Structural Basis of BRCC36 Function in DNA Repair and Immune Regulation.

In mammals, ∼100 deubiquitinases act on ∼20,000 intracellular ubiquitination sites. Deubiquitinases are commonly regarded as constitutively active, with limited regulatory and targeting capacity. The BRCA1-A and BRISC complexes serve in DNA double-strand break repair and immune signaling and contain the lysine-63 linkage-specific BRCC36 subunit that is functionalized by scaffold subunits ABRAXAS and ABRO1, respectively. The molecular basis underlying BRCA1-A and BRISC function is currently unknown. Here we show that in the BRCA1-A complex structure, ABRAXAS integrates the DNA repair protein RAP80 and provides a high-affinity binding site that sequesters the tumor suppressor BRCA1 away from the break site. In the BRISC structure, ABRO1 binds SHMT2α, a metabolic enzyme enabling cancer growth in hypoxic environments, which we find prevents BRCC36 from binding and cleaving ubiquitin chains. Our work explains modularity in the BRCC36 DUB family, with different adaptor subunits conferring diversified targeting and regulatory functions.

De novo transcriptome assembly of mouse male germ cells reveals novel genes, stage-specific bidirectional promoter activity, and noncoding RNA expression.

In mammals, the adult testis is the tissue with the highest diversity in gene expression. Much of that diversity is attributed to germ cells, primarily meiotic spermatocytes and postmeiotic haploid spermatids. Exploiting a newly developed cell purification method, we profiled the transcriptomes of such postmitotic germ cells of mice. We used a de novo transcriptome assembly approach and identified thousands of novel expressed transcripts characterized by features distinct from those of known genes. Novel loci tend to be short in length, monoexonic, and lowly expressed. Most novel genes have arisen recently in evolutionary time and possess low coding potential. Nonetheless, we identify several novel protein-coding genes harboring open reading frames that encode proteins containing matches to conserved protein domains. Analysis of mass-spectrometry data from adult mouse testes confirms protein production from several of these novel genes. We also examine overlap between transcripts and repetitive elements. We find that although distinct families of repeats are expressed with differing temporal dynamics during spermatogenesis, we do not observe a general mode of regulation wherein repeats drive expression of nonrepetitive sequences in a cell type-specific manner. Finally, we observe many fairly long antisense transcripts originating from canonical gene promoters, pointing to pervasive bidirectional promoter activity during spermatogenesis that is distinct and more frequent compared with somatic cells.

Transcriptome and translatome co-evolution in mammals.

Gene-expression programs define shared and species-specific phenotypes, but their evolution remains largely uncharacterized beyond the transcriptome layer1. Here we report an analysis of the co-evolution of translatomes and transcriptomes using ribosome-profiling and matched RNA-sequencing data for three organs (brain, liver and testis) in five mammals (human, macaque, mouse, opossum and platypus) and a bird (chicken). Our within-species analyses reveal that translational regulation is widespread in the different organs, in particular across the spermatogenic cell types of the testis. The between-species divergence in gene expression is around 20% lower at the translatome layer than at the transcriptome layer owing to extensive buffering between the expression layers, which especially preserved old, essential and housekeeping genes. Translational upregulation specifically counterbalanced global dosage reductions during the evolution of sex chromosomes and the effects of meiotic sex-chromosome inactivation during spermatogenesis. Despite the overall prevalence of buffering, some genes evolved faster at the translatome layer-potentially indicating adaptive changes in expression; testis tissue shows the highest fraction of such genes. Further analyses incorporating mass spectrometry proteomics data establish that the co-evolution of transcriptomes and translatomes is reflected at the proteome layer. Together, our work uncovers co-evolutionary patterns and associated selective forces across the expression layers, and provides a resource for understanding their interplay in mammalian organs.

SUMOylated PRC1 controls histone H3.3 deposition and genome integrity of embryonic heterochromatin.

