title: Neutralization of Mu and C.1.2 SARS-CoV-2 Variants by Vaccine-elicited Antibodies in Individuals With and Without Previous History of Infection

abstract: Recently identified SARS-CoV-2 variants Mu and C.1.2 have mutations in the receptor binding domain and N- and C-terminal domains that might confer resistance to natural and vaccine-elicited antibody. Analysis with pseudotyped lentiviruses showed that viruses with the Mu and C.1.2 spike proteins were partially resistant to neutralization by antibodies in convalescent sera and those elicited by mRNA and adenoviral vector-based vaccine-elicited antibodies. Virus with the C.1.2 variant spike, which is heavily mutated, was more neutralization-resistant than that of any of variants of concern. The resistance of the C.1.2 spike was caused by a combination of the RBD mutations N501Y, Y449H and E484K and the NTD mutations. Although Mu and C.1.2 were partially resistant to neutralizing antibody, neutralizing titers elicited by mRNA vaccination remained above what is found in convalescent sera and thus are likely to remain protective against severe disease. The neutralizing titers of sera from infection-experienced BNT162b2-vaccinated individuals, those with a history of previous SARS-CoV-2 infection, were as much as 15-fold higher than those of vaccinated individuals without previous infection and effectively neutralized all of the variants. The findings demonstrate that individuals can raise a broadly neutralizing humoral response by generating a polyclonal response to multiple spike protein epitopes that should protect against current and future variants.


title: The unique pattern of mannose-capped lipoarabinomannan expression in Mycobacterium tuberculosis with different drug resistant profiles following isoniazid stress

abstract:
Tuberculosis (TB) is a global health problem caused by Mycobacterium tuberculosis (MTB) infection. The main problem of TB treatment is the emergence of drug resistance, which can occur by inappropriate of antibiotic used. Isoniazid (INH) is the first-line anti-TB drug that inhibits mycolic acid synthesis, an important part of the mycobacterial cell wall. Mannose-capped lipoarabinomannan (ManLAM) is an essential cell wall part that plays a role as an immunomodulator and acts as a virulence factor. In this study, MTB clinical isolates with different drug resistant profiles were used to determine the expression of ManLAM related genes including pimB, mptA, mptC, dprE1, dprE2 and embC by qRT-PCR. Stress-related genes including hspX, tgs1, and sigE were determined by multiplex real-time PCR with probe assay. Sanger sequencing of ManLAM related genes and genes associated with drug resistance (inhA, katG, and rpoB) were analyzed. In response to INH, the expression pattern of ManLAM related genes was different among four strains. Interestingly, MDR-TB markedly up-regulated ManLAM related genes greater than others. Stress-related genes hspX and tgs1 were significantly upregulated in MDR response to INH, whereas sigE was significantly upregulated in MDR response to RIF and INH-R. DprE1 is crucial for MTB and it is a valuable target for anti-TB drugs. RIF-R and MDR isolates show C[->]T mutation at nucleotide position 459 of the dprE1 gene leading to the same amino acid at codon 153. Codon usage analysis for DprE1 showed that RIF-R and MDR preferred ACT codon over drug sensitive strains. This work provides the expression pattern of ManLAM related genes and stress responder genes, which are key factors in the interaction between MTB and host. Moreover, ManLAM is a possible factor that plays an important role in the adaptive mechanism and the drug resistance mechanism of mycobacteria.

Author summaryThe adaptive mechanism of mycobacteria in response to stressors is an important strategy to promote their virulence and pathogenesis. This study determined the effect of antibiotic stress on Mycobacterium tuberculosis (MTB) focusing on mannose-capped lipoarabinomannan (ManLAM), which is one of the virulence factors that modulate host immune response. Multiplex real-time PCR with probe assay targeting stress responder genes and qRT-PCR targeting ManLAM related genes were performed. Isoniazid acts as a stressor to induce stress response in mycobacteria, as shown in the up-regulation of stress-related genes including hspX, tgs1, and sigE. The expression pattern of ManLAM related genes in drug resistant and drug sensitive-MTB in response to INH was different, causing a unique pattern. ManLAM related genes respond to isoniazid mostly in drug resistant strains and are present at high expression levels in INH-R and MDR. The results suggest that ManLAM is one factor involved in the adaptive mechanism of MTB response to antibiotic stress and probably associated with the emergence of MTB drug resistance. This work provides new insights into the adaptive mechanism of mycobacterial response to isoniazid that will improve understanding of how mycobacteria develop drug resistance.

