title: Mutation-induced Changes in the Receptor-binding Interface of the SARS-CoV-2 Delta Variant B.1.617.2 and Implications for Immune Evasion

abstract: While the vaccination efforts against SARS-CoV-2 infections are ongoing worldwide, new genetic variants of the virus are emerging and spreading. Following the initial surges of the Alpha (B.1.1.7) and the Beta (B.1.351) variants, a more infectious Delta variant (B.1.617.2) is now surging, further deepening the health crises caused by the pandemic. The sharp rise in cases attributed to the Delta variant has made it especially disturbing and is a variant of concern. Fortunately, current vaccines offer protection against known variants of concern, including the Delta variant. However, the Delta variant has exhibited some ability to dodge the immune system as it is found that neutralizing antibodies from prior infections or vaccines are less receptive to binding with the Delta spike protein. Here, we investigated the structural changes caused by the mutations in the Delta variants receptor-binding interface and explored the effects on binding with the ACE2 receptor as well as with neutralizing antibodies. We find that the receptor-binding {beta}-loop-{beta} motif adopts an altered but stable conformation causing separation in some of the antibody binding epitopes. Our study shows reduced binding of neutralizing antibodies and provides a possible mechanism for the immune evasion exhibited by the Delta variant.

category: biophysics


title: ARF4-mediated Retrograde Trafficking Drives Chemoresistance in Glioblastoma

abstract: Glioblastoma (GBM) is the most common type of adult malignant brain tumor, with a median survival of only 21 months. This is partly due to the high rate of resistance to conventional therapy, including temozolomide (TMZ), leading to recurrence rates close to 100%. It still remains unknown what drives the development of this resistance. To identify the unknown genes driving the development of this resistance, we performed a genome-wide CRISPR knockout screen comparing a DMSO-treated population with a TMZ-treated population over 14 days. We identified 4 previously unstudied genes - ARF4, PLAA, SPTLC1, and PIGK - that showed significant elevations in expression in recurrent tumors in patient datasets, along with significant survival benefits corresponding to low gene expression. Further investigation of ARF4, known to be involved in retrograde trafficking, allowed us to identify a mechanism of resistance that is mediated by increased retrograde transport of EGFR into the nucleus. Ultimately, our CRISPR-Cas9 screen has identified a promising therapeutic target, ARF4, which may drive GBMs high resistance to chemotherapy.

category: cancer biology


title: Phenotypic plasticity and genetic control in colorectal cancer evolution

abstract: Cancer evolution is driven by natural selection acting upon phenotypic trait variation. However, the extent to which phenotypic variation within a tumour is a consequence of intra-tumour genetic heterogeneity remains undetermined. Here we show that colorectal cancer cells frequently have highly plastic phenotypic traits in vivo in patient tumours. We measured the degree to which trait variation reflects genetic ancestry by quantifying the phylogenetic signal of gene expression across 297 samples with multi-region paired whole genome and transcriptome sequencing collected from 27 primary colorectal cancers. Within-tumour phylogenetic signal for genes and pathways was detected only infrequently, suggesting that the majority of intra-tumour variation in gene expression programmes was not strongly heritable. Expression quantitative trait loci analyses (eQTL) identified a small number of putative mechanisms of genetic control of gene expression due to the cis-acting coding, non-coding and structural genetic alteration, but most gene expression variation was not explained by our genetic analysis. Leveraging matched chromatin-accessibility sequencing data, enhancer mutations with cis regulatory effects on gene expression were associated with a change in chromatin accessibility, indicating that non-coding variation can have phenotypic consequence through modulation of the 3D architecture of the genome. This study maps the evolution of transcriptional variation during cancer evolution, highlighting that intra-tumour phenotypic plasticity is pervasive in colorectal malignancies, and may play key roles in further tumour evolution, from metastasis to therapy resistance.


title: Botrytis cinerea BcPTP1 is a late infection phase, cysteine rich protein cytotoxic effector

abstract: Botrytis cinerea is a broad-host-range necrotrophic phytopathogen responsible for serious crops diseases. To facilitate infection, B. cinerea secretes a large number of effectors that induce plant cell death. In screening secretome data of B. cinerea during infection stage, we identified a phytotoxic protein (BcPTP1) that can also induce immune resistance in plants. BcPTP1 is a small (90 aa), cysteine rich protein without any known domains. Transiently expression of BcPTP1 in leaves caused chlorosis that intensifies with time and eventually lead to cell death. Point mutations in eight of the 10 cysteine residues of BcPTP1 abolished the toxic effect, however residual toxic activity remained after heating the peptide, suggesting contribution of unknown epitopes to protein phytotoxic effect. The transcript level of the bcptp1 gene was low during the first 36 h after inoculation and increased sharply upon transition to the late infection stage, suggesting a role of BcPTP1 in lesion spreading. While statistically insignificant, deletion of the bcptp1 gene led to slightly smaller lesions on bean leaves. Further analyses indicated that BcPTP1 is internalized into plant cells after secreting into the apoplast and its phytotoxic effect is negatively regulated by the receptor-like kinases BAK1 and SOBIR1. Collectively, our findings show that BcPTP1 is a virulence factor that toxifies the host cells and facilitates lesion spreading during the late infection stage.

category: pathology


title: A mosaic adeno-associated virus vector as a versatile tool that exhibits high levels of transgene expression and neuron specificity in primate brain

abstract: Recent emphasis has been placed on gene transduction mediated through recombinant adeno-associated virus (AAV) vector to manipulate activity of neurons and their circuitry in the primate brain. In the present study, we created a novel AAV vector of which capsid was composed of capsid proteins derived from the serotypes 1 and 2 (AAV1 and AAV2). Following the injection into the frontal cortex of macaque monkeys, this mosaic vector, termed AAV2.1 vector, was found to exhibit the excellence in transgene expression (for the AAV1 vector) and neuron specificity (for the AAV2 vector) simultaneously. To explore its applicability to chemogenetic manipulation and in vivo calcium imaging, the AAV2.1 vector expressing excitatory DREADDs or GCaMP was injected into the striatum or the visual cortex of macaque monkeys, respectively. Our results have defined that such vectors secure intense and stable expression of the target proteins and yield conspicuous modulation and imaging of neuronal activity.

category: neuroscience


title: One mucosal administration of a live attenuated recombinant COVID-19 vaccine protects non-human primates from SARS-CoV-2

abstract: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 global pandemic. SARS-CoV-2 is an enveloped RNA virus that relies on its trimeric surface glycoprotein, spike, for entry into host cells. Here we describe the COVID-19 vaccine candidate MV-014-212, a live attenuated, recombinant human respiratory syncytial virus (RSV) expressing a chimeric SARS-CoV-2 spike as the only viral envelope protein. MV-014-212 was attenuated and immunogenic in African green monkeys (AGMs). One mucosal administration of MV-014-212 in AGMs protected against SARS-CoV-2 challenge, reducing by more than 200- fold the peak shedding of SARS-CoV-2 in the nose. MV-014-212 elicited mucosal immunity in the nose and neutralizing antibodies in serum that exhibited cross-neutralization against two virus variants of concern. Intranasally delivered, live attenuated vaccines such as MV-014-212 entail low-cost manufacturing suitable for global deployment. MV-014-212 is currently in phase 1 clinical trials as a single-dose intranasal COVID-19 vaccine.

category: immunology


title: SARS-CoV-2 Spike Pseudoviruses: A Useful tool to study virus entry and address emerging neutralization escape phenotypes

abstract: SARS-CoV-2 genetic variants are emerging around the globe. Unfortunately, several SARS-CoV-2 variants, especially, variants of concern (VOC) are less susceptible to neutralization by the convalescent and post-vaccination sera, raising concerns of increased disease transmissibility and severity. Recent data suggests the SARS-CoV-2 neutralizing anti-body levels as a good correlate of vaccine mediated protection. However, currently used BSL3 based virus micro-neutralization (MN) assays are more laborious, time consuming and expensive, underscoring the need for BSL2 based, cost effective neutralization assays against SARS-CoV-2 variants. In light of this unmet need, we have developed a BSL2 pseudovirus based neutralization assay (PBNA) in cells expressing Angiotensin Converting Enzyme-2 (ACE2) receptor for SARS-CoV-2. The assay is reproducible (R2=0.96), demonstrates a good dynamic range and high sensitivity. Our data suggests that the biological Anti-SARS-CoV-2 research reagents such as NIBSC 20/130 show lower neutralization against B.1.351 RSA and B1.1.7 UK VOC, whereas a commercially available monoclonal antibody MM43 retains activity against both these variants. SARS-CoV-2 Spike Pseudovirus based neutralization assays for VOC would be useful tools to measure the neutralization ability of candidate vaccines in both preclinical models and clinical trials and further help develop effective prophylactic countermeasures against emerging neutralization escape phenotypes.

category: microbiology


title: Susceptibilities of human ACE2 genetic variants in coronavirus infection

abstract: The COVID-19 pandemic, caused by SARS-CoV-2, has resulted in more than 1603 million cases worldwide and 3.4 million deaths (as of May 2021), with varying incidences and death rates among regions/ethnicities. Human genetic variation can affect disease progression and outcome, but little is known about genetic risk factors for SARS-CoV-2 infection. The coronaviruses SARS-CoV, SARS-CoV-2 and HCoV-NL63 all utilize the human protein angiotensin-converting enzyme 2 (ACE2) as the receptor to enter cells. We hypothesized that the genetic variability in ACE2 may contribute to the variable clinical outcomes of COVID-19. To test this hypothesis, we first conducted an in silico investigation of single-nucleotide polymorphisms (SNPs) in the coding region of ACE2 gene. We then applied an integrated approach of genetics, biochemistry and virology to explore the capacity of select ACE2 variants to bind coronavirus spike protein and mediate viral entry. We identified the ACE2 D355N variant that restricts the spike protein-ACE2 interaction and consequently limits infection both in vitro and in vivo. In conclusion, ACE2 polymorphisms could modulate susceptibility to SARS-CoV-2, which may lead to variable disease severity.


title: A nationwide genomic study of clinical Klebsiella pneumoniae in Norway 2001-2015: Introduction and spread of ESBL facilitated by CG15 and CG307

abstract:
SynopsisO_ST_ABSObjectiveC_ST_ABSWe have used the nationwide Norwegian surveillance program on resistant microbes in humans (NORM) to address longitudinal changes in the population structure K. pneumoniae isolates during 2001-15, encompassing the emergence and spread of ESBL-producing Enterobacterales (ESBL-E) in Norway.

