title: No broad decline of breeding monarch butterflies in North America: implications for conservation efforts

abstract: Many insects are in clear decline, with monarch butterflies (Danaus plexippus) drawing particular attention as a flagship species. Falling numbers of overwintering monarchs are well documented, but there has been debate regarding population trends of summer breeding populations. Here, we compile a series of long-term monarch monitoring datasets, some which are analyzed here for the first time, that reveal highly variable responses across the migratory geographic range, but no broad net decline in numbers of breeding monarchs. We also did not find evidence that sampling biased towards natural sites was masking declines at disturbed sites. Overall, our results suggest a robust resiliency in summer populations that thus far has allowed recovery from losses during the winter. Thus, monarchs may not require as much breeding habitat restoration as once thought, and focus should be on conserving the fall and spring migration.


title: Opposite effects of nutrient enrichment and herbivory by an alien snail on growth of an invasive macrophyte and native macrophytes

abstract: Human-mediated introduction of plant and animal species into biogeographic ranges where they did not occur before has been so pervasive globally that many ecosystems are now co-invaded by multiple alien plant and animal species. Although empirical evidence of invaders modifying recipient ecosystems to the benefit of other aliens is accumulating, these interactions remain underexplored and underrepresented in heuristic models of invasion success. Many freshwater ecosystems are co-invaded by aquatic macrophytes and mollusks and at the same time experience nutrient enrichment from various sources. However, studies are lacking that test how nutrient enrichment and co-invasion by alien herbivores and plant species can interactively affect native plant communities in aquatic habitats. To test such effects, we performed a freshwater mesocosm experiment in which we grew a synthetic native macrophyte community of three species under two levels of nutrient enrichment (enrichment vs. no-enrichment) treatment and fully crossed with two levels of competition from an invasive macrophyte Myriophyllum aquaticum (competition vs. no-competition), and two levels of herbivory by an invasive snail Pomacea canaliculata (herbivory vs. no-herbivory) treatments. Results show that herbivory by the invasive snail enhanced above-ground biomass yield of the invasive macrophyte. Moreover, the invasive herbivore preferentially fed on biomass of the native macrophytes over that of the invasive macrophyte. However, nutrient enrichment reduced above-ground biomass yield of the invasive macrophyte. Our results suggest that eutrophication of aquatic habitats that are already invaded by M. aquaticum may slow down invasive spread of the invasive macrophyte. However, herbivory by the invasive snail P. canaliculata may enhance invasive spread of M. aquaticum in the same habitats. Broadly, our study underscores the significance of considering several factors and their interaction when assessing the impact of invasive species, especially considering that many habitats experience co-invasion by plants and herbivores and simultaneously undergo varous other disturbances including nutrient enrichment.

category: ecology


title: Transcriptome-wide association study in UK Biobank Europeans identifies associations with blood cell traits.

abstract: Previous genome-wide association studies (GWAS) of hematological traits have identified over 10,000 distinct trait-specific risk loci, but the underlying causal mechanisms at these loci remain incompletely characterized. We performed a transcriptome-wide association study (TWAS) of 29 hematological traits in 399,835 UK Biobank (UKB) participants of European ancestry using gene expression prediction models trained from whole blood RNA-seq data in 922 individuals. We discovered 557 TWAS signals associated with hematological traits distinct from previously discovered GWAS variants, including 10 completely novel gene-trait pairs corresponding to 9 unique genes. Among the 557 associations, 301 were available for replication in a cohort of 141,286 participants of European ancestry from the Million Veteran Program (MVP). Of these 301 associations, 199 replicated at a nominal threshold ( = 0.05) and 108 replicated at a strict Bonferroni adjusted threshold ( = 0.05/301). Using our TWAS results, we systematically assigned 4,261 out of 16,900 previously identified hematological trait GWAS variants to putative target genes. Compared to coloc, our TWAS results show reduced specificity and increased sensitivity to assign variants to target genes.

category: genetics


title: Novel design of imputation-enabled SNP arrays for breeding and research applications supporting multi-species hybridisation

abstract:
Array-based SNP genotyping platforms have low genotype error and missing data rates compared to genotyping-by-sequencing technologies. However, design decisions used to create array-based SNP genotyping assays for both research and breeding applications are critical to their success. We describe a novel approach applicable to any animal or plant species for the design of cost-effective imputation-enabled SNP genotyping arrays with broad utility and demonstrate its application through the development of the Infinium Wheat Barley 40K SNP array. We show the approach delivers high-quality and high-resolution data for wheat and barley, including when samples are jointly hybridised. The new array aims to maximally capture haplotypic diversity in globally diverse wheat and barley germplasm while minimising ascertainment bias. Comprising mostly biallelic markers designed to be species-specific and single-copy, it permits highly accurate imputation in diverse germplasm to improve statistical power for GWAS and genomic selection. The SNP content captures tetraploid wheat (A- and B-genome) and Ae. tauschii (D-genome) diversity and delineates synthetic and tetraploid wheat from other wheats, as well as tetraploid species and subgroups. The content includes SNP tagging key trait loci in wheat and barley and that directly connect to other genotyping platforms and legacy datasets. The utility of the array is enhanced through the web-based tool Pretzel (https://plantinformatics.io/) which enables the arrays content to be visualised and interrogated interactively in the context of numerous genetic and genomic resources to more seamlessly connect research and breeding. The array is available for use by the international wheat and barley community.

Short summaryDesigning SNP genotyping arrays for closely related species with broad applicability in both research and breeding is challenging. Here we describe a novel generic approach to select SNP content for such arrays and demonstrate its utility in wheat and barley to: O_LIcapture haplotypic diversity while minimising ascertainment bias;
C_LIO_LIaccurately impute to high SNP density in diverse germplasm;
C_LIO_LIgenerate high-quality high-resolution genotypic data; and
C_LIO_LIjointly hybridise samples to the same bead chip array.
C_LI


title: Quantifying RNA Synthesis at Rate-Limiting Steps of Transcription Using Nascent RNA-Sequencing Data

abstract:
Nascent RNA-sequencing tracks transcription at nucleotide resolution. The genomic distribution of engaged transcription complexes, in turn, uncovers functional genomic regions. Here, we provide data-analytical steps to 1) identify transcribed regulatory elements de novo genome-wide, 2) quantify engaged transcription complexes at enhancers, promoter-proximal regions, divergent transcripts, gene bodies and termination windows, and 3) measure distribution of transcription machineries and regulatory proteins across functional genomic regions. This protocol follows RNA synthesis and genome-regulation in mammals, as demonstrated in human K562 erythroleukemia cells.

For complete details on the use and execution of this protocol, please refer to Vihervaara, et al., 2021.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=128 HEIGHT=200 SRC="FIGDIR/small/454856v1_ufig1.gif" ALT="Figure 1">
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category: genomics


title: Identification and in silico comparison of transposable elements of Solanum tuberosum subsp. andigena from two localities of Peru

abstract: The potato is one of the main crops in Peru, being one of the pillars in the countrys economy. It also has a cultural importance, being a basic element of Peruvian gastronomy and an emblematic crop of the Peruvian highlands, where small farmers over time have been able to conserve and cultivate 6048 native varieties. In Peru, around 300,000 hectares of potatoes are planted annually, and in 2019 Peru remained the main potato producer in Latin America, registering an annual production of 5.3 million tons. Therefore, it is necessary to study and characterize the genome of this very important tuber for the country. Although there are many potato genomic studies in the world and in Peru; these have been based on the search for genes of importance, genetic diversity, etc. and little has been deeply studied about the transposable elements (ETs) that are part of its genome. It is known that in many eukaryotes, transposable elements are powerful drivers of genome evolution, and they occupy a considerable part in plant genomes, so with these studies, more could be understood about how these ETs have intervened in the evolution of the potato in Peru; thus, how these mechanisms would help in the future to the survival of the species in scenarios of climate change, among others. This study proposes the identification and comparison of the transposable elements of the only 2 complete genomes of potatoes grown in Peru, found in the NCBI database, one from the department of Puno and the other from Cusco. There are various approaches to search for ETs in genomes, but for a complete and reliable annotation, it has been shown that the best strategy is the adoption of combined approaches, in this study, we will use 2 approaches, one based on homology, with the program RepeatMasker, and the other de novo, with the RepeatModeler2 program.

category: bioinformatics


title: Highly accurate local functional fingerprints and their stability

abstract: Individual subjects can be accurately identified in a database based on their functional connectome (FC), i.e. the whole-brain patterns of synchrony in fluctuations of brain activation as measured with functional MRI (fMRI). Accurate identification requires a representation of brain function to be simultaneously reliable (i.e. low intraindividual variability) and idiosyncratic (i.e. high interindividual variability), hence calling it a "fingerprint". Importantly, several studies have shown lower test-retest reliability (i.e. higher intraindividual variability) of the global FC compared to local representations of brain function such as regional homogeneity (ReHo) and (fractional) amplitude of low-frequency fluctuations ((f)ALFF). Therefore, here, with resting-state and task fMRI-data from the Human Connectome Project and the enhanced Nathan Kline Institute, we demonstrate that the local functional fingerprint, and especially regional homogeneity (ReHo), achieves near-perfect identification accuracies using four different ways to quantify identification. This finding is replicable across various parcellations as well as resilient to confounding effects. Moreover, using a machine-learning setup, we show that the small intraindividual ReHo fingerprint variability across sessions is meaningful for explaining individual-level intelligence. Further analyses reveal that the attention networks and the Default Mode Network contributed the most to the individual "uniqueness" of the ReHo fingerprint. Last, with another publicly available dataset, we show that ReHo is also stable in individuals with schizophrenia and that its (in)stability across sessions relates to intelligence scores. Altogether, our findings suggest that the ReHo fingerprint is a good candidate for further exploration of applicability in precision medicine.

