title: Genomic and Spectral Visual Adaptation in Southern Leopard Frogs during the Ontogenetic Transition from Aquatic to Terrestrial Light Environments

abstract: Many animals have complex life cycles where larval and adult forms have distinct ecologies and habitats that impose different demands on their sensory systems. While the adaptive decoupling hypothesis predicts reduced genetic correlations between life stages, how sensory systems adapt across life stages at the molecular level is not well understood. Frogs are a compelling system to study this question in because most species rely on vision as both aquatic tadpoles and terrestrial adults, but these habitats present vastly different light environments. Here we used whole eye transcriptome sequencing to investigate differential expression between aquatic tadpoles and terrestrial juveniles of the southern leopard frog (Lithobates sphenocephalus). Because visual physiology changes with light levels, we also tested how constant light or dark exposure affected gene expression. We found 42% of genes were differentially expressed in the eyes of tadpoles versus juveniles, versus 5% for light/dark exposure. Analyses targeting a curated set of visual genes revealed significant differential expression between life stages in genes that control aspects of visual function and development, including spectral sensitivity and lens composition. Light/dark exposure had a significant effect on a smaller set of visual genes. Finally, microspectrophotometry of photoreceptors confirmed shifts in spectral sensitivity predicted by the expression results, consistent with adaptation to distinct light environments. Overall, we identified extensive expression-level differences in the eyes of tadpole and juvenile frogs related to observed morphological and physiological changes through metamorphosis, and corresponding adaptive shifts to optimize vision in aquatic versus terrestrial environments.


title: Selective stimulation of the ferret abdominal vagus nerve with multi-contact nerve cuff electrodes

abstract: Dysfunction and diseases of the gastrointestinal (GI) tract are a major driver of medical care. The vagus nerve innervates and controls multiple organs of the GI tract and vagus nerve stimulation (VNS) could provide a means for affecting GI function and treating disease. However, the vagus nerve also innervates many other organs throughout the body, and off-target effects of VNS could cause major side effects such as changes in blood pressure. In this study, we aimed to achieve selective stimulation of populations of vagal afferents using a multi-contact cuff electrode wrapped around the abdominal trunks of the vagus nerve. Four-contact nerve cuff electrodes were implanted around the dorsal (N=3) or ventral (N=3) abdominal vagus nerve in six ferrets, and the response to stimulation was measured via a 32-channel microelectrode array (MEA) inserted into the nodose ganglion. Selectivity was characterized by the ability to evoke responses in MEA channels through one bipolar pair of cuff contacts but not through the other bipolar pair. We demonstrated that is was possible to selectively activate subpopulations of vagal afferents using abdominal VNS. Additionally, we quantified the conduction velocity of evoked responses to determine what types of nerve fibers (i.e. A{delta} vs. C) responded to stimulation. We also quantified the spatial organization of evoked responses in the nodose MEA to determine if there is somatotopic organization of the neurons in that ganglion. Finally, we demonstrated in a separate set of three ferrets that stimulation of the abdominal vagus via a four-contact cuff could selectively alter gastric myoelectric activity, suggesting that abdominal VNS can potentially be used to control GI function.

category: bioengineering


title: Harnessing the Power of Antibodies to Fight Bone Metastasis

abstract: Over the past 20 years, antibody-based therapies have proved to be of great value in cancer treatment. Despite the clinical success of these biopharmaceuticals, reaching targets in the bone micro-environment has proved to be difficult perhaps due to the relatively low vascularization of bone tissue and the presence of physical barriers that impair drug penetration. Here, we have used an innovative bone targeting (BonTarg) technology to generate a first-in-class bone-targeting anti-body. Moreover, we have used two xenograft models to demonstrate the enhanced therapeutic efficacy of this bone-targeting antibody against bone metastases, compared to the efficacy of traditional antibodies. Our strategy involves the use of pClick antibody conjugation technology to chemically couple the bone-targeting moiety bisphosphonate to the human epidermal growth factor receptor 2 (HER2)-specific antibody trastuzumab. Bisphosphonate modification of therapeutic antibodies results in delivery of higher conjugate concentrations to the bone metastatic niche, relative to other tissues. In both HER2-positive and negative xenograft mice models, this strategy provides enhanced inhibition of experimental bone metastases as well as multi-organ secondary metastases that arise from the bone lesions. Specific delivery of therapeutic antibodies to the bone therefore represents a promising strategy for the treatment of bone metastatic cancers and other bone diseases.

category: cancer biology


title: Relevance of network topology for the dynamics of biological neuronal networks

abstract: Complex random networks provide a powerful mathematical framework to study high-dimensional physical and biological systems. Several features of network structure (e.g. degree correlation, average path length, clustering coefficient) are correlated with descriptors of network dynamics and function. However, it is not clear which features of network structure relate to the dynamics of biological neuronal networks (BNNs), characterized by non-linear nodes with high in- and out degrees, but being weakly connected and communicating in an event-driven manner, i.e. only when neurons spike. To better understand the structure-dynamics relationship in BNNs, we analysed the structure and dynamics of > 9, 000 BNNs with different sizes and topologies. In addition, we also studied the effect of network degeneration on neuronal network structure and dynamics. Surprisingly, we found that the topological class (random, small-world, scale-free) was not an indicator of the BNNs activity state as quantified by the firing rate, network synchrony and spiking regularity. In fact, we show that different network topologies could result in similar activity dynamics. Furthermore, in most cases, the network activity changes did not depend on the rules according to which neurons or synapses were pruned from the networks. The analysis of dynamics and structure of the networks we studied revealed that the effective synaptic weight (ESW) was the most crucial feature in predicting the statistics of spiking activity in BNNs. ESW also explained why different synapse and neuron pruning strategies resulted in almost identical effects on the network dynamics. Thus, our findings provide new insights into the structure-dynamics relationships in BNNs. Moreover, we argue that network topology and rules by which BNNs degenerate are irrelevant for BNN activity dynamics. Beyond neuroscience, our results suggest that in large networks with non-linear nodes, the effective interaction strength among the nodes, instead of the topological network class, may be a better predictor of the network dynamics and information flow.

category: neuroscience


title: Biochemical Methane Potential (BMP) from sugarcane biorefinery residues: maximizing their use by co-digestion

abstract: This is a pioneer study evaluating the methane (CH4) production potential from residues of integrated 1st (vinasse and filter cake) and 2nd (deacetylation pretreatment liquor from straw) generation (1G2G) sugarcane biorefinery, providing a fully chemical characterization of them and their relation with the anaerobic digestion (AD) process. Small-scale assays provided fundamentals for basing the co-digestion optimization by assessing the optimal co-substrates synergistic conditions. Biochemical Methane Potential (BMP) tests showed co-digestion enhanced CH4 yield of isolated substrates, reaching up to 605 NmLCH4 gVS-1. The association of vinasse and deacetylation liquor as co-substrates increased the BMP by ~38% mostly by nutritionally benefiting the methanogenic activity. The kinetic analysis confirmed that the deacetylation liquor was the co-substrate responsible for improving the CH4 production in the co-digestion systems due to the highest CH4 conversion rate. The alkaline characteristic of the liquor (pH~12) also prevented alkalizing from being added to the co-digestion, an input that normally makes the process economically unfeasible to implement on an industrial scale due to the large quantities required for buffering the reactor. The filter cake had the lowest BMP (262 NmLCH4 gVS-1) and digestibility ([≤] 40%), further limited by the required stirring to improve the mass transfer of biochemical reactions. The present study drives towards more sustainable use of vinasse, the most voluminous waste from the sugarcane industry, and lignin-rich residues derived from pre-treatment alkaline methods, aiming at an energy-efficient utilization, by at least 16% when compared to the traditional vinasse AD. The experimental and modeling elements from this work indicated the lignin-rich liquor is the main responsible for putting the co-digestion as a disruptive technological arrangement within the 1G2G sugarcane biorefineries, reinforcing the biogas production as the hub of the bioeconomy in the agroindustrial sector.


title: Anaerobic Thermophilic Co-digestion of Sugarcane Biorefinery Residues Enhances Biogas Production


title: Use of lignocellulosic residue from 2G ethanol production to enhance methane production through co-digestion


title: Use of lignocellulosic residue from second-generation ethanol production to enhance methane production through co-digestion


title: VAMP2 AND SYNAPTOTAGMINS ARE RELATIVELY IMMOBILE ON CHROMAFFIN GRANULE MEMBRANES: IMPLICATIONS FOR MEMBRANE FUSION AND FUSION PORE EXPANSION

abstract:
Although many of the proteins of secretory granules have been identified, little is known about their molecular organization and diffusion characteristics. Granule-plasma membrane fusion can only occur when proteins that enable fusion are present at the granule-plasma membrane contact. Thus, the mobility of granule membrane proteins may be an important determinant of fusion pore formation and expansion. To address this issue, we measured the mobility of (fluorophore-tagged) vesicle associated membrane protein 2 (VAMP2), synaptotagmin 1 (Syt1), and synaptotagmin 7 (Syt7) in chromaffin granule membranes in living chromaffin cells. We used a method that is not limited by standard optical resolution. A bright flash of strongly decaying evanescent field ([~]80 nm exponential decay constant) produced by total internal reflection (TIR) was used to photobleach GFP-labeled proteins in the granule membrane. Fluorescence recovery occurs as unbleached protein in the granule membrane distal from the glass interface diffuses into the more bleached proximal regions, thereby enabling the measurement of diffusion coefficients. The studies revealed that VAMP2, Syt1, and Syt7 are relatively immobile in chromaffin granules membranes with diffusion constants of [≤] 3 x 10-10 cm2/s. Utilizing these diffusion parameters and the known density of VAMP2 and Syt 1 on synaptic vesicles, we estimated the time required for these proteins to arrive at a nascent fusion site to be tens of milliseconds. We propose that the mobilities of secretory granule SNARE and Syt proteins, heretofore unappreciated factors, influence the kinetics of exocytosis and protein discharge.

