The role of anatomical connection strength for interareal communication in macaque cortex What is the relationship between anatomical connection strength and rhythmic synchronization? Simultaneous recordings of 15 cortical areas in two macaque monkeys show that interareal networks are functionally organized in spatially distinct modules with specific synchronization frequencies, i.e. frequency-specific functional connectomes. We relate the functional interactions between 91 area pairs to their anatomical connection strength defined in a separate cohort of twenty six subjects. This reveals that anatomical connection strength predicts rhythmic synchronization and vice-versa, in a manner that is specific for frequency bands and for the feedforward versus feedback direction, even if interareal distances are taken into account. These results further our understanding of structure-function relationships in large-scale networks covering different modality-specific brain regions and provide strong constraints on mechanistic models of brain function. Because this approach can be adapted to non-invasive techniques, it promises to open new perspectives on the functional organization of the human brain.
Boosting L-type Ca2+ channel activity in tuberous sclerosis mitigates excess glutamate receptors Tuberous sclerosis complex (TS) is a dominant, multisystem disorder with devastating neurological symptoms. Approximately 85% of TS patients suffer from epilepsy over their lifespan and roughly 25-50% of those patients develop Autism Spectrum Disorder (1, 2). Current seizure therapies are effective in some, but not all, and often have significant risk factors associated with their use (1, 3). Thus, there is a critical need for new medication development or drug repositioning. Herein, we leveraged proteomic signatures of epilepsy and ASD, often comorbid in TS, to utilize an in silco approach to identify new drug therapies for TS-related seizures. We have discovered that activation of L-type voltage dependent calcium channels (L-VDCC) by Bay-K8644 (BayK) in a preclinical mouse model of TS rescues the excess expression of ionotropic, AMPA-subtype glutamate (GluA) receptors. As added proof of BayK working through L-VDCC to regulate GluA levels, we found that increasing expression of alpha2delta2 (2{delta}2), an auxiliary calcium channel subunit that boosts L-VDCC surface expression, similarly lowers the surface expression of dendritic GluA in TS. These BayK-induced molecular alterations may potentially improve the quality of life of humans suffering from TS.

