title: The calcium channel Orai1 is required for osteoblast development: studies in a chimeric mouse with variable in vivo Runx-cre deletion of Orai-1

abstract: The calcium-selective ion channel Orai1 has a complex role in bone homeostasis, with defects in both bone production and resorption detected in Orai1 germline knock-out mice. To determine whether Orai1 has a direct, cell-intrinsic role in osteoblast differentiation and function, we bred Orai1 flox/flox (Orai1f/f) mice with Runx2-cre mice to eliminate its expression in osteoprogenitor cells. Interestingly, Orai1 was expressed in a mosaic pattern in Orai1f/f-Runx2-cre bone. Specifically, antibody labeling for Orai1 in vertebral sections was uniform in wild type animals, but patchy regions in Orai1f/f-Runx2-cre bone revealed Orai1 loss while in other areas expression persisted. Nevertheless, by micro-CT, bones from Orai1f/f-Runx2-cre mice showed reduced bone mass overall, with impaired bone formation identified by dynamic histomorphometry. Cortical surfaces of Orai1f/f-Runx2-cre vertebrae however exhibited patchy defects. In cell culture, Orai1-negative osteoblasts showed profound reductions in store-operated Ca2+ entry, exhibited decreased alkaline phosphatase activity, and had markedly impaired substrate mineralization. We conclude that defective bone formation observed in the absence of Orai1 reflects an intrinsic role for Orai1 in differentiating osteoblasts.


title: Neural correlates of multisensory enhancement in audiovisual narrative speech perception: a fMRI investigation

abstract: This fMRI study investigated the effect of seeing articulatory movements of a speaker while listening to a naturalistic narrative stimulus. It had the goal to identify regions of the language network showing multisensory enhancement under synchronous audiovisual conditions. We expected this enhancement to emerge in regions known to underlie the integration of auditory and visual information such as the posterior superior temporal gyrus as well as parts of the broader language network, including the semantic system. To this end we presented 53 participants with a continuous narration of a story in auditory alone, visual alone, and both synchronous and asynchronous audiovisual speech conditions while recording brain activity using BOLD fMRI. We found multisensory enhancement in an extensive network of regions underlying multisensory integration and parts of the semantic network as well as extralinguistic regions not usually associated with multisensory integration, namely the primary visual cortex and the bilateral amygdala. Analysis also revealed involvement of thalamic brain regions along the visual and auditory pathways more commonly associated with early sensory processing. We conclude that under natural listening conditions, multisensory enhancement not only involves sites of multisensory integration but many regions of the wider semantic network and includes regions associated with extralinguistic sensory, perceptual and cognitive processing.

category: neuroscience


title: Loss of Neurodevelopmental Gene CASK Disrupts Neural Connectivity in Human Cortical Excitatory Neurons

abstract:
Loss-of-function (LOF) mutations in CASK cause severe developmental phenotypes, including microcephaly with pontine and cerebellar hypoplasia, X-linked intellectual disability, and autism. Unraveling the pathogenesis of CASK-related disorders has been challenging due to limited human cellular models to study the dynamic roles of this molecule during neuronal and synapse development. Here, we generated CASK knockout (KO) isogenic cell lines from human embryonic stem cells (hESCs) using CRISPR/Cas9 and examined gene expression, morphometrics, and synaptic function of induced neuronal cells during development. While young (immature) CASK KO neurons show robust neuronal outgrowth, mature CASK KO neurons displayed severe defects in synaptic transmission and synchronized burst activity without compromising neuronal morphology and synapse numbers. In developing human cortical neurons, CASK functioned to promote both structural integrity and establishment of cortical excitatory neuronal networks. These results lay the foundation for future studies to identify suppressors of such phenotypes relevant to human patients.

HighlightsO_LICASK LOF mutations increase neuronal complexity in immature developing neurons
C_LIO_LICASK LOF does not alter synapse formation and neurite complexity in mature neurons
C_LIO_LISynaptic transmission and network synchronicity are compromised in CASK KO neurons
C_LIO_LIDifferential gene expression analysis reveals enrichment of synaptic gene networks in mature CASK KO neurons
C_LI


title: Mineralized coleoid cranial cartilage from the Late Triassic Polzberg Konservat-Lagerstätte (Austria)

abstract: Although hyaline cartilage is widely distributed in various invertebrate groups such as sabellid polychaetes, molluscs (cephalopods, gastropods) and a chelicerate arthropod group (horseshoe crabs), the enigmatic relationship and distribution of cartilage in taxonomic groups remains to be explained. It can be interpreted as a convergent trait in animal evolution and thus does not seem to be a vertebrate invention. Due to the poor fossil record of cartilaginous structures, occurrences of mineralized fossil cartilages are important for evolutionary biology and paleontology. Although the biochemical composition of recent cephalopod cartilage differs from vertebrate cartilage, histologically the cartilages of these animal groups resemble one another remarkably. In this study we present fossil material from the late Triassic Polzberg Konservat-Lagerstatte near Lunz am See (Lower Austria, Northern Calcareous Alps). A rich Carnian fauna is preserved here, whereby a morphogroup (often associated with coleoid remains) of black, amorphous appearing fossils still remained undetermined. These multi-elemental, mirroring fossils show remarkable similarities to recent cephalopod cartilage. We examined the conspicuous micro- and ultrastructure of these enigmatic fossils by thin-sectioning and Scanning Electron Microscopy (SEM). The geochemical composition analyzed by Microprobe and Energy Dispersive X-ray Spectroscopy (SEM-EDX) revealed carbonisation as the taphonomic pathway for this fossil group. 3D preservation of this otherwise degradable soft tissue can be explained by the mineralization processes that may take place in particular environments. Fifty-nine specimens from the Polzberg locality and five specimens from Cave del Predil (formerly Raibl, Julian Alps, Italy) underwent extensive studies. The detected grooves are interpreted to be muscular attachment areas, and the preserved branched system of canaliculi is comparable to a channel system that is also present in recent coleoid cartilage. The new findings on these long-known enigmatic structures strongly point to the preservation of cranial cartilage belonging to the belemnoid Phragmoteuthis bisinuata.

category: paleontology


title: Dynamic S-acylation of STIM1 is required for store-operated Ca 2+ entry

abstract: Many cell surface stimuli cause calcium release from endoplasmic reticulum (ER) stores to regulate cellular physiology. Upon ER calcium store depletion, the ER-resident protein STIM1 physically interacts with plasma membrane protein Orai1 to induce calcium release-activated calcium (CRAC) currents that conduct calcium influx from the extracellular milieu. Although the physiological relevance of this process is well established, the mechanism supporting the assembly of these proteins is incompletely understood. Earlier we demonstrated a previously unknown post-translational modification of Orai1 with long chain fatty acids, known as S-acylation. We found that S-acylation of Orai1 is dynamically regulated in a stimulus-dependent manner and essential for its function as a calcium channel. Here we show that STIM1 is also rapidly and transiently S-acylated at cysteine 437 upon ER calcium store depletion. S-acylation of STIM1 is required for the assembly of STIM1 into puncta with Orai1 and full CRAC channel function. Together with the S-acylation of Orai1, our data suggest that stimulus-dependent S-acylation of CRAC channel components Orai1 and STIM1 is a critical mechanism facilitating CRAC channel assembly and function.

category: cell biology


title: Microsecond Melting and Revitrification of Cryo Samples - Protein Structure and Beam-Induced Motion

abstract:
We have recently introduced a novel approach to time-resolved cryo-electron microscopy (cryo-EM) that involves melting a cryo sample with a laser beam to allow protein dynamics to briefly occur in liquid, before trapping the particles in their transient configurations by rapidly revitrifying the sample. With a time-resolution of just a few microseconds, this approach is notably fast enough to study domain motions that are typically associated with the activity of proteins, but which have previously remained inaccessible. Here, we add crucial details to the characterization of our method. We show that single-particle reconstructions of apoferritin and cowpea chlorotic mottle virus (CCMV) from revitrified samples are indistinguishable from those in conventional samples, demonstrating that melting and revitrification leaves the particles intact and that they do not undergo structural changes within the spatial resolution afforded by our instrument. We also characterize how rapid revitrification affects the properties of the ice, showing that revitrified samples exhibit comparable amounts of beam-induced motion. Our results pave the way for microsecond time-resolved studies of the conformational dynamics of proteins and open up new avenues to study the vitrification process and address beam-induced specimen movement.

SynopsisMicrosecond melting and revitrification of cryo samples preserves the structure of embedded particles. The beam-induced motion of revitrified samples is comparable to that of conventional cryo samples.

category: biophysics


title: The V-type H+-ATPase is targeted in anti-diuretic hormone control of the Malpighian (renal) tubules in Aedes aegypti

abstract:
Like other insects, secretion by mosquito MTs is driven by the V-type H+-ATPase (VA), which is densely packed in the apical membrane of principal cells. The anti-diuretic neurohormone CAPA inhibits tubule secretion stimulated by select diuretic hormones; however, the cellular effectors of this inhibitory signaling cascade remain unclear. Herein, we show that the VA inhibitor bafilomycin selectively inhibits serotonin (5HT)- and calcitonin-related diuretic hormone (DH31)-stimulated secretion. VA activity increases in DH31-treated MTs, whereas CAPA abolishes this increase. A critical feature of VA activation involves the reversible association of the cytosolic (V1) and membrane (Vo) complexes and results revealed higher V1 protein abundance in membrane fractions of DH31-treated MTs whereas CAPA significantly decreased V1 protein abundance in membrane fractions while increasing it in cytosolic fractions. Immunolocalization of V1 was observed strictly in the apical membrane of MTs treated with DH31 alone whereas immunoreactivity was dispersed following CAPA treatment. Elevated levels of cGMP in CAPA-treated MTs suggests activation of a phosphodiesterase (PDE), resulting in decreased levels of DH31-stimulated cAMP. Therefore, CAPA inhibition of MTs involves reducing VA activity and promotes complex dissociation hindering secretion. Collectively, these findings reveal a key target necessary for hormone-mediated inhibition of MTs required in countering diuresis providing a deeper understanding of this critical physiological process.