Material and methodsAmong blood (n= 6124) and urinary tract (n=5496) surveillance isolates from 2001-15, we used Illumina technology to whole genome sequence 201 ESBL-producing isolates from blood (n=130) and urine (n=71), and 667 non-ESBL isolates from blood. Complete genomes for four isolates were resolved with Oxford Nanopore sequencing.

ResultsIn a highly diverse collection, Klebsiella variicola ssp. variicola caused a quarter of Klebsiella pneumoniae species complex bacteraemias. ESBL-production was limited to K. pneumoniae sensu stricto (98.5 %). A diverse ESBL population of 57 clonal groups (CGs) were dominated by multidrug resistant CG307 (17%), CG15 (12%), CG70 (6%), CG258 (5%) and CG45 (5%) carrying blaCTX-M-15. Yersiniabactin was significantly more common in ESBL-positive (37.8%) compared to non-ESBL K. pneumoniae sensu stricto isolates (12.7%), indicating convergence of virulence and resistance determinants

Moreover, we found a significant lower prevalence of yersinabactin (3.0 %, 37.8 % and 17.3 %), IncFIB (58.7 %, 87.9 % and 79.4 %) and IncFII plasmid replicons (40.5 %, 82.8 % and 54.2%) in K. variicola ssp. variicola compared to ESBL- and non-ESBL K. pneumoniae sensu stricto, respectively.

ConclusionThe increase in Norwegian KpSC ESBLs during 2010-15 was driven by blaCTX-M-15 carrying CG307 and CG15. K. variicola ssp. variicola was a frequent cause of invasive KpSC infection, but rarely carried ESBL.


title: Integrative multi-omics landscape of non-structural protein 3 of severe acute respiratory syndrome coronaviruses

abstract: The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is currently a global pandemic. Extensive investigations have been performed to study the clinical and cellular effects of SARS-CoV-2 infection. Mass spectrometry-based proteomics studies have revealed the cellular changes due to the infection and identified a plethora of interactors for all SARS-CoV-2 components, except for the longest non-structural protein 3 (NSP3). Here, we expressed the full-length NSP3 proteins of SARS-CoV and SARS-CoV-2 to investigate their unique and shared functions using multi-omics methods. We conducted interactome, phosphoproteome, ubiquitylome, transcriptome, and proteome analyses of NSP3-expressing cells. We found that NSP3 plays essential roles in cellular functions such as RNA metabolism and immune response such as NF-{kappa}B signal transduction. Interestingly, we showed that SARS-CoV-2 NSP3 has both endoplasmic reticulum and mitochondrial localizations. In addition, SARS-CoV-2 NSP3 is more closely related to mitochondrial ribosomal proteins, whereas SARS-CoV NSP3 is related to the cytosolic ribosomal proteins. In summary, our multi-omics studies of NSP3 enhance our understanding of the functions of NSP3 and offer valuable insights for the development of anti-SARS strategies.

category: cell biology


title: The effect of periodic disturbances and carrying capacity on the significance of selection and drift in complex bacterial communities

abstract: Understanding how periodical disturbances affect the community assembly processes is vital for predicting temporal dynamics in microbial communities. The effect of dilutions as disturbances are poorly understood. We used a marine bacterial community to investigate the effect of disturbance (+/-) and carrying capacity (high/low) over 50 days in a dispersal-limited 2x2 factorial crossover study in triplicates. The communitys disturbance regime was crossed halfway. We modelled the rate of change in community composition between replicates and used this rate to quantify selection and ecological drift. The disturbed communities increased in Bray-Curtis similarity with 0.011{+/-}0.0045 (Period 1) and 0.0092{+/-}0.0080 day-1 (Period 2), indicating that selection dominated community assembly. The undisturbed communities decreased in similarity at a rate of -0.015{+/-}0.0038 day-1 in Period 1 and were stable in Period 2 at 0.00050{+/-}0.0040 day-1, suggesting drift structured community assembly. Interestingly, carrying capacity had minor effects on community dynamics. This study is the first to show that stochastic effects are suppressed by periodical disturbances resulting in exponential growth periods due to density-independent biomass loss and resource input. The increased contribution of selection as a response to disturbances implies that ecosystem prediction is achievable.

category: ecology


title: Low-coverage sequencing in a deep intercross of the Virginia body weight lines provides insight to the polygenic genetic architecture of growth: novel loci revealed by increased power and improved genome-coverage

abstract: Genetic dissection of highly polygenic traits is a challenge, in part due to the power necessary to confidently identify loci with minor effects. Experimental crosses are valuable resources for mapping such traits. Traditionally, genome-wide analyses of experimental crosses have targeted major loci using data from a single generation, often the F2, with additional, later generation individuals being generated for replication and fine-mapping. Here, we aim to confidently identify minor-effect loci contributing to the highly polygenic basis of the long-term, divergent bi-directional selection responses for 56-day body weight in the Virginia chicken lines. To achieve this, a powerful strategy was developed to make use of data from all generations (F2-F18) of an advanced intercross line, developed by crossing the low and high selected lines after 40 generations of selection. A cost-efficient low-coverage sequencing based approach was used to obtain high-confidence genotypes in 1Mb bins across 99.3% of the chicken genome for >3,300 intercross individuals. In total, 12 genome-wide significant and 10 additional suggestive QTL for 56-day body weight were mapped, with only two of these QTL reaching genome-wide, and one suggestive, significance in analyses of the F2 generation. Five of the significant, and four of the suggestive, QTL were among the 20 loci reaching a 20% FDR-threshold in previous analyses of data from generation F15. The novel, minor-effect QTL mapped here were generally mapped due to an overall increase in power by integrating data across generations, with minor contributions from increased genome-coverage and improved marker information content. Significant and suggestive QTL now explain >60% of the difference between the parental lines, three times more than the previously reported significant QTL. Making integrated use of all available samples from multiple generations in experimental crosses is now economically feasible using the low-cost, sequencing-based genotyping strategies outlined here. Our empirical results illustrate the value of this strategy for mapping novel minor-effect loci contributing to complex traits to provide a more confident, comprehensive view of the individual loci that form the genetic basis of the highly polygenic, long-term selection responses for 56-day body weight in the Virginia chicken lines.

category: genetics


title: Real-time genomic surveillance during the 2021 re-emergence of the yellow fever virus in Rio Grande do Sul State, Brazil

abstract: The yellow fever virus (YFV) re-emergence in Rio Grande do Sul, Brazil, raised big concerns and led the state to declare a Public Health Emergency of State Importance. Here, we generated near-complete genomes from the ongoing outbreak in Southern Brazil, aiming to better understand the phylogenetic aspects and also spatio-temporal dynamics of the virus. Our findings highlight the path and dispersion in Rio Grande do Sul and that YFV was reintroduced from Sao Paulo to the Rio Grande do Sul state through Parana and Santa Catarina states, at the end of 2020.

category: genomics


title: Identification of Novel Long non-coding RNAs and Expression Analyses in Rectal Neuroendocrine Tumors

abstract: Altered expression of long-non-coding RNAs (lncRNAs) is associated with various pathological conditions, including cancers. LncRNAs associated with rectal neuroendocrine tumors (rNETs) remain unexplored. Here, we report the identification of 418 high confidence novel lncRNAs by analyzing publicly available RNA-Seq datasets of primary rNETs and rNETs with lymph node or liver metastases. Novel lncRNAs had basic characteristics similar to the known lncRNAs and showed individual-specific expressions. Expression analysis in the normal rectal mucosa and pancreas NETs (pNETs) and small intestine NETs (siNETs) revealed high expression of two lncRNAs, namely MSTRG30689.1 and MSTRG341161.1. Functional characterization revealed that 9 lncRNAs could work as the ce-RNA and one as miRNAs precursor. Cis- and trans- analysis of the 9 PCG-associated lncRNAs revealed that these lncRNAs could regulate various important cancer-related genes. Further, an integrated regulatory mechanism for selected cancer hallmark genes such as SMAD3, ZEB1, TCF4 and ATM through lncRNAs is predicted. Taken together, our analyses reveal common and unique lncRNAs associated with rNETs and their metastatic variants, their PCG targets, and predict mechanisms of lncRNA mediated regulation of target PCGs.


title: The Role of Context Conditioning in the Reinstatement of Responding to an Alcohol-Predictive Conditioned Stimulus

abstract: Re-exposure to an unconditioned stimulus (US) can reinstate extinguished conditioned responding elicited by a conditioned stimulus (CS). We tested the hypothesis that the reinstatement of responding to an appetitive CS is driven by an excitatory association formed between the US and the context that the US was ingested in during US re-exposure. Male, Long-Evans rats were acclimated to drinking alcohol (15%, v/v) in the home-cage, then trained to associate an auditory CS with an alcohol-US that was delivered into a fluid port for oral intake. During subsequent extinction sessions, the CS was presented as before, but without alcohol. After extinction, rats were re-exposed to alcohol as in training, but without the CS (alcohol re-exposure). 24 h later at test, the CS was presented as in training, but without alcohol. First, we tested the effect of extinguishing the context-alcohol association, formed during alcohol re-exposure, on reinstatement. Conducting four context extinction sessions across four days (spaced extinction) after the alcohol re-exposure session did not impact reinstatement. However, four context extinction sessions conducted across two days (massed extinction) prevented reinstatement. Next, we conducted alcohol re-exposure in a context that either differed from, or was the same as, the test context. One alcohol re-exposure session in a different context did not affect reinstatement, however, three alcohol re-exposure sessions in a different context significantly reduced reinstatement during the first CS trial. These results partially support the view that a context-US association formed during US re-exposure drives the reinstatement of responding to an appetitive, alcohol-predictive CS.

