Mammalian E-type cyclins control chromosome pairing, telomere stability and CDK2 localization in male meiosis.

Loss of function of cyclin E1 or E2, important regulators of the mitotic cell cycle, yields viable mice, but E2-deficient males display reduced fertility. To elucidate the role of E-type cyclins during spermatogenesis, we characterized their expression patterns and produced additional deletions of Ccne1 and Ccne2 alleles in the germline, revealing unexpected meiotic functions. While Ccne2 mRNA and protein are abundantly expressed in spermatocytes, Ccne1 mRNA is present but its protein is detected only at low levels. However, abundant levels of cyclin E1 protein are detected in spermatocytes deficient in cyclin E2 protein. Additional depletion of E-type cyclins in the germline resulted in increasingly enhanced spermatogenic abnormalities and corresponding decreased fertility and loss of germ cells by apoptosis. Profound meiotic defects were observed in spermatocytes, including abnormal pairing and synapsis of homologous chromosomes, heterologous chromosome associations, unrepaired double-strand DNA breaks, disruptions in telomeric structure and defects in cyclin-dependent-kinase 2 localization. These results highlight a new role for E-type cyclins as important regulators of male meiosis.

The DNA damage checkpoint protein RAD9A is essential for male meiosis in the mouse.

In mitotic cells, RAD9A functions in repairing DNA double-strand breaks (DSBs) by homologous recombination and facilitates the process by cell cycle checkpoint control in response to DNA damage. DSBs occur naturally in the germline during meiosis but whether RAD9A participates in repairing such breaks is not known. In this study, we determined that RAD9A is indeed expressed in the male germ line with a peak of expression in late pachytene and diplotene stages, and the protein was found associated with the XY body. As complete loss of RAD9A is embryonic lethal, we constructed and characterized a mouse strain with Stra8-Cre driven germ cell-specific ablation of Rad9a beginning in undifferentiated spermatogonia in order to assess its role in spermatogenesis. Adult mutant male mice were infertile or sub-fertile due to massive loss of spermatogenic cells. The onset of this loss occurs during meiotic prophase, and there was an increase in the numbers of apoptotic spermatocytes as determined by TUNEL. Spermatocytes lacking RAD9A usually arrested in meiotic prophase, specifically in pachytene. The incidence of unrepaired DNA breaks increased, as detected by accumulation of γH2AX and DMC1 foci on the axes of autosomal chromosomes in pachytene spermatocytes. The DNA topoisomerase IIß-binding protein 1 (TOPBP1) was still localized to the sex body, albeit with lower intensity, suggesting that RAD9A may be dispensable for sex body formation. We therefore show for the first time that RAD9A is essential for male fertility and for repair of DNA DSBs during meiotic prophase I.

Genetic modulation of the HTR2A gene reduces anxiety-related behavior in mice.

The expanding field of precision gene editing using CRISPR/Cas9 has demonstrated its potential as a transformative technology in the treatment of various diseases. However, whether this genome-editing tool could be used to modify neural circuits in the central nervous system (CNS), which are implicated in complex behavioral traits, remains uncertain. In this study, we demonstrate the feasibility of noninvasive, intranasal delivery of adeno-associated virus serotype 9 (AAV9) vectors containing CRISPR/Cas9 cargo within the CNS resulting in modification of the HTR2A receptor gene. In vitro, exposure to primary mouse cortical neurons to AAV9 vectors targeting the HT2RA gene led to a concentration-dependent decrease in spontaneous electrical activity following multielectrode array (MEA) analysis. In vivo, at 5 weeks postintranasal delivery in mice, analysis of brain samples revealed single base pair deletions and nonsense mutations, leading to an 8.46-fold reduction in mRNA expression and a corresponding 68% decrease in the 5HT-2A receptor staining. Our findings also demonstrate a significant decrease in anxiety-like behavior in treated mice. This study constitutes the first successful demonstration of a noninvasive CRISPR/Cas9 delivery platform, capable of bypassing the blood-brain barrier and enabling modulation of neuronal 5HT-2A receptor pathways. The results of this study targeting the HTR2A gene provide a foundation for the development of innovative therapeutic strategies for a broad range of neurological disorders, including anxiety, depression, attentional deficits, and cognitive dysfunction.

Esrrb Regulates Specific Feed-Forward Loops to Transit From Pluripotency Into Early Stages of Differentiation.