Significance StatementThe V-type H+-ATPase (VA) proton pump, provides the driving force for transepithelial ion and fluid secretion in insect Malpighian tubules (MTs). While studies have shown that diuretic stimulation activates various signaling pathways that culminate with increased VA activity, our understanding of anti-diuretic signaling and its potential regulation involving the VA remains rudimentary. Here we show that a CAPA neuropeptide acts through the NOS/cGMP/PKG pathway that inhibits VA activity, supporting the notion that anti-diuretic regulation is mediated through reversible dissociation of the VA complexes. Our results demonstrate a critical role of VA inhibition and trafficking necessary for anti-diuretic signaling, advancing our understanding of the complex neuroendocrine regulatory mechanisms of the MTs in an important human disease vector mosquito.

category: physiology


title: Insulin/IGF Signaling Regulates Presynaptic Glutamate Release in Aversive Olfactory Learning

abstract: Information flow through neural circuits is continuously modified by context-dependent learning. In the nematode Caenorhabditis elegans, pairing specific odors with food deprivation results in aversion to the odor. Here we identify cell-specific mechanisms of insulin/IGF receptor signaling that integrate sensory information with food context during aversive olfactory learning. Using a conditional allele of the insulin/IGF receptor DAF-2, we show that aversive learning to butanone, an odor sensed only by the AWCON olfactory neuron, requires DAF-2 in AWCON. Learning requires an axonally-localized DAF-2c isoform and the insulin receptor substrate (IRS) protein IST-1, but is partly independent of the FoxO transcription factor DAF-16. Upon food deprivation, the unconditioned stimulus for learning, DAF-2 expression increases post-transcriptionally through an insulin- and ist-1-dependent process. Aversive learning suppresses odor-regulated glutamate release from AWCON in wild-type animals but not in ist-1 mutants, suggesting that localized insulin signaling drives presynaptic depression to generate an aversive memory.


title: PyZebrascope: an open-source platform for brain-wide neural activity imaging in zebrafish

abstract: Understanding how neurons interact across the brain to control animal behaviors is one of the central goals in neuroscience. Recent developments in fluorescent microscopy and genetically-encoded calcium indicators led to the establishment of whole-brain imaging methods in zebrafish, which records neural activity across a brain-wide volume with single-cell resolution. Pioneering studies of whole-brain imaging used custom light-sheet microscopes, and their operation relied on commercially developed and maintained software that is not available globally. Hence it has been challenging to disseminate and develop the technology in the research community. Here, we present PyZebrascope, an open-source Python platform designed for neural activity imaging in zebrafish using light-sheet microscopy. PyZebrascope has intuitive user interfaces and implements essential features for whole-brain imaging, such as two orthogonal excitation beams and eye damage prevention. Its modular architecture allows the inclusion of advanced algorithms for microscope control and image processing. As a proof of concept, we implemented an automatic algorithm for maximizing the image resolution in the brain by precisely aligning the excitation beams to the image focal plane. PyZebrascope enables whole-brain neural activity imaging in fish behaving in a virtual reality environment with a stable high data throughput and low CPU and memory consumption. Thus, PyZebrascope will help disseminate and develop light-sheet microscopy techniques in the neuroscience community and advance our understanding of whole-brain neural dynamics during animal behaviors.


title: KIBRA Regulates AMPA Receptor Expression, Synaptic Plasticity, and Memory in an Age-Dependent Manner

abstract:
The biological mechanisms supporting age-dependent changes in learning and memory remain elusive. While a growing body of human literature implicates KIBRA in memory and neurodevelopmental disorders, KIBRAs molecular function and contribution to maturation of synaptic function and cognition remain poorly understood. Despite being expressed throughout early postnatal development, germline deletion of KIBRA impairs synaptic plasticity selectively in adult rodents. However, it is unclear whether KIBRA facilitates proper brain maturation necessary for adult plasticity or whether it plays a distinct role in plasticity in the adult brain. Here, using an inducible KIBRA knockout mouse, we demonstrate that acutely deleting KIBRA in adult forebrain neurons impairs both spatial memory and long-term potentiation (LTP). The deficits in LTP correlate with an adult-selective decrease in extrasynaptic AMPA receptors under basal conditions. We also identify a novel role for KIBRA in LTP-induced AMPAR upregulation. In contrast, acute deletion of KIBRA in juvenile forebrain neurons did not affect LTP and had minimal effects on basal AMPAR expression. These data suggest that KIBRA serves a unique role in adult hippocampal function through regulation of basal and activity-dependent AMPAR proteostasis that supports synaptic plasticity.

Significance StatementSynaptic plasticity supported by trafficking of postsynaptic AMPA receptors is a conserved mechanism underlying learning and memory. The nature and efficacy of learning and memory undergo substantial changes during childhood and adolescent development, but the mechanisms underlying this cognitive maturation remain poorly understood. Here, we demonstrate that the human memory- and neurodevelopmental disorder-associated gene KIBRA facilitates memory and hippocampal synaptic plasticity selectively in the adult hippocampus. Furthermore, we show that selective loss of KIBRA from adult but not juvenile neurons reduces expression of extrasynaptic AMPA receptors and prevents LTP-induced increases in AMPAR expression. Overall, our results suggest that KIBRA participates in cellular and molecular processes that become uniquely necessary for memory and synaptic plasticity in early adulthood.


title: Quantifying the economic impact of Peristenus relictus establishment on host Lygus hesperus populations in California organic strawberry

abstract: Lygus hesperus is a key pest in California-grown strawberries, in part because it lacks specialized natural enemies. In response, the specialist European nymphal parasitoid Peristenus relictus was released, and subsequently established, along the California Central Coast. A simulation model, based on field-collected L. hesperus immigration rates and 16 years of parasitism data, compared simulated pest populations with and without parasitism by P. relictus. After 10 years, L. hesperus population reductions exceeded 95% in weeds and alfalfa, and 70% in strawberry. These simulated results were in agreement with field-based P. relictus longitudinal studies. The averted value loss associated with L. hesperus, due to the establishment of P. relictus, is estimated at $1,901 per acre in organic strawberry.

category: ecology


title: Uncertainties of cell number estimation in cyanobacterial colonies and the potential use of sphere packing

abstract:
Cyanobacteria are notorious bloom formers causing various water quality concerns, such as toxin production, extreme diurnal variation of oxygen, or pH, etc., therefore, their monitoring is essential to protect the ecological status of aquatic systems. Cyanobacterial cell counts and biovolumes are currently being used in water management and water quality alert systems. In this study, we investigated the accuracy of colonial biovolume and cell count estimation approaches used in the everyday practice. Using real like 3-dimensional images of cyanobacterial colonies, we demonstrated that their shape cannot be approximated by ellipsoids. We also showed that despite the significant relationship between overall colony volume and cell biovolumes because of the considerable scatter of cell count data the regressions give biased estimates for cyanobacterial cell counts. We proposed a novel approach to estimate cell counts in colonies that was based on the random close sphere packing method. This method provided good results only in those cases when overall colony volumes could be accurately measured. The visual investigation of colonies done by skilled experts has given precise but lower estimates for cell counts. Estimation results of several experts were surprisingly good which suggest that this capability can be improved, and estimation bias can be reduced to the level acceptable for water quality estimations.

HighlightsCyanobacterial colony - cell biovolume relationships provide biased estimates for cellbiovolumes.
Sphere packing approach provides good cellcount estimates if colony volumes are accurately measured.
Considering cyanobacterial colonies as ellipsoids gives inaccurate volume estimates.
Skilled experts slightly underestimate the cellcounts but dispersion of their estimates is low.


title: Natural tolerance to transposition is associated with Myc-regulation and DNA repair

abstract: Transposable elements (TE) are mobile genetic parasites whose unregulated activity in the germline causes DNA damage and sterility. While the regulation of TE mobilization by hosts is studied extensively, little is known about mechanisms that could allow germline cells to persist in the face of genotoxic stress imposed by active transposition. Such tolerance mechanisms are predicted to be beneficial when new TEs invade and host repression has not yet evolved. Here we use hybrid dysgenesis--a sterility syndrome of Drosophila caused by transposition of invading DNA transposons--to uncover genetic variants that confer tolerance to transposition. Using a panel of highly recombinant inbred lines of Drosophila melanogaster, we identified two linked quantitative trait loci (QTL), that determine tolerance in young and old females, respectively. Through transcriptomic and phenotypic comparisons, we provide evidence that young tolerant females exhibit enhanced repair of double-stranded breaks, explaining their ability to withstand high germline transposition rates. We furthermore identify the germline differentiation factor brat as an independent tolerance factor, whose activity may promote germline maintenance in aging dysgenic females. Together, our work reveals the diversity of potential tolerance mechanisms across development, as well as tolerant variants that may be beneficial in the context of P-element transposition.

category: genomics


title: Natural tolerance to transposition is associated with double-strand break repair and germ-cell differentiation


title: SpecDB: A Relational Database for Archiving Biomolecular NMR Spectra Data

abstract:
NMR is a valuable experimental tool in the structural biologists toolkit to elucidate the structures, functions, and motions of biomolecules. The progress of machine learning, particularly in structural biology, reveals the critical importance of large, diverse, and reliable datasets in developing new methods and understanding in structural biology and science more broadly. Protein NMR research groups produce large amounts of data, and there is renewed interest in organizing this data to train new, sophisticated machine learning architectures to improve biomolecular NMR analysis pipelines. The foundational data type in NMR is the free-induction decay (FID). There are opportunities to build sophisticated machine learning methods to tackle long-standing problems in NMR data processing, resonance assignment, dynamics analysis, and structure determination using NMR FIDs. Our goal in this study is to provide a lightweight, broadly available tool for archiving FID data as it is generated at the spectrometer, and grow a new resource of FID data and associated metadata. This study presents a relational schema for storing and organizing the metadata items that describe an NMR sample and FID data, which we call Spectra Database (SpecDB). SpecDB is implemented in SQLite and includes a Python software library providing a command-line application to create, organize, query, backup, share, and maintain the database. This set of software tools and database schema allow users to store, organize, share, and learn from NMR time domain data. SpecDB is freely available under an open source license at https://github.rpi.edu/RPIBioinformatics/SpecDB.

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category: bioinformatics


title: Rapid Knowledgebase Construction and Hypotheses Generation Using Extractive Literature Search

abstract:
As knowledgebases become increasingly important for structuring vast amounts of scientific knowledge and making it accessible to researchers, their construction entails expensive multi-year projects involving teams of bio-curators, computer scientists, or both. This restricts the coverage of existing knowledgebases to a limited set of popular topics, leaving a long tail of more specialized interests uncovered.