category: neuroscience


title: Spatially localized fMRI metrics as predictive andhighly distinct state-independent fingerprints


title: Natural Killer cell division regulates FcεRIγ expression downstream of mTOR activity

abstract: The expansion of human Fc{varepsilon}RI{gamma}-/low (FcR{gamma}-/low) natural killer (NK) cells accrues during viral infections; however, the molecular mechanisms regulating FcR{gamma} expression is not well defined and can have implication for host protection and NK cell immunotherapy. Our analysis of NK cell subsets in lung transplant patients during rapamycin treatment revealed significantly lower FcR{gamma} levels in the NK cell population. Moreover, lower FcR{gamma} levels in healthy donors were associated with low mTORC1/C2 activity and low T-bet expression. Cell division suppression by rapamycin or TGF{beta} suppressed FcR{gamma} upregulation during IL-2 receptor stimulation, whereas promoting NK cell division by co-inhibiting FOXO1 activity restored FcR{gamma} upregulation. These results suggest that the human FcR{gamma}-/low NK cell phenotype is associated with cell division suppression and reduced mTOR activity.

category: immunology


title: Localization of T cell clonotypes using spatial transcriptomics

abstract: Spatial transcriptomics is an emerging technology that measures gene expression while preserving spatial information. Here, we present a method to determine localization of specific T cell clones by obtaining T cell receptor (TCR) sequences from spatial transcriptomics assays. Our method uses an existing commercial spatial transcriptomics platform and open-source software for analysis, allowing simple and inexpensive integration with archived samples and existing laboratory workflows. Using human brain metastasis samples, we show that TCR sequences are readily obtained from human tumor tissue and that these sequences are recapitulated by single-cell sequencing methods. This technique will permit detailed studies of the spatial organization of the human T cell repertoire, such as the identification of tumor-infiltrating and tumor-excluded T cell clones.


title: SARS-CoV-2 variants of concern have acquired mutations associated with an increased spike cleavage

abstract: For efficient cell entry and membrane fusion, SARS-CoV-2 spike (S) protein needs to be cleaved at two different sites, S1/S2 and S2 by different cellular proteases such as furin and TMPRSS2. Polymorphisms in the S protein can affect cleavage, viral transmission, and pathogenesis. Here, we investigated the role of arising S polymorphisms in vitro and in vivo to understand the emergence of SARS-CoV-2 variants. First, we showed that the S:655Y is selected after in vivo replication in the mink model. This mutation is present in the Gamma Variant Of Concern (VOC) but it also occurred sporadically in early SARS-CoV-2 human isolates. To better understand the impact of this polymorphism, we analyzed the in vitro properties of a panel of SARS-CoV-2 isolates containing S:655Y in different lineage backgrounds. Results demonstrated that this mutation enhances viral replication and spike protein cleavage. Viral competition experiments using hamsters infected with WA1 and WA1-655Y isolates showed that the variant with 655Y became dominant in both direct infected and direct contact animals. Finally, we investigated the cleavage efficiency and fusogenic properties of the spike protein of selected VOCs containing different mutations in their spike proteins. Results showed that all VOCs have evolved to acquire an increased spike cleavage and fusogenic capacity despite having different sets of mutations in the S protein. Our study demonstrates that the S:655Y is an important adaptative mutation that increases viral cell entry, transmission, and host susceptibility. Moreover, SARS-COV-2 VOCs showed a convergent evolution that promotes the S protein processing.

category: microbiology


title: Identification of Potent Small Molecule Inhibitors of SARS-CoV-2 Entry

abstract: The severe acute respiratory syndrome coronavirus 2 responsible for COVID-19 remains a persistent threat to mankind, especially for the immunocompromised and elderly for which the vaccine may have limited effectiveness. Entry of SARS-CoV-2 requires a high affinity interaction of the viral spike protein with the cellular receptor angiotensin-converting enzyme 2. Novel mutations on the spike protein correlate with the high transmissibility of new variants of SARS-CoV-2, highlighting the need for small molecule inhibitors of virus entry into target cells. We report the identification of such inhibitors through a robust high-throughput screen testing 15,000 small molecules from unique libraries. Several leads were validated in a suite of mechanistic assays, including whole cell SARS-CoV-2 infectivity assays. The main lead compound, Calpeptin, was further characterized using SARS-CoV-1 and the novel SARS-CoV-2 variant entry assays, SARS-CoV-2 protease assays and molecular docking. This study reveals Calpeptin as a potent and specific inhibitor of SARS-CoV-2 and some variants.


title: Rapid assessment of SARS-CoV-2 evolved variants using virus-like particles

abstract:
Newly evolved SARS-CoV-2 variants are driving ongoing outbreaks of COVID-19 around the world. Efforts to determine why these viral variants have improved fitness are limited to mutations in the viral spike (S) protein and viral entry steps using non-SARS-CoV-2 viral particles engineered to display S. Here we show that SARS-CoV-2 virus-like particles can package and deliver exogenous transcripts, enabling analysis of mutations within all structural proteins and rapid dissection of multiple steps in the viral life cycle. Identification of an RNA packaging sequence was critical for engineered transcripts to assemble together with SARS-CoV-2 structural proteins S, nucleocapsid (N), membrane (M) and envelope (E) into non-replicative SARS-CoV-2 virus-like particles (SC2-VLPs) that deliver these transcripts to ACE2- and TMPRSS2-expressing cells. Using SC2-VLPs, we tested the effect of 30 individual mutations within the S and N proteins on particle assembly and entry. While S mutations unexpectedly did not affect these steps, SC2-VLPs bearing any one of four N mutations found universally in more-transmissible viral variants (P199L, S202R, R203M and R203K) showed increased particle production and up to 10-fold more reporter transcript expression in receiver cells. Our study provides a platform for rapid testing of viral variants outside a biosafety level 3 setting and identifies viral N mutations and viral particle assembly as mechanisms to explain the increased spread of current viral variants, including Delta (N:R203M).

One-Sentence SummaryR203M substitution within SARS-CoV-2 N, found in delta variant, improves RNA packaging into virus-like particles by 10-fold.


title: Virucidal activity of CPC-containing oral rinses against SARS-CoV-2 variants and are active in the presence of human saliva

abstract: The role of human saliva in aerosol-based transmission of SARS-CoV-2 has highlighted the need to understand the potential of oral hygiene products to inactivate the virus. Here we examined the efficacy of mouthwashes containing cetylpyridinium chloride (CPC) or chlorhexidine (CHX) in inactivating SARS-CoV-2. After 30 seconds contact under standard aqueous conditions CPC mouthwashes achieved a [≥]4.0log10 PFU/mL reduction in SARS-CoV-2 (USA-WA1/2020) titres whereas comparable products containing CHX achieved <2.0log10 PFU/mL reduction. Further testing with CPC mouthwashes demonstrated efficacy against multiple SARS-CoV-2 variants, with inactivation below the limit of detection observed against the Alpha (B.1.1.7), Beta (B.1.351) and Gamma (P.1) variants. Virucidal efficacy of CPC mouthwash was also observed in the presence of human saliva with the product delivering [≥]4.0log10 PFU/mL reduction in SARS-CoV-2 titres after 30 seconds providing additional evidence for the virucidal efficacy of CPC mouthwashes under simulated physiological conditions. Together these data suggest CPC-based mouthwashes are effective at inactivating SARS-CoV-2 and further supports the use of mouthwash to mitigate the risk of transmission during dentistry procedures.


title: A new approach to integrate phylogenetic structure and partner availability to study biotic specialization in ecological networks

abstract: Biotic specialization holds information about the assembly, evolution and stability of biological communities. Phylogenetic diversity metrics have been used to quantify biotic specialization, but their current implementations do not adequately account for the availability of the interacting partners. Also, the overdispersed pattern of phylogenetic specialization has been misinterpreted as an attribute of generalists. We developed an approach that resolves these issues by accounting for partner availability to quantify the phylogenetic structure of specialization (i.e., clustered, overdispersed, or random) in ecological networks. We showed that our approach avoids biases of previous methods. We also implemented it on empirical networks of host-parasite, avian seed-dispersal, lichenized fungi- cyanobacteria and coral-dinoflagellate interactions. We found a large proportion of taxa that interact with phylogenetically random partners, in some cases to a larger extent than detected with an existing method that does not account for partner availability. We also found many taxa that interact with phylogenetically clustered partners, while taxa with overdispersed partners were rare. Our results highlight the important role of randomness in shaping interaction networks, even in highly intimate symbioses, and provide a much-needed quantitative framework to assess the role that evolutionary history and symbiotic specialization play in shaping patterns of biodiversity.


title: Robust bacterial co-occurence community structures are independent of r- and K-selection history

abstract: Selection for bacteria which are K-strategists instead of r-strategists has been shown to improve fish health and survival in aquaculture. We considered an experiment where microcosms were inoculated with natural seawater and the selection regime was switched from K-selection (by continuous feeding) to r-selection (by pulse feeding) and vice versa. We found the networks of significant co-occurrences to contain clusters of taxonomically related bacteria having positive associations. Comparing this with the time dynamics, we found that the clusters most likely were results of similar niche preferences of the involved bacteria. In particular, the distinction between r- or K-strategists was evident. Each selection regime seemed to give rise to a specific pattern, to which the community converges regardless of its prehistory. Furthermore, the results proved robust to parameter choices in the analysis, such as the filtering threshold, level of random noise, replacing absolute abundances with relative abundances, and the choice of similarity measure. Even though our data and approaches cannot directly predict ecological interactions, our approach provides insights on how the selection regime affects the composition and workings of the microbial community, providing a basis for aquaculture experiments targeted at eliminating opportunistic fish pathogens.


title: Pandemic-Scale Phylogenomics Reveals Elevated Recombination Rates in the SARS-CoV-2 Spike Region

abstract: Accurate and timely detection of recombinant lineages is crucial for interpreting genetic variation, reconstructing epidemic spread, identifying selection and variants of interest, and accurately performing phylogenetic analyses. During the SARS-CoV-2 pandemic, genomic data generation has exceeded the capacities of existing analysis platforms, thereby crippling real-time analysis of viral recombination. Low SARS-CoV-2 mutation rates make detecting recombination difficult. Here, we develop and apply a novel phylogenomic method to exhaustively search a nearly comprehensive SARS-CoV-2 phylogeny for recombinant lineages. We investigate a 1.6M sample tree, and identify 606 recombination events. Approximately 2.7% of sequenced SARS-CoV-2 genomes have recombinant ancestry. Recombination breakpoints occur disproportionately in the Spike protein region. Our method empowers comprehensive real time tracking of viral recombination during the SARS-CoV-2 pandemic and beyond.

category: evolutionary biology


title: Relative model selection can be sensitive to multiple sequence alignment uncertainty

abstract:
BackgroundMultiple sequence alignments (MSAs) represent the fundamental unit of data inputted to most comparative sequence analyses. In phylogenetic analyses in particular, errors in MSA construction have the potential to induce further errors in downstream analyses such as phylogenetic reconstruction itself, ancestral state reconstruction, and divergence time estimation. In addition to providing phylogenetic methods with an MSA to analyze, researchers must also specify a suitable evolutionary model for the given analysis. Most commonly, researchers apply relative model selection to select a model from candidate set and then provide both the MSA and the selected model as input to subsequent analyses. While the influence of MSA errors has been explored for most stages of phylogenetics pipelines, the potential effects of MSA uncertainty on the relative model selection procedure itself have not been explored.