category: animal behavior and cognition


title: Artificial Intelligence-rationalized balanced PPARα/γ dual agonism resets the dysregulated macrophage processes in inflammatory bowel disease

abstract: A computational platform, the Boolean network explorer (BoNE), has recently been developed to infuse AI-enhanced precision into drug discovery; it enables querying and navigating invariant Boolean Implication Networks of disease maps for prioritizing high-value targets. Here we used BoNE to query an Inflammatory Bowel Disease (IBD)-map and prioritize a therapeutic strategy that involves dual agonism of two nuclear receptors, PPAR/{gamma}. Balanced agonism of PPAR/{gamma} was predicted to modulate macrophage processes, ameliorate colitis in network-prioritized animal models,  reset the gene expression network from disease to health, and achieve a favorable therapeutic index that tracked other FDA-approved targets. Predictions were validated using a balanced and potent PPAR/{gamma}-dual agonist (PAR5359) in two pre-clinical murine models, i.e., Citrobacter rodentium-induced infectious colitis and DSS-induced colitis. Using a combination of selective inhibitors and agonists, we show that balanced dual agonism promotes bacterial clearance more efficiently than individual agonists, both in vivo and in vitro. PPARa is required and its agonism is sufficient to induce the pro-inflammatory cytokines and cellular ROS, which are essential for bacterial clearance and immunity, whereas PPARg-agonism blunts these responses, delays microbial clearance and induces the anti-inflammatory cytokine, IL10; balanced dual agonism achieved controlled inflammation while protecting the gut barrier and  reversal of the transcriptomic network. Furthermore, dual agonism reversed the defective bacterial clearance observed in PBMCs derived from IBD patients. These findings not only deliver a macrophage modulator for use as barrier-protective therapy in IBD, but also highlight the potential of BoNE to rationalize combination therapy.

category: bioinformatics


title: Unveiling Mutation Effects on the Structural Dynamics of the Main Protease from SARS-CoV-2 with Hybrid Simulation Methods

abstract:
The main protease of SARS-CoV-2 (called Mpro or 3CLpro) is essential for processing polyproteins encoded by viral RNA. Macromolecules adopt several favored conformations in solution depending on their structure and shape, determining their dynamics and function. Integrated methods combining the lowest-frequency movements obtained by Normal Mode Analysis (NMA), and the faster movements from Molecular Dynamics (MD), and data from biophysical techniques, are necessary to establish the correlation between complex structural dynamics of macromolecules and their function. In this article, we used a hybrid simulation method to sample the conformational space to characterize the structural dynamics and global motions of WT SARS-CoV-2 Mpro and 48 mutants, including several mutations that appear in P.1, B.1.1.7, B.1.351, B.1.525 and B.1.429+B.1.427 variants. Integrated Hybrid methods combining NMA and MD have been useful to study the correlation between the complex structural dynamics of macromolecules and their functioning mechanisms. Here, we applied this hybrid approach to elucidate the effects of mutation in the structural dynamics of SARS-CoV-2 Mpro, considering their flexibility, solvent accessible surface area analyses, global movements, and catalytic dyad distance. Furthermore, some mutants showed significant changes in their structural dynamics and conformation, which could lead to distinct functional properties.

HighlightsO_LISingle surface mutations lead to changes in Mpro structural dynamics.
C_LIO_LIMutants can be more stable than WT according to the structural dynamics properties.
C_LIO_LIMpromutants can present a distinct functionality in relation to the wild-type.
C_LIO_LIPotential viral markers for more pathogenic or transmissible SARS-CoV-2 variants.
C_LI


title: Xenogeneic Fibroblasts Inhibit The Growth Of Breast And Ovarian Cancer Cell Lines In Co-Culture

abstract: Cell-based therapies cure some hematologic malignancies, although little information exists on solid cancer cell responses. The study objective was to test the hypothesis that xenogeneic fibroblasts can inhibit the growth of human cancer cell lines in vitro. Seven human cell lines (pancreatic cancer HPAF II; brain cancer U-87 MG; fibrosarcoma; ovarian cancer OVCAR3 and SKOV3; and breast cancer MCF7 and MDA-MB231) were co-cultured with two xenogeneic fibroblast cell lines (CV-1;monkey, Cercopithecus aethiops and DF-1; chicken, Gallus gallus) in a transwell culture system. Cancer cell proliferation was assessed colorimetrically. Different concentrations of breast and ovarian cancer cells were tested. Gene expression induced by DF-1 xenogeneic fibroblasts was assessed by RNAseq of MCF7 breast cancer cells. The proliferation of the majority of the cancer cell lines was altered by co-culture with xenogeneic fibroblasts. Cell proliferation was increased (4-17%) by CV-1; DF-1 increased brain cancer cell proliferation (16%), decreased breast and ovarian cancer cell growth (15 and 26% respectively) but did not affect fibrosarcoma and pancreatic cancer cells. When the initial cancer cell concentrations were lowered 4-fold, growth inhibition of breast and ovarian cancer increased more than 2-fold. DF-1 fibroblasts induced significant differential expression of 484 genes in MCF7 breast cancer cells; 285 genes were down-regulated and 199 genes were upregulated compared to control. Genes involved in the immune response were the major downregulated entities. RNAseq results were validated by qRT-PCR of 12 genes. The results show that xenogeneic fibroblasts can alter the growth and gene expression of cancer cells in vitro. This suggests a potentially novel investigational approach to the control of cancer cell growth.


title: Single-dose respiratory mucosal delivery of next-generation viral-vectored COVID-19 vaccine provides robust protection against both ancestral and variant strains of SARS-CoV-2

abstract: The emerging SARS-CoV-2 variants of concern (VOC) increasingly threaten the effectiveness of current first-generation COVID-19 vaccines that are administered intramuscularly and are designed to only target the spike protein. There is thus a pressing need to develop next-generation vaccine strategies to provide more broad and long-lasting protection. By using adenoviral vectors (Ad) of human and chimpanzee origin, we developed Ad-vectored trivalent COVID-19 vaccines expressing Spike-1, Nucleocapsid and RdRp antigens and evaluated them following single-dose intramuscular or intranasal immunization in murine models. We show that respiratory mucosal immunization, particularly with chimpanzee Ad-vectored vaccine, is superior to intramuscular immunization in induction of the three-arm immunity, consisting of local and systemic antibody responses, mucosal tissue-resident memory T cells, and mucosal trained innate immunity. We further show that single-dose intranasal immunization provides robust protection against not only the ancestral strain of SARS-CoV-2, but also two emerging VOC, B.1.1.7 and B.1.351. Our findings indicate that single-dose respiratory mucosal delivery of an Ad-vectored multivalent vaccine represents an effective next-generation COVID-19 vaccine strategy against current and future VOC. This strategy has great potential to be used not only to boost first-generation vaccine-induced immunity but also to expand the breadth of protective T cell immunity at the respiratory mucosa.


title: TGF--dependent Lymphoid Tissue Residency of Stem-like T cells Limits the Response to Tumor Vaccine

abstract: Stem-like CD8+ T cells represent the key subset responding to multiple tumor immunotherapies, including tumor vaccination. However, the signals that control the differentiation of stem-like T cells are not entirely known. Most previous investigations on stem-like T cells are focused on tumor infiltrating T cells (TIL). The behavior of stem-like T cells in other tissues remains to be elucidated. Tissue-resident memory T cells (TRM) are often defined as a non-circulating T cell population residing in non-lymphoid tissues. TILs carrying TRM features are associated with better tumor control. Here, we found that stem-like CD8+ T cells differentiated into TRMs in a TGF-{beta} and tumor antigen dependent manner almost exclusively in tumor draining lymph node (TDLN). TDLN-resident stemlike T cells were negatively associated with the response to tumor vaccine. In other words, after tumor vaccine, TDLN stem-like T cells transiently lost TRM features, differentiated into migratory effectors and exerted tumor control.


title: Comparison of Neutralizing Antibody Titers Elicited by mRNA and Adenoviral Vector Vaccine against SARS-CoV-2 Variants

abstract: The increasing prevalence of SARS-CoV-2 variants has raised concerns regarding possible decreases in vaccine efficacy. Here, neutralizing antibody titers elicited by mRNA-based and an adenoviral vector-based vaccine against variant pseudotyped viruses were compared. BNT162b2 and mRNA-1273-elicited antibodies showed modest neutralization resistance against Beta, Delta, Delta plus and Lambda variants whereas Ad26.COV2.S-elicited antibodies from a significant fraction of vaccinated individuals were of low neutralizing titer (IC50 <50). The data underscore the importance of surveillance for breakthrough infections that result in severe COVID-19 and suggest the benefit of a second immunization following Ad26.COV2.S to increase protection against the variants.


title: Rosin Soap Exhibits Virucidal Activity

abstract:
Chemical methods of virus inactivation are used routinely to prevent viral transmission in both a personal hygiene capacity but also in at-risk environments like hospitals. Several  virucidal products exist, including hand soaps, gels and surface disinfectants. Resin acids, which can be derived from Tall oil produced from trees, have been shown to exhibit anti-bacterial activity. However, whether these products or their derivatives have virucidal activity is unknown. Here, we assessed the capacity of Rosin soap to inactivate a panel of pathogenic mammalian viruses in vitro. We show that Rosin soap can inactivate the human enveloped viruses: influenza A virus (IAV), respiratory syncytial virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For IAV, rosin soap could provide a 100,000-fold reduction in infectivity. However, Rosin soap failed to affect the non-enveloped encephalomyocarditis virus (EMCV). The inhibitory effect of Rosin soap against IAV infectivity was dependent on its concentration but not dependent on incubation time nor temperature. Together, we demonstrate a novel chemical inactivation method against enveloped viruses, which could be of use in preventing virus infections in certain settings.

ImportanceViruses remain a significant cause of human disease and death, most notably illustrated through the current Covid-19 pandemic. Control of virus infection continues to pose a significant global health challenge to the human population. Viruses can spread through multiple routes, including via environmental and surface contamination where viruses can remain infectious for days. Methods to inactivate viruses on such surfaces may help mitigate infection. Here we present evidence identifying a novel  virucidal product in Rosin soap, which is produced from Tall oil from coniferous trees. Rosin soap was able to rapidly and potently inactivate influenza virus and other enveloped viruses.


title: Dickeya solani D s0432-1 produces an arsenal of secondary metabolites with anti-prokaryotic and anti-eukaryotic activities against bacteria, yeasts, fungi, and aphids.

abstract: The necrotrophic plant pathogenic bacterium Dickeya solani is a new invader of potato agrosystem in Europe. All isolated strains of D. solani contain several large polyketide/fatty acid/non-ribosomal peptide synthetase clusters. Analogy with genes described in other bacteria, suggests that two clusters are involved in the production of secondary metabolites of the oocydin and zeamine family. In this study, we constructed by an approach of reverse genetics mutants affected in the three secondary metabolite clusters ssm, ooc and zms in order to compare the phenotype of the D. solani strain D s0432-1 with its derived mutants. We demonstrated that the zeamine cluster zms inhibits growth of gram-positive and gram-negative bacteria. It is also implicated in a toxicity against aphids. The oocydin cluster ooc inhibits growth of fungi of the phylum Ascomycota. Finally, we unveiled the function of a new secondary metabolite cluster ssm (for solani secondary metabolite), only conserved in some Dickeya species. This cluster produces a secondary metabolite inhibiting yeasts. D. solani therefore produces several molecules that are toxic to a wide range of living and potentially interacting organisms, from bacteria to insects. The expression of these secondary metabolite pathways could contribute to the rapid spread of D. solani in Europe.


title: Single-cell RNA Sequencing Reveals the Temporal Diversity and Dynamics of Cardiac Immunity after Myocardial Infarction

abstract:
Myocardial infarction (MI) is strongly associated with the temporal regulation of cardiac immunity. However, a variety of current clinical trials have failed because of the lack of post-MI immunomodulating/anti-inflammatory targets. We performed single-cell RNA sequencing analysis of cardiac Cd45+ immune cell at 0, 3, 7, and 14 days after injury in a mouse left anterior descending coronary artery ligation model. Major immune cell populations, distinct subsets, and dynamic changes were identified. Macrophages (Mo)/monocytes were most abundant, peaking at 3 days after infarction. Mo-5 and Mo-6 were the predominant infiltrated subsets at this time point, with strong expression of inflammatory factors. Further analysis demonstrated that suppressing these sets attenuated pathological MI progression by preventing subsequent leukocyte extravasation and adverse remodeling. We also detected abundant apoptotic neutrophils and a profibrotic macrophage subset on days 7 and 14 respectively. These results provide a basis for developing cell type- and time-specific interventions in MI.

