title: Efficient Hydrophilic Small Molecule Delivery To Retinal Cell Lines Using Solid Lipid Nanoparticle Containing Gel Core


title: Machine learning approaches to predict the plant-associated phenotype of Xanthomonas strains

abstract: The genus Xanthomonas has long been considered to consist predominantly of plant pathogens, but over the last decade there has been an increasing number of reports on non-pathogenic and endophytic members. As Xanthomonas species are prevalent pathogens on a wide variety of important crops around the world, there is a need to distinguish between these plant-associated phenotypes. To date a large number of Xanthomonas genomes have been sequenced, which enables the application of machine learning (ML) approaches on the genome content to predict this phenotype. Until now such approaches to the pathogenomics of Xanthomonas strains have been hampered by the fragmentation of information regarding strain pathogenicity over many studies. Unification of this information into a single resource was therefore considered to be an essential step. Mining of 39 papers considering both plant-associated phenotypes, allowed for a phenotypic classification of 578 Xanthomonas strains. For 65 plant-pathogenic and 53 non-pathogenic strains the corresponding genomes were available and de novo annotated for the presence of Pfam protein domains used as features to train and compare three ML classification algorithms; CART, Lasso and Random Forest. Recursive feature extraction provided further insights into the virulence enabling factors, but also yielded domains linked to traits not present in pathogenic strains.

category: microbiology


title: Efficient Alcoholic Conversion of Glycerol by Engineered Saccharomyces cerevisiae

abstract:
Glycerol is an eco-friendly solvent that enhances plant biomass decomposition via glycerolysis in many pretreatment methods. Nonetheless, the lack of efficient conversion of glycerol by natural Saccharomyces cerevisiae hinders its use in these methods. Here, we have aimed to develop a complete strategy for the generation of efficient glycerol-converting yeast by modifying the oxidation of cytosolic nicotinamide adenine dinucleotide (NADH) by an O2-dependent dynamic shuttle, while abolishing both glycerol phosphorylation and biosynthesis. By following a vigorous glycerol oxidation pathway, the engineered strain increased the conversion efficiency (CE) to up to 0.49 g ethanol/g glycerol (98% of theoretical CE), with production rate > 1 g{middle dot}L{middle dot}h, when glycerol was supplemented in a single fed-batch fermentation in a rich medium. Furthermore, the engineered strain fermented a mixture of glycerol and glucose, producing > 86 g/L bioethanol with 92.8% CE. To our knowledge, this is the highest ever reported titer in this field. Notably, this strategy changed conventional yeast from a non-grower on minimal medium containing glycerol to a fermenting strain with productivity of 0.25-0.5 g{middle dot}L{middle dot}h and 84-78% CE, which converted 90% of the substrate to products. Our findings may improve the utilization of glycerol in several eco-friendly biorefinery approaches.

IMPORTANCEWith the development of efficient lignocellulosic biorefineries, glycerol has attracted attention as an eco-friendly biomass-derived solvent that can enhance the dissociation of lignin and cell wall polysaccharides during the pretreatment process. Co-conversion of glycerol with the sugars released from biomass after glycerolysis increases the resources for ethanol production and lowers the burden of component separation. However, titer productivity hinders the industrial application of this process. Therefore, generation of efficient glycerol-fermenting yeast will promote the applicability of integrated biorefineries. Furthermore, glycerol is an important carbon source for the production of various chemicals. Hence, control of the metabolic flux of yeast grown on glycerol will contribute to the generation of cell factories that produce chemicals such as ethanol from glycerol, which will boost biodiesel and bioethanol industries. Additionally, the use of glycerol-fermenting yeast will reduce global warming and generation of agricultural waste, leading to the establishment of a sustainable society.


title: Phase variable expression of pdcB, a phosphodiesterase influences sporulation in Clostridioides difficile

abstract: Clostridioides difficile is the causative agent of antibiotic-associated diarrhea and is the leading cause of nosocomial infection in developed countries. An increasing number of C. difficile infections are attributed to hypervirulence strains that produce more toxins and spores. C. difficile spores are the major factor for the transmission and persistence of the organism. Previous studies have identified global regulators that influence sporulation in C. difficile. This study discovered that PdcB, a phosphodiesterase to influence sporulation in C. difficile UK1 strain positively. Through genetic and biochemical assays, we have shown that phase variable expression of pdcB results in hypo- and hyper-sporulation phenotype. In the "ON" orientation, the identified promotor is the right orientation to drive the expression of pdcB. Production of PdcB phosphodiesterase reduces the intracellular cyclic-di-GMP concentration, resulting in hyper-sporulation phenotype. The OFF orientation of pdcB switch or mutating pdcB results in increased cyclic-di-GMP and hypo-sporulating phenotype. Additionally, we demonstrated that CodY binds to the upstream region of pdcB to represses its expression, and CodY mediated repression is relieved by the DNA inversion.


title: Cellular calcium levels influenced by NCA-2 impact circadian period determination in Neurospora

abstract:
Intracellular calcium signaling has been implicated in control of a variety of circadian processes in animals and plants but its role in microbial clocks has remained largely cryptic. To examine the role of intracellular Ca2+ in the Neurospora clock we screened knockouts of calcium transporter genes and identified a gene encoding a calcium exporter, nca-2, uniquely as having significant period effects. Loss of NCA-2 results in an increase in cytosolic calcium level, and this leads to hyper-phosphorylation of core clock components, FRQ and WC-1, and a short period as measured by both the core oscillator and overt clock. Genetic analyses showed that mutations in certain frq phospho-sites, and in Ca2+-calmodulin-dependent kinase (camk-2), are epistatic to nca-2 in controlling the pace of the oscillator. These data are consistent with a model in which elevated intracellular Ca+2 leads to increased activity of CAMK-2 leading to enhanced FRQ phosphorylation, accelerated closure of the circadian feedback loop, and a shortened circadian period length. At a mechanistic level some CAMKs undergo more auto-phosphorylations in {Delta}nca-2, consistent with high calcium in the {Delta}nca-2 mutant influencing the enzymatic activity of CAMKs. NCA-2 interacts with multiple proteins including CSP-6, a protein known to be required for circadian output. Most importantly, expression of nca-2 is circadian clock-controlled at both the transcriptional and translational levels, and this in combination with the period effects seen in strains lacking NCA-2, firmly places calcium signaling within the larger circadian system where it acts as both an input to and output from the core clock.

ImportanceCircadian rhythms are based on cell-autonomous, auto-regulatory, feedback loops formed by interlocked positive and negative arms, and they regulate myriad molecular and cellular processes in most eukaryotes including fungi. Intracellular calcium signaling is also a process that impacts a broad range of biological events in most eukaryotes. Clues have suggested that calcium signaling can influence circadian oscillators through multiple pathways; however, mechanistic details have been lacking in microorganisms. Building on prior work describing calcium transporters in the fungus Neurospora, one such transporter, NCA-2, was identified as a regulator of circadian period length. Increased intracellular calcium levels caused by loss of NCA-2 results in over-activation of calcium-responsive protein kinases, in turn leading to a shortened circadian period length. Importantly, expression of NCA-2 is itself controlled by the molecular clock. In this way calcium signaling can be seen as providing both input to and output from the circadian system.

category: molecular biology


title: Gaseous Signaling Compounds (Hydrogen Sulfide and Nitric Oxide) and Their Relative Roles in Affecting Anaerobic HeLa 229 Cell Viability

abstract: Metabolic pathways supporting long-term anaerobic cell viability have not been identified. The effect NO and H2S pathway effectors have on HeLa 229 cell viability was measured after 10 days anaerobic incubation. The addition of arginine or xanthine (NO pathway precursors) consistently increased HeLa cell viability by 13.1- and 4.4-fold, respectively. Allopurinol, a xanthine oxidase inhibitor, also increased viability, as compared to control levels. In contrast, inhibition of iNOS by 1400W increased cell viability by 79-fold. Regarding the H2S pathway, precursor cysteine enhanced viability by 9.8-fold with the greatest number of viable cells measured in response to the presence of a H2S donor (GYY4137), or an inhibitor of glutathione synthesis, propargylglycine (40- and 85-fold, respectively). These results demonstrate that the constitutive level of cell viability after extended (10 days) growth without oxygen can be modulated by affecting NO or H2S generating pathways.

category: cell biology


title: Interactions between TULP3 tubby domain cargo site and ARL13B amphipathic helix promote lipidated protein transport to cilia

abstract: The tubby family protein-TULP3 coordinates with the intraflagellar transport complex-A (IFT-A) in trafficking certain transmembrane proteins to cilia. These transmembrane cargoes have short motifs that are necessary and sufficient for TULP3-mediated trafficking. However, whether TULP3 regulates trafficking of membrane-associated proteins is not well understood. Here we show that TULP3 is required for transport of the atypical GTPase ARL13B into cilia, and for ciliary enrichment of ARL13B-dependent farnesylated and myristoylated proteins. ARL13B transport requires TULP3 binding to IFT-A core but not to phosphoinositides, unlike transmembrane cargo transport that requires binding to both by TULP3. A conserved lysine in TULP3s tubby domain mediates direct ARL13B binding and trafficking of lipidated and transmembrane cargoes. An N-terminal amphipathic helix in ARL13B flanking the palmitoylation site mediates binding to TULP3 and directs trafficking to cilia even in absence of palmitoylation and RVxP sorting motif. Therefore, TULP3 transports transmembrane proteins and ARL13B into cilia by capture of short sequences through a shared tubby domain site.


title: Soft limbal niche maintains stem cell compartmentalization and function through YAP

abstract: Stem cells (SCs) decision to self-renew or differentiate largely depends on the external control of their niche. However, the complex mechanisms that underlie this crosstalk are poorly understood. To address this question, we focused on the corneal epithelial SC model in which the SC niche, known as the limbus, is spatially segregated from the differentiation compartment. We report that the unique biomechanical property of the limbus supports the nuclear localization and function of Yes-associated protein (YAP), a putative mediator of the mechanotransduction pathway. Perturbation of tissue stiffness or YAP activity affects SC function as well as tissue integrity under homeostasis and significantly inhibited the regeneration of the SC population following SC depletion. In vitro experiments revealed that substrates with the rigidity of the corneal differentiation compartment inhibit YAP localization and induce differentiation, a mechanism that is mediated by the TGFbeta-SMAD2/3 pathway. Taken together, these results indicate that SC sense biomechanical niche signals and that manipulation of mechano-sensory machinery or its downstream biochemical output may bear fruits in SC expansion for regenerative therapy.