ResultsWe assessed the consistency of relative model selection when presented with multiple perturbed versions of a given MSA. We find that while relative model selection is mostly robust to MSA uncertainty, in a substantial proportion of circumstances, relative model selection identifies distinct best-fitting models from different MSAs created from the same set of sequences. We find that this issue is more pervasive for nucleotide data compared to amino-acid data. However, we also find that it is challenging to predict whether relative model selection will be robust or sensitive to uncertainty in a given MSA.

ConclusionsWe find that that MSA uncertainty can affect virtually all steps of phylogenetic analysis pipelines to a greater extent than has previously been recognized, including relative model selection.


title: Relative model selection of evolutionary substitution models can be sensitive to multiple sequence alignment uncertainty


title: Sparse Sonomyography-based Estimation of Isometric Force: A Comparison of Methods and Features

abstract: Noninvasive methods for estimation of joint and muscle forces have widespread clinical and research applications. Surface electromyography or sEMG provides a measure of the neural activation of muscles which can be used to estimate the force produced by the muscle. However, sEMG based measures of force suffer from poor signal-to-noise ratio and limited spatiotemporal specificity. In this paper, we propose an ultrasound imaging or sonomyography-based approach for estimating continuous isometric force from a sparse set of ultrasound scanlines. Our approach isolates anatomically relevant features from A-mode ultrasound signals, greatly reducing the dimensionality of the feature space and the computational complexity involved in traditional ultrasound-based methods. We evaluate the performance of four regression methodologies for force prediction using the reduced feature set. We also evaluate the feasibility of a practical wearable sonomyography-based system by simulating the effect of transducer placement and varying the number of transducers used in force prediction. Our results demonstrate that Gaussian process regression models outperform other regression methods in predicting continuous force levels from just four equispaced transducers and are tolerant to speckle noise. These findings will aid in the design of wearable sonomyography-based force prediction systems with robust, computationally efficient operation.

category: bioengineering


title: But Mouse, you are not alone: On some severe acute respiratory syndrome coronavirus 2 variants infecting mice

abstract: In silico predictions combined with in vitro, in vivo and in situ observations collectively suggest that mouse adaptation of the SARS-CoV-2 virus requires an aromatic substitution in position 501 or position 498 (but not both) of the spike proteins receptor binding domain. This effect could be enhanced by mutations in positions 417, 484, and 493 (especially K417N, E484K, Q493K and Q493R), and to a lesser extent by mutations in positions 486 and 499 (such as F486L and P499T). Such enhancements due to more favourable binding interactions with residues on the complementary angiotensin-converting enzyme 2 (ACE2) interface, are however, unlikely to sustain mouse infectivity on their own based on theoretical and experimental evidence to date. Our current understanding thus points to the Alpha, Beta, Gamma, and Omicron variants of concern infecting mice, while Delta and  Delta Plus lack a similar biomolecular basis to do so. This paper identifies eleven countries (Brazil, Chile, Djibouti, Haiti, Malawi, Mozambique, Reunion, Suriname, Trinidad and Tobago, Uruguay and Venezuela) where targeted local field surveillance of mice is encouraged because they may have come in contact with humans who had the virus with adaptive mutation(s). It also provides a systematic methodology to analyze the potential for other animal reservoirs and their likely locations.


title: Super-resolution imaging of highly curved membrane structures in giant vesicles encapsulating molecular condensates

abstract: Molecular crowding is an inherent feature of the cell interior. Synthetic cells as provided by giant unilamellar vesicles (GUVs) encapsulating macromolecules (polyethylene-glycol and dextran) represent an excellent mimetic system to study membrane transformations associated with molecular crowding and protein condensation. Similarly to cells, such GUVs loaded with macromolecules exhibit highly curved structures such as internal nanotubes. In addition, upon liquid-liquid phase separation as inside living cells, the membrane of GUVs encapsulating an aqueous two-phase system deforms to form apparent kinks at the contact line of the interface between the two aqueous phases. These structures, nanotubes and kinks, have dimensions below optical resolution and if resolved, can provide information about material properties such as membrane spontaneous curvature and intrinsic contact angle describing the wettability contrast of the encapsulated phases to the membrane. Previous experimental studies were based on conventional optical microscopy which cannot resolve these membrane and wetting properties. Here, we studied these structures with super-resolution microscopy, namely stimulated emission depletion (STED) microscopy, together with microfluidic manipulation. We demonstrate the cylindrical nature of the nanotubes with unprecedented detail based on the superior resolution of STED and automated data analysis. The spontaneous curvature deduced from the nanotube diameters is in excellent agreement with theoretical predictions. Furthermore, we were able to resolve the membrane "kink" structure as a smoothly curved membrane demonstrating the existence of the intrinsic contact angle. We find very good agreement between the directly measured values and the theoretically predicted ones based on the apparent contact angles on the micrometer scale. During different stages of cellular events, biomembranes undergo a variety of shape transformations such as the formation of buds and nanotubes regulated by membrane necks. We demonstrate that these highly curved membrane structures are amenable to STED imaging and show that such studies provide important insights in the membrane properties and interactions underlying cellular activities.

category: biophysics


title: Integrated transcriptomic meta-analysis provides novel insights into breast cancer molecular subtypes

abstract: Breast cancer is the most common cancer in women worldwide. There are four major breast cancer subtypes (luminal A, luminal B, HER2-enriched and triple-negative/TNBC). TNBC is the most aggressive form with the worst prognosis. However, the differences among the subtypes have not been completely established at the molecular level, thereby limiting therapeutic and diagnostic options for TNBC. We performed a meta-analysis of microarray and RNA-sequencing data to identify candidate genes with an expression-based association in each molecular subtype of breast cancer. The protein interaction network of the candidate genes was analyzed to discover functionally significant gene clusters and hub genes. Potential therapeutic candidates for TNBC were explored through gene-miRNA interactions. We identified 316, 347, 382, and 442 candidate genes in luminal A, luminal B, HER2 and TNBC subtypes, respectively. A total of 135 (26 up- and 109 down-regulated) candidate genes were shared by all four subtypes. Functional analysis of the candidate genes indicated up-regulation of  cell cycle and  p53 signaling pathways and down-regulation of multiple signaling pathways. COL10A1 was found to be highly up-regulated in all subtypes. It may be a good target for research towards multiple types of applications, including therapeutics. KIF4A, a commonly up-regulated X-chromosome gene was significantly associated with the survival of breast cancer patients. Protein interaction network and centrality analysis revealed that low-moderately differentially regulated genes play an important role in functional cascades across proteins in breast cancer subtypes and may be potential candidates for therapeutics. Targeting FN1 (fibronectin 1), the key up-regulated hub gene by miR-1271 5p may be an important molecular event to be targeted for potential therapeutic application in TNBC.

category: cancer biology


title: A spatio-temporal model to reveal oscillatorphenotypes in molecular clocks: Parameterestimation elucidates circadian gene transcriptiondynamics in single-cells

abstract: We propose a stochastic distributed delay model together with a Markov random field prior and a measurement model for bioluminescence-reporting to analyse spatiotemporal gene expression in intact networks of cells. The model describes the oscillating time evolution of molecular mRNA counts through a negative transcriptional-translational feedback loop encoded in a chemical Langevin equation with a probabilistic delay distribution. The model is extended spatially by means of a multiplicative random effects model with a first order Markov random field prior distribution. Our methodology effectively separates intrinsic molecular noise, measurement noise, and extrinsic noise and phenotypic variation driving cell heterogeneity, while being amenable to parameter identification and inference. Based on the single-cell model we propose a novel computational stability analysis that allows us to infer two key characteristics, namely the robustness of the oscillations, i.e. whether the reaction network exhibits sustained or damped oscillations, and the profile of the regulation, i.e. whether the inhibition occurs over time in a more distributed versus a more direct manner, which affects the cells ability to phase-shift to new schedules. We show how insight into the spatio-temporal characteristics of the circadian feedback loop in the suprachiasmatic nucleus (SCN) can be gained by applying the methodology to bioluminescence-reported expression of the circadian core clock gene Cry1 across mouse SCN tissue. We find that while (almost) all SCN neurons exhibit robust cell-autonomous oscillations, the parameters that are associated with the regulatory transcription profile give rise to a spatial division of the tissue between the central region whose oscillations are resilient to perturbation in the sense that they maintain a high degree of synchronicity, and the dorsal region which appears to phase shift in a more diversified way as a response to large perturbations and thus could be more amenable to entrainment.

category: systems biology


title: Metabolic reprogramming of cytotoxic T lymphocytes by high glucose enhances pancreatic beta cell apoptosis via TRAIL

abstract: Cytotoxic T lymphocytes (CTLs) are involved in development of diabetes. However, the impact of excessive glucose on CTL-mediated antigen-independent killing remains elusive. Here, we report that TNF-related apoptosis inducing ligand (TRAIL) is substantially up- regulated in CTLs in environments with high glucose (HG) both in vitro and in vivo. The PI3K- Akt-NF{kappa}B axis and non-mitochondrial reactive oxygen species are essential in HG-induced TRAIL upregulation in CTLs. TRAILhigh CTLs induce apoptosis of pancreatic beta cell line 1.4E7. Metformin and Vitamin D synergistically reduce HG-enhanced expression of TRAIL in CTLs and coherently protect 1.4E7 cells from TRAIL-mediated apoptosis. Notably, in patients with diabetes, correlation between Vitamin D concentrations in plasma and glucose levels is linked to HG-enhanced TRAIL expression on CTLs. Microarray data reveal that OXCT2, an important enzyme in ketone body catabolism, is a promising target in response to vitamin D. Our work not only reveals a novel mechanism of CTL involvement in progression of diabetes, but also establishes CTLs as a target for combined metformin and vitamin D therapy to protect pancreatic beta cells of diabetic patients.