Chromatin integrity is essential for cellular homeostasis. Polycomb group proteins modulate chromatin states and transcriptionally repress developmental genes to maintain cell identity. They also repress repetitive sequences such as major satellites and constitute an alternative state of pericentromeric constitutive heterochromatin at paternal chromosomes (pat-PCH) in mouse pre-implantation embryos. Remarkably, pat-PCH contains the histone H3.3 variant, which is absent from canonical PCH at maternal chromosomes, which is marked by histone H3 lysine 9 trimethylation (H3K9me3), HP1, and ATRX proteins. Here, we show that SUMO2-modified CBX2-containing Polycomb Repressive Complex 1 (PRC1) recruits the H3.3-specific chaperone DAXX to pat-PCH, enabling H3.3 incorporation at these loci. Deficiency of Daxx or PRC1 components Ring1 and Rnf2 abrogates H3.3 incorporation, induces chromatin decompaction and breakage at PCH of exclusively paternal chromosomes, and causes their mis-segregation. Complementation assays show that DAXX-mediated H3.3 deposition is required for chromosome stability in early embryos. DAXX also regulates repression of PRC1 target genes during oogenesis and early embryogenesis. The study identifies a novel critical role for Polycomb in ensuring heterochromatin integrity and chromosome stability in mouse early development.

Looking with new eyes: advanced microscopy and artificial intelligence in reproductive medicine.

Microscopy has long played a pivotal role in the field of assisted reproductive technology (ART). The advent of artificial intelligence (AI) has opened the door for new approaches to sperm and oocyte assessment and selection, with the potential for improved ART outcomes.

Dual DNA staining enables isolation of multiple sub-types of post-replicative mouse male germ cells.

During spermatogenesis, mammalian male germ cells undergo multiple developmental processes, including meiosis and post-meiotic differentiation (spermiogenesis). To understand the transitions between different cellular states it is essential to isolate pure populations of cells at different stages of development. Previous approaches enabled the isolation of cells from different stages of meiotic prophase I, but techniques to sub-fractionate unfixed, post-meiotic spermatids have been lacking. Here we report the development of a protocol enabling simultaneous isolation of cells at different stages of meiotic prophase and post-meiotic differentiation from testes of adult mice. This approach builds on existing fluorescence activated cell sorting protocols designed to purify cells in different stages of meiotic prophase I. By utilizing the specific spectral properties that two different DNA dyes (Hoechst 33342 and SYTO 16) exhibit when bound to chromatin of different stage male germ cells, we obtain highly pure populations of cells in relatively large numbers. This FACS protocol will enable immunocytological and molecular characterization studies of fractionated meiotic and haploid germ cells from both wild type and genetically mutant animals.

A Gene Regulatory Program for Meiotic Prophase in the Fetal Ovary.

The chromosomal program of meiotic prophase, comprising events such as laying down of meiotic cohesins, synapsis between homologs, and homologous recombination, must be preceded and enabled by the regulated induction of meiotic prophase genes. This gene regulatory program is poorly understood, particularly in organisms with a segregated germline. We characterized the gene regulatory program of meiotic prophase as it occurs in the mouse fetal ovary. By profiling gene expression in the mouse fetal ovary in mutants with whole tissue and single-cell techniques, we identified 104 genes expressed specifically in pre-meiotic to pachytene germ cells. We characterized the regulation of these genes by 1) retinoic acid (RA), which induces meiosis, 2) Dazl, which is required for germ cell competence to respond to RA, and 3) Stra8, a downstream target of RA required for the chromosomal program of meiotic prophase. Initial induction of practically all identified meiotic prophase genes requires Dazl. In the presence of Dazl, RA induces at least two pathways: one Stra8-independent, and one Stra8-dependent. Genes vary in their induction by Stra8, spanning fully Stra8-independent, partially Stra8-independent, and fully Stra8-dependent. Thus, Stra8 regulates the entirety of the chromosomal program but plays a more nuanced role in governing the gene expression program. We propose that Stra8-independent gene expression enables the stockpiling of selected meiotic structural proteins prior to the commencement of the chromosomal program. Unexpectedly, we discovered that Stra8 is required for prompt down-regulation of itself and Rec8. Germ cells that have expressed and down-regulated Stra8 are refractory to further Stra8 expression. Negative feedback of Stra8, and subsequent resistance to further Stra8 expression, may ensure a single, restricted pulse of Stra8 expression. Collectively, our findings reveal a gene regulatory logic by which germ cells prepare for the chromosomal program of meiotic prophase, and ensure that it is induced only once.

