMC) shortly after constitution of the tribunal. It is explicitly stated in the guidelines that the CMC aims to agree procedural directions to be followed at the outset to ensure a prudent and cost-effective determination of a dispute. A comprehensive CMC matrix setting out detailed particulars of the items to be discussed and agreed at the CMC is included as Annex 1 to the guidelines.

Further, NOMA's procedural timetable and time limits are shorter than those in ad hoc arbitrations based on the rules of the NAA. For instance, the time limit for appointment of arbitrators (provided the parties cannot agree) is reduced from one month to 21 days. The following default time limits for submissions of pleadings are also included:

There are no similar time limits under the NAA, as these are left to the parties to agree or to be directed by the arbitrators at their discretion. Moreover, under NOMA arbitration rules, oral hearings must take place:

By comparison, there are no time limits for the scheduling of hearings under the NAA.

NOMA is also currently working on a separate set of rules for small claims or fast-track proceedings aimed at streamlining the determination of smaller, low-value claims. These rules, which are expected to be launched shortly, will also seek to further limit costs and reduce applicable time limits.

While the NAA offers limited rules on evidence, NOMA offers detailed rules in that regard. The NOMA Rules on the Taking of Evidence are set out as Annex 2 to the NOMA Guidelines.

Comment

NOMA arbitration has many benefits, making it an attractive option for contracting parties. There has been increased uptake in NOMA arbitration both in terms of contracts specifically providing for NOMA as the dispute resolution mechanism and as something which the parties agree to if and when a dispute arises. This is unsurprising as NOMA first and foremost represents a Nordic best practice of conducting arbitration, with greater emphasis on transparency and efficiency.(1)

Endnotes

(1) The current suggested wording for a NOMA arbitration clause is as follows:

Nordic Arbitration Recommended Arbitration Clause

ARBITRATION CLAUSE AND GOVERNING LAW

This agreement shall be governed by and construed in accordance with [insert governing law] law.

Any dispute arising out of or in connection with this agreement, including any disputes regarding the existence, breach, termination or validity thereof, shall be finally settled by arbitration under the rules of arbitration procedure adopted by the Nordic Offshore and Maritime Arbitration Association ("Nordic Arbitration") in force at the time when such arbitration proceedings are commenced. Nordic Arbitration's Best Practice Guidelines shall be taken into account.

The place of arbitration shall be [insert city and country] and the language of the arbitration

shall be [insert Danish, Norwegian, Swedish or English].

The arbitration tribunal shall be composed of three (3) arbitrators.

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Benefits of NOMA versus ad hoc arbitration in shipping and offshore contracts - Lexology

Installation Team Needed for Danish Offshore Wind Farms – Offshore WIND

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Vattenfall has issued a tender for installation works on two wind farms in the Danish North Sea Vesterhav Syd and Vesterhav Nord.

The tender is divided into three lots and covers the installation of the foundations, the cables, and the wind turbines.

The contracts are scheduled to run from July 2021 to the end of 2023. The tender will remain open until 20 October.

Vesterhav SydandVesterhav Nordwill comprise a total of 41 Siemens Gamesa 8.4 MW turbines with a combined capacity of 344.4 MW. The wind farms are located nine and eight kilometres off the Danish western coast, respectively.

Vattenfall had initially planned to commission the two wind farms in 2020, but the developerhad to delaythe two projects after the Danish Energy Agency initiated new environmental impact assessment (EIA) processes.

The Swedish energy company subsequently canceled several ongoing tenders related to the projects, including the ones for the installation works.

Vattenfallhas since changedthe layout of the wind farms, pushing the wind turbines as far offshore as possible.

Back in May, the Danish Energy Agencyopeneda public consultation on the new EIA reports, with the final permits expected to be issued in September or October.

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Installation Team Needed for Danish Offshore Wind Farms - Offshore WIND

Gear up for the change! W&T Offshore Inc. (WTI) has hit the volume of 921184 – The InvestChronicle

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Lets start up with the current stock price of W&T Offshore Inc. (WTI), which is $2.18 to be very precise. The Stock rose vividly during the last session to $2.24 after opening rate of $2.23 while the lowest price it went was recorded $2.17 before closing at $2.18.

Recently in News on August 5, 2020, W&T Offshore Announces Second Quarter 2020 Results. W&T Offshore, Inc. (NYSE: WTI) (W&T or the Company) today reported operational and financial results for the second quarter 2020. You can read further details here

W&T Offshore Inc. had a pretty Dodgy run when it comes to the market performance. The 1-year high price for the companys stock is recorded $6.10 on 01/03/20, with the lowest value was $1.07 for the same time period, recorded on 03/18/20.