Graphic abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/452902v1_ufig1.gif" ALT="Figure 1">
View larger version (53K):
org.highwire.dtl.DTLVardef@1d7c810org.highwire.dtl.DTLVardef@18f9c9dorg.highwire.dtl.DTLVardef@17325f7org.highwire.dtl.DTLVardef@c1a4e7_HPS_FORMAT_FIGEXP  M_FIG C_FIG Temporal regulation of cardiac immunity is strongly associated with the onset, progression, and outcomes of MI. Our study of time-series scRNA-seq on the whole cardiac immune cells isolated from the LAD ligation mice and the tanshinone IIA treated mice shed light on the underlying pathology of MI.


title: Quantitative single molecule RNA-FISH and RNase-free cell wall digestion in Neurospora crassa.

abstract: Single molecule RNA-FISH (smFISH) is a valuable tool for analysis of mRNA spatial patterning in fixed cells that is underutilized in filamentous fungi. A primary complication for fixed-cell imaging in filamentous fungi is the need for enzymatic cell wall permeabilization, which is compounded by considerable variability in cell wall composition between species. smFISH adds another layer of complexity due to a requirement for RNase free conditions. Here, we describe the cloning, expression, and purification of a chitinase suitable for supplementation of a commercially available RNase-free enzyme preparation for efficient permeabilization of the Neurospora cell wall. We further provide a method for smFISH in Neurospora which includes a tool for generating numerical data from images that can be used in downstream customized analysis protocols.


title: Single-nucleus cross-tissue molecular reference maps to decipher disease gene function

abstract: Understanding the function of genes and their regulation in tissue homeostasis and disease requires knowing the cellular context in which genes are expressed in tissues across the body. Single cell genomics allows the generation of detailed cellular atlases in human tissues, but most efforts are focused on single tissue types. Here, we establish a framework for profiling multiple tissues across the human body at single-cell resolution using single nucleus RNA-Seq (snRNA-seq), and apply it to 8 diverse, archived, frozen tissue types (three donors per tissue). We apply four snRNA-seq methods to each of 25 samples from 16 donors, generating a cross-tissue atlas of 209,126 nuclei profiles, and benchmark them vs. scRNA-seq of comparable fresh tissues. We use a conditional variational autoencoder (cVAE) to integrate an atlas across tissues, donors, and laboratory methods. We highlight shared and tissue-specific features of tissue-resident immune cells, identifying tissue-restricted and non-restricted resident myeloid populations. These include a cross-tissue conserved dichotomy between LYVE1- and HLA class II-expressing macrophages, and the broad presence of LAM-like macrophages across healthy tissues that is also observed in disease. For rare, monogenic muscle diseases, we identify cell types that likely underlie the neuromuscular, metabolic, and immune components of these diseases, and biological processes involved in their pathology. For common complex diseases and traits analyzed by GWAS, we identify the cell types and gene modules that potentially underlie disease mechanisms. The experimental and analytical frameworks we describe will enable the generation of large-scale studies of how cellular and molecular processes vary across individuals and populations.


title: Host phylogeny drives the structure and diversity of insect microbiota

abstract: As for most of the life that inhabits our planet, microorganisms play an essential role in the fitness of insects, including nutrition, reproduction, defence, and many other functions. More recently, we assisted in an exponential growth of studies describing the taxonomical composition of bacterial communities across insects phylogeny. However, there is still an outstanding question that needs to be answered: which factors contribute most to shape insects microbiomes? This study tries to find an answer to this question by taking advantage of publicly available sequencing data and reanalysing over 4,000 samples of insect-associated bacterial communities under a common framework. Results suggest that insect taxonomy has a wider impact on the structure and diversity of their associated microbial communities than the other factors considered (diet, sex, life stage, sample origin, and treatment). However, when specifically testing for signatures of co-diversification of insect species and their microbiota, analyses found weak support for this, suggesting that while insect species strongly drive the structure and diversity of insect microbiota, the diversification of those microbial communities did not follow their hosts phylogeny. Furthermore, a parallel survey of the literature highlights several methodological limitations that need to be considered in future research endeavours.


title: Host species identity shapes the diversity and structure of insect microbiota


title: Characterization of vaginal microbial enzymes identifies amylopullulanases that support growth of Lactobacillus crispatus on glycogen

abstract: The healthy human vaginal microbiota is generally dominated by Lactobacilli, and the transition to a more diverse community of anaerobic microbes is associated with a number of health risks. While the mechanisms underlying the stability of Lactobacillus-dominated vaginal communities are not fully understood, competition for nutrients is a likely contributing factor. Glycogen secreted by epithelial cells is widely believed to support the growth of vaginal microbes. However, the mechanism by which bacteria access sugars from this complex polymer is unclear, with evidence to support a role for both microbial and human enzymes. To shed light on the potential contribution from microbial enzymes, here we biochemically characterize six glycogen-degrading enzymes predicted to be secreted by vaginal bacteria and confirm their ability to support the growth of an amylase-deficient strain of L. crispatus on glycogen. We reveal significant differences in the pH tolerance between enzymes from different organisms, suggesting the adaptation of Lactobacilli to an acidic vaginal environment. Using a simple assay specific for the microbial enzymes, we confirm their presence in cervicovaginal lavage samples. Finally, we demonstrate the selective inhibition of glycogen-degrading enzymes from two vaginal microbes associated with dysbiosis. This work provides biochemical evidence to support the role of vaginal bacterial amylase enzymes in the breakdown of glycogen, providing insight into factors that shape the vaginal microbiota and highlighting the possibility of manipulating community structure via non-antibiotic small molecules.

category: biochemistry


title: Loss of the H4 lysine methyltransferase KMT5B drives tumorigenic phenotypes by depleting H3K27me3 at loci otherwise retained in H3K27M mutant DIPG cells

abstract: DIPG are characterised by histone H3K27M mutations, resulting in global loss of the repressive mark H3K27me3, although certain key loci are retained. We recently identified subclonal loss-of-function mutations in the H4 lysine methyltransferase KMT5B to be associated with enhanced invasion/migration, but the mechanism by which this occurred was unclear. Here we use integrated ChIP-seq, ATAC-seq and RNA-seq on patient-derived, subclonal and CRISPR-Cas9-KD DIPG cells to show that loss of KMT5B/C causes depletion of these retained H3K27me3 loci via changes in chromatin accessibility, causing a raft of transcriptional changes which promote tumorigenesis. De-repression occurred at bivalent loci marked by H3K4me3, driving increased transcriptional heterogeneity and elevated gene expression associated with increased invasion, abrogated DNA repair and mesenchymal transition, along with a markedly altered secretome. These data suggest a previously unrecognised trans-histone (H4/H3) interaction in DIPG cells with a potentially profound effect on their diffusely infiltrating phenotype.


title: Gata2a maintains cebpa and npm1a in haematopoietic stem cells to sustain lineage differentiation and genome stability

abstract: The transcription factor Gata2 is required to produce and maintain haematopoietic stem and progenitor cells (HSPCs) in development and adult haematopoiesis. Mutations in GATA2 lead to GATA2 deficiency syndrome and predispose patients to acquire leukaemia. Here we use zebrafish gata2a enhancer deletion mutants and single cell transcriptomics to understand how GATA2 mediates survival and differentiation of haematopoietic stem cells in GATA2 deficiency. Gata2a mutants show marrow failure, neutropenia, B-lymphopenia and erythrocytosis from 6 months post-fertilization (mpf). Single cell transcriptional profiling of the adult kidney marrow demonstrated that HSPCs express elevated expression of erythroid- and decreased expression of myeloid genes, including cebpa. This is associated with a lineage skewing towards the erythroid fate at the expense of the myeloid fate. Thus, Gata2a is required to initiate and maintain lineage priming in HSPCs, favouring myeloid differentiation. Gata2a regulates expression of multiple targets associated with replication and DNA damage repair (DDR), including npm1a, a zebrafish NPM1 orthologue. Accordingly, mutant marrow cells show increased DNA damage associated with progressive loss of npm1a expression with age. This effect was replicated by inhibiting NPM1 activity in murine HPC7 progenitor cells. We propose that the impaired DDR leads to marrow failure in GATA2 deficiency. This leads to increased genomic instability in the surviving HSPCs, favouring acquisition of secondary leukaemogenic mutations.


title: The long non-coding RNA H19 drives the proliferation of diffuse intrinsic pontine glioma with H3K27 mutation

abstract: Diffuse intrinsic pontine glioma (DIPG) is an incurable paediatric malignancy. Identifying molecular drivers of DIPG progression is of utmost importance. Long non-coding RNAs (lncRNAs) represent a large family of disease- and tissue-specific transcripts, whose functions have not been yet elucidated in DIPG. Here, we study the oncogenic role of the development-associated H19 lncRNA in DIPG. Bioinformatic analyses of clinical datasets were used to measure the expression of H19 lncRNA in paediatric high-grade gliomas (pedHGG). Expression and sub-cellular location of H19 lncRNA was validated in DIPG cell lines. Locked nucleic acid antisense oligonucleotides were designed to test the function of H19 in DIPG cells. We found that H19 expression was higher in DIPG vs normal brain tissue and other pedHGGs. H19 knockdown resulted in decreased cell proliferation and survival in DIPG cells. Mechanistically, H19 buffers let-7 microRNAs, resulting in up-regulation of oncogenic let-7 target (e.g SULF2, OSMR). H19 is the first functionally characterized lncRNA in DIPG and a promising therapeutic candidate to treat this incurable cancer.


title: OSTEOPONTIN DEFICIENCY LEADS TO THE RESOLUTION OF PROSTATIC FIBROSIS AND INFLAMMATION

abstract:
Fibrogenic and inflammatory processes in the prostate are linked to the development of lower urinary tract symptoms (LUTS) in men. Our previous studies identified that osteopontin (OPN), a pro-fibrotic cytokine, is abundant in the prostate of men with LUTS and its secretion is stimulated by inflammatory cytokines potentially to drive fibrosis. This study investigates whether the lack of OPN ameliorates inflammation and fibrosis in the mouse prostate.

