Second Heart Field-derived Cells Contribute to Angiotensin II-mediated Ascending Aortopathies BackgroundThe ascending aorta is a common location for aneurysm and dissection. This aortic region is populated by a mosaic of medial and adventitial cells that are embryonically derived from either the second heart field (SHF) or the cardiac neural crest. SHF-derived cells populate areas that coincide with the spatial specificity of thoracic aortopathies. The purpose of this study was to determine whether and how SHF-derived cells contribute to ascending aortopathies.

MethodsAscending aortic pathologies were examined in patients with sporadic thoracic aortopathies and angiotensin II (AngII)-infused mice. Ascending aortas without overt pathology from AngII-infused mice were subjected to mass spectrometry assisted proteomics, and molecular features of SHF-derived cells were determined by single cell transcriptomic analyses. Genetic deletion of either low-density lipoprotein receptor-related protein 1 (Lrp1) or transforming growth factor-{beta} receptor 2 (Tgfbr2) in SHF- derived cells was conducted to examine the impact of SHF-derived cells on vascular integrity.

ResultsPathologies in human ascending aortic aneurysmal tissues were predominant in outer medial layers and adventitia. This gradient was mimicked in mouse aortas following AngII infusion that was coincident with the distribution of SHF-derived cells. Proteomics indicated that brief AngII infusion, prior to overt pathology, evoked downregulation of SMC proteins and differential expression of extracellular matrix proteins, including several LRP1 ligands. LRP1 deletion in SHF-derived cells augmented AngII-induced ascending aortic aneurysm and rupture. Single cell transcriptomic analysis revealed that brief AngII infusion decreased Lrp1 and Tgfbr2 mRNA abundance in SHF-derived cells and induced a unique fibroblast population with low abundance of Tgfbr2 mRNA. SHF-specific Tgfbr2 deletion led to embryonic lethality at E12.5 with dilatation of the outflow tract and retroperitoneal hemorrhage. Integration of proteomic and single cell transcriptomics results identified plasminogen activator inhibitor 1 (PAI1) as the most increased protein in SHF-derived SMCs and fibroblasts during AngII infusion. Immunostaining revealed a transmural gradient of PAI1 in both ascending aortas of AngII-infused mice and human ascending aneurysmal aortas that mimicked the gradient of medial and adventitial pathologies.

ConclusionSHF-derived cells exert a critical role in maintaining vascular integrity through LRP1 and TGF-{beta} signaling associated with increases of aortic PAI1.



O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/930917v6_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@185e3d9org.highwire.dtl.DTLVardef@131d702org.highwire.dtl.DTLVardef@11afa42org.highwire.dtl.DTLVardef@242786_HPS_FORMAT_FIGEXP  M_FIG C_FIG Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABS- SHF-derived SMCs and fibroblasts associate with AngII-induced aortic pathologies.
- AngII induces a distinct fibroblast sub-cluster that is less abundant for mRNAs related to major extracellular components and TGF{beta} ligands and receptors, but more abundant for proliferative genes.
- TGFBR2 deletion in SHF-derived cells are embryonic lethal with significant dilatation of the outflow tract in mice.
- SHF-specific deletion of LRP1 leads to aortic pathologies in mice, supporting the importance of SHF-derived cells in maintaining ascending aortic wall integrity.


What are the clinical implications?- Heterogeneity of the embryonic origins of SMCs and fibroblasts contributes to complex mechanisms of vasculopathy formation, which should be considered when investigating the pathogenesis of thoracic aortopathies.
Fatal COVID-19 and non-COVID-19 Acute Respiratory Distress Syndrome is Associated with Incomplete Alveolar Type 1 Epithelial Cell Differentiation from the Transitional State Without Fibrosis ARDS due to COVID-19 and other etiologies results from injury to the alveolar epithelial cell (AEC) barrier resulting in noncardiogenic pulmonary edema, which causes acute respiratory failure; clinical recovery requires epithelial regeneration. During physiologic regeneration in mice, AEC2s proliferate, exit the cell cycle, and transiently assume a transitional state before differentiating into AEC1s; persistence of the transitional state is associated with pulmonary fibrosis in humans. It is unknown whether transitional cells emerge and differentiate into AEC1s without fibrosis in human ARDS and why transitional cells differentiate into AEC1s during physiologic regeneration but persist in fibrosis. We hypothesized that incomplete but ongoing AEC1 differentiation from transitional cells without fibrosis may underlie persistent barrier permeability and fatal acute respiratory failure in ARDS. Immunostaining of postmortem ARDS lungs revealed abundant transitional cells in organized monolayers on alveolar septa without fibrosis. They were typically cuboidal or partially spread, sometimes flat, and occasionally expressed AEC1 markers. Immunostaining and/or interrogation of scRNAseq datasets revealed that transitional cells in mouse models of physiologic regeneration, ARDS, and fibrosis express markers of cell cycle exit but only in fibrosis express a specific senescence marker. Thus, in severe, fatal early ARDS, AEC1 differentiation from transitional cells is incomplete, underlying persistent barrier permeability and respiratory failure, but ongoing without fibrosis; senescence of transitional cells may be associated with pulmonary fibrosis.