Significance StatementCausal mechanisms of Tuberous Sclerosis (TS)-associated neurological disorders are under-characterized and treatment options are lacking. Using a computational approach of mTOR/DJ-1 target mRNAs to predict new medications, we report that boosting L-type voltage-dependent Ca2+ channel (L-VDCC) activity in a preclinical TS mouse model that exhibits a deficit in dendritic L-VDCC activity ameliorates key molecular pathologies that are predicted to underlie seizures. Restoring the mTOR/DJ-1 pathway upstream of L-VDCC, therefore, may serve as a new therapeutic avenue to mitigate seizures and mortality in TS.
A humanized nanobody phage display library yields potent binders of SARS CoV-2 spike Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants. Deciphering the potential niche of two novel black yeast fungi from a biological soil crust based on their genomes, phenotypes, and melanin regulation Black yeasts are polyextremotolerant fungi that contain high amounts of melanin in their cell wall and maintain a primarily yeast form. These fungi grow in xeric, nutrient deplete environments which implies that they require highly flexible metabolisms and the ability to form lichen-like mutualisms with nearby algae and bacteria. However, the exact ecological niche and interactions between these fungi and their surrounding community is not well understood. We have isolated two novel black yeast fungi of the genus Exophiala: E. viscosium and E. limosus, which are from dryland biological soil crusts. A combination of whole genome sequencing and various phenotyping experiments have been performed on these isolates to determine their fundamental niches within the biological soil crust consortium. Our results reveal that these Exophiala spp. are capable of utilizing a wide variety of carbon and nitrogen sources potentially from symbiotic microbes, they can withstand many abiotic stresses, and can potentially provide UV resistance to the crust community in the form of secreted melanin. Besides the identification of two novel species within the genus Exophiala, our study also provides new insight into the production and regulation of melanin in extremotolerant fungi. Define protein variant functions with high-complexity mutagenesis libraries and enhanced mutation detection software ASMv1.0 Open reading frame (ORF) variant libraries have advanced our ability to query the functions of a large number of variants of a protein simultaneously in a single experiment. Variant libraries targeting full-length ORFs typically consists of all possible single-amino-acid substitutions and a stop codon at each amino-acid position. Because a variant differs from the template ORF by a single codon variation, variant quantification presents the most profound challenge to this technology. Efforts such as dividing a library into sub-libraries for direct sequencing, or tag-directed subassembly are practical only for small ORFs. Our approach, however, features an enhanced variant-calling tool that processes massive shotgun sequencing data, allowing one to screen a single variant library for ORFs sized up to [~]3600 bases. The variant-calling tool detects variants reliably, and also presents variant annotations in datafiles enabling analyses that have reshaped our strategies governing library design, screen deconvolution, sequencing and sequencing data analysis. Tuft cell-produced cysteinyl leukotrienes and IL-25 synergistically initiate lung type 2 inflammation Aeroallergen sensing by airway epithelial cells can trigger pathogenic immune responses leading to chronic type 2 inflammation, the hallmark of airway diseases such as asthma. Airway tuft cells are specialized chemosensory epithelial cells and the dominant source of the epithelial cytokine IL-25 in the trachea and of cysteinyl leukotrienes (CysLTs) in the naive murine nasal mucosa. The interaction of IL-25 and CysLTs and the contribution of tuft cell-derived CysLTs to the development of allergen-triggered inflammation in the airways has not been clarified. Here we show that inhalation of LTC4 in combination with a subthreshold dose of IL-25 leads to dramatic synergistic induction of type 2 inflammation throughout the lungs, causing rapid eosinophilia, dendritic cell (DC) and inflammatory type 2 innate lymphoid cell (ILC2) expansion, and goblet cell metaplasia. While lung eosinophilia is dominantly mediated through the classical CysLT receptor CysLT1R, type 2 cytokines and activation of innate immune cells require signaling through both CysLT1R and CysLT2R. Tuft cell-specific deletion of the terminal enzyme requisite for CysLT production, Ltc4s, was sufficient to reduce both the innate immune response in the lung - eosinophilia, KLRG1+ ILC2 activation and DC recruitment - and the systemic immune response in the draining lymph nodes after inhalation of the mold aeroallergen Alternaria. Our findings identify surprisingly potent synergy of CysLTs and IL-25 downstream of aeroallergen-trigged activation of airway tuft cells leading to a highly polarized type 2 immune response and further implicate airway tuft cells as powerful modulators of type 2 immunity in the lungs.

One Sentence SummaryTuft cells produce two highly synergistic mediators: LTC4 and IL-25 to cooperatively induce allergen-driven airway inflammation. An integrative approach leads to the discovery of a novel anti-leukemic peptide from human milk Chemotherapy in childhood leukemia is associated with late morbidity in leukemic survivors, while certain patient subsets are relatively resistant to standard chemotherapy. It is therefore important to identify new agents with sensitivity and selectivity towards leukemic cells, while having less systemic toxicity. Peptide-based therapeutics has gained much attention during the last few years. Here, we used an integrative workflow combining mass spectrometric peptide library construction, in silico anticancer peptide screening, and in vitro leukemic cell studies to discover a novel anti-leukemic peptide having 3+charges and alpha-helical structure, namely HMP-S7, from human breast milk. HMP-S7 showed cytotoxic activity against four distinct leukemic cell lines in a dose-dependent manner but had no effect on solid malignancies or representative normal cells. HMP-S7 induced leukemic cell death by penetrating the plasma membrane to enter the cytoplasm and cause leakage of lactate dehydrogenase, thus acting in a membranolytic manner. Importantly, HMP-S7 exhibited anti-leukemic effect against patient-derived leukemic cells ex vivo. In conclusion, HMP-S7 is a selective anti-leukemic peptide with promise which requires further validation in preclinical and clinical studies.