We instilled uropathogenic E. coli (UTI89) or saline (control) transurethrally to C57BL/6J (WT) or Spp1tm1Blh/J (OPN-KO) mice and collected the prostates one or 8 weeks later. We found that OPN mRNA and protein expression were significantly induced by E. coli-instillation in the dorsal prostate (DP) after one week in WT mice. Deficiency in OPN expression led to decreased inflammation and fibrosis and the prevention of urinary dysfunction after 8 weeks. RNAseq analysis identified that E. coli-instilled WT mice expressed increased levels of inflammatory and fibrotic marker RNAs compared to OPN-KO mice including Col3a1, Dpt, Lum and Mmp3 which were confirmed by RNAscope.

Our results indicate that OPN is induced by inflammation and prolongs the inflammatory state; genetic blockade of OPN accelerates recovery after inflammation, including a resolution of prostate fibrosis.


title: Redox regulation by TXNRD3 during epididymal maturation underlies capacitation-associated mitochondrial activation and sperm motility in mice

abstract: During epididymal transit, redox remodeling protects mammalian spermatozoa, preparing them for survival in the subsequent journey to fertilization. However, molecular mechanisms of redox regulation in sperm development and maturation remain largely elusive. In this study, we report that TXNRD3, a thioredoxin reductase family member particularly abundant in elongating spermatids at the site of mitochondrial sheath formation, regulates redox homeostasis to support male reproduction. Using Txnrd3-/- mice, our biochemical, ultrastructural, and live cell imaging analyses revealed impairments in sperm morphology and motility under conditions of TXNRD3 deficiency. We reveal that mitochondria develop more defined cristae during capacitation in wild type sperm. Absence of TXNRD3 alters redox status in both the head and tail during sperm maturation and capacitation, resulting in defective mitochondrial ultrastructure and activity under capacitating conditions. These findings provide insights into molecular mechanisms of redox homeostasis and bioenergetics during sperm maturation, capacitation, and fertilization.

category: physiology


title: Cellular and behavioral effects of altered NaV1.2 sodium channel ion permeability in Scn2aK1422E mice

abstract:
Genetic variants in SCN2A, encoding the NaV1.2 voltage-gated sodium channel, are associated with a range of neurodevelopmental disorders with overlapping phenotypes. Some variants fit into a framework wherein gain-of-function missense variants that increase neuronal excitability lead to infantile epileptic encephalopathy, while loss-of-function variants that reduce neuronal excitability lead to developmental delay and/or autism spectrum disorder with or without co- morbid seizures. One unique case less easily classified using this binary paradigm is the de novo missense variant SCN2A p.K1422E, associated with infant-onset developmental delay, infantile spasms, and features of autism spectrum disorder. Prior structure-function studies demonstrated that K1422E substitution alters ion selectivity of NaV1.2, conferring Ca2+ permeability, lowering overall conductance, and conferring resistance to tetrodotoxin (TTX). Based on heterologous expression of K1422E, we developed a compartmental neuron model that predicted mixed effects on channel function and neuronal activity. We also generated Scn2aK1422E mice and characterized effects on neurons and neurological/neurobehavioral phenotypes. Dissociated neurons from heterozygous Scn2aK1422E/+ mice exhibited a novel TTX-resistant current with a reversal potential consistent with mixed ion permeation. Cortical slice recordings from Scn2aK1442E/+ tissue demonstrated impaired action potential initiation and larger Ca2+ transients at the axon initial segment during the rising phase of the action potential, suggesting mixed effects on channel function. Scn2aK1422E/+ mice exhibited rare spontaneous seizures, interictal EEG abnormalities, altered response to induced seizures, reduced anxiety-like behavior and alterations in olfactory-guided social behavior. Overall, Scn2aK1422E/+ mice present with phenotypes similar yet distinct from Scn2a knockout models, consistent with mixed effects of K1422E on NaV1.2 channel function.

Significance StatementThe early-onset epilepsy variant SCN2A-p.K1422E displays unique biophysical properties in vitro. To model the impact of this rare variant, we generated Scn2aK1422E mice. Neurons from heterozygous Scn2aK1422E/+ mice showed functional deficits similar to the loss-of-function effects observed in the Scn2a haploinsufficiency model, as well as gain-of-function effects specific to the K1422E variant. There is also some overlap in neurobehavioral phenotypes between Scn2aK1422E/+ and Scn2a haploinsufficient mice. However, Scn2aK1422E/+ mice exhibited unique epilepsy-related phenotypes, including epileptiform events and seizures. Scn2aK1422E/+ mice serve as a useful platform to investigate phenotypic complexity of SCN2A-associated disorders.


title: Granulocyte-colony stimulating factor (G-CSF) enhances cocaine effects in the nucleus accumbens via a dopamine release-based mechanism

abstract: Cocaine use disorder is associated with alterations in immune function including altered expression of multiple peripheral cytokines in humans - several of which correlate with drug use. Individuals suffering from cocaine use disorder show altered immune system responses to drug-associated cues, highlighting the interaction between the brain and immune system as a critical factor in the development and expression of cocaine use disorder. We have previously demonstrated in animal models that cocaine use upregulates expression of granulocyte colony stimulating factor (G-CSF) - a pleiotropic cytokine - in the serum and the nucleus accumbens (NAc). G-CSF signaling has been causally linked to behavioral responses to cocaine across multiple behavioral domains. The goal of this study was to define whether increases in G-CSF alter the pharmacodynamic effects of cocaine on the dopamine system and whether this occurs via direct mechanisms within local NAc microcircuits. We find that systemic G-CSF injection increases cocaine effects on dopamine terminals. The enhanced dopamine levels in the presence of cocaine occur through a release-based mechanism, rather than through effects on the dopamine transporter - as uptake rates were unchanged following G-CSF treatment. Critically, this effect could be recapitulated by acute bath application of G-CSF to dopamine terminals, an effect that was occluded by prior G-CSF treatment, suggesting a similar mechanistic basis for direct and systemic exposures. This work highlights the critical interaction between the immune system and psychostimulant effects that can alter drug responses and may play a role in vulnerability to cocaine use disorder.


title: Self-sensitisation of Staphylococcus aureus to the antimicrobial factors found in human blood.

abstract: Introductory ParagraphFor opportunistic pathogens, the switch from a commensal to an invasive lifestyle is often considered an accidental event. But with plentiful opportunity, what leads one accidental event to result in an invasive infection, and another not to? And how much of this apparent stochasticity is driven by bacterial factors? To answer these questions, here we focussed on the major human pathogen Staphylococcus aureus, which can both reside asymptomatically as a member of our respiratory microbiome, or become invasive and cause infections as severe as bacteraemia. Survival upon exposure to the antibacterial factors found in serum is a critical aspect of their ability to cause bacteraemia, and across a collection of 300 clinical isolates we found there to be significant variability in this capability. Utilising a GWAS approach we have uncovered the genetic basis of much of this variability through the identification and functional verification of a number of new polymorphic loci that affect serum survival: tcaA, tarK, gntR, ilvC, arsB, yfhO, and pdhD. The expression of one of these genes, tcaA, was found to be induced upon exposure to serum, while simultaneously enhancing the sensitivity of S. aureus to serum through a process involving the ligation of wall teichoic acids into the cell wall. As blood-stage infections are a transmission dead-end for the bacteria, that S. aureus actively responds to serum to produce a protein which specifically limits their ability to survive in this environment demonstrates that the switch from the commensal to the invasive lifestyle is complex, and that TcaA may contribute to the long-term success of S. aureus by restricting the bacteria to their more readily transmissible commensal state.


title: Pathotyping of Newcastle disease virus: A novel single BsaHI digestion method of detection and differentiation of avirulent strains (lentogenic and mesogenic vaccine strains) from virulent virus

abstract: We provide a novel single restriction enzyme (RE) (BsaHI) digestion approach for detecting distinct pathotypes of the Newcastle disease virus (NDV). After scanning 4000 F gene nucleotide sequences in the NCBI database, a single RE (BsaHI) digesting site was discovered in the cleavage site. APMV-I "F gene" Class II specific primer-based reverse transcriptase PCR (RT-PCR) was utilized to amplify a 535 bp fragment, which was then digested with a single RE (BsaHI) for pathotyping avian NDV field isolates and pigeon paramyxovirus-1 isolates. The avirulent (lentogenic and mesogenic strains) produce 189 and 346 bp fragments, respectively, but the result in velogenic strains remains undigested with 535 bp fragments. In addition, 45 field NDV isolates and 8 vaccine strains were used to confirm the approach. The sequence-based analysis also agrees with the data obtained utilizing the single RE (BsaHI) digestion approach. The proposed technique had the potential to distinguish between avirulent and virulent strains in a short space of time, making it valuable in NDV surveillance and monitoring research.


title: Key substitutions in the spike protein of SARS-CoV-2 variants can predict resistance to monoclonal antibodies, but other substitutions can modify the effects

abstract:
Mutations in the spike protein of SARS-CoV-2 variants can compromise the effectiveness of therapeutic antibodies. Most clinical-stage therapeutic antibodies target the spike receptor binding domain (RBD), but variants often have multiple mutations in several spike regions. To help predict antibody potency against emerging variants, we evaluated 25 clinical-stage therapeutic antibodies for neutralization activity against 60 pseudoviruses bearing spikes with single or multiple substitutions in several spike domains, including the full set of substitutions in B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.429 (Epsilon), B.1.526 (Iota), A.23.1 and R.1 variants. We found that 14 of 15 single antibodies were vulnerable to at least one RBD substitution, but most combination and polyclonal therapeutic antibodies remained potent. Key substitutions in variants with multiple spike substitutions predicted resistance, but the degree of resistance could be modified in unpredictable ways by other spike substitutions that may reside outside of the RBD. These findings highlight the importance of assessing antibody potency in the context of all substitutions in a variant and show that epistatic interactions in spike can modify virus susceptibility to therapeutic antibodies.