A cross biomes bacterial diversity shed light on ocean-atmosphere microbial transmission. Microbes are ubiquitous in the oceans and the atmosphere, playing essential roles in biogeochemical processes. The bio-exchanges between the two environments can provide important insights into microbial distribution and diversity but are still not well understood. We simultaneously surveyed the genomic diversity of airborne and marine bacterial communities across 15 000 kilometers in the Atlantic and Pacific oceans. Higher variability of microbial community composition was observed in the atmosphere than in the ocean surface waters. In addition, a greater similarity was observed between oceans than their overlaying atmosphere, and between atmospheric samples than with the ocean beneath. We additionally detected a higher coverage rate and relative abundance of marine bacteria in the Pacific atmosphere as compared to the Atlantic, while the dominant fraction in the Atlantic atmosphere was annotated as soil-associated bacteria. This study advances our understanding of microbial dispersion in the ocean, the atmosphere, and the exchange between them, as well as their potential impact on microbial composition, ecology, and biogeochemistry. The immunopeptidome from a genomic perspective: Establishing immune-relevant regions for cancer vaccine design A longstanding disconnect between the growing number of MHC Class I immunopeptidomic studies and genomic medicine hinders cancer vaccine design. We develop COD-dipp to genomically map the full spectrum of detected canonical and non-canonical (non-exonic) MHC Class I antigens from 26 cancer studies. We demonstrate that patient mutations in regions overlapping physically identified antigens better predict immunotherapy response when compared to neoantigen predictions. We suggest a vaccine design approach using 140,966 highly immune-visible regions of the genome annotated by their expression and haplotype frequency in the human population. These regions tend to be highly conserved, mutated in cancer and harbor 7.8 times more immunogenicity. Intersecting pan-cancer mutations with these immune surveilled regions revealed a potential to create off-the-shelf multi-epitope vaccines against public neoantigens. Here we release COD-dipp, a cancer vaccine toolkit as a web-application (https://www.proteogenomics.ca/COD-dipp) and open-source high-throughput resource. Reconstitution of minimal motility system based on Spiroplasma swimming by expressing two bacterial actins in synthetic minimal bacterium Motility is one of the most important features of life, but its evolutionary origin remains unknown. In this study, we focused on Spiroplasma, commensal, or parasitic bacteria. They swim by switching the helicity of a ribbon-like cytoskeleton that comprises six proteins, each of which evolved from a nucleosidase and bacterial actin called MreB. We expressed these proteins in a synthetic, non-motile minimal bacterium, JCVI-syn3.0B, whose reduced genome was computer-designed and chemically synthesized. The synthetic bacterium exhibited swimming motility with features characteristic of Spiroplasma swimming. Moreover, some combinations of the two proteins produced a helical cell shape and swimming, suggesting that the swimming originated from the differentiation and coupling of bacterial actins, and we also obtained a minimal system for motility of the synthetic bacterium.