title: Biomechanical property of limbal niche maintains stemness through YAP


title: Bycatch mitigation could prevent strong changes in the ecological strategies of seabird communities across the globe

abstract: Fisheries bycatch, the incidental mortality of non-target species, is a major threat to seabirds worldwide. Mitigating bycatch is an important factor to reduce seabird population declines and consequent changes in ocean trophic dynamics and ecosystem functioning. However, it remains an open question how and where mitigating bycatch at a global scale may conserve seabird traits and the ecological strategies that traits represent. Here we combine a dataset of species traits and distribution ranges for 341 seabirds with spatially resolved fishing effort data for gillnet, longline, trawl, and purse seine gears to: (1) understand spatial variation in seabird community traits; and (2) test whether mitigating fisheries bycatch may prevent shifts in traits of seabird communities and loss of ecological strategies. We find distinct spatial variation in the community weighted mean of five seabird traits (clutch size, body mass, generation length, foraging guild, and diet guild). Furthermore, our analysis suggests that successful bycatch mitigation could prevent strong shifts in the traits of seabird communities across the globe particularly in the North Atlantic and Southern Oceans. Specifically, changes in dominant foraging and diet guilds, and shifts towards communities with faster reproductive speeds (larger clutch sizes and shorter generation lengths) and smaller body masses could be avoided. Therefore, bycatch mitigation may have important indirect benefits for sustaining ecosystem functioning, as mediated by species traits. Incorporating species traits into management actions will provide valuable tools for marine spatial planning and when evaluating the success of conservation initiatives.

category: ecology


title: Binding affinity landscapes constrain the evolution of broadly neutralizing anti-influenza antibodies

abstract: Over the past two decades, several broadly neutralizing antibodies (bnAbs) that confer protection against diverse influenza strains have been isolated1,2. Structural and biochemical characterization of these bnAbs has provided molecular insight into how they bind distinct antigens1. However, our understanding of the evolutionary pathways leading to bnAbs, and thus how best to elicit them, remains limited. Here, we measure equilibrium dissociation constants of combinatorially complete mutational libraries for two naturally isolated influenza bnAbs3-5 (CR-9114, 16 mutations; CR-6261, 11 mutations), reconstructing all possible intermediates back to the unmutated germline sequences. We find that these two libraries exhibit strikingly different patterns of breadth: while many variants of CR-6261 display moderate affinity to diverse antigens, those of CR-9114 display appreciable affinity only in specific, nested combinations. By examining the extensive pairwise and higher-order epistasis between mutations, we find key sites with strong synergistic interactions that are highly similar across antigens for CR-6261 and different for CR-9114. Together, these features of the binding affinity landscapes strongly favor sequential acquisition of affinity to diverse antigens for CR-9114, while the acquisition of breadth to more similar antigens for CR-6261 is less constrained. These results, if generalizable to other bnAbs, may explain the molecular basis for the widespread observation that sequential exposure favors greater breadth6-8, and such mechanistic insight will be essential for predicting and eliciting broadly protective immune responses.

category: evolutionary biology


title: Identification of multiple TAR DNA Binding Protein retropseudogene lineages during the evolution of primates

abstract: The TAR DNA Binding Protein (TARDBP) gene has gained attention in biomedicine after the discovery of several pathogenic mutations. The lack of knowledge about its evolutionary history contrasts with a large number of studies in the biomedical area. This study aimed to investigate the retrotransposition evolutionary dynamics associated with this gene in primates. We identified retropseudogenes that originated in the ancestors of anthropoids, catarrhines, and lemuriformes, i.e. the strepsirrhine clade that inhabit Madagascar. We also found species-specific retropseudogenes in the philippine tarsier, Bolivian squirrel monkey, capuchin monkey and vervet. Although retropseudogenes are not able to produce a functional protein, we can not rule out that they may represent genetic material upon which evolution acts on, especially with regulatory functions.


title: An improved hidden Markov model for the characterization of homozygous-by-descent segments in individual genomes

abstract: Inbreeding results from the mating of related individuals and has negative consequences because it brings together deleterious variants in one individual. Genomic estimates of the inbreeding coefficients are preferred to pedigree-based estimators as they measure the realized inbreeding levels and they are more robust to pedigree errors. Several methods identifying homozygous-by-descent (HBD) segments with hidden Markov models (HMM) have been recently developed and are particularly valuable when the information is degraded or heterogeneous (e.g., low-fold sequencing, low marker density, heterogeneous genotype quality or variable marker spacing). We previously developed a multiple HBD class HMM where HBD segments are classified in different groups based on their length (e.g., recent versus old HBD segments) but we recently observed that for high inbreeding levels with many HBD segments, the estimated contributions might be biased towards more recent classes (i.e., associated with large HBD segments) although the overall estimated level of inbreeding remained unbiased. We herein propose a new model in which the HBD classification is modeled in successive nested levels with decreasing expected HBD segment lengths, the underlying exponential rates being directly related to the number of generations to the common ancestor. The non-HBD classes are now modeled as a mixture of HBD segments from later generations and shorter non-HBD segments (i.e., both with higher rates). The new model has improved statistical properties and performs better on simulated data compared to our previous version. We also show that the parameters of the model are easier to interpret and that the model is more robust to the choice of the number of classes. Overall, the new model results in an improved partitioning of inbreeding in different HBD classes and should be preferred.

category: genetics


title: An hidden Markov model to estimate homozygous-by-descent probabilities associated with nested layers of ancestors


title: Dissection of multiple sclerosis genetics identifies B and CD4 T cells as driver cell subsets

abstract: Multiple sclerosis (MS) is an autoimmune condition of the central nervous system with a well-characterized genetic background. Prior analyses of MS genetics have identified broad enrichments across peripheral immune cells, yet the driver immune subsets are unclear. We utilized chromatin accessibility data across hematopoietic cells to identify cell type-specific enrichments of MS genetic signals. We found that CD4 T and B cells were independently enriched for MS genetics and further refined the driver subsets to Th17 and memory B cells, respectively. We replicated our findings in data from untreated and treated MS patients and found that immunomodulatory treatments suppress chromatin accessibility at driver cell types. Integration of statistical fine-mapping and chromatin interactions nominated numerous putative causal genes, illustrating complex interplay between shared and cell-specific genes. Our study highlights how careful integration of genetics and epigenetics can provide fine-scale insights into causal cell types and nominate new genes and pathways for disease.


title: Optimized liquid and gas phase fractionation increase HLA-peptidome coverage for primary cell and tissue samples

abstract: Mass spectrometry is the most effective method to directly identify peptides presented on HLA molecules. However, current standard approaches often require many millions of cells for input material to achieve high coverage of the immunopeptidome and are therefore not compatible with the often limited amounts of tissue available from clinical tumor samples. Here, we evaluated microscaled basic reversed-phase fractionation to separate HLA peptide samples off-line followed by ion mobility coupled to LC-MS/MS for analysis. The combination of these two separation methods enabled identification of 20% to 50% more peptides compared to samples analyzed without either prior fractionation or use of ion mobility alone. We demonstrate coverage of HLA immunopeptidomes with up to 8,107 distinct peptides starting with as few as 100 million cells or 150 milligrams of wet weight tumor tissue. This increased sensitivity can improve HLA binding prediction algorithms and enable detection of clinically relevant epitopes such as neoantigens.

category: biochemistry


title: On the sparsity of fitness functions and implications for learning

abstract: Fitness functions map biological sequences to a scalar property of interest. Accurate estimation of these functions yields biological insight and sets the foundation for model-based sequence design. However, the amount of fitness data available to learn these functions is typically small relative to the large combinatorial space of sequences; characterizing how much data is needed for accurate estimation remains an open problem. There is a growing body of evidence demonstrating that empirical fitness functions display substantial sparsity when represented in terms of epistatic interactions. Moreover, the theory of Compressed Sensing provides scaling laws for the number of samples required to exactly recover a sparse function. Motivated by these results, we develop a framework to study the sparsity of fitness functions sampled from a generalization of the NK model, a widely-used random field model of fitness functions. In particular, we present results that allow us to test the effect of the Generalized NK (GNK) models interpretable parameters--sequence length, alphabet size, and assumed interactions between sequence positions--on the sparsity of fitness functions sampled from the model and, consequently, the number of measurements required to exactly recover these functions. We validate our framework by demonstrating that GNK models with parameters set according to structural considerations can be used to accurately approximate the number of samples required to recover two empirical protein fitness functions and an RNA fitness function. In addition, we show that these GNK models identify important higher-order epistatic interactions in the empirical fitness functions using only structural information.

category: bioinformatics


title: VP-Detector: A 3D convolutional neural network for automated macromolecule localization and classification in cryo-electron tomograms

abstract:
MotivationCryo-electron tomography (Cryo-ET) with sub-tomogram averaging (STA) is indispensable when studying macromolecule structures and functions in their native environments. However, current tomographic reconstructions suffer the low signal-to-noise (SNR) ratio and the missing wedge artifacts. Hence, automatic and accurate macromolecule localization and classification become the bottleneck problem for structural determination by STA. Here, we propose a 3D multi-scale dense convolutional neural network (MSDNet) for voxel-wise annotations of tomograms. Weighted focal loss is adopted as a loss function to solve the class imbalance. The proposed network combines 3D hybrid dilated convolutions (HDC) and dense connectivity to ensure an accurate performance with relatively few trainable parameters. 3D HDC expands the receptive field without losing resolution or learning extra parameters. Dense connectivity facilitates the re-use of feature maps to generate fewer intermediate feature maps and trainable parameters. Then, we design a 3D MSDNet based approach for fully automatic macromolecule localization and classification, called VP-Detector (Voxel-wise Particle Detector). VP-Detector is efficient because classification performs on the pre-calculated coordinates instead of a sliding window.