Characterization of pluripotent states, in which cells can both self-renew or differentiate, with the irreversible loss of pluripotency, are important research areas in developmental biology. Although microRNAs (miRNAs) have been shown to play a relevant role in cellular differentiation, the role of miRNAs integrated into gene regulatory networks and its dynamic changes during these early stages of embryonic stem cell (ESC) differentiation remain elusive. Here we describe the dynamic transcriptional regulatory circuitry of stem cells that incorporate protein-coding and miRNA genes based on miRNA array expression and quantitative sequencing of short transcripts upon the downregulation of the Estrogen Related Receptor Beta (Esrrb). The data reveals how Esrrb, a key stem cell transcription factor, regulates a specific stem cell miRNA expression program and integrates dynamic changes of feed-forward loops contributing to the early stages of cell differentiation upon its downregulation. Together these findings provide new insights on the architecture of the combined transcriptional post-transcriptional regulatory network in embryonic stem cells.

Osmolal and anion gaps after acute self-poisoning with agricultural formulations of the organophosphorus insecticides profenofos and diazinon: A pilot study.

Self-poisoning with organophosphorus (OP) insecticides is an important means of global self-harm. The insecticides are formulated with solvents that may also contribute to toxicity. We set up a study to detect changes in osmolal and anion gaps following ingestion of OP insecticides. We recruited consecutive patients admitted to a Teaching Hospital, Sri Lanka, with a history of OP self-poisoning. The osmolal and anion gaps were calculated on admission and at 4, 24 and 72 h post-ingestion together with ethanol concentration. Forty-nine patients were recruited (28 profenofos, 10 diazinon, one coumaphos, one chlorpyrifos, one phenthoate and eight unknown OP). Only modest increases in osmolal and anion gaps were noted. Small rises in osmolal gap above the upper limit of normal were noted in 16/49 (32.7%) of all cases, 9/28 (32.1%) profenofos cases and 4/10 (40.0%) diazinon cases. The anion gap was raised in 24/49 (49.0%) of all cases, 15/28 (53.6%) profenofos cases and 5/10 (50.0%) diazinon cases. We observed a trend for a fall in osmolal gap during the first 24 h, followed by an increase up to 72 h. There was no correlation between the anion gap and serum lactate concentration, indicating that a lactic acidosis was not responsible for the anion gap. Formate, which could have explained the increased gap, was not detected in any of the samples; ketoacids (beta-hydroxybutyrate and acetoacetate) were not measured. This pilot study found that profenofos and diazinon poisoning caused only modest increases in the osmolal and anion gaps in a minority of cases.

An Esrrb and Nanog Cell Fate Regulatory Module Controlled by Feed Forward Loop Interactions.

Cell fate decisions during development are governed by multi-factorial regulatory mechanisms including chromatin remodeling, DNA methylation, binding of transcription factors to specific loci, RNA transcription and protein synthesis. However, the mechanisms by which such regulatory "dimensions" coordinate cell fate decisions are currently poorly understood. Here we quantified the multi-dimensional molecular changes that occur in mouse embryonic stem cells (mESCs) upon depletion of Estrogen related receptor beta (Esrrb), a key pluripotency regulator. Comparative analyses of expression changes subsequent to depletion of Esrrb or Nanog, indicated that a system of interlocked feed-forward loops involving both factors, plays a central part in regulating the timing of mESC fate decisions. Taken together, our meta-analyses support a hierarchical model in which pluripotency is maintained by an Oct4-Sox2 regulatory module, while the timing of differentiation is regulated by a Nanog-Esrrb module.

Administration of high titer convalescent anti-SARS-CoV-2 plasma: From donor selection to monitoring recipient outcomes.

Early in the SARS-CoV-2 pandemic, convalescent plasma (CP) therapy was proposed as a treatment for severely ill patients. We conducted a CP treatment protocol under the Mayo Clinic Extended Access Program at University Hospital Brooklyn (UHB). Potential donors were screened with a lateral flow assay (LFA) for IgM and IgG antibodies against the SARS-CoV-2 S1 receptor-binding domain (RBD). Volunteers that were LFA positive were tested with an ELISA to measure IgG titers against the RBD. Subjects with titers of at least 1:1024 were selected to donate. Most donors with positive LFA had acceptable titers and were eligible to donate. Out of 171 volunteers, only 65 tested positive in the LFA (38.0%), and 55 (32.2%) had titers of at least 1:1024. Before our donation program started, 31 CP units were procured from the New York Blood Center (NYBC). Among the 31 CP units that were obtained from the NYBC, 25 units (80.6%) were positive in the LFA but only 12 units (38.7%) had titers of at least 1:1024. CP was administered to 28 hospitalized COVID-19 patients. Patients who received low titer CP, high titer CP and patients who did not receive CP were followed for 45 days after presentation. Severe adverse events were not associated with CP transfusion. Death was a less frequent outcome for patients that received high titer CP (>1:1024) 38.6% mortality, than patients that received low titer CP (≤1:1024) 77.8% mortality.

Implementation of a pooled surveillance testing program for asymptomatic SARS-CoV-2 infections in K-12 schools and universities.