One-Sentence SummaryThe minimal system comprised two bacterial actins that provided cell helicity and swimming to the synthetic minimal bacterium. Inhibitors of ERp44, PDIA1, and AGR2 induce disulfide-mediated oligomerization of Death Receptors 4 and 5 and cancer cell death Breast cancer mortality remains unacceptably high, indicating a need for safer and more effective therapeutic agents. Disulfide bond Disrupting Agents (DDAs) were previously identified as a novel class of anticancer compounds that selectively kill cancers that overexpress the Epidermal Growth Factor Receptor (EGFR) or its family member HER2. DDAs kill EGFR+ and HER2+ cancer cells via the parallel downregulation of EGFR, HER2, and HER3 and activation/oligomerization of Death Receptors 4 and 5 (DR4/5). However, the mechanisms by which DDAs mediate these effects are unknown. Affinity purification analyses employing biotinylated-DDAs reveal that the Protein Disulfide Isomerase (PDI) family members AGR2, PDIA1, and ERp44 are DDA target proteins. Further analyses demonstrate that shRNA-mediated knockdown of AGR2 and ERp44, or expression of ERp44 mutants, enhance basal and DDA-induced DR5 oligomerization. DDA treatment of breast cancer cells disrupts PDIA1 and ERp44 mixed disulfide bonds with their client proteins. Together, the results herein reveal DDAs as the first small molecule, active site inhibitors of AGR2 and ERp44, and demonstrate roles for AGR2 and ERp44 in regulating the activity, stability, and localization of DR4 and DR5, and activation of Caspase 8. Plasmodium DDI1 is a potential dual-therapeutic target and essential chromatin-associated protein DDI1 proteins are involved in a variety of cellular processes, including proteasomal degradation of specific proteins. All DDI1 proteins contain a ubiquitin-like (UBL) domain and a retroviral aspartyl protease (RVP) domain. Some DDI1 proteins also contain a ubiquitin-associated (UBA) domain. The three domains confer distinct activities to DDI1 proteins. The presence of RVP domain makes DDI1 a potential target of HIV protease inhibitors, which also block the development of malaria parasites. Hence, we investigated the DDI1 of malaria parasites to identify its roles during parasite development and potential as a therapeutic target. DDI1 proteins of Plasmodium and other Apicomplexan parasites share the UBL-RVP domain architecture, and some also contain the UBA domain. Plasmodium DDI1 is expressed across all the major life cycle stages and is essential, as conditional depletion of DDI1 protein in the mouse malaria parasite Plasmodium berghei and the human malaria parasite Plasmodium falciparum compromised parasite development. Infection of mice with DDI1 knock-down P. berghei was self-limiting and protected the recovered mice from subsequent infection with homologous as well as heterologous parasites, indicating potential of DDI1 knock-down parasites as a whole organism vaccine. P. falciparum DDI1 (PfDDI1) is associated with chromatin and DNA-protein crosslinks. PfDDI1-depleted parasites accumulated DNA-protein crosslinks and showed enhanced susceptibility to DNA damaging chemicals, indicating a role of PfDDI1 in removal of DNA-protein crosslinks. Knock-down of PfDDI1 increased susceptibility to the retroviral protease inhibitor lopinavir and antimalarial artemisinin, which suggests that simultaneous inhibition of DDI1 could potentiate antimalarial activity of these drugs. Hence, Plasmodium DDI1 could be a dual-therapeutic target for malaria control. A map of binary SARS-CoV-2 protein interactions implicates host immune regulation and ubiquitination Key steps in viral propagation, immune suppression, and pathology are mediated by direct, binary, physical interactions between viral and host proteins. To understand the biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we generated an unbiased systematic map of binary interactions between viral and host proteins, complementing previous co-complex association maps by conveying more direct mechanistic understanding and potentially enabling targeted disruption of direct interactions. To this end, we deployed two parallel strategies, identifying 205 virus-host and 27 intraviral binary interactions amongst 171 host and 19 viral proteins, and confirming high quality of these interactions via a calibrated orthogonal assay. Host proteins interacting with SARS-CoV-2 proteins are enriched in various cellular processes, including immune signaling and inflammation, protein ubiquitination, and membrane trafficking. Specific subnetworks provide new hypotheses related to viral modulation of host protein homeostasis and T-cell regulation. The binary virus-host protein interactions we identified can now be prioritized as targets for therapeutic intervention. More generally, we provide a resource of systematic maps describing which SARS-CoV-2 and human proteins interact directly. Motor cortical output for skilled forelimb movement is selectively distributed across projection neuron classes The interaction of descending neocortical outputs and subcortical premotor circuits is critical for shaping the skilled movements. Two broad classes of motor cortical output projection neurons provide input to many subcortical motor areas: pyramidal tract neurons (PT), which project throughout the neuraxis; and intratelencephalic neurons (IT), which project within cortex and subcortical striatum. It is unclear whether these classes are functionally in series or whether separable components of descending motor control signals are distributed across these distinct classes of projection neurons. Here we combine large-scale neural recordings across all layers of motor cortex with cell-type specific perturbations to study cortically-dependent mouse motor behaviors: kinematically-variable manipulation of a joystick and a kinematically-precise reach-to-grasp. We find that striatum-projecting IT neuron activity preferentially represents amplitude whereas pons-projecting PT neurons preferentially represent the variable direction of forelimb movements. Thus, separable components of descending motor cortical commands are distributed across motor cortical projection cell classes.