ImportanceTherapeutic antibodies are effective in preventing severe disease from SARS-CoV-2 infection (COVID-19), but their effectiveness may be reduced by virus variants with mutations affecting the spike protein. To help predict resistance to therapeutic antibodies in emerging variants, we profiled resistance patterns of 25 antibody products in late stages of clinical development against a large panel of variants that include single and multiple substitutions found in the spike protein. We found that the presence of a key substitution in variants with multiple spike substitutions can predict resistance against a variant, but that other substitutions can affect the degree of resistance in unpredictable ways. These finding highlight complex interactions among substitutions in the spike protein affecting virus neutralization and potentially virus entry into cells.


title: Structural model of PORCN illuminates disease-associated variants and drug binding sites

abstract: Wnt signaling is essential for normal development and is a therapeutic target in cancer. The enzyme PORCN, or porcupine, is a membrane-bound O-acyltransferase (MBOAT) that is required for the post-translational modification of all Wnts, adding an essential mono-unsaturated palmitoleic acid to a serine on the tip of Wnt hairpin 2. Inherited mutations in PORCN cause focal dermal hypoplasia, and therapeutic inhibition of PORCN slows the growth of Wnt-dependent cancers. Here, based on homology to mammalian MBOAT proteins we develop and validate a molecular structural model of PORCN. The model accommodates palmitoleoyl-CoA and Wnt hairpin 2 in two tunnels in the conserved catalytic core, shedding light on the catalytic mechanism. The model predicts how previously uncharacterized human variants of uncertain significance can alter PORCN function. Drugs including ETC-159, IWP-L6 and LGK-974 dock in the PORCN catalytic site, providing insights into PORCN pharmacologic inhibition. This structural model provides mechanistic insights into PORCN substrate recognition and catalysis as well as the inhibition of its enzymatic activity and can facilitate the development of improved inhibitors and the understanding of disease relevant PORCN mutants.

category: molecular biology


title: Small-molecule cocktails induce the differentiation of human adipose-derived mesenchymal stem cells into hepatocyte-like cells

abstract: At present, liver transplantation and hepatocyte therapy are common methods for the treatment of end-stage liver diseases, but they are restricted due to the shortage of liver donors and the safety and effectiveness of hepatocyte sources. Human adipose-derived mesenchymal stem cells (HAD-MSCs) have been applied to efficiently and stably induce phenotypic and functional liver cells or tissues in vitro due to their advantages such as wide sources and easy access to materials. In this study, the HAD-MSCs liver differentiation induction system was established and optimized based on the "cocktail method" of chemical small molecule compounds. We used HAD-MSCs as seed cells and gradually obtained mature hepatoid cells with normal phenotype and function after induction with small molecule compounds and growth factor system in vitro. The hepatoid cells induced by the two groups showed high similarity in phenotype and functional characteristics of mature hepatocytes. The differentiation system of human adipose mesenchymal stem cells into hepatocytes induced by small-molecule compounds in vitro was successfully constructed. This study will lay a foundation for the optimization of liver differentiation strategies and provide a reliable source of functional liver cells for clinical studies of liver diseases.


title: Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy

abstract: Removal of damaged organelles via the process of selective autophagy constitutes a major form of cellular quality control. Damaged organelles are recognized by a dedicated surveillance machinery, leading to the assembly of an autophagosome around the damaged organelle, prior to fusion with the degradative lysosomal compartment. Lysosomes themselves are also prone to damage and are degraded through the process of lysophagy. While early steps involve recognition of ruptured lysosomal membranes by glycan-binding Galectins and ubiquitylation of transmembrane lysosomal proteins, many steps in the process, and their inter-relationships, remain poorly understood, including the role and identity of cargo receptors required for completion of lysophagy. Here, we employ quantitative organelle capture and proximity biotinylation proteomics of autophagy adaptors, cargo receptors, and Galectins in response to acute lysosomal damage, thereby revealing the landscape of lysosomal proteome remodeling during lysophagy. Among proteins dynamically recruited to damaged lysosomes were ubiquitin-binding autophagic cargo receptors. Using newly developed lysophagic flux reporters including Lyso-Keima, we demonstrate that TAX1BP1, together with its associated kinase TBK1, are both necessary and sufficient to promote lysophagic flux in both Hela cells and induced neurons (iNeurons). While the related receptor OPTN can drive damage-dependent lysophagy when overexpressed, cells lacking either OPTN or CALCOCO2 still maintain significant lysophagic flux in HeLa cells. Mechanistically, TAX1BP1-driven lysophagy requires its N-terminal SKICH domain, which binds both TBK1 and the autophagy regulatory factor RB1CC1, and requires upstream ubiquitylation events for efficient recruitment and lysophagic flux. These results identify TAX1BP1 as a central component in the lysophagy pathway and provide a proteomic resource for future studies of the lysophagy process.


title: A high-throughput assay for directly monitoring nucleolar rRNA biogenesis

abstract: Studies of the regulation of nucleolar function are critical for ascertaining clearer insights into the basic biological underpinnings of ribosome biogenesis, and for future development of therapeutics to treat cancer and ribosomopathies. A number of high-throughput primary assays based on morphological alterations of the nucleolus can indirectly identify hits affecting ribosome biogenesis. However, there is a need for a more direct high-throughput assay for nucleolar function to further evaluate hits. Previous reports have monitored nucleolar RNA biogenesis using 5-ethynyl uridine (5-EU) in low-throughput. We report a miniaturized, high-throughput 5-EU assay for nucleolar function which enables specific calculation of nucleolar rRNA biogenesis inhibition, based on co-staining of the nucleolar protein fibrillarin (FBL). The assay utilizes two siRNA controls, a negative non-targeting siRNA control (siNT) and a positive siRNA control targeting POLR1A (siPOLR1A), and specifically quantifies median 5-EU signal within nucleoli. Maximum nuclear 5-EU signal can also be used to monitor the effects of putative small molecule inhibitors of RNAP1, like BMH-21, or other treatment conditions that cause FBL dissociation. We validate the 5-EU assay on 68 predominately nucleolar hits from a high-throughput primary screen, showing that 58/68 hits significantly inhibit nucleolar rRNA biogenesis. Our new method establishes direct quantification of nucleolar function in high-throughput, facilitating closer study of ribosome biogenesis in health and disease.


title: Predicting the Conformational Variability of Oncogenic GTP-bound G12D Mutated KRas-4B Proteins at Cell Membranes

abstract: KRas proteins are the largest family of mutated Ras isoforms, participating in a wide variety of cancers. Due to their importance, large effort is being carried out on drug development by small-molecule inhibitors. However, understanding protein conformational variability remains a challenge in drug discovery. In the case of the Ras family, their multiple conformational states can affect the binding of potential drug inhibitors. To overcome this challenge, we propose a computational framework based on combined all-atom Molecular Dynamics and Metadynamics simulations able to accurately access conformational variants of the target protein. We tested the methodology using a G12D mutated GTP bound oncogenic KRas-4B protein located at the interface of a DOPC/DOPS/cholesterol model anionic cell membrane. Two main orientations of KRas-4B at the anionic membrane have been obtained and explored. The corresponding angles have been taken as reliable reaction coordinates so that free-energy landscapes have been obtained by well-tempered metadynamics simulations, revealing the local and global minima of KRas-4B binding to the cell membrane, unvealing reactive paths of the system between the two preferential orientations and highlighting opportunities for targeting the unique metastable states through the identification of druggable pockets.


title: A novel nanoluciferase transgenic reporter to measure proteinuria in zebrafish

abstract:
The zebrafish is an important animal system for modelling human diseases. This includes kidney dysfunction as the embryonic kidney (pronephros) shares considerable molecular and morphological homology with the human nephron. Zebrafish also have a high fecundity, with females capable of laying 200-300 eggs per week, thereby facilitating chemical and mutation screening. A key clinical indicator of kidney disease is proteinuria, but a high-throughput readout of proteinuria in the zebrafish is lacking. Coupling the advantages of the zebrafish system with a tool to measure proteinuria will advance the scope for testing the efficacy of drugs to treat kidney diseases. Here, we generated a stable transgenic zebrafish line using the l-fabp10 liver-specific promoter to over-express a nanoluciferase molecule fused with the D3 domain of Receptor-Associated-Protein (RAP) to create NL-D3. In the healthy state, NL-D3 is excreted, but when embryos were treated with chemicals that affected either proximal tubular reabsorption (cisplatin, gentamicin) or glomerular filtration (angiotensin II, Hanks Balanced Salt Solution, Bovine Serum Albumin), NL-D3 presence in the urine increased. Similarly, depletion of several gene products associated with kidney disease (nphs1, nphs2, lrp2a, ocrl, col4a3, col4a4, and col4a5) also induced NL-D3 proteinuria. Furthermore, we found that treating col4a4 depleted zebrafish larvae (a model of Alport syndrome) with captopril reduced proteinuria. Our findings confirm the use of the NL-D3 transgenic zebrafish as a robust and quantifiable proteinuria reporter. Given the feasibility of high-throughput assays in zebrafish, this novel reporter will permit screening for drugs that ameliorate proteinuria and thereby prioritise candidates for further translational studies.