TeaserIn silico screening of naturally occurring human milk peptides discovers a new anticancer peptide that kills leukemic cells in vitro and ex vivo. A terrain treadmill to study animal locomotion through large obstacles A major challenge to understanding locomotion in complex 3-D terrain with large obstacles is to create tools for controlled, systematic lab experiments. Existing terrain arenas only allow observations at small spatiotemporal scales ([~]10 body length, [~]10 stride cycles). Here, we create a terrain treadmill to enable high-resolution observations of animal locomotion through large obstacles over large spatiotemporal scales. An animal moves through modular obstacles on an inner sphere, while a rigidly-attached, concentric, transparent outer sphere rotated with the opposite velocity via closed-loop feedback to keep the animal on top. During sustained locomotion, a discoid cockroach moved through pillar obstacles for 25 minutes ({approx} 2500 strides) over 67 m ({approx} 1500 body lengths), and was contained within a radius of 4 cm (0.9 body length) for 83% of the duration, even at speeds of up to 10 body length/s. The treadmill enabled observation of diverse locomotor behaviors and quantification of animal-obstacle interaction.

Summary StatementA device keeps a small animal on top of a sphere while it traverses obstacles, creating a "terrain treadmill" to study locomotion over a long time and distance at high-resolution. Force correction analysis method for derivation of multidimensional free energy landscapes from adaptively-biased replica simulations A methodology is proposed for the calculation of multidimensional free-energy landscapes of molecular systems, based on analysis of multiple Molecular Dynamics trajectories wherein adaptive biases have been applied to enhance the sampling of different collective variables. In this approach, which we refer to as Force Correction Analysis Method (FCAM), local averages of the total and biasing forces are evaluated post-hoc, and the latter are subtracted from the former to obtain unbiased estimates of the mean force across collective-variable space. Multidimensional free-energy surfaces and minimum free-energy pathways are then derived from integration of the mean force landscape through kinetic Monte Carlo algorithm. To evaluate the proposed method, a series of numerical tests and comparisons with existing approaches were carried out for small molecules, peptides, and proteins, based on all-atom trajectories generated with standard, concurrent and replica-exchange Metadynamics in collective-variable spaces ranging from one- to six-dimensional. The tests confirm the correctness of the FCAM formulation and demonstrate that calculated mean forces and free energies converge rapidly and accurately, outperforming other methods used to unbias this kind of simulation data.



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C_FIG Ineffectual AEC1 differentiation from KRT8hi transitional state without fibrosis is associated with fatal COVID-19 ARDS 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. Allosteric activation of SARS-CoV-2 RdRp by remdesivir triphosphate and other phosphorylated nucleotides The catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), Nsp12, has a unique NiRAN domain that transfers nucleoside monophosphates to the Nsp9 protein. The NiRAN and RdRp modules form a dynamic interface distant from their catalytic sites and both activities are essential for viral replication. We report that codon-optimized (for the pause-free translation) Nsp12 exists in inactive state in which NiRAN/RdRp interactions are broken, whereas translation by slow ribosomes and incubation with accessory Nsp7/8 subunits or NTPs partially rescue RdRp activity. Our data show that adenosine and remdesivir triphosphates promote synthesis of A-less RNAs, as does ppGpp, while amino acid substitutions at the NiRAN/RdRp interface augment activation, suggesting that ligand binding to the NiRAN catalytic site modulates RdRp activity. The existence of allosterically-linked nucleotidyl transferase sites that utilize the same substrates has important implications for understanding the mechanism of SARS-CoV-2 replication and design of its inhibitors.