title: High glucose enhances antigen-independent CTL killing via TRAIL


title: Optical nanoscopy reveals SARS-CoV-2-induced remodeling of human airway cells

abstract: A better understanding of host cell remodeling by the coronavirus SARS-CoV-2 is urgently needed to understand viral pathogenesis and guide drug development. Expression profiling and electron microscopy have frequently been used to study virus-host interactions, but these techniques do not readily enable spatial, sub-cellular and molecular analysis of specific cellular compartments. Here, we use diffraction-unlimited fluorescence microscopy to analyze how SARS-CoV-2 infection exploits and repurposes the subcellular architecture of primary human airway cells. Using STED nanoscopy, we detect viral entry factors along the motile cilia of ciliated cells and visualize key aspects of the viral life cycle. Using Tenfold Robust Expansion (TREx) microscopy, we analyze the extensively remodeled three-dimensional ultrastructure of SARS-CoV-2-infected ciliated cells and uncover Golgi fragmentation, emergence of large and atypical multivesicular bodies enclosing viral proteins, ciliary clustering, and remodeling of the apical surface. These results demonstrate a broadly applicable strategy to study how viruses reorganize host cells with spatial and molecular specificity and provide new insights into SARS-CoV-2 infection in primary human cell models.

category: cell biology


title: Evaluating the efficacy of a consumer-centric method for ecological sampling: Using bonobo (Pan paniscus) feeding patterns as an instrument for tropical forest characterization

abstract:
O_LICharacteristics of food availability and distribution are a key component of a species ecology. Objective measurement of food resources, such as vegetation plot sampling, do not consider aspects of selection by the consumer and therefore may produce imprecise measures of availability. Further, in most animal ecology research, traditional ecological surveying often is time-intensive and supplementary to ongoing behavioral observation. We propose a method to integrate ecological sampling of an animals environment into existing behavioral data collection systems by using the consumer as the surveyor. Here, we introduce the consumer-centric method (CCM) of assessing resource availability for its ability to measure food resource abundance, distribution, and dispersion. This method catalogues feeding locations observed during behavioral observation and uses aggregated data to characterize these ecological metrics.
C_LIO_LIWe evaluated the CCM relative to traditional vegetation plot surveying using accumulated feeding locations across three years visited by a tropical frugivore, the bonobo (Pan paniscus), and compared it with data derived from over 200 vegetation plots across their 50km2+ home range.
C_LIO_LIWe demonstrate that food species abundance estimates derived from the CCM are comparable to those derived from traditional vegetation plot sampling after approximately 600 observation days or 60 spatially explicit feeding locations. The agreement between the methods further improved when accounting for aspects of consumer selectivity in objective vegetation plot sampling (e.g., size minima). Estimates of density from CCM correlated with plot-derived estimates and were relatively insensitive to home range inclusion and other species characteristics, but were sensitive to sampling frequency (e.g., consumption frequency). Agreement between the methods in relative distribution of resources performed better across species than expected by chance, although measures of dispersion correlated poorly.
C_LIO_LIWe demonstrate that while providing a robust measure to quantify local food availability, the CCM has an advantage over traditional sampling methods as it incorporates sampling biases relevant to the consumer. Therefore, as this method can be incorporated into existing observational data collection and does not require additional ecological surveying, it serves as a promising method for behavioral ecological data collection for animal species who re-use space and consume immobile food items.
C_LI


title: Inferring inter- and intrachromosomal Dobzhansky-Muller incompatibilities                from imbalanced segregation of recombinant haplotypes in hybrid populations

abstract:
Dobzhansky-Muller incompatibilities (DMIs) are a major component of reproductive isolation between species. DMIs imply negative epistasis, and are exposed when two diverged populations hybridize. Mapping the locations of DMIs has largely relied on classical genetic mapping. Approaches to date are hampered by low power and the challenge of identifying DMI loci on the same chromosome, because strong initial linkage of parental haplotypes weakens statistical tests. Here, we propose new statistics to infer negative epistasis from haplotype frequencies in hybrid populations. When two divergent populations hybridize, the variance in heterozygosity at two loci decreases faster with time at DMI loci than at random pairs of loci. When two populations hybridize at near-even admixture proportions, the deviation of the observed variance from its expectation becomes negative for the DMI pair. This negative deviation enables us to detect intermediate to strong negative epistasis both within and between chromosomes. In practice, the detection window in hybrid populations depends on the demographic scenario, the recombination rate, and the strength of epistasis. When the initial proportion of the two parental populations is uneven, only strong DMIs can be detected with our method unless migration prevents parental haplotypes from being lost. We use the new statistics to infer candidate DMIs from three hybrid populations of swordtail fish. We identify numerous new DMI candidates, some of which are inferred to interact with several loci within and between chromosomes. Moreover, we discuss our results in the context of an expected enrichment in intrachromosomal over interchromosomal DMIs.

Author SummaryGenetic incompatibility in the form of (Bateson-)Dobzhansky-Muller incompatibilities (DMIs) is an important component of reproductive isolation between species. However, the evolutionary role of DMIs during the process of speciation is contentious. DMIs occur when two or more genetic variants interact to reduce their carriers fitness. Once recombination combines incompatible variants in hybrids, selection acts to remove these variants from the population. One step towards addressing the evolutionary role of DMIs is to quantify the prevalence of DMIs in incipient and hybridizing species. Here, we present statistics that are sensitive to the resulting recombinant imbalance and that can indicate the location of DMIs in hybrid genomes in various demographic scenarios. We use simulations to show that the time window during which a DMI is detectable depends on its genomic location, the severity of the DMI, and the populations demography. Importantly, our statistic distinguishes genetic associations arising due to physical linkage from those arising due to gene interactions, which allows for the inference of both inter- and intrachromosomal DMIs. Applying our statistics to three hybrid populations of swordtail fish, we confirm previously known DMIs and identify new candidate incompatibilities.


title: Imbalanced segregation of recombinant haplotypes in hybrid populations reveals inter- and intrachromosomal Dobzhansky-Muller incompatibilities


title: cageminer: an R/Bioconductor package to prioritize candidate genes by integrating GWAS and gene coexpression networks

abstract:
SummaryAlthough genome-wide association studies (GWAS) identify variants associated with traits of interest, they often fail in identifying causative genes underlying a given phenotype. Integrating GWAS and gene coexpression networks can help prioritize high-confidence candidate genes, as the expression profiles of trait-associated genes can be used to mine novel candidates. Here, we present cageminer, the first R package to prioritize candidate genes through the integration of GWAS and coexpression networks. Genes are considered high-confidence candidates if they pass all three filtering criteria implemented in cageminer, namely physical proximity to SNPs, coexpression with known trait-associated genes, and significant changes in expression levels in conditions of interest. Prioritized candidates can also be scored and ranked to select targets for experimental validation. By applying cageminer to a real data set, we demonstrate that it can effectively prioritize candidates, leading to >99% reductions in candidate gene lists.

Availability and implementationThe package is available at Bioconductor (http://bioconductor.org/packages/cageminer).


title: Canine oral melanoma genomic and transcriptomic study defines two molecular subgroups with different therapeutical targets

abstract: Mucosal melanoma (MM) is a rare and aggressive clinical cancer that occurs mostly in the head, neck, and anogenital regions. Despite recent advances in genetics and the development of revolutionary treatments, such as immunotherapy, the prognosis for MM remains poor. Canine MM shares several clinical, histological, and genetic features with its human counterpart, offering a relevant spontaneous and immunocompetent model to decipher the genetic bases and explore treatment options for human MM. We performed an integrative genomic and transcriptomic analysis of 32 canine MM samples, which allowed us to identify two molecular subgroups differing in microenvironment and structural variant (SV) content. The overexpression of genes related to the microenvironment and T-cell response was associated with tumors harboring a lower content of structural variants, whereas the overexpression of pigmentation-related pathways and oncogenes such as TERT were associated with a high SV burden. To detail the SVs, especially those with focal amplifications, whole-genome sequencing was performed on four canine MM cell lines. We showed that focal amplifications characterized complex chromosomal rearrangements targeting oncogenes such as MDM2 or CDK4 and a recurrently amplified region on canine chromosome 30, comprising the genes TRPM7, GABPB1, USP8, and SPPL2A, were candidate oncogenes for MM. We showed that the copy numbers of these genes were significantly correlated with their expression levels. Finally, we demonstrated that the genes TRPM7, GABPB1, and SPPL2A play a role in cell proliferation; thus, these may be considered new candidate oncogenes for human MM. Our findings suggest the existence of two MM molecular subgroups that may benefit from dedicated therapies, such as immune checkpoint inhibitors or targeted therapies. These results illustrate the relevance of dog models for deciphering genetic mechanisms in spontaneous MM, along with the potential to screen for efficient targeted therapies for rare and aggressive cancers in humans.


title: NO-stressed Y. pseudotuberculosis have decreased cell division rates in the mouse spleen

abstract: Fluorescence dilution approaches can detect bacterial cell division events, and can detect if there are differential rates of cell division across individual cells within a population. This approach typically involves inducing expression of a fluorescent protein, and then tracking partitioning of fluorescence into daughter cells. However, fluorescence can be diluted very quickly within a rapidly replicating population, such as pathogenic bacterial populations replicating within host tissues. To overcome this limitation, we have generated two revTetR reporter constructs, where either mCherry or yellow fluorescent protein (YFP) is constitutively expressed, and repressed by addition of tetracyclines, resulting in fluorescence dilution within defined timeframes. We show that fluorescent signals are diluted in replicating populations, and that signal accumulates in growth-inhibited populations, including during nitric oxide exposure. Furthermore, we show that tetracyclines can be delivered to the mouse spleen during Yersinia pseudotuberculosis infection, and defined a drug concentration that results in even exposure of cells to tetracyclines. We then used this system to visualize bacterial cell division within defined timeframes post-inoculation. We detected growth attenuation of the revTetR-mCherry strains within mouse tissues, however data suggested heightened NO exposure correlated with heightened mCherry signal. We were able to restore normal bacterial growth with revTetR-YFP, and use this strain to show that heightened NO exposure correlated with heightened YFP signal, indicating decreased cell division rates within this subpopulation in vivo. This revTetR reporter will provide a critical tool for future studies to identify and isolate slowly replicating bacterial subpopulations from host tissues.


