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Funder: Donation from the Estate of Dr Hans Salvisberg, Funder: Nomis Foundation; Id: http://dx.doi.org/10.13039/501100008483, A defining characteristic of mammalian prions is their capacity for self-sustained propagation. Theoretical considerations and experimental evidence suggest that prion propagation is modulated by cell-autonomous and non-autonomous modifiers. Using a novel quantitative phospholipase protection assay (QUIPPER) for high-throughput prion measurements, we performed an arrayed genome-wide RNA interference (RNAi) screen aimed at detecting cellular host-factors that can modify prion propagation. We exposed prion-infected cells in high-density microplates to 35,364 ternary pools of 52,746 siRNAs targeting 17,582 genes representing the majority of the mouse protein-coding transcriptome. We identified 1,191 modulators of prion propagation. While 1,151 modified the expression of both the pathological prion protein, PrPSc , and its cellular counterpart, PrPC , 40 genes selectively affected PrPSc . Of the latter 40 genes, 20 augmented prion production when suppressed. A prominent limiter of prion propagation was the heterogeneous nuclear ribonucleoprotein Hnrnpk. Psammaplysene A (PSA), which binds Hnrnpk, reduced prion levels in cultured cells and protected them from cytotoxicity. PSA also reduced prion levels in infected cerebellar organotypic slices and alleviated locomotor deficits in prion-infected Drosophila melanogaster expressing ovine PrPC . Hence, genome-wide QUIPPER-based perturbations can discover actionable cellular pathways involved in prion propagation. Further, the unexpected identification of a prion-controlling ribonucleoprotein suggests a role for RNA in the generation of infectious prions.

Species conservation requires a clear understanding of habitat availability and subsequent use of those habitats. In cases where species declines have occurred and gone undetected by conservation managers, habitat alteration, fragmentation, and loss are often the largest contributors. River fragmentation often results in altered flow regimes, subsequently impacting the availability of riverine habitats. Blue sucker (Cycleptus elongatus) is associated with riffle and run habitat, which is especially impacted when river flows are altered. The goal of this research was to identify the extent of blue sucker habitat and mobility of the species in the Colorado River, Texas. To understand habitat selection and use, blue suckers (N = 49) were surgically implanted with telemetry tags. During 2015–2017, thirty-eight attempts were completed to relocate individuals. Optimized hotspot analysis identified three river reaches critical for blue suckers that accounted for 20% of the study area. Blue suckers used these locations year-round including during spawning. Habitats used by blue sucker were composed of gravel, cobble, boulder, and bedrock typically in riffle and run habitat. Mobility, as measured by home range size, increased as riffle density decreased. Larger home ranges were presumably necessary to find habitat to complete aspects of their life history. Results of this study suggest suitable habitats are limited throughout the fragmented riverscape. Conservation action in the form of habitat construction or increased stream connectivity through barrier mitigation could have positive impacts on the future of blue suckers in the lower Colorado River, Texas.

Myelin, multilayered lipid-rich membrane extensions formed by oligodendrocytes around neuronal axons, is essential for fast and efficient action potential propagation in the central nervous system. Initially thought to be a static and immutable process, myelination is now appreciated to be a dynamic process capable of responding to and modulating neuronal function throughout life. While the importance of this type of plasticity, called adaptive myelination, is now well accepted, we are only beginning to understand the underlying cellular and molecular mechanisms by which neurons communicate experience-driven circuit activation to oligodendroglia and precisely how changes in oligodendrocytes and their myelin refine neuronal function. Here, we review recent findings addressing this reciprocal relationship in which neurons alter oligodendroglial form and oligodendrocytes conversely modulate neuronal function.

Proper timing of gene expression is central to lymphocyte development and differentiation. Lymphocytes often delay gene activation for hours to days after the onset of signaling components, which act on the order of seconds to minutes. Such delays play a prominent role during the intricate choreography of developmental events and during the execution of an effector response. Though a number of mechanisms are sufficient to explain timing at short timescales, it is not known how timing delays are implemented over long timescales that may span several cell generations. Based on the literature, we propose that a class of cis-regulatory elements, termed "timing enhancers," may explain how timing delays are controlled over these long timescales. By considering chromatin as a kinetic barrier to state switching, the timing enhancer model explains experimentally observed dynamics of gene expression where other models fall short. In this review, we elaborate on features of the timing enhancer model and discuss the evidence for its generality throughout development and differentiation. We then discuss potential molecular mechanisms underlying timing enhancer function. Finally, we explore recent evidence drawing connections between timing enhancers and genetic risk for immunopathology. We argue that the timing enhancer model is a useful framework for understanding how cis-regulatory elements control the central dimension of timing in lymphocyte biology.

The SWI/SNF chromatin remodeling complexes control accessibility of chromatin to transcriptional and co-regulatory machineries. Chromatin remodeling plays important roles in normal physiology and diseases, particularly cancer. The ARID1A-containing SWI/SNF complex is commonly mutated and thought to be a key tumor suppressor in hepatocellular carcinoma (HCC), but its regulation in response to oncogenic signals remains poorly understood. mTOR is a conserved central controller of cell growth and an oncogenic driver of HCC. Remarkably, cancer mutations in mTOR and SWI/SNF complex are mutually exclusive in human HCC tumors, suggesting that they share a common oncogenic function. Here we report that mTOR complex 1 (mTORC1) interact with ARID1A and regulates ubiquitination and proteasomal degradation of ARID1A protein. The mTORC1-ARID1A axis promoted oncogenic chromatin remodeling and YAP-dependent transcription, thereby enhancing liver cancer cell growth in vitro and tumor development in vivo. Conversely, excessive ARID1A expression counteracted AKT-driven liver tumorigenesis in vivo. Moreover, dysregulation of this axis conferred resistance to mTOR-targeted therapies. These findings demonstrate that the ARID1A-SWI/SNF complex is a regulatory target for oncogenic mTOR signaling, which is important for mTORC1-driven hepatocarcinogenesis with implications for therapeutic interventions in HCC.

Human cytomegalovirus (HCMV) induces long-lasting T-cell immune responses that control but do not clear infection. Typical responses involve private T-cell clones, expressing T-cell antigen receptors (TCRs) unique to a person, and public T-cell clones with identical TCRs active in different people. Here, we report the development of a pretherapeutic immunostimulation modality against HCMV for human T cells, CD3 copotentiation, and the clonal analysis of its effects in recall assays at single-cell resolution. CD3 copotentiation of human T cells required identification of an intrinsically inert anti-CD3 Fab fragment that conditionally augmented signaling only when TCR was coengaged with antigen. When applied in recall assays, CD3 copotentiation enhanced the expansion of both public and private T-cell clones responding to autologous HLA-A2(+) antigen-presenting cells and immunodominant NLVPMVATV (NLV) peptide from HCMV pp65 protein. Interestingly, public vs private TCR expression was associated with distinct clonal expansion signatures in response to recall stimulus. This implied that besides possible differences in their generation and selection in an immune response, public and private T cells may respond differently to pharmacoimmunomodulation. Furthermore, a third clonal expansion profile was observed upon CD3 copotentiation of T-cell clones from HLA-A2(−) donors and 1 HLA-A2(+) presumed-uninfected donor, where NLV was of low intrinsic potency. We conclude that human T-cell copotentiation can increase the expansion of different classes of T-cell clones responding to recall antigens of different strengths, and this may be exploitable for therapeutic development against chronic, persistent infections such as HCMV.