Retinoic acid activates two pathways required for meiosis in mice.

In all sexually reproducing organisms, cells of the germ line must transition from mitosis to meiosis. In mice, retinoic acid (RA), the extrinsic signal for meiotic initiation, activates transcription of Stra8, which is required for meiotic DNA replication and the subsequent processes of meiotic prophase. Here we report that RA also activates transcription of Rec8, which encodes a component of the cohesin complex that accumulates during meiotic S phase, and which is essential for chromosome synapsis and segregation. This RA induction of Rec8 occurs in parallel with the induction of Stra8, and independently of Stra8 function, and it is conserved between the sexes. Further, RA induction of Rec8, like that of Stra8, requires the germ-cell-intrinsic competence factor Dazl. Our findings strengthen the importance of RA and Dazl in the meiotic transition, provide important details about the Stra8 pathway, and open avenues to investigate early meiosis through analysis of Rec8 induction and function.

Licensing of gametogenesis, dependent on RNA binding protein DAZL, as a gateway to sexual differentiation of fetal germ cells.

Mammalian oocytes and spermatozoa derive from fetal cells shared by the sexes. These primordial germ cells (PGCs) migrate to the developing somatic gonad, giving rise to oocytes or spermatozoa. These opposing sexual fates are determined not by the PGCs' own sex chromosome constitution (XX or XY), but by the sexual identity of the fetal gonad that they enter. We asked whether PGCs undergo a developmental transition that enables them to respond to feminizing or masculinizing cues from fetal ovary or testis. We conducted in vivo genetic studies of DAZL, an RNA-binding protein expressed in both ovarian and testicular germ cells. We found that germ cells in C57BL/6 Dazl--deficient fetuses-whether XX or XY--migrate to the gonad but do not develop either male or female features. Instead, they remain in a sexually undifferentiated state similar to that of migrating PGCs. Thus, germ cells in C57BL/6 Dazl-deficient fetuses do not respond to sexual cues from ovary or testis, whereas the earlier processes of germ cell specification and migration are unaffected. We propose that PGCs of both XX and XY fetuses undergo licensing, an active developmental transition that enables the resultant gametogenesis-competent cells to respond to feminizing or masculinizing cues produced by the fetal ovary or testis and hence to embark on oogenesis or spermatogenesis. In C57BL/6 mice, Dazl is required for licensing. Licensing serves as a gateway from the embryonic processes shared between the sexes--germ cell specification and migration--to the sex-specific pathways of oogenesis and spermatogenesis.

Mir-290-295 deficiency in mice results in partially penetrant embryonic lethality and germ cell defects.

Mir-290 through mir-295 (mir-290-295) is a mammalian-specific microRNA (miRNA) cluster that, in mice, is expressed specifically in early embryos and embryonic germ cells. Here, we show that mir-290-295 plays important roles in embryonic development as indicated by the partially penetrant lethality of mutant embryos. In addition, we show that in surviving mir-290-295-deficient embryos, female but not male fertility is compromised. This impairment in fertility arises from a defect in migrating primordial germ cells and occurs equally in male and female mutant animals. Male mir-290-295(-/-) mice, due to the extended proliferative lifespan of their germ cells, are able to recover from this initial germ cell loss and are fertile. Female mir-290-295(-/-) mice are unable to recover and are sterile, due to premature ovarian failure.

Isolation of Mouse Germ Cells by FACS Using Hoechst 33342 and SYTO16 Double Staining.

