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Alzheimer’s disease (AD) is the most common form of dementia that is initiated by extracellular plaque deposition and intraneuronal hyperphosphorylated tau aggregates. Reactive astrocytosis and microgliosis are secondary cellular responses to pathology that have gained increasing attention. Variants of the microglia receptor TREM2 increase AD risk and activation of “disease-associated microglia” (DAM) is dependent on TREM2 in mouse models of AD. To investigate global transcriptomic changes during AD pathology, we surveyed gene expression changes associated with AD pathology and TREM2 in Ab-driven 5XFAD mouse model and human AD by single nucleus RNA sequencing. We confirmed the presence of Trem2-dependent DAM and identified a novel Serpina3n+C4b+ reactive oligodendrocyte population in mice. Interestingly, remarkably different glial phenotypes were evident in human AD. Microglia signature was reminiscent of IRF8-driven reactive microglia in peripheral nerve injury. Oligodendrocyte signatures suggested impaired axonal myelination and metabolic adaptation to neuronal degeneration. Astrocyte profiles indicated weakened metabolic coordination with neurons. Notably, the reactive phenotype of microglia was less palpable in TREM2 R47H and R62H carriers than in non-carriers, demonstrating a TREM2 requirement in both mouse and human AD, despite the marked species-specific differences.

Background: Provider burnout remains a serious problem facing medical training programs and has been shown to affect more than half of internal medicine residents. In addition to broader efforts to revamp a health care system that contributes to this epidemic, exposure to the medical humanities offers potential to promote engagement, resilience, and restoration of meaning in residents' daily lives. Objective: We aim to create a reproducible, evidence-based workshop utilizing artful thinking routines to prepare trainees to combat burnout with reflection, perspective-taking, and community-building. Methods: A single, 4-hour workshop for senior internal medicine residents, centered on visual artistic analysis, was offered in June 2017 at the Philadelphia Museum of Art. Pre- and post-workshop burnout metrics and survey evaluation data were analyzed using a mixed-methods approach. Results: Workshop participation was offered to 29 internal medicine residents, of whom 17 (59%) participated. All survey respondents (n=13) rated the workshop as excellent and would recommend it to colleagues. Moderate decreases in the observed frequencies of both high emotional exhaustion scores (64.7% before the workshop to 55.5% following the workshop) and high depersonalization scores (70.6% before the workshop to 55.5% following the workshop) were observed. Conclusions: While results are preliminary in nature, the workshop was received favorably and demonstrated modest decreases in emotional exhaustion and depersonalization. We are encouraged to explore and repeat this workshop with modifications to identify its optimal position in the broader landscape of emerging wellness curricula.

Starshade concepts must be stowed within rocket fairings for launch and then deployed in space. The in-plane deployment accuracy must be on the order of hundreds of micrometers for sufficient starlight suppression to enable the detection and study of Earth-like exoplanets around nearby Sun-like stars. We describe tests conducted to demonstrate deployment repeatability of two key structural subsystems of the “furled” starshade architecture—the petal and the inner disk. Together, the petals and the inner disk create the in-plane shape of a starshade. Test articles to represent the petal and inner disk subsystems were constructed at relevant scales for a 26-m-diameter starshade. These test articles were subjected to stowage-and-deployment cycles and their shapes were measured. The measured performance—tens of parts per million of petal strain after deployment, and hundreds of micrometers of inner disk deployment accuracy—was found to be within required allocations.