Significance StatementThe zebrafish has become an important system for modelling kidney disease. However, proteinuria, an important clinical indicator of kidney dysfunction, is not easily detected in zebrafish. Here, we describe a transgenic line that uses a nanoluciferase reporter to enable detection of proteinuria in multiple models of glomerular and proximal tubular kidney disease in the zebrafish. In this system proteinuria can be accurately measured in a high-throughput manner and will enable the screening of drugs that affect glomerular filtration or protein re-uptake in the proximal tubule.


title: SOX4 facilitates PGR protein stability and FOXO1 expression conducive for human endometrial decidualization

abstract: The establishment of receptive endometrium in human necessitates appropriate decidualization of stromal cells, which involves steroids regulated periodic transformation of endometrial stromal cells during menstrual cycle. Insufficient decidualization of endometrium contributes to not only the failure of embryo implantation and unexplained infertility, but also the occurrence of recurrent spontaneous abortion, intrauterine growth retardation, preeclampsia, and other clinical gynecological diseases. However, the potential molecular regulatory mechanism underlying the initiation and maintenance of decidualization in humans is yet to be fully elucidated. In this investigation, we document that SOX4 is a key regulator of human endometrial stromal cells (hESCs) decidualization by directly regulating PRL and FOXO1 expression as revealed by whole genomic binding of SOX4 assay and RNA-Seq. Besides, our immunoprecipitation and mass spectrometry results unravel that SOX4 modulates progesterone receptor (PGR) stability through repressing E3 ubiquitin ligase HERC4 mediated degradation. More importantly, we provide evidence that dysregulated SOX4-HERC4-PGR axis is a potential cause of defective decidualization and recurrent implantation failure (RIF) in IVF patients. In summary, this study evidences that SOX4 is a new and critical regulator for human endometrial decidualization, and provides insightful information for the pathology of decidualization-related infertility and will pave the way for pregnancy improvement.


title: Chemical inhibition of lycopene β-cyclases unmask operation of phytoene synthase 2 in ripening tomato fruits

abstract:
In ripening tomato fruits, the leaf-specific carotenoids biosynthesis mediated by phytoene synthase 2 (PSY2) is replaced by a fruit-specific pathway by the expression of two chromoplast-specific genes: phytoene synthase 1 (PSY1) and lycopene-{beta}-cyclase (CYCB). Though both PSY1 and PSY2 genes express in tomato fruits, the functional role of PSY2 is not known. To decipher whether PSY2-mediated carotenogenesis operates in ripening fruits, we blocked the in vivo activity of lycopene-{beta}-cyclases in fruits of several carotenoids and ripening mutants by CPTA (2-(4-Chlorophenylthio)triethylamine hydrochloride), an inhibitor of lycopene-{beta}-cyclases. The CPTA-treatment induced accumulation of lycopene in leaves, immature-green and ripening fruits. Even, in psy1 mutants V7 and r that are deficient in fruit-specific carotenoid biosynthesis, CPTA triggered lycopene accumulation but lowered the abscisic acid level. Differing from fruit-specific carotenogenesis, CPTA-treated V7 and r mutant fruits accumulated lycopene but not phytoene and phytofluene. The lack of phytoene and phytofluene accumulation was reminiscent of PSY2-mediated leaf-like carotenogenesis, where phytoene and phytofluene accumulation is never seen. The lycopene accumulation was associated with the partial transformation of chloroplasts to chromoplasts bearing thread-like crystalline structures. Our study uncovers the operation of a parallel carotenogenesis pathway mediated by PSY2 that provides precursors for abscisic acid biosynthesis in ripening tomato fruits.

Significance statementIt is believed that in ripening tomato fruits phytoene synthase 2 that drives carotenoid biosynthesis in leaves is redundant. Contrary to this, we show that in phytoene synthase 1 mutant fruit that is bereft of lycopene, the chemical inhibition of lycopene {beta}-cyclases triggers lycopene accumulation. Our results uncover that phytoene synthase 2 remains functional in ripening fruits and provides precursors for abscisic acid formation in fruits.

category: plant biology


title: Phytoene synthase 2 in tomato fruits remains functional and contributes to abscisic acid formation


title: Singlet oxygen leads to structural changes to chloroplasts during degradation in the Arabidopsis thaliana plastid ferrochelatase two mutant

abstract: During photosynthesis, chloroplasts can produce large amounts of reactive oxygen species (ROS), particularly under stressful conditions. Along with other nutrients, chloroplasts also contain 80% of a leafs nitrogen supply. For these reasons, chloroplasts are prime targets for cellular degradation to protect cells from photo-oxidative damage and to redistribute nutrients to sink tissues. Multiple chloroplast degradation pathways have been described and are induced by photo-oxidative stress and nutrient starvation. However, the mechanisms by which damaged or senescing chloroplasts are identified, transported to the central vacuole, and ultimately degraded are not well characterized. Here, we investigated the subcellular structures involved with degrading chloroplasts induced by the ROS singlet oxygen (1O2) in the Arabidopsis thaliana plastid ferrochelatase two (fc2) mutant. Using a three-dimensional serial-block face electron microscopy analysis, we show up to 35% of degrading chloroplasts in fc2 mutants protrude into the central vacuole. While the location of a chloroplast within a cell had no effect on the likelihood of its degradation, chloroplasts in spongy mesophyll cells were degraded at a higher rate than those in palisade mesophyll cells. To determine if degrading chloroplasts have unique structural characteristics allowing them to be distinguished from healthy chloroplasts, we analyzed fc2 seedlings grown under different levels of photo-oxidative stress. A clear correlation was observed between chloroplast swelling, 1O2-signaling, and the state of degradation. Finally, plastoglobule enzymes involved in chloroplast disassembly were shown to be upregulated while plastoglobules increased their association with the thylakoid grana, implicating an interaction between 1O2-induced chloroplast degradation and senescence pathways.


title: Ultra-lightweight living structural material for enhanced stiffness and environmental sensing

abstract: Natural materials such as bone, wood, and bamboo can inspire the fabrication of stiff, lightweight structural materials. Biofilms are one of the most dominant forms of life in nature. However, little is known about their physical properties as a structural material. Here we report an Escherichia coli biofilm having a Youngs modulus close to 10 GPa with ultra-low density, indicating a high-performance structural material. The mechanical and structural characterization of the biofilm and its components illuminates its adaptable bottom-up design, consisting of lightweight microscale cells covered by a dense network of amyloid nanofibrils on the surface. We engineered E. coli such that 1) carbon nanotubes assembled on the biofilm, enhancing its stiffness to over 30 GPa, or that 2) the biofilm sensitively detected heavy metal as an example of an environmental toxin. These demonstrations offer new opportunities for developing responsive living structural materials to serve many real-world applications.

category: synthetic biology


title: The ASC Speck And NLRP3 Inflammasome Function Are Spatially And Temporally Distinct

abstract: Although considered the ternary inflammasome structure, whether the singular, perinuclear NLRP3:ASC speck is synonymous with the NLRP3 inflammasome is unclear. Herein we report that the NLRP3:ASC speck is not required for nigericin-induced inflammasome activation, but facilitates and maximizes IL-1{beta} processing. Further, the NLRP3 agonists H2O2 and MSU elicited IL-1{beta} maturation without inducing specks. Notably, caspase-1 activity is spatially distinct from the speck, occurring at multiple cytoplasmic sites. Additionally, caspase-1 activity negatively regulates speck frequency and speck size while speck numbers and IL-1{beta} processing are negatively correlated, cyclical, and can be uncoupled by NLRP3 mutations or inhibiting microtubule polymerization. Finally, when specks are present, caspase-1 is likely activated after leaving the speck structure. Thus, the speck is not the NLRP3 inflammasome itself, but is instead a dynamic structure which may amplify the NLRP3 response to weak stimuli by facilitating the formation and release of small NLRP3:ASC complexes which in turn activate caspase-1.


title: Quantification of spatial subclonal interactions enhancing the invasive phenotype of paediatric glioma

abstract: Intra-tumour heterogeneity is an intrinsic property of all cancers. In some cases, such variation can be maintained by interactions between tumour subclones with distinct molecular and phenotypic characteristics. In paediatric gliomas, interactions can take the form of enhanced invasive phenotype, a hallmark of these malignancies. However, subclonal interactions are hard to quantify and difficult to distinguish from spatial confounding factors and experimental bias. Here we combine spatial computational modelling of cellular interactions and invasion, with co-evolution experiments of clonally disassembled primary glioma lines derived at autopsy. We design a Bayesian inference framework to quantify spatial subclonal interactions between molecular and phenotypically distinct lineages with different patterns of invasion. We show how this approach could discriminate genuine subclonal interactions where one clone enhanced the invasive phenotype of another, from apparent interactions that were only due to the complex dynamics of subclones growing in space. This study provides a new approach for the identification and quantification of spatial subclonal interactions in cancer.


title: The zebra finch auditory cortex reconstructs occluded syllables in conspecific song

abstract: Sensory input provides incomplete and often misleading information about the physical world. To compensate, the brain uses internal models to predict what the inputs should be from context, experience, and innate biases (Liberman and Mattingly, 1985; Komatsu, 2006; Gilbert and Sigman, 2007; Heald and Nusbaum, 2014). For example, when speech is interrupted by noise, humans perceive the missing sounds behind the noise (Miller and Licklider, 1950; Warren, 1970; Samuel, 1996), a perceptual illusion known as phonemic (or auditory) restoration. The neural mechanisms allowing the auditory system to generate predictions that override ascending sensory information remain poorly understood. Here, we show that the zebra finch (Taeniopygia guttata) exhibits auditory restoration of conspecific song both in a behavioral task and in neural recordings from the equivalent of auditory cortex. Decoding the responses of a population of single units to occluded songs reveals the spectrotemporal structure of the missing syllables. Surprisingly, restoration occurs under anesthesia and for songs that the bird has not heard. These results show that an internal model of the general structure of conspecific vocalizations can bias sensory processing without attention.


title: The amplitude of fNIRS hemodynamic response in the visual cortex unmasks autistic traits in typically developing children

abstract: Autistic traits represent a continuum dimension across the population, with autism spectrum disorder (ASD) being the extreme end of the distribution. Accumulating evidence shows that neuroanatomical and neurofunctional profiles described in relatives of ASD individuals reflect an intermediate neurobiological pattern between the clinical population and healthy controls. This suggests that quantitative measures detecting autistic traits in the general population represent potential candidates for the development of biomarkers identifying early pathophysiological processes associated with ASD. Functional near-infrared spectroscopy (fNIRS) has been extensively employed to investigate neural development and function. In contrast, the potential of fNIRS to define reliable biomarkers of brain activity has been barely explored. Features of non-invasiveness, portability, ease of administration and low-operating costs make fNIRS a suitable instrument to assess brain function for differential diagnosis, follow-up, analysis of treatment outcomes and personalized medicine in several neurological conditions. Here, we introduce a novel standardized procedure with high entertaining value to measure hemodynamic responses (HDR) in the occipital cortex of adult subjects and children. We found that the variability of evoked HDR correlates with the autistic traits of children, assessed by the Autism-Spectrum Quotient. Interestingly, HDR amplitude was especially linked to social and communication features, representing the core symptoms of ASD. These findings establish a quick and easy strategy for measuring visually-evoked cortical activity with fNIRS that optimize the compliance of young subjects, setting the background for testing the diagnostic value of fNIRS visual measurements in the ASD clinical population.