One-sentence summarySeparable components of cortical motor commands are distributed across distinct glutamatergic projection neuron cell-types. "Adopt a Microorganism" methodology enhances the discourse richness of high school students in both hybrid learning and remote education The Internet has changed the way teachers and students access information and build knowledge. The recent COVID-19 pandemic has created challenges for both teachers and students and a demand for new methodologies of remote learning. In the life sciences, mixing online content with practical activities represents an even greater challenge. In microbiology, the implementation of an active teaching methodology, the #Adopt project, based on the social network Facebook(R), represents an excellent option for connecting remote education with classroom activities. In 2020, the version applied in high school, "Adopt a Microorganism", was adapted to meet the demands of emergency remote education owing to the suppression of face-to-face activities caused by the pandemic. In the present study, we assessed how the change in methodology impacted the discourse richness of students from high school integrated with technical education in the Business Administration program of the Federal Institute of Sao Paulo, Sorocaba Campus. Three questionnaires related to the groups of microorganisms (Archaea, Bacteria, Virus, Fungi, and Protozoan) were applied. The students responses in the 2019 and 2020 classes were compared concerning content richness and multiplicity of concepts through the application of the Shannon diversity index, an approach that is generally used to assess biodiversity in different environments. The observed results suggest that remote learning provided students with a conceptual basis and richness of content equivalent to that achieved by students subjected to the hybrid teaching model. In conclusion, this study suggests that the #Adopt project methodology increases students discourse richness in microbiology even without face-to-face traditional classes. The transmission of African Animal Trypanosomiasis (Nagana) in two districts in the forest zone of Ghana The African Animal Trypanosomiasis (nagana) is caused by several species of trypanosome species which results in significant clinical diseases. This consequently leads to huge economic losses. We carried out a cross-sectional survey to investigate the composition of vectors, parasite diversity and intensities in two districts in the Eastern region of Ghana. Cytochrome c oxidase subunit 1 (COI) and internal transcribed spacer (ITS1) dependent PCR assays identified tsetse fly species and trypanosome parasites circulating in the area. A total of 229 tsetse flies, 65 pigs and 20 were sampled. Female and male flies 155 (51.8%), 74 (32.3%) respectively were collected with biconical traps. The overall vector density of 4.3 flies/trap/day was observed. Typanosome prevalence of 58.9% (95% CI: 52.5-65.1), 46.2% (95% CI: 34.6-58.1) and 0.0% (95% CI: 0.0-16.1) in tsetse flies, pigs and cattle respectively were detected. Trypanosoma congolense was the predominant species with a prevalence of 80.7% (95% CI: 73.3-86.5) in flies and 60.0% (95% CI: 42.3-75.4). There was evidence of multiple trypanosome infection with T. congolense/T. simiae occurring highest with prevalence of 38.0% (95% CI: 30.7-46.9). The parasite prevalence in pigs across the communities was high with significant differences associated between locations (Chi2 = 28.06, 95% CI: 0.05-0.81, P=0.0009). Blood meal with flies revealed feeding on both domestic Sus scrofa domesticus (pig) and Phacochoerus africanus (warthog). Tsetse flies in this area remain highly infected with trypanosomes and continue to transmit the parasites to livestock and human populations in the communities. Testing for phylogenetic signal in single-cell RNA-seq data AO_SCPLOWBSTRACTC_SCPLOWPhylogenetic methods are emerging as a useful tool to understand cancer evolutionary dynamics, including tumor structure, heterogeneity, and progression. Most currently used approaches utilize either bulk whole genome sequencing (WGS) or single-cell DNA sequencing (scDNA-seq) and are based on calling copy number alterations and single nucleotide variants (SNVs). Here we explore the potential of single-cell RNA sequencing (scRNA-seq) to reconstruct cancer evolutionary dynamics. scRNA-seq is commonly applied to explore differential gene expression of cancer cells throughout tumor progression. The method exacerbates the single-cell sequencing problem of low yield per cell with uneven expression levels. This accounts for low and uneven sequencing coverage and makes SNV detection and phylogenetic analysis challenging. In this paper, we demonstrate for the first time that scRNA-seq data contains sufficient evolutionary signal and can be utilized in phylogenetic analyses. We explore and compare results of such analyses based on both expression levels and SNVs called from scRNA-seq data. Both techniques are shown to be useful for reconstructing phylogenetic relationships between cells, reflecting the clonal composition of a tumor. Both standardized expression values and SNVs appear to be equally capable of