Alteration of flow regimes by anthropogenic activities is one of the primary environmental problems in riverine systems. Understanding how hydrologic conditions can affect ontogenetic habitat shifts of imperiled fishes is important in order to develop conservation and management strategies for each life-history stage. We examined relationships between the abundance of young-of-the-year (YOY) Rio Grande Blue Sucker and various abiotic variables in the Trans-Pecos region of the Rio Grande in Texas, USA. We used open N-mixture modeling to better understand the factors affecting ontogenetic habitat shifts of the imperiled aridland river fish. In addition, we examined differences in Rio Grande Blue Sucker total length among three mesohabitat types (pool, riffle, and run). The results of open N-mixture modeling suggested that as pool area increased, the abundance of YOY Rio Grande Blue Sucker increased. Total length of YOY Rio Grande Blue Sucker also significantly differed among the three mesohabitat types. The total lengths of YOY Rio Grande Blue Sucker in pool habitats were lower than in other mesohabitats, suggesting that YOY Rio Grande Blue Sucker undergo ontogenetic habitat shifts into greater current velocity habitats as they grow. The habitat associations we documented support the growing body of research emphasizing the importance of maintaining sufficient and appropriately timed flows to avoid prolonged low flows that limit habitat availability for native fish species during sensitive life stages in the Rio Grande and other aridland rivers.

Theiler's murine encephalomyelitis virus (TMEV) infection of the CNS is cleared in C57BL/6 mice by a CD8 T cell response restricted by the MHC class I molecule H-2Db. The identity and function of the APC(s) involved in the priming of this T cell response is (are) poorly defined. To address this gap in knowledge, we developed an H-2Db LoxP-transgenic mouse system using otherwise MHC class I–deficient C57BL/6 mice, thereby conditionally ablating MHC class I–restricted Ag presentation in targeted APC subpopulations. We observed that CD11c+ APCs are critical for early priming of CD8 T cells against the immunodominant TMEV peptide VP2121-130. Loss of H-2Db on CD11c+ APCs mitigates the CD8 T cell response, preventing early viral clearance and immunopathology associated with CD8 T cell activity in the CNS. In contrast, animals with H-2Db–deficient LysM+ APCs retained early priming of Db:VP2121-130 epitope–specific CD8 T cells, although a modest reduction in immune cell entry into the CNS was observed. This work establishes a model enabling the critical dissection of H-2Db–restricted Ag presentation to CD8 T cells, revealing cell-specific and temporal features involved in the generation of CD8 T cell responses. Employing this novel system, we establish CD11c+ cells as pivotal to the establishment of acute antiviral CD8 T cell responses against the TMEV immunodominant epitope VP2121-130, with functional implications both for T cell–mediated viral control and immunopathology.

Background In the last decade, tuberculosis (TB) incidence among Inuit in the Canadian Arctic has been rising. Our aim was to better understand the transmission dynamics of TB in this remote region of Canada using whole-genome sequencing. Methods Isolates from patients who had culture-positive pulmonary TB in Iqaluit, Nunavut, between 2009 and 2015 underwent whole-genome sequencing (WGS). The number of transmission events between cases within clusters was calculated using a threshold of a ≤3 single nucleotide polymorphism (SNP) difference between isolates and then combined with detailed epidemiological data using a reproducible novel algorithm. Social network analysis of epidemiological data was used to support the WGS data analysis. Results During the study period, 140 Mycobacterium tuberculosis isolates from 135 cases were sequenced. Four clusters were identified, all from Euro-American lineage. One cluster represented 62% of all cases that were sequenced over the entire study period. In this cluster, 2 large chains of transmission were associated with 3 superspreading events in a homeless shelter. One of the superspreading events was linked to a nonsanctioned gambling house that resulted in further transmission. Shelter to nonshelter transmission was also confirmed. An algorithm developed for the determination of transmission events demonstrated very good reproducibility (κ score .98, 95% confidence interval, .97–1.0). Conclusions Our study suggests that socioeconomic factors, namely residing in a homeless shelter and spending time in a gambling house, combined with the superspreading event effect may have been significant factors explaining the rise in cases in this predominantly Inuit Arctic community.

Primary cutaneous γδ T cell lymphomas (PCGDTLs) represent a heterogeneous group of uncommon but aggressive cancers. Herein, we perform genome-wide DNA, RNA, and T cell receptor (TCR) sequencing on 29 cutaneous γδ lymphomas. We find that PCGDTLs are not uniformly derived from Vδ2 cells. Instead, the cell-of-origin depends on the tissue compartment from which the lymphomas are derived. Lymphomas arising from the outer layer of skin are derived from Vδ1 cells, the predominant γδ cell in the epidermis and dermis. In contrast, panniculitic lymphomas arise from Vδ2 cells, the predominant γδ T cell in the fat. We also show that TCR chain usage is non-random, suggesting common antigens for Vδ1 and Vδ2 lymphomas respectively. In addition, Vδ1 and Vδ2 PCGDTLs harbor similar genomic landscapes with potentially targetable oncogenic mutations in the JAK/STAT, MAPK, MYC, and chromatin modification pathways. Collectively, these findings suggest a paradigm for classifying, staging, and treating these diseases., Cutaneous gamma-delta T cell lymphomas are rare but aggressive cancers. Here, by DNA, RNA and T cell receptor sequencing, the authors elucidate the molecular ontogeny by revising the cell of origin and identifying potentially targetable mutations.

Breast tumors develop under environmental pressures with phenotypically variant cells generated by mutation and epigenetic changes providing the substrate for clonal selection. Chromosomal mutations are a feature of spontaneous breast tumors in the BALB-neuT mouse model. However, there is little evidence that specific changes in chromosomal structure or ploidy confer selective advantage in these spontaneous tumors. The prominent exception is a consistent loss of chromosome 4, a little understood feature common to several mouse tumors. We previously measured heterozygosity in spontaneous breast tumors from FVB X BALB-neuT F1 mice using Illumina’s Golden Gate assay. In addition to the expected gain and loss of heterozygosity (LOH) evident throughout the genomes of a cohort of spontaneous tumors, the assay also detected wide-spread stochastic pseudo-“LOH” at 600 discrete loci spread throughout the genome in unique patterns among the tumors. Several of these positions were examined by sequence analysis revealing no deviations from the WT sequences, suggesting the detected “LOH” may have been generated by epigenetic modification of DNA which altered sequence detection. While, epigenetically modified DNA templates recapitulated the observed “LOH” signals, no canonical CpG motifs were present in the majority of the 600 loci probed, suggesting that an unusual DNA modification could be responsible for the unexpected wide-spread stochastic structural changes in the breast tumor DNA. We next assessed whether these putative epigenetic changes in DNA structure might impact gene regulation, and have reported that the stochastic pattern observed as “LOH” in DNA was recapitulated in the transcriptomes in unique patterns among the tumors. A direct correlation between the number of “LOH” variants and the down regulation of hundreds of non-polymorphic genes in the transcriptome also was noted. Furthermore, pathway analyses of the genes exhibiting changes in allelic ratios in these tumors revealed significant enrichments within gene networks regulating tissue homeostasis and antigen presentation, providing strong evidence that the perturbations in gene expression translated into selectable tumor cell phenotypes. We now have extended this study to examine the relationship between the magnitude of change in the expression of genes mapping within the pathways regulating tissue homeostasis (Molecular Mechanisms of Cancer). A remarkable feature of the flagged genes is that the magnitude of change in gene expression was not great in each case, yet the biological consequences were strongly reflected in the evolutionary history of the tumors. Importantly, the polymorphisms marking the parental alleles are mostly silent, not altering the structure of the encoded products. Therefore magnitude and timing of gene expression are the likely determinants of phenotypic variation. Each tumor contained several outliers within the pathways regulating tissue homeostasis, suggesting that the integration of multiple small perturbations in the expression of genes comprising functional networks could influence the biology properties of the tumor cells. Overall expression of the loci marked by allelic outliers was significantly below the average expression found among tumors in the cohort highlighting the importance of down regulation of one allele in the establishment of selectable traits. We find a similar direct correlation of multiple small changes in the transcriptome of normal lymphocytes with immune response phenotypes, suggesting this principle of integration of multiple small deviations in gene expression applies widely to the phenotypes of normal cells, tumors, and by extension to organismic traits. Citation Format: Larry R Pease, Sara J Felts, Adam D Scheid, Xiaojia Tang, Krishna R Kalari. Phenotypes of breast tumor cells and normal lymphocytes are determined by the integration of minor changes in expression of multiple genes: A new dimension in quantitative inheritance [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-09-03.