Significance StatementIn eukaryotic cells, secretory vesicles fuse with the plasma membrane to secrete chemical transmitters, hormones and proteins that enable diverse physiological functions including neurotransmission. Fusion proteins need to be assembled at the fusion site in sufficient number in order to enable membrane fusion. However, the diffusion characteristics of fusogenic proteins on secretory vesicles remained unknown. Here we used a novel method not limited by standard optical resolution to measure the diffusion of VAMP2 and synaptotagmins on chromaffin granule membranes. We found they have limited mobility. The time required for these proteins to reach the granule-plasma membrane contact site suggests that their limited mobility likely influences the kinetics of membrane fusion and subsequent fusion pore expansion.

category: biophysics


title: Interacting brains revisited: A cross-brain network neuroscience perspective

abstract: Elucidating the neural basis of social behavior is a long-standing challenge in neuroscience. Such endeavors are driven by attempts to extend the isolated perspective on the human brain by considering interacting persons brain activities, but a theoretical and computational framework for this purpose is still in its infancy. Here, we posit a comprehensive framework based on bipartite graphs for interbrain networks and address whether they provide meaningful insights into the neural underpinnings of social interactions. First, we show that the nodal density of such graphs exhibits nonrandom properties. While the current analyses mostly rely on global metrics, we encode the regions roles via matrix decomposition to obtain an interpretable network representation yielding both global and local insights. With Bayesian modeling, we reveal how synchrony patterns seeded in specific brain regions contribute to global effects. Beyond inferential inquiries, we demonstrate that graph representations can be used to predict individual social characteristics, outperforming functional connectivity estimators for this purpose. In the future, this may provide a means of characterizing individual variations in social behavior or identifying biomarkers for social interaction and disorders.


title: CheSPI: Chemical shift Secondary structure Population Inference

abstract: NMR chemical shifts (CSs) are delicate reporters of local protein structure, and recent advances in random coil CS (RCCS) prediction and interpretation now offer the compelling prospect of inferring small populations of structure from small deviations from RCCSs. Here, we present CheSPI, a simple and efficient method that provides unbiased and sensitive aggregate measures of local structure and disorder. It is demonstrated that CheSPI can predict even very small amounts of residual structure and robustly delineate subtle differences into four structural classes for intrinsically disordered proteins. For structured regions and proteins, CheSPI can assign up to eight structural classes, which coincide with the well-known DSSP classification. The program is freely available, and can either be invoked from URL www.protein-nmr.org as a web implementation, or run locally from command line as a python program. CheSPI generates comprehensive numeric and graphical output for intuitive annotation and visualization of protein structures. A number of examples are provided.

category: biochemistry


title: Kinetic Interval Measurement: A Tool to Characterize Thermal Reversion Dynamics of Light-switchable Fluorescent Proteins

abstract: Light-switchable proteins like Light-Oxygen-Voltage (LOV) domains can be used to control protein interactions and have been applied in vivo to manipulate cell behavior. The switching between dark and light state of LOV domains depends on temperature or their chemical microenvironment and can be tuned by point mutations. Here, we present a method called Kinetic Interval Measurement (KIM) to quantify the thermal reversion dynamics of light-switchable proteins by using a custom microplate reader. We show that this versatile method can be used to determine the reversion half-life of the excited state of LOV proteins in a reproducible, fast and simple manner consuming only small amounts of protein. The sensitivity of the method allows to report on changes in temperature and imidazole concentration as well as the photoswitching dynamics of LOV proteins in living cells.


title: KLF2 is a therapeutic target for COVID-19 induced endothelial dysfunction

abstract: Coronavirus disease 2019 (COVID-19) is regarded as an endothelial disease (endothelialitis) with its mechanism being incompletely understood. Emerging evidence has demonstrated that the endothelium represents the Achilles' heel in COVID-19 patients and that endothelial dysfunction precipitates COVID-19 and accompanying multi-organ injuries. Thus, pharmacotherapies targeting endothelial dysfunction have potential to ameliorate COVID-19 and its cardiovascular complications. Primary human umbilical vein endothelial cells (HUVECs) and human pulmonary microvascular endothelial cells (HPMECs) were treated with serum from control subjects or COVID-19 patients. Downstream monocyte adhesion and associated gene/protein expression was evaluated in endothelial cells exposed to COVID-19 patient serum in the presence of KLF2 activator (Atorvastatin) or KLF2 overexpression by an adenoviral vector. Here, we demonstrate that the expression of KLF2 was significantly reduced and monocyte adhesion was increased in endothelial cells treated with COVID-19 patient serum due to elevated levels of pro-adhesive molecules, ICAM1 and VCAM1. IL-1{beta} and TNF-, two cytokines observed in cytokine release syndrome in COVID-19 patients, decreased KLF2 gene expression. Next-generation RNA-sequencing data showed that atorvastatin treatment leads to a cardiovascular protective transcriptome associated with improved endothelial function (vasodilation, anti-inflammation, antioxidant status, anti-thrombosis/-coagulation, anti-fibrosis and reduced angiogenesis). Treatment of HPMECs with atorvastatin or KLF2 adenovirus ameliorate COVID-19 serum-induced increase in endothelial inflammation and monocyte adhesion by increasing KLF2 expression. Altogether, the present study demonstrates that genetic and pharmacological activation of KLF2 represses COVID-19 associated endothelial dysfunction, heralding a potentially new direction to treat endothelialitis accompanying COVID-19.

category: pharmacology and toxicology


title: Losartan Increases Survival of the Fbn1mgR/mgR Mouse Model of Marfan Syndrome in and Age-Dependent Manner

abstract: Clinical trials investigating angiotensin receptor blockers (ARB) for attenuation of thoracic aortic aneurysm in people with Marfan syndrome have demonstrated variable efficacy. The primary objective of this study was to determine whether the age of mice at the time of losartan initiation affected mortality in fibrillin-1 hypomorphic (Fbn1mgR/mgR) mice. Male (n=40) and female (n=28) Fbn1mgR/mgR mice were randomized to receive losartan in drinking water (0.6 g/L) starting at either 24 or 50 days of age. Controls included Fbn1mgR/mgR mice (20M, 14F) and wild type (15M, 15F) littermates who were not administered the drug. Mortality of Fbn1mgR/mgR males receiving losartan at postnatal day 24 (P24) was not different from wild type controls (p=0.138). Survival of Fbn1mgR/mgR males administered losartan at P50 was not different compared to Fbn1mgR/mgR males receiving no drug (p=0.194) and decreased compared to wild type mice (p=0.002). Survival analysis after P50 demonstrated increased survival of Fbn1mgR/mgR males administered losartan at P50 compared to Fbn1mgR/mgR mice receiving no drug (p=0.017). Age is a critical variable that affects the therapeutic efficacy of losartan in male Fbn1mgR/mgR mice. Since overall mortality in female Fbn1mgR/mgR mice was lower than in male Fbn1mgR/mgR mice, a survival benefit with losartan was not detected in females.

category: physiology


title: A neural circuit basis for bilateral olfactory input-enhanced chemosensory avoidance navigation

abstract: Our understanding of how bilaterian animals utilize parallel input channels from paired sensory organs to optimize chemosensory behavior and the underlying neural circuit mechanisms are limited. Here we developed microfluidics-based behavioral and brainwide imaging platforms to study the neural integration of binasal inputs and chemosensory avoidance in larval zebrafish. We show that larval zebrafish efficiently escape from cadaverine-carrying streams by making more frequent swim bouts and larger undirected turns. Binasal inputs are strictly required for the nasal input-dependent component of klinokinesis, while each nasal input additively enhances angular orthokinesis. Throughout brain regions, including those along the olfactory processing pathways, a distributed neural representation with a wide spectrum of ipsilateral-contralateral nasal stimulus selectivity is maintained. Nonlinear sensory information gain with bilateral signal convergence is especially prominent in neurons weakly encoding unilateral cadaverine stimulus, and associated with stronger activation of sensorimotor neurons in the downstream brain regions. Collectively, these results provide insights into how the vertebrate model sums parallel input signals to guide chemosensory avoidance behavior.


title: A neural circuit basis for binasal input-enhanced chemosensory avoidance


title: Behavioral deficits, learning impairment, and enhanced hippocampal excitability in co-isogenic PrnpZH3/ZH3 mice

abstract:
BackgroundThe cellular prion protein (PrPC) has been associated with numerous cellular processes, such as cell differentiation and neurotransmission. Moreover, it was recently demonstrated that some functions were misattributed to PrPC since results were obtained from mouse models with genetic artifacts. Here we elucidate the role of PrPC in the hippocampal circuitry and its related functions, like learning and memory, using the new strictly co-isogenic Prnp0/0 mouse (PrnpZH3/ZH3). Behavioral and operant conditioning tests were performed to evaluate memory and learning capabilities. In vivo electrophysiological recordings were carried out at CA3-CA1 synapses in living behaving mice, and spontaneous neuronal firing and network formation were monitored in primary neuronal cultures of PrnpZH3/ZH3 vs. wild-type mice.

ResultsResults showed decreased motility, impaired operant conditioning learning, and anxiety-related behavior in PrnpZH3/ZH3 animals. PrPC absence enhanced susceptibility to high-intensity stimulations and kainate-induced seizures. However, long-term potentiation (LTP) was not enhanced in the PrnpZH3/ZH3 hippocampus. In addition, we observed a delay in neuronal maturation and network formation in PrnpZH3/ZH3 cultures.