HighlightsO_LICodon-optimization of Nsp12 triggers misfolding and activity loss
C_LIO_LISlow translation, accessory Nsp7 and Nsp8 subunits, and NTPs rescue Nsp12
C_LIO_LINon-substrate nucleotides activate RNA chain synthesis, likely via NiRAN domain
C_LIO_LICrosstalk between two Nsp12 active sites that bind the same ligands
C_LI 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 migration: Associations between wing, and bill morphology and migration in kingbirds (Tyrannus) Morphology is closely linked to locomotion and diet in animals. In animals that undertake long-distance migrations, limb-morphology is under selection to maximize mobility and minimize energy expenditure. Migratory behaviors also interact with diet, such that migratory animals tend to be dietary generalists, while sedentary taxa tend to be dietary specialists. Despite a hypothesized link between migration status and morphology, phylogenetic comparative studies have yielded conflicting findings. We tested for evolutionary associations between migratory status and limb and bill morphology across kingbirds, a pan-American genus of birds with migratory, partially migratory, and sedentary taxa. Migratory kingbirds had longer wings, in agreement with expectations if selection favors improved aerodynamics for long-distance migration. We also found an association between migratory status and bill shape, such that more migratory taxa had wider, deeper, and shorter bills compared to sedentary taxa. However, there was no difference in intraspecific morphological variation among migrants, partial migrants, and residents, suggesting that dietary specialization has evolved independently of migration strategy. The evolutionary links between migration, diet, and morphology in kingbirds uncovered here further strengthen ecomorphological associations that underlie long-distance seasonal movements in animals. Embedded 3D printing in self-healing annealable composites for precise patterning of functionally mature human neural constructs Human in vitro models of neural tissue with controllable cellular identity, tunable microenvironment, and defined spatial arrangement are needed to facilitate studies of brain development and disease. Towards this end, embedded printing in jammed microgel supports (i.e., granular gels) holds great promise as it allows precise and programmable patterning of extremely soft and compliant tissue constructs. However, in contrast to the vast material landscape available for bulk hydrogels, granular printing support formulations are restricted to a handful of materials without the ability for facile adjustment of biofunctional properties of the cellular microenvironment. Therefore, there has been a need for novel materials that take advantage of versatile biomimicry of bulk hydrogels while providing high-fidelity support for embedded printing akin to granular gels. To address this need, we present a modular platform for bioengineering of neuronal networks via direct embedded 3D printing of human stem cells inside Self-Healing Annealable Particle-Extracellular matrix (SHAPE) composites. SHAPE composites consist of soft microgels immersed in viscous extracellular-matrix solution to enable precise freeform patterning of human stem cells and consequent generation and long-term maintenance of mature subtype-specific neurons that extend projections within the volume of the annealed support. The developed approach further allows multi-ink deposition, live spatial and temporal monitoring of oxygen levels, as well as creation of vascular channels. Due to its modularity, SHAPE biomanufacturing toolbox not only offers a solution for functional modeling of mechanically sensitive neural constructs, but also has potential to be applied to a wide range of biomaterials with different crosslinking mechanisms to model tissues and diseases where recapitulation of complex architectural features and topological cues is essential. The impact of housing conditions on mesenchymal stromal/stem cell populations differ between adipose tissue and skeletal muscle BackgroundIn pigs, the ratio between lean mass and fat mass in the carcass determines production efficiency and is strongly influenced by the number and size of cells in tissues. During growth, the increase in the number of cells results from the recruitment of different populations of multipotent mesenchymal stromal/stem cells (MSCs) residing in the tissues. We hypothesized that the impact of hygiene of housing conditions during growth on the proportions of MSCs in adipose tissue and skeletal muscle may differ between pigs with different residual feed intake (RFI), a measure of feed efficiency.