Inhibició de l’ATR; Resposta de danys a l’ADN; Càncer de pròstata Inhibición de ATR; Respuesta al daño del ADN; Cancer de prostata ATR inhibition; DNA damage response; Prostate cancer Background Deleterious ATM alterations are found in metastatic prostate cancer (PC); PARP inhibition has antitumour activity against this subset, but only some ATM loss PCs respond. Objective To characterise ATM-deficient lethal PC and to study synthetic lethal therapeutic strategies for this subset. Design, setting, and participants We studied advanced PC biopsies using validated immunohistochemical (IHC) and next-generation sequencing (NGS) assays. In vitro cell line models modified using CRISPR-Cas9 to impair ATM function were generated and used in drug-sensitivity and functional assays, with validation in a patient-derived model. Outcome measurements and statistical analysis ATM expression by IHC was correlated with clinical outcome using Kaplan-Meier curves and log-rank test; sensitivity to different drug combinations was assessed in the preclinical models. Results and limitations Overall, we detected ATM IHC loss in 68/631 (11%) PC patients in at least one biopsy, with synchronous and metachronous intrapatient heterogeneity; 46/71 (65%) biopsies with ATM loss had ATM mutations or deletions by NGS. ATM IHC loss was not associated with worse outcome from advanced disease, but ATM loss was associated with increased genomic instability (NtAI:number of subchromosomal regions with allelic imbalance extending to the telomere, p = 0.005; large-scale transitions, p = 0.05). In vitro, ATM loss PC models were sensitive to ATR inhibition, but had variable sensitivity to PARP inhibition; superior antitumour activity was seen with combined PARP and ATR inhibition in these models. Conclusions ATM loss in PC is not always detected by targeted NGS, associates with genomic instability, and is most sensitive to combined ATR and PARP inhibition. We gratefully acknowledge research funding for this work from Cancer Research UK, Prostate Cancer UK, the Movember Foundation through the London Movember Centre of Excellence ( CEO13_2-002 ), the Prostate Cancer Foundation (including Young Investigator Awards to Joaquin Mateo, Pasquale Rescigno, and Adam Sharp), Stand Up To Cancer, and the UK Department of Health through an Experimental Cancer Medicine Centre grant. Professor Johann de Bono is a National Institute for Health Research (NIHR) Senior Investigator; research at the Royal Marsden Hospital is supported by a Biomedical Research Centre grant. Part of this work was also funded by a Deparment of Defense CDMRP Impact Award (W81XWH-18-1-0756) to Joaquin Mateo and by Instituto de Salud Carlos III through Grant FI19/00280 to Sara Arce-Gallego, Grant CP19/00170 to Nicolás Herranz, and Grant PI18/01384 to Joaquin Mateo. The authors affiliated to VHIO acknowledge the “la Caixa” Foundation ( ID 100010434 ) for funding under agreement LCF/PR/PR17/51120011 and funding from Fundacion FERO and Moventia . This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement 837900 . The funding organisations had no role in the design, conduction or data analysis of this project, neither in the manuscript preparation.

Although PARP inhibitors (PARPi) target homologous recombination defective tumours, drug resistance frequently emerges, often via poorly understood mechanisms. Here, using genome-wide and high-density CRISPR-Cas9 “tag-mutate-enrich” mutagenesis screens, we identify close to full-length mutant forms of PARP1 that cause in vitro and in vivo PARPi resistance. Mutations both within and outside of the PARP1 DNA-binding zinc-finger domains cause PARPi resistance and alter PARP1 trapping, as does a PARP1 mutation found in a clinical case of PARPi resistance. This reinforces the importance of trapped PARP1 as a cytotoxic DNA lesion and suggests that PARP1 intramolecular interactions might influence PARPi-mediated cytotoxicity. PARP1 mutations are also tolerated in cells with a pathogenic BRCA1 mutation where they result in distinct sensitivities to chemotherapeutic drugs compared to other mechanisms of PARPi resistance (BRCA1 reversion, 53BP1, REV7 (MAD2L2) mutation), suggesting that the underlying mechanism of PARPi resistance that emerges could influence the success of subsequent therapies., The mechanisms of PARP inhibitor (PARPi) resistance are poorly understood. Here the authors employ a CRISPR mutagenesis approach to identify PARP1 mutants causing PARPi resistance and find that PARP1 mutations are tolerated in BRCA1 mutated cells, suggesting alternative resistance mechanisms.