ConclusionIn conclusion, PrPC mediates synaptic function and protects the synapse from excitotoxic insults. Its deletion might evoke a susceptible epileptogenic brain that would fail to perform highly cognitive-demanding tasks such as associative learning and anxiety-like behaviors.


title: Daily orexinergic modulation of the rat superficial layers of the superior colliculus - implications for intrinsic clock activities in the visual system

abstract: The orexinergic system delivers excitation for multiple brain centres to facilitate behavioural arousal, with its malfunction resulting in narcolepsy, somnolence, and notably, visual hallucinations. Since the circadian clock underlies the daily arousal, a timed coordination is expected between the orexin system and its target subcortical visual system, including the superior colliculus (SC). Here, we use a combination of electrophysiological, immunohistochemical, and molecular approaches across 24 h, together with the neuronal tract tracing methods in rodents to elucidate the daily coordination between the orexin system and the superficial layers of the SC. We find the daily orexinergic innervation onto the SC, coinciding with the daily silencing of spontaneous firing in this visual brain area. We identify autonomous daily and circadian expression of clock genes in the SC, which may underlie these day-night changes. Additionally, we establish the lateral hypothalamic origin of orexin innervation to the SC and that the SC neurons robustly respond to orexin A via OX2 receptor in both excitatory and GABAA receptor-dependent inhibitory manners. Together, our evidence supports that the clock coordination between the orexinergic input and its response in the SC provides arousal-related excitation needed to detect sparse visual information during the behaviourally active phase.


title: STAG proteins promote cohesin ring loading at R-loops.

abstract: Most studies of cohesin function consider the Stromalin Antigen (STAG/SA) proteins as core complex members given their ubiquitous interaction with the cohesin ring. Here, we provide functional data to support the notion that the SA subunit is not a mere passenger in this structure, but instead plays a key role in cohesins localization to diverse biological processes and promotes loading of the complex at these sites. We show that in cells acutely depleted for RAD21, SA proteins remain bound to chromatin and interact with CTCF, as well as a wide range of RNA binding proteins involved in multiple RNA processing mechanisms. Accordingly, SA proteins interact with RNA and are localised to endogenous R-loops where they act to suppress R-loop formation. Our results place SA proteins on chromatin upstream of the cohesin complex and reveal a role for SA in cohesin loading at R-loops which is independent of NIPBL, the canonical cohesin loader. We propose that SA takes advantage of this structural R-loop platform to link cohesin loading and chromatin structure with diverse genome functions. Since SA proteins are pan-cancer targets, and R-loops play an increasingly prevalent role in cancer biology, our results have important implications for the mechanistic understanding of SA proteins in cancer and disease.

category: cell biology


title: Multiple PDZ Domain Protein Maintains Patterning of the Apical Cytoskeleton in Sensory Hair Cells

abstract: Sound transduction occurs in the hair bundle, the apical compartment of sensory hair cells in the inner ear. The hair bundle is formed of stereocilia aligned in rows of graded heights. It was previously shown that the GNAI-GPSM2 complex is part of a developmental blueprint that defines the polarized organization of the apical cytoskeleton in hair cells, including stereocilia distribution and elongation. Here we report a novel and critical role for Multiple PDZ domain (MPDZ) protein during apical hair cell morphogenesis. We show that MPDZ is enriched at the hair cell apical membrane, and required there to maintain the proper segregation of apical blueprints proteins, including GNAI-GPSM2. Loss of the blueprint coincides with misaligned stereocilia in Mpdz mutants, and results in permanently misshapen hair bundles. Graded molecular and structural defects along the cochlea can explain the profile of hearing loss in Mpdz mutants, where deficits are most severe at high frequencies.

category: developmental biology


title: Early embryogenesis and organogenesis in the annelid Owenia fusiformis

abstract:
BackgroundAnnelids are a diverse group of segmented worms within Spiralia, whose embryos exhibit spiral cleavage and a variety of larval forms. While most modern embryological studies focus on species with unequal spiral cleavage nested in Pleistoannelida (Sedentaria + Errantia), a few recent studies looked into Owenia fusiformis, a member of the sister group to all remaining annelids and thus a key lineage to understand annelid and spiralian evolution and development. However, the timing of early cleavage and detailed morphogenetic events leading to the formation of the idiosyncratic mitraria larva of O. fusiformis remain largely unexplored.

ResultsO. fusiformis undergoes equal spiral cleavage where the first quartet of animal micromeres are slightly larger than the vegetal macromeres. Cleavage results in a coeloblastula approximately five hours post fertilization (hpf) at 19 {degrees}C. Gastrulation occurs via invagination and completes four hours later, with putative mesodermal precursors and the chaetoblasts appearing 10 hpf at the dorsoposterior side. Soon after, at 11 hpf, the apical tuft emerges, followed by the first neurons (as revealed by the expression of elav1 and synaptotagmin1) in the apical organ and the prototroch by 13 hpf. Muscles connecting the chaetal sac to various larval tissues develop around 18 hpf and by the time the mitraria is fully formed at 22 hpf, there are FMRFamide+ neurons in the apical organ and prototroch, the latter forming a prototrochal ring. As the mitraria feeds, it grows in size and the prototroch expands through active proliferation. The larva becomes competent after [~]3 weeks post fertilization at 15 {degrees}C, when a conspicuous juvenile rudiment has formed ventrally.

ConclusionsO. fusiformis embryogenesis is similar to that of other equal spiral cleaving annelids, supporting that equal cleavage is associated with the formation of a coeloblastula, gastrulation via invagination, and a feeding trochophore-like larva in Annelida. The nervous system of the mitraria larva forms earlier and is more complex than previously recognised and develops from anterior to posterior, which is likely an ancestral condition to Annelida. Altogether, our study identifies the major developmental events during O. fusiformis ontogeny, defining a conceptual framework for future investigations.


title: Unique functions for Notch4 in murine embryonic lymphangiogenesis

abstract: In mice, embryonic dermal lymphatic development is well-understood and used to study gene functions in lymphangiogenesis. Notch signaling is an evolutionarily conserved pathway that modulates cell fate decisions, which has been shown to both inhibit and promote dermal lymphangiogenesis. Here, we demonstrate distinct roles for Notch4 signaling versus canonical Notch signaling in embryonic dermal lymphangiogenesis. Actively growing embryonic dermal lymphatics expressed NOTCH1, NOTCH4 and DLL4 which correlated with Notch activity. In lymphatic endothelial cells (LECs), DLL4 activation of Notch induced a subset of Notch effectors and lymphatic genes, which were distinctly regulated by Notch1 and Notch4 activation. Treatment of LECs with VEGF-A or VEGF-C upregulated Dll4 transcripts, and differentially and temporally regulated the expression of Notch1 and Hes/Hey genes. Mice nullizygous for Notch4 had an increase in the closure of the lymphangiogenic fronts which correlated with reduced vessel caliber in the maturing lymphatic plexus at E14.5, and reduced branching at E16.5. Activation of Notch4 suppressed LEC migration in a wounding assay significantly more than Notch1, suggesting a dominant role for Notch4 in regulating LEC migration. Unlike Notch4 nulls, inhibition of canonical Notch signaling by expressing a dominant negative form of MAML1 (DNMAML) in Prox1+ LECs led to increased lymphatic density consistent with an increased in LEC proliferation, described for the loss of LEC Notch1. Moreover, loss of Notch4 did not affect LEC canonical Notch signaling. Thus, we propose that Notch4 signaling and canonical Notch signaling have distinct functions in the coordination of embryonic dermal lymphangiogenesis.


title: A conserved paint box underlies color pattern diversity in Estrildid finches

abstract: Many animals exhibit typical color patterns that have been linked to key adaptive functions, yet the developmental mechanisms establishing these crucial designs remain unclear. Here, we surveyed color distribution in the plumage across a large number of passerine finches. Despite extreme apparent pattern diversity, we identified a small set of conserved color regions whose combinatory association can explain all observed patterns. We found these domains are instructed by early embryonic landmarks, and through profiling and comparative analyses produced a molecular map marking putative color domains in the developing skin. This revealed cryptic pre-patterning common to differently colored species, uncovering a simple molecular landscape underlying extensive color pattern variation.


title: Oxford Nanopore sequencing-based protocol to detectCpG methylation in human mitochondrial DNA

abstract:
Methylation on CpG residues is one of the most important epigenetic modifications of nuclear DNA, regulating gene expression. Methylation of mitochondrial DNA (mtDNA) has been studied using whole genome bisulfite sequencing (WGBS), but recent evidence has uncovered major technical issues which introduce a potential bias during methylation quantification. Here, we validate the technical concerns with WGBS, and then develop and assess the accuracy of a protocol for variant-specific methylation identification using long-read Oxford Nanopore Sequencing. Our approach circumvents mtDNA-specific confounders, while enriching for native full-length molecules over nuclear DNA. Variant calling analysis against Illumina deep re-sequencing showed that all expected mtDNA variants can be reliably identified. Methylation calling revealed negligible mtDNA methylation levels in multiple human primary and cancer cell lines. In conclusion, our protocol enables the reliable analysis of epigenetic modifications of mtDNA at single-molecule level at single base resolution, with potential applications beyond methylation.

MotivationAlthough whole genome bisulfite sequencing (WGBS) is the gold-standard approach to determine base-level CpG methylation in the nuclear genome, emerging technical issues raise questions about its reliability for evaluating mitochondrial DNA (mtDNA) methylation. Concerns include mtDNA strand asymmetry rendering the C-rich light strand disproportionately vulnerable the chemical modifications introduced with WGBS. Also, short-read sequencing can result in a co-amplification of nuclear sequences originating from ancestral mtDNA with a high nucleotide similarity. Lastly, calling mtDNA alleles with varying proportions (heteroplasmy) is complicated by the C-to-T conversion introduced by WGBS on unmethylated CpGs. Here, we propose an alternative protocol to quantify methyl-CpGs in mtDNA, at single-molecule level, using Oxford Nanopore Sequencing (ONS). By optimizing the standard ONS library preparation, we achieved selective enrichment of native mtDNA and accurate single nucleotide variant and CpG methylation calling, thus overcoming previous limitations.

category: genomics


title: Waves of sumoylation support transcription dynamics during adipocyte differentiation

abstract: Tight control of gene expression networks required for adipose tissue formation and plasticity is essential for adaptation to energy needs and environmental cues. However, little is known about the mechanisms that orchestrate the dramatic transcriptional changes leading to adipocyte differentiation. We investigated the regulation of nascent transcription by the sumoylation pathway during adipocyte differentiation using SLAMseq and ChIPseq. We discovered that the sumoylation pathway has a dual function in differentiation; it supports the initial downregulation of pre-adipocyte-specific genes, while it promotes the establishment of the mature adipocyte transcriptional program. By characterizing sumoylome dynamics in differentiating adipocytes by mass spectrometry, we found that sumoylation of specific transcription factors like Ppar{gamma}/RXR and their co-factors is associated with the transcription of adipogenic genes. Our data demonstrate that the sumoylation pathway coordinates the rewiring of transcriptional networks required for formation of functional adipocytes. This study also provides an in-depth resource of gene transcription dynamics, SUMO-regulated genes and sumoylation sites during adipogenesis.


title: Distinguishing dopamine and calcium responses using XNA-nanotube sensors for improved neurochemical sensing

abstract:
To date, the engineering of single-stranded DNA-SWCNT (DNA-SWCNT) optical biosensors have largely focused on creating sensors for new applications with little focus on optimising existing sensors for in vitro and in vivo conditions. Recent studies have shown that nanotube fluorescence can be severely impacted by changes in local cation concentrations. This is particularly problematic for neurotransmitter sensing applications as spatial and temporal fluctuations in the concentration of cations, such as Na+, K+, or Ca2+, play a central role in neuromodulation. This can lead to inaccuracies in the determination of neurotransmitter concentrations using DNA-SWCNT sensors, which limits their use for detecting and treating neurological diseases.