reconstructing a similar pattern of phylogenetic relationship. This pattern is stable even when phylogenetic uncertainty is taken in account. Our results open up a new direction of somatic phylogenetics based on scRNA-seq data. Further research is required to refine and improve these approaches to capture the full picture of somatic evolutionary dynamics in cancer. ZIKV disrupts placental ultrastructure and drug transporter expression in mice Congenital Zika virus (ZIKV) infection can induce fetal brain abnormalities. Here, we investigated whether maternal ZIKV infection affects placental physiology and metabolic transport potential and impacts the fetal outcome, regardless of viral presence in the fetus at term. Low (103 PFU-ZIKVPE243; low ZIKV) and high (5x107 PFU-ZIKVPE243; high ZIKV) virus titers were injected into immunocompetent (ICompetent C57BL/6) and immunocompromised (ICompromised A129) mice at gestational day (GD) 12.5 for tissue collection at GD18.5 (term). High ZIKV elicited fetal death rates of 66% and 100%, whereas low ZIKV induced fetal death rates of 0% and 60% in C57BL/6 and A129 dams, respectively. All surviving fetuses exhibited intrauterine growth restriction (IUGR) and decreased placental efficiency. High-ZIKV infection in C57BL/6 and A129 mice resulted in virus detection in maternal spleens and placenta, but only A129 fetuses presented virus RNA in the brain. Nevertheless, pregnancies in both strains produced fetuses with decreased head sizes (p<0.05). Low-ZIKV-A129 dams had higher IL-6 and CXCL1 levels (p<0.05), and their placentas showed increased CCL-2 and CXCL-1 contents (p<0.05). In contrast, low-ZIKV-C57BL/6 dams had an elevated CCL2 serum level and increased type I and II IFN expression in the placenta. Notably, less abundant microvilli and mitochondrial degeneration were evidenced in the placental labyrinth zone (Lz) of ICompromised and high-ZIKV-ICompetent mice but not in low-ZIKV-C57BL/6 mice. In addition, decreased placental expression of the drug transporters P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) and the lipid transporter Abca1 was detected in all ZIKV-infected groups, but Bcrp and Abca1 were only reduced in ICompromised and high-ZIKV ICompetent mice. Our data indicate that gestational ZIKV infection triggers specific proinflammatory responses and affects placental turnover and transporter expression in a manner dependent on virus concentration and maternal immune status. Placental damage may impair proper fetal-maternal exchange function and fetal growth/survival, likely contributing to congenital Zika syndrome. Electrocommunication signals indicate motivation to compete during dyadic interactions of an electric fish Animals across species compete for limited resources. While in some species competition behavior is solely based on own abilities, others assess their opponents to facilitate these interactions. Using cues and communication signals, contestants gather information about their opponent, adjust their behavior accordingly, and can thereby avoid high costs of escalating fights. We tracked electrocommunication signals, in particular "rises", and agonistic behaviors of the gymnotiform electric fish Apteronotus leptorhynchus in staged competition experiments. A larger body size relative to the opponent was the sole significant predictor for winners. Sex and the frequency of the continuously emitted electric field were only mildly influencing competition outcome. In males, correlations of body-size and winning were stronger than in females and, especially when losing against females, communication and agonistic interactions were enhanced, hinting towards males being more motivated to compete. Fish that lost competitions emitted the majority of rises in dependence on the competitors relative size and sex. The emission of rises was costly since it provoked ritualized biting or chasing behaviors by the other fish. Despite winners being accurately predictable based on rise numbers already after the initial 25 minutes, losers continued to emit rises. The number of rises emitted by losers and the duration of chasing behaviors depended in similar ways on physical attributes of contestants. The detailed evaluation of these correlations hint towards A. leptorhynchus adjusting their competition behavior according to mutual assessment, where rises could signal a losers motivation to continue assessment through ritualized fighting. The contributions from the progenitor genomes of the mesopolyploid Brassiceae are evolutionarily distinct but functionally compatible The members of the tribe Brassiceae share a whole genome triplication (WGT), and one proposed model for its formation is a "two-step" pair of hybridizations producing hexaploid descendants. However, evidence for this model is incomplete, and the evolutionary and functional constraints that drove evolution after the hexaploidy are even less understood. Here we report a new genome sequence of Crambe hispanica, a species sister to most sequenced Brassiceae. Using this new genome and three others that share the hexaploidy, we traced the history of gene loss after the WGT using POInT (the Polyploidy Orthology Inference Tool). We confirm