title: Mitochondrial Respiratory Chain Protein Co-Regulation in the Human Brain

abstract: Mitochondrial respiratory chain (RC) function requires the stoichiometric interaction among dozens of proteins but their co-regulation has not been defined in the human brain. Here, using quantitative proteomics across three independent cohorts we systematically characterized the co-regulation patterns of mitochondrial RC proteins in the human dorsolateral prefrontal cortex (DLPFC). Whereas the abundance of RC protein subunits that physically assemble into stable complexes were correlated, indicating their co-regulation, RC assembly factors exhibited modest co-regulation. Within complex I, nuclear DNA-encoded subunits exhibited >2.5-times higher co-regulation than mitochondrial (mt)DNA-encoded subunits. Moreover, mtDNA copy number was unrelated to mtDNA-encoded subunits abundance, suggesting that mtDNA content is not limiting. Alzheimers disease (AD) brains exhibited reduced abundance of complex I RC subunits, an effect largely driven by a 2-4% overall lower mitochondrial protein content. These findings provide foundational knowledge to identify molecular mechanisms contributing to age- and disease-related erosion of mitochondrial function in the human brain.


title: Network expansion of genetic associations defines a pleiotropy map of human cell biology

abstract: Proteins that interact within molecular networks tend to have similar functions and when perturbed influence the same organismal traits. Interaction networks can be used to expand the list of likely trait associated genes from genome-wide association studies (GWAS). Here, we used improvements in SNP-to-gene mapping to perform network based expansion of trait associated genes for 1,002 human traits showing that this recovers known disease genes or drug targets. The similarity of network expansion scores identifies groups of traits likely to share a common genetic basis as well as the biological processes underlying this. We identified 73 pleiotropic gene modules linked to multiple traits that are enriched in genes involved in processes such as protein ubiquitination and RNA processing. We show examples of modules linked to human diseases enriched in genes with pathogenic variants found in patients or relevant mouse knock-out phenotypes and can be used to map targets of approved drugs for repurposing opportunities. Finally, we illustrate the use of the network expansion scores to study genes at inflammatory bowel disease (IBD) GWAS loci, and implicate IBD-relevant genes with strong functional and genetic support.


title: Genomic insights into the differentiated population admixture structure and demographic history of North East Asians

abstract: North China and South Siberia, mainly populated by Altaic-speaking populations, possess extensive ethnolinguistic diversity and serve as the crossroad for the initial peopling of America and western-eastern trans-continental communication. Yet, the complex scenarios of genetic origin, population structure, and admixture history of North-East Asia remain to be fully characterized, especially for Mongolic people in China with a genome-wide perspective. Thus, we genotyped genome-wide SNPs for 510 individuals from 38 Chinese Mongolic, Tungusic, and Sinitic populations to explore the sharing alleles and haplotypes within the studied groups and following merged it with 3508 modern and ancient Eurasian individuals to reconstruct the deep evolutionary and natural selection history of northern East Asians. We identified significant substructures within Altaic-speaking populations with the primary common ancestry linked to the Neolithic northern East Asians: Western Turkic people harbored more western Eurasian ancestry; Northern Mongolic people in Siberia and eastern Tungusic people in Amur River Basin (ARB) possessed dominant Neolithic Mongolian Plateau (MP) or ARB ancestry; Southern Mongolic people in China owned obvious genetic impact from Neolithic Yellow River Basin (YRB) farmers. Additionally, we found the differentiated admixture history between western and eastern Mongolians and geographically close Northeast Hans: the former received a genetic impact from western Eurasians and the latter retained the dominant YRB and ARB Neolithic ancestry. Moreover, we demonstrated that Kalmyk people from the northern Caucasus Mountain possessed a strong genetic affinity with Neolithic MP people, supporting the hypothesis of their eastern Eurasian origin and long-distance migration history. We also illuminated that historic pastoral empires in the MP contributed considerably to the gene pool of northern Mongolic people but rarely to southern ones. We finally found natural signatures in Mongolians associated with alcohol metabolism. Generally, our results not only illuminated that complex population migration and admixture of Neolithic ancestral sources from the MP or ARB played an important role in the spread of Altaic-speaking populations and Proto-Altaic language, which partly supported the Northeast Asia-origin hypothesis, but also demonstrated that the observed multi-sources of genetic diversity contributed significantly to the modern existing extensive ethnolinguistic diversity in North-East Asia.


title: Data-Driven Strain Design Using Aggregated Adaptive Laboratory Evolution Mutational Data

abstract:
Microbes are being engineered for an increasingly large and diverse set of applications. However, the designing of microbial genomes remains challenging due to the general complexity of biological system. Adaptive Laboratory Evolution (ALE) leverages natures problem-solving processes to generate optimized genotypes currently inaccessible to rational methods. The large amount of public ALE data now represents a new opportunity for data-driven strain design. This study presents a novel and first of its kind meta-analysis workflow to derive data-driven strain designs from aggregate ALE mutational data using rich mutation annotations, statistical and structural biology methods. The mutational dataset consolidated and utilized in this study contained 63 Escherichia coli K-12 MG1655 based ALE experiments, described by 93 unique environmental conditions, 357 independent evolutions, and 13,957 observed mutations. High-level trends across the entire dataset were established and revealed that ALE-derived strain designs will largely be gene-centric, as opposed to non-coding, and a relatively small number of variants (approx. 4) can significantly alter cellular states and provide benefits which range from an increase in fitness to a complete necessity for survival. Three novel experimentally validated designs relevant to metabolic engineering applications are presented as use cases for the workflow. Specifically, these designs increased growth rates with glycerol as a carbon source through a point mutation to glpK and a truncation to cyaA or increased tolerance to toxic levels of isobutyric acid through a pykF truncation. These results demonstrate how strain designs can be extracted from aggregated ALE data to enhance strain design efforts.

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category: bioengineering


title: Construction of functional biliary epithelial branched networks with predefined geometry using digital light stereolithography

abstract: Cholangiocytes, biliary epithelial cells, are known to spontaneously self-organize into spherical cysts with a central lumen. In this work, we explore a promising biocompatible stereolithographic approach to encapsulate cholangiocytes into geometrically-controlled 3D hydrogel structures to guide them towards the formation of branched tubular networks. We demonstrate that within the appropriate mix of hydrogels, normal rat cholangiocytes can proliferate, migrate and organize into branched tubular structures, form walls consisting of a cell monolayer, transport fluorescent dyes into the luminal space and show markers of epithelial maturation such as primary cilia. The resulting structures have dimensions typically found in the intralobular and intrahepatic bile ducts and are stable for weeks, without any requirement of bulk supporting material, thereby offering total access to the basal side of these biliary epithelial constructs.


title: Cell minor-axis length is a critical feature for breast cancer cell migration on straight, wavy, loop and grid microfibre patterns

abstract: Cell migration plays an important role in physiological and pathological processes where the fibrillar morphology of extracellular matrice (ECM) could regulate the migration dynamics. To mimic the morphological characteristics of fibrillar matrix structures, low-voltage continuous electrospinning was adapted to construct straight, wavy, looped and gridded fibre patterns made of polystyrene (of fibre diameter ca. 3 m). With microfibres deposited onto non-passivated surfaces, cells were permitted to explore their different shapes in response to the directly-adhered fibre, as well as to the neighbouring patterns. For all the patterns studied, analysing cellular migration dynamics of MDA-MB-231 (a highly migratory breast cancer cell line) demonstrated a switch in behaviour when the pattern features approach the upper limit of the cell minor axis. Our findings suggest that, although cells dynamically adjust their shapes in response to different fibrillar environments during migration, their ability to divert from an existing fibre track is limited by the size along the cell minor axis. We therefore conclude that the upper limit of cell minor axis might act as a guide for the design of microfibre patterns for different purposes of cell migration.


title: Absolute copy number fitting from shallow whole genome sequencing data

abstract: Low-coverage or shallow whole genome sequencing (sWGS) approaches can efficiently detect somatic copy number aberrations (SCNAs) at low cost. This is clinically important for many cancers, in particular cancers with severe chromosomal instability (CIN) that frequently lack actionable point mutations and are characterised by poor disease outcome. Absolute copy number (ACN), measured in DNA copies per cancer cell, is required for meaningful comparisons between copy number states, but is challenging to estimate and in practice often requires manual curation. Using a total of 60 cancer cell lines, 148 patient-derived xenograft (PDX) and 142 clinical tissue samples, we evaluate the performance of available tools for obtaining ACN from sWGS. We provide a validated and refined tool called Rascal (relative to absolute copy number scaling) that provides improved fitting algorithms and enables interactive visualisation of copy number profiles. These approaches are highly applicable to both pre-clinical and translational research studies on SCNA-driven cancers and provide more robust ACN fits from sWGS data than currently available tools.


title: A Quantitative Paradigm for Water Assisted Proton Transport Through Proteins and Other Confined Spaces

abstract:
Water assisted proton transport through confined spaces influences many phenomena in biomolecular and nanomaterial systems. In such cases, the water molecules that fluctuate in the confined pathways provide the environment and the medium for the hydrated excess proton migration via Grotthuss shuttling. However, a definitive collective variable (CV) that accurately couples the hydration and the connectivity of the proton wire with the proton translocation has remained elusive. To address this important challenge - and thus to define a new quantitative paradigm for facile proton transport in confined spaces - a CV is derived in this work from graph theory, which is verified to accurately describe water wire formation and breakage coupled to the proton translocation in carbon nanotubes and the Cl-/H+ antiporter protein, ClC-ec1. Significant alterations in the conformations and thermodynamics of water wires are uncovered after introducing an excess proton into them. Large barriers in the proton translocation free energy profiles are found when water wires are defined to be disconnected according to the new CV, even though the pertinent confined space is still reasonably well hydrated and - by the simple measure of the mere existence of a water structure - the proton transport would have been predicted to be facile via that oversimplified measure. In this new paradigm, however, the simple presence of water is not sufficient for inferring proton translocation since an excess proton itself is able to drive hydration and, additionally, the water molecules themselves must be adequately connected to facilitate any successful proton transport.

Significance StatementAs first proposed more than 200 years ago by Grotthuss, proton transport is enabled by a chemical bond-breaking and bond-making proton hopping mechanism through water networks or "wires", often contained within confined systems such as protein channels or nanotubes. Herein, concepts from graph theory are utilized in order to define a new continuously differentiable collective variable (CV) for water wire connectivity and facile proton transport. As such, the water connectivity can be explicitly quantified via free energy sampling, to both qualitatively and quantitatively describe the thermodynamics and kinetics of water-facilitated proton transport via Grotthuss hopping - something that has been lacking since the first conceptual identification of this key chemical process in Nature.

category: biophysics