ResultsWe evaluated the VP-Detector on simulated tomograms. Compared to the state-of-the-art methods, our method achieved a competitive performance on localization with the highest F1-score. We also demonstrated that the weighted focal loss improves the classification of hard classes. We trained the network on a part of training sets to prove the availability of training on relatively small datasets. Moreover, the experiment shows that VP-Detector has a fast particle detection speed, which costs less than 14 minutes on a test tomogram.

Contactzsh@amss.ac.cn, xfcui@email.sdu.edu.cn, zhangfa@ict.ac.cn

Supplementary informationSupplementary data are available at Bioinformatics online.


title: A Neural Model of RNA Splicing: Learning Motif Distances with Self-Attention and Toeplitz Max Pooling

abstract: Alternative RNA splicing is an important regulator of tissue development and specificity, and a relevant mechanism in cancer progression. There exists a strong motivation to disentangle the rules that govern RNA splicing, in part because this knowledge may one day yield new clinically relevant diagnostic tools and therapeutics. It is no easy to task to reverse engineer how the splicesome machinery choreographs the removal and addition of RNA elements following transcription. Here, we propose an interpretable neural network called the Toeplitz ATtention Architecture (TATA), which learns distance-dependent motif interactions through a novel Toeplitz max pool layer that captures the relative distance between interacting CNN filters. TATA is a completely transparent "clear-box" solution: every model parameter is human-interpretable. We validate TATA on simulated data, then apply it to real data to identify putative cis-regulatory elements that interact with primary RNA splice sites.


title: nf-LO: A scalable, containerised workflow for genome-to-genome lift over

abstract:
The increasing availability of new genome assemblies often comes with an impaired amount of associated genomic annotations, limiting the range of studies that can be performed. A common workaround is to lift over annotations from better annotated genomes. However, generating the files required to perform a liftover is computationally and labour intensive and only a limited number are currently publicly available.

Here we present nf-LO (nextflow-LiftOver), a containerised and scalable Nextflow pipeline that enables liftovers within and between any species for which assemblies are available. nf-LO will consequently facilitates data interpretation across a broad range of genomic studies.


title: Nanofluidic chips for cryo-EM structure determination from picoliter sample volumes

abstract: Cryogenic electron microscopy has become an essential tool for structure determination of biological macromolecules. In practice, the difficulty to reliably prepare samples with uniform ice thickness still represents a barrier for routine high-resolution imaging and limits the current throughput of the technique. We show that a nanofluidic sample support with well-defined geometry can be used to prepare cryo-EM specimens with reproducible ice thickness from picoliter sample volumes. The sample solution is contained in electron-transparent nanochannels that provide uniform thickness gradients without further optimisation and eliminate the potentially destructive air-water interface. We demonstrate the possibility to perform high-resolution structure determination with three standard protein specimens. Nanofabricated sample supports bear potential to automate the cryo-EM workflow, and to explore new frontiers for cryo-EM applications such as time-resolved imaging and high-throughput screening.

category: biophysics


title: The molecular mechanism of load adaptation by branched actin networks

abstract: Branched actin networks are self-assembling molecular motors that move biological membranes and drive many important cellular processes. Load forces slow the growth and increase the density of these networks, but the molecular mechanisms governing this force response are not well understood. Here we use single-molecule imaging and AFM cantilever deflection to measure how applied forces affect each step in branched actin network assembly. Unexpectedly, force slows the rate of filament nucleation by promoting the interaction of nucleation promoting factors with actin filament ends, limiting branch formation. This inhibition is countered by an even larger force-induced drop in the rate of filament capping, resulting in a shift in the balance between nucleation and capping that increases network density. Remarkably, the force dependence of capping is identical to that of filament elongation because they require the same size gap to appear between the filament and load for insertion. These results provide direct evidence that Brownian Ratchets generate force and govern the load adaptation of branched actin networks.


title: Estimating the Dynamic Range of Quantitative Single-Molecule Localization Microscopy

abstract: In recent years, there have been significant advances in quantifying molecule copy number and protein stoichiometry with single-molecule localization microscopy (SMLM). However, as the density of fluorophores per diffraction-limited spot increases, distinguishing between detection events from different fluorophores becomes progressively more difficult, affecting the accuracy of such measurements. Although essential to the design of quantitative experiments, the dynamic range of SMLM counting techniques has not yet been studied in detail. Here we provide a working definition of the dynamic range for quantitative SMLM in terms of the relative number of missed localizations or blinks, and explore the photophysical and experimental parameters that affect it. We begin with a simple two-state model of blinking fluorophores, then extend the model to incorporate photobleaching and temporal binning by the detection camera. From these models, we first show that our estimates of the dynamic range agree with realistic simulations of the photoswitching. We find that the dynamic range scales inversely with the duty cycle when counting both blinks and localizations. Finally, we validate our theoretical approach on dSTORM datasets of photo-switching Alexa647 dyes. Our results should help guide researchers in designing and implementing SMLM-based molecular counting experiments.


title: Relationships between endogenous circadian period, physiological and cognitive parameters and sex in aged non-human primates

abstract:
The biological clock exhibits circadian rhythms, with an endogenous period tau close to 24h. The circadian resonance theory proposes that lifespan is reduced when endogenous period goes far from 24h. It has been suggested that daily resetting of the circadian clock to the 24h external photoperiod might induce marginal costs that would accumulate over time and forward accelerate aging and affect fitness. In this study, we aimed to evaluate the link between the endogenous period and biomarkers of aging in order to investigate the mechanisms of the circadian resonance theory. We studied 39 middle-aged and aged Microcebus murinus, a nocturnal non-human primate whose endogenous period is about 23.1h, measuring the endogenous period of locomotor activity, as well as several physiological and behavioral parameters (rhythm fragmentation and amplitude, energetic expenditure, oxidative stress, insulin-like growth factor-1 (IGF-1) concentrations and cognitive performances) in both males and females. We found that aged males with tau far from 24h displayed increased oxidative stress. We also demonstrated a positive correlation between tau and IGF-1 concentrations, as well as learning performances, in males and females. Together these results suggest that a great deviation of tau from 24h leads to increased biomarkers of age-related impairments.

Summary StatementThis manuscript describes how the misalignment between the endogenous circadian period and the 24h of the environmental periodicity impacts aging biomarkers and accelerates cognitive decline.

category: physiology


title: Systemic gene therapy with thymosin β4 alleviates glomerular injury in mice

abstract: Plasma ultrafiltration in the kidney occurs across glomerular capillaries, which are surrounded by epithelial cells called podocytes. Podocytes have a unique shape maintained by a complex cytoskeleton, which becomes disrupted in glomerular disease resulting in defective filtration and albuminuria. Lack of endogenous thymosin {beta}4 (TB4), an actin sequestering peptide, exacerbates glomerular injury and disrupts the organisation of the podocyte actin cytoskeleton, however, the effect of exogenous TB4 therapy on podocytopathy is unknown. Here, through interrogating single-cell RNA-sequencing data of isolated glomeruli we demonstrate that Adriamycin, a toxin which injures podocytes and leads to leakage of albumin in the urine of mice, results in reduced levels of podocyte TB4. Systemic administration of an adeno-associated virus vector encoding TB4 prevented Adriamycin-induced podocyte loss and albuminuria. Adriamycin injury was associated with disorganisation of the actin cytoskeleton in vitro, which was ameliorated by exogenous TB4. Furthermore, Adriamycin administration in mice was associated with increased prevalence of podocyte vesicles, a mechanism by which albumin may leak into the urine, which was also prevented by TB4. Collectively, we propose that TB4 gene therapy prevents podocyte injury and maintains glomerular filtration via modulation of the podocyte cytoskeleton thus presenting a novel treatment strategy for glomerular disease.


title: Distinct changes in mature root and growing root tip proteomes underlie physiological responses of bread wheat to salinity stress

abstract: The impact of salinity on wheat plants is often studied by analysis of shoot responses, even though the main mechanism of tolerance is shoot Na+ exclusion. There is a need to understand the molecular responses of root tissues that directly experience rising NaCl concentrations. We have combined analysis of root growth, ion content and respiration with proteome responses in wheat root tip and mature root tissues under saline conditions. We find significant changes in translation and protein synthesis, energy metabolism and amino acid metabolism in a root tissue specific manner. Translation and protein synthesis related proteins showed significant decreases in abundance only in root tips, as did most of the glycolytic enzymes and selected TCA cycle enzymes and ATP synthase subunits. This selective root tip proteome response indicates protein synthesis capacity and energy production were impaired under salt stress, correlating with the anatomical response of roots and reduced root tip respiration rate. Wheat roots respond directly to soil salinity, therefore shoot responses such as reduction in shoot growth and photosynthetic capacity need to be considered in light of these effects.

category: plant biology


title: A rapid and reliable multiplexed LC-MS/MS method for simultaneous analysis of six monoamines from brain tissues

abstract: Monoamines are a class of neuromodulators that are crucial for a variety of brain functions, including control of mood, movement, sleep and cognition. From mammals to insects, the nervous system is enriched in monoamines such as dopamine, serotonin and melatonin, analytes which range from being highly polar to non-polar. Here we developed a method using liquid chromatography coupled with mass spectrometry (LC-MS) to quantify in a single run the amounts of six distinct monoamines in extracts from dissected Drosophila and mouse brain tissues. The measured monoamines were dopamine (DA), serotonin (also known as 5-hydroxytryptamine (5-HT)), octopamine (OA, an insect equivalent of norepinephrine), tyramine (TA), melatonin (MT) and N-acetyl-hydroxy-serotonin (NAS). The analytical range of these monoamines was between 0.25 to 5.0 ng/mL.

category: neuroscience


title: Nonsense-mediated mRNA decay utilizes complementary mechanisms to suppress mRNA and protein accumulation

abstract: Nonsense-mediated mRNA decay (NMD) is an essential, highly conserved quality control pathway that detects and degrades mRNAs containing premature termination codons (PTCs). Although the essentiality of NMD is frequently ascribed to its prevention of truncated protein accumulation, the extent to which NMD actually suppresses proteins encoded by NMD-sensitive transcripts is less well-understood than NMD-mediated suppression of mRNA. Here, we describe a reporter system that permits accurate quantification of both mRNA and protein levels via stable integration of paired reporters encoding NMD-sensitive and NMD-insensitive transcripts into the AAVS1 safe harbor loci in human cells. We use this system to demonstrate that NMD suppresses proteins encoded by NMD-sensitive transcripts by up to [~]8-fold more than the mRNA itself. Our data indicate that NMD limits the accumulation of proteins encoded by NMD substrates by mechanisms beyond mRNA degradation, such that even when NMD-sensitive mRNAs escape destruction, their encoded proteins are still effectively suppressed.