In the adult mouse testis, germ cells of various developmental cell states co-exist. FACS isolation of cells stained with the DNA dye Hoechst 33342 has been used for many years to sub-divide these cells based on their DNA content. This approach provides an efficient way to obtain broad categories of male germ cells: pre-meiotic spermatogonia, meiotic spermatocytes and post-meiotic spermatids. The addition of a red filter for Hoechst staining enables further sub-division of spermatocytes depending on sub-stages of meiotic prophase. However, separation of different stage spermatids using Hoechst staining alone is not possible. We recently reported a methodology, combining Hoechst staining with a second DNA dye (SYTO16) that enables the further separation of these cells into three sub-populations: round, early elongating, and late elongating spermatids (Gill et al., Cytometry A 101:529-536, 2022). This method makes it possible to obtain rapidly and simply pure fractions of male germ cells from multiple developental stages from the same animal.

Investigating nicotinic acetylcholine receptor expression in neonicotinoid resistant Myzus persicae FRC.

The peach-potato aphid Myzus persicae is a pest of many commercial crops due to its polyphagous nature of feeding and has a well-documented history of acquiring resistance to insecticides. In 2009 a strain (M. persicae FRC) emerged in southern France with a point mutation (R81T) at the nicotinic acetylcholine receptor (nAChR), the target site for neonicotinoids such as imidacloprid. This point mutation was associated with the loss of the high affinity imidacloprid binding site (pM Kd), with the single remaining binding site (low nM Kd) highly overexpressed compared to laboratory controls (Bass et al., 2011 [1]). Here we report that after 2years of continuous selection in the glass house environment with neonicotinoids, the total level of IMD-sensitive nAChRs (low nM Kd) in M. persicae FRC is now comparable to laboratory controls (pM and low nM Kd). Interestingly, despite this large reduction in IMD-sensitive nAChRs, this was not associated with any significant alteration in NNIC-lethality. Additionally, sustained absence of neonicotinoid-selection did not alter nAChR protein levels. We suggest that alterations in nAChR protein expression level described in the original characterisation of the field-isolated M. persicae FRC is unlikely to have been a direct consequence of the R81T mutation. Rather, we speculate that nAChR expression in aphids is likely influenced by as yet unknown conditions in the natural field environment that are absent in the laboratory setting.

Comparing music- and food-evoked autobiographical memories in young and older adults: A diary study.

Previous research has found that music brings back more vivid and emotional autobiographical memories than various other retrieval cues. However, such studies have often been low in ecological validity and constrained by relatively limited cue selection and predominantly young adult samples. Here, we compared music to food as cues for autobiographical memories in everyday life in young and older adults. In two separate four-day periods, 39 younger (ages 18-34) and 39 older (ages 60-77) adults recorded their music- and food-evoked autobiographical memories in paper diaries. Across both age groups, music triggered more frequent autobiographical memories, a greater proportion of involuntary memories, and memories rated as more personally important in comparison to food cues. Age differences impacted music- and food-evoked memories similarly, with older adults consistently recalling older and less specific memories, which they rated as more positive, vivid, and rehearsed. However, young and older adults did not differ in the number or involuntary nature of their recorded memories. This work represents an important step in understanding the phenomenology of naturally occurring music-evoked autobiographical memories across adulthood and provides new insights into how and why music may be a more effective trigger for personally valued memories than certain other everyday cues.

Altered DNA methylation in estrogen-responsive repetitive sequences of spermatozoa of infertile men with shortened anogenital distance.

BACKGROUND: It has been suggested that antenatal exposure to environmental endocrine disruptors is responsible for adverse trends in male reproductive health, including male infertility, impaired semen quality, cryptorchidism and testicular cancer, a condition known as testicular dysgenesis syndrome. Anogenital distance (AGD) is an anthropomorphic measure of antenatal exposure to endocrine disruptors, with higher exposure levels leading to shortened AGD. We hypothesized that exposure to endocrine disruptors could lead to changes in DNA methylation during early embryonic development, which could then persist in the sperm of infertile men with shortened AGD. RESULTS: Using fluorescence activated cell sorting based on staining with either YO-PRO-1 (YOPRO) or chromomycin-3 (CMA3), we isolated four sperm fractions from eleven infertile men with short AGD and ten healthy semen donors. We examined DNA methylation in these sorted spermatozoa using reduced representation bisulfite sequencing. We found that fractions of spermatozoa from infertile men stained with CMA3 or YOPRO were more likely to contain transposable elements harboring an estrogen receptor response element (ERE). Abnormal sperm (as judged by high CMA3 or YOPRO staining) from infertile men shows substantial hypomethylation in estrogenic Alu sequences. Conversely, normal sperm fractions (as judged by low CMA3 or YO-PRO-1 staining) of either healthy donors or infertile patients were more likely to contain hypermethylated Alu sequences with ERE. CONCLUSIONS: Shortened AGD, as related to previous exposure to endocrine disruptors, and male infertility are accompanied by increased presence of hormonal response elements in the differentially methylated regulatory sequences of the genome of sperm fractions characterized by chromatin decondensation and apoptosis.