We present a methodology and a supporting tool to allow individual researchers or small teams, without background in bio-curation or computer science, to mine the scientific literature and construct ad-hoc, personalized, and literature-anchored knowledgebases, that are tailored around their specific research interests and support their scientific goals. The time investment involved in creating a knowledgebase ranges from a few hours to a few weeks, depending on the desired coverage and accuracy.

We demonstrate the methodology by constructing knowledgebases for different purposes: a high-level overview of challenges and controversies in a field (the cancer frontiers knowledgebase); a mapping of main concepts and interactions in a field, to support lab-internal hypothesis generation (tissue engineering and regeneration, cancer surgery and radiotherapy knowledgebases); and a comprehensive and accurate knowledgebase designated as an online up-to-date resource for the wider research community (the cell specific drug delivery knowledgebase). In each case we show how the structured knowledgebase, coupled with effective visualizations, facilitates effective data exploration, hypothesis generation and meta-analysis.

We implement the method as part of an open source web-based platform for knowledgebase construction, available publicly and freely at https://spike-kbc.apps.allenai.org.


title: Epilepsy-associated increase in gonadotropin-releasing hormone neuron firing in diestrous female mice is independent of chronic seizure burden severity

abstract: Reproductive endocrine disorders are common comorbidities of temporal lobe epilepsy (TLE). Our previous studies using the intrahippocampal kainic acid (IHKA) mouse model of TLE demonstrated that many females show prolonged estrous cycles and hypothalamic gonadotropin-releasing hormone (GnRH) neurons exhibit elevated firing during diestrus. However, it is unknown whether the degree of change in GnRH neuron activity is dependent on epilepsy severity. Here, we used 24/7 in vivo EEG and in vitro electrophysiological recordings in acute brain slices to assess GnRH neuron firing in relation to chronic seizure burden in diestrous female mice at two months after IHKA injection. We found that percentage of time in seizure activity in the 24 hours prior to slice preparation is an accurate proxy of overall seizure burden. Firing rates of GnRH neurons from EEG-recorded IHKA mice were increased in comparison to controls, but no relationships were found between GnRH neuron firing and seizure burden measured in vivo. The independence of GnRH neuron firing rate in relation to seizure burden was unaffected by GnRH neuron soma location or estrous cycle length. Furthermore, GnRH neuron firing rates were not yet different from control values when measured 1 month after injection when epileptogenesis is already complete in IHKA mice. This result suggests a potential window for intervention to prevent epilepsy-associated GnRH neuron dysfunction. These findings indicate that the severity of epilepsy and the degree of downstream disruption to GnRH neuron activity are independent, suggesting that susceptibility to reproductive endocrine comorbidities is driven by other risk factors.


title: Omicron-Based Vaccine Candidate Elicits Potent Neutralizing Antibodies in the Animal Model

abstract:
The B.1.1.529 (Omicron) variant of SARS-CoV-2 with multiple novel mutations has reduced the neutralization potential of vaccinated individuals sera. World Health Organization has suggested that a vaccination strategy based on repeated booster doses is unlikely to be sustainable. The objective of this commentary was to investigate the safety and neutralizing antibody induction of the Omicron-based SARS-CoV-2 vaccine candidate, BIV1-CovIran Plus, against the Omicron variant on mice and guinea pig models.

After isolation and characterization, the Omicron variant underwent chemical inactivation, purification, and was then formulated with alum adjuvant. A full human dose of BIV1-CovIran Plus was injected intraperitoneally to five female mice and two Guinea pigs for abnormal toxicity reactions evaluation and pathologic investigations. For potency evaluation, four groups of ten mice received two doses of BIV1-CovIran Plus or phosphate-buffered-saline at 7-day and 14-day intervals. The conventional virus-neutralizing test was conducted on sera acquired from vaccinated mice groups seven days after the second injection.

There was no evidence of abnormal clinical symptoms macroscopic or microscopic tissue alterations among the animal models. In all samples from the study group that received two doses of BIV1-CovIran Plus at a 7-day interval, the sera at [≥]1/32 times dilution would neutralize the Omicron variant SARS-CoV-2. Similarly, the sera of all samples from the study group, which received two doses of BIV1-CovIran Plus at a 14-day interval, at [≥]1/64 times dilution, would neutralize the Omicron variant SARS-CoV-2. Moreover, six out of ten (60%) of the samples in this group would neutralize the Omicron variant of SARS-CoV-2 at 1/128 times dilution. CPE formation was observed in all samples from the control group, and no neutralizing activity was detected at any sera dilutions.

BIV1-CovIran Plus was well-tolerated in the animal models, and no safety concerns were raised. Moreover, the vaccine candidate elicited protective neutralization against the Omicron variant. Future reports will focus on the use of the updated vaccine as a booster dose and the potency of the vaccine candidate on other SARS-CoV-2 variants.

category: immunology


title: Increased Potency and Breadth of SARS-CoV-2 Neutralizing Antibodies After a Third mRNA Vaccine Dose

abstract: The omicron variant of SARS-CoV-2 infected very large numbers of SARS-CoV-2 vaccinated and convalescent individuals1-3. The penetrance of this variant in the antigen experienced human population can be explained in part by the relatively low levels of plasma neutralizing activity against Omicron in people who were infected or vaccinated with the original Wuhan-Hu-1 strain4-7. The 3rd mRNA vaccine dose produces an initial increase in circulating anti-Omicron neutralizing antibodies, but titers remain 10-20-fold lower than against Wuhan-Hu-1 and are, in many cases, insufficient to prevent infection7. Despite the reduced protection from infection, individuals that received 3 doses of an mRNA vaccine were highly protected from the more serious consequences of infection8. Here we examine the memory B cell repertoire in a longitudinal cohort of individuals receiving 3 mRNA vaccine doses9,10. We find that the 3rd dose is accompanied by an increase in, and evolution of, anti-receptor binding domain specific memory B cells. The increase is due to expansion of memory B cell clones that were present after the 2nd vaccine dose as well as the emergence of new clones. The antibodies encoded by these cells showed significantly increased potency and breadth when compared to antibodies obtained after the 2nd vaccine dose. Notably, the increase in potency was especially evident among newly developing clones of memory cells that differed from the persisting clones in targeting more conserved regions of the RBD. Overall, more than 50% of the analyzed neutralizing antibodies in the memory compartment obtained from individuals receiving a 3rd mRNA vaccine dose neutralized Omicron. Thus, individuals receiving 3 doses of an mRNA vaccine encoding Wuhan-Hu-1, have a diverse memory B cell repertoire that can respond rapidly and produce antibodies capable of clearing even diversified variants such as Omicron. These data help explain why a 3rd dose of an mRNA vaccine that was not specifically designed to protect against variants is effective against variant-induced serious disease.


title: SARS-CoV-2-specific T cells associate with reduced lung function and inflammation in pulmonary post-acute sequalae of SARS-CoV-2

abstract:
As of January 2022, at least 60 million individuals are estimated to develop post-acute sequelae of SARS-CoV-2 (PASC) after infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While elevated levels of SARS-CoV-2-specific T cells have been observed in non-specific PASC, little is known about their impact on pulmonary function which is compromised in the majority of these individuals. This study compares frequencies of SARS-CoV-2-specific T cells and inflammatory markers with lung function in participants with pulmonary PASC and resolved COVID-19 (RC). Compared to RC, participants with respiratory PASC had up to 34-fold higher frequencies of IFN-{gamma}- and TNF--producing SARS-CoV-2-specific CD4+ and CD8+ T cells in peripheral blood and elevated levels of plasma CRP and IL-6. Importantly, in PASC participants the frequency of TNF--producing SARS-CoV-2-specific CD4+ and CD8+ T cells, which exhibited the highest levels of Ki67 indicating they were activity dividing, correlated positively with plasma IL-6 and negatively with measures of lung function, including forced expiratory volume in one second (FEV1), while increased frequencies of IFN-{gamma}-producing SARS-CoV-2-specific T cells associated with prolonged dyspnea. Statistical analyses stratified by age, number of comorbidities and hospitalization status demonstrated that none of these factors affect differences in the frequency of SARS-CoV-2 T cells and plasma IL-6 levels measured between PASC and RC cohorts. Taken together, these findings demonstrate elevated frequencies of SARS-CoV-2-specific T cells in individuals with pulmonary PASC are associated with increased systemic inflammation and decreased lung function, suggesting that SARS-CoV-2-specific T cells contribute to lingering pulmonary symptoms. These findings also provide mechanistic insight on the pathophysiology of PASC that can inform development of potential treatments to reduce symptom burden.

Author SummaryLong COVID-19 or post-acute sequelae of SARS-CoV-2 (PASC) impacts 20-30% of those infected with SARS-CoV-2 and is characterized by COVID-19 symptoms exceeding 4 weeks from symptom onset. While those with PASC experience a wide variety of persistent symptoms including shortness of breath, cough, chest pain, irregular heartbeat, brain fog, fatigue, and intermittent fever, lung-related conditions are the most common. Although, infection with SARS-CoV-2 is clearly the inciting factor for PASC, the mechanisms responsible for long-term lung dysfunction are unclear and current treatments are ineffective at resolving pulmonary symptoms. Generalized PASC has been associated with SARS-CoV-2-specific T cells, a component of adaptive immunity, suggesting that residual virus may persist. Here, we investigated the frequency and function of virus-specific T cells in the blood of individuals with pulmonary PASC and correlated their presence with systemic inflammation and lung function. Our findings demonstrated that T cells specific for SARS-CoV-2 are elevated in the blood of those with pulmonary PASC and are associated with increased IL-6, a cytokine strongly associated with COVID-19 severity, and decreased lung function. These findings provide mechanistic insight into the pathophysiology of pulmonary PASC needed for the development of new treatments to improve quality of life for those affected.