title: Computational toolbox for ultrastructural quantitative analysis of filament networks in cryo-ET data

abstract: A precise quantitative description of the ultrastructural characteristics underlying biological mechanisms is often key to their understanding. This is particularly true for dynamic extra- and intracellular filamentous assemblies, playing a role in cell motility, cell integrity, cytokinesis, tissue formation and maintenance. For example, genetic manipulation or modulation of actin regulatory proteins frequently manifests in changes of the morphology, dynamics, and ultrastructural architecture of actin filament-rich cell peripheral structures, such as lamellipodia or filopodia. However, the observed ultrastructural effects often remain subtle and require sufficiently large datasets for appropriate quantitative analysis. The acquisition of such large datasets has been enabled by recent advances in high-throughput cryo-electron tomography (cryo-ET) methods. However, this also necessitates the development of complementary approaches to maximize the extraction of relevant biological information. We have developed a computational toolbox for the semi-automatic quantification of filamentous networks from cryo-ET datasets to facilitate the analysis and cross-comparison of multiple experimental conditions. GUI-based components simplify the manipulation of data and allow users to obtain a large number of ultrastructural parameters describing filamentous assemblies. We demonstrate the feasibility of this workflow by analyzing cryo-ET data of untreated and chemically perturbed branched actin filament networks and that of parallel actin filament arrays. In principle, the computational toolbox presented here is applicable for data analysis comprising any type of filaments in regular (i.e. parallel) or random arrangement. We show that it can ease the identification of key differences between experimental groups and facilitate the in-depth analysis of ultrastructural data in a time-efficient manner.


title: A method for single molecule localization microscopy of tissues reveals nonrandom distribution of nuclear pores in Drosophila

abstract: Single Molecule Localisation Microscopy (SMLM) can provide nanoscale resolution in thin samples but has rarely been applied to tissues, because of high background from out of focus emitters. Here we describe a line scanning microscope that provides optical sectioning for SMLM in tissues. Imaging endogenously-tagged nucleoporins and F-actin on this system using DNA- and peptide-PAINT routinely gives 30nm resolution or better at depths greater than 20 {micro}m. This revealed that the nuclear pores are nonrandomly distributed in most Drosophila tissues, in contrast to cultured cells. Lamin Dm0 shows a complementary localisation to the nuclear pores, suggesting that it corrals the pores. Furthermore, ectopic expression of the tissue-specific Lamin C distributes the nuclear pores more randomly, whereas lamin C mutants enhance nuclear pore clustering, particularly in muscle nuclei. Since nucleoporins interact with specific chromatin domains, nuclear pore clustering could regulate chromatin organisation locally and contribute to the disease phenotypes caused by human Lamin A/C laminopathies.


title: Language barriers in global bird conservation

abstract: Multiple languages being spoken within a species distribution can impede communication among conservation stakeholders, the compilation of scientific information, and the development of effective conservation actions. Here, we investigate the number of official languages spoken within the distributions of 10,863 bird species to identify which ones might be particularly affected by consequences of language barriers. We show that 1587 species have 10 languages or more spoken within their distributions. Threatened, migratory and wide-ranging species have especially many languages spoken within their distribution. Particularly high numbers of species with many languages within their distribution are found in Eastern Europe, Russia and central and western Asia. Global conservation efforts would benefit from implementing guidelines to overcome language barriers, especially in regions with high species and language diversity.


title: Species' traits and exposure as a future lens for quantifying seabird bycatch vulnerability in global fisheries

abstract: Fisheries bycatch, the incidental mortality of non-target species, is a global threat to seabirds and a major driver of their declines worldwide. Identifying the most vulnerable species is core to developing sustainable fisheries management strategies that aim to improve conservation outcomes. To advance this goal, we present a preliminary vulnerability framework that integrates dimensions of species exposure, sensitivity, and adaptive capacity to fisheries bycatch to classify species into five vulnerability classes. The framework combines species traits and distribution ranges for 341 seabirds, along with a spatially resolved fishing effort dataset. Overall, we find most species have high vulnerability scores for the sensitivity and adaptive capacity dimensions. By contrast, exposure is more variable across species, and thus the median scores calculated within seabird families is low. We further find 46 species have high exposure to fishing activities, but are not identified as vulnerable to bycatch, whilst 133 species have lower exposure, but are vulnerable to bycatch. Thus, the framework has been valuable for revealing patterns between and within the vulnerability dimensions. Still, further methodological development, additional traits, and greater availability of threat data are required to advance the framework and provide a new lens for quantifying seabird bycatch vulnerability that complements existing efforts, such as the International Union for Conservation of Nature (IUCN) Red List.


title: Phase separation on cell surface: a mechanism of basic fibroblast growth factor signal transduction with heparan sulphate

abstract: Liquid-liquid phase separation (LLPS) driven by weak, multivalent interactions among biomolecules is an important means of cellular compartmentation and plays a central role in cellular processes including stress resistance, RNA processing and other cellular activities. Coordination of the condensates and inner membrane was recently revealed, mediating intracellular processes like cell signalling and cargo trafficking. Intracellular LLPS has been observed extensively in vivo, whereas LLPS in extracellular compartments has not been reported under physiological conditions. Here we show, for the first time, that basic fibroblast growth factor (bFGF) undergoes LLPS on the cell surface by interacting with heparan sulphate proteoglycans (HSPG) and the phase transition is required for effective downstream signalling. The condensation is driven by multivalent interactions between bFGF and sulpho-groups on heparan sulphate (HS), and dimerization and oligomerization of bFGF promote the LLPS process. Compared with free bFGF, phase separated bFGF with HS showed higher thermo stability, providing a potential mechanism for the preservation of bFGF activity. Furthermore, we have found that downstream signalling is triggered by phase separation of a ternary complex formed by bFGF, HSPGs and FGFR on cell surface. Our results revealed a molecular mechanism that HS can serve as a platform to promote extracellular proteins like bFGF to condensate on outer membrane, consequently coordinating the signal transduction activities. This novel finding expands the horizons of phase separation in vivo, providing a new dimension on how HSPG may regulate extracellular protein behaviour and cell signalling.


title: Self-Supervised Pretraining for Transferable Quantitative Phase Image Cell Segmentation

abstract:
In this paper, U-Net-based method for robust adherent cell segmentation for quantitative phase microscopy image is designed and optimised. We designed and evaluated four specific post-processing pipelines. To increase the transferability to different cell types, non-deep learning transfer with adjustable parameters is used in the post-processing step. Additionally, we proposed a self-supervised pretraining technique using nonlabelled data, which is trained to reconstruct multiple image distortions and improved the segmentation performance by from 0.67 to 0.70 of Object-wise Intersection over Union. Moreover, we publish a new dataset of manually labelled images suitable for this task together with the unlabelled data for self-supervised pretraining.

Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

HighlightsO_LIFour strategies for instance cell segmentation with U-Net were compared.
C_LIO_LISpecialised post-processing pipelines with tunable/optimizable parameters were designed for each segmentation strategy.
C_LIO_LITransferability to different cell types by optimisation of post-processing parameters was tested.
C_LIO_LIThe proposed self-supervised pretraining method improved both segmentation performance and transferability to different cell types.
C_LIO_LIA new manually labelled quantitative phase imaging dataset for cell segmentation with unlabelled data for self-supervised pretraining was created.
C_LI


title: CellMap: Characterizing the types and composition of iPSC-derived cells from RNA-seq data

abstract: Induced pluripotent stem cell (iPSC) derived cell types are increasingly employed as in vitro model systems for drug discovery. For these studies to be meaningful, it is important to understand the reproducibility of the iPSC-derived cultures and their similarity to equivalent endogenous cell types. Single-cell and single-nucleus RNA sequencing (RNA-seq) are useful to gain such understanding, but they are expensive and time consuming, while bulk RNA-seq data can be generated quicker and at lower cost. In silico cell type decomposition is an efficient, inexpensive, and convenient alternative that can leverage bulk RNA-seq to derive more fine-grained information about these cultures. We developed CellMap, a computational tool that derives cell type profiles from publicly available single-cell and single-nucleus datasets to infer cell types in bulk RNA-seq data from iPSC-derived cell lines.


title: Effects of vapor exposure to Δ9-tetrahydrocannabinol (THC) in the Maine Lobster (Homarus americanus)

abstract:
RationaleDespite a long history of use in synaptic physiology, the lobster has been a neglected model for behavioral pharmacology. A restaurateur proposed that exposing lobster to cannabis smoke reduces anxiety and pain during the cooking process. It is unknown if lobster gill respiration in air would result in significant {Delta}9-tetrahydrocannabinol (THC) uptake and whether this would have any detectable behavioral effects.

ObjectiveThe primary goal was to determine tissue THC levels in the lobster after exposure to THC vapor. Secondary goals were to determine if THC vapor altered locomotor behavior or nociception.

MethodsTissue samples were collected (including muscle, brain and hemolymph) from Homarus americanus (N=3 per group) following 30 or 60 minutes of exposure to vapor generated by an e-cigarette device using THC (100 mg/mL in a propylene glycol vehicle). Separate experiments assessed locomotor behavior and hot water nociceptive responses following THC vapor exposure.

ResultsTHC vapor produced duration-related THC levels in all tissues examined. Locomotor activity was decreased (distance, speed, time-mobile) by 30 min inhalation of THC. Lobsters exhibit a temperature-dependent withdrawal response to immersion of tail, antennae or claws in warm water; this is novel evidence of thermal nociception for this species. THC exposure for 60 minutes had only marginal effect on nociception under the conditions assessed.