Lack of effect of P-glycoprotein inhibition on renal clearance of dicloxacillin in patients with cystic fibrosis.

STUDY OBJECTIVE: To determine whether upregulation of P-glycoprotein is responsible for the enhanced renal clearance of dicloxacillin in patients with cystic fibrosis. DESIGN: Single-center, prospective, open-label, randomized, three-part crossover pharmacokinetic study. SETTING: General clinical research center. SUBJECTS: Eleven patients with cystic fibrosis and 11 age-matched healthy volunteers. INTERVENTION: All subjects received a single oral dose of dicloxacillin 500 mg alone, dicloxacillin 500 mg plus probenecid (an organic anion transport inhibitor) 1 g, and dicloxacillin 500 mg plus cyclosporine (a P-glycoprotein inhibitor) 5 mg/kg; each treatment was separated by a washout period of 48 hours. A bolus dose of iothalamate meglumine 456 mg was administered on each study day as a marker of glomerular filtration. MEASUREMENTS AND MAIN RESULTS: Blood and urine samples were taken serially up to 6 hours after each dose. Pharmacokinetics of dicloxacillin and iothalamate were determined by using compartmental and noncompartmental methods. Quantitative polymerase chain reaction was performed on peripheral blood mononuclear cells to measure expression of multidrug resistance 1 (MDR1) messenger RNA (mRNA). Genotyping for ABCB1 was performed to determine the presence of single nucleotide polymorphisms (exons 21 and 26). In both healthy subjects and patients with cystic fibrosis, compared with dicloxacillin alone, coadministration with probenecid produced a significantly lower renal clearance of dicloxacillin, whereas coadministration with cyclosporine resulted in no significant change; renal clearance was not significantly different between the two study groups. No correlation was found between MDR1 mRNA expression and renal clearance of dicloxacillin. The renal excretion of dicloxacillin was significantly greater in subjects with the ABCB1 exon 26 TT polymorphism when compared with subjects with the CT genotype. CONCLUSION: We found no significant difference in the pharmacokinetics of dicloxacillin between patients with cystic fibrosis and healthy volunteers. Renal clearance of dicloxacillin was significantly reduced in the presence of probenecid but not with cyclosporine, suggesting that the rate-limiting step in tubular secretion of dicloxacillin is uptake mediated by the organic anion transporter, and not P-glycoprotein inhibition.

Pharmacokinetics of Ibuprofen in children with cystic fibrosis.

An exaggerated inflammatory response is responsible for the decline of lung function in patients with cystic fibrosis (CF). Ibuprofen is a potent anti-inflammatory agent that demonstrates inhibition of neutrophil activity in vitro at concentrations between 50 and 100 mg/L, whereas lower concentrations result in an increase in inflammatory mediators. Significant decline in the rate of deterioration of pulmonary function and increased nutritional status were observed in children with CF who were administered long-term high-dosage ibuprofen therapy. As with many other drugs, CF patients appear to exhibit altered pharmacokinetics of ibuprofen (reduced bioavailability, increased volume of distribution, and more rapid clearance) when compared with healthy controls. However, the absence of studies with intravenous ibuprofen as well as protein binding measurements in patients with CF currently limits the ability to compare the pharmacokinetics with those in other populations. Current studies indicate that there is high interpatient variability in ibuprofen pharmacokinetics among CF patients. Some of this variability can be explained by differences in ibuprofen formulation administered. Therapeutic drug monitoring of high-dosage ibuprofen therapy is recommended because of the biphasic response to inflammatory mediators demonstrated in vitro as well as the high interpatient variability in pharmacokinetics. Due to the differences in absorption characteristics between ibuprofen formulations, the timing of obtaining blood samples for pharmacokinetic analysis is critical. Maximum a posteriori Bayesian analysis has been shown to provide more accurate and precise estimates of the pharmacokinetic parameters of ibuprofen in children with CF, and may also be a useful tool to further investigate the relationship between measures of drug exposure and efficacy/toxicity outcomes.