title: A novel consensus-based computational pipeline for rapid screening of antibody therapeutics for efficacy against SARS-CoV-2 variants of concern including omicron variant

abstract: Multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to evolve carrying flexible amino acid substitutions in the spike proteins receptor binding domain (RBD). These substitutions modify the binding of the SARS-CoV-2 to human angiotensin-converting enzyme 2 (hACE2) receptor and have been implicated in altered host fitness, transmissibility and efficacy against antibody therapeutics and vaccines. Reliably predicting the binding strength of SARS-CoV-2 variants RBD to hACE2 receptor and neutralizing antibodies (NAbs) can help assessing their fitness, and rapid deployment of effective antibody therapeutics, respectively. Here, we introduced a two-step computational framework with three-fold validation that first identified dissociation constant as a reliable predictor of binding affinity in hetero-dimeric and -trimeric protein complexes. The second step implements dissociation constant as descriptor of the binding strengths of SARS-CoV-2 variants RBD to hACE2 and NAbs. Then, we examined several variants of concern (VOCs) such as Alpha, Beta, Gamma, Delta, and Omicron and demonstrated that these VOCs RBD bind to the hACE2 with enhanced affinity. Furthermore, the binding affinity of Omicron variants RBD was reduced with majority of the RBD-directed NAbs, which is highly consistent with the experimental neutralization data. By studying the atomic contacts between RBD and NAbs, we revealed the molecular footprints of four NAbs (GH-12, P2B-1A1, Asarnow_3D11, and C118) -- that may likely neutralize the recently emerged omicron variant -- facilitating enhanced binding affinity. Finally, our findings suggest a computational pathway that could aid researchers identify a range of current NAbs that may be effective against emerging SARS-CoV-2 variants.


title: Introducing CARATE: Finally speaking chemistry.

abstract: AO_SCPLOWBSTRACTC_SCPLOWComputer-Aided Drug Design is advancing to a new era. Recent developments in statistical modelling, including Deep Learning, Machine Learning and high throughput simulations, enable workflows and deductions not achievable 20 years ago. The key interaction for many small molecules is via bio-molecules. The interaction between a small molecule and a biological system therefore manifests itself at multiple time and length scales. While the human chemist quite intuitively grasps the concept of multiple scales, most of the computer technologies do not relate multiple scales easily. Therefore, numerous methods in the realm of computational sciences have been developed. However, up to now it was not clear that the problem of multiple scales is not only a mere matter of computational abilities but even more a matter of accurate representation. Because of the amount of already performed simulations at various scales, deep learning (DL) becomes a viable approach to speed up computations by approximating simulations in a data-driven way. However, to accurately approximate the physical (simulated) properties of a given compound, an accurate, uniform representation is mandatory. Therefore, the biochemical and pharmaceutical encoder (CARATE) is introduced. Furthermore, the regression and classification abilities of CARATE are evaluated against benchmarking datasets (ZINC, ALCHEMY, MCF-7, MOLT-4, YEAST, Enzymes, Proteins) and compared to other baseline approaches.


title: Characterization of TR-107, a Novel Chemical Activator of the Human Mitochondrial Protease ClpP

abstract: We recently described the identification of a new class of small molecule activators of the mitochondrial protease ClpP. These compounds synthesized by Madera Therapeutics showed increased potency of cancer growth inhibition over the related compound ONC201. In this study, we describe chemical optimization and characterization of the next generation of highly potent and selective small molecule ClpP activators (TR compounds) and demonstrate their efficacy against breast cancer models in vitro and in vivo. One of these compounds (TR-107) with excellent potency, specificity and drug-like properties was selected for further evaluation. Examination of TR-107 effects in triple-negative breast cancer (TNBC) cell models showed growth inhibition in the low nanomolar range, equipotent to paclitaxel, in a ClpP-dependent manner. TR-107 reduced specific mitochondrial proteins including OXPHOS and TCA cycle components, in a time, dose and ClpP-dependent manner. Seahorse XF analysis and glucose deprivation experiments confirmed inactivation of OXPHOS and demonstrated an increased dependence on glycolysis following TR-107 exposure. The pharmacokinetic properties of TR-107 were compared to other known ClpP activators including ONC201 and ONC212. TR-107 displayed excellent exposure and serum t1/2 after oral administration. The antitumor response to TR-107 was investigated using human TNBC MDA-MB-231 cell line-induced xenograft tumors. Oral administration of TR-107 resulted in reduction in tumor volume and extension of survival in the treated compared with vehicle control mice. In summary, we describe the identification of highly potent new ClpP agonists with improved efficacy against TNBC, through targeted inactivation of OXPHOS and disruption of mitochondrial metabolism.

category: pharmacology and toxicology


title: Virological characteristics of SARS-CoV-2 BA.2 variant

abstract: Soon after the emergence and global spread of a new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron lineage, BA.1 (ref1, 2), another Omicron lineage, BA.2, has initiated outcompeting BA.1. Statistical analysis shows that the effective reproduction number of BA.2 is 1.4-fold higher than that of BA.1. Neutralisation experiments show that the vaccine-induced humoral immunity fails to function against BA.2 like BA.1, and notably, the antigenicity of BA.2 is different from BA.1. Cell culture experiments show that BA.2 is more replicative in human nasal epithelial cells and more fusogenic than BA.1. Furthermore, infection experiments using hamsters show that BA.2 is more pathogenic than BA.1. Our multiscale investigations suggest that the risk of BA.2 for global health is potentially higher than that of BA.1.

category: microbiology


title: SARS-CoV-2 Omicron-specific mRNA vaccine induces potent and broad antibody responses in vivo

abstract: The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has high transmissibility and recently swept the globe. Due to the extensive number of mutations, this variant has high level of immune evasion, which drastically reduced the efficacy of existing antibodies and vaccines. Thus, it is important to test an Omicron-specific vaccine, evaluate its immune response against Omicron and other variants, and compare its immunogenicity as boosters with existing vaccine designed against the reference wildtype virus (WT). Here, we generated an Omicron-specific lipid nanoparticle (LNP) mRNA vaccine candidate, and tested its activity in animals, both alone and as a heterologous booster to existing WT mRNA vaccine. Our Omicron-specific LNP-mRNA vaccine elicited strong and specific antibody response in vaccination-naive mice. Mice that received two-dose WT LNP-mRNA, the one mimicking the commonly used Pfizer/Moderna mRNA vaccine, showed a >40-fold reduction in neutralization potency against Omicron variant than that against WT two weeks post second dose, which further reduced to background level >3 months post second dose. As a booster shot for two-dose WT mRNA vaccinated mice, a single dose of either a homologous booster with WT LNP-mRNA or a heterologous booster with Omicron LNP-mRNA restored the waning antibody response against Omicron, with over 40-fold increase at two weeks post injection as compared to right before booster. Interestingly, the heterologous Omicron LNP-mRNA booster elicited neutralizing titers 10-20 fold higher than the homologous WT booster against the Omicron variant, with comparable titers against the Delta variant. All three types of vaccination, including Omicron mRNA alone, WT mRNA homologous booster, and Omicron heterologous booster, elicited broad binding antibody responses against SARS-CoV-2 WA-1, Beta, and Delta variants, as well as other Betacoronavirus species such as SARS-CoV, but not Middle East respiratory syndrome coronavirus (MERS-CoV). These data provided direct proof-of-concept assessments of an Omicron-specific mRNA vaccination in vivo, both alone and as a heterologous booster to the existing widely-used WT mRNA vaccine form.


title: Omicron-specific mRNA vaccination alone and as a heterologous booster against SARS-CoV-2


title: BinSPreader: refine binning results for fuller MAG reconstruction

abstract:
Despite the recent advances in high-throughput sequencing, analysis of the metagenome of the whole microbial population still remains a challenge. In particular, the metagenome-assembled genomes (MAGs) are often fragmented due to interspecies repeats, uneven coverage and vastly different strain abundance. MAGs are usually constructed via a dedicated binning process that uses different features of input data in order to cluster contigs that might belong to the same species. This process has some limitations and therefore binners usually discard input contigs that are shorter than several kilobases. Therefore, binning of even simple metagenome assemblies can miss a decent fraction of contigs and resulting MAGs oftentimes do not contain important conservative sequences that might be of great interest of researcher.

In this work we present BO_SCPLOWINC_SCPLOWSPO_SCPLOWREADERC_SCPLOW -- a novel binning refiner tool that exploits the assembly graph topology and other connectivity information to refine the existing binning, correct binning errors, propagate binning from longer contigs to shorter contigs and infer contigs belonging to multiple bins. Furthermore, BO_SCPLOWINC_SCPLOWSPO_SCPLOWREADERC_SCPLOW can split input reads in accordance with the resulting binning, predicting reads potentially belonging to multiple MAGs. We show that BO_SCPLOWINC_SCPLOWSPO_SCPLOWREADERC_SCPLOW could effectively complete the binning, increasing the completeness of the bins without sacrificing the purity and could predict contigs belonging to several MAGs.


title: Salinity induces discontinuous protoxylem by a DELLA-dependent mechanism promoting salt tolerance in Arabidopsis seedlings

abstract:
O_LISalinity is detrimental to plants and developmental adjustments limiting salt uptake and transport is therefore important for acclimation to high salt. These parameters may be influenced by xylem morphology, however, how plant root xylem development is affected by salt stress remains unclear.
C_LIO_LIThrough detailed phenotypic analyses, molecular and genetic techniques, we demonstrate that salt causes distinct effects on Arabidopsis seedling root xylem, and reveal underlying molecular mechanisms.
C_LIO_LISalinity causes intermittent inhibition of protoxylem cell differentiation, generating protoxylem gaps, in Arabidopsis and other eudicot seedlings. The extent of protoxylem gaps positively correlates with salt tolerance. Reduced gibberellin signaling is required for protoxylem gap formation. Mutant analyses reveal that the xylem differentiation regulator VASCULAR RELATED NAC DOMAIN 6 (VND6), along with secondary cell wall-producing and cell wall modifying enzymes, including EXPANSIN A1 (EXP1), are involved in protoxylem gap formation, in a DELLA-dependent manner.
C_LIO_LISalt stress impacts seedling survival and formation of protoxylem gaps is a means of enhancing salt tolerance. Salt stress likely reduces levels of bioactive gibberellins, stabilizing DELLAs, which in turn activate multiple factors modifying protoxylem differentiation. Formation of protoxylem gaps is induced in diverse eudicot species suggesting that this is an evolutionary conserved response for salt acclimation in seedlings.
C_LI

category: plant biology


title: Exploring volume mediated bystander-inhibition in a neuron model of the Locus coeruleus

abstract:
The Locus coeruleus (LC) modulates various neuronal circuits throughout the brain. Its unique architectural organization encompasses a net of axonal innervation that spans the entire brain; while its somatic core is highly compact. Recent research revealed an unexpected cellular input specificity within the nucleus that can give rise to various network states that either broadcast norepinephrine signals throughout the brain or pointedly modulate specific brain areas. Such adaptive input-output functions likely surpass our existing network models that build upon a given synaptic wiring configuration between neurons. As the distances between noradrenergic neurons in the core of the LC are unusually small, neighboring neurons could theoretically impact each other via volume transmission of NE. We therefore set out to investigate if such interaction could be mediated through noradrenergic alpha2-receptors in a spiking neuron model of the LC. We validate our model of LC neurons through comparison with experimental patch-clamp data and identify key variables that impact alpha2-mediated inhibition of neighboring LC neurons. Our simulation confirms a reliable autoinhibition of LC neurons after episodes of high neuronal activity that continue even after neuronal activity subsided. Also, dendro-somatic synapses inhibit spontaneous spiking in the somatic compartment of connected neurons in our model. We determined the exact position of hundreds of LC neurons in the mouse brain stem via a tissue clearing approach and, based on this, further determined that 25 percent of noradrenergic neurons have a neighboring LC neuron within less than a 25-micrometer radius. By modeling NE diffusion, we estimate that more than 15 percent of alpha2-adrenergic receptors fraction can bind NE within such diffusion radius. Our spiking neuron model of LC neurons predicts that repeated or long-lasting episodes of high neuronal activity induce partitioning of the gross LC network, and reduces the spike rate in neighboring neurons at distances smaller than 25 micrometers.