Price records that include history of low and high prices in the period of 52 weeks can tell a lot about the stocks existing status and the future performance. Presently, W&T Offshore Inc. shares are logging -64.18% during the 52-week period from high price, and 104.21% higher than the lowest price point for the same timeframe. The stocks price range for the 52-week period managed to maintain the performance between $1.07 and $6.10.

The companys shares, operating in the sector of Energy managed to top a trading volume set approximately around 921184 for the day, which was evidently lower, when compared to the average daily volumes of the shares.

When it comes to the year-to-date metrics, the W&T Offshore Inc. (WTI) recorded performance in the market was -60.79%, having the revenues showcasing -22.14% on a quarterly basis in comparison with the same period year before. At the time of this writing, the total market value of the company is set at 302.19M, as it employees total of 291 workers.

During the last month, 0 analysts gave the W&T Offshore Inc. a BUY rating, 0 of the polled analysts branded the stock as an OVERWEIGHT, 0 analysts were recommending to HOLD this stock, 0 of them gave the stock UNDERWEIGHT rating, and 0 of the polled analysts provided SELL rating.

According to the data provided on Barchart.com, the moving average of the company in the 100-day period was set at 2.51, with a change in the price was noted +0.08. In a similar fashion, W&T Offshore Inc. posted a movement of +3.77% for the period of last 100 days, recording 5,749,521 in trading volumes.

Raw Stochastic average of W&T Offshore Inc. in the period of last 50 days is set at 16.05%. The result represents improvement in oppose to Raw Stochastic average for the period of the last 20 days, recording 6.36%. In the last 20 days, the companys Stochastic %K was 3.62% and its Stochastic %D was recorded 11.93%.

Lets take a glance in the erstwhile performances of W&T Offshore Inc., multiple moving trends are noted. Year-to-date Price performance of the companys stock appears to be encouraging, given the fact the metric is recording -60.79%. Additionally, trading for the stock in the period of the last six months notably deteriorated by -9.92%, alongside a downfall of -48.34% for the period of the last 12 months. The shares increased approximately by -7.63% in the 7-day charts and went down by -2.68% in the period of the last 30 days. Common stock shares were lifted by -22.14% during last recorded quarter.

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Gear up for the change! W&T Offshore Inc. (WTI) has hit the volume of 921184 - The InvestChronicle

How US Ports Can Capitalize on the Offshore Wind Boom – The Maritime Executive

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Block Island Wind, the first commercial offshore wind development in the U.S. (file image)

By Jay Borkland 08-27-2020 12:57:13

With over 30 gigawatts (GW) of planned electric power generating capacity being installed on the U.S. East Coast continental shelf over the next decade and a half, offshore wind presents a major opportunity for seaboard states to generate green jobs in the decades ahead.

If current projections are realized, the Gulf Coast and the West Coast of the U.S. will see similar growth, pushing the U.S. installed capacity to match, and even exceed, that of Europe by 2050. The next ten years will see a significant uptick in the construction of major offshore wind farms with between two and four GW per year coming online in the years to 2030 and beyond.

To build these megafarms which will include turbines with blades dwarfing a jumbo jets wingspan there will need to be a rapid upgrade to port infrastructure in key states in addition to building a new fleet of Jones Act compliant vessels to construct and service them. To put this in context, each gigawatt installed represents billions of dollars of net expenditure and if states and ports play it right, these investments will flow into the U.S. green collar supply chain.

So far, the U.S. has 30MW installed, with Vineyard Winds 804MW Park City project off Connecticut scheduled to commence construction in 2021.

These projects have attracted significant interest from the European supply chain, which is seeing the U.S. as the hot new market that will reward those that can innovate appropriately. Each offshore wind farm represents a major opportunity for the nearest state to win contracts for every element of the wind farm from permitting and surveys through to manufacturing, construction, installation, operations and maintenance. The U.S. has the advantage of having the European experience to learn from, including the many mistakes that were made along the way.

In this respect the U.S. has a natural advantage, with U.S.-specific regulations and competitor nations distanced by vast oceans. The U.S. is on a path to developing a robust and uniquely American offshore wind industry that benefits from all the experience of the European offshore wind developers with the added benefit of American ingenuity, innovation and local knowledge.

Floating future

Looking ahead, to 2030 and beyond, it seems likely that floating offshore wind structures will become a growing segment within the offshore wind mix.

The vast majority of current developments have been in waters of up to 50 meters deep, where fixed bottom infrastructure makes the most sense. But the growing demand for new sites in Europe and in deep water nations such as Japan (and along our own U.S. West Coast and the Gulf of Maine) means floating wind comes next.

Already, the Bureau of Ocean Energy Management (BOEM) has identified areas off of California and the Gulf of Maine as two potential areas where floating wind is likely to be the technology of choice, and their development could bring real benefits to the U.S.