MethodsAt the age of 12 weeks, Large White pigs from two lines divergently selected for low and high RFI were housed in two contrasting hygiene conditions (good vs poor). After six weeks, pigs were slaughtered (n = 30; 5-9/group). Samples of subcutaneous adipose tissue and longissimus skeletal muscle were collected, and cells from the stromal vascular fraction (SVF), which includes mesenchymal stromal/stem cell populations, were isolated from each tissue. Adipose and muscle cell populations from the SVF were phenotyped by flow cytometry using antibodies that targeted different cell surface markers (CD45 to separate hematopoietic cells from MSCs; CD34, CD38, CD56 and CD140a to identify MSC populations with adipogenic and/or myogenic potential).

ResultsAdipose tissue and muscle shared some common MSC populations although MSC diversity was higher in muscle than in adipose tissue. In muscle, the CD45-CD56+CD34-CD140a+ and CD45-CD56+CD34+CD140a+ cell populations were abundant. Of these two cell populations, only the proportions of CD45-CD56+CD34+CD140a+ cells increased (P < 0.05) in pigs housed in poor hygiene as compared with pigs in good hygiene conditions. For the CD45-CD56-CD34- cell population, present in low proportion, there was an interaction between hygiene condition and genetic line (P < 0.05) with a decrease in low RFI pigs housed in poor hygiene conditions. In adipose tissue, the two abundant MSC populations were CD45-CD56-CD34- and CD45-CD56+CD34-. The proportion of CD45-CD56-CD34- cells increased (P < 0.05) whereas the proportion of CD45-CD56+CD34- tended to decrease (P < 0.1) in pigs housed in poor conditions. This study shows that the proportions of some MSC populations were affected by hygiene of housing conditions in a tissue-dependent manner in pigs of both RFI lines. It suggests that MSCs may play a significant role in adipose tissue and skeletal muscle homeostasis and may influence later growth and body composition in growing animals. "Neighbourhood watch" model: embryonic epiblast cells assess positional information in relation to their neighbours In many developing and regenerating systems, tissue pattern is established through gradients of informative morphogens, but we know little about how cells interpret these. Using experimental manipulation of early chick embryos including misexpression of an inducer (VG1 or ACTIVIN) and an inhibitor (BMP4), we test two alternative models for their ability to explain how the site of primitive streak formation is positioned relative to the rest of the embryo. In one model, cells read morphogen concentrations cell-autonomously. In the other, cells sense changes in morphogen status relative to their neighbourhood. We find that only the latter model can account for the experimental results, including some counter-intuitive predictions. This mechanism (which we name "neighbourhood watch" model) illuminates the classic "French Flag Problem" and how positional information is interpreted by a sheet of cells in a large developing system.