As these volume-mediate neighboring effects are challenging to test with the current methodology, our findings can guide future experimental approaches to test this phenomenon and its physiological consequences.


title: Spike-dependent dynamic partitioning of the Locus coeruleus network through noradrenergic volume release in a simulation of nucleus core


title: Predicting vertical ground reaction forces from 3D accelerometery using reservoir computers leads to accurate gait event detection

abstract:
Accelerometers are low-cost measurement devices that can readily be used outside the lab. However, determining isolated gait events from accelerometer signals, especially foot-off events during running, is an open problem. We outline a two-step approach where machine learning serves to predict vertical ground reaction forces from accelerometer signals, followed by force-based event detection.

We collected shank accelerometer signals and ground reaction forces from five adults during comfortable walking and running on an instrumented treadmill. We trained one common reservoir computer using segmented data using both walking and running data. Despite being trained on just a small number of strides, this reservoir computer predicted vertical ground reaction forces in continuous gait with excellent quality. The subsequent foot contact and foot off event detection proved highly accurate when compared to the golden standard based on co-registered ground reaction forces. Our proof-of-concept illustrates the capacity of combining accelerometery with machine learning for detecting isolated gait events irrespective of mode of locomotion.


title: Population based selection shapes the T cell receptor repertoire during thymic development

abstract: One of the feats of adaptive immunity is its ability to recognize foreign pathogens while sparing the self. During maturation in the thymus, T cells are selected through the binding properties of their antigen-specific T-cell receptor (TCR), through the elimination of both weakly (positive selection) and strongly (negative selection) self-reactive receptors. However, the impact of thymic selection on the TCR repertoire is poorly understood. Here, we use transgenic Nur77-mice expressing a T-cell activation reporter to study the repertoires of thymic T cells at various stages of their development, including cells that do not pass selection. We combine high-throughput repertoire sequencing with statistical inference techniques to charactarize the selection of the TCR in these distinct subsets. We find small but significant differences in the TCR repertoire parameters between the maturation stages, which recapitulate known differentiation pathways leading to the CD4+ and CD8+ subtypes. These differences can be simulated by simple models of selection acting linearly on the sequence features. We find no evidence of specific sequences or sequence motifs or features that are suppressed by negative selection. These results are consistent with a collective or statistical model for T-cell specificity, where negative selection biases the repertoire away from self recognition, rather than ensuring lack of self-reactivity at the single-cell level.


title: Clock Work: Deconstructing the Epigenetic Clock Signals in Aging, Disease, and Reprogramming

abstract: Epigenetic clocks have come to be regarded as powerful tools for estimating aging. However, a major drawback in their application is our lack of mechanistic understanding. We hypothesize that uncovering the underlying biology is difficult due to the fact that epigenetic clocks are multifactorial composites: They are comprised of disparate parts, each with their own causal mechanism and functional consequences. Thus, only by deconstructing epigenetic clock signals will it be possible to glean biological insight. Here we clustered 5,717 clock CpGs into twelve distinct modules using multi-tissue and in-vitro datasets. We show that epigenetic clocks are comprised of different proportions of modules, which may explain their discordance when simultaneously applied in a given study. We also observe that epigenetic reprogramming does not  reset the entire clock and instead the observed rejuvenation is driven by a subset of modules. Overall, two modules stand-out in terms of their unique features. The first is one of the most responsive to epigenetic reprogramming; is the strongest predictor of all-cause mortality; and shows increases with in vitro passaging up until senescence burden begins to emerge. The light-second module is moderately responsive to reprogramming; is very accelerated in tumor vs. normal tissues; and tracks with passaging in vitro even as population doublings decelerate. Overall, we show that clock deconstruction can identify unique DNAm alterations and facilitate our mechanistic understanding of epigenetic clocks.

category: genetics


title: Characterization of altered molecular mechanisms in Parkinson disease through cell type-resolved multi-omics analyses

abstract:
Parkinsons disease (PD) is a progressive neurodegenerative disorder. However, cell type-dependent transcriptional regulatory programs responsible for PD pathogenesis remain elusive. Here, we establish transcriptomic and epigenomic landscapes of the substantia nigra (SN) by profiling 87,733 nuclei obtained from healthy controls and PD patients. Our multi-omic data integration provides functional annotation of 128,724 cis-regulatory elements (cREs) and uncovers cell-type specific dysregulated cREs with a strong transcriptional influence on genes implicated in PD. The establishment of high-resolution three-dimensional chromatin contact maps identifies 656 target genes of dysregulated cREs and genetic risk loci, including both novel candidates and known PD risk genes. Notably, these new PD candidate genes exhibit modular gene expression patterns with unique molecular signatures in distinct cell types. Thus, our single-cell transcriptome and epigenome uncover cell type-specific disrupted transcriptional regulations in PD.

TeaserSingle-cell transcriptome and epigenome uncover cell type-specific disrupted transcriptional regulations in Parkinsons disease.


title: Experimental and analytical framework for ''mix-and-read'' assays based on split luciferase

abstract: The use of immunodetection assays including the widely used enzyme-linked immunosorbent assay (ELISA) in applications such as point-of-care detection is often limited by the need for protein immobilization and multiple binding and washing steps. Here, we describe an experimental and analytical framework for the development of simple and modular "mix-and-read" enzymatic complementation assays based on split luciferase that enable sensitive detection and quantification of analytes in solution. In this assay, two engineered protein binders targeting non-overlapping epitopes on the target analyte were each fused to non-active fragments of luciferase to create biosensor probes. Binding proteins to two model targets, lysozyme and Sso6904, were isolated from a combinatorial library of Sso7d mutants using yeast surface display. In the presence of the analyte, probes were brought into close proximity, reconstituting enzymatic activity of luciferase and enabling detection of low picomolar concentrations of the analyte by chemiluminescence. Subsequently, we constructed an equilibrium binding model that relates binding affinities of the binding proteins for the target, assay parameters such as the concentrations of probes used, and assay performance (limit of detection and concentration range over which the target can be quantified). Overall, our experimental and analytical framework provide the foundation for the development of split luciferase assays for detection and quantification of various targets.

category: bioengineering


title: Efficient Bayesian inference for mechanistic modelling with high-throughput data

abstract: Bayesian methods are routinely used to combine experimental data with detailed mathematical models to obtain insights into physical phenomena. However, the computational cost of Bayesian computation with detailed models has been a notorious problem. Moreover, while high-throughput data presents opportunities to calibrate sophisticated models, comparing large amounts of data with model simulations quickly becomes computationally prohibitive. Inspired by the method of Stochastic Gradient Descent, we propose a minibatch approach to approximate Bayesian computation. Through a case study of a high-throughput imaging scratch assay experiment, we show that reliable inference can be performed at a fraction of the computational cost of a traditional Bayesian inference scheme. By applying a detailed mathematical model of single cell motility, proliferation and death to a data set of 118 gene knockdowns, we characterise functional subgroups of gene knockdowns, each displaying its own typical combination of local cell density-dependent and -independent motility and proliferation patterns. By comparing these patterns to experimental measurements of cell counts and wound closure, we find that density-dependent interactions play a crucial role in the process of wound healing.


title: scShapes: A statistical framework for identifying distribution shapes in single-cell RNA-sequencing data

abstract:
BackgroundSingle cell RNA sequencing (scRNA-seq) methods have been advantageous for quantifying cell-to-cell variation by profiling the transcriptomes of individual cells. For scRNA-seq data, variability in gene expression reflects the degree of variation in gene expression from one cell to another. Analyses that focus on cell-cell variability therefore are useful for going beyond changes based on average expression and instead, identifying genes with homogenous expression versus those that vary widely from cell to cell.

ResultsWe present a novel statistical framework scShapes for identifying differential distributions in single-cell RNA-sequencing data using generalized linear models. Most approaches for differential gene expression detect shifts in the mean value. However, as single cell data are driven by over-dispersion and dropouts, moving beyond means and using distributions that can handle excess zeros is critical. scShapes quantifies gene-specific cell-to-cell variability by testing for differences in the expression distribution while flexibly adjusting for covariates if required. We demonstrate that scShapes identifies subtle variations that are independent of altered mean expression and detects biologically-relevant genes that were not discovered through standard approaches.