Herein, we present new approaches using locked nucleic acid (LNA) to engineer SWCNT sensors with improved stability towards cation-induced fluorescence changes. By incorporating LNA bases into the (GT)15-DNA sequence, we create sensors that are not only more resistant towards undesirable fluorescence modulation in the presence of Ca2+ but that also retain their capabilities for the label-free detection of dopamine. The synthetic biology approach presented in this work therefore serves as a complementary means for enhancing nanotube optoelectronic behavior, unlocking previously unexplored possibilities for developing nano-bioengineered sensors with augmented capabilities.


title: quincunx: an R package to query, download and wrangle PGS Catalog data

abstract:
MotivationThe Polygenic Score (PGS) Catalog is a recently established open database of published polygenic scores that, to date, has collected, curated, and made available 721 polygenic scores from over 133 publications. The PGS Catalog REST API is the only method allowing programmatic access to this resource.

ResultsHere, we describe quincunx, an R package that provides the first client interface to the PGS Catalog REST API. quincunx enables users to query and quickly retrieve, filter and integrate metadata associated with polygenic scores, as well as polygenic scoring files in tidy table format.

Availabilityquincunx is freely available under an MIT License, and can be accessed from https://github.com/maialab/quincunx.

category: bioinformatics


title: High Light and High Temperature Reduce Photosynthesis via Different Mechanisms in the C4 Model Setaria viridis

abstract: C4 plants frequently experience damaging high light (HL) and high temperature (HT) conditions in native environments, which reduce growth and yield. However, the mechanisms underlying these stress responses in C4 plants have been under-explored, especially the coordination between mesophyll (M) and bundle sheath (BS) cells. We investigated how the C4 model plant Setaria viridis responded to a four-hour HL or HT treatment at the photosynthetic, transcriptomic, and ultrastructural levels. Although we observed a comparable reduction of photosynthetic efficiency in HL- or HT-treated leaves, detailed analysis of multi-level responses revealed important differences in key pathways and M/BS specificity responding to HL and HT. We provide a systematic analysis of HL and HT responses in S. viridis, reveal different acclimation strategies to these two stresses in C4 plants, discover unique light/temperature responses in C4 plants in comparison to C3 plants, and identify potential targets to improve abiotic stress tolerance in C4 crops.

category: plant biology


title: Preprints in motion: tracking changes between posting and journal publication

abstract: Amidst the COVID-19 pandemic, preprints in the biomedical sciences are being posted and accessed at unprecedented rates, drawing widespread attention from the general public, press and policymakers for the first time. This phenomenon has sharpened longstanding questions about the reliability of information shared prior to journal peer review. Does the information shared in preprints typically withstand the scrutiny of peer review, or are conclusions likely to change in the version of record? We assessed preprints from bioRxiv and medRxiv that had been posted and subsequently published in a journal through 30th April 2020, representing the initial phase of the pandemic response. We utilised a combination of automatic and manual annotations to quantify how an article changed between the preprinted and published version. We found that the total number of figure panels and tables changed little between preprint and published articles. Moreover, the conclusions of 7.2% of non-COVID-19-related and 17.2% of COVID-19-related abstracts undergo a discrete change by the time of publication, but the majority of these changes do not qualitatively change the conclusions of the paper.

category: scientific communication and education


title: Preprints in motion: tracking changes between preprint posting and journal publication during a pandemic


title: Antigenic waves of virus-immune co-evolution

abstract: The evolution of many microbes and pathogens, including circulating viruses such as seasonal influenza, is driven by immune pressure from the host population. In turn, the immune systems of infected populations get updated, chasing viruses even further away. Quantitatively understanding how these dynamics result in observed patterns of rapid pathogen and immune adaptation is instrumental to epidemiological and evolutionary forecasting. Here we present a mathematical theory of co-evolution between immune systems and viruses in a finite-dimensional antigenic space, which describes the cross-reactivity of viral strains and immune systems primed by previous infections. We show the emergence of an antigenic wave that is pushed forward and canalized by cross-reactivity. We obtain analytical results for shape, speed, and angular diffusion of the wave. In particular, we show that viral-immune co-evolution generates a new emergent timescale, the persistence time of the waves direction in antigenic space, which can be much longer than the coalescence time of the viral population. We compare these dynamics to the observed antigenic turnover of influenza strains, and we discuss how the dimensionality of antigenic space impacts on the predictability of the evolutionary dynamics. Our results provide a concrete and tractable framework to describe pathogen-host co-evolution.

category: evolutionary biology


title: Target enrichment and extensive population sampling help untangle the recent, rapid radiation of Oenothera sect. Calylophus

abstract: Oenothera sect. Calylophus is a North American group of 13 recognized taxa in the evening primrose family (Onagraceae) with an evolutionary history that may include independent origins of bee pollination, edaphic endemism, and permanent translocation heterozygosity. Like other groups that radiated relatively recently and rapidly, taxon boundaries within Oenothera sect. Calylophus have remained challenging to circumscribe. In this study, we used target enrichment, flanking non-coding regions, summary coalescent methods, tests for gene flow modified for target-enrichment data, and morphometric analysis to reconstruct phylogenetic hypotheses, evaluate current taxon circumscriptions, and examine character evolution in Oenothera sect. Calylophus. Because sect. Calylophus comprises a clade with a relatively restricted geographic range, we were able to extensively sample across the range of geographic and morphological diversity in the group. We found that the combination of exons and flanking non-coding regions led to improved support for species relationships. We reconstructed potential hybrid origins of some accessions and note that if processes such as hybridization are not taken into account, the number of inferred evolutionary transitions may be artificially inflated. We recovered strong evidence for multiple origins of the evolution of bee pollination from ancestral hawkmoth pollination, the evolution of edaphic specialization on gypsum, and permanent translocation heterozygosity. This study applies newly emerging techniques alongside dense infraspecific sampling and morphological analyses to effectively address a relatively common but recalcitrant problem in evolutionary biology.


title: A Multiscale Multicellular Spatiotemporal Model of Local Influenza Infection and Immune Response

abstract: Respiratory viral infections pose a serious public health concern, from mild seasonal influenza to pandemics like those of SARS-CoV-2. Spatiotemporal dynamics of viral infection impact nearly all aspects of the progression of a viral infection, like the dependence of viral replication rates on the type of cell and pathogen, the strength of the immune response and localization of infection. Mathematical modeling is often used to describe respiratory viral infections and the immune response to them using ordinary differential equation (ODE) models. However, ODE models neglect spatially-resolved biophysical mechanisms like lesion shape and the details of viral transport, and so cannot model spatial effects of a viral infection and immune response. In this work, we develop a multiscale, multicellular spatiotemporal model of influenza infection and immune response by combining non-spatial ODE modeling and spatial, cell-based modeling. We employ cellularization, a recently developed method for generating spatial, cell-based, stochastic models from non-spatial ODE models, to generate much of our model from a calibrated ODE model that describes infection, death and recovery of susceptible cells and innate and adaptive responses during influenza infection, and develop models of cell migration and other mechanisms not explicitly described by the ODE model. We determine new model parameters to generate agreement between the spatial and original ODE models under certain conditions, where simulation replicas using our model serve as microconfigurations of the ODE model, and compare results between the models to investigate the nature of viral exposure and impact of heterogeneous infection on the time-evolution of the viral infection. We found that using spatially homogeneous initial exposure conditions consistently with those employed during calibration of the ODE model generates far less severe infection, and that local exposure to virus must be multiple orders of magnitude greater than a uniformly applied exposure to all available susceptible cells. This strongly suggests a prominent role of localization of exposure in influenza A infection. We propose that the particularities of the microenvironment to which a virus is introduced plays a dominant role in disease onset and progression, and that spatially resolved models like ours may be important to better understand and more reliably predict future health states based on susceptibility of potential lesion sites using spatially resolved patient data of the state of an infection. We can readily integrate the immune response components of our model into other modeling and simulation frameworks of viral infection dynamics that do detailed modeling of other mechanisms like viral internalization and intracellular viral replication dynamics, which are not explicitly represented in the ODE model. We can also combine our model with available experimental data and modeling of exposure scenarios and spatiotemporal aspects of mechanisms like mucociliary clearance that are only implicitly described by the ODE model, which would significantly improve the ability of our model to present spatially resolved predictions about the progression of influenza infection and immune response.

category: systems biology


title: DNA-Encoded Multivalent Display of Protein Tetramers on Phage: Synthesis and In Vivo Aplications

abstract:
Phage display links phenotype of displayed polypeptides with DNA sequence in phage genome and offers a universal method for discovery of proteins with novel properties. Injection of phage-displayed libraries in living organisms further provides a unique and powerful approach to optimize biochemical, pharmacological and biological properties of the displayed peptides, antibodies and other proteins in vivo. However, over 60% of the proteome is comprised of multi-domain proteins, and display of large multi-subunit proteins on phages remains a challenge. Majority of protein display systems are based on monovalent phagemid constructs but methods for robust display of multiple copies of large proteins are scarce. Here, we describe a DNA-encoded display of a [~]200 kDa tetrameric protein tetrameric L-asparaginase on M13 phage produced by ligation of SpyCatcher-Asparaginase fusion (ScA) to prospectively barcoded phage clones displaying SpyTag peptide. Starting from the SpyTag display on p3 minor coat protein or p8 major coat protein yielded constructs with five copies of ScA displayed on p3 (ScA5-phage) and 50 copies of ScA on p8 protein (ScA50-phage). ScA remained active after conjugation. It could be easily produced directly from lysates of bacteria that express ScA. Display constructs of different valency can be injected into mice and analyzed by deep-sequencing of the DNA barcodes associated phage clones. In these multiplexed studies, we observed a density-dependent clearance rate in vivo. A known clearance mechanism of L-asparaginase is endocytosis by phagocytic cells. Our observations, thus, link the increase in density of the displayed protein with the increased rate of the endocytosis by cells in vivo. In conclusion, we demonstrate that a multivalent display of L-asparaginase on phage could be used to study the circulation life of this protein in vivo and such approach opens the possibility to use DNA sequencing to investigate multiplexed libraries of other multi-subunit proteins in vivo.