Summary Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) latently infects approximately one-fourth of the world’s population. The immune mechanisms that govern progression from latent (LTBI) to active pulmonary TB (PTB) remain poorly defined. Experimentally Mtb-infected non-human primates (NHP) mirror the disease observed in humans and recapitulate both PTB and LTBI. We characterized the lung immune landscape in NHPs with LTBI and PTB using high-throughput technologies. Three defining features of PTB in macaque lungs include the influx of plasmacytoid dendritic cells (pDCs), an Interferon (IFN)-responsive macrophage population, and activated T cell responses. In contrast, a CD27+ Natural killer (NK) cell subset accumulated in the lungs of LTBI macaques. This NK cell population was also detected in the circulation of LTBI individuals. This comprehensive analysis of the lung immune landscape will improve the understanding of TB immunopathogenesis, providing potential targets for therapies and vaccines for TB control.

Summary Systematic understanding of immune aging on a whole-body scale is currently lacking. We characterized age-associated alterations in immune cells across multiple mouse organs using single-cell RNA and antigen receptor sequencing and flow cytometry-based validation. We defined organ-specific and common immune alterations and identified a subpopulation of age-associated granzyme K (GZMK)-expressing CD8+ T (Taa) cells that are distinct from T effector memory (Tem) cells. Taa cells were highly clonal, had specific epigenetic and transcriptional signatures, developed in response to an aged host environment, and expressed markers of exhaustion and tissue homing. Activated Taa cells were the primary source of GZMK, which enhanced inflammatory functions of non-immune cells. In humans, proportions of the circulating GZMK+CD8+ T cell population that shares transcriptional and epigenetic signatures with mouse Taa cells increased during healthy aging. These results identify GZMK+ Taa cells as a potential target to address age-associated dysfunctions of the immune system.

Chikungunya virus (CHIKV), an emerging alphavirus, has infected millions of people. However, the factors modulating disease outcome remain poorly understood. Here, we show in germ-free mice or in oral antibiotic-treated conventionally housed mice with depleted intestinal microbiomes that greater CHIKV infection and spread occurs within 1 day of virus inoculation. Alteration of the microbiome alters TLR7-MyD88 signaling in plasmacytoid dendritic cells (pDCs) and blunts systemic production of type I interferon (IFN). Consequently, circulating monocytes express fewer IFN-stimulated genes and become permissive for CHIKV infection. Reconstitution with a single bacterial species, Clostridium scindens, or its derived metabolite, the secondary bile acid deoxycholic acid, can restore pDC- and MyD88-dependent type I IFN responses to restrict systemic CHIKV infection and transmission back to vector mosquitoes. Thus, symbiotic intestinal bacteria modulate antiviral immunity and levels of circulating alphaviruses within hours of infection through a bile acid-pDC-IFN signaling axis, which affects viremia, dissemination, and potentially transmission.

In 1972, nineteen-year-old Romas Kalanta set himself on fire in Soviet Lithuania leaving a note saying, “only the system is responsible for my death.” Kalanta’s “dead body” was employed by the Lithuanian popular front Sąjūdis in 1989 as a powerful symbol of the nation’s oppression and suffering under Soviet rule. Just over a decade later, however, Kalanta was no longer useful as a national symbol, primarily due to the manner of his death. By 2002, Kalanta’s act of suicide discouraged rather than encouraged identification with him, and the issue of mental illness continued to taint his potential as a national hero. Instead, emphasis shifted to the street demonstrations by young people that followed his funeral. In this case, the live bodies of young people engaged in popular protest became a more powerful symbol than Kalanta, as Lithuanians claimed the moral value of civil resistance and a “Sixties” youth culture with the West. В 1972 г. девятнадцатилетний Ромас Каланта, житель советской Лит-вы, совершил самосожжение. В оставленной им записке говорилось: “Только систему следует винить в моей смерти”. В 1989 г. литовский народный фронт “Саюдис” превратил “мертвое тело” Каланты в важ-ный символ национального притеснения и страданий при советской власти. Однако уже через десять лет фигура Каланты потеряла значи-мость национального символа. К 2002 г. факт самоубийства осложнял идентификацию с ним, а подозрения в психическом расстройстве Ка-ланты бросали пятно на его потенциал национального героя. Теперь акцент делался на уличных демонстрациях, организованных молодыми людьми после похорон Каланты. По мере того, как литовцы осозна-вали моральную ценность гражданского сопротивления и начинали ассоциировать молодежную культуру 1960-х с “Западом”, живые тела молодых людей, участвовавших в протестах, приобретали большую символическую значимость, чем мертвое тело Каланты.