title: Interbacterial transfer of carbapenem resistance and large antibiotic resistance islands by natural transformation in pathogenic Acinetobacter

abstract:
Acinetobacter baumannii infection poses a major health threat with recurrent treatment failure due to antibiotic resistance, notably to carbapenems. While genomic analyses of clinical strains indicate that homologous recombination plays a major role in the acquisition of antibiotic resistance genes, the underlying mechanisms of horizontal gene transfer often remain speculative. Our understanding of the acquisition of antibiotic resistance is hampered by the lack of experimental systems able to reproduce genomic observations. We here report the detection of recombination events occurring spontaneously in mixed bacterial populations and which can result in the acquisition of resistance to carbapenems. We show that natural transformation is the main driver of intra-, but also inter-strain recombination events between A. baumannii clinical isolates and pathogenic species of Acinetobacter. We observed that interbacterial natural transformation in mixed populations is more efficient at promoting the acquisition of large resistance islands (AbaR4, AbaR1) than when the same bacteria are supplied with large amounts of purified genomic DNA. Importantly, analysis of the genomes of the recombinant progeny revealed large recombination tracts (from 13 to 123 kb) similar to those observed in the genome of clinical isolates. Moreover, we highlight that transforming DNA availability is a key determinant of the rate of recombinants and results from both spontaneous release and interbacterial predatory behavior. In the light of our results, natural transformation should be considered as a leading mechanism of genome recombination and horizontal gene transfer of antibiotic resistance genes in Acinetobacter baumannii.

ImportanceAcinetobacter baumannii is a multidrug resistant pathogen responsible for difficult-to-treat hospital-acquired infections. Understanding the mechanisms leading to the emergence of the multi-drug resistance in this pathogen is today crucial. Horizontal gene transfer is assumed to largely contribute to this multidrug resistance. However, in A. baumannii, the mechanisms leading to genome recombination and the horizontal transfer of resistance genes are poorly understood. We bring experimental evidence that natural transformation, a horizontal gene transfer mechanism recently highlighted in A. baumannii, allows the highly efficient interbacterial transfer of genetic elements carrying resistance to last line antibiotic carbapenems. Importantly, we demonstrated that natural transformation, occurring in mixed populations of Acinetobacter, enables the transfer of large resistance island mobilizing multiple resistance genes.


title: One Health or Three? Transmission modelling of Klebsiella isolates reveals ecological barriers to transmission between humans, animals and the environment

abstract: The Klebsiella group is highly diverse both genetically and ecologically, being commonly recovered from humans, livestock, plants, soil, water, and wild animals. Many species are opportunistic pathogens, and can harbour diverse classes of antimicrobial resistance (AMR) genes. K. pneumoniae is responsible for a high public-health burden, due in part to the rapid spread of health-care associated clones that are non-susceptible to carbapenems. Klebsiella thus represents a highly pertinent taxon for assessing the risk to public health posed by animal and environmental reservoirs. Here we report an analysis of 6548 samples and 3,482 genome sequences representing 15 Klebsiella species sampled over a 15-month period from a wide range of clinical, community, animal and environmental settings in and around the city of Pavia, in the northern Italian region of Lombardy. Despite carbapenem-resistant clones circulating at a high frequency in the hospitals, we find no genotypic or phenotypic evidence for non-susceptibility to carbapenems outside of the clinical environment. The non-random distribution of species and strains across sources point to ecological barriers that are likely to limit AMR transmission. Although we find evidence for occasional transmission between settings, hierarchical modelling and intervention analysis suggests that direct transmission from the multiple non-human (animal and environmental) sources included in our sample accounts for less than 1% of hospital disease, with the vast majority of clinical cases originating from other humans.


title: σ28-dependent small RNAs regulate timing of flagella biosynthesis

abstract: Flagella are important for bacterial motility as well as for pathogenesis. Synthesis of these structures is energy intensive and, while extensive transcriptional regulation has been described, little is known about the posttranscriptional regulation. Small RNAs (sRNAs) are widespread posttranscriptional regulators, most base pairing with mRNAs to affect their stability and/or translation. Here we describe four UTR-derived sRNAs (UhpU, MotR, FliX and FlgO) whose expression is controlled by the flagella sigma factor {sigma}28 (fliA) in Escherichia coli. Interestingly, MotR and FliX have opposing effects on flagellin protein levels, flagella number and cell motility, with MotR accelerating flagella synthesis and FliX decelerating flagella synthesis. Unlike most sRNAs, MotR and FliX base pair within the coding sequences of target mRNAs. They also uniquely act on ribosomal protein mRNAs thus coordinating flagella synthesis with ribosome production. The study shows how sRNA-mediated regulation can overlay a complex network enabling nuanced control of flagella synthesis.


title: A Cell Culture Model of BK Polyomavirus Persistence, Genome Recombination, and Reactivation

abstract:
BK Polyomavirus (BKPyV) is a small non-enveloped DNA virus that establishes a ubiquitous, asymptomatic, and lifelong persistent infection in at least 80% of the worlds population. In some immunosuppressed transplant recipients, BKPyV reactivation causes polyomavirus-associated nephropathy and hemorrhagic cystitis. We report a novel in vitro model of BKPyV persistence and reactivation using a BKPyV natural host cell line. In this system, viral genome loads remain constant for various times post-establishment of persistent infection, during which BKPyV undergoes extensive random genome recombination. Certain recombination events result in viral DNA amplification and protein expression, resulting in production of viruses with enhanced replication ability.

ImportanceBK polyomavirus (BKPyV) generally establishes a persistent subclinical infection in healthy individuals but can cause severe disease in transplant recipients. While an in vitro model to study acute replication exists, no practical model with which to study BKPyV persistence is currently available. We established a BKPyV persistence model in cell culture. Our model reveals that the virus can persist for varying periods of time before random recombination of the viral genome leads to enhanced replication.


title: First observation of Aedes albopictus in the Tshuapa province (Boende) of the Democratic Republic of the Congo

abstract: In May-June 2021, we detected Aedes albopictus adults near the central hospital in Boende, the capital city of the Tshuapa province of the Democratic Republic of the Congo (DRC). We identified the mosquitoes using morphological and molecular techniques (COI barcoding). This is the first report of this species in the DRC outside of Kinshasa and Kongo Central. Given the central location of Boende in the Congo Basin, our finding suggests that the vector might also have spread to other cities which are located upstream of the Congo River and its major tributaries. Because Aedes albopictus is an important vector for human arbovirus transmission, we highlight the need to investigate its distribution range and to update disease risk maps in Central Africa.


title: Evolution-Inspired Engineering of Anthracycline Methyltransferases

abstract: Streptomyces soil bacteria produce hundreds of anthracycline anticancer agents with a relatively conserved set of genes. This diversity depends on the rapid evolution of biosynthetic enzymes to acquire novel functionalities. Previous work has identified S-adenosyl-L-methionine-dependent methyltransferase-like proteins that catalyze either 4-O-methylation, 10-decarboxylation or 10-hydroxylation, with additional differences in substrate specificities. Here we focused on four protein regions to generate chimeric enzymes using sequences from four distinct subfamilies to elucidate their influence in catalysis. Combined with structural studies we managed to depict factors that influence gain-of-hydroxylation, loss-of-methylation and substrate selection. The engineering expanded the catalytic repertoire to include novel 9,10-elimination activity, and 4-O-methylation and 10-decarboxylation of unnatural substrates. The work provides an instructive account on how the rise of diversity of microbial natural products may occur through subtle changes in biosynthetic enzymes.

category: biochemistry


title: Evidence for a trap-and-flip mechanism in a proton-dependent lipid transporter

abstract: Transport of lipids across membranes is fundamental for diverse biological pathways in cells. Multiple ion-coupled transporters participate in lipid translocation, but their mechanisms remain largely unknown. Major facilitator superfamily (MFS) lipid transporters play central roles in cell wall synthesis, brain development and function, lipids recycling, and cell signaling. Recent structures of MFS lipid transporters revealed overlapping architectural features pointing towards a common mechanism. Here we used cysteine disulfide trapping, molecular dynamics simulations, mutagenesis analysis, and transport assays in vitro and in vivo, to investigate the mechanism of LtaA, a proton-dependent MFS lipid transporter essential for lipoteichoic acids synthesis in the pathogen Staphylococcus aureus. We reveal that LtaA displays asymmetric lateral openings with distinct functional relevance and that cycling through outward- and inward-facing conformations is essential for transport activity. We demonstrate that while the entire amphipathic central cavity of LtaA contributes to lipid binding, its hydrophilic pocket dictates substrate specificity. We propose that LtaA catalyzes lipid translocation by a  trap-and-flip mechanism that might be shared among MFS lipid transporters.


title: Knockout of murine Lyplal1 confers sex-specific protection against diet-induced obesity

abstract: Human genome-wide association studies found SNPs near LYPLAL1 that have sex-specific effects on fat distribution and metabolic traits. To determine whether altering LYPLAL1 affects obesity and metabolic disease we created and characterized a mouse knockout of Lyplal1. Here we show that CRISPR-Cas9 whole-body Lyplal1 knockout (KO) mice fed a high fat, high sucrose (HFHS) diet showed sex-specific differences in weight gain and fat accumulation. Female, not male, KO mice weighed less than WT mice, had reduced body fat percentage, white fat mass, and adipocyte diameter not accounted for by changes in metabolic rate. Female, but not male, KO mice had increased serum triglycerides, decreased aspartate, and alanine aminotransferase. Lyplal1 KO mice of both sexes have reduced liver triglycerides and steatosis. These diet-specific effects resemble the effects of SNPs near LYPLAL1 in humans, suggesting that LYPLAL1 has an evolutionary conserved sex-specific effect on adiposity. This murine model can be used to study this novel gene-by-sex-by-diet interaction to elucidate the metabolic effects of LYPLAL1 on human obesity.