Summary statementIn a large developing system, the chick embryo before gastrulation, cells interpret gradients of positional signals relative to their neighbours to position the primitive streak, establishing bilateral symmetry. Comparing genome-based estimates of relatedness for use in pedigree-based conservation management Researchers have long debated which estimator of relatedness best captures the degree of relationship between two individuals. In the genomics era, this debate continues, with relatedness estimates being sensitive to the methods used to generate markers, marker quality, and levels of diversity in sampled individuals. Here, we compare six commonly used genome-based relatedness estimators (kinship genetic distance (KGD), Wang Maximum Likelihood (TrioML), Queller and Goodnight (Rxy), Kinship INference for Genome-wide association studies (KING-robust), and Pairwise Relatedness (RAB), allele-sharing co-ancestry (AS)) across five species bred in captivity-including three birds and two mammals-with varying degrees of reliable pedigree data, using reduced-representation and whole genome resequencing data. Genome-based relatedness estimates varied widely across estimators, sequencing methods, and species, yet the most consistent results for known first order relationships were found using Rxy, RAB, and AS. However, AS was found to be less consistently correlated with known pedigree relatedness than either Rxy or RAB. Our combined results indicate there is not a single genome-based estimator that is ideal across different species and data types. To determine the most appropriate genome-based relatedness estimator for each new dataset, we recommend assessing the relative: (1) correlation of candidate estimators with known relationships in the pedigree and (2) precision of candidate estimators with known first-order relationships. These recommendations are broadly applicable to conservation breeding programs, particularly where genome-based estimates of relatedness can complement and complete poorly pedigreed populations. Given a growing interest in the application of wild pedigrees, our results and are also applicable to in-situ wildlife management. A tool for assessing alignment of biomedical data repositories with open, FAIR, citation and trustworthy principles Increasing attention is being paid to the operation of biomedical data repositories in light of efforts to improve how scientific data is handled and made available for the long term. Multiple groups have produced recommendations for functions that biomedical repositories should support, with many using requirements of the FAIR data principles as guidelines. However, FAIR is but one set of principles that has arisen out of the open science community. They are joined by principles governing open science, data citation and trustworthiness, all of which are important aspects for biomedical data repositories to support. Together, these define a framework for data repositories that we call OFCT: Open, FAIR, Citable and Trustworthy. Here we developed an instrument using the open source PolicyModels toolkit that attempts to operationalize key aspects of OFCT principles and piloted the instrument by evaluating eight biomedical community repositories listed by the NIDDK Information Network (dkNET.org). Repositories included both specialist repositories that focused on a particular data type or domain, in this case diabetes and metabolomics, and generalist repositories that accept all data types and domains. The goal of this work was both to obtain a sense of how much the design of current biomedical data repositories align with these principles and to augment the dkNET listing with additional information that may be important to investigators trying to choose a repository, e.g., does the repository fully support data citation? The evaluation was performed from March to November 2020 through inspection of documentation and interaction with the sites by the authors. Overall, although there was little explicit acknowledgement of any of the OFCT principles in our sample, the majority of repositories provided at least some support for their tenets. DCIS genomic signatures define biology and correlate with clinical outcome: a Human Tumor Atlas Network (HTAN) analysis of TBCRC 038 and RAHBT cohorts Ductal carcinoma in situ (DCIS) is the most common precursor of invasive breast cancer (IBC), with variable propensity for progression. We have performed the first multiscale, integrated profiling of DCIS with clinical outcomes by analyzing 677 DCIS samples from 481 patients with 7.1 years median follow-up from the Translational Breast Cancer Research Consortium (TBCRC) 038 study and the Resource of Archival Breast Tissue (RAHBT) cohorts. We made observations on DNA, RNA, and protein expression, and generated a de novo clustering scheme for DCIS that represents a fundamental transcriptomic organization at this early stage of breast neoplasia. Distinct stromal expression patterns and immune cell compositions were identified. We found RNA expression patterns that correlate with later events. Our multiscale approach employed in situ methods to generate a spatially resolved atlas of breast precancers, where complementary modalities can be directly compared and correlated with conventional pathology findings, disease states, and clinical outcome.

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C_LIO_LIFour stroma-specific signatures identified.
C_LIO_LIutcome analysis identifies pathways involved in DCIS progression.
C_LIO_LICNAs characterize high risk of distant relapse IBC subtypes observed in DCIS.
C_LI Resetting of the 24-nt siRNA landscape in rice zygotes BackgroundThe zygote, a totipotent stem cell, constitutes a critical stage of the life cycle of sexually reproducing organisms. It is produced by the fusion of two differentiated cells -- the egg and sperm, which in plants have radically different siRNA transcriptomes from each other and from multicellular embryos. Due to technical challenges, the epigenetic changes that accompany the zygotic transition are poorly understood.

ResultsHere, we characterized the small RNA transcriptome of rice zygotes. We found widespread redistribution of 24-nt siRNAs relative to gametes, including absence of sperm signature siRNAs, reduction at egg signature siRNA loci, and upregulation at seedling signature siRNA loci. Loci with reduced siRNAs in zygote relative to egg were gene-distal and heterochromatic, while loci with increased siRNAs relative to egg had a similar genomic distribution to canonical siRNA loci. Although both egg and zygote siRNA loci had higher mCHH level in wildtype than in drm2 embryo, zygote but not egg siRNA loci were associated with hypermethylation in mature embryo. A small fraction of siRNA loci (~1%) called siren loci accounted for 60% of all siRNAs within zygote siRNA loci, that likely arose from maternal carryover as they had similarly abundant siRNAs in egg; these siren loci were not associated with embryo hypermethylation.