During adaptive radiation, diversification within clades is limited by adaptation to the available ecological niches, and this may drive patterns of both trait and species diversity. However, adaptation to disparate niches may result in varied impacts on the timing, pattern and rate of morphological evolution. In this study, we examined the relationship between feeding ecology and functional diversification across a diverse clade of freshwater fishes, the Neotropical cichlids. Species dietary niches were ordinated via multivariate analysis of stomach content data. We investigated changes in the rate and pattern of morphological diversification associated with feeding, including dietary niche and degree of dietary specialization. A major division in dietary niche space was observed between predators that consume fish and macroinvertebrates vs. other groups with diets dominated by small invertebrates, detritus or vegetation. These trophic niches were strongly associated with groupings defined by functional morphospace. Clades within the piscivore/macroinvertivore group rarely transitioned to other dietary niches. Comparatively, high dietary specialization enhanced functional diversification, driving the evolution of more extreme morphologies. Divergent patterns of trophic diversification among Neotropical cichlids appear to derive from different performance demands in regional abiotic and biotic environments associated with biogeographical history.

Upon antigen exposure, naïve B cells expressing B cell receptors (BCR) able to bind antigen can undergo robust proliferation and differentiation that can result in the production of antibody-secreting and memory B cells. The factors determining whether an individual naïve B cell will proliferate following antigen encounter remains unclear. In this study, we found that polyclonal naïve murine B cell populations specific for a variety of foreign antigens express high levels of the orphan nuclear receptor Nur77, which is known to be up-regulated downstream of BCR signaling as a result of cross-reactivity with self-antigens in vivo. Similarly, a fraction of naïve human B cells specific for clinically-relevant antigens derived from respiratory syncytial virus (RSV) and HIV-1 also exhibited an IgM(LOW) IgD(+) phenotype, which is associated with self-antigen cross-reactivity. Functionally, naïve B cells expressing moderate levels of Nur77 are most likely to proliferate in vivo following antigen injection. Together, our data indicate that BCR cross-reactivity with self-antigen is a common feature of populations of naïve B cells specific for foreign antigens and a moderate level of cross-reactivity primes individual cells for optimal proliferative responses following antigen exposure.

Background Less than 5% of medulloblastoma (MB) patients survive following failure of contemporary radiation-based therapies. Understanding the molecular drivers of medulloblastoma relapse (rMB) will be essential to improve outcomes. Initial genome-wide investigations have suggested significant genetic divergence of the relapsed disease. Methods We undertook large-scale integrated characterization of the molecular features of rMB—molecular subgroup, novel subtypes, copy number variation (CNV), and driver gene mutation. 119 rMBs were assessed in comparison with their paired diagnostic samples (n = 107), alongside an independent reference cohort sampled at diagnosis (n = 282). rMB events were investigated for association with outcome post-relapse in clinically annotated patients (n = 54). Results Significant genetic evolution occurred over disease-course; 40% of putative rMB drivers emerged at relapse and differed significantly between molecular subgroups. Non-infant MBSHH displayed significantly more chromosomal CNVs at relapse (TP53 mutation-associated). Relapsed MBGroup4 demonstrated the greatest genetic divergence, enriched for targetable (eg, CDK amplifications) and novel (eg, USH2A mutations) events. Importantly, many hallmark features of MB were stable over time; novel subtypes (>90% of tumors) and established genetic drivers (eg, SHH/WNT/P53 mutations; 60% of rMB events) were maintained from diagnosis. Critically, acquired and maintained rMB events converged on targetable pathways which were significantly enriched at relapse (eg, DNA damage signaling) and specific events (eg, 3p loss) predicted survival post-relapse. Conclusions rMB is characterised by the emergence of novel events and pathways, in concert with selective maintenance of established genetic drivers. Together, these define the actionable genetic landscape of rMB and provide a basis for improved clinical management and development of stratified therapeutics, across disease-course.

The Streptococcus pyogenes cell envelope protease (SpyCEP) is a vital virulence factor in streptococcal pathogenesis. Despite its key role in disease progression and strong association with invasive disease, little is known about the enzymatic function beyond the ELR+ CXC chemokine substrate range. We utilised multiple SpyCEP constructs to interrogate the protein domains and catalytic residues necessary for enzyme function. We leveraged high-throughput mass spectrometry to describe the Michaelis-Menton parameters of active SpyCEP, revealing a Michaelis-Menton constant (KM) of 53.49 nM and a turnover of 1.34 molecules per second, for the natural chemokine substrate CXCL8.Unexpectedly, we found that an N-terminally-truncated SpyCEP C-terminal construct consisting of only the H279 and S617 catalytic dyad had specific CXCL8 cleaving activity, albeit with a reduced substrate turnover of 2.45 molecules per hour, representing a ~2000-fold reduction in activity. In contrast, the KM of the C-terminal SpyCEP construct and full-length enzyme did not differ. We conclude that the SpyCEP C-terminus plays a key role in substrate binding and recognition with key implications for both current and future streptococcal vaccine designs.

Severe cardiovascular complications can occur in coronavirus disease of 2019 (COVID-19) patients. Cardiac damage is attributed mostly to the aberrant host response to acute respiratory infection. However, direct infection of cardiac tissue by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also occurs. We examined here the cardiac tropism of SARS-CoV-2 in spontaneously beating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). These cardiomyocytes express the angiotensin-converting enzyme 2 (ACE2) receptor but not the transmembrane protease serine 2 (TMPRSS2) that mediates spike protein cleavage in the lungs. Nevertheless, SARS-CoV-2 infection of hiPSC-CMs was prolific; viral transcripts accounted for about 88% of total mRNA. In the cytoplasm of infected hiPSC-CMs, smooth-walled exocytic vesicles contained numerous 65- to 90-nm particles with canonical ribonucleocapsid structures, and virus-like particles with knob-like spikes covered the cell surface. To better understand how SARS-CoV-2 spreads in hiPSC-CMs, we engineered an expression vector coding for the spike protein with a monomeric emerald-green fluorescent protein fused to its cytoplasmic tail (S-mEm). Proteolytic processing of S-mEm and the parental spike were equivalent. Live cell imaging tracked spread of S-mEm cell-to-cell and documented formation of syncytia. A cell-permeable, peptide-based molecule that blocks the catalytic site of furin and furin-like proteases abolished cell fusion. A spike mutant with the single amino acid change R682S that disrupts the multibasic furin cleavage motif was fusion inactive. Thus, SARS-CoV-2 replicates efficiently in hiPSC-CMs and furin, and/or furin-like-protease activation of its spike protein is required for fusion-based cytopathology. This hiPSC-CM platform enables target-based drug discovery in cardiac COVID-19. IMPORTANCE Cardiac complications frequently observed in COVID-19 patients are tentatively attributed to systemic inflammation and thrombosis, but viral replication has occasionally been confirmed in cardiac tissue autopsy materials. We developed an in vitro model of SARS-CoV-2 spread in myocardium using induced pluripotent stem cell-derived cardiomyocytes. In these highly differentiated cells, viral transcription levels exceeded those previously documented in permissive transformed cell lines. To better understand the mechanisms of SARS-CoV-2 spread, we expressed a fluorescent version of its spike protein that allowed us to characterize a fusion-based cytopathic effect. A mutant of the spike protein with a single amino acid mutation in the furin/furin-like protease cleavage site lost cytopathic function. Of note, the fusion activities of the spike protein of other coronaviruses correlated with the level of cardiovascular complications observed in infections with the respective viruses. These data indicate that SARS-CoV-2 may cause cardiac damage by fusing cardiomyocytes.