ConclusionsThis analysis also draws attention to genes that switch distribution shapes from a unimodal distribution to a zero-inflated distribution and raises open questions about the plausible biological mechanisms that may give rise to this, such as transcriptional bursting. Overall, the results from scShapes helps to expand our understanding of the role that gene expression plays in the transcriptional regulation of a specific perturbation or cellular phenotype. Our framework scShapes is incorporated into Bioconductor R package (https://github.com/Malindrie/scShapes).


title: uniPort: a unified computational framework for single-cell data integration with optimal transport

abstract: Single-cell data integration can provide a comprehensive molecular view of cells. Here we introduce uniPort, a unified single-cell data integration framework which combines a coupled Variational Autoencoder (coupled-VAE) and Minibatch Unbalanced Optimal Transport (Minibatch-UOT). It leverages both highly variable common and dataset-specific genes for integration and is scalable to large-scale and partially overlapping datasets. uniPort jointly embeds heterogeneous single-cell multi-omics datasets into a shared latent space. It can further construct a reference atlas for online prediction across datasets. Meanwhile, uniPort provides a flexible label transfer framework to deconvolute spatial heterogeneous data using optimal transport space, instead of embedding latent space. We demonstrate the capability of uniPort by integrating a variety of datasets, including single-cell transcriptomics, chromatin accessibility and spatially resolved transcriptome data. uniPort software is available at https://github.com/caokai1073/uniPort.


title: R-based method for quantitative analysis of biofilm thickness by using Confocal Laser Scanning Microscopy

abstract:
Microscopy is mostly the method of choice to analyse biofilms. Due to the high local heterogeneity of biofilms, single and punctual analyses only give an incomplete insight into the local distribution of biofilms. In order to retrieve statistically significant results a quantitative method for biofilm thickness measurements was developed based on confocal laser scanning microscopy and the programming language R. The R-script allows the analysis of large image volumes with little hands-on work and outputs statistical information on homogeneity of surface coverage and overall biofilm thickness. The applicability of the script was shown in microbial fuel cell experiments. It was found that G. sulfurreducens responds differently to poised anodes of different material so that the optimum potential for MFC on poised ITO anodes had to be identified with respect to maximum current density, biofilm thickness and MFC start-up time. Thereby, a positive correlation between current density and biofilm thickness was found, but with no direct link to the applied potential. The optimum potential turned out to be +0.1 V vs SHE. The script proved to be a valuable stand-alone tool to quantify biofilm thickness in a statistically valid manner, which is required in many studies.

Practical applicationBiofilm communities are ubiquitous. They can be found in every habitat in which water, nutrients and a colonisable surface are present. Depending on the surface, biofilms can cause economic losses due to bio-corrosion (pipelines and ship walls are prominent examples) or are a severe threat to human health when important medical devices or body tissues are colonised [1]. Desirable biofilms are catalytic biofilms how they are used in bioelectrochemical production processes, for example. In all cases, quantitative and qualitative biofilm analysis is necessary in order to prevent or promote biofilm formation. In bioelectrochemistry quantitative biofilm analysis is essential to link productivity (current or chemicals) with biomass deposited on the electrode. Microscopic analysis (e.g. with CLSM) of stained biofilms allows the recording of high volumes of image data but often image analysis then remains at a qualitative stage. In terms of biofilm thickness determination this limits analysis to an estimated thickness of a small amount of images, mostly. The presented R-script allows the calculation of biofilm thickness based on a larger amount of image sets and allows conclusions on the homogeneity of biofilm coverage on the electrode surface. The script is a stand-alone tool if only biofilm thickness should be determined and does not require any image segmentation or processing.


title: Designing human Sphingosine-1-phosphate lyases using a temporal Dirichlet variational autoencoder

abstract:
Enzymatic deficiencies cause the accumulation of toxic levels of substrates in a cell and are associated with life-threatening pathologies. Restoring physiological enzymes levels by injecting a recombinant version of the defective enzyme could provide a viable therapeutic option. However, these enzyme replacement therapies have had limited success, as the recombinant enzymes are less catalytically active, cause immune response and are difficult to manufacture. Moreover, the vast sequence design space makes finding enzymes with desired therapeutic properties extremely challenging.

Here, we present a new enzyme engineering framework, which builds on recent advances in deep learning, variational calculus and natural language processing, to design variants of human enzymes with biochemical features comparable to the wild type protein as a way to rapidly build targeted libraries for downstream screening. We applied our method to design variants of human Sphyngosine-1-phosphate lyase (HsS1PL) as potential therapeutic treatments for nephrotic syndrome type 14 (NPHS14), and characterized their biochemical properties through extensive sequence and molecular dynamics analyses.


title: Dexamethasone Inhibits Cytokine-Induced, DUOX2-Related VEGF-A Expression and DNA damage in Human Pancreatic Cancer Cells and Growth of Pancreatic Cancer Xenografts

abstract: Previously, we demonstrated that pro-inflammatory cytokines enhance dual oxidase 2 (DUOX2)-dependent production of reactive oxygen species by human pancreatic ductal carcinoma (PDAC) cells, and that DUOX2 expression is significantly increased in patients with early stages of PDAC. In genetically-engineered mouse models of PDAC, dexamethasone (Dex) decreases formation of pancreatic intraepithelial neoplasia (PanIn) foci as well as PDAC invasiveness. Herein, we report that Dex, in a concentration- and time-dependent fashion, inhibited pro-inflammatory cytokine (IFN-{gamma}/LPS/IL-17A/IL-4)-mediated enhancement of DUOX2 expression in BxPC-3, CFPAC-1, and AsPC-1 human PDAC cell lines, as well as DUOX2-induced DNA damage. The inhibitory effects of Dex were abolished by pre-treatment with the Dex antagonist RU-486. Examination of the human DUOX2 promoter in silico revealed a putative negative glucocorticoid receptor (GR) binding element (IRnGRE). Western analysis, using nuclear extracts from Dex-treated PDAC cells, demonstrated that Dex activated the glucocorticoid receptor in PDAC cell nuclei in the presence of certain co-repressors, such as NCoR-1/2 and histone deacetylases (HDAC1, 2, and 3). Dex produced no anti-proliferative effects on PDAC cells in vitro. However, Dex significantly decreased the growth of BxPC-3 xenografts while decreasing inflammatory and immune cell infiltration of the microenvironment, as well as the mRNA expression of DUOX2 and VEGF-A, in BxPC-3 tumors. In contrast, Dex had no effect on the growth of xenografts developed from MIA-PaCa cells that are unresponsive to pro-inflammatory cytokines in culture. In summary, these studies suggest that suppression of inflammation-related DUOX2 expression by Dex could diminish the oxidative milieu supporting PDAC growth and development.


title: Interpretation of southern hemisphere humpback whale diet  via stable isotopes; implications of tissue-specific analysis

abstract: Blubber and skin are commonly used tissues in stable isotope analysis for the purpose of investigating cetacean diet. Critical comparison of tissue-specific isotopic signals is, however, lacking resulting in uncertainty surrounding the representativeness and therefore utility of different tissues for accurate determination of recent foraging. This study used remotely biopsied blubber and skin tissues from southern hemisphere humpback whales for strategic comparison of {delta}13C and {delta}15N values. Samples were collected 2008-2018 as part of long-term monitoring under the Humpback Whale Sentinel Program. Blubber tissues were lipid-extracted prior to analysis, whilst mathematical lipid-correction was performed on skin samples. Isotopic values from paired blubber and skin samples from the same individuals were compared to assess whether tissues could be used interchangeably for isotope analysis and dietary interpretation. Significant differences were observed for both {delta}13C and {delta}15N, flagging previously undocumented methodological considerations, and the need for method validation and standardisation in application of these approaches. This study therefore advances methodological aspects of cetacean dietary analysis. This is of elevated importance in the context of rapidly changing ocean ecosystems.


title: Nitric oxide negatively regulates gibberellin signaling to coordinate growth and salt tolerance in Arabidopsis

abstract: In response to dynamically altered environments, plants must finely coordinate the balance between growth and stress responses for their survival. However, the underpinning regulatory mechanisms remain largely elusive. The phytohormone gibberellin promotes growth via a derepression mechanism by proteasomal degradation of the DELLA transcription repressors. Conversely, the stress-induced burst of nitric oxide (NO) enhances stress tolerance, largely relaying on NO-mediated S-nitrosylation, a redox-based posttranslational modification. Here, we show that S-nitrosylation of Cys-374 in the Arabidopsis RGA protein, a key member of DELLAs, inhibits its interaction with the F-box protein SLY1, thereby preventing its proteasomal degradation under salinity condition. The accumulation of RGA consequently retards growth but enhances salt tolerance. We propose that NO negatively regulates gibberellin signaling via S-nitrosylation of RGA to coordinate the balance of growth and stress responses when challenged by adverse environments.


title: OsIRO3 negatively regulates Fe homeostasis by repressing the expression of OsIRO2

abstract: Iron (Fe) is crucial for crop productivity and quality. However, Fe deficiency is prevalent worldwide, especially in alkaline soil. Plants have evolved sophisticated mechanisms to withstand Fe deficiency conditions. Oryza sativa IRON-RELATED BHLH TRANSCRIPTION FACTOR 3 (OsIRO3/OsbHLH63) has been identified as a negative regulator of Fe deficiency response signaling, however, the underlying mechanism remains unclear. In the present study, we constructed two iro3 mutants which generated leaves with necrotic lesions under Fe deficient conditions. Loss-of-function of OsIRO3 caused upregulation of Fe deficiency-associated genes in the root under Fe deficient conditions. Fe concentration measurement showed that the iro3 mutants had increased shoot Fe concentration only under Fe deficient conditions. Further analysis revealed that OsIRO3 directly regulated the expression of IRON-RELATED BHLH TRANSCRIPTION FACTOR 2 (OsIRO2) which encodes a positive regulator of Fe uptake system. Protein interaction tests indicated that OsIRO3 interacted with OsPRI1 and OsPRI2. Further investigation demonstrated that OsIRO3 repressed the transactivation of OsPRI1 and OsPRI2 towards OsIRO2. OsIRO3 contains an EAR motif which recruits the TOPLESS/TOPLESS-RELATED (OsTPL/OsTPRs) corepressors. Mutation of the EAR motif attenuated the repression ability of OsIRO3. This work sheds light on the molecular mechanism by which OsIRO3 modulates Fe homeostasis in rice.


title: Faster growth enhances low carbon fuel and chemical production through gas fermentation

abstract: Gas fermentation offers both fossil carbon-free sustainable production of fuels and chemicals and recycling of gaseous and solid waste using gas-fermenting microbes. Bioprocess development, systems-level analysis of biocatalyst metabolism, and engineering of cell factories are advancing the widespread deployment of the commercialised technology. Acetogens are particularly attractive biocatalysts but effects of the key physiological parameter - specific growth rate () - on acetogen metabolism and the gas fermentation bioprocess have not been established yet. Here, we investigate the -dependent bioprocess performance of the model-acetogen Clostridium autoethanogenum in CO and syngas (CO+CO2+H2) grown chemostat cultures and assess systems-level metabolic responses using gas analysis, metabolomics, transcriptomics, and metabolic modelling. We were able to obtain steady-states up to  ~2.8 day-1 (~0.12 h-1) and show that faster growth supports both higher yields and productivities for reduced by-products ethanol and 2,3-butanediol. Transcriptomics data revealed differential expression of 1,337 genes with increasing  and suggest that C. autoethanogenum uses transcriptional regulation to a large extent for facilitating faster growth. Metabolic modelling showed significantly increased fluxes for faster growing cells that were, however, not accompanied by gene expression changes in key catabolic pathways for CO and H2 metabolism. Cells thus seem to maintain sufficient "baseline" gene expression to rapidly respond to CO and H2 availability without delays to kick-start metabolism. Our work advances understanding of transcriptional regulation in acetogens and shows that faster growth of the biocatalyst improves the gas fermentation bioprocess.