ConclusionsTaken together, our results indicate re-distribution of siRNAs in rice zygotes towards the canonical vegetative profile, that are consistent with the initiation of resetting of the gametic epigenome before the first embryonic division. Effect of urbanization and its environmental stressors on the intraspecific variation of flight functional traits in two bumblebee species The way urbanization shapes the intraspecific variation of pollinator functional traits is little understood. However, this topic is relevant for investigating ecosystem services and pollinator health. Here, we studied how urbanization affects the functional traits of workers in two bumblebee species (Bombus terrestris and B. pascuorum) sampled in 37 sites along a gradient of urbanization in North Italy (an area of 1800 km2 including the metropolitan context of Milan and other surrounding capital districts). Namely, we investigated the role played by land-use composition, configuration, temperature, flower resource abundance, and air pollutants on the variation of traits related to flight performance (i.e., body size, wing shape and size fluctuating asymmetry). These traits are relevant because they are commonly used as indicators of stress during insect development. The functional traits of the two bumblebees responded idiosyncratically to urbanization. Urban temperatures were associated with smaller wing sizes in B. pascuorum and with more accentuated fluctuating asymmetry of wing size in B. terrestris. Moreover, flower abundance correlated with bigger wings in B. terrestris and with less asymmetric wing sizes in B. pascuorum. Other traits did not vary significantly and other urban variables played minor effects. These patterns highlight that environmental stressors linked to urbanization negatively impact traits related to flight performance and development stability of these species with possible consequences on the pollination service they provide.

Overall, this study found species-specific variation patterns in syntopic taxa, expanding our understanding about the effects of anthropic disturbance in shaping relevant functional traits of pollinator model species. 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. Cell-type specific circadian bioluminescence rhythms in Dbp reporter mice Circadian rhythms are endogenously generated physiological and molecular rhythms with a cycle length of about 24 h. Bioluminescent reporters have been exceptionally useful for studying circadian rhythms in numerous species. Here, we report development of a reporter mouse generated by modification of a widely expressed and highly rhythmic gene encoding D-site albumin promoter binding protein (Dbp). In this line of mice, firefly luciferase is expressed from the Dbp locus in a Cre-recombinase-dependent manner, allowing assessment of bioluminescence rhythms in specific cellular populations. A mouse line in which luciferase expression was Cre-independent was also generated. The Dbp reporter alleles do not alter Dbp gene expression rhythms in liver or circadian locomotor activity rhythms. In vivo and ex vivo studies show the utility of the reporter alleles for monitoring rhythmicity. Our studies reveal cell-type specific characteristics of rhythms among neuronal populations within the suprachiasmatic nuclei ex vivo. In vivo studies show Dbp-driven bioluminescence rhythms in the liver of Albumin-Cre;DbpKI/+ "liver reporter" mice. After a shift of the lighting schedule, locomotor activity achieved the proper phase relationship with the new lighting cycle more rapidly than hepatic bioluminescence did. As previously shown, restricting food access to the daytime altered the phase of hepatic rhythmicity. Our model allowed assessment of the rate of recovery from misalignment once animals were provided with food ad libitum. These studies confirm the previously demonstrated circadian misalignment following environmental perturbations and reveal the utility of this model for minimally invasive, longitudinal monitoring of rhythmicity from specific mouse tissues. A Bayesian method to cluster single-cell RNA sequencing data using Copy Number Alterations MotivationCancers are composed by several heterogeneous subpopulations, each one harbouring different genetic and epigenetic somatic alterations that contribute to disease onset and therapy response. In recent years, copy number alterations leading to tumour aneuploidy have been identified as potential key drivers of such populations, but the definition of the precise makeup of cancer subclones from sequencing assays remains challenging. In the end, little is known about the mapping between complex copy number alterations and their effect on cancer phenotypes.