SF-1 is a master regulator of steroidogenesis whose expression is critical for normal adrenal and gonadal organogenesis. Strict maintenance of SF-1 levels is essential, and mutations causing under- or overexpression result in congenital adrenal and gonadal defects or hyperplasia, respectively. Data from transgenic mouse models points to a network of transcription factors responsible for stringent regulation of Sf-1 expression during development, which bind to intronic enhancer elements in addition to the basal promoter to specifically modulate transcription in each Sf-1-expressing tissue. Furthermore, analysis of the role of SF-1 in adrenal tumourigenesis implies that improper developmental regulation of Sf-1 expression may have postnatal consequences separate from the well-documented developmental defects.

Rationale: Vascular smooth muscle cell (VSMC) proliferation causes intimal thickening in atherosclerosis and restenosis. Previously, we demonstrated that Wnt/β-catenin signaling upregulates VSMC proliferation in vitro. Objective: We examined this pathway in vivo and investigated the involvement of specific Wnt proteins in VSMC proliferation. Methods and Results: Left carotid arteries of TOPgal (β-catenin signaling reporter) transgenic mice were ligated to induce intimal thickening. β-Catenin signaling was induced in the media and intima at 3 and 28 days after ligation, respectively, and was associated with VSMC proliferation and cyclin D1 expression. In vitro, a Wnt agonist promoted mouse VSMC proliferation, whereas Wnt inhibitory factor (WIF)-1 retarded platelet-derived growth factor-BB (PDGF-BB)–induced VSMC proliferation. Microarray analysis and quantitative PCR detected a significant induction of Wnt2 and Wnt4 mRNA in PDGF-BB-treated (proliferating) VSMCs compared to quiescent VSMCs. Western blotting revealed this increase was only translated into protein for Wnt4. Specific silencing RNA knockdown of Wnt4, but not Wnt2, significantly reduced VSMC proliferation. Recombinant Wnt4, but not Wnt2, significantly increased VSMC proliferation by ≈2-fold and silencing RNA knockdown revealed this is via Frizzled 1. Immunohistochemistry showed that increased Wnt4 protein correlated with VSMC proliferation and cyclin D1 expression ( P P +/− compared to Wnt4 +/+ mice. Conclusions: This study demonstrates that Wnt/β-catenin signaling occurs in proliferating VSMCs during intimal thickening and indicates that this is a result of Wnt4 upregulation.

The mammalian prostate arises from the urogenital sinus under the influence of testicular androgens. Few factors have been identified to be important in the early stages of prostate development. Here we review the role of the transcription factor Sox9 in prostate development. Sox9 is a member of the Sox gene family that plays an important role during embryogenesis in the cellular differentiation of various tissues, including testicular Sertoli cells, neural crest cells and chondrocytes. This gene is expressed in the epithelia of all mouse prostatic lobes from the initial stages of their development. Mice with a prostate specific deletion of Sox9 showed a lack of ventral prostate development and abnormal anterior prostate differentiation. In depth analysis of these mutant animals suggested that Sox9 is required for the early differentiation of the prostate bud epithelia, consistent with the function of this factor in other developmental processes. These studies also revealed different phases of prostate bud development. These phases were characterized by being dependent on different molecular pathways and having lobe specific properties. Future studies on the identification of pathways regulated by Sox9 will provide insight into the molecular networks required for prostate epithelia differentiation.