the two-step formation model and infer that there was a significant temporal gap between those two allopolyploidizations, with about a third of the gene losses from the first two subgenomes occurring prior to the arrival of the third. We also, for the 90,000 individual genes in our study, make parental "subgenome" assignments, inferring, with measured uncertainty, which of the progenitor genomes of the allohexaploidy each gene derives from. We further show that each subgenome has a statistically distinguishable rate of homoeolog losses. There is little indication of functional distinction between the three subgenomes: the individual subgenomes show no patterns of functional enrichment, no excess of shared protein-protein or metabolic interactions between their members, and no biases in their likelihood of having experienced a recent selective sweep. We propose a "mix and match" model of allopolyploidy, where subgenome origin drives homoeolog loss propensities but where genes from different subgenomes function together without difficulty. An alternative UPF1 isoform drives conditional remodeling of nonsense-mediated mRNA decay The nonsense-mediated mRNA decay (NMD) pathway monitors translation termination to degrade transcripts with premature stop codons and regulate thousands of human genes. Here we show that an alternative mammalian-specific isoform of the core NMD factor UPF1, termed UPF1LL, enables condition-dependent remodeling of NMD specificity. Previous studies indicate that the extension of a conserved regulatory loop in the UPF1LL helicase core confers a decreased propensity to dissociate from RNA upon ATP hydrolysis relative to the major UPF1 isoform, designated UPF1 SL. Using biochemical and transcriptome-wide approaches, we find that UPF1LL overcomes the protective RNA binding proteins PTBP1 and hnRNP L to preferentially bind and down-regulate long 3UTRs normally shielded from NMD. Unexpectedly, UPF1LL supports induction of NMD on new populations of substrate mRNAs in response to activation of the integrated stress response and impaired translation efficiency. Thus, while canonical NMD is abolished by moderate translational repression, UPF1LL activity is enhanced, providing a mechanism to rapidly rewire NMD specificity in response to cellular stress. Intermittent Bulk Release of Human Cytomegalovirus Human Cytomegalovirus (HCMV) can infect a variety of cell types by using virions of varying glycoprotein compositions. It is still unclear how this diversity is generated, but spatio-temporally separated envelopment and egress pathways might play a role. So far, one egress pathway has been described in which HCMV particles are individually enveloped into small vesicles and are subsequently exocytosed continuously. However, some studies have also found enveloped virus particles inside multivesicular structures but could not link them to productive egress or degradation pathways.

We used a novel 3D-CLEM workflow allowing us to investigate these structures in HCMV morphogenesis and egress at high spatio-temporal resolution. We found that multiple envelopment events occurred at individual vesicles leading to multiviral bodies (MViBs), which subsequently traversed the cytoplasm to release virions as intermittent bulk pulses at the plasma membrane to form extracellular virus accumulations (EVAs). Our data support the existence of a novel bona fide HCMV egress pathway, which opens the gate to evaluate divergent egress pathways in generating virion diversity. Orientation dependent proton transverse relaxation in the human brain white matter: The magic angle effect on a cylindrical helix PurposeTo overcome some limitations of prior orientation-dependent proton transverse relaxation formalisms in white matter (WM) with a novel framework based on the generalized magic angle effect function.

MethodsA cylindrical helix model was developed embracing anisotropic rotational and translational diffusion of restricted molecules in human brain WM, with the former characterized by an axially symmetric system. Transverse relaxation rates R2 and [Formula] were divided into isotropic [Formula] and anisotropic parts, [Formula], with  denoting an open angle and{varepsilon} 0 an orientation ({Phi}) offset from DTI-derived primary diffusivity direction. The proposed framework (Fit A) was compared with prior models without{varepsilon} 0 on previously published water and methylene proton transverse relaxation rates from developing, healthy, and pathological WM at 3T. Goodness of fit was represented by root-mean-square error (RMSE). F-test and linear correlation were used with statistical significance set to P [&le;] 0.05.

ResultsFit A significantly (P<0.01) outperformed prior models as demonstrated by reduced RMSEs, e.g., 0.349 vs. 0.724 in myelin water. Fitted{varepsilon} 0 was in good agreement with calculated{varepsilon} 0 from directional diffusivities. Compared with those from healthy adult, the fitted [Formula], and  from neonates were substantially reduced but{varepsilon} 0 increased, consistent with incomplete myelination. Significant positive [Formula] and negative ( and [Formula]) correlations were found with aging (demyelination) in elderly.