The aggregation of huntingtin fragments with expanded polyglutamine repeat regions (HttpolyQ) that cause Huntington’s disease depends on the presence of a prion with an amyloid conformation in yeast. As a result of this relationship, HttpolyQ aggregation indirectly depends on Hsp104 due to its essential role in prion propagation. We find that HttQ103 aggregation is directly affected by Hsp104 with and without the presence of [RNQ(+)] and [PSI(+)] prions. When we inactivate Hsp104 in the presence of prion, yeast cells have only one or a few large HttQ103 aggregates rather than numerous smaller aggregates. When we inactivate Hsp104 in the absence of prion, there is no significant aggregation of HttQ103, whereas with active Hsp104, HttQ103 aggregates accumulate slowly due to the severing of spontaneously nucleated aggregates by Hsp104. We do not observe either effect with HttQ103P, which has a polyproline-rich region downstream of the polyglutamine region, because HttQ103P does not spontaneously nucleate and Hsp104 does not efficiently sever the prion-nucleated HttQ103P aggregates. Therefore, the only role of Hsp104 in HttQ103P aggregation is to propagate yeast prion. In conclusion, because Hsp104 efficiently severs the HttQ103 aggregates but not HttQ103P aggregates, it has a marked effect on the aggregation of HttQ103 but not HttQ103P.

The cellular prion protein PrPC is necessary for prion replication, and its reduction greatly increases life expectancy in animal models of prion infection. Hence the factors controlling the levels of PrPC may represent therapeutic targets against human prion diseases. Here we performed an arrayed whole-transcriptome RNA interference screen to identify modulators of PrPC expression. We cultured human U251-MG glioblastoma cells in the presence of 64’752 unique siRNAs targeting 21’584 annotated human genes, and measured PrPC using a one-pot fluorescence-resonance energy transfer immunoassay in 51’128 individual microplate wells. This screen yielded 743 candidate regulators of PrPC. When downregulated, 563 of these candidates reduced and 180 enhanced PrPC expression. Recursive candidate attrition through multiple secondary screens yielded 54 novel regulators of PrPC, 9 of which were confirmed by CRISPR interference as robust regulators of PrPC biosynthesis and degradation. The phenotypes of 6 of the 9 candidates were inverted in response to transcriptional activation using CRISPRa. The RNA-binding post-transcriptional repressor Pumilio-1 was identified as a potent limiter of PrPC expression through the degradation of PRNP mRNA. Because of its hypothesis-free design, this comprehensive genetic-perturbation screen delivers an unbiased landscape of the genes regulating PrPC levels in cells, most of which were unanticipated, and some of which may be amenable to pharmacological targeting in the context of antiprion therapies., Author summary The cellular prion protein (PrPC) acts as both, the substrate for prion formation and mediator of prion toxicity during the progression of all prion diseases. Suppressing the levels of PrPC is a viable therapeutic strategy as PRNP null animals are resistant to prion disease and the knockout of PRNP is not associated with any severe phenotypes. Motivated by the scarcity of knowledge regarding the molecular regulators of PrPC biosynthesis and degradation, which might serve as valuable targets to control its expression, here, we present a cell-based genome wide RNAi screen in arrayed format. The screening effort led to the identification of 54 regulators, nine of which were confirmed by an independent CRISPR-based method. Among the final nine targets, we identified PUM1 as a regulator of PRNP mRNA by acting on the 3’UTR promoting its degradation. The newly identified factors involved in the life cycle of PrPC provided by our study may also represent themselves as therapeutic targets for the intervention of prion diseases.

Bacteria possess the ability to evolve varied and ingenious strategies to outwit the host immune system, instigating an evolutionary arms race. Proteases are amongst the many weapons employed by bacteria, which specifically cleave and neutralize key signalling molecules required for a coordinated immune response. In this article, we focus on a family of S8 subtilisin-like serine proteases expressed as cell-envelope proteases (CEPs) by group A and group B streptococci. Two of these proteases known as Streptococcus pyogenes CEP (SpyCEP) and C5a peptidase cleave the chemokine CXCL8 and the complement fragment C5a, respectively. Both CXCL8 and C5a are potent neutrophil-recruiting chemokines, and by neutralizing their activity, streptococci evade a key defence mechanism of innate immunity. We review the mechanisms by which CXCL8 and C5a recruit neutrophils and the characterization of SpyCEP and C5a peptidase, including both in vitro and in vivo studies. Recently described structural insights into the function of this CEP family are also discussed. We conclude by examining the progress of prototypic vaccines incorporating SpyCEP and C5a peptidase in their preparation. Since streptococci-producing SpyCEP and C5a peptidase are responsible for a considerable global disease burden, targeting these proteases by vaccination strategies or by small-molecule antagonists should provide protection from and promote the resolution of streptococcal infections.

Understanding the degree of intraspecific variation within and among populations is a key aspect of predicting the capacity of a species to respond to anthropogenic disturbances. However, intraspecific variation is usually assessed at either limited temporal, but broad spatial scales or vice versa, which can make assessing changes in response to long‐term disturbances challenging. We evaluated the relationship between the longitudinal gradient of changing flow regimes and land use/land cover patterns since 1980 and morphological variation of Guadalupe Bass Micropterus treculii throughout the Colorado River Basin of central Texas. The Colorado River Basin in Texas has experienced major alterations to the hydrologic regime due to changing land‐ and water‐use patterns. Historical collections of Guadalupe Bass prior to rapid human‐induced change present the unique opportunity to study the response of populations to varying environmental conditions through space and time. Morphological differentiation of Guadalupe Bass associated with temporal changes in flow regimes and land use/land cover patterns suggests that they are exhibiting intraspecific trait variability, with contemporary individuals showing increased body depth, in response to environmental alteration through time (specifically related to an increase in herbaceous land cover, maximum flows, and the number of low pulses and high pulses). Additionally, individuals from tributaries with increased hydrologic alteration associated with urbanization or agricultural withdrawals tended to have a greater distance between the anal and caudal fin. These results reveal trait variation that may help to buffer populations under conditions of increased urbanization and sprawl, human population growth, and climate risk, all of which impose novel selective pressures, especially on endemic species like Guadalupe Bass. Our results contribute an understanding of the adaptability and capacity of an endemic population to respond to expected future changes based on demographic or climatic projection.