Genes previously implicated in mammalian sexual development have either a male- or female-specific role. The signaling molecule WNT4 has been shown to be important in female sexual development. Lack of Wnt4 gives rise to masculinization of the XX gonad and we showed previously that the role of WNT4 was to inhibit endothelial and steroidogenic cell migration into the developing ovary. Here we show that Wnt4 also has a function in the male gonad. We find that Sertoli cell differentiation is compromised in Wnt4 mutant testes and that this defect occurs downstream of the testis-determining gene Sry but upstream of Sox9 and Dhh, two early Sertoli cell markers. Genetic analysis shows that this phenotype is primarily due to the action of WNT4 within the early genital ridge. Analysis of different markers identifies the most striking difference in the genital ridge at early stages of its development between wild-type and Wnt4 mutant embryos to be a significant increase of steroidogenic cells in the Wnt4 −/− gonad. These results identify WNT4 as a new factor involved in the mammalian testis determination pathway and show that genes can have a specific but distinct role in both male and female gonad development.

How the physical differences between the sexes develop is an intriguing process. In mammals, the first important stage in sex determination occurs during fetal life, when the male-determining Y chromosome expresses a gene called sex determining region Y ( SRY ), which acts on the bipotential gonad to initiate testis (instead of ovary) formation ( 1 ). Landmark experiments by Jost in the 1950s showed that the development of male-specific structures is established by factors produced from the emerging testis, namely androgens and anti-Mullerian hormone (AMH) ( 2 ). This suggested that the formation of male characteristics is an active process, whereas female-specific differentiation is passive. On page [717][1] of this issue, Zhao et al. ( 3 ) show that female development is also an active process. [1]: /lookup/doi/10.1126/science.aai9136

DAX1, which encodes an unusual member of the nuclear hormone-receptor superfamily, is a gene that may be responsible for a sex-reversal syndrome in humans, referred to as dosage-sensitive sex reversal, in which XY individuals carrying duplications of Xp21, part of the small arm of the X chromosome, develop as females. XY mice carrying extra copies of mouse Dax1 as a transgene show delayed testis development when the gene is expressed at high levels, but do not normally show sex reversal. Complete sex reversal occurs, however, when the transgene is tested against weak alleles of the sex-determining Y-chromosome gene Sry. These results show that DAX1 is largely, if not solely, responsible for dosage-sensitive sex reversal and provide a model for early events in mammalian sex determination, when precise levels and timing of gene expression are critical.

Mammalian sex determination occurs in the gonad of the developing embryo. This process is dependent on the Y-chromosome-encoded Sry gene that acts in the somatic cells of the genital ridge. The transient nature of Sry gene expression suggests that it acts as a switch from one cell fate to another. One of the roles of Sry is to initiate the differentiation of Sertoli cells, which are the first cell type of the testis to be formed. Two genes are thought to be important in Sertoli cell differentiation and function, Sox9, an Sry-related gene, and SF-1, a nuclear hormone receptor. Sox9 is expressed in Sertoli cells throughout development of the mouse embryo, and inactivating mutations in this gene in humans give rise to XY females. SF-1 is also expressed in Sertoli cells and is thought to activate the AMH gene--an early marker of these cells. DAX-1, an X-linked member of the nuclear hormone superfamily, is a candidate for a human condition in which duplication of regions of the X chromosome results in XY females. Expression of this gene during mouse development is associated with ovary development and is down-regulated in the differentiating testis. Mutations in DAX-1 in humans have shown that this gene is not necessary for testis development. The properties of the DAX-1 gene suggest that it is important in ovary determination and might therefore be antagonistic to the action of the Sry gene.

In many species, females have two X chromosomes whereas males have only one. To compensate for this difference, genes on the female X chromosomes are switched off. But how does an organism count how many chromosomes it has? A study in the nematode worm has just identified a protein called SEX-1, which represses a gene,xol-1, that is involved in both dosage compensation and sex determination.