Abstract Graphic

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/432100v1_ufig1.gif" ALT="Figure 1">
View larger version (27K):
org.highwire.dtl.DTLVardef@18edf0org.highwire.dtl.DTLVardef@ad55c6org.highwire.dtl.DTLVardef@1da47d8org.highwire.dtl.DTLVardef@1a2ec32_HPS_FORMAT_FIGEXP  M_FIG C_FIG

category: molecular biology


title: Widespread cryptic variation in genetic architecture between the sexes

abstract: The majority of the genome is shared between the sexes, and it is expected that the genetic architecture of most traits is shared as well. This common architecture has been viewed as a major source of constraint on the evolution of sexual dimorphism (SD). SD is nonetheless common in nature, leading to assumptions that it results from differential regulation of shared genetic architecture. Here, we study the effect of thousands of gene knock-out mutations on 202 mouse phenotypes to explore how regulatory variation affects SD. We show that many traits are dimorphic to some extent, and that a surprising proportion of knock-outs have sex-specific phenotypic effects. Many traits, regardless whether they are monomorphic or dimorphic, harbor cryptic differences in genetic architecture between the sexes, resulting in sexually discordant phenotypic effects from sexually concordant regulatory changes. This provides an alternative route to dimorphism through sex-specific genetic architecture, rather than differential regulation of shared architecture.


title: Condensates in RNA Repeat Sequences are Heterogeneously Organized and Exhibit Reptation-like Dynamics

abstract: Although it is known that RNA undergoes liquid-liquid phase separation (LLPS), the interplay between the molecular driving forces and the emergent features of the condensates, such as their morphologies and dynamical properties, is not well understood. We introduce a coarse-grained model to simulate phase separation of trinucleotide repeat RNAs, which are implicated in neurological disorders such as Huntington disease and amyotrophic lateral sclerosis. After establishing that the simulations reproduce key experimental findings (length and concentration dependence of the phase transition in (CAG)n repeats), we show that once recruited inside the liquid droplets, the monomers transition from hairpin-like structures to extended states. Interactions between the monomers in the condensates result in the formation of an intricate and dense intermolecular network, which severely restrains the fluctuations and mobilities of the RNAs inside large droplets. In the largest densely packed high viscosity droplets, the mobility of RNA chains is best characterized by reptation, reminiscent of the dynamics in polymer melts. Our work provides a microscopic framework for understanding liquid-liquid phase separation in RNA, which is not easily discernible in current experiments.


title: Microstates and power envelope hidden Markov modeling probe bursting brain activity at different timescales

abstract: State modeling of whole-brain electroencephalography (EEG) or magnetoencephalography (MEG) allows to investigate transient, recurring neurodynamical events. Two widely-used techniques are the microstate analysis of EEG signals and hidden Markov modeling (HMM) of MEG power envelopes. Both reportedly lead to similar state lifetimes on the 100 ms timescale, suggesting a common neural basis. We addressed this issue by using simultaneous MEG/EEG recordings at rest and comparing the spatial signature and temporal activation dynamics of microstates and power envelope HMM states obtained separately from EEG and MEG. Results showed that microstates and power envelope HMM states differed both spatially and temporally. Microstates tend to exhibit spatio-temporal locality, whereas power envelope HMM states disclose network-level activity with 100-200 ms lifetimes. Further, MEG microstates do not correspond to the canonical EEG microstates but are better interpreted as split HMM states. On the other hand, both MEG and EEG HMM states involve the (de)activation of similar functional networks. Microstate analysis and power envelope HMM thus appear sensitive to neural events occurring over different spatial and temporal scales. As such, they represent complementary approaches to explore the fast, sub-second scale bursting electrophysiological dynamics in spontaneous human brain activity.


title: Comparative analysis of 16S rRNA gene and metagenome sequencing in pediatric gut microbiomes

abstract: The colonization of the human gut microbiome begins at birth, and, over time, these microbial communities become increasingly complex. Most of what we currently know about the human microbiome, especially in early stages of development, was described using culture-independent sequencing methods that allow us to identify the taxonomic composition of microbial communities using genomic techniques, such as amplicon or shotgun metagenomic sequencing. Each method has distinct tradeoffs, but there has not been a direct comparison of the utility of these methods in stool samples from very young children, which have different features than those of adults. We compared the effects of profiling the human infant gut microbiome with 16S rRNA amplicon versus shotgun metagenomic sequencing techniques in 130 fecal samples; younger than 15, 15-30, and older than 30 months of age. We demonstrate that observed changes in alpha-diversity and beta-diversity with age occur to similar extents using both profiling methods. We also show that 16S rRNA profiling identified a larger number of genera and we find several genera that are missed or underrepresented by each profiling method. We present the link between alpha diversity and shotgun metagenomic sequencing depth for children of different ages. These findings provide a guide for selecting an appropriate method and sequencing depth for the three studied age groups.

category: microbiology


title: A genome-wide circular RNA transcriptome in Rat

abstract:
BackgroundCircular RNAs are a novel class of non-coding RNAs that backsplice from 5 donor site and 3 acceptor site to form a circular structure. A number of circRNAs have been discovered in model organisms including human, mouse, Drosophila, among other organisms. There are a few candidate-based studies on circular RNAs in rat, a well studied model organism. The availability of a recent dataset of transcriptomes encompassing 11 tissues, 4 developmental stages and 2 genders motivated us to explore the landscape of circular RNAs in the organism.

MethodologyIn order to understand the difference among different pipelines, we have used the same bodymap RNA sequencing dataset. A number of pipelines have been published to identify the backsplice junctions for the discovery of circRNAs but studies comparing these tools have suggested that a combination of tools would be a better approach to identify high-confidence circular RNAs. We employed 5 different combinations of tools including tophat_CIRCexplorer2, segemehl_CIRCexplorer2, star_CIRCexplorer, Bowtie2_findcirc and Bowtie2_findcirc (noHisat2) to identify circular RNAs from the dataset.

ResultsOur analysis identified a number of tissue-specific, developmental stage specific and gender specific circular RNAs. We further independently validated 16 circRNA junctions out of 24 selected candidates in 5 tissue samples. We additionally estimated the quantitative expression of 5 circRNA candidates using real-time PCR and our analysis suggests 3 candidates as tissue-enriched

ConclusionThis study is one of the most comprehensive studies that provides a circular RNA transcriptome as well as to understand the difference among different computational pipelines in Rat.


title: Intrinsic and growth-mediated cell and matrix specialization during meniscus tissue assembly

abstract: The incredible mechanical strength and durability of mature fibrous tissues and their extremely limited turnover and regenerative capacity underscores the importance of proper matrix assembly during early postnatal growth. In tissues with composite extracellular matrix (ECM) structures, such as the adult knee meniscus, fibrous (Collagen-I rich) and cartilaginous (Collagen-II, proteoglycan-rich) matrix components are regionally segregated to the outer and inner portions of the tissue. While this spatial variation in composition is appreciated to be functionally important for resisting complex mechanical loads associated with gait, the establishment of these specialized zones is poorly understood. To address this issue, the following study tracked the growth of the murine meniscus from its embryonic formation through its first month of growth, encompassing the critical time-window during which animals begin to ambulate and weight bear. Using histological analysis, region specific high-throughput qPCR, and Col-1 and Col-2 fluorescent reporter mice, we found that matrix and cellular features defining specific tissue zones were already present at birth, before continuous weight-bearing had occurred. These differences were further refined with postnatal growth and maturation, resulting in specialization of mature tissue regions. Taken together, this work establishes a detailed timeline of the concurrent spatiotemporal changes that occur at both the cellular and matrix level throughout meniscus maturation. The findings of this study provide a framework for investigating the reciprocal feedback between cells and their evolving microenvironments during assembly of a mechanically robust fibrocartilage tissue, thus providing insight into mechanisms of tissue degeneration and effective regenerative strategies.


title: A Ratio-Dependent Model of Replicator-Genetic Parasite Coevolution Demonstrates Instability of the Parasite-Free State

abstract: Nearly all organisms on earth are hosts to diverse genetic parasites including viruses and various types of mobile genetic elements. The emergence and persistence of genetic parasites was hypothesized to be an intrinsic feature of biological evolution. Here we examine this proposition by analysis of a ratio-dependent Lotka-Volterra type model of replicator(host)-parasite coevolution where the evolutionary outcome depends on the ratio of the host and parasite numbers. In a large, unbounded domain of the space of the model parameters, which include the replicator carrying capacity, the damage inflicted by the parasite, the replicative advantage of the parasites, and its mortality rate, the parasite-free equilibrium takes the form of a saddle and accordingly is unstable. Therefore, the evolutionary outcome is either the stable coexistence of the replicator and the parasite or extinction of both. Thus, the results of ratio-dependent model analysis are compatible with the hypothesis that genetic parasites are inherent to life.


title: Ku70 suppresses alternative end-joining in G1-arrested progenitor B cells

abstract:
Classical nonhomologous end-joining (C-NHEJ) repairs DNA double-stranded breaks (DSBs) throughout interphase but predominates in G1-phase when homologous recombination is unavailable. Complexes containing the Ku70/80 ("Ku") and XRCC4/Ligase IV (Lig4) core C-NHEJ factors are required, respectively, for sensing and joining DSBs. While XRCC4/Ligase IV are absolutely required for joining RAG1/2-endonucease ("RAG")-initiated DSBs during V(D)J recombination in G1-phase progenitor lymphocytes, cycling cells deficient for XRCC4/Ligase IV also can join chromosomal DSBs by alternative end-joining (A-EJ) pathways. Restriction of V(D)J recombination by XRCC4/Ligase IV-mediated joining has been attributed to RAG shepherding V(D)J DSBs exclusively into the C-NHEJ pathway. Here, we report that A-EJ of DSB ends generated by RAG1/2, Cas9:gRNA and Zinc finger endonucleases in Lig4-deficient G1-arrested progenitor B cell lines is suppressed by Ku. Thus, while diverse DSBs remain largely as free broken ends in Lig4-deficient G1-arrested progenitor B cells, deletion of Ku70 increases DSB rejoining and translocation levels to those observed in Ku70-deficient counterparts. Correspondingly, while RAG-initiated V(D)J DSB joining is abrogated in Lig4-deficient G1-arrested progenitor B cell lines, joining of RAG-generated DSBs in Ku70-deficient and Ku70/Lig4 double-deficient lines occurs through a translocation-like A-EJ mechanism. Thus, in G1-arrested, Lig4-deficient progenitor B cells are functionally end-joining suppressed due to Ku-dependent blockage of A-EJ, potentially, in association with G1-phase down-regulation of Ligase1. Finally, we suggest that differential impacts of Ku-deficiency versus Lig4-deficiency on V(D)J recombination, neuronal apoptosis, and embryonic development results from Ku-mediated inhibition of A-EJ in the G1 cell cycle phase in Lig4-defcient developing lymphocyte and neuronal cells.