Severe cardiovascular complications can occur in coronavirus disease of 2019 (COVID-19) patients. Cardiac damage is attributed mostly to a bystander effect: the aberrant host response to acute respiratory infection. However, direct infection of cardiac tissue by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also occurs. We examined here the cardiac tropism of SARS-CoV-2 in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) that beat spontaneously. These cardiomyocytes express the angiotensin I converting-enzyme 2 (ACE2) receptor and a subset of the proteases that mediate spike protein cleavage in the lungs, but not transmembrane protease serine 2 (TMPRSS2). Nevertheless, SARS-CoV-2 infection was productive: viral transcripts accounted for about 88% of total mRNA. In the cytoplasm of infected hiPSC-CM, smooth walled exocytic vesicles contained numerous 65-90 nm particles with typical ribonucleocapsid structures, and virus-like particles with knob-like spikes covered the cell surface. To better understand the mechanisms of SARS-CoV-2 spread in hiPSC-CM we engineered an expression vector coding for the spike protein with a monomeric emerald-green fluorescent protein fused to its cytoplasmic tail (S-mEm). Proteolytic processing of S-mEm and the parental spike were equivalent. Live cell imaging tracked spread of S-mEm signal from cell to cell and documented formation of syncytia. A cell-permeable, peptide-based molecule that blocks the catalytic site of furin abolished cell fusion. A spike mutant with the single amino acid change R682S that inactivates the furin cleavage site was fusion inactive. Thus, SARS-CoV-2 can replicate efficiently in hiPSC-CM and furin activation of its spike protein is required for fusion-based cytopathology. This hiPSC-CM platform provides an opportunity for target-based drug discovery in cardiac COVID-19.Author SummaryIt is unclear whether the cardiac complications frequently observed in COVID-19 patients are due exclusively to systemic inflammation and thrombosis. Viral replication has occasionally been confirmed in cardiac tissue, but rigorous analyses are restricted to rare autopsy materials. Moreover, there are few animal models to study cardiovascular complications of coronavirus infections. To overcome these limitations, we developed an in vitro model of SARS-CoV-2 spread in induced pluripotent stem cell-derived cardiomyocytes. In these cells, infection is highly productive: viral transcription levels exceed those documented in permissive transformed cell lines. To better understand the mechanisms of SARS-CoV-2 spread we expressed a fluorescent version of its spike protein that allowed to characterize a fusion-based cytopathic effect. A mutant of the spike protein with a single amino acid mutation in the furin cleavage site lost cytopathic function. The spike protein of the Middle East Respiratory Syndrome (MERS) coronavirus drove cardiomyocyte fusion with slow kinetics, whereas the spike proteins of SARS-CoV and the respiratory coronavirus 229E were inactive. These fusion activities correlated with the level of cardiovascular complications observed in infections with the respective viruses. These data indicate that SARS-CoV-2 has the potential to cause cardiac damage by fusing cardiomyocytes.

In a phase 1b study of acalabrutinib (a covalent Bruton tyrosine kinase (BTK) inhibitor) in combination with vistusertib (a dual mTORC1/2 inhibitor) in patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL), multiple ascending doses of the combination as intermittent or continuous schedules of vistusertib were evaluated. The overall response rate was 12% (3/25). The pharmacodynamic (PD) profile for acalabrutinib showed that BTK occupancy in all patients was >95%. In contrast, PD analysis for vistusertib showed variable inhibition of phosphorylated 4EBP1 (p4EBP1) without modulation of AKT phosphorylation (pAKT). The pharmacokinetic (PK)/PD relationship of vistusertib was direct for TORC1 inhibition (p4EBP1) but did not correlate with TORC2 inhibition (pAKT). Cell-of-origin subtyping or next-generation sequencing did not identify a subset of DLBCL patients with clinical benefit; however, circulating tumor DNA dynamics correlated with radiographic response. These data suggest that vistusertib does not modulate targets sufficiently to add to the clinical activity of acalabrutinib monotherapy. Clinicaltrials.gov identifier: NCT03205046.

Summary Neuropathological and experimental evidence suggests that the cell-to-cell transfer of α-synuclein has an important role in the pathogenesis of Parkinson’s disease (PD). However, the mechanism underlying this phenomenon is not fully understood. We undertook a small interfering RNA (siRNA), genome-wide screen to identify genes regulating the cell-to-cell transfer of α-synuclein. A genetically encoded reporter, GFP-2A-αSynuclein-RFP, suitable for separating donor and recipient cells, was transiently transfected into HEK cells stably overexpressing α-synuclein. We find that 38 genes regulate the transfer of α-synuclein-RFP, one of which is ITGA8, a candidate gene identified through a recent PD genome-wide association study (GWAS). Weighted gene co-expression network analysis (WGCNA) and weighted protein-protein network interaction analysis (WPPNIA) show that those hits cluster in networks that include known PD genes more frequently than expected by random chance. The findings expand our understanding of the mechanism of α-synuclein spread., Graphical abstract, Highlights • 38 genes regulate the cell-to-cell transfer of α-synuclein • Those genes participate in the same networks as known Parkinson’s disease (PD) genes • Knockdown of selected genes affects mitochondrial ΔΨm similarly to recessive PD genes • The “prionoid” hypothesis likely has an important role in the pathogenesis of PD, Kara et al. undertook a high-throughput screen that identified 38 genes that regulate the cell-to-cell transfer of α-synuclein. Those genes participate in the same networks as known Parkinson’s disease Mendelian and risk genes. This work provides a framework for a gene-discovery strategy in complex diseases.

In previous research, stall-housing growing horses resulted in decreased bone mineral content (BMC) of the third metacarpal (MCIII) compared to pasture-housing. To determine whether stall confinement negatively impacts bone, regardless of age, and whether the effects could be reversed upon return to pasture, 12 mature horses (5-15 years) and 12 yearlings were pair-matched by age and gender and randomly assigned to one of two treatment groups: pasture (PT) or stall (ST). Horses on PT remained there for the 84-d study, while horses on ST spent the first 28 d in stalls and the remaining 56 d on pasture. Radiographs and blood samples were taken on d 0 for baseline determinants. Radiographs and blood were taken every 7 d for radiographic photodensitometry estimates of BMC and analysis of osteocalcin (OC) and C-telopeptide (CTX-1)-markers of bone formation and degradation. There were no differences in BMC between treatments at the medial cortex of MCIII in mature horses. There were treatment (P=0.05) and day (P

Stereotactic radiosurgery (SRS) has become a primary option for management for both newly diagnosed vestibular schwannomas (VS), as well as VS that enlarge after initial observation. A retrospective review of our prospectively maintained data base found 871 patients who underwent Gamma knife® SRS as their initial (primary) management between 1987 and 2008. Follow-up ranged from 1–25 years (median = 5.2 years) Median tumor volume was 0.9 cc (0.02–36) and median margin dose was 13 Gy (12–25). Progression free survival (PFS) after SRS was 97% at 3 years, 95% at 5 years, and 94% at 10 years. Freedom from delayed surgical resection was found in 98.7% of patients. Smaller tumor volume was significantly associated with improved PFS. There were 326 patients with serviceable hearing (Gardner–Robertson 1 or 2) at the time of SRS with audiological follow-up of ≥ 1 year. Serviceable hearing preservation rates after SRS were 89.8% at 1 year, 76.9% at 3 years, 68.4% at 5 years, 62.5% at 7 years, and 51.4% at 10 years. Factors associated with improved serviceable hearing preservation included younger age, Gardner-Robertson grade 1 at SRS, and absence of subjective complaints of dysequilibrium or vertigo (vestibulopathy). Fifty-one patients (5.8%) developed trigeminal neuropathy. Fourteen (1.6%) developed a transient House-Brackmann grade 2 or 3 facial neuropathy. In this report with extended follow-up, primary SRS achieved tumor growth control in 94% of patients. Optimization of long- term cranial nerve outcomes remains an important achievement of this management strategy for VS.