category: microbiology


title: Major complex trait for early de novo programming 'CoV-MAC-TED' detected in human nasal epithelial cells infected by two SARS-CoV-2 variants is promising for designing therapeutic strategies

abstract:
BackgroundEarly metabolic reorganization was only recently recognized as essentially integrated part of immunology. In this context, unbalanced ROS/RNS levels that connected to increased aerobic fermentation, which linked to alpha-tubulin-based cell restructuration and control of cell cycle progression, was identified as major complex trait for early de novo programming ( CoV-MAC-TED) during SARS-CoV-2 infection. This trait was highlighted as critical target for developing early anti-viral/anti-SARS-CoV-2 strategies. To obtain this result, analyses had been performed on transcriptome data from diverse experimental cell systems. A call was released for wide data collection of the defined set of genes for transcriptome analyses, named  ReprogVirus, which should be based on strictly standardized protocols and data entry from diverse virus types and variants into the  ReprogVirus Platform. This platform is currently under development. However, so far an in vitro cell system from primary target cells for virus attacks that could ideally serve for standardizing data collection of early SARS-CoV-2 infection responses was not defined.

ResultsHere, we demonstrate transcriptome level profiles of the most critical  ReprogVirus gene sets for identifying  CoV-MAC-TED in cultured human nasal epithelial cells. Our results (a) validate  Cov-MAC-TED as crucial trait for early SARS-CoV-2 reprogramming for both tested virus variants and (b) demonstrate its relevance in cultured human nasal epithelial cells.

ConclusionIn vitro-cultured human nasal epithelial cells proved to be appropriate for standardized transcriptome data collection in the  ReprogVirus Platform. Thus, this cell system is highly promising to advance integrative data analyses by help of Artificial Intelligence methodologies for designing anti-SARS-CoV-2 strategies.

category: cell biology


title: Transcriptome data from human nasal epithelial cells infected by H3N2 influenza virus indicate early unbalanced ROS/RNA levels, temporarily increased aerobic fermentation linked to enhanced α-tubulin and rapid energy-dependent IRF9-marked immunization

abstract: BackgroundTranscriptome studies of a selected gene set (ReprogVirus) had identified unbalanced ROS/RNS levels, which connected to increased aerobic fermentation that linked to alpha-tubulin-based cell restructuration and cell cycle control, as a major complex trait for early de novo programming (CoV-MAC-TED) upon SARS-CoV-2 infection. Recently, CoV-MAC-TED was confirmed as promising marker by using primary target human nasal epithelial cells (NECs) infected by two SARS-CoV-2 variants with different effects on disease severity. To further explore this marker/cell system as a standardized tool for identifying anti-viral targets in general, testing of further virus types is required. Results: Transcriptome level profiles of H3N2 influenza-infected NECs indicated ROS/RNS level changes and increased transcript accumulation of genes related to glycolysis, lactic fermentation and -tubulin at 8 hours post infection. These early changes linked to energy-dependent, IRF9-marked rapid immunization. However, ReprogVirus-marker genes indicated the absence of initial cell cycle progress, which contrasted our findings during infections with two SARS-CoV-2 variants, where cell cycle progress was linked to delayed IRF9 response. Our results point to the possibility of CoV-MAC-TED-assisted, rapid individual host cell response identification upon virus infections. Conclusion: The complex trait CoV-MAC-TED can identify similar and differential early responses of SARS-CoV-2 and influenza H3N2 viruses. This indicates its appropriateness to search for anti-viral targets in view of therapeutic design strategies. For standardization, human NECs can be used. This marker/cell system is promising to identify differential early cell responses upon viral infections also depending on cell origins.


title: Inhibition of adrenergic b1-AR/Gas signaling promotes cardiomyocyte proliferation through activation of RhoA-YAP axis