ConclusionsVapor exposure of lobsters, using an e-cigarette based model, produces dose-dependent THC levels in all tissues and reduces locomotor activity. Hot water nociception was temperature dependent, but only minimal anti-nociceptive effect of THC exposure was confirmed.

category: pharmacology and toxicology


title: Vapor exposure to Δ9-tetrahydrocannabinol (THC) slows locomotion of the Maine Lobster (Homarus americanus)


title: Targeting the nociceptive somatosensory system with AAV9 and AAV2retro viral vectors

abstract: Adeno-associated viral (AAV) vectors allow for site-specific and time-dependent genetic manipulation of neurons. However, for successful implementation of AAV vectors, major consideration must be given to the selection of viral serotype and route of delivery for efficient gene transfer into the cell type being investigated. Here we compare the transduction pattern of neurons in the somatosensory system following injection of AAV9 or AAV2retro in the parabrachial complex of the midbrain, the spinal cord dorsal horn, the intrathecal space, and the colon. Transduction was evaluated based on Cre-dependent expression of tdTomato in transgenic reporter mice, following delivery of AAV9 or AAV2retro carrying identical constructs that drive the expression of Cre/GFP. The pattern of distribution of tdTomato expression indicated notable differences in the access of the two AAV serotypes to primary afferent neurons via peripheral delivery in the colon and to spinal projections neurons via intracranial delivery within the parabrachial complex. Additionally, our results highlight the superior sensitivity of detection of neuronal transduction based on reporter expression relative to expression of viral products.


title: Cellular heterogeneity in DNA alkylation repair as a trade-off between cell survival and genetic plasticity

abstract: DNA repair mechanisms fulfil a dual role, as they are essential for cell survival and genome maintenance. Here, we studied how cells regulate the interplay between DNA repair and mutation. We focused on the Escherichia coli adaptive response that increases resistance to DNA alkylation damage. Combination of single-molecule imaging and microfluidic-based single-cell microscopy showed that noise in the gene activation timing of the master regulator Ada is accurately propagated to generate a distinct subpopulation of cells in which all proteins of the adaptive response are absent. Although lack of these proteins causes extreme sensitivity to alkylation stress, cellular heterogeneity in DNA alkylation repair provides a functional benefit by increasing the evolvability of the whole population. We demonstrated this by monitoring the dynamics of nascent mutations during alkylation stress as well as the frequency of fixed mutations that are generated by the distinct subpopulations of the adaptive response. This highlighted that evolvability is a trade-off between mutability and cell survival. Stochastic modulation of DNA repair capacity by the adaptive response solves this trade-off through the generation of a viable hypermutable subpopulation of cells that acts as a source of genetic diversity in a clonal population.


title: ATP disrupts lipid binding equilibrium to drive retrograde transport critical for bacterial outer membrane asymmetry

abstract:
The hallmark of the Gram-negative bacterial envelope is the presence of the outer membrane (OM). The OM is asymmetric, comprising lipopolysaccharides (LPS) in the outer leaflet and phospholipids (PLs) in the inner leaflet; this critical feature confers permeability barrier function against external insults, including antibiotics. To maintain OM lipid asymmetry, the OmpC-Mla system is believed to remove aberrantly localized PLs from the OM and transport them to the inner membrane (IM). Key to the system in driving lipid trafficking is the MlaFEDB ABC transporter complex in the IM, but mechanistic details, including transport directionality, remain enigmatic. Here, we develop a sensitive point-to-point in vitro lipid transfer assay that allows direct tracking of [14C]-labelled PLs between the periplasmic chaperone MlaC and MlaFEDB reconstituted into nanodiscs. We reveal that MlaC spontaneously transfers PLs to the IM transporter in an MlaD-dependent manner that can be further enhanced by coupled ATP hydrolysis. In addition, we show that MlaD is important for modulating productive coupling between ATP hydrolysis and such retrograde PL transfer. We further demonstrate that spontaneous PL transfer also occurs from MlaFEDB to MlaC, but such anterograde movement is instead abolished by ATP hydrolysis. Our work uncovers a model where PLs reversibly partition between two lipid binding sites in MlaC and MlaFEDB, and ATP binding and/or hydrolysis shift this equilibrium to ultimately drive retrograde PL transport by the OmpC-Mla system. These mechanistic insights will inform future efforts towards discovering new antibiotics against Gram-negative pathogens.

Significance StatementBiological membranes define cellular boundaries, allow compartmentalization, and represent a prerequisite for life. In Gram-negative bacteria, the outer membrane (OM) prevents entry of toxic substances, conferring intrinsic resistance against many antibiotics. This barrier function requires unequal distribution of lipids across the OM bilayer, yet how such lipid asymmetry is maintained is not well understood. In this study, we established the directionality of lipid transport for a conserved membrane protein complex, and uncovered mechanistic insights into how ATP powers such transport from the OM to the inner membrane. Our work provides fundamental understanding of lipid trafficking within the Gram-negative double-membrane envelope in the context of OM lipid asymmetry, and highlights the importance of targeting lipid transport processes for future antibiotics development.


title: Cdk5 drives formation of heterogeneous pancreatic neuroendocrine tumors

abstract: Pancreatic neuroendocrine tumors (PanNETs) are a heterogeneous population of neoplasms that arise from hormone-secreting islet cells of the pancreas and have increased markedly in incidence over the past four decades. Non-functional PanNETs, which occur more frequently than hormone-secreting tumors, are often not diagnosed until later stages of tumor development and have poorer prognoses. Development of successful therapeutics for PanNETs has been slow, partially due to a lack of diverse animal models for pre-clinical testing. Here, we report development of an inducible, conditional mouse model of PanNETs by using a bitransgenic system for regulated expression of the aberrant activator of Cdk5, p25, specifically in {beta}-islet cells. This model produces a heterogeneous population of PanNETs that includes a subgroup of well-differentiated, non-functional tumors. The utility of this model is enhanced by ability to form tumor-derived allografts. Production of these tumors demonstrates the causative potential of aberrantly active Cdk5 for generation of PanNETs. Further, we show that human PanNETs express Cdk5 pathway components, are dependent on Cdk5 for growth, and share genetic and transcriptional overlap with the INS-p25OE model. This new model of PanNETs will facilitate molecular delineation of Cdk5-dependent PanNETs and the development of new targeted therapeutics.

category: cancer biology


title: Phototropin-mediated perception of light direction in Arabidopsis leaves regulates blade flattening

abstract:
One conserved feature among angiosperms is the development of flat thin leaves. This developmental pattern optimizes light capture and gas exchange for photosynthesis. The blue light receptors phototropins are required for leaf flattening, with the null phot1phot2 mutant showing downwards curled leaves in Arabidopsis. However, key aspects of their function in leaf development remain unknown. Here, we performed a detailed spatiotemporal characterization of phototropin function in Arabidopsis leaves. We found that phototropins perceive light direction in the leaf blade, and similar to their role in hypocotyls they control the spatial pattern of auxin signaling possibly modulating auxin transport, to ultimately regulate cell expansion. Phototropin signaling components in the leaf partially differ from hypocotyls. Moreover, the light response on the upper (adaxial) and lower (abaxial) sides of the leaf blade suggest a partially distinct requirement of phototropin signaling components on each side. In particular, NON PHOTOTROPIC HYPOCOTYL 3 (NPH3) showed an adaxial-specific function. In addition, we show a prominent role of PHYTOCHROME KINASE SUBSTRATE 3 (PKS3) in leaf flattening. Among the auxin transporters tested, PINs and AUX/LAX influence the response most prominently. Overall, our results show that directional blue light perception by the phototropins is a key aspect of leaf development, integrating endogenous and exogenous signals.

One sentence summaryPhototropins perceive light direction in the leaf and control the auxin signaling pattern to regulate blade flattening.


title: Rhizobiales commensal bacteria promote Arabidopsis thaliana root growth via host sulfated peptide pathway

abstract: Root-associated commensal bacteria that belong to the order Rhizobiales, which also contains symbiotic and pathogenic bacteria, promote primary root growth of Arabidopsis thaliana. However, the molecular mechanism underlying this root growth promotion (RGP) activity remained unclear. Here, we conducted a transcriptomic analysis of A. thaliana roots inoculated with root-associated commensal bacteria of Rhizobiales and sister lineages and revealed common and strain/lineage-specific transcriptional response, possibly mediated by WRKY and ANAC family of transcription factors. We showed that the observed common response was also partly triggered by a wide range of non-pathogenic bacteria, fungi, and a multikingdom synthetic community (SynCom). This response was characterized by a down-regulation of genes related to intracellular redox regulation, suggesting distinctive redox status between pathogenic and non-pathogenic interactions. By integrating with developmental and cell biological experiments, we identified a crucial role of TYROSYLPROTEIN SULFOTRANSFERASE (TPST) in Rhizobiales RGP. Conversely, none of the known TPST-dependent sulfated peptide pathways appeared to be required for this activity, suggesting an unidentified component in the protein sulfation pathway targeted by Rhizobiales RGP. Finally, we show that TPST is needed for RGP exerted by Rhizobiales but not Pseudomonadales isolates, delineating lineage-specific mechanisms to manipulate host root development.


title: Sorghum Root Epigenetic Landscape During Limiting Phosphorus Conditions

abstract:
Efficient acquisition and use of available phosphorus from the soil is crucial for plant growth, development, and yield. With an ever-increasing acreage of croplands with suboptimal available soil phosphorus, genetic improvement of sorghum germplasm for enhanced phosphorus acquisition from soil is crucial to increasing agricultural output and reducing inputs, while confronted with a growing world population and uncertain climate. Sorghum bicolor is a globally important commodity for food, fodder, and forage. Known for robust tolerance to heat, drought, and other abiotic stresses, its capacity for optimal phosphorus use efficiency (PUE) is still being investigated for optimized root system architectures (RSA). Whilst a few RSA-influencing genes have been identified in sorghum and other grasses, the epigenetic impact on expression and tissue-specific activation of candidate PUE genes remains elusive. Here, we present transcriptomic, epigenetic, and regulatory network profiling of RSA modulation in the BTx623 sorghum background in response to limiting phosphorus (LP) conditions. We show that during LP, sorghum RSA is remodeled to increase root length and surface area, likely enhancing its ability to acquire P. Global DNA 5-methylcytosine and H3K4 and H3K27 trimethylation levels decrease in response to LP, while H3K4me3 peaks and DNA hypomethylated regions contain recognition motifs of numerous developmental and nutrient responsive transcription factors that display disparate expression patterns between different root tissues (primary root apex, elongation zone, and lateral root apex). Suggesting that epigenetic shifts during growth on LP results in targeted gene expression in a tissue-specific manner that optimizes the RSA for improved P uptake.