category: systems biology


title: Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission

abstract: The fluorescent glutamate indicator iGluSnFR enables imaging of neurotransmission with genetic and molecular specificity. However, existing iGluSnFR variants exhibit saturating activation kinetics and are excluded from post-synaptic densities, limiting their ability to distinguish synaptic from extrasynaptic glutamate. Using a multi-assay screen in bacteria, soluble protein, and cultured neurons, we generated novel variants with improved kinetics and signal-to-noise ratios. We also developed surface display constructs that improve iGluSnFRs nanoscopic localization to post-synapses. The resulting indicator, iGluSnFR3, exhibits rapid non-saturating activation kinetics and reports synaptic glutamate release with improved linearity and increased specificity versus extrasynaptic signals in cultured neurons. In mouse visual cortex, imaging of iGluSnFR3 at individual boutons reported single electrophysiologically-observed action potentials with high specificity versus non-synaptic transients. In vibrissal sensory cortex Layer 4, we used iGluSnFR3 to characterize distinct patterns of touch-evoked feedforward input from thalamocortical boutons and both feedforward and recurrent input onto L4 cortical neuron dendritic spines.


title: Translaminar Recurrence from Layer 5 Suppresses Superficial Cortical Layers

abstract: Information flow in the sensory cortex has been described as a predominantly feedforward sequence with deep layers as the output structure. Although recurrent excitatory projections from layer 5 (L5) to superficial L2/3 have been identified by anatomical and physiological studies, their functional impact on sensory processing remains unclear. Here, we use layer-selective optogenetic manipulations in the primary auditory cortex to demonstrate that feedback inputs from L5 suppress the activity of superficial layers, contrary to the prediction from their excitatory connectivity. This suppressive effect is predominantly mediated by translaminar circuitry through intratelencephalic (IT) neurons, with an additional contribution of subcortical projections by pyramidal tract (PT) neurons. Furthermore, L5 activation sharpened tone-evoked responses of superficial layers in both frequency and time domains, indicating its impact on cortical spectro-temporal integration. Together, our findings challenge the classical view of feedforward cortical circuitry and reveal a major contribution of inhibitory recurrence in shaping sensory representations.


title: Increased low-frequency brain responses to music after psilocybin therapy for depression

abstract: Psychedelic-assisted psychotherapy with psilocybin is an emerging therapy with great promise for depression, and modern psychedelic therapy (PT) methods incorporate music as a key element. Music is an effective emotional/hedonic stimulus that could also be useful in assessing changes in emotional responsiveness following psychedelic therapy. Brain responses to music were assessed before and after PT using functional Magnetic Resonance Imaging (fMRI) and ALFF (Amplitude of Low Frequency Fluctuations) analysis methods. Nineteen patients with treatment-resistant depression underwent two treatment sessions involving administration of psilocybin, with MRI data acquired one week prior and the day after completion of the second of two psilocybin dosing sessions. Comparison of music-listening and resting-state scans revealed significantly greater ALFF in bilateral superior temporal cortex for the post-treatment music scan, and in the right ventral occipital lobe for the post-treatment resting-state scan. ROI analyses of these clusters revealed a significant effect of treatment in the superior temporal lobe for the music scan only. Somewhat consistently, voxelwise comparison of treatment effects showed relative increases for the music scan in the bilateral superior temporal lobes and supramarginal gyrus, and relative decreases in the medial frontal lobes for the resting-state scan. ALFF in these music-related clusters was significantly correlated with intensity of subjective effects felt during the dosing sessions. These data suggest a specific effect of PT on the brains response to a hedonic stimulus (music), implying an elevated responsiveness to music after psilocybin therapy that was related to subjective drug effects felt during dosing.


title: Revisiting the origin of interleukin 1 (IL-1) based on biological activities of IL-1 in anamniotes and their sub-functionalization in amniotes

abstract: The cytokine Interleukin 1 (IL-1) is an evolutionary innovation of vertebrates. Fish and amphibia have one IL1 gene, while mammals have two copies of IL1, IL1A and IL1B, with distinct expression patterns and differences in their proteolytic activation. Our current understanding of the evolutionary history of IL-1 is mainly based on phylogenetic analyses, but this approach provides no information on potentially different functions of IL-1 homologs, and it remains unclear which biological activities identified for IL-1 and IL-1{beta} in mammals are present in lower vertebrates. Here, we use in vitro and in vivo experimental models to examine the expression patterns and cleavage of IL-1 proteins from various species. We found that IL-1 in the teleost medaka shares the transcriptional patterns of mammalian IL-1, and its processing also resembles that of mammalian IL-1, which is sensitive to cysteine protease inhibitors specific for the calpain and cathepsin families. By contrast, IL-1 proteins in reptiles also include biological properties of IL-1{beta}. Therefore, we propose that duplication of the ancestral IL1 gene led to segregation of expression patterns and protein processing that characterizes the two extant forms of IL-1 in mammals.


title: The BNT162b2 mRNA SARS-CoV-2 vaccine induces transient afucosylatedIgG1 in naive but not antigen-experienced vaccinees

abstract:
The onset of severe SARS-CoV-2 infection is characterized by the presence of afucosylated IgG1 responses against the viral spike (S) protein, which can trigger exacerbated inflammatory responses. Here, we studied IgG glycosylation after BNT162b2 SARS-CoV-2 mRNA vaccination to explore whether vaccine-induced S protein expression on host cells also generates afucosylated IgG1 responses. SARS-CoV-2 naive individuals initially showed a transient afucosylated anti-S IgG1 response after the first dose, albeit to a lower extent than severely ill COVID-19 patients. In contrast, previously infected, antigen-experienced individuals had low afucosylation levels, which slightly increased after immunization. Afucosylation levels after the first dose correlated with low fucosyltransferase 8 (FUT8) expression levels in a defined plasma cell subset. Remarkably, IgG afucosylation levels after primary vaccination correlated significantly with IgG levels after the second dose. Further studies are needed to assess efficacy, inflammatory potential, and protective capacity of afucosylated IgG responses.

One sentence summaryA transient afucosylated IgG response to the BNT162b2 mRNA vaccine was observed in naive but not in antigen-experienced individuals, which predicted antibody titers upon the second dose.


title: Linking CFTR modulators to opportunistic bacterial infections in cystic fibrosis

abstract: Cystic fibrosis transmembrane conductance regulator (CFTR) modulators improve clinical outcomes with variable efficacy in patients with cystic fibrosis (CF). However, changes produced by bacterial persistence and adaptation in addition to antibiotic regimens could influence CFTR modulator efficacy and vice versa and hence clinical outcomes. We first evaluated the effects of ivacaftor (IVA), lumacaftor (LUM), tezacaftor, elexacaftor and elexacaftor/tezacaftor/ivacaftor (ETI), alone or combined with antibiotics, on sequential Staphylococcus aureus and Pseudomonas aeruginosa CF isolates. IVA and ETI showed the most potent direct antimicrobial activity against S. aureus, while P. aeruginosa was not affected. Additive effects or synergies were observed between the CFTR modulators and antibiotics against both S. aureus and P. aeruginosa, independently of the stage of colonization. IVA and LUM were the most effective in potentiating antibiotic activity against S. aureus, while IVA and ETI enhanced mainly polymyxins activity against P. aeruginosa. Next, we evaluated the effect of P. aeruginosa pneumonia on the pharmacokinetics of IVA in mice. The time-concentration curves of IVA and its metabolites in plasma, lung and epithelial lining fluid were influenced by P. aeruginosa infection. The area under the concentration-time curve showed that airway exposure to IVA was greater in infected than non-infected mice. These results suggest that CFTR modulators can have direct antimicrobial properties and/or enhance antibiotic activity against S. aureus and P. aeruginosa. Furthermore, bacterial infection impacts the IVA concentration and airway exposure, potentially affecting its efficacy. Our findings suggest optimizing host- and pathogen-directed drug regimens to improve efficacy for personalized treatment.


title: NaCl Triggers the Sessile-to-Motile Transition of Bacillus subtilis

abstract: Various chemical cues are known to alter the motile and sessile states of bacteria differentially and, in turn, the formation of biofilms. However, the underlying mechanisms at the cellular and molecular level remain less understood, which severely limits our ability to control biofilms. Here, we systematically studied the effects of NaCl on the dynamics of biofilm formation across various length scales and the associated changes in the regulation of gene expression in an undomesticated natural isolate of Bacillus subtilis. Interestingly, NaCl induced significant changes in the architecture of pellicles and yielded systematic increase in lateral expansion rates of biofilms when grown on an agar surface. At the microscopic level, both in the presence and absence of NaCl, bacteria displayed super-diffusive motion at times lesser than a second. However, at larger delay times, we observed an intriguing NaCl-induced transition from sub-diffusion behavior of individual bacterial cells to rapid diffusion behavior. In addition, NaCl reduced the dynamical heterogeneity of the bacterial cells within the biofilm. The reduced heterogeneity and the increased flagellation in a subpopulation of cells in the presence of NaCl corroborates well with the observed higher motility of the cells. Further, the cellular uptake of NaCl resulted in the downregulation of several genes underlying the formation of biofilms, revealing the role of chemical cues like NaCl in controlling the gene regulatory circuit underlying the sessile to motile transition. Our study opens a new avenue to decipher the competitive advantage provided to the subcellular populations by NaCl due to lifestyle switch in Bacillus subtilis.