abstract: The regeneration potential of the mammalian heart is incredibly limited, as cardiomyocyte proliferation ceases shortly after birth. {beta}-adrenergic receptor ({beta}-AR) blockade has been shown to improve heart functions in response to injury; however, the underlying mechanisms remain poorly understood. Here we inhibited {beta}-AR signaling in the heart using a cardiomyocyte specific {beta}1-adrenergic receptor ({beta}1-AR) blocker (metoprolol) to examine its role in heart maturation and regeneration at the neonatal stage. We found that metoprolol robustly enhanced cardiomyocyte proliferation and promoted cardiac regeneration post myocardial infarction, resulting in reduced scar formation and improved cardiac function. Moreover, the increased cardiomyocyte proliferation was also induced by the genetic deletion of Gnas, the gene encoding G protein alpha subunit (Gs), a downstream effector of {beta}-AR. Genome wide transcriptome analysis revealed that the cardiomyocytes of {beta}-blocker treated and Gnas cKO hearts maintained an immature proliferating status even at the young-adult age, and that the loss of Gs function enhanced the activity of the Hippo-effector YAP, which is associated with immature cardiomyocyte proliferation. Moreover, the increased YAP activity is modulated by RhoA signaling. Our pharmacological and genetic studies reveal a previously unrecognized {beta}1-AR-Gs-YAP signaling axis for regulating cardiac regeneration. These results suggest that inhibiting {beta}-AR-Gs signaling promotes the regenerative capacity and extends the cardiac regenerative window in mice by activating YAP-mediated transcriptional programs. Thus, targeting {beta}-AR-Gs signaling may serve as a novel therapeutic target for the treatment of ischemic heart.

category: developmental biology


title: Characterization of adenine phosphoribosyltransferase (APRT) activity in Trypanosoma brucei brucei: only one of the two isoforms is kinetically active

abstract:
Human African Trypanosomiasis (HAT), also known as sleeping sickness, is a Neglected Tropical Disease endemic to 36 African countries, with approximately 70 million people currently at risk for infection. Current therapeutics are suboptimal due to toxicity, adverse side effects, and emerging resistance. Thus, both effective and affordable treatments are urgently needed. The causative agent of HAT is the protozoan Trypanosoma brucei ssp. Annotation of the T. brucei genome confirms previous observations that T. brucei is a purine auxotroph. Incapable of de novo purine synthesis, these protozoan parasites rely on purine phosphoribosyltransferases to salvage purines from their hosts for the synthesis of purine monophosphates. Complete and accurate genome annotations in combination with the identification and characterization of the catalytic activity of purine salvage enzymes enables the development of target-specific therapies in addition to providing a deeper understanding of purine metabolism in T. brucei. In trypanosomes, purine phosphoribosyltransferases represent promising drug targets due to their essential and central role in purine salvage. Enzymes involved in adenine and adenosine salvage, such as adenine phosphoribosyltransferases (APRTs, EC 2.4.2.7), are of particular interest for their potential role in the activation of adenine and adenosine-based pro-drugs. Analysis of the T. brucei genome shows two putative aprt genes: APRT1 (Tb927.7.1780) and APRT2 (Tb927.7.1790). Here we report studies of the catalytic activity of each putative APRT, revealing that of the two T. brucei putative APRTs, only APRT1 is kinetically active, thereby signifying a genomic misannotation of Tb927.7.1790 (putative APRT2). Reliable genome annotation is necessary to establish potential drug targets and identify enzymes involved in adenine and adenosine-based prodrug activation.

Author SummaryNeglected Tropical Diseases, are defined by the World Health Organization as a diverse group of 20 different diseases that disproportionally affect the worlds poorest populations, including Human African Trypanosomiasis (HAT). HAT is endemic to 36 African countries and approximately 70 million people worldwide are currently at risk for infection. Current therapeutics are suboptimal due to toxicity, adverse side effects, and emerging resistance. The causative agent of HAT is Trypanosoma brucei ssp. Unlike humans, these protozoan parasites rely on purine phosphoribosyltransferases to salvage purine bases from their hosts for the synthesis of DNA and RNA. Here we report on the characterization of adenine phosphoribosyltransferase (APRT) activity in Trypanosoma brucei brucei. Our studies reveal that of the two putative APRTs, only APRT1 is kinetically active under physiological conditions. Accurate genome annotations in combination with the characterization of purine salvage enzymes allows the development of target-specific therapies. Enzymes involved in adenine and adenosine salvage, such as APRTs, are of particular interest for their potential role in the activation of adenine and adenosine-based pro-drugs.

category: biochemistry


title: Parameter Sensitivity and Experimental Validation for Fractional-Order Dynamical Modeling of Neurovascular Coupling

abstract:
GoalNeurovascular coupling is a fundamental mechanism linking neural activity to cerebral blood flow (CBF) response. Modeling this coupling is very important to understand brain functions, yet challenging due to the complexity of the involved phenomena. One key feature that different studies have reported is the time delay that is inherently present between the neural activity and cerebral blood flow, which has been described by adding a delay parameter in standard models. An alternative approach was recently proposed where the framework of fractional-order modeling is employed to characterize the complex phenomena underlying the neurovascular. Thanks to its nonlocal property, a fractional derivative is suitable for modeling delayed and power-law phenomena.