SummaryIn response to low P, epigenetic and transcriptional changes stimulate lateral root growth in Sorghum bicolor BTx623, increasing the root surface area for enhanced "mining" of P from the soil.


title: Maternally inherited piRNAs direct transient heterochromatin formation at active transposons during early Drosophila embryogenesis

abstract: The PIWI-interacting RNA (piRNA) pathway controls transposon expression in animal germ cells, thereby ensuring genome stability over generations. In Drosophila, piRNAs are intergenerationally inherited through the maternal lineage, and this has demonstrated importance in the specification of piRNA source loci and in silencing of I- and P-elements in the germ cells of daughters. Maternally inherited Piwi protein enters somatic nuclei in early embryos prior to zygotic genome activation and persists therein for roughly half of the time required to complete embryonic development. To investigate the role of the piRNA pathway in the embryonic soma, we created a conditionally unstable Piwi protein. This enabled maternally deposited Piwi to be cleared from newly laid embryos within 30 minutes and well ahead of the activation of zygotic transcription. Examination of RNA and protein profiles over time, and correlation with patterns of H3K9me3 deposition, suggests a role for maternally deposited Piwi in attenuating zygotic transposon expression in somatic cells of the developing embryo. In particular, robust deposition of piRNAs targeting roo, an element whose expression is mainly restricted to embryonic development, results in the deposition of transient heterochromatic marks at active roo insertions. We hypothesize that roo, an extremely successful mobile element, may have adopted a lifestyle of expression in the embryonic soma to evade silencing in germ cells.


title: An Issue of Concern: Unique Truncated ORF8 Protein Variants of SARS-CoV-2

abstract: Open reading frame 8 (ORF8) protein is one of the most evolving accessory proteins in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19). It was previously reported that the ORF8 protein inhibits presentation of viral antigens by the major histocompatibility complex class I (MHC-I) and interacts with host factors involved in pulmonary inflammation. The ORF8 protein assists SARS-CoV-2 to evade immunity and replication. Among many contributing mutations, Q27STOP, a mutation in the ORF8 protein defines the B.1.1.7 lineage of SARS-CoV-2, which is engendering the second wave of COVID-19. In the present study, 47 unique truncated ORF8 proteins (T-ORF8) due to the Q27STOP mutations were identified among 49055 available B.1.1.7 SARS-CoV-2 sequences. The results show that only one of the 47 T-ORF8 variants spread to over 57 geo-locations in North America, and other continents which includes Africa, Asia, Europe and South America. Based on various quantitative features such as amino acid homology, polar/non-polar sequence homology, Shannon entropy conservation, and other physicochemical properties of all specific 47 T-ORF8 protein variants, a collection of nine possible T-ORF8 unique variants were defined. The question of whether T-ORF8 variants work similarly to ORF8 has yet to be investigated. A positive response to the question could exacerbate future COVID-19 waves, necessitating severe containment measures.


title: Impact of Glycosylation on SARS-CoV-2 Infection and Broadly Protective Vaccine Design

abstract:
A major challenge to end the pandemic caused by SARS-CoV-2 is to develop a broadly protective vaccine. As the key immunogen, the spike protein is frequently mutated with conserved epitopes shielded by glycans. Here, we reveal that spike glycosylation has site-differential effects on viral infectivity and lung epithelial cells generate spike with more infective glycoforms. Compared to the fully glycosylated spike, immunization of spike protein with N-glycans trimmed to the monoglycosylated state (Smg) elicits stronger immune responses and better protection for hACE2 transgenic mice against variants of concern. In addition, a broadly neutralizing monoclonal antibody was identified from the Smg immunized mice, demonstrating that removal of glycan shields to better expose the conserved sequences is an effective and simple approach to broad-spectrum vaccine development.

One-Sentence SummaryRemoving glycan shields to expose conserved epitopes is an effective approach to develop a broad-spectrum SARS-CoV-2 vaccine.


title: Impact of glycosylation on a broad-spectrum vaccine against SARS-CoV-2


title: Resf1 supports embryonic stem cell self-renewal and effective germline entry

abstract: Retroelement silencing factor 1 (RESF1) interacts with the key regulators of mouse embryonic stem cells (ESCs) OCT4 and NANOG, and its absence results in sterility of mice. However, the function of RESF1 in ESCs and germ line specification is poorly understood. In this study, we used Resf1 knockout cell lines to determine the requirements of RESF1 for ESCs self-renewal and for in vitro specification of ESCs into primordial germ cell-like cells (PGCLCs). We found that deletion of Resf1 in ESCs cultured in serum and LIF reduces self-renewal potential whereas episomal expression of RESF1 has a modest positive effect on ESC self-renewal. In addition, RESF1 is not required for the capacity of NANOG and its downstream target ESRRB to drive self-renewal in the absence of LIF. However, Resf1 deletion reduces efficiency of PGCLC differentiation in vitro. These results identify Resf1 as a novel player in the regulation of pluripotent stem cells and germ cell specification.

category: developmental biology


title: Loss of Resf1 reduces the efficiency of embryonic stem cell self-renewal and germline entry


title: Identification of site-specific evolutionary trajectories shared across human betacoronaviruses

abstract: Comparison of evolution among related viruses can provide insights into shared adaptive processes, for example following host switching to a mutual host species. Whilst phylogenetic methods can help identify mutations that may be important for evolutionary processes such as adaptation to a new host, these can be enhanced by positioning candidate mutations to known functional sites on protein structures. Over the past two decades, three zoonotic betacoronaviruses have significantly impacted human public health: SARS-CoV-1, MERS-CoV and SARS-CoV-2, whilst two other betacoronaviruses, HKU1 and OC43, have circulated endemically in the human population for over 100 years. In this study, we use a comparative approach to prospectively search for potentially evolutionarily-relevant mutations within the Orf1ab and S genes across betacoronavirus species that have demonstrated sustained human-to-human transmission (HKU1, OC43, SARS-CoV-1 and SARS-CoV-2). We used a combination of molecular evolution methods to identify 30 sites that display evidence of homoplasy and/or stepwise evolution, that may be suggestive of adaptation across emerging and endemic betacoronaviruses. Of these, seven sites also display evidence of being selectively relevant. Drawing upon known protein structure data, we find that four of the identified mutations [18121 (exonuclease/27), 21623 (spike/21), 21635 (spike/25) and 23948 (spike/796), in SARS-CoV-2 genome coordinates] are proximal to regions of known functionality. Our results provide a molecular-level context for common evolutionary pathways that betacoronaviruses may undergo during adaptation to the human host.


title: Assembly and levels of P-TEFb depend on reversiblephosphorylation of cyclinT1

abstract: The positive transcription elongation factor b (P-TEFb) is a critical co-activator for transcription of most cellular and viral genes, including those of HIV. While P-TEFb is regulated by 7SK snRNA in proliferating cells, it is absent in quiescent and terminally differentiated cells, which has remained unexplored. In these cells, we found that CycT1 not bound to CDK9 is rapidly degraded. Moreover, productive CycT1:CDK9 interactions require phosphorylation of two threonine residues (Thr143 and Thr149) in CycT1 by PKC. Conversely, PP1 dephosphorylates these sites. Thus, PKC inhibitors or removal of PKC by chronic activation results in P-TEFb disassembly and CycT1 degradation. This finding not only recapitulates P-TEFb depletion in resting CD4+ T cells but also in anergic T cells. Importantly, our studies reveal mechanisms of P-TEFb inactivation underlying T cell quiescence, anergy, and exhaustion as well as proviral latency and terminal differentiation of cells.


title: Listeria monocytogenes utilizes the ClpP1/2 proteolytic machinery for fine-tuned substrate degradation under heat stress

abstract: Listeria monocytogenes exhibits two ClpP isoforms (ClpP1/ClpP2) which assemble into a heterooligomeric complex with enhanced proteolytic activity. Herein, we demonstrate that the formation of this complex depends on temperature and reaches a maximum ratio of about 1:1 at heat shock conditions, while almost no complex formation occurred below 4{degrees}C. In order to decipher the role of the two isoforms at elevated temperatures, we constructed L. monocytogenes ClpP1, ClpP2 and ClpP1/2 knockout strains and analyzed their protein regulation in comparison to the wild type (WT) strain via whole proteome mass-spectrometry (MS) at 37 {degrees}C and 42 {degrees}C. While {Delta}clpP1 strain only altered the expression of very few proteins, {Delta}clpP2 and {Delta}clpP1/2 strains revealed the dysregulation of many proteins at both temperatures. These effects were corroborated by crosslinking co-immunoprecipitation MS analysis. Thus, while ClpP1 serves as a mere enhancer of protein degradation in the heterocomplex, ClpP2 is essential for ClpX binding and thus functions as a gatekeeper for substrate entry. Applying an integrated proteomic approach combining whole proteome and co-immunoprecipitation datasets, several putative ClpP2 substrates were identified in the context of different temperatures and discussed with regards to their function in cellular pathways such as the SOS response.


title: Purification of functional Plasmodium falciparum tubulin allows for the identification of parasite-specific microtubule inhibitors

abstract: Cytoskeletal proteins are essential for parasite proliferation, growth, and transmission, and therefore represent promising drug targets. While {beta}-tubulin, the molecular building block of microtubules, is an established drug target in a variety of cancers, we still lack substantial knowledge of the biochemistry of parasite tubulins, which would allow us to exploit the structural divergence between parasite and human tubulins. Indeed, mechanistic insights have been limited by the lack of purified, functional parasite tubulin. In this study, we isolated Plasmodium falciparum tubulin that is assembly-competent and shows specific microtubule dynamics in vitro. We further present mechanistic evidence that two compounds selectively interact with parasite over host microtubules and inhibit Plasmodium microtubule polymerization at substoichiometric compound concentrations. The ability of compounds to selectively disrupt protozoan microtubule growth without affecting human microtubules provides the exciting possibility for the targeted development of novel antimalarials.