BACKGROUND: The negative impact of continued school closures during the height of the COVID-19 pandemic warrants the establishment of cost-effective strategies for surveillance and screening to safely reopen and monitor for potential in-school transmission. Here, we present a novel approach to increase the availability of repetitive and routine COVID-19 testing that may ultimately reduce the overall viral burden in the community. METHODS: We implemented a testing program using the SalivaClear࣪ pooled surveillance method that included students, faculty and staff from K-12 schools (student age range 5-18 years) and universities (student age range >18 years) across the country (Mirimus Clinical Labs, Brooklyn, NY). The data analysis was performed using descriptive statistics, kappa agreement, and outlier detection analysis. FINDINGS: From August 27, 2020 until January 13, 2021, 253,406 saliva specimens were self-collected from students, faculty and staff from 93 K-12 schools and 18 universities. Pool sizes of up to 24 samples were tested over a 20-week period. Pooled testing did not significantly alter the sensitivity of the molecular assay in terms of both qualitative (100% detection rate on both pooled and individual samples) and quantitative (comparable cycle threshold (Ct) values between pooled and individual samples) measures. The detection of SARS-CoV-2 in saliva was comparable to the nasopharyngeal swab. Pooling samples substantially reduced the costs associated with PCR testing and allowed schools to rapidly assess transmission and adjust prevention protocols as necessary. In one instance, in-school transmission of the virus was determined within the main office and led to review and revision of heating, ventilating and air-conditioning systems. INTERPRETATION: By establishing low-cost, weekly testing of students and faculty, pooled saliva analysis for the presence of SARS-CoV-2 enabled schools to determine whether transmission had occurred, make data-driven decisions, and adjust safety protocols. We provide strong evidence that pooled testing may be a fundamental component to the reopening of schools by minimizing the risk of in-school transmission among students and faculty. FUNDING: Skoll Foundation generously provided funding to Mobilizing Foundation and Mirimus for these studies.

Precise hit: adeno-associated virus in gene targeting.

Vectors based on the adeno-associated virus (AAV) have attracted much attention as potent gene-delivery vehicles, mainly because of the persistence of this non-pathogenic virus in the host cell and its sustainable therapeutic gene expression. However, virus infection can be accompanied by potentially mutagenic random vector integration into the genome. A novel approach to AAV-mediated gene therapy based on gene targeting through homologous recombination allows efficient, high-fidelity, non-mutagenic gene repair in a host cell.

Homologous recombination is required for AAV-mediated gene targeting.

High frequencies of gene targeting can be achieved by infection of mammalian cells with recombinant adeno-associated virus (rAAV) vectors [D. W. Russell and R. K. Hirata (1998) Nature Genet., 18, 325-330; D. W. Russell and R. K. Hirata (2000) J. Virol., 74, 4612-4620; R. Hirata et al. (2002) Nat. Biotechnol., 20, 735-738], but the mechanism of targeting is unclear and random integration often occurs in parallel. We assessed the role of specific DNA repair and recombination pathways in rAAV gene targeting by measuring correction of a mutated enhanced green fluorescent protein (EGFP) gene in cells where homologous recombination (HR) or non-homologous end-joining (NHEJ) had been suppressed by RNAi. EGFP-negative cells were transduced with rAAV vectors carrying a different inactivating deletion in the EGFP, and in parallel with rAAV vectors carrying red fluorescent protein (RFP). Expression of RFP accounted for viral transduction efficiency and long-term random integration. Approximately 0.02% of the infected GFP-negative cells were stably converted to GFP positive cells. Silencing of the essential NHEJ component DNA-PK had no significant effect on the frequency of targeting at any time point examined. Silencing of the SNF2/SWI2 family members RAD54L or RAD54B, which are important for HR, reduced the rate of stable rAAV gene targeting approximately 5-fold. Further, partial silencing of the Rad51 paralogue XRCC3 completely abolished stable long-term EGFP expression. These results show that rAAV gene targeting requires the Rad51/Rad54 pathway of HR.

Modified penetrance of coding variants by cis-regulatory variation contributes to disease risk.

Coding variants represent many of the strongest associations between genotype and phenotype; however, they exhibit inter-individual differences in effect, termed 'variable penetrance'. Here, we study how cis-regulatory variation modifies the penetrance of coding variants. Using functional genomic and genetic data from the Genotype-Tissue Expression Project (GTEx), we observed that in the general population, purifying selection has depleted haplotype combinations predicted to increase pathogenic coding variant penetrance. Conversely, in cancer and autism patients, we observed an enrichment of penetrance increasing haplotype configurations for pathogenic variants in disease-implicated genes, providing evidence that regulatory haplotype configuration of coding variants affects disease risk. Finally, we experimentally validated this model by editing a Mendelian single-nucleotide polymorphism (SNP) using CRISPR/Cas9 on distinct expression haplotypes with the transcriptome as a phenotypic readout. Our results demonstrate that joint regulatory and coding variant effects are an important part of the genetic architecture of human traits and contribute to modified penetrance of disease-causing variants.