ConclusionThe developed framework can better characterize orientation dependences from a wide range of proton transverse relaxation measurements in human brain WM, shedding new light on myelin microstructural alterations at the molecular level. An automated workflow for multiplexed single-cell proteomics sample preparation at unprecedented sensitivity. The analysis of single-cell proteomes has recently become a viable complement to transcriptomics and genomics studies. Proteins are the primary driver of cellular functionality and mRNA levels are often an unreliable proxy of such. Therefore, the global analysis of the proteome is essential to study cellular identities. Multiplexed and label-free mass spectrometry-based approaches with single-cell resolution have lately attributed surprising heterogeneity to presumed homogenous cell populations. Even though specialized experimental designs and instrumentation have demonstrated remarkable advances, the efficient sample preparation of single cells still lag. Here, we introduce the proteoCHIP, a universal option for single-cell proteomics sample preparation at surprising sensitivity and throughput. The automated processing using a commercial system combining single-cell isolation and picoliter dispensing, the cellenONE(R), reduces final sample volumes to low nanoliters submerged in a hexadecane layer simultaneously eliminating error-prone manual sample handling and overcoming evaporation. The specialized proteoCHIP design allows direct injection of single cells via a standard autosampler resulting in around 1,500 protein groups per analytical run at remarkable reporter ion signal to noise while reducing or eliminating the carrier proteome. We identified close to 2,600 proteins across 170 multiplexed single cells from two highly similar human cell types. This dedicated loss-less workflow allows distinguishing in vitro co-differentiated cell types of self-organizing cardiac organoids based on indicative markers across 150 single cells. In-depth characterization revealed enhanced cellular motility of cardiac endothelial cells and sarcomere organization in cardiomyocytes. Our versatile and automated sample preparation has not only proven to be easily adaptable but is also sufficiently sensitive to drive biological applications of single-cell proteomics. Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. Morphology of immatures of the thelytokous ant, Monomorium triviale Wheeler (Formicidae: Myrmicinae: Solenopsidini) with descriptions of the extraordinary last-instar queen larvae The ant genus Monomorium is one of the most species-rich but taxonomically problematic groups in the hyperdiverse subfamily Myrmicinae. An East Asian species, M. triviale Wheeler, produces both reproductive queens and sterile workers via obligate thelytokous parthenogenesis. Here, we describe the immature forms of M. triviale based on light and scanning electron microscopy observations, with a note on the striking caste dimorphism in the last larval instar. The last-instar queen larvae were easily recognized by their large size, "aphaenogastroid" body shape, and rows of doorknob-like tubercles on the lateral and dorsal body surface. This type of queen-specific structure has not been found in ants in general, let alone congeneric species found in Japan. In stark contrast to the queen larvae, worker larvae showed a "pheidoloid" body shape and a body surface similar to other ants. The worker larvae were estimated to have three instars, consistent with previously described congeners. The pupae of both castes had no cocoon, a characteristic commonly described in other Myrmicinae species. In total, the developmental period from egg to adult worker averaged 59 days under 25{degrees}C. We discuss possible functions of the tubercles of queen larvae based on previous studies. Multi-omic analysis of the Arabidopsis clock activator mutant rve 4 6 8 reveals connections to carbohydrate metabolism and proteasome regulation Plants are able to sense changes in their light environments, such as the onset of day and night, as well as anticipate these changes in order to adapt and survive. Central to this ability is the plant circadian clock, a molecular circuit that precisely orchestrates plant cell processes over the course of a day. REVEILLE proteins (RVEs) are recently discovered members of the plant circadian circuitry that activate the evening complex and PRR genes to maintain regular circadian oscillation. The RVE 8 protein and its two homologs, RVE 4 and 6, have been shown to limit the length of the circadian period, with rve 4 6 8 triple-knockout plants possessing an elongated period along with increased leaf surface area, biomass, cell size and delayed flowering relative to wild-type Col-0 plants. Here, using a multi-omics approach consisting of phenomics, transcriptomics, proteomics, and metabolomics we draw novel connections between RVE8-like proteins and a number of core plant cell processes. In particular, we reveal that loss of RVE8-like proteins results in altered carbohydrate, organic acid and lipid metabolism, including a starch excess phenotype at dawn. We further demonstrate that rve 4 6 8 plants have lower levels of 20S proteasome subunits and possess significantly reduced proteasome activity, potentially explaining the increase in cell-size observed in RVE8-like mutants. Overall, this robust, multi-omic dataset, provides substantial new insights into the far reaching impact RVE8-like proteins have on the diel plant cell environment. SARS-CoV-2 variants resist antibody neutralization and broaden host ACE2 usage New SARS-CoV-2 variants continue to emerge from the current