title: Mechanism of Tripeptide Trimming by γ-Secretase

abstract:
The membrane-embedded {gamma}-secretase complex processively cleaves within the transmembrane domain of amyloid precursor protein (APP) to produce 37-to-43-residue amyloid {beta}-peptides (A{beta}) of Alzheimers disease (AD). Despite its importance in pathogenesis, the mechanism of processive proteolysis by {gamma}-secretase remains poorly understood. Here, mass spectrometry and western blotting were used to quantify the efficiency of the first tripeptide trimming step (A{beta}49[->]A{beta}46) of wildtype (WT) and familial AD (FAD) mutant APP substrate. In comparison to WT APP, the efficiency of this first trimming step was similar for the I45F, A42T and V46F APP FAD mutants, but substantially diminished for the I45T and T48P mutants. In parallel with biochemical experiments, all-atom simulations using a novel Peptide Gaussian accelerated molecular dynamics (Pep-GaMD) method were applied to investigate tripeptide trimming of A{beta}49 by {gamma}-secretase. The starting structure was active {gamma}-secretase bound to A{beta}49 and APP intracellular domain (AICD), as generated from our previous study that captured activation of {gamma}-secretase for the initial endoproteolytic cleavage of APP (Bhattarai et al., ACS Cent Sci, 2020, 6:969-983). Pep-GaMD simulations captured remarkable structural rearrangements of both the enzyme and substrate, in which hydrogen-bonded catalytic aspartates and water became poised for tripeptide trimming of A{beta}49 to A{beta}46. These structural changes required a positively charged N-terminus of endoproteolytic coproduct AICD, which could dissociate during conformational rearrangements of the protease and A{beta}49. The simulation findings were highly consistent with biochemical experimental data. Taken together, our complementary biochemical experiments and Pep-GaMD simulations have enabled elucidation of the mechanism of tripeptide trimming by {gamma}-secretase.

Significance statementProduction of amyloid {beta}-peptide (A{beta}) of Alzheimers disease (AD) from its precursor protein requires a series of proteolytic events carried out by the membrane-embedded {gamma}-secretase complex. Mutations in the substrate and enzyme that produce A{beta} cause hereditary AD and these mutations affect tripeptide trimming of initially formed long A{beta} peptides by {gamma}-secretase. Little is known about the structural mechanism of this trimming process. Here, we have developed a molecular dynamic model for the first step of trimming (A{beta}49 to A{beta}46) by {gamma}-secretase. Conformational changes in the enzyme-substrate complex to set up this trimming step require the presence of N-terminally charged endoproteolytic cleavage co-product. Computational effects of AD-causing mutations in A{beta}49 on the trimming process were validated through biochemical experiments.


title: Mechanism of Tethered Agonist-Mediated Signaling by Polycystin-1

abstract:
Polycystin-1 (PC1) is an important unusual G-protein-coupled receptor (GPCR) with 11 transmembrane (TM) domains and its mutations account for 85% of cases of autosomal dominant polycystic kidney disease (ADPKD). PC1 shares multiple characteristics with Adhesion GPCRs. These include a GPCR proteolysis site that autocatalytically divides these proteins into extracellular, N-terminal and membrane-embedded, C-terminal (CTF) fragments, and a tethered agonist (TA) within the N-terminal stalk of the CTF that is suggested to activate signaling. However, the mechanism by which a TA can activate PC1 is not known. Here, we have combined functional cellular signaling experiments of PC1 CTF expression constructs encoding wild type, stalkless and three different ADPKD stalk variants with all-atom Gaussian accelerated molecular dynamics (GaMD) simulations to investigate TA-mediated signaling activation. Correlations of residue motions and free-energy profiles calculated from the GaMD simulations correlated with the differential signaling abilities of wild type and stalk variants of PC1 CTF. They suggested an allosteric mechanism involving residue interactions connecting the stalk, Tetragonal Opening for Polycystins (TOP) domain and putative pore loop in TA-mediated activation of PC1 CTF. Key interacting residues such as N3074-S3585 and R3848-E4078 predicted from the GaMD simulations were validated by new mutagenesis experiments. Together, these complementary analyses have provided novel insights into a TA-mediated activation mechanism of PC1 CTF signaling, which will be important for future rational drug design targeting PC1.

Significance StatementMutations of polycystin-1 (PC1) are the major cause (85% of cases) of autosomal dominant polycystic kidney disease (ADPKD), which is the fourth leading cause of kidney failure. PC1 is thought to function as an atypical GPCR, yet the mechanism by which PC1 regulates G-protein signaling remains poorly understood. A significant portion of ADPKD mutations of PC1 encode a protein with defects in maturation or reduced function that may be amenable to functional rescue. In this work, we have combined complementary biochemical and cellular assay experiments and accelerated molecular simulations, which revealed a novel allosteric transduction pathway in activation of the PC1 CTF. Our findings shall facilitate future rational drug design efforts targeting the PC1 signaling function.


title: Physiological Magnesium Concentrations Increase Fidelity of Diverse Reverse Transcriptases from HIV-1, HIV-2, and Foamy Virus, but not MuLV or AMV

abstract: Reverse transcriptases (RTs) are typically assayed in vitro using optimized Mg2+ concentrations ([~]5-10 mM) several-fold higher than physiological cellular free Mg2+ ([~]0.5 mM). Analysis of fidelity using lacZ-based -complementation assays showed that tested HIV RTs, including HIV-1 from subtype B (HXB2-derived), HIV-2, subtype A/E, and several drug-resistant HXB2 derivatives all showed significantly higher fidelity using physiological Mg2+. This also occurred with prototype foamy virus (PFV) RT. In contrast, Moloney murine leukemia virus (MuLV) and avian myeloblastosis virus (AMV) RTs demonstrated equivalent fidelity in both low and high Mg2+. In 0.5 mM Mg2+, all RTs demonstrated {approx} equal fidelity, except for PFV RT which showed higher fidelity. A Next Generation Sequencing (NGS) approach that used barcoding to accurately determine mutation rates and profiles was used to examine the types of mutations made by HIV-1 (subtype B, wild type) in low (0.5 mM) and high (6 mM) Mg2+ with DNA or RNA that coded for lacZ. Unlike the -complementation assay, which is dependent on LacZ activity, the NGS assay scores mutations at all positions and of every type. Consistent with -complementation assays, a [~]4-fold increase in mutations was observed in high Mg2+. These findings help explain why HIV RT displays lower fidelity in vitro (with high Mg2+ concentrations) than other RTs (e.g., MuLV and AMV), yet cellular fidelity for these viruses is comparable. Establishing in vitro conditions that accurately represent RTs activity in cells is pivotal to determining the contribution of RT and other factors to the mutation profile observed with HIV.


title: Structure of PINK1 reveals autophosphorylation dimer and provides insights into binding to the TOM complex

abstract: Mutations in PINK1 causes autosomal-recessive Parkinsons disease. Mitochondrial damage results in PINK1 import arrest on the Translocase of the Outer Mitochondrial Membrane (TOM) complex, resulting in the activation of its ubiquitin kinase activity by autophosphorylation and initiation of Parkin-dependent mitochondrial clearance. Herein we report crystal structures of the entire cytosolic domain of insect PINK1. Our structures reveal a dimeric autophosphorylation complex targeting phosphorylation at the invariant Ser205 (human Ser228). The dimer interface requires insert 2, which is unique to PINK1. The structures also reveal how an N-terminal helix binds to the C-terminal extension and provide insights into stabilization of PINK1 on the core TOM complex.


title: Microalgal protein AstaP is a potent carotenoid solubilizer and delivery module with a broad carotenoid binding repertoire

abstract: Carotenoids are lipophilic substances with many biological functions, from coloration to photoprotection. Being potent antioxidants, carotenoids have multiple biomedical applications, including the treatment of neurodegenerative disorders and retina degeneration. Nevertheless, the delivery of carotenoids is substantially limited by their poor solubility in the aqueous phase. Natural water-soluble carotenoproteins can facilitate this task, necessitating studies on their ability to uptake and deliver carotenoids. One such promising carotenoprotein, AstaP (Astaxanthin-binding protein), was recently identified in eukaryotic microalgae, but its structure and functional properties remained largely uncharacterized. By using a correctly folded recombinant protein, here we show that AstaP is an efficient carotenoid solubilizer that can stably bind not only astaxanthin but also zeaxanthin, canthaxanthin, and, to a lesser extent, {beta}-carotene, i.e. carotenoids especially valuable to human health. AstaP accepts carotenoids provided as acetone solutions or embedded in membranes, forming carotenoid-protein complexes with an apparent stoichiometry of 1:1. We successfully produced AstaP holoproteins in specific carotenoid-producing strains of Escherichia coli, proving it is amenable to cost-efficient biotechnology processes. Regardless of the carotenoid type, AstaP remains monomeric in both apo- and holoforms, while its rather minimalistic mass ([~]20 kDa) makes it an especially attractive antioxidant delivery module. In vitro, AstaP transfers different carotenoids to the liposomes and to unrelated proteins from cyanobacteria, which can modulate their photoactivity and/or oligomerization. These findings expand the toolkit of the characterized carotenoid-binding proteins and outline the perspective of the use of AstaP as a unique monomeric antioxidant nanocarrier with an extensive carotenoid-binding repertoire.


title: Gadolinium-free Magnetic Resonance Imaging of the Liver using an Oatp1-targeted Manganese(III) Porphyrin

abstract: Controversy surrounding gadolinium-based contrast agents (GBCAs) have rendered their continued utility highly contentious, but the liver-specific GBCA Gd(III) ethoxybenzyl-diethylene triamine pentaacetic acid (Gd(III)-EOB-DTPA) remains in use because it provides unique diagnostic information that could not be obtained by any other means. To address the need for an alternative liver-specific MRI contrast agent, we synthesized Mn(III) 20-(4-ethoxyphenyl) porphyrin-5,10,15-tricarboxylate (Mn(III)TriCP-PhOEt), which exhibited significantly higher r1 relaxivity than Gd(III)-EOB-DTPA, and targeted organic anion-transporting polypeptide 1 (Oatp1) channels as a biomarker of hepatocyte viability. Mn(III)TriCP-PhoEt increased the r1 relaxation rate of cells expressing rodent Oatp1a1 and human Oatp1b3, relative to control cells not expressing these liver channels. In mice, Mn(III)TriCP-PhoEt resulted in significant and specific increases in liver signal intensity on T1-weighted images, and significant decreases in liver T1 time relative to precontrast measurements. Our findings suggest that Mn(III)TriCP-PhOEt operates as a specific and sensitive MR contrast agent for in vivo liver imaging.