title: Strain-dependent induction of primary bile acid 7-dehydroxylation by cholic acid

abstract: Bile acids (BAs) are steroid-derived molecules with important roles in digestion, the maintenance of host metabolism and immunomodulation. Primary BAs are synthesised by the host and their concentrations tightly regulated, while secondary BAs are produced by the gut microbiome through transformation of the former. Regulation of microbial production of secondary BAs is not well understood, particularly the production of 7-dehydroxylated BAs, which are the most potent agonists for host BA receptors. The 7-dehydroxylation of cholic acid (CA) is well established and is linked to the expression of a bile acid-inducible (bai) operon. However, little to no 7-dehydroxylation has been reported for other host-derived BAs (e.g., chenodeoxycholic acid, CDCA or the mouse ursodeoxycholic acid, UDCA) and it is unknown whether they induce bai gene expression. Here, we demonstrate that the 7-dehydroxylation of CDCA and UDCA by Clostridium scindens is induced by CA suggesting that CA-dependent transcriptional regulation of 7-dehydroxylation is generalisable to CDCA and UDCA. Nevertheless, this effect is strain-specific and the murine isolate Extibacter muris did not respond to CA exposure, suggesting that bai genes are regulated differently in this strain. Moreover, the accessory gene baiJ was only upregulated in the Clostridium scindens ATCC 35704 strain, implying mechanistic differences amongst isolates. Interestingly, the C. scindens strains were also capable of 7-DH-ing muricholic acids (MCAs) to a limited extent. This study shows novel 7-dehydroxylation activity in vitro as a result of CA-driven induction and suggests that it is likely that a different BA plays the same role in the murine gut.


title: The interplay between sulfur metabolism and desulfurization profile in Rhodococcus: Unraveling the role of the transsulfuration pathway

abstract: Biodesulfurization (BDS) is a process that selectively removes sulfur from dibenzothiophene and its derivatives. Several natural biocatalysts have been isolated, all harboring the highly conserved desulfurization operon dszABC. Even though the desulfurization phenotype is known to be significantly repressed by methionine, cysteine, and inorganic sulfate, the available information on the metabolic regulation of gene expression is still limited. In this study, scarless knockouts of the sulfur metabolism-related cbs and metB genes are constructed in the desulfurizing strain Rhodococcus sp. IGTS8. We provide sequence analyses for both enzymes of the reverse transsulfuration pathway and report their involvement in the sulfate- and methionine-dependent repression of the biodesulfurization phenotype, based on desulfurization assays in the presence of different sulfur sources. Additionally, the positive effect of cbs and metB gene deletions on dsz gene expression in the presence of both sulfate and methionine, but not cysteine, is uncovered and highlighted.


title: ATPase activity of B. subtilis RecA affects the dynamic formation of RecA filaments at DNA double strand breaks

abstract: RecA plays a central role in DNA repair and is a main actor involved in homologous recombination (HR). In vivo, RecA forms filamentous structures termed "threads", which are essential for HR, but whose nature is still ill defined. We show that RecA from Bacillus subtilis having lower ATP binding activity can still form nucleoprotein filaments in vitro, and still retains most of wild type RecA activity in vivo. Contrarily, loss of ATPase activity strongly reduces formation of nucleoprotein filaments in vitro, and effectivity to repair double strand breaks (DSBs) in vivo. While lowered ATP-binding activity only moderately affected RecA dynamics, loss of ATPase activity lead to a large reduction of the formation of threads, as well as of their dynamic changes observed in a seconds-scale. Single molecule tracking of RecA revealed incorporation of freely diffusing and non-specifically DNA-bound molecules into filaments upon induction of a single DSB. This change of dynamics was highly perturbed in the absence of ATPase activity, revealing that filamentous forms of RecA as well as their dynamics depend on ATPase activity. Our data suggest that RecA/ssDNA filaments change in subcellular localization and length involving ATP-driven homology search.


title: A live-attenuated SARS-CoV-2 vaccine candidate with accessory protein deletions

abstract: We report a live-attenuated SARS-CoV-2 vaccine candidate with (i) re-engineered viral transcriptional regulator sequences and (ii) deleted open-reading-frames (ORF) 3, 6, 7, and 8 ({Delta}3678). The {Delta}3678 virus replicates about 7,500-fold lower than wild-type SARS-CoV-2 on primary human airway cultures, but restores its replication on interferon-deficient Vero-E6 cells that are approved for vaccine production. The {Delta}3678 virus is highly attenuated in both hamster and K18-hACE2 mouse models. A single-dose immunization of the {Delta}3678 virus protects hamsters from wild-type virus challenge and transmission. Among the deleted ORFs in the {Delta}3678 virus, ORF3a accounts for the most attenuation through antagonizing STAT1 phosphorylation during type-I interferon signaling. We also developed an mNeonGreen reporter {Delta}3678 virus for high-throughput neutralization and antiviral testing. Altogether, the results suggest that {Delta}3678 SARS-CoV-2 may serve as a live-attenuated vaccine candidate and a research tool for potential biosafety level-2 use.


title: A global lipid map reveals host dependency factors conserved across SARS-CoV-2 variants

abstract: A comprehensive understanding of host dependency factors for SARS-CoV-2 remains elusive. We mapped alterations in host lipids following SARS-CoV-2 infection using nontargeted lipidomics. We found that SARS-CoV-2 rewires host lipid metabolism, altering 409 lipid species up to 64-fold relative to controls. We correlated these changes with viral protein activity by transfecting human cells with each viral protein and performing lipidomics. We found that lipid droplet plasticity is a key feature of infection and that viral propagation can be blocked by small-molecule glycerolipid biosynthesis inhibitors. We found that this inhibition was effective against the main variants of concern (alpha, beta, gamma, and delta), indicating that glycerolipid biosynthesis is a conserved host dependency factor that supports this evolving virus.


title: Diverse innate immune factors protect yeast from lethal viral pathogenesis

abstract: In recent years, newly characterized antiviral systems have been found to be remarkably conserved from bacteria to mammals, revealing the potential to gain unique insight into these systems through the study of microbial organisms1. Despite the enthusiasm generated by these findings, the key microbial model organism Saccharomyces cerevisiae (budding yeast) has been minimally exploited for studies of viral defense, primarily because it is not infected with exogenously transmitted viruses. However, most yeast strains are infected with an endogenous double stranded RNA (dsRNA) virus called L-A, and previous studies have identified conserved antiviral systems that attenuate L-A replication2. Although these systems do not lead to eradication of L-A, we show here that they collaboratively prevent massive over-proliferation of L-A that leads to proteostatic stress and lethality. Exploiting this new insight, we asked if the powerful genetic screening methods available in yeast could be used to identify additional conserved antiviral systems. Validating this approach, we discovered antiviral functions for the yeast homologs of polyA-binding protein (PABPC1) and the La-domain containing protein Larp1, which are both involved in viral innate immunity in humans3,4. We also identify new antiviral functions for the RNA exonucleases REX2 and MYG1, both of which have distinct but poorly characterized human and bacterial homologs5-8. These findings highlight the potential of yeast as a powerful model system for studies of the ubiquitous host-virus arms race.


title: A model of persistent post SARS-CoV-2 induced lung disease for target identification and testing of therapeutic strategies

abstract: COVID-19 survivors develop post-acute sequelae of SARS-CoV-2 (PASC), but the mechanistic basis of PASC-associated lung abnormalities suffers from a lack of longitudinal samples. Mouse-adapted SARS-CoV-2 MA10 produces an acute respiratory distress syndrome (ARDS) in mice similar to humans. To investigate PASC pathogenesis, studies of MA10-infected mice were extended from acute disease through clinical recovery. At 15-120 days post-virus clearance, histologic evaluation identified subpleural lesions containing collagen, proliferative fibroblasts, and chronic inflammation with tertiary lymphoid structures. Longitudinal spatial transcriptional profiling identified global reparative and fibrotic pathways dysregulated in diseased regions, similar to human COVID-19. Populations of alveolar intermediate cells, coupled with focal upregulation of pro-fibrotic markers, were identified in persistently diseased regions. Early intervention with antiviral EIDD-2801 reduced chronic disease, and early anti-fibrotic agent (nintedanib) intervention modified early disease severity. This murine model provides opportunities to identify pathways associated with persistent SARS-CoV-2 pulmonary disease and test countermeasures to ameliorate PASC.


title: Monoacylglycerol O-acyltransferase 1 is required for adipocyte differentiation in vitro but does not affect adiposity in mice

abstract:
ObjectiveMonoacylglycerol O-acyltransferase 1 (Mogat1), a lipogenic enzyme that converts monoacylglycerol to diacylglycerol, is highly expressed in adipocytes and may regulate lipolysis by re-esterifying fatty acids released during times when lipolytic rates are low. However, the role of Mogat1 in regulating adipocyte fat storage during differentiation and diet-induced obesity is relatively understudied.

MethodsHere we generated adipocyte-specific Mogat1 knockout mice and subjected them to a high-fat diet to determine the effects of Mogat1 deficiency on diet-induced obesity. We also used Mogat1 floxed mice to develop preadipocyte cell lines wherein Mogat1 could be conditionally knocked out to study adipocyte differentiation in vitro.

ResultsIn preadipocytes, we found that Mogat1 knockout at the onset of preadipocyte differentiation prevented the accumulation of glycerolipids and reduced the differentiation capacity of preadipocytes. However, the loss of adipocyte Mogat1 did not affect weight gain or fat mass induced by high-fat diet in mice. Furthermore, loss of Mogat1 in adipocytes did not affect plasma lipid or glucose concentrations or insulin tolerance.

ConclusionsThese data suggest Mogat1 may play a role in adipocyte differentiation in vitro but not adipose tissue expansion in response to nutrient overload in mice.

STUDY IMPORTANCEO_ST_ABSWhat is already known?C_ST_ABSO_LIAdipose tissue expansion through adipocyte precursor cell differentiation is critical for proper lipid storage during nutrient overload.
C_LIO_LIMonoacylglycerol O-acyltransferase 1 (Mogat1), a lipogenic enzyme, is highly induced during adipocyte differentiation of human and mouse precursor cells and is reduced in patients with obesity and metabolic dysfunction.
C_LI

What does this study add?O_LIMogat1 deletion during early adipocyte differentiation reduces differentiation capacity, adipogenic gene expression and lowers glycerolipid content of differentiated adipocytes.
C_LIO_LIAdipocyte Mogat1 expression is dispensable for adiposity and metabolic outcomes high-fat fed mice and suggests compensation from other glycerolipid synthesis enzymes.
C_LI

How might these results change the direction of research?O_LIUnderstanding the molecular mechanisms of glycerolipid metabolism healthy adipose tissue expansion.
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category: molecular biology