MethodsIn this study, we analyzed and validated an effective fractional-order for the effective modeling and characterization of the neurovascular coupling mechanism. To show the added value of the fractional order parameters of the proposed model, we perform a parameter sensitivity analysis of the fractional model compared to its integer counterpart. Moreover, the model was validated using neural activity-CBF data related to both event and block design experiments that were acquired using electrophysiology and laser Doppler flowmetry recordings, respectively.

ResultsThe validation results show the aptitude and flexibility of the fractional-order paradigm in fitting a more comprehensive range of well-shaped CBF response behaviors while maintaining a low model complexity. Comparison with the standard integer-order models shows the added value of the fractional-order parameters in capturing various key determinants of the cerebral hemodynamic response, e.g., post-stimulus undershoot.

ConclusionsThis investigation authenticates the ability and adaptability of the fractional-order framework to characterize a wider range of well-shaped cerebral blood flow responses while preserving low model complexity through a series of unconstrained and constrained optimizations.

Impact StatementThe present study proposes a novel fractional-order framework for modeling neurovascular coupling. A parameter sensitivity analysis demonstrates the potential flexibility, and effectiveness of the fractional-order paradigm in reconstructing the cerebral hemodynamics with manageable complexity; and a real experimental validation analysis demonstrates the ability of the model in modeling a wider range of well-shaped CBF responses.

category: bioengineering


title: Discovery of Potent Triple Inhibitors of Both SARS-CoV-2 Proteases and Human Cathepsin L

abstract: There are currently no FDA approved inhibitors of SARS-CoV-2 viral proteases with specific treatment for post-exposure of SARS-CoV-2. Here, we discovered inhibitors containing thiuram disulfide or dithiobis-(thioformate) tested against three key proteases in SARS CoV-2 replication including SARS CoV-2 Main Protease (Mpro), SARS CoV-2 Papain Like Protease (PLpro), and human cathepsin L. The use of thiuram disulfide and dithiobis-(thioformate) covalent inhibitor warheads was inspired by disulfiram, a currently prescribed drug commonly used to treat chronic alcoholism that at the present time is in Phase 2 clinical trials against SARS-CoV-2. At the maximal allowed dose, disulfiram is associated with adverse effects. Our goal was to find more potent inhibitors that target both viral proteases and one essential human protease to reduce the dosage and minimize the adverse effects associated with these agents. We found that compounds coded as RI175, JX 06, and RI172 are the most potent inhibitors from an enzymatic assay against SARS-CoV-2 Mpro, SARS-CoV-2 PLpro, and human cathepsin L with IC50s of 330, 250 nM, and 190 nM about 4.5, 17, and 11.5-fold more potent than disulfiram, respectively. The identified protease inhibitors in this series were also tested against SARS CoV-2 in a cell-based and toxicity assay and were shown to have similar or greater antiviral effect than disulfiram. The identified triple protease inhibitors and their derivatives are promising candidates for treatment of the Covid-19 virus and related variants.

category: pharmacology and toxicology


title: Microtubule dynamics and the evolution of mitochondrial populations in fission yeast cells: A kinetic Monte Carlo study

abstract:
Mitochondrial populations in cells are maintained by cycles of fission and fusion events. Perturbation of this balance has been observed in several diseases such as cancer and neurodegeneration. In fission yeast cells, the association of mitochondria with microtubules inhibits mitochondrial fission, [1] illustrating the intricate coupling between mitochondria and the dynamic population of microtubules within the cell. In order to understand this coupling, we carried out kinetic Monte Carlo (KMC) simulations to predict the evolution of mitochondrial size distributions for different cases; wild-type cells, cells with short and long microtubules, and cells without microtubules. Comparison are made with mitochondrial distributions reported in experiments with fission yeast cells. Using experimentally determined mitochondrial fission and fusion frequencies, simulations implemented without the coupling of microtubule dynamics predicted an increase in the mean number of mitochondria, equilibrating within 50 s. The mitochondrial length distribution in these models also showed a higher occurrence of shorter mitochondria, implying a greater tendency for fission, similar to the scenario observed in the absence of microtubules and cells with short microtubules. Interestingly, this resulted in overestimating the mean number of mitochondria and underestimating mitochondrial lengths in cells with wild-type and long microtubules. However, coupling mitochondrias fission and fusion events to the microtubule dynamics effectively captured the mitochondrial number and size distributions in wild-type and cells with long microtubules. Thus, the model provides greater physical insight into the temporal evolution of mitochondrial populations in different microtubule environments, allowing one to study both the short-time evolution as observed in the experiments (<5 minutes) as well as their transition towards a steady-state (>15 minutes). Our study illustrates the critical role of microtubules in mitochondrial dynamics and that coupling their growth and shrinkage dynamics is critical to predicting the evolution of mitochondrial populations within the cell.