title: Genomic Surveillance of COVID-19 Variants with Language Models and Machine Learning

abstract: The global efforts to control COVID-19 are threatened by the rapid emergence of novel SARS-CoV-2 variants that may display undesirable characteristics such as immune escape, increased transmissibility or pathogenicity. Early prediction for emergence of new strains with these features is critical for pandemic preparedness. We present Strainflow, a supervised and causally predictive model using unsupervised latent space features of SARS-CoV-2 genome sequences. Strainflow was trained and validated on 0.9 million sequences for the period December, 2019 to June, 2021 and the frozen model was prospectively validated from July, 2021 to December, 2021. Strainflow captured the rise in cases two months ahead of the Delta and Omicron surges in most countries including the prediction of a surge in India as early as beginning of November, 2021. Entropy analysis of Strainflow unsupervised embeddings clearly reveals the explore-exploit cycles in genomic feature-space, thus adding interpretability to the deep learning based model. We also conducted codon-level analysis of our model for interpretability and biological validity of our unsupervised features. Strainflow application is openly available as an interactive web-application for prospective genomic surveillance of COVID-19 across the globe.


title: Evolutionary Inference Predicts Novel ACE2 Protein Interactions Relevant to COVID-19 Pathologies

abstract: Angiotensin-converting enzyme 2 (ACE2) is the cell receptor that the coronavirus SARS-CoV-2 binds to and uses to enter and infect human cells. COVID-19, the pandemic disease caused by the coronavirus, involves diverse pathologies beyond those of a respiratory disease, including micro-thrombosis (micro-clotting), cytokine storms, and inflammatory responses affecting many organ systems. Longer-term chronic illness can persist for many months, often well after the pathogen is no longer detected. A better understanding of the proteins that ACE2 interacts with can reveal information relevant to these disease manifestations and possible avenues for treatment. We have undertaken an approach to predict candidate ACE2 interacting proteins which uses evolutionary inference to identify a set of mammalian proteins that "coevolve" with ACE2. The approach, called evolutionary rate correlation (ERC), detects proteins that show highly correlated evolutionary rates during mammalian evolution. Such proteins are candidates for biological interactions with the ACE2 receptor. The approach has uncovered a number of key ACE2 protein interactions of potential relevance to COVID-19 pathologies. Some proteins have previously been reported to be associated with severe COVID-19, but are not currently known to interact directly with ACE2, while additional predicted novel ACE2 interactors are of potential relevance to the disease. Using reciprocal rankings of protein ERCs, we have identified strongly interconnected ACE2 associated protein networks relevant to COVID-19 pathologies. ACE2 has clear connections to coagulation pathway proteins, such as Coagulation Factor V and fibrinogen components FGG, FGB, and FGA, the latter possibly mediated through ACE2 connections to Clusterin (which clears misfolded extracellular proteins) and GPR141 (whose functions are relatively unknown). ACE2 also connects to proteins involved in cytokine signaling and immune response (e.g. IFNAR2, XCR1, and TLR8), and to Androgen Receptor (AR). The ERC prescreening approach has also elucidated possible functions for previously uncharacterized proteins and possible new functions for well-characterized ones. Suggestions are made for the validation of ERC predicted ACE2 protein interactions. We propose that ACE2 has novel protein interactions that are disrupted during SARS-CoV-2 infection, contributing to the spectrum of COVID-19 pathologies.


title: Probing DNA-protein interactions using single-molecule diffusivity contrast

abstract: Single-molecule fluorescence investigations of protein-nucleic acid interactions require robust means to identify the binding state of individual substrate molecules in real time. Here we show that diffusivity contrast, widely used in fluorescence correlation spectroscopy at the ensemble level and in single-particle tracking on individual (but slowly diffusing) species, can be used as a general readout to determine the binding state of single DNA molecules with unlabeled proteins in solution. We first describe the technical basis of drift-free single-molecule diffusivity measurements in an Anti-Brownian ELetrokinetic (ABEL) trap. We then cross-validate our method with protein-induced fluorescence enhancement (PIFE), a popular technique to detect protein binding on nucleic acid substrates with single-molecule sensitivity. We extend an existing hydrodynamic modeling framework to link measured diffusivity to particular DNA-protein structures and obtain good agreement between the measured and predicted diffusivity values. Finally, we show that combining diffusivity contrast with PIFE allows simultaneous mapping of binding stoichiometry and location on individual DNA-protein complexes, potentially enhancing single-molecule views of relevant biophysical processes.


title: Infection of brain pericytes underlying neuropathology of COVID-19 patients

abstract:
A wide range of neurological manifestations have been associated with the development of COVID-19 following SARS-CoV-2 infection. However, the etiology of the neurological symptomatology is still largely unexplored.

Here, we used state-of-the-art multiplexed immunostaining of human brains (n = 6 COVID-19, median age = 69,5 years; and n = 7 control, median age = 68 years), and demonstrated that expression of the SARS-CoV-2 receptor ACE2 is restricted to a subset of neurovascular pericytes. Strikingly, neurological symptoms were exclusive to, and ubiquitous in, patients that exhibited moderate to high ACE2 expression in peri-vascular cells. Viral particles were identified in the vascular wall and paralleled by peri-vascular inflammation, as signified by T cell and macrophage infiltration. Furthermore, fibrinogen leakage indicated compromised integrity of the blood-brain barrier. Notably, cerebrospinal fluid from an additional 16 individuals (n = 8 COVID-19, median age = 67 years; and n = 8 control, median age = 69,5 years) exhibited significantly lower levels of the pericyte marker PDGFR{beta} in SARS-CoV-2-infected cases, indicative of disrupted pericyte homeostasis.

We conclude that pericyte infection by SARS-CoV-2 underlies virus entry into the privileged central nervous system space, as well as neurological symptomatology due to peri-vascular inflammation and a locally compromised blood-brain barrier.

category: pathology


title: A photo-switchable yeast isocitrate dehydrogenase to control metabolic flux through the citric acid cycle

abstract: For various research questions in metabolism, it is highly desirable to have means available, with which the flux through specific pathways can be perturbed dynamically, in a reversible manner, and at a timescale that is consistent with the fast turnover rates of metabolism. Optogenetics, in principle, offers such possibility. Here, we developed an initial version of a photo-switchable isocitrate dehydrogenase (IDH) aimed at controlling the metabolic flux through the citric acid cycle in budding yeast. By inserting a protein-based light switch (LOV2) into computationally identified active/regulatory-coupled sites of IDH and by using in vivo screening in Saccharomyces cerevisiae, we obtained a number of IDH enzymes whose activity can be switched by light. Subsequent in-vivo characterization and optimization resulted in an initial version of photo-switchable (PS) IDH. While further improvements of the enzyme are necessary, our study demonstrates the efficacy of the overall approach from computational design, via in vivo screening and characterization. It also represents one of the first few examples, where optogenetics were used to control the activity of a metabolic enzyme.

category: synthetic biology


title: Interneuron subtypes enable independent modulation of excitatory and inhibitory firing rates after sensory deprivation

abstract: Diverse interneuron subtypes determine how cortical circuits process sensory information depending on their connectivity. Sensory deprivation experiments are ideally suited to unravel the plasticity mechanisms which shape circuit connectivity, but have yet to consider the role of different inhibitory subtypes. We investigate how synaptic changes due to monocular deprivation affect the firing rate dynamics in a microcircuit network model of the visual cortex. We demonstrate that, in highly recurrent networks, deprivation-induced plasticity generates fundamentally different activity changes depending on interneuron composition. Considering parvalbumin-positive (PV+) and somatostatin-positive (SST+) interneuron subtypes can capture the experimentally observed independent modulation of excitatory and inhibitory activity during sensory deprivation when SST+ feedback is sufficiently strong. Our model also applies to whisker deprivation in the somatosensory cortex revealing that these mechanisms are general across sensory cortices. Therefore, we provide a mechanistic explanation for the differential role of interneuron subtypes in regulating cortical dynamics during deprivation-induced plasticity.


title: Loss-of-function variants in the schizophrenia risk gene SETD1A alter neuronal network activity in human neurons through cAMP/PKA pathway

abstract: Heterozygous loss-of-function (LoF) mutations in SETD1A, which encodes a subunit of histone H3 lysine 4 methyltransferase, were shown to cause a novel neurodevelopmental syndrome and increase the risk for schizophrenia. We generated excitatory/inhibitory neuronal networks from human induced pluripotent stem cells with a SETD1A heterozygous LoF mutation (SETD1A+/-) using CRISPR/Cas9. Our data show that SETD1A haploinsufficiency resulted in morphologically increased dendritic complexity and functionally increased bursting activity. This network phenotype was primarily driven by SETD1A haploinsufficiency in glutamatergic neurons. In accordance with the functional changes, transcriptomic profiling revealed perturbations in gene sets associated with glutamatergic synaptic function. At the molecular level, we identified specific changes in the cAMP/PKA pathway pointing toward a hyperactive cAMP pathway in SETD1A+/- neurons. Finally, by pharmacologically targeting the cAMP pathway we were able to rescue the network deficits in SETD1A+/- cultures. Our results demonstrate a link between SETD1A and the cAMP-dependent pathway in human neurons.


title: AAV-BR1 targets endothelial cells in the retina to reveal their morphological diversity and to deliver Cx43.

abstract: Endothelial cells (ECs) are key players in the development and maintenance of the vascular tree, the establishment of the blood brain barrier and control of blood flow. Disruption in ECs is an early and active component of vascular pathogenesis. However, our ability to selectively target ECs in the CNS for identification and manipulation is limited. Here, in the mouse retina, a tractable model of the CNS, we utilized a recently developed AAV-BR1 system to identify distinct classes of ECs along the vascular tree using a GFP reporter. We then developed an inducible EC-specific ectopic Connexin 43 (Cx43) expression system using AAV-BR1-CAG-DIO-Cx43-P2A-DsRed2 in combination with a mouse line carrying inducible CreERT2 in ECs. We targeted Cx43 because its loss has been implicated in microvascular impariment in numerous diseases such as diabetic retinopathy and vascular edema. GFP-labeled ECs were numerous, evenly distributed along the vascular tree and their morphology was polarized with respect to the direction of blood flow. After tamoxifen induction, ectopic Cx43 was specifically expressed in ECs. Similarly to endogenous Cx43, ectopic Cx43 was localized at the membrane contacts of ECs and it did not affect tight junction proteins. The ability to enhance gap junctions in ECs provides a precise and potentially powerful tool to treat microcirculation deficits, an early pathology in numerous diseases.