However you look at it, offshore wind is certain to become a major and growing part of the U.S. energy paradigm in the decades ahead. This will bring massive economic opportunity to the supply chain and, if the lessons of Europe can be learned, those states which move fast and coordinate well will prosper the most. As Europe moves aggressively beyond the 18.5 GW already installed, ramping up to 75GW by 2050, the cost of energy from offshore wind continues to tumble, and it looks as if future offshore wind farms will be able to operate tariff-free and still be competitive with energy derived from other, more traditional sources.

Ports will be key

In the European experience, the leading offshore wind countries learned that the industry and the supply chain will develop around those places where the best port infrastructure exists; such as the mega ports of Hull in the UK and Bremerhaven in Germany on the northern North Sea.

The competitive drive to bring down cost has led to larger and larger machines with GEs Haliade-X 12MW paving new directions for the industry, and the new Siemens 14 MW monster turbine preparing to wade into the industry turbine wars.

These machines and the components that accompany them, require highly specialized ports with appropriate quaysides, extremely high load bearing capacities, large laydown areas, and specialized vessels, bulkheads and cranes.

Port facilities that can support offshore wind deployment have been identified up and down the coast and are actively being developed for offshore wind use. Facilities such as Brayton Point (MA), ProvPort and South Quay (RI), Arthur Kill, the South Brooklyn Marine Terminal, and the Port of Coeymans (NY), Paulsboro (NJ), New London (CT), Sparrows Point (MD), and Portsmouth Marine Terminal (VA) are all getting ready and preparing for the shipping and handling of hundreds of giant super-heavy components.

However, no U.S .port has the vast acreage that developed at the major European hubs. On the East Coast we anticipate the need for between four to six large ports to build and service the turbines, while every state that has a wind farm will require a smaller, specialized port aligned to the long term 25-year operations and maintenance phase.

By working closely with port owners, state governments, manufacturers and the supply chain, developers can use an integrated approach that offers a compelling service to wind farm developers and at the same time maximize the benefit for the U.S. economy.

Of course, visibility of the offshore wind pipeline is key ports will not commit millions of dollars in investment without some idea of the opportunity ahead. Here the Bureau of Ocean Energy Management (BOEM) leasing rounds give a tremendous view of the bigger picture and increasing confidence in the ten-year pipeline. Major developers all want a toehold in the nascent U.S. market, and LR is already working with a number of them to offer a concierge service to help them navigate local waters.

In the U.S., our team at LR have already compiled a detailed database of more than a hundred eastern seaboard ports with potential to capitalize on the offshore wind boom. Our personnel have been with the U.S. east coast ports development for offshore wind since the beginning, having acted as the lead for the design, permitting, and owners engineer oversight of the construction of the New Bedford Marine Commerce Terminal in Massachusetts.

Jay Borkland is the head of clean energy at Lloyds Register (LR) USA.

The opinions expressed herein are the author's and not necessarily those of The Maritime Executive.

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How US Ports Can Capitalize on the Offshore Wind Boom - The Maritime Executive

Alleged rapist Tariq Ramadan to launch centre teaching feminism – The National

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Tariq Ramadan is set to launch a centre to teach ethics and feminism next month despite being suspended from his Oxford University post over rape allegations.

Mr Ramadan, 58, was given a leave of absence from his post at Oxford University after he was charged in France with raping a feminist activist in 2012 and a disabled woman in 2009.

In February this year he was charged with two further rapes.

The Islamic scholar, who is the grandson of the founder of Egypts Muslim Brotherhood, has now announced he was launching an educational centre to teach topics including ethics and feminism.

Courses will be taught by a team of teachers around the themes of religion, spirituality, humanity, psychology, feminism, ecology, teaching, ethics, colonialism, racism, law, economy

Taraq Ramadan

The news has been met with anger from those who have called the case the #MeToo of the Muslim world.

Do we laugh or cry? The intellectual dishonesty, hypocrisy and cynicism of #TariqRamadan have absolutely no limits, teacher and researcher Anne-Marie Picard said on Twitter.

The married father-of-four made the announcement about the project on his Facebook page.

He says he will be opening Chifa, an international research and training centre for students based in France, in October.

The topics in the curriculum include religion, spirituality, humanism and law, as well as ethics and feminism.

Courses will be taught by a team of teachers around the themes of religion, spirituality, humanity, psychology, feminism, ecology, teaching, ethics, colonialism, racism, law, economy, etc. You will be able to sign up for face-to-face and distance classes, basic classes or more targeted modules, he wrote.

The centre is based in France but the teaching platform is international. Teachers are all graduates in their respective fields and are, voluntarily, of different origins with various training courses, he said.

Not all are Muslims. The curriculum is led by a steering committee such as the Centre for Research and Training itself. There will be a certificate for those who have completed the three-year training. We haven't thought about connecting our centre to an official institute yet but there might be some leads to this in the future.