global pandemic, some of which can replicate faster and with greater transmissibility and pathogenicity. In particular, UK501Y.V1 identified in UK, SA501Y.V2 in South Africa, and BR501Y.V3 in Brazil are raising serious concerns as they spread quickly and contain spike protein mutations that may facilitate escape from current antibody therapies and vaccine protection. Here, we constructed a panel of 28 SARS-CoV-2 pseudoviruses bearing single or combined mutations found in the spike protein of these three variants, as well as additional nine mutations that within or close by the major antigenic sites in the spike protein identified in the GISAID database. These pseudoviruses were tested against a panel of monoclonal antibodies (mAbs), including some approved for emergency use to treat SARS-CoV-2 infection, and convalescent patient plasma collected early in the pandemic. SA501Y.V2 pseudovirus was the most resistant, in magnitude and breadth, against mAbs and convalescent plasma, followed by BR501Y.V3, and then UK501Y.V1. This resistance hierarchy corresponds with Y144del and 242-244del mutations in the N-terminal domain as well as K417N/T, E484K and N501Y mutations in the receptor binding domain (RBD). Crystal structural analysis of RBD carrying triple K417N-E484K-N501Y mutations found in SA501Y.V2 bound with mAb P2C-1F11 revealed a molecular basis for antibody neutralization and escape. SA501Y.V2 and BR501Y.V3 also acquired substantial ability to use mouse and mink ACE2 for entry. Taken together, our results clearly demonstrate major antigenic shifts and potentially broadening the host range of SA501Y.V2 and BR501Y.V3, which pose serious challenges to our current antibody therapies and vaccine protection. Notch signaling via Hey1 and Id2b regulates Muller glia's regenerative response to retinal injury Zebrafish Muller glia (MG) respond to retinal injury by suppressing Notch signaling and producing progenitors for retinal repair. A certain threshold of injury-derived signal must be exceeded in order to engage MG in a regenerative response (MGs injury-response threshold). Pan-retinal Notch inhibition expands the zone of injury-responsive MG at the site of focal injury, suggesting that Notch signaling regulates MGs injury-response threshold. We found that Notch signaling enhanced chromatin accessibility and gene expression at a subset of regeneration-associated genes in the uninjured retina. Two Notch effector genes, hey1 and id2b, were identified that reflect bifurcation of the Notch signaling pathway, and differentially regulate MGs injury-response threshold and proliferation of MG-derived progenitors. Furthermore, Notch signaling component gene repression in the injured retina suggests a role for Dll4, Dlb, and Notch3 in regulating Notch signaling in MG and epistasis experiments confirm that the Dll4/Dlb-Notch3-Hey1/Id2b signaling pathway regulates MGs injury-response threshold and proliferation. Identifying and correcting for misspecifications in GWAS summary statistics and polygenic scores Publicly available genome-wide association studies (GWAS) summary statistics exhibit uneven quality, which can impact the validity of follow-up analyses. First, we present an overview of possible misspecifications that come with GWAS summary statistics. Then, in both simulations and real data analyses, we show that additional information such as imputation INFO scores, allele frequencies, and per-variant sample sizes in GWAS summary statistics can be used to detect possible issues and correct for misspecifications in the GWAS summary statistics. One important motivation for us is to improve the predictive performance of polygenic scores built from these summary statistics. Unfortunately, due to the lack of reporting standards for GWAS summary statistics, this additional information is not systematically reported. We also show that using well-matched LD references can improve model fit and translate into more accurate prediction. Finally, we discuss how to make polygenic score methods such as lassosum and LDpred2 more robust to these misspecifications to improve their predictive power. Drug screening at single-organoid resolution via bioprinting and interferometry High-throughput drug screening is an established approach to investigate tumor biology and identify therapeutic leads. Traditional platforms for high-throughput screening use two-dimensional cultures of immortalized cell lines which do not accurately reflect the biology of human tumors. More clinically relevant model systems, such as three-dimensional tumor organoids, can be difficult to screen and scale. For example, manually seeded organoids coupled to destructive endpoint assays allow for the characterization of response to treatment, but do not capture the transitory changes and intra-sample heterogeneity underlying clinically observed resistance to therapy. We therefore developed a pipeline to generate bioprinted tumor organoids linked to label-free, real-time imaging via high-speed live cell interferometry (HSLCI) and machine learning-based quantitation of individual organoids. Bioprinting cells gives rise to 3D organoid structures that preserve tumor histology and gene expression. HSLCI imaging in tandem with machine learning-based image segmentation and organoid classification tools enables accurate, label-free parallel mass measurements for thousands of bioprinted organoids. We demonstrate that our method quantitatively identifies individual organoids as insensitive, transiently sensitive, or persistently sensitive to specific treatments. This opens new avenues for rapid, actionable therapeutic selection using automated tumor organoid screening.