title: The extracellular ATP receptor P2RX7 imprints a pro-memory transcriptional signature in effector CD8+ T cells

abstract: Development of central memory (Tcm) and resident memory (Trm) CD8+ T cells, which respectively promote immunity in the circulation and in barrier tissues, is not completely understood. Tcm and Trm cells may arise from common precursors, however their fate-inducing signals are elusive. We found that virus-specific effector CD8+ T cells display heterogeneous expression of the extracellular ATP sensor P2RX7. P2RX7-high expression is confined, at peak effector phase, to CD62L+ memory precursors which preferentially form Tcm cells. Among early effector CD8+ T cells, asymmetrical P2RX7 distribution correlated with distinct transcriptional signatures, with P2RX7-high cells enriched for memory and tissue-residency sets. P2RX7-high early effectors preferentially form both Tcm and Trm cells. Defective Tcm and Trm formation in P2RX7 deficiency is significantly reverted when the transcriptional repressor Zeb2 is ablated. Our study indicates that unequal P2RX7 upregulation in effector CD8+ T cells is a foundational element of the early Tcm/Trm fate.

category: immunology


title: Divergent early antibody responses define COVID-19 disease trajectories

abstract:
A damaging inflammatory response is strongly implicated in the pathogenesis of severe COVID-19 but mechanisms contributing to this response are unclear. In two prospective cohorts, early non-neutralizing, afucosylated, anti-SARS-CoV-2 IgG predicted progression from mild, to more severe COVID-19. In contrast to the antibody structures that predicted disease progression, antibodies that were elicited by mRNA SARS-CoV-2 vaccines were low in Fc afucosylation and enriched in sialylation, both modifications that reduce the inflammatory potential of IgG. To study the biology afucosylated IgG immune complexes, we developed an in vivo model which revealed that human IgG-Fc{gamma}R interactions can regulate inflammation in the lung. Afucosylated IgG immune complexes induced inflammatory cytokine production and robust infiltration of the lung by immune cells. By contrast, vaccine elicited IgG did not promote an inflammatory lung response. Here, we show that IgG-Fc{gamma}R interactions can regulate inflammation in the lung and define distinct lung activities associated with the IgG that predict severe COVID-19 and protection against SARS-CoV-2.

One Sentence SummaryDivergent early antibody responses predict COVID-19 disease trajectory and mRNA vaccine response and are functionally distinct in vivo.


title: Structurally and functionally distinct early antibody responses predict COVID-19 disease trajectory and mRNA vaccine response


title: An infection-activated redox switch promotes tumor growth

abstract: Oxidative stress is a defining feature of most cancers, including those that stem from carcinogenic infections1. Reactive oxygen species (ROS) can drive tumor formation2-4, yet the molecular oxidation events that contribute to tumorigenesis are largely unknown. Here we show that inactivation of a single, redox-sensitive cysteine in the host protease legumain, which is oxidized during infection with the gastric cancer-causing bacterium Helicobacter pylori, accelerates tumor growth. By using chemical proteomics to map cysteine reactivity in human gastric cells, we determined that H. pylori infection induces oxidation of legumain at Cys219. Legumain oxidation, which is enhanced by the ROS-promoting bacterial oncoprotein CagA, dysregulates intracellular legumain processing and localization and decreases legumain activity in H. pylori-infected cells. We further show that the site-specific loss of Cys219 reactivity increases tumor growth and mortality in a xenograft model. Our findings establish a link between the precise oxidation of a host protein and tumorigenic signaling during bacterial infection and demonstrate the importance of oxidative post-translational modifications in tumor growth.

category: microbiology


title: The Plasmodium NOT1-G Paralogue Acts as an Essential Nexus for Sexual Stage Maturation and Parasite Transmission

abstract: Productive transmission of malaria parasites hinges upon the execution of key transcriptional and post-transcriptional regulatory events. While much is now known about how specific transcription factors activate or repress sexual commitment programs, far less is known about the production of a preferred mRNA homeostasis following commitment and through the host-to-vector transmission event. Here we show that Plasmodium parasites have taken the unique approach to duplicate the NOT1 scaffold protein of the CAF1/CCR4/Not complex in order to dedicate one paralogue for essential transmission functions. Moreover, this NOT1-G paralogue is central to the sex-specific functions previously associated with its interacting partners, as deletion of not1-g in Plasmodium yoelii leads to a comparable or complete arrest phenotype for both male and female parasites. We show that, consistent with its role in other eukaryotes, PyNOT1-G localizes to cytosolic puncta throughout much of the Plasmodium life cycle. PyNOT1-G is essential to both the complete maturation of male gametes and to the continued development of the fertilized zygote originating from female parasites. Comparative transcriptomics of wild-type and pynot1-g- parasites shows that loss of PyNOT1-G leads to transcript dysregulation preceding and during gametocytogenesis, and shows that PyNOT1-G acts to preserve mRNAs that are critical to sexual and early mosquito stage development. Finally, we demonstrate that the tristetraprolin-binding domain, which acts as the typical organization platform for RNA decay (TTP) and RNA preservation (ELAV/HuR) factors is dispensable for PyNOT1-Gs essential blood stage functions but impacts host-to-vector transmission. Together, we conclude that Plasmodium has created and adapted a NOT1-G paralogue to fulfill the complex transmission requirements of both male and female parasites.


title: Characterization of gut microbiome and metabolome in Helicobacter pylori patients in an underprivileged community in the United States

abstract:
BackgroundHelicobacter pylori, a bacterium that infects approximately half of the worlds population, is associated with various gastrointestinal diseases, including peptic ulcers, non-ulcer dyspepsia, gastric adenocarcinoma, and gastric lymphoma. To combat the increasing antibiotic resistance of H. pylori, the need for new therapeutic strategies has become more pressing. Characterization of the interactions between H. pylori and the fecal microbiome, as well as the mechanisms underlying these interactions, may offer new therapeutic approaches. Exploration of changes in fatty acid metabolism associated with H. pylori-mediated alterations of the fecal microbiome may also reveal strategies to help prevent progression to neoplasia.

AimTo characterize the gut microbiome and metabolome in H. pylori patients in a socioeconomically challenged and underprivileged inner-city community.

MethodsStool samples from 19 H. pylori patients and 16 control subjects were analyzed. 16S rRNA gene sequencing was performed on normalized pooled amplicons using the Illumina MiSeq System using a MiSeq reagent kit v2. Alpha and beta diversity analyses were performed in QIIME 2. Non-targeted fatty acid analysis of the samples was carried out using gas chromatography-mass spectrometry (GC-MS), which measures the total content of 30 fatty acids in stool after conversion into their corresponding fatty acid methyl esters. Multi-dimensional scaling (MDS) was performed on Bray-Curtis distance matrices created from both the metabolomics and microbiome datasets and a Procrustes test was performed on the metabolomics and microbiome MDS coordinates.

ResultsFecal microbiome analysis showed that alpha diversity was lowest in H. pylori patients over 40 years of age compared to control subjects of similar age group. Beta diversity analysis of the samples revealed significant differences in microbial community structure between H. pylori patients and control subjects. Thirty-eight and six taxa had lower and higher relative abundance in H. pylori patients, respectively. Taxa that were enriched in H. pylori patients included Atopobium, Gemellaceae, Micrococcaceae, Gemellales and Rothia (R. mucilaginosa). Notably, relative abundance of the phylum Verrucomicrobia was decreased in H. pylori patients compared to control subjects, suggesting disruption of the gut mucosal environment by H. pylori. Procrustes analysis showed a significant relationship between the microbiome and metabolome datasets. Stool samples from H. pylori patients showed increases in several fatty acids including the polyunsaturated fatty acids (PUFAs) 22:4n6, 22:5n3, 20:3n6 and 22:2n6, while decreases were noted in other fatty acids including the PUFA 18:3n6. The pattern of changes in fatty acid concentration correlated to the Bacteroidetes:Firmicutes ratio determined by 16S rRNA gene analysis.

ConclusionAn individualized understanding of gut microbiome features among H. pylori patients will pave the way for improved community impact, reduced healthcare burdens of repeated treatment, and decreased mounting resistance.


title: Salmonella Typhimurium uses anaerobic respiration to overcome propionate-mediated colonization resistance

abstract: The gut microbiota benefits the host by limiting enteric pathogen expansion (colonization resistance) partially via the production of inhibitory metabolites. Propionate, a short-chain fatty acid produced by microbiota members, is proposed to mediate colonization resistance against Salmonella enterica serovar Typhimurium (S. Tm). Here, we show that S. Tm overcomes the inhibitory effects of propionate by using it as a carbon source for anaerobic respiration. We determined that propionate metabolism provides an inflammation-dependent colonization advantage to S. Tm during infection. Such benefit was abolished in the intestinal lumen of Salmonella-infected germ-free mice. Interestingly, S. Tm propionate-mediated intestinal expansion was restored when germ-free mice were monocolonized with Bacteroides thetaiotaomicron (B. theta), a prominent propionate producer in the gut, but not when mice were monocolonized with a propionate production-deficient B. theta strain. Taken together, our results reveal a novel strategy used by S. Tm to mitigate colonization resistance by metabolizing microbiota-derived propionate.


title: Structural basis for target-site selection in RNA-guided DNA transposition systems

abstract:
CRISPR-associated transposition systems allow guide RNA-directed integration of a single DNA insertion in one orientation at a fixed distance from a programmable target sequence. We define the mechanism explaining this process by characterizing the transposition regulator, TnsC, from a Type V-K CRISPR-transposase system using cryo-EM. Polymerization of ATP-bound TnsC helical filaments explains how polarity information is passed to the transposase. Our Cryo-EM structure of TniQ-TnsC reveals that TniQ caps the TnsC filament, establishing a universal mechanism for target information transfer in Tn7/Tn7-like elements. Transposase-driven disassembly establishes delivery of the element only to unused protospacers. Finally, structures with the transition state mimic, ADP{middle dot}AlF3, reveals how TnsC transitions to define the fixed point of insertion. These mechanistic findings provide the underpinnings for engineering CRISPR-associated transposition systems for research and therapeutic applications.

One Sentence SummaryCryo-EM studies reveals the role of the AAA+ regulator TnsC for target-site selection in CRISPR-associated transposition systems.

category: molecular biology