The ball-gall fly Eurosta solidaginis is considered a classic example of host-race formation in herbivorous insects, with host-associated races evolving at least twice, including the well-known pair on Solidago altissima and S. gigantea. Yet E. solidaginis has been occasionally observed galling other Solidago species. Here, we explore the origins of E. solidaginis on Solidago rugosa. We hypothesize that flies associated with S. rugosa are derived from the S. altissima host-race, and ask whether S. rugosa-associated flies have initiated host-race formation. We compared genetic variation among seventeen E. solidaginis populations collected from S. rugosa, S. altissima, and S. gigantea, in an adjacent COI/COII region of the mitochondrial genome. Across the study area, E. solidaginis flies from S. rugosa were as diverged from S. altissima-flies as they were from S. gigantea-flies (pairwise ΦPT 0.075 and 0.78 respectively) but S. altissima and S. gigantea-flies appeared considerably less diverged (0.002). This pattern was driven by the majority of flies, regardless of host-plant, sharing the same haplotype across the study area. However, we detected several site/region-specific haplotypes, not shared among host species. At the local site scale we were able to distinguish S. gigantea-associated fly haplotypes from either S. altissima or S. rugosa haplotypes, but the majority of S. altissima and S. rugosa-flies shared the same haplotype locally. These patterns of haplotype diversity support existing evidence of host-associated divergence in S. altissima- and S. gigantea-associated flies, and suggest that S. rugosa flies are either the same host-race as, or are recently derived from, S. altissima flies. Successful development on S. rugosa and the use of S. rugosa in the absence of use on sympatric S. altissima suggests that E. solidaginis either has a single oligophagous race using both S. altissima and S. rugosa or is in the earliest stages of host-associated differentiation.

Small cell lung cancer (SCLC) accounts for 15% of lung cancers and is almost always linked to inactivating RB1 and TP53 mutations. SCLC frequently responds, albeit briefly, to chemotherapy. The canonical function of the RB1 gene product RB1 is to repress the E2F transcription factor family. RB1 also plays both E2F-dependent and E2F-independent mitotic roles. We performed a synthetic lethal CRISPR/Cas9 screen in an RB1−/− SCLC cell line that conditionally expresses RB1 to identify dependencies that are caused by RB1 loss and discovered that RB1−/− SCLC cell lines are hyperdependent on multiple proteins linked to chromosomal segregation, including Aurora B kinase. Moreover, we show that an Aurora B kinase inhibitor is efficacious in multiple preclinical SCLC models at concentrations that are well tolerated in mice. These results suggest that RB1 loss is a predictive biomarker for sensitivity to Aurora B kinase inhibitors in SCLC and perhaps other RB1−/− cancers. Significance: SCLC is rarely associated with actionable protooncogene mutations. We did a CRISPR/Cas9-based screen that showed that RB1−/− SCLC are hyperdependent on AURKB, likely because both genes control mitotic fidelity, and confirmed that Aurora B kinase inhibitors are efficacious against RB1−/− SCLC tumors in mice at nontoxic doses. See related commentary by Dick and Li, p. 169. This article is highlighted in the In This Issue feature, p. 151

The cellular prion protein (PrPC ) is best known for its misfolded disease-causing conformer, PrPS c . Because the availability of PrPC is often limiting for prion propagation, understanding its regulation may point to possible therapeutic targets. We sought to determine to what extent the human microRNAome is involved in modulating PrPC levels through direct or indirect pathways. We probed PrPC protein levels in cells subjected to a genome-wide library encompassing 2019 miRNA mimics using a robust time-resolved fluorescence-resonance screening assay. Screening was performed in three human neuroectodermal cell lines: U-251 MG, CHP-212 and SH-SY5Y. The three screens yielded 17 overlapping high-confidence miRNA mimic hits, 13 of which were found to regulate PrPC biosynthesis directly via binding to the PRNP 3'UTR, thereby inducing transcript degradation. The four remaining hits (miR-124-3p, 192-3p, 299-5p and 376b-3p) did not bind either the 3'UTR or CDS of PRNP, and were therefore deemed indirect regulators of PrPC . Our results show that multiple miRNAs regulate PrPC levels both directly and indirectly. These findings may have profound implications for prion disease pathogenesis and potentially also for their therapy. Furthermore, the possible role of PrPC as a mediator of Aβ toxicity suggests that its regulation by miRNAs may also impinge on Alzheimer's disease. This article is protected by copyright. All rights reserved

A phytoplasma was initially detected in Dypsis poivriana by nested and real-time PCR from the botanical gardens in Cairns, Queensland, Australia in 2017. Further surveys in the Cairns region identified phytoplasma infections in eight additional dying ornamental palm species (Euterpe precatoria, Cocos nucifera, Verschaffeltia splendida, Brassiophoenix drymophloeodes, Burretiokentia hapala, Cyrtostachys renda, Reinhardtia gracilis, Carpoxylon macrospermum), a Phoenix species, a Euterpe species and two native palms (Archontophoenix alexandrae). Analysis of 16S rRNA gene sequences showed that this phytoplasma is distinct as it shared less than 97.5 % similarity with all other ‘Candidatus Phytoplasma’ species. At 96.3 % similarity, the most closely related formally described member of the provisional 'Ca. Phytoplasma' genus was 'Ca. Phytoplasma noviguineense', a novel taxon from the island of New Guinea found in monocotyledonous plants. It was slightly more closely related (96.6–96.8 %) to four palm-infecting strains from the Americas, which belong to strain group 16SrIV and which have not been assigned to a formal 'Candidatus Phytoplasma’ species taxon. Phylogenetic analysis of the 16S rRNA gene and ribosomal protein genes of the phytoplasma isolate from a dying coconut palm revealed that the phytoplasma represented a distinct lineage within the phytoplasma clade. As the nucleotide identity with other phytoplasmas is less than 97.5 % and the phylogenetic analyses show that it is distinct, a novel taxon 'Candidatus Phytoplasma dypsidis' is proposed for the phytoplasma found in Australia. Strain RID7692 (GenBank accession no. MT536195) is the reference strain. The impact and preliminary aspects of the epidemiology of the disease outbreak associated with this novel taxon are described.

Proper regulation of genome architecture and activity is essential for the development and function of multicellular organisms. Histone modifications, acting in combination, specify these activity states at individual genomic loci. However, the methods used to study these modifications often require either a large number of cells or are limited to targeting one histone mark at a time. Here, we developed a new method called Single Cell Evaluation of Post-TRanslational Epigenetic Encoding (SCEPTRE) that uses Expansion Microscopy (ExM) to visualize and quantify multiple histone modifications at non-repetitive genomic regions in single cells at a spatial resolution of ∼75 nm. Using SCEPTRE, we distinguished multiple histone modifications at a single housekeeping gene, quantified histone modification levels at multiple developmentally-regulated genes in individual cells, and evaluated the relationship between histone modifications and RNA polymerase II loading at individual loci. We find extensive variability in epigenetic states between individual gene loci hidden from current population-averaged measurements. These findings establish SCEPTRE as a new technique for multiplexed detection of combinatorial chromatin states at single genomic loci in single cells., Graphical Abstract Graphical AbstractSingle cell evaluation of post-translational epigenetic encoding (SCEPTRE) uses expansion microscopy to combine DNA in situ labeling with immunofluorescence and quantify histone mark levels at individual loci within cells.

Mycobacterium chelonae is a type of nontuberculous mycobacteria most commonly associated with skin and soft tissue infections. We present a case of recurrent M. chelonae pulmonary infection presenting with severe weight loss. After recurrence, sputum cultures remained positive for 2 years despite appropriate antibiotics. Cultures only became negative after the addition of intravenous imipenem and jejunostomy feeds. The rarity of M. chelonae pulmonary infection means that optimal treatment regimens have not yet been fully established but a regimen of clarithromycin plus an additional antibiotic has been recommended1. The prognosis of such infections also remains unclear but lower rates of macrolide resistance suggest that the prognosis may be better than the closely related species M. abscessus. Although its benefit has not been proven, nutrition supplementation, including percutaneous enteral feeding, can be considered for refractory NTM infection in underweight patients.