Author summaryMitochondria are semi-autonomous organelles that undergo fission and fusion to facilitate quality control and exchange of mitochondrial mass within the cell. Impaired mitochondrial fusion and fission dynamics are associated with disease states such as cancer and neurodegeneration. Recent experiments in fission yeast cells revealed a reduction in mitochondrial fission events when mitochondria were bound to the microtubules and longer microtubules shifted the mitochondrial population to longer lengths. In a distinct departure from earlier reports [2-16], we develop a generic framework to study the evolution of the mitochondrial population in fission yeast cells to predict the observed mitochondrial population by coupling the microtubule and mitochondrial dynamics. Using kinetic Monte Carlo (KMC) simulations we predict the temporal evolution of mitochondria in both the mutated and wild-type states of microtubules in fission yeast cells. The mitochondrial population evolves due to multiple fission and fusion reactions occurring between mitochondrial species of various lengths. Several models with varying complexity have been developed to study mitochondrial evolution, and predictions of the mitochondrial populations agree well with experimental data on fission yeast cells without microtubules and cells with short, wild-type and long microtubules. These set of microtubule states are consistent with not only the microtubule dynamics typically observed in cells under different physiological stimuli such as mitosis and disease states but also the stable microtubule states obtained through post-translational modification of  and {beta} tubulin subunits of microtubules. Our study reveals that the temporal evolution of mitochondrial populations is an intrinsic function of the state of microtubules which modulates the fission and fusion frequencies to maintain mitochondrial homeostasis within cells.

category: systems biology


title: An intranasally administrated SARS-CoV-2 beta variant subunit booster vaccine prevents beta variant viral replication in rhesus macaques

abstract: Emerging of SARS-CoV-2 variants and waning of vaccine/infection-induced immunity poses threats to curbing the COVID-19 pandemic. An effective, safe, and convenient booster vaccine will be needed. We hypothesized that a variant-modified mucosal booster vaccine might induce local immunity to prevent SARS-CoV-2 infection at the port of entry. The beta-variant is hardest to cross-neutralize. Herein we assessed the protective efficacy of an intranasal booster composed of beta variant-spike protein S1 with IL-15 and TLR agonists in previously immunized macaques. The macaques were first vaccinated with Wuhan strain S1 with the same adjuvant. One year later, negligibly detectable SARS-CoV-2-specific antibody remained. Nevertheless, the booster induced vigorous humoral immunity including serum- and bronchoalveolar lavage (BAL)-IgG, secretory nasal- and BAL-IgA, and neutralizing antibody against the original strain and/or beta variant. Beta-variant S1-specifc CD4+ and CD8+ T cell responses were also elicited in PBMC and BAL. Following SARS-CoV-2 beta variant challenge, the vaccinated group demonstrated significant protection against viral replication in the upper and lower respiratory tracts, with almost full protection in the nasal cavity. The fact that one intranasal beta-variant booster administrated one year after the first vaccination provoked protective immunity against beta variant infections may inform future SARS-CoV-2 booster design and administration timing.

category: immunology


title: Post-entry, spike-dependent replication advantage of B.1.1.7 and B.1.617.2 over B.1 SARS-CoV-2 in an ACE2-deficient human lung cell line

abstract: Epidemiological data demonstrate that SARS-CoV-2 variants of concern (VOC) B.1.1.7 and B.1.617.2 are more transmissible and infections are associated with a higher mortality than non-VOC virus infections. Phenotypic properties underlying their enhanced spread in the human population remain unknown. B.1.1.7 virus isolates displayed inferior or equivalent spread in most cell lines and primary cells compared to an ancestral B.1 SARS-CoV-2, and were outcompeted by the latter. Lower