Significance StatementAlternative end-joining (A-EJ) is implicated in oncogenic translocations and mediating DNA double-strand break (DSB) repair in cycling cells when classical nonhomologous endjoining (C-NHEJ) factors of the C-NHEJ Ligase complex are absent. However, V(D)J recombination-associated DSBs that occur in G1 cell cycle-phase progenitor lymphocytes are joined exclusively by the C-NHEJ pathway. Until now, however, the overall mechanisms that join general DSBs in G1-phase progenitor B cells had not been fully elucidated. Here, we report that Ku, a core C-NHEJ double-strand break recognition complex, directs repair of a variety of different targeted DSBs towards C-NHEJ and suppresses A-EJ in G1-phase cells. We suggest this Ku activity explains how Ku-deficiency can rescue the neuronal development and embryonic lethality phenotype of Ligase 4-deficient mice.

category: genetics


title: Modulating the properties of DNA-SWCNT sensors using chemically modified DNA

abstract:
Properties of SWCNT-based sensors such as brightness and detection capabilities strongly depend on the characteristics of the wrapping used to suspend the nanotubes. In this study, we explore ways to modify the properties of DNA-SWCNT sensors by using chemically modified DNA sequences, with the aim of creating sensors more suitable for use in in vivo and in vitro applications. We show that both the fluorescence intensity and sensor reactivity are strongly impacted not only by the chemical modification of the DNA but also by the method of preparation. In the absence of modifications, the sensors prepared using MeOH-assisted surfactant exchange exhibited higher overall fluorescence compared to those prepared by direct sonication. However, we demonstrate that the incorporation of chemical modifications in the DNA sequence could be used to enhance the fluorescence intensity of sonicated samples. We attribute these improvements to both a change in dispersion efficiency as well as to a change in SWCNT chirality distribution.

Furthermore, despite their higher intensities, the response capabilities of sensors prepared by MeOH-assisted surfactant exchange were shown to be significantly reduced compared to their sonicated counterparts. Sonicated sensors exhibited a globally higher turn-on response towards dopamine compared to the exchanged samples, with modified samples retaining their relative intensity enhancement. As the increases in fluorescence intensity were achieved without needing to alter the base sequence of the DNA wrapping or to add any exogenous compounds, these modifications can - in theory - be applied to nearly any DNA sequence to increase the brightness and penetration depths of a variety of DNA-SWCNT sensors without affecting biocompatibility or reducing the near-limitless sequence space available. This makes these sensors an attractive alternative for dopamine sensing in vitro and in vivo by enabling significantly higher penetration depths and shorter laser exposure times.

category: bioengineering


title: Prediction of nanographene binding-scores to trout cellular receptors and cytochromes

abstract: To address the increasing concerns surrounding possible impacts of graphene-related materials on the aquatic environment, this study focused on computational predictions of binding between models of graphenes in the nm size range (nanographenes, nGs) and the aryl-hydrocarbon receptor (tAHR) and P450 cytochromes (tCYPs) of rainbow trout (Oncorhynchus mykiss). The tAHR plays a key role in the induction of detoxifying and early immune responses and tCYPs are essential for detoxifying planar hydrophobic chemicals such as nGs. After 3D modelling of those trout proteins, docking algorithms predicted the size-dependance profiles of nGs binding-scores to tAHR and tCYPs in the low nM range (high binding-affinities). Virtual oxidations of nGs to nGOs (carboxy-, epoxy-and/or hydroxy-oxidations) further lowered the corresponding binding-scores in level/type-oxidation manners. Among all the tCYPs, the tCYP3AR (the equivalent to human CYP3A4) was identified as a potential key interaction enzyme for nGs because of its lower binding-scores. These results implicate a possible processing pathway to be further probed through in vitro and in vivo experimentation. Together the information generated can be pivotal for the design of safer graphene-related materials for a variety of applications and help to understand their detoxification in aquatic vertebrates.


title: Interaural Time Difference Tuning in the Rat Inferior Colliculus is Predictive of Behavioral Sensitivity

abstract: Recent studies have shown that rats are a useful model for binaural cochlear implant (CI) research, with behavioral sensitivity to interaural time differences (ITDs) of CI stimuli which are better than those of human patients. Here, we characterize ITD tuning in the rat inferior colliculus (IC) and explore whether quality of tuning can predict behavioral performance. We recorded IC responses to stimuli of varying pulse rates and envelope types and quantified both mutual information (MI) and neural d' as measures of ITD sensitivity. Neural d' values paralleled behavioral ones, declining with increasing click rates or when envelopes changed from rectangular to Hanning windows. While MI values increased with experience, neural d' did not. However, neural d' values correlated much better with behavioral performance than MI. Thus, neural d' appears to be a particularly well suited to predicting how stimulus parameters will impact behavioral performance.

category: neuroscience


title: Young and Undamaged rMSA Improves the Longevity of Mice

abstract: Improvement of longevity is an eternal dream of human beings. Here we report that a single protein recombinant mouse serum albumin (rMSA) improved the lifespan and healthspan of C57BL/6N mice. The median lifespan extensions were 17.6% for female and 20.3% for male, respectively. The grip strength of rMSA-treated female and male mice increased by 29.6% and 17.4%, respectively. Meanwhile, the percentage of successful escape increased 23.0% in rMSA-treated male mice using the Barnes Maze test. The rMSA used in this study is young and almost undamaged. We define the concept "young and undamaged" to any protein without any unnecessary modifications by four parameters: intact free thiol (if any), no advanced glycation end-product, no carbonylation, and no homocysteinylation. Here "young and undamaged" rMSA is much younger and less damaged than the endogenous serum albumin from young mice at 1.5 months of age. We predict that young and undamaged proteins altogether can further improve the longevity.


title: Mechanical mapping of mammalian follicle development using Brillouin microscopy

abstract: In early mammalian development, the maturation of follicles containing the immature oocytes is an important biological process as the functional oocytes provide the bulk genetic and cytoplasmic materials for successful reproduction. Despite recent work demonstrating the regulatory role of mechanical stress in oocyte growth, quantitative studies of ovarian mechanical properties remain lacking both in vivo and ex vivo. In this work, we quantify the material properties of ooplasm, follicles and connective tissues in intact mouse ovaries at distinct stages of follicle development using Brillouin microscopy, a non-invasive tool to probe mechanics in three-dimensional (3D) tissues. We find that the ovarian cortex and its interior stroma have distinct material properties associated with extracellular matrix deposition, and that intra-follicular mechanical compartments emerge during follicle maturation. Our work provides a novel approach to study the role of mechanics in follicle morphogenesis and pave the way for future understanding of mechanotransduction in reproductive biology, with potential implications for infertility diagnosis and treatment.

category: biophysics


title: Rigid-Body Fitting to Atomic Force Microscopy Images for Inferring Probe Shape and Biomolecular Structure

abstract:
Atomic force microscopy (AFM) can visualize functional biomolecules near the physiological condition, but the observed data are limited to the surface height of specimens. Since the AFM images highly depend on the probe tip shape, for successful inference of molecular structures from the measurement, the knowledge of the probe shape is required, but is often missing. Here, we developed a method of the rigid-body fitting to AFM images, which simultaneously finds the shape of the probe tip and the placement of the molecular structure via an exhaustive search. First, we examined four similarity scores via twin-experiments for four test proteins, finding that the cosine similarity score generally worked best, whereas the pixel-RMSD and the correlation coefficient were also useful. We then applied the method to two experimental high-speed-AFM images inferring the probe shape and the molecular placement. The results suggest that the appropriate similarity score can differ between target systems. For an actin filament image, the cosine similarity apparently worked best. For an image of the flagellar protein FlhAC, we found the correlation coefficient gave better results. This difference may partly be attributed to the flexibility in the target molecule, ignored in the rigid-body fitting. The inferred tip shape and placement results can be further refined by other methods, such as the flexible fitting molecular dynamics simulations. The developed software is publicly available.

Author SummaryObservation of functional dynamics of individual biomolecules is important to understand molecular mechanisms of cellular phenomena. High-speed (HS) atomic force microscopy (AFM) is a powerful tool that enables us to visualize the real-time dynamics of working biomolecules under near-physiological conditions. However, the information available by the AFM images is limited to the two-dimensional surface shape detected via the force to the probe. While the surface information is affected by the shape of the probe tip, the probe shape itself cannot be directly measured before each AFM measurement. To overcome this problem, we have developed a computational method to simultaneously infer the probe tip shape and the molecular placement from an AFM image. We show that our method successfully estimates the effective AFM tip shape and visualizes a structure with a more accurate placement. The estimation of a molecular placement with the correct probe tip shape enables us to obtain more insights into functional dynamics of the molecule from HS-AFM images.


title: Non-canonical role of Hippo tumor suppressor serine/threonine kinase 3 STK3 in prostate cancer

abstract:
Serine/threonine kinase 3 (STK3) is an essential member of the highly conserved Hippo Tumor suppressor pathway which regulates Yes 1 Associated protein (YAP1) and TAZ. STK3 and its paralog STK4 initiate a phosphorylation cascade that regulate YAP1/TAZ activation and degradation, which is important for regulated cell growth and organ size. Deregulation of this pathway leads to hyper-activation of YAP1 in various cancers. Counter to the canonical tumor suppression role of STK3, we report that in the context of prostate cancer (PC), STK3 has a pro-tumorigenic role. Our investigation started with the observation that STK3, but not STK4, is frequently amplified in PC. A high STK3 expression is associated with decreased overall survival and positively correlates with androgen receptor (AR) activity in metastatic castrate resistant PC. XMU-MP-1, an STK3/4 inhibitor, slowed cell proliferation, spheroid growth and matrigel invasion in multiple models. Genetic depletion of STK3 decreased proliferation in several PC cell lines. In a syngeneic allograft model, STK3 loss slowed tumor growth kinetics in vivo and biochemical analysis suggest a mitotic growth arrest phenotype. To further probe the role of STK3 in PC, we identified and validated a new set of selective STK3 inhibitors, with enhanced kinase selectivity relative XMU-MP-1, that inhibited tumor spheroid growth and invasion. Consistent with the canonical role, inhibition of STK3 induced cardiomyocyte growth and had chemo-protective effects. Our results contend that STK3 has a non-canonical role in PC progression and inhibition of STK3 may have therapeutic potential for PC that merits further investigation.