Chemokines play diverse and fundamental roles in the immune system and human disease, which has prompted their structural and functional characterisation. Production of recombinant chemokines that are folded and bioactive is vital to their study but is limited by the stringent requirements of a native N-terminus for receptor activation and correct disulphide bonding required to stabilise the chemokine fold. Even when expressed as fusion proteins, overexpression of chemokines in E. coli tends to result in the formation of inclusion bodies, generating the additional steps of solubilisation and refolding. Here we present a novel method for producing soluble chemokines in relatively large amounts via a simple two-step purification procedure with no requirements for refolding. CXCL8 produced by this method has the correct chemokine fold as determined by NMR spectroscopy and in chemotaxis assays was indistinguishable from commercially available chemokines. We believe that this protocol significantly streamlines the generation of recombinant chemokines.

Genetic association data from national biobanks and large-scale association studies have provided new prospects for understanding the genetic evolution of complex traits and diseases in humans. In turn, genomes from ancient human archaeological remains are now easier than ever to obtain, and provide a direct window into changes in frequencies of trait-associated alleles in the past. This has generated a new wave of studies aiming to analyse the genetic component of traits in historic and prehistoric times using ancient DNA, and to determine whether any such traits were subject to natural selection. In humans, however, issues about the portability and robustness of complex trait inference across different populations are particularly concerning when predictions are extended to individuals that died thousands of years ago, and for which little, if any, phenotypic validation is possible. In this review, we discuss the advantages of incorporating ancient genomes into studies of trait-associated variants, the need for models that can better accommodate ancient genomes into quantitative genetic frameworks, and the existing limits to inferences about complex trait evolution, particularly with respect to past populations.

International audience; Archaeological evidence indicates that pig domestication had begun by ∼10,500 y before the present (BP) in the Near East, and mitochondrial DNA (mtDNA) suggests that pigs arrived in Europe alongside farmers ∼8,500 y BP. A few thousand years after the introduction of Near Eastern pigs into Europe, however, their characteristic mtDNA signature disappeared and was replaced by haplotypes associated with European wild boars. This turnover could be accounted for by substantial gene flow from local Euro-pean wild boars, although it is also possible that European wild boars were domesticated independently without any genetic contribution from the Near East. To test these hypotheses, we obtained mtDNA sequences from 2,099 modern and ancient pig samples and 63 nuclear ancient genomes from Near Eastern and European pigs. Our analyses revealed that European domestic pigs dating from 7,100 to 6,000 y BP possessed both Near Eastern and European nuclear ancestry, while later pigs possessed no more than 4% Near Eastern ancestry, indicating that gene flow from European wild boars resulted in a near-complete disappearance of Near East ancestry. In addition, we demonstrate that a variant at a locus encoding black coat color likely originated in the Near East and persisted in European pigs. Altogether, our results indicate that while pigs were not independently domesticated in Europe, the vast majority of human-mediated selection over the past 5,000 y focused on the genomic fraction derived from the European wild boars, and not on the fraction that was selected by early Neolithic farmers over the first 2,500 y of the domestication process. domestication | evolution | gene flow | Neolithic

Viruses spread between hosts through particles, but within hosts, viral genomes can spread from cell to cell through fusion, evading antiviral defenses and obviating costly infectious virion production1-3. Billions of electromechanically coupled cardiomyocytes (CMs) make myocardium inherently vulnerable to pathological electromechanical short circuits caused by intercellular viral spread 4-6. Beyond respiratory illness, COVID-19 affects the heart7 and cardiac injury and arrhythmias are serious public health concerns8-12. By studying myocardium of a young woman who died suddenly, diagnosed postmortem with COVID-19, we discovered highly focal myocardial SARS-CoV-2 infection spreading from one CM to another through intercellular junctions identified by highly concentrated sarcolemmal t-tubule viral spike glycoprotein. SARS-CoV-2 permissively infected beating human induced pluripotent stem cell (hiPSC)-CMs building multinucleated cardiomyotubes (CMTs) through cell type-specific fusion driven by proteolytically-activated spike glycoprotein. Recombinant spike glycoprotein, co-localizing to sarcolemma and sarcoplasmic reticulum, produced multinucleated CMTs with pathological structure, electrophysiology and Ca2+ excitation-contraction coupling. Blocking cleavage, a peptide-based protease inhibitor neutralized SARS-CoV-2 spike glycoprotein pathogenicity. We conclude that SARS-CoV-2 spike glycoprotein, efficiently primed, activated and strategically poised during biosynthesis, can exploit the CM’s inherent membranous connectivities to drive heart damage directly, uncoupling clinically common myocardial injury from lymphocytic myocarditis, often suspected but rarely confirmed in COVID-19.

Cutaneous T-cell lymphomas (CTCLs) are a clinically heterogeneous collection of lymphomas of the skin-homing T cell. To identify molecular drivers of disease phenotypes, we assembled representative samples of CTCLs from patients with diverse disease subtypes and stages. Via DNA/RNA-sequencing, immunophenotyping, and ex vivo functional assays, we identified the landscape of putative driver genes, elucidated genetic relationships between CTCLs across disease stages, and inferred molecular subtypes in patients with stage-matched leukemic disease. Collectively, our analysis identified 86 putative driver genes, including 19 genes not previously implicated in this disease. Two mutations have never been described in any cancer. Functionally, multiple mutations augment T-cell receptor–dependent proliferation, highlighting the importance of this pathway in lymphomagenesis. To identify putative genetic causes of disease heterogeneity, we examined the distribution of driver genes across clinical cohorts. There are broad similarities across disease stages. Many driver genes are shared by mycosis fungoides (MF) and Sezary syndrome (SS). However, there are significantly more structural variants in leukemic disease, leading to highly recurrent deletions of putative tumor suppressors that are uncommon in early-stage skin-centered MF. For example, TP53 is deleted in 7% and 87% of MF and SS, respectively. In both human and mouse samples, PD1 mutations drive aggressive behavior. PD1 wild-type lymphomas show features of T-cell exhaustion. PD1 deletions are sufficient to reverse the exhaustion phenotype, promote a FOXM1-driven transcriptional signature, and predict significantly worse survival. Collectively, our findings clarify CTCL genetics and provide novel insights into pathways that drive diverse disease phenotypes.

Purpose To conduct quantitative analysis of astrocytic glial fibrillary acidic protein (GFAP), actin and nuclei distribution in mouse optic nerve (ON) and investigate changes in the measured features after 3 days of ocular hypertension (OHT). Method Serial cross-sections of 3-day microbead-induced OHT and control ONs were fluorescently labelled and imaged using confocal microscope. Eighteen structural features were measured from the acquired images, including GFAP coverage, actin area fraction, process thickness, and aspect ratio of cell nucleus. The measured features were analyzed for variations with axial locations along ON and radial zones transverse to ON, as well as for the correlations with degree of intraocular pressure (IOP) change. Results The most significant changes in structural features after 3-day OHT occurred in the unmyelinated ON region (R1), and the changes were greater with greater IOP elevation. Although the GFAP, actin, axonal, and ON areas all increased in 3-day OHT ONs in R1 (P ≤ 0.004 for all), the area fraction of GFAP actually decreased (P = 0.02), the actin area fraction was stable and individual axon compartments were unchanged in size. Within R1, the number of nuclear clusters increased (P < 0.001), but the mean size of nuclear clusters was smaller (P = 0.02) and the clusters became rounder (P < 0.001). In all cross-sections of control ONs, astrocytic processes were thickest in the rim zone compared with the central and peripheral zones (P ≤ 0.002 for both), whereas the overall process width in R1 decreased after 3 days of OHT (P < 0.001). Conclusions The changes in structure elucidated IOP-generated alterations that underlie astrocyte mechanotranslational responses relevant to glaucoma.