Despite hundreds of people showing enthusiasm for his endeavour, his initiative has been met with outrage.

French-Swiss journalist Ian Hamel wrote on Twitter: Harvey Weinstein hadnt even thought about it, but Tariq Ramadan himself dared to create a training centre giving courses in feminism and ethics! Do you want to know what this individual is capable of? .

Others have accused him of using it to target women.

A recruiting centre? Is he getting old to hunt on his own? Daniel Fleury, another journalist, said on Twitter.

The rape investigations into Mr Ramadan are still continuing.

Last year he portrayed himself as a victim of a political witch hunt in a book he published.

There is a desire to pursue Tariq Ramadan at all costs and against the evidence, his lawyer Emmanuel Marsigny said in February as new charges were added.

This case is becoming grotesque.

The new charges involve two women who themselves had not originally filed complaints, but both allege they were involved in brutal sexual relationships with Mr Ramadan one from November-December 2015 and the other in March 2016.

When the first allegations were made in 2018, Mr Ramadan was held for nine months in France before being granted bail.

Mr Ramadan denies the charges and insists all his relationships have been consensual.

Being charged in France does not necessarily mean a suspect will end up on trial, as a case can still be dropped for lack of evidence.

Oxford University said he had taken a leave of absence by mutual agreement.

"The university has consistently acknowledged the gravity of the allegations against Professor Ramadan, while emphasising the importance of fairness and the principles of justice and due process, it said.

An agreed leave of absence implies no presumption or acceptance of guilt and allows Professor Ramadan to address the extremely serious allegations made against him, all of which he categorically denies, while meeting our principal concern addressing heightened and understandable distress and putting first the well-being of our students and staff."

The National has conducted an investigation into the UKs Muslim charity sector for an exclusive three-part series.

Part one reveals how Islamic Relief Worldwide employees have praised extremists online.

Part two unveils the intense regulatory scrutiny facing the sector.

Part three reveals how the dismissed boss of one of the sectors biggest charities is on the comeback trail.

Updated: September 2, 2020 07:00 PM

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Alleged rapist Tariq Ramadan to launch centre teaching feminism - The National

TAIJI Medical Supplies Inc., a US PPE Manufacturer, Announces Expansion of Manufacturing Capacity Amid Expected Surge of COVID-19 and Influenza Cases…

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Made in the USA masks feature better quality while putting Americans back to work.

LINCOLNTON, N.C. (PRWEB) September 02, 2020

TAIJI Medical Supplies, Inc. (TMS), a United States manufacturer of Personal Protective Equipment (PPE) has expanded their manufacturing capacity of face masks including Level I, Level II surgical and children's masks for an expected surge in COVID-19 and influenza cases in the upcoming Fall and Winter months.

The Centers for Disease Control just released a study entitled, "Effectiveness of Cloth Masks for Protection Against Severe Acute Respiratory Syndrome Coronavirus 2," which concluded with alarming findings with regard to cloth masks. Part of the conclusion of the study stated: "The filtration, effectiveness, fit, and performance of cloth masks are inferior to those of medical masks and respirators. Cloth mask use should not be mandated for healthcare workers, who should as a priority be provided proper respiratory protection. Cloth masks are a more suitable option for community use when medical masks are unavailable.

But, TMS is making medical grade masks available to everyone.

Meizong Yin, President and CEO of TAIJI Medical Supplies Inc. stated, We have successfully installed, tested and made operational numerous adult and childrens mask-making machines to serve the public, commercial and healthcare markets. TMS is ready to meet the surge demand for masks in the upcoming months.

TMS is also one of the very few companies in the U.S. that has multiple in-house machines that manufacture the core material used in filtration masks: melt-blown fabric.

We have refined our manufacturing technique, overall processes and use higher quality materials to make our masks more comfortable for extended periods of wear, Yin continued. We feel confident that we can compete with any country on price and we certainly can deliver faster than any overseas manufacturer because there's no issues when you control the supply chain and manufacturing process."

About TAIJI Medical Supplies, Inc.

TAIJI Medical Supplies, Inc. (TMS), based in Lincolnton, NC, is an American company that designs, manufactures and distributes Personal Protective Equipment (PPE) directly and through a dedicated network of third-party distributors. TMS chose to address the growing PPE supply / demand issues through building their corporate and manufacturing teams in Lincolnton, North Carolina. Extensive industrial experience, deep supply chain roots and American ingenuity have allowed TMS to commercialize rapidly, thus resolving supply chain problems while bringing Americans back to work that were displaced because of the COVID-19 Pandemic.

For general or sales inquiries, please use the contact info in this release.

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TAIJI Medical Supplies Inc., a US PPE Manufacturer, Announces Expansion of Manufacturing Capacity Amid Expected Surge of COVID-19 and Influenza Cases...

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