SignificanceOur findings illuminate a new actionable target for PC therapy that would traditionally be overlooked due to its canonical role as a tumor suppressor in other cancer types.

category: cancer biology


title: Multimodal-Neural Predictive Models of Children's General Intelligence That Are Stable Across Two Years of Development

abstract: Childhood intelligence is strikingly predictive of major life outcomes. Understanding the brain underpinnings of early-life intelligence is of prime clinical and public health importance, but has so far remained elusive. Here, we demonstrate that it is possible to arrive at models of the brain that are both predictive and explanatory of childhood intelligence. For this we leverage the unique power of the large-scale, longitudinal, multimodal MRI data from the Adolescent Brain Cognitive Development (ABCD) study (n [~]11k) and create a novel predictive modeling framework that integrates machine-learning and structural-equation-based methodologies. Our predictive models combine six MRI modalities (task-fMRI from three tasks, resting-state fMRI, structural MRI, DTI) using machine-learning. In terms of prediction, our models achieve an unprecedented longitudinal association (r=.41) with childhood intelligence across two years in unseen data. We found fronto-parietal networks during a working-memory task to drive childhood-intelligence prediction. In terms of explanation, our models significantly explain variance in childhood intelligence due to (1) key socio-demographic and psychological factors (proportion mediated=18.65% [17.29%-20.12%]) and (2) genetic liability, as reflected by the polygenic score of cognitive ability (proportion mediated=15.6% [11%-20.7%]). In summary, our work shows that novel models of human multimodal neuroimaging data are powerful in helping us predict and explain variation in childhood intelligence.


title: Longitudinally stable, brain-based predictive models explain the relationships ofchildhood intelligence with socio-demographic, psychological and genetic factors


title: Structure-based prediction of KDAC6 substrates validated by enzymatic assay reveals determinants of promiscuity and detects new potential substrates

abstract: Histone deacetylases play important biological roles well beyond the deacetylation of histone tails. In particular, HDAC6 is involved in multiple cellular processes such as apoptosis, cytoskeleton reorganization, and protein folding, affecting substrates such as -tubulin, Hsp90 and cortactin proteins. We have applied a biochemical enzymatic assay to measure the activity of HDAC6 on a set of candidate unlabeled peptides. These served for the calibration of a structure-based substrate prediction protocol, Rosetta FlexPepBind, previously used for the successful substrate prediction of HDAC8 and other enzymes. A proteome-wide screen of reported acetylation sites using our calibrated protocol together with the enzymatic assay provide new peptide substrates and avenues to novel potential functional regulatory roles of this promiscuous, multi-faceted enzyme. In particular, we propose novel regulatory roles of HDAC6 in tumorigenesis and cancer cell survival via the regulation of the EGFR/Akt pathway activation process. The calibration process and comparison of the results between HDAC6 and HDAC8 highlight structural differences that explain the established promiscuity of HDAC6.


title: Unraveling the role of MADS transcription factor complexes in apple tree dormancy using sequential DAP-seq

abstract:
BackgroundThe effect of global warming on dormancy and flowering patterns of crop trees threatens world-wide fruit production and food security. In Rosaceous tree species, it is believed that a group of genes encoding MADS transcription factors (TFs) controls temperature-mediated dormancy cycle. These genes are similar to SHORT VEGETATIVE PHASE (SVP) from Arabidopsis thaliana and referred as DORMANCY-ASSOCIATED MADS-BOX (DAM) genes.

ResultsBy making use of apple tree (Malus x domestica) as a model for Rosaceous species, we have investigated the function of MADS TFs during the dormancy cycle. We found that MdDAM and other dormancy related MADS TFs form multimeric complexes with MdSVPa, and that MdSVPa is essential for the transcriptional complex activity. Then, for the first time in non-model plant species, we performed sequential DNA Affinity Purification sequencing (seq-DAP-seq) to define the genome-wide binding sites of these MADS TF complexes. Target genes associated with the binding sites were identified by combining seq-DAP-seq data with transcriptomics datasets obtained by the inducible glucocorticoid receptor expression system, and reanalyzing preexisting data related to dormancy cycle in apple trees.

ConclusionWe have determined a gene regulatory network formed by MdSVPa-containing complexes that regulate the dormancy cycle in apple trees in response to environmental cues. Key genes identified with our genomic approach and the elucidated regulatory relationships provide leads for breeding fruit trees better adapted to changing climate conditions. Moreover, we provide novel molecular evidence on the evolutionary functional segregation between DAM and SVP proteins in the Rosaceae family.


title: A cannabinoid receptor agonist shows anti-inflammatory and survival properties in human SARS-CoV-2-infected iPSC-derived cardiomyocytes

abstract: Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which can infect several organs, especially impacting respiratory capacity. Among the extrapulmonary manifestations of COVID-19 is myocardial injury, which is associated with a high risk of mortality. Myocardial injury, caused directly or indirectly by SARS-CoV-2 infection, can be triggered by inflammatory processes that cause damage to the heart tissue. Since one of the hallmarks of severe COVID-19 is the "cytokine storm", strategies to control inflammation caused by SARS-CoV-2 infection have been considered. Cannabinoids are known to have anti-inflammatory properties by negatively modulating the release of pro-inflammatory cytokines. Herein, we investigated the effects of the cannabinoid agonist WIN 55,212-2 (WIN) in human iPSC-derived cardiomyocytes (hiPSC-CMs) infected with SARS-CoV-2. WIN did not modify angiotensin-converting enzyme II protein levels, nor reduced viral infection and replication in hiPSC-CMs. On the other hand, WIN reduced the levels of interleukins 6, 8, 18 and tumor necrosis factor-alpha (TNF-) released by infected cells, and attenuated cytotoxic damage measured by the release of lactate dehydrogenase (LDH). Our findings suggest that cannabinoids should be further explored as a complementary therapeutic tool for reducing inflammation in COVID-19 patients.


title: WIN 55,212-2 shows anti-inflammatory and survival properties in human iPSC-derived cardiomyocytes infected with SARS-CoV-2


title: Investigating crosstalk among PTMs provides novel insight into the structural basis underlying the differential effects of Nt17 PTMs on mutant Httex1 aggregation

abstract:
Post-translational modifications (PTMs) within the first 17 amino acids (Nt17) of the Huntingtin protein (Htt) have been shown to inhibit the aggregation and attenuate the toxicity of mutant Htt proteins in vitro and in various models of Huntingtons disease. Our groups previous studies suggested that the Nt17 PTM code is a combinatorial code that involves a complex interplay between different PTMs. Here, we expand on these studies by investigating the effect of methionine 8 oxidation (oxM8) and crosstalk between this PTM and either lysine 6 acetylation (AcK6) or threonine 3 phosphorylation (pT3) on the aggregation of mutant Httex1. We show that M8 oxidation delays but does not inhibit the aggregation and has no effect on the final morphologies of mutant Httex1 aggregates. This delay in aggregation kinetics could be attributed to the transient accumulation of oligomeric aggregates, which disappear upon the formation of Httex1 oxM8 fibrils. Interestingly, the presence of both oxM8 and AcK6 resulted in dramatic inhibition of Httex1 fibrillization, whereas the presence of oxM8 did not influence the aggregation inhibitory effect of pT3. To gain insight into the structural basis underlying these proteins aggregation properties, we investigated the impact of each PTM and the combination of these PTMs on the conformational properties of the Nt17 peptide by circular dichroism spectroscopy and molecular dynamics simulation. These studies show that M8 oxidation decreases the helicity of the Nt17 in the presence or absence of PTMs and provides novel insight into the structural basis underlying the effects of different PTMs on mutant Httex1 aggregation. PTMs that lower the mutant Httex1 aggregation rate (oxM8, AcK6/oxM8, pT3, pT3/oxM8, and phosphorylation at Serine 13) result in stabilization and increased population of a short N-terminal helix (first eight residues) in Nt17 or decreased abundance of other helical forms, including long helix and short C-terminal helix. PTMs that did not alter the aggregation of mutant Httex1 exhibit a similar distribution of helical conformation as the unmodified peptides. These results show that the relative abundance of N- vs. C-terminal helical conformations and long helices, rather than the overall helicity of Nt17, better explains the effect of different Nt17 PTMs on mutant Httex1; thus, explaining the lack of correlation between the effect of PTMs on the overall helicity of Nt17 and mutant Httex1 aggregation in vitro. Taken together, our results provide novel structural insight into the differential effects of single PTMs and crosstalk between different PTMs in regulating mutant Httex1 aggregation.