ResultsWe introduce CONGAS, a Bayesian probabilistic method to phase bulk DNA and single-cell RNA measurements from independent assays. CONGAS jointly identifies clusters of single cells with subclonal copy number alterations, and differences in RNA expression. The model builds statistical priors leveraging bulk DNA sequencing data, does not require a normal reference and scales fast thanks to a GPU backend and variational inference. We test CONGAS on both simulated and real data, and find that it can determine the tumour subclonal composition at the single-cell level together with clone-specific RNA phenotypes in tumour data generated from both 10x and Smart-Seq assays.

AvailabilityCONGAS is available as 2 packages: CONGAS (https://github.com/caravagnalab/congas), which implements the model in Python, and RCONGAS (https://caravagnalab.github.io/rcongas/), which provides R functions to process inputs, outputs, and run CONGAS fits. The analysis of real data and scripts to generate figures of this paper are available via RCONGAS; code associated to simulations is available at https://github.com/caravagnalab/rcongas_test.

Contactgcaravagna@units.it

Supplementary informationSupplementary data are available at Bioinformatics online. Genome-wide association study of musical beat synchronization demonstrates high polygenicity Moving in synchrony to the beat is a fundamental component of musicality. Here, we conducted a genome-wide association study (GWAS) to identify common genetic variants associated with beat synchronization in 606,825 individuals. Beat synchronization exhibited a highly polygenic architecture, with sixty-nine loci reaching genome-wide significance (p<5x10-8) and SNP-based heritability (on the liability scale) of 13%-16%. Heritability was enriched for genes expressed in brain tissues, and for fetal and adult brain-specific gene regulatory elements, underscoring the role of central nervous system-expressed genes linked to the genetic basis of the trait. We performed validations of the self-report phenotype (through internet-based experiments) and of the GWAS (polygenic scores for beat synchronization were associated with patients algorithmically classified as musicians in medical records of a separate biobank). Genetic correlations with breathing function, motor function, processing speed, and chronotype suggest shared genetic architecture with beat synchronization and provide avenues for new phenotypic and genetic explorations. Discovery of Aminoglycosides as First in Class, Nanomolar Inhibitors of Heptosyltransferase I A clinically relevant inhibitor for Heptosyltransferase I (HepI) has been sought after for many years and while many have designed novel small-molecule inhibitors, these compounds lack the bioavailability and potency necessary for therapeutic use. Extensive characterization of the HepI protein has provided valuable insight into the dynamic motions necessary for catalysis that could be targeted for inhibition. With the help of molecular dynamic simulations, aminoglycoside antibiotics were shown to be putative inhibitors for HepI and in this study, they were experimentally determined to be the first in-class nanomolar inhibitors of HepI with the best inhibitor demonstrating a Ki of 600 +/- 90 nM. Detailed kinetic analyses were performed to determine the mechanism of inhibition while circular dichroism spectroscopy, intrinsic tryptophan fluorescence, docking, and MD simulations were used to corroborate kinetic experimental findings. Kinetic analysis methods include Lineweaver-Burk, Dixon, Cornish-Bowden and Mixed-Model of Inhibition which allowed for unambiguous assignment of inhibition mechanism for each inhibitor. In this study, we show that neomycin and kanamycin b are competitive inhibitors against the sugar acceptor substrate while tobramycin exhibits a mixed inhibitory effect and streptomycin is non-competitive. MD simulations also allowed us to suggest that the inhibitors bind tightly and inhibit catalytic dynamics due to a major desolvation penalty of the enzyme active site. While aminoglycosides have long been known as a class of potent antibiotics, they also have been scientifically shown to impact cell membrane stability, and we propose that inhibition of HepI contributes to this effect by disrupting lipopolysaccharide biosynthesis.