title: Ribosome elongation kinetics of consecutively charged residues are coupled to electrostatic force

abstract:
The speed of protein synthesis can dramatically change when consecutively charged residues are incorporated into an elongating nascent protein by the ribosome. The molecular origins of this class of allosteric coupling remain unknown. We demonstrate, using multi-scale simulations, that positively charged residues generate large forces that pull the P-site amino acid away from the A-site amino acid. Negatively charged residues generate forces of similar magnitude but opposite direction. And that these conformational changes, respectively, raise and lower the transition state barrier height to peptide bond formation, explaining how charged residues mechanochemically alter translation speed. This mechanochemical mechanism is consistent with in vivo ribosome profiling data exhibiting a proportionality between translation speed and the number of charged residues, experimental data characterizing nascent chain conformations, and a previously published cryo-EM structure of a ribosome-nascent chain complex containing consecutive lysines. These results expand the role of mechanochemistry in translation, and provide a framework for interpreting experimental results on translation speed.

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title: A single layer artificial neural network type architecture with molecular engineered bacteria for complex conventional and reversible computing

abstract: Here, we adapted the basic concept of artificial neural networks (ANN) and experimentally demonstrate a broadly applicable single layer ANN type architecture with molecular engineered bacteria to perform complex irreversible computing like multiplexing, de-multiplexing, encoding, decoding, majority functions, and reversible computing like Feynman and Fredkin gates. The encoder and majority functions and reversible computing were experimentally implemented within living cells for the first time. We created molecular-devices, which worked as artificial neuro-synapses in bacteria, where input chemical signals were linearly combined and processed through a non-linear activation function to produce fluorescent protein outputs. To create such molecular devices, we established a set of rules by corelating truth tables, mathematical equations of ANN, and molecular-device design, which unlike molecular computing, does not require circuit diagram and the equation directly correlates the design of the molecular-device. To our knowledge this is the first adaptation of ANN type architecture with engineered cells. This work may have significance in new platform for biomolecular computing, reversible computing and in transforming living cells as ANN-enabled hardware.

category: synthetic biology


title: Limited variation between SARS-CoV-2-infected individuals in domain specificity and relative potency of the antibody response against the spike glycoprotein

abstract:
The spike protein of SARS-CoV-2 is arranged as a trimer on the virus surface, composed of three S1 and three S2 subunits. Infected and vaccinated individuals generate antibodies against spike, which can neutralize the virus. Most antibodies target the receptor-binding domain (RBD) and N-terminal domain (NTD) of S1; however, antibodies against other regions of spike have also been isolated. The variation between infected individuals in domain specificity of the antibodies and in their relative neutralization efficacy is still poorly characterized. To this end, we tested serum and plasma samples from 85 COVID-19 convalescent subjects using 7 immunoassays that employ different domains, subunits and oligomeric forms of spike to capture the antibodies. Samples were also tested for their neutralization of pseudovirus containing SARS-CoV-2 spike and of replication-competent SARS-CoV-2. We observed strong correlations between the levels of NTD- and RBD-specific antibodies, with a fixed ratio of each type to all anti-spike antibodies. The relative potency of the response (defined as the measured neutralization efficacy relative to the total level of spike-targeting antibodies) also exhibited limited variation between subjects, and was not associated with the overall amount of anti-spike antibodies produced. Accordingly, the ability of immunoassays that use RBD, NTD and different forms of S1 or S1/S2 as capture antigens to estimate the neutralizing efficacy of convalescent samples was largely similar. These studies suggest that host-to-host variation in the polyclonal response elicited against SARS-CoV-2 spike is primarily limited to the quantity of antibodies generated rather than their domain specificity or relative neutralization potency.

IMPORTANCEInfection by SARS-CoV-2 elicits antibodies against various domains of the spike protein, including the RBD, NTD and S2. Different infected individuals generate vastly different amounts of anti-spike antibodies. By contrast, as we show here, there is a remarkable similarity in the properties of the antibodies produced. Different individuals generate the same proportions of antibodies against each domain of the spike protein. Furthermore, the relationship between the amount of anti-spike antibodies produced and their neutralization efficacy of SARS-CoV-2 is highly conserved. Therefore, the observed variation in the neutralizing activity of the antibody response in COVID-19 convalescent subjects is caused by differences in the amounts of antibodies rather than their recognition properties or relative antiviral activity. These findings suggest that COVID-19 vaccine strategies that focus on enhancing the overall level of the antibodies will likely elicit a more uniformly efficacious protective response.


title: The Low Complexity Domain of the FUS RNA Binding Protein Self-assembles via the Mutually Exclusive Use of Two Distinct Cross-β Cores

abstract:
The low complexity (LC) domain of the fused in sarcoma (FUS) RNA binding protein self-associates in a manner causing phase separation from an aqueous environment. Incubation of the FUS LC domain under physiologically normal conditions of salt and pH leads to rapid formation of liquid-like droplets that mature into a gel-like state. Both examples of phase separation have enabled reductionist biochemical assays allowing discovery of an N-terminal region of 56 residues that assembles into a labile, cross-{beta} structure. Here we provide evidence of a non-overlapping, C-terminal region of the FUS LC domain that also forms specific cross-{beta} interactions. We propose that biologic function of the FUS LC domain may operate via the mutually exclusive use of these N- and C-terminal cross-{beta} cores. Neurodegenerative disease-causing mutations in the FUS LC domain are shown to imbalance the two cross-{beta} cores, offering an unanticipated concept of LC domain function and dysfunction.

Significance StatementSingle amino acid changes causative of neurologic disease often map to the cross-{beta} forming regions of low complexity (LC) domains. All such mutations studied to date lead to enhanced avidity of cross-{beta} interactions. The LC domain of the fused in sarcoma (FUS) RNA binding protein contains three different regions that are capable of forming labile cross-{beta} interactions. Here we describe the perplexing effect of amyotrophic lateral sclerosis (ALS)-causing mutations localized to the LC domain of FUS to substantially weaken its ability to form one of its three cross-{beta} interactions. An understanding of how these mutations abet uncontrolled polymerization of the FUS LC domain may represent an important clue as to how LC domains achieve their proper biological function.


title: Multi-layered chromatin proteomics identifies cell vulnerabilities in DNA repair

abstract:
The DNA damage response (DDR) is essential to maintain genome stability, and its deregulation predisposes to carcinogenesis while encompassing attractive targets for cancer therapy. Chromatin governs the DDR via interplay among all chromatin layers including DNA, histones post-translational modifications (hPTMs), and chromatin-associated proteins. Here we employ multi-layered proteomics to characterize chromatin-mediated interactions of repair proteins, signatures of hPTMs, and the DNA-bound proteome during DNA double-strand break repair at high temporal resolution. We functionally attribute novel chromatin-associated proteins to repair by non-homologous end-joining or homologous recombination (HR) revealing histone reader ATAD2, microtubule organizer TPX2 and histone methyltransferase G9A as regulators of HR and PARP inhibitor sensitivity. Furthermore, we dynamically profile numerous hPTMs at {gamma}H2AX-mononucleosomes during the DDR. Integration of these complementary data implicated G9A-mediated monomethylation of H3K56 in HR. Collectively, we provide a dynamic chromatin-centered view of DDR, while representing a valuable resource for the use of PARP inhibitors in cancer.

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title: Conjugates for use in peptide therapeutics: a systematic review and meta-analysis

abstract: While peptides can be excellent therapeutics for several conditions, their limited in vivo half-lives have been a major bottleneck in the development of therapeutic peptides. Conjugating the peptide to an inert chemical moiety is a strategy that has repeatedly proven to be successful in extending the half-life of some therapeutics. This systematic review and meta-analysis was conducted to examine the available literature and assess it in an unbiased manner to determine which conjugates, both biological and synthetic, provide the greatest increase in therapeutic peptide half-life. Systematic searches run on PubMed, Scopus and SciFinder databases resulted in 845 studies pertaining to the topic, 16 of these were included in this review after assessment against pre-specified inclusion criteria registered on PROSPERO (#CRD42020222579). The most common reasons for exclusion were non-IV administration and large peptide size. Of the 16 studies that were included, a diverse suite of conjugates that increased half-life from 0.1 h to 33.57 h was identified. Amongst these peptides, the largest increase in half-life was seen when conjugated with glycosaminoglycans. A meta-analysis of studies that contained fatty acid conjugates indicated that acylation contributed to a statistically significant extension of half-life. Additionally, another meta-analysis followed by a sensitivity analysis suggested that conjugation with specifically engineered recombinant peptides might contribute to a more efficient extension of peptide half-life as compared to PEGylation. Moreover, we confirmed that while polyethylene glycol is a good synthetic conjugate, its chain length likely has an impact on its effectiveness in extending half-life. Furthermore, we found that most animal studies do not include as much detail when reporting findings as compared to human studies. Inclusion of additional experimental detail on aspects such as independent assessment and randomization may be an easily accomplished strategy to drive more conjugated peptides towards clinical studies.