A review of rivastigmine: a reversible cholinesterase inhibitor.

BACKGROUND: Rivastigmine tartrate is a reversible cholinesterase inhibitor indicated for the symptomatic treatment of mild to moderate dementia. It was approved by the US Food and Drug Administration for the treatment of Alzheimer's disease (AD) on April 21, 2000. OBJECTIVE: The purpose of this review was to summarize the background on dementia of the Alzheimer type and the pharmacokinetic properties, efficacy and tolerability profiles, clinical applications, adverse effects (AEs), drug interactions, and pharmacoeconomics of rivastigmine. METHODS: A literature search was conducted using MEDLINE (1995-2002), EMBASE Geriatrics and Gerontology (1995-2002), the National Institutes of Health Alzheimer's Disease Education and Resource Center Combined Health Information Database, and Google. Search terms included rivastigmine, Exelon, ENA 713, and ENA-713. The bibliographies of retrieved articles also were searched for relevant articles. RESULTS: In clinical trials, rivastigmine has improved or maintained cognitive function, global function (ie, activities of daily living [ADLs]), and behavior in patients with mild to moderate AD for up to 52 weeks. AEs are generally mild to moderate and primarily affect the gastrointestinal (GI) tract. Clinically significant drug interactions with rivastigmine have thus far not been reported. Treatment with rivastigmine for up to 2 years may reduce the cost of caring for patients with AD. Cost savings are minimal during the first year, particularly for those with mild disease, but increase during the second year of treatment. Cost savings occur earlier for those with moderate AD. Most savings are realized from a delay in the need for institutionalization. CONCLUSIONS: Rivastigmine has been shown to improve or maintain patients' performance in 3 major domains: cognitive function, global function (ADLs), and behavior. The efficacy and tolerability of rivastigmine have been proved by numerous clinical trials, with the most prominent AE being GI irritation.

Stability of advanced life support drugs in the field.

PURPOSE: The effects of wide temperature variations on the stability of atropine, epinephrine, and lidocaine stored under field conditions in advanced life support (ALS) paramedic units were evaluated. METHODS: Vehicles from various ALS paramedic units were selected throughout Los Angeles County, California, including desert, marine, and helicopter-based divisions. A temperature-recording device was placed in the compartment where drugs are stored and used to record and store temperature data at 15-minute intervals. Three autoinjector-style syringes of atropine, epinephrine, and lidocaine were taken from stock for each ALS unit and placed in each vehicle, while three control syringes were stored in the laboratory under controlled conditions. Six samples of each drug were withdrawn at time 0 and on days 5, 10, 15, 30, and 45. Samples were analyzed using high-performance liquid chromatography. Stock solutions, created using analytical grade atropine, epinephrine, and lidocaine, were used to construct 5-point standard curves to determine the drug concentration of each sample. RESULTS: Seven sites exceeded 104 degrees F (40 degrees C) for as little as 30 minutes and as long as 795 minutes. Ten of the sites achieved a mean kinetic temperature (MKT) above 77 degrees F (25 degrees C), with the highest MKT calculated being 84.1 degrees F (28.9 degrees C) over a 45-day period. There was no evidence of drug degradation at any site, at any temperature, or at any time point. CONCLUSION: Atropine, epinephrine, and lidocaine can be stored at temperatures of up to 84.1 degrees F (28.9 degrees C) for up to 45 days and tolerate temperature spikes of up to 125 degrees F (51.7 degrees C) for a cumulative time of 795 minutes (13.25 hours) without undergoing degradation.