Dopamine (DA) signaling is central in hypothesized causal paths linking the influence of social and environmental variables with cognition, behavior and affective states, including vulnerability to drug dependence. Here, we study whether change in one’s social rank induces DA and norepinephrine (NE) changes using a novel pig model with a social-ranking-and-re-ranking protocol to investigate social context influences on catecholamine concentrations in cerebrospinal fluid (CSF) and blood. For two weeks, 16 recently weaned male piglets were socially housed in four groups, with video-recordings for social rank assessments (α, β, γ, and δ); CSF and blood were obtained from these stable social groups. Next, all four α were housed together, as were all four β, etc., again with video recording for blinded social ranking. CSF and blood samples were collected at three time points: prior to initial social housing, following social housing and following re-organization. Regression analyses disclosed a positive relationship between changes in social rank and post-rank change in CSF levels of DA; one unit increase of social rank predicted a 17.4 pg/ml increase in CSF dopamine concentrations (95% CI= 1.2, 33.7). Compared to piglets with downward shifts in ranks (i.e., high-to-low), piglets with upward shifts (i.e., low-to-high) had a statistically significant greater increase in CSF DA levels. No relationship was observed for CSF NE or blood concentrations of DA or NE at any phase of this experiment. This work, using a novel pig model, adds new evidence on alteration of the brain dopaminergic system induced by social rank change.

Many international guidance documents for deriving water quality guideline values recommend the use of chronic toxicity data. For the tropical fish northern trout gudgeon, Mogurnda mogurnda, 96-h acute and 28-d chronic toxicity tests have been developed, but both tests have drawbacks. The 96-h toxicity test is acute and has a lethal endpoint; hence it is not a preferred method for guideline value derivation. The 28-d method has a sublethal (growth) endpoint, but is highly resource intensive and is high risk in terms of not meeting quality control criteria. The present study aimed to determine the feasibility of a 7-d larval growth toxicity test as an alternative to the 96-h survival and 28-d growth tests. Once the method was successfully developed, derived toxicity estimates for uranium, magnesium, and manganese were compared with those for other endpoints and tests lengths within the literature. As a final validation of the 7-d method, the sensitivity of the 7-d growth endpoint was compared with those of 14-, 21-, and 28-d exposures. Fish growth rate, based on length, over 7 d was significantly more sensitive compared with existing acute toxicity endpoints for magnesium and manganese, and was similarly sensitive to existing chronic toxicity endpoints for uranium. For uranium, the sensitivity of the growth endpoint over the 4 exposure periods was similar, suggesting that 7 d as an exposure duration is sufficient to provide an indication of longer term chronic growth effects. The sensitivity of the 7-d method, across the 3 metals tested, highlights the benefit of utilizing the highly reliable short-term 7-d chronic toxicity test method in future toxicity testing using M. mogurnda. Environ Toxicol Chem 2021;40:1596-1605. © 2021 Commonwealth of Australia. Environmental Toxicology and Chemistry © 2021 SETAC.

Breast cancer (BC) is the second leading cause of cancer deaths among women. DEK is a known oncoprotein that is highly expressed in over 60% of breast cancers and is an independent marker of poor prognosis. However, the molecular mechanisms by which DEK promotes tumor progression are poorly understood. To identify novel oncogenic functions of DEK, we performed RNA-Seq analysis on isogenic Dek-knockout and complemented murine BC cells. Gene ontology analyses identified gene sets associated with immune system regulation and cytokine-mediated signaling and differential cytokine and chemokine expression was confirmed across Dek-proficient versus Dek-deficient cells. By exposing murine bone marrow-derived macrophages (BMDM) to tumor cell conditioned media (TCM) to mimic a tumor microenvironment, we showed that Dek-expressing breast cancer cells produce a cytokine milieu, including up-regulated Tslp and Ccl5 and down-regulated Cxcl1, Il-6, and GM-CSF, that drives the M2 polarization of macrophages. We validated this finding in primary murine mammary tumors and show that Dek expression in vivo is also associated with increased expression of M2 macrophage markers in murine tumors. Using TCGA data, we verified that DEK expression in primary human breast cancers correlates with the expression of several genes identified by RNA-Seq in our murine model and with M2 macrophage phenotypes. Together, our data demonstrate that by regulating the production of multiple secreted factors, DEK expression in BC cells creates a potentially immune suppressed tumor microenvironment, particularly by inducing M2 tumor associated macrophage (TAM) polarization.

Harmful algal bloom (HAB) dinoflagellate species Karlodinium veneficum and Prorocentrum cordatum (prev. P. minimum) are commonly found in Chesapeake Bay during the late spring and early summer months, coinciding with the spawning season of the eastern oyster (Crassostrea virginica). Unexplained larval oyster mortalities at regional commercial hatcheries prompted screening of oyster hatchery water samples for these HAB species. Both HAB species were found in treated hatchery water during the oyster spawning season, sometimes exceeding bloom cell concentrations (≥ 1,000 cells/mL). To investigate the potential for these HAB species, independently or in co-exposure, to affect larval oyster mortality and activity, 96-h laboratory single and dual HAB bioassays with seven-day-old oyster larvae were performed. Treatments for the single HAB bioassay included fed and unfed controls, K. veneficum at 1,000; 5,000; 10,000; and 50,000 cells/mL, P. cordatum at 100; 5,000; 10,000; and 50,000 cells/mL. Subsequently, the 1,000 cells/mL K. veneficum and 50,000 cells/mL P. cordatum treatments were combined in a co-exposure treatment for the dual HAB bioassay. At all cell concentrations tested, K. veneficum swarmed oyster larvae and caused significant larval oyster mortality by 96 h (Karlo1,000: 21 ± 5%; Karlo5,000: 93 ± 2%; Karlo10,000: 85 ± 3%; Karlo50,000: 83 ± 5%, SE). In contrast, there was no significant difference in larval oyster mortality between the control treatments and any of the P. cordatum treatments by 96 h. By 24 h, larval oysters were significantly less active (immotile) in the presence of either HAB species as compared to control treatments (e.g., Karlo1,000: 37.8 ± 4.1%; Proro100: 47.3 ± 7.4%; Fed: 10.8 ± 3.2%; Unfed: 10.1 ± 4.9%, SE). In the dual HAB bioassay, larval oyster mortality associated with 1,000 cells/mL K. veneficum (44 ± 9%, SE) was not changed by the addition of 50,000 cells/mL P. cordatum (55 ± 7%, SE), demonstrating that K. veneficum was primarily responsible for the observed mortality. This study demonstrated that even low cell concentrations of K. veneficum and P. cordatum are harmful to larval oysters, and could contribute to reductions in oyster hatchery production through impacts on this critical life stage.

Numerous pairs of evolutionarily divergent mammalian species have been shown to produce hybrid offspring. In some cases, F 1 hybrids are able to produce F 2 s through matings with F 1 s. In other instances, the hybrids are only able to produce offspring themselves through backcrosses with a parent species owing to unisexual sterility (Haldane's Rule). Here, we explicitly tested whether genetic distance, computed from mitochondrial and nuclear genes, can be used as a proxy to predict the relative fertility of the hybrid offspring resulting from matings between species of terrestrial mammals. We assessed the proxy's predictive power using a well-characterized felid hybrid system, and applied it to modern and ancient hominins. Our results revealed a small overlap in mitochondrial genetic distance values that distinguish species pairs whose calculated distances fall within two categories: those whose hybrid offspring follow Haldane's Rule, and those whose hybrid F 1 offspring can produce F 2 s. The strong correlation between genetic distance and hybrid fertility demonstrated here suggests that this proxy can be employed to predict whether the hybrid offspring of two mammalian species will follow Haldane's Rule.