category: pharmacology and toxicology


title: Microarray Analysis of Potential Biomarkers for brachial plexus avulsion caused neuropathic pain

abstract: Nerve injury-induced neuropathic pain remains a challenging clinical problem due to a lack of satisfactory treatment. Pain after BPA (Brachial Plexus Avulsion) is resistant to most traditional pain relief treatments due to the lack of understanding of the cellular or molecular mechanism of pain development. The present study aimed to investigate the expression of mRNA in the brachial plexus avulsion neuropathic pain model and analyze biological functions. Sprague-Dawley rats were treated with complete brachial plexus avulsion. An animal behavior test was carried out to distinguish the pain group from the control group. In this study, a microarray mRNA assay and reverse transcriptase quantitative polymerase chain reaction (RT-PCR) was conducted. The whole blood was collected from two groups for Microarray mRNA analysis. The predicted mRNA targets were studied by gene ontology analysis and pathway analysis. The PIK3CB, HRAS, and JUN genes were verified by RT-PCR. In total, differentially expressed genes(DEGs) were identified between individuals with or without neuropathic pain (case and control), and A biological processes were enriched. We identified 3 targeted mRNAs, including PIK3CB, HRAS, and JUN, which may be potential biomarkers for BPA-caused NP. The results showed that PIK3CB, HRAS, and JUN gene expression was increased in the control group but decreased in the neuropathic pain group. The PIK3CB gene was part of the Neurotrophin signaling pathway. The function of the HRAS gene was synergetic in the aspect of axon guidance and the Neurotrophin signaling pathway. The JUN gene participates in axon regeneration. These results suggest that PIK3CB, HRAS, and JUN genes might become potential biomarkers for the prediction of and new targets for the prevention and treatment of neuropathic pain after BPA. These findings indicate that mRNA expression changes in the blood may play an important role in the development of NP after BPA, which is of theoretical and clinical importance for future research and clinical-treatment strategies.


title: Vaccination with B.1.1.7, B.1.351 and P.1 variants protects mice from challenge with wild type SARS-CoV-2

abstract:
Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been highly efficient in protecting against coronavirus disease 2019 (COVID-19). However, the emergence of viral variants that are more transmissible and, in some cases, escape from neutralizing antibody responses has raised concerns. Here, we evaluated recombinant protein spike antigens derived from wild type SARS-CoV-2 and from variants B.1.1.7, B.1.351 and P.1 for their immunogenicity and protective effect in vivo against challenge with wild type SARS-CoV-2 in the mouse model. All proteins induced high neutralizing antibodies against the respective viruses but also induced high cross-neutralizing antibody responses. The decline in neutralizing titers between variants was moderate, with B.1.1.7 vaccinated animals having a maximum fold reduction of 4.8 against B.1.351 virus. P.1 induced the most cross-reactive antibody responses but was also the least immunogenic in terms of homologous neutralization titers. However, all antigens protected from challenge with wild type SARS-CoV-2 in a mouse model.

Author SummaryThe emergence of variants of SARS-CoV-2 has led to an urgency to study whether vaccines will lead to cross-protection against these variants. Here, we demonstrate that vaccination with spike proteins of various variants leads to cross-neutralizing responses, as well as protection in a mouse model against wild type SARS-CoV-2.


title: Th1/17 Cells Infiltrate Murine Cytomegalovirus-Infected Renal Allografts via Virus-Induced CCL20 and Promote Th1 Cells through IL-17A.

abstract: Cytomegalovirus (CMV) infection is associated with renal allograft failure by unknown mechanisms. In a murine renal transplant model, murine CMV (MCMV) induces intragraft infiltration of Th17 cells co-expressing Th1 cytokines, IFN-{gamma} and TNF-, but only a minority of intragraft Th17 cells are specific for MCMV antigens. Instead, MCMV promotes viral antigen-independent Th17 cell recruitment via CCL20-CCR6 and CXCL10-CXCR3 interactions. Th17 cells correlate directly with Th1 cell frequencies and inversely with Tregs in MCMV infected grafts. Pharmacologic inhibition of IL-17A reduces intragraft Th17 cells and neutrophils, increases Tregs, and reduces total but not MCMV-specific Th1 cells. Among a clinical renal transplant cohort with acute rejection, patients with CMV DNAemia had significantly higher serum IL-17A quantities compared to those without CMV DNAemia. Together, these findings indicate that CMV infection upregulates Th17 cell activity during acute rejection and suggests that inhibition of IL-17A may ameliorate CMV-associated allograft injury without impairing antiviral Th1 cells.


title: Entamoeba histolytica develops resistance to complement deposition and lysis after acquisition of human complement regulatory proteins through trogocytosis

abstract:
Entamoeba histolytica is the cause of amoebiasis. The trophozoite (amoeba) form of this parasite is capable of invading the intestine, and can disseminate through the bloodstream to other organs. The mechanisms that allow amoebae to evade complement deposition during dissemination have not been well characterized. We previously discovered a novel complement-evasion mechanism employed by E. histolytica. E. histolytica ingests small bites of living human cells in a process termed trogocytosis. We demonstrated that amoebae were protected from lysis by human serum following trogocytosis of human cells, and that amoebae acquired and displayed human membrane proteins from the cells they ingested. Here, we aimed to define how amoebae are protected from complement lysis after performing trogocytosis. We found that amoebae were protected from complement lysis after ingestion of both human Jurkat T cells and red blood cells, and that the level of protection correlated with the amount of material ingested. Trogocytosis of human cells led to a reduction in deposition of C3b on the surface of amoebae. We asked whether display of human complement regulators is involved in amoebic protection, and found that CD59 was displayed by amoebae after trogocytosis. Deletion of a single complement regulatory protein, CD59 or CD46, from Jurkat cells was not sufficient to alter amoebic protection from lysis, suggesting that multiple, redundant complement regulators mediate amoebic protection. However, exogeneous expression of CD46 or CD55 in amoebae was sufficient to confer protection from lysis. These studies shed light on a novel strategy for immune evasion by a pathogen.

IMPORTANCEEntamoeba histolytica is the cause of amoebiasis, a diarrheal disease of global importance. While infection is often asymptomatic, the trophozoite (amoeba) form of this parasite is capable of invading and ulcerating the intestine, and can disseminate through the bloodstream to other organs. Understanding how E. histolytica evades the complement system during dissemination is of great interest. Here we demonstrate for the first time that amoebae that have performed trogocytosis (nibbling of human cells) resist deposition of the complement protein C3b. Amoebae that have performed trogocytosis display the complement regulatory protein CD59. Overall, our studies suggest that acquisition and display of multiple, redundant complement regulators is involved in amoebic protection from complement lysis. These findings shed light on a novel strategy for immune evasion by a pathogen. Since other parasites use trogocytosis for cell killing, our findings may apply to the pathogenesis of other infections.


title: Nuclear Lamina binds the EBV genome during latency and regulates viral gene expression

abstract:
The Epstein Barr virus (EBV) infects almost 95% of the population worldwide. While typically asymptomatic, EBV latent infection is associated with several malignancies of epithelial and lymphoid origin in immunocompromised individuals. In latently infected cells, the EBV genome persists as a chromatinized episome that expresses a limited set of viral genes in different patterns, referred to as latency types, which coincide with varying stages of infection and various malignancies. We have previously demonstrated that latency types correlate with differences in the composition and structure of the EBV episome. Several cellular factors, including the nuclear lamina, regulate chromatin composition and architecture. While the interaction of the viral genome with the nuclear lamina has been studied in the context of EBV lytic reactivation, the role of the nuclear lamina in controlling EBV latency has not been investigated. Here, we report that the nuclear lamina is an essential epigenetic regulator of the EBV episome. We observed that in B cells, EBV infection affects the composition of the nuclear lamina by inducing the expression of lamin A/C, but only in EBV+ cells expressing the Type III latency program. Using ChIP-Seq, we determined that lamin B1 and lamin A/C bind the EBV genome, and their binding correlates with deposition of the histone repressive mark H3K9me2. By RNA-Seq, we observed that knock-out of lamin A/C in B cells alters EBV gene expression. Our data indicate that the interaction between lamins and the EBV episome contributes to the epigenetic control of viral gene expression during latency, suggesting a restrictive function of the nuclear lamina as part of the host response against viral DNA entry into the nucleus.

AUTHOR SUMMARYEpstein-Barr virus (EBV) is a common herpesvirus that establishes a lifelong latent infection in a small fraction of B cells of the infected individuals. In most cases, EBV infection is asymptomatic; however, especially in the context of immune suppression, EBV latent infection is associated with several malignancies. In EBV+ cancer cells, latent viral gene expression plays an essential role in sustaining the cancer phenotype. We and others have established that epigenetic modifications of the viral genome are critical to regulating EBV gene expression during latency. Understanding how the EBV genome is epigenetically regulated during latent infection may help identify new specific therapeutic targets for treating EBV+ malignancies. The nuclear lamina is involved in regulating the composition and structure of the cellular chromatin. In the present study, we determined that the nuclear lamina binds the EBV genome during latency, influencing viral gene expression. Depleting one component of the nuclear lamina, lamin A/C, increased the expression of latent EBV genes associated with cellular proliferation, indicating that the binding of the nuclear lamina with the viral genome is essential to control viral gene expression in infected cells. Our data show for the first time that the nuclear lamina may be involved in the cellular response against EBV infection by restricting viral gene expression.


title: The Nuclear Lamina binds the EBV genome during latency and regulates viral gene expression


title: A Herpes Simplex Virus Type-1-derived influenza vaccine induces balanced adaptive immune responses and protects mice from lethal influenza virus challenge.

abstract: Influenza virus is a major respiratory viral pathogen responsible for the deaths of hundreds of thousands worldwide each year. Current vaccines provide protection primarily by inducing strain-specific antibody responses with the requirement of a match between vaccine strains and circulating strains. It has been suggested that anti-influenza T-cell responses, in addition to antibody responses may provide the broadest protection against different flu strains. Therefore, to address this urgent need, it is desirable to develop a vaccine candidate with an ability to induce balanced adaptive immunity including cell mediated immune responses. A live viral vector technology should exhibit safety, immunogenicity, effectiveness in the presence of pre-existing immunity, and the ability to induce mucosal immune responses. Here, we used VC2, an established Herpes Simplex Virus type 1 vaccine vector, to express the influenza HA protein. We show that this virus is capable of generating potent and specific anti-influenza humoral and cell-mediated immune responses. We further show that a single vaccination with the VC2-derived influenza vaccine protects mice from lethal challenge with influenza virus. Our data support the continued development of VC2-derived influenza vaccines for protection of human populations from both seasonal and pandemic strains of influenza. Finally, our results support the potential of VC2-derived vaccines as a platform for the rapid development of vaccines against emerging and established pathogens, particularly respiratory pathogens.