TOC Figure

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/432155v1_ufig1.gif" ALT="Figure 1">
View larger version (33K):
org.highwire.dtl.DTLVardef@15dbf84org.highwire.dtl.DTLVardef@1c8a50corg.highwire.dtl.DTLVardef@1cbe0c6org.highwire.dtl.DTLVardef@120e0a_HPS_FORMAT_FIGEXP  M_FIG C_FIG


title: A Transient Metabolic State In Melanoma Persister Cells Mediated By Chemotherapeutic Treatments

abstract: Persister cells are defined as the small fraction of quiescent cells in a bulk cancer cell population that can tolerate unusually high levels of drugs. Persistence is a transient state that poses an important health concern in cancer therapy. The mechanisms associated with persister phenotypes are highly diverse and complex, and many aspects of persister cell physiology remain to be explored. We applied a melanoma cell line and panel of chemotherapeutic agents to show that melanoma persister cells are not necessarily preexisting dormant cells or stem cells; in fact, they may be induced by cancer chemotherapeutics. Our metabolomics analysis and phenotype microarray assays further demonstrated that the levels of Krebs cycle molecules are significantly lower in the melanoma persister subpopulation than in the untreated bulk cell population due to increased utilization rates in persisters. Our data indicate that this observed metabolic remodeling is transient, as the consumption rates of Krebs cycle metabolites are significantly reduced in the progenies of persisters. Given that the mitochondrial electron transport chain (ETC) is more active in the persister subpopulation than in the bulk cancer cell population, we also verified that targeting ETC activity can reduce melanoma persistence. The reported metabolic remodeling feature seems to be a conserved characteristic of melanoma persistence, as it has been observed in various melanoma persister subpopulations derived from a diverse range of chemotherapeutics. Elucidating a global metabolic mechanism that contributes to persister survival and reversible switching will ultimately foster the development of novel cancer therapeutic strategies.


title: Analysis Of Molecular Networks In The Cerebellum In Chronic Schizophrenia: Modulation By Early Postnatal Life Stressors In Murine Models

abstract: Despite the growing importance of the cortico-cerebellar-thalamo-cortical circuit in schizophrenia, limited information is available regarding altered molecular networks in cerebellum. To identify altered protein networks, we conducted proteomic analysis of grey matter of postmortem cerebellar cortex in chronic schizophrenia subjects (n=12) and healthy individuals (n=14) followed by an extensive bioinformatic analysis. Two double-hit postnatal stress murine models for SZ were used to validate the most robust candidates. The models were maternal deprivation combined with an additional stressor: social isolation or chronic restraint stress. We found that the individual proteomic profile allowed the segregation of most schizophrenia cases from healthy individuals. We found 250 proteins with altered levels. This group was enriched in proteins related to mental disorders, mitochondrial disease, stress, and a number of biological functions including energy, immune response, axonal cytoskeletal organization and vesicle-mediated transport. Network analysis identified three modules: energy metabolism, neutrophil degranulation and a mixed module of mainly axonal-related functions. We analysed the most robust candidates in the networks in two double-hit stress murine models. METTL7A from the degranulation pathway was reduced in both models, while NDUFB9 from the energy network and CLASP1 from the axonal module decreased in only one model. This work provides evidence for altered energy, immune and axonal-related networks in the cerebellum in schizophrenia, suggesting that the accumulation of molecular errors, some by an early postnatal stress exposure, could lead to a failure in the normal cerebellar functions, impairing synaptic response and the defence mechanisms of this region against external harmful injuries in schizophrenia.


title: Synchrony of mind and body are distinct in mother-child dyads

abstract: Hyperscanning studies have begun to unravel the brain mechanisms underlying social interaction, indicating a functional role for interpersonal neural synchronization (INS), yet the mechanisms that drive INS are poorly understood. While interpersonal synchrony is considered a multimodal phenomenon, it is not clear how different biological and behavioral synchrony markers are related to each other. The current study, thus, addresses whether INS is functionally-distinct from synchrony in other systems - specifically the autonomic nervous system (ANS) and motor behavior. To test this, we used a novel methodological approach, based on concurrent functional near-infrared spectroscopy-electrocardiography, recorded while N = 34 mother-child and stranger-child dyads (child mean age 14 years) engaged in cooperative and competitive tasks. Results showed a marked differentiation between neural, ANS and behavioral synchrony. Importantly, only in the neural domain was higher synchrony for mother-child compared to stranger-child dyads observed. Further, ANS and neural synchrony were positively related during competition but not during cooperation. These results suggest that synchrony in different behavioral and biological systems may reflect distinct processes. Mother-child INS may arise due to neural processes related to social affiliation, which go beyond shared arousal and similarities in behavior.


title: Extracellular Delivery of Functional Mitochondria Reverses the Dysfunction of CD4+ T Cells in Aging

abstract: Mitochondrial dysfunction alters cellular metabolism, increases oxidative stress, and may be principal to the dysregulated signaling and function of CD4+ T lymphocytes in the elderly. Mitochondria influence adaptive immune responses through glucose and fatty acid metabolism, calcium buffering, and redox signaling. By transferring young mitochondria into CD4+ T cells from old mice, we investigated whether aging-associated mitochondrial dysfunction could be abrogated and whether such transfer changed CD4+ T cell function. Our results show that mitochondrial transfer improved the redox status and function of CD4+ T cells from old mice ex vivo. These findings support the notion that mitochondria can serve as targets of therapeutic intervention in aging.

category: immunology


title: Comprehensive survey of microsporidia reveals extensive ecological and phenotypic diversity

abstract: Microsporidia are a large group of fungal-related obligate intracellular parasites. Though many microsporidia species have been identified over the past 160 years, there is a lacking depiction of the full diversity of this phylum. To systematically describe the characteristics of these parasites, we created a database of 1,440 species and their attributes, including the hosts they infect and spore characteristics. We find that microsporidia have been reported to infect 16 metazoan and four protozoan phyla, with smaller phyla being underrepresented. Most species are only reported to infect a single host, but those that are generalists are also more likely to infect a broader set of host tissues. Strikingly, polar tubes are 3-fold longer in species that infect tissues besides the intestine, suggesting that polar tube length is a determinant of tissue specificity. Phylogenetic analysis revealed four clades which each contain microsporidia infecting hosts from all major habitats. Although related species are more likely to infect similar hosts, we observe examples of changes in host specificity and convergent evolution. Taken together, our results show that microsporidia display vast diversity in their morphology and the hosts they infect, illustrating the flexibility of these parasites to evolve new traits.


title: Generation of a microsporidia species attribute database and analysis of the extensive ecological and phenotypic diversity of microsporidia


title: DpaA detaches Braun's lipoprotein from peptidoglycan

abstract:
Gram-negative bacteria have a unique cell envelope with a lipopolysaccharide-containing outer membrane that is tightly connected to a thin layer of peptidoglycan. The tight connection between the outer membrane and peptidoglycan is needed to maintain the outer membrane as an impermeable barrier for many toxic molecules and antibiotics. Enterobacteriaceae such as Escherichia coli covalently attach the abundant outer membrane-anchored lipoprotein Lpp (Brauns lipoprotein) to tripeptides in peptidoglycan, mediated by the transpeptidases LdtA, LdtB and LdtC. LdtD and LdtE are members of the same family of LD-transpeptidases but they catalyse a different reaction, the formation of 3-3 cross-links in the peptidoglycan. The function of the sixth homologue in E. coli, LdtF remains unclear, although it has been shown to become essential in cells with inhibited LPS export to the outer membrane. We now show that LdtF hydrolyses the Lpp-peptidoglycan linkage, detaching Lpp from peptidoglycan, and have renamed LdtF to peptidoglycan meso-diaminopimelic acid protein amidase A (DpaA). We show that the detachment of Lpp from peptidoglycan is beneficial for the cell under certain stress conditions and that the deletion of dpaA allows frequent transposon inactivation in the lapB (yciM) gene, whose product down-regulates lipopolysaccharide biosynthesis. DpaA-like proteins have characteristic sequence motifs and are present in many Gram-negative bacteria of which some have no Lpp, raising the possibility that DpaA has other substrates in these species. Overall, our data show that the Lpp-peptidoglycan linkage in E. coli is more dynamic than previously appreciated.

IMPORTANCEGram-negative bacteria have a complex cell envelope with two membranes and a periplasm containing the peptidoglycan layer. The outer membrane is firmly connected to the peptidoglycan by highly abundant proteins. The outer membrane-anchored Brauns lipoprotein (Lpp) is the most abundant protein in E. coli and about one third of the Lpp molecules become covalently attached to tripeptides in peptidoglycan. The attachment of Lpp to peptidoglycan stabilizes the cell envelope and is crucial for the outer membrane to function as a permeability barrier for a range of toxic molecules and antibiotics. So far the attachment of Lpp to peptidoglycan has been considered to be irreversible. We have now identified an amidase, DpaA, which is capable of detaching Lpp from PG and we show that the detachment of Lpp is important under certain stress conditions. DpaA-like proteins are present in many Gram-negative bacteria and may have different substrates in these species.


title: TOR complex 2 promotes proteasome dependent Pub1 (NEDD4 family E3 ligase) turnover in fission yeast.

abstract: Cells respond to changing nutrient environments by adjusting the abundance of surface nutrient transporters and receptors. This can be achieved by modulating ubiquitin-dependent endocytosis, which in part is regulated by the NEDD4 family of E3 ligases. Here we report novel regulation of Pub1, a fission yeast Schizosaccharomyces pombe member of the NEDD4-family of E3 ligases. We show that nitrogen stress inhibits Pub1 function, thereby increasing the abundance of the amino acid transporter Aat1 at the plasma membrane and enhancing sensitivity to the toxic arginine analogue canavanine. We show that TOR complex 2 (TORC2) signalling negatively regulates Pub1, thus TORC2 mutants under nutrient stress have decreased Aat1 at the plasma membrane and are resistant to canavanine. Inhibition of TORC2 signalling increases Pub1 phosphorylation, and this is dependent on Gsk3 activity. Addition of the Tor inhibitor Torin1 increases phosphorylation of Pub1 at serine 199 (S199) by 2.5-fold, and Pub1 protein levels in S199A phospho-ablated mutants are reduced. S199 is conserved in NEDD4 and is located immediately upstream of a WW domain required for protein interaction. Together, we describe how the major TORC2 nutrient-sensing signalling network regulates environmental control of Pub1 to modulate the abundance of nutrient transporters.


title: Environmental control of Pub1 (NEDD4 family E3 ligase) in S. pombe is regulated byTORC2 and Gsk3.


title: Inhibition of SARS-CoV-2 infection in human cardiomyocytes by targeting the Sigma-1 receptor disrupts cytoskeleton architecture and contractility

abstract: Heart dysfunction, represented by conditions such as myocarditis and arrhythmia, has been reported in COVID-19 patients. Therapeutic strategies focused on the cardiovascular system, however, remain scarce. The Sigma-1 receptor (S1R) has been recently proposed as a therapeutic target because its inhibition reduces SARS-CoV-2 replication. To investigate the role of S1R in SARS-CoV-2 infection in the heart, we used human cardiomyocytes derived from induced pluripotent stem cells (hiPSC-CM) as an experimental model. Here we show that the S1R antagonist NE-100 decreases SARS-CoV-2 infection and viral replication in hiPSC-CMs. Also, NE-100 reduces cytokine release and cell death associated with infection. Because S1R is involved in cardiac physiology, we investigated the effects of NE-100 in cardiomyocyte morphology and function. We show that NE-100 compromises cytoskeleton integrity and reduces beating frequency, causing contractile impairment. These results show that targeting S1R to challenge SARS-CoV-2 infection may be a useful therapeutic strategy but its detrimental effects in vivo on cardiac function should not be ignored.