Duplications of a chromosome Xp21 locus DSS (dosage sensitive sex reversal) are associated with male to female sex reversal. An unusual member of the nuclear hormone receptor superfamily, DAX1, maps to the DSS critical region and is responsible for X-linked adrenal hypoplasia congenita. Here we describe the isolation of the mouse Dax1 gene and its pattern of expression during development. Expression was detected in the first stages of gonadal and adrenal differentiation and in the developing hypothalamus. Moreover, Dax1 expression is down-regulated coincident with overt differentiation in the testis, but persists in the developing ovary. Comparison of the predicted protein products of the human and mouse genes show that specific domains are evolving rapidly. Our results suggest a basis for adrenal insufficiency and hypogonadotropic hypogonadism in males affected by adrenal hypoplasia congenita and are consistent with a role for DAX1 in gonadal sex determination.

Retroviruses can capture cellular sequences and express them as oncogenes. Capture has been proposed to be a consequence of the inefficiency of polyadenylation of the viral genome that allows the packaging of cellular sequences flanking the integrated provirus in virions; after transfer into virions, these sequences could be incorporated into the viral genome by illegitimate recombination during reverse transcription. As a test for this hypothesis, a tissue culture system was developed that mimics the transduction process and allows the analysis and quantitation of capture events in a single step. In this model, transduction of sequences adjacent to a provirus depends on the formation of readthrough transcripts and their transmission in virions and leads to various recombinant structures whose formation is independent of sequence similarity at the crossover site. Thus, all events in the transduction process can be attributed to the action of reverse transcriptase on readthrough transcripts without involving deletions of cellular DNA.

The mammalian prostate arises from the urogenital sinus and few factors have been identified to be important in the early stages of prostate development. In this study we show that the transcription factor Sox9 is expressed in the epithelia of all mouse prostatic lobes from the initial stages of their development. We used a conditional approach with mice expressing Cre recombinase under the control of Nkx3.1 regulatory sequences to delete Sox9 from the developing prostate. Mice with a prostate specific deletion of Sox9 showed a lack of ventral prostate development and abnormal anterior prostate differentiation. Analysis of these mutant animals revealed an early loss of expression of genes specific to the prostate epithelia such as Nkx3.1 and Shh and a marked reduction in proliferation in the ventral prostate but not in other lobes. Fgf signalling, through the MAPK pathway, has been shown to be important in prostate development and a lobe specific phenotype was reported for a prostate specific Fgfr2 mutant mouse model. Here we show that the levels of Fgfr2 and Sprouty2, a downstream target of Fgf signalling, were severely reduced in the ventral prostate of Sox9 mutant animals but not in other lobes. Prostate organ culture studies with a Mek inhibitor, U0126, and a Fgf receptor inhibitor, SU5402, indicate that the timing of expression of Cre in the mutant animals could account for the lobe specific phenotype in the Sox9 and Fgfr2 mutants. These studies imply that Sox9 is required for the early differentiation of the prostate bud epithelia.

Steroidogenic cells of the adrenal and gonad are thought to be derived from a common primordium that divides into separate tissues during embryogenesis. In this paper, we show that cells with mixed adrenal and Leydig cell properties are found dispersed in the insterstitium of the embryonic and adult mouse testis. They express the adrenal markers Cyp11b1 and Cyp21 and respond to ACTH. Consistent with these properties, we show that the embryonic testis produces the adrenal steroid corticosterone. These cells also express Cyp17 and respond to hCG stimulation but do not express the Leydig specific marker Insl3 showing that they are a population of steroidogenic cells distinct from Leydig cells. Based on their properties, we refer to these cells as adrenal-like cells of the testis and propose that they are the mouse equivalent of the precursors of human adrenal rests, tumors found primarily in male patients with congenital adrenal hyperplasia. Organ culture studies show that ACTH-responsive cells are present at the gonad/mesonephros border and seem to migrate into the XY but not the XX gonad during development. Consistent with this, using transgenic Cyp11a1 reporter mice, we definitively show that steroidogenic cells can migrate from the mesonephros into the XY gonad. We also show that the region between the mesonephros and the gonad harbors steroidogenic cell precursors that are repressed by the presence of the mesonephros. We propose that this region is the source of the adrenal-like cells that migrate into the testis as it develops and are activated when Leydig cells differentiate. These studies reveal the complex nature of steroidogenic cell differentiation during urogenital development.