Role of cyclins in controlling progression of mammalian spermatogenesis.

Cyclins are key regulators of the mammalian cell cycle, functioning primarily in concert with their catalytic partners, the cyclin-dependent kinases (Cdks). While their function during mitosis in somatic cells has been extensively documented, their function during both mitosis and meiosis in the germ line is poorly understood. From the perspective of cell cycle regulation there are several aspects of mammalian spermatogenesis that suggest unique modes of regulation and hence, possible unique functions for the cyclins. This review will summarize our current understanding of cyclin expression and function in the male germ line, with particular focus on the A and E type cyclins in the mouse model. While the focus is on mammalian spermatogenesis, we note contrasts with similar functions in the female germ line when relevant and also draw upon observations in other model systems to provide further insight.

Clamping down on mammalian meiosis.

The RAD9A-RAD1-HUS1 (9-1-1) complex is a PCNA-like heterotrimeric clamp that binds damaged DNA to promote cell cycle checkpoint signaling and DNA repair. While various 9-1-1 functions in mammalian somatic cells have been established, mounting evidence from lower eukaryotes predicts critical roles in meiotic germ cells as well. This was investigated in 2 recent studies in which the 9-1-1 complex was disrupted specifically in the mouse male germline through conditional deletion of Rad9a or Hus1. Loss of these clamp subunits led to severely impaired fertility and meiotic defects, including faulty DNA double-strand break repair. While 9-1-1 is critical for ATR kinase activation in somatic cells, these studies did not reveal major defects in ATR checkpoint pathway signaling in meiotic cells. Intriguingly, this new work identified separable roles for 9-1-1 subunits, namely RAD9A- and HUS1-independent roles for RAD1. Based on these studies and the high-level expression of the paralogous proteins RAD9B and HUS1B in testis, we propose a model in which multiple alternative 9-1-1 clamps function during mammalian meiosis to ensure genome maintenance in the germline.

Sp1 transcription factor and GATA1 cis-acting elements modulate testis-specific expression of mouse cyclin A1.

Cyclin A1 is a male germ cell-specific cell cycle regulator that is essential for spermatogenesis. It is unique among the cyclins by virtue of its highly restricted expression in vivo, being present in pachytene and diplotene spermatocytes and not in earlier or later stages of spermatogenesis. To begin to understand the molecular mechanisms responsible for this narrow window of expression of the mouse cyclin A1 (Ccna1) gene, we carried out a detailed analysis of its promoter. We defined a 170-bp region within the promoter and showed that it is involved in repression of Ccna1 in cultured cells. Within this region we identified known cis-acting transcription factor binding sequences, including an Sp1-binding site and two GATA1-binding sites. Neither Sp1 nor GATA1 is expressed in pachytene spermatocytes and later stages of germ cell differentiation. Sp1 is readily detected at earlier stages of spermatogenesis. Site-directed mutagenesis demonstrated that neither factor alone was sufficient to significantly repress expression driven by the Ccna1 promoter, while concurrent binding of Sp1, and most likely GATA1 and possibly additional factors was inhibitory. Occupancy of Sp1 on the Ccna1 promoter and influence of GATA1-dependent cis-acting elements was confirmed by ChIP analysis in cell lines and most importantly, in spermatogonia. In contrast with many other testis-specific genes, the CpG island methylation status of the Ccna1 promoter was similar among various tissues examined, irrespective of whether Ccna1 was transcriptionally active, suggesting that this regulatory mechanism is not involved in the restricted expression of Ccna1.

Tdrd6 is required for spermiogenesis, chromatoid body architecture, and regulation of miRNA expression.

BACKGROUND: Chromatoid bodies (CBs) are characteristic spermatid organelles, which were suggested to function in RNA storage and small RNA processing but whose functions remain largely unknown. CB components include Mili, Miwi, and Tudor domain proteins such as Tdrd6, whose contribution to CB structure and function is elusive. RESULTS: We determined gametogenesis stage- and male-specific expression and localization of Tdrd6, identified a C-terminally truncated form as predominant after meiosis I, and demonstrated direct physical interaction of Tdrd6 with the CB components Mili and Miwi. Development from round into elongated spermatids is abrogated in Tdrd6(-/-) mice. Their round spermatids bear "ghost" CBs, whose architecture is greatly disrupted. Mael, Miwi, and Mvh do not localize to the Tdrd6-deficient CBs, but retrotransposons are not significantly activated. However, more than 50 miRNAs are more abundant in Tdrd6(-/-) testes, as are exemplary pre- and pri-miRNAs. CONCLUSION: We conclude that Tdrd6 is essential for spermiogenesis, for CB structure, and for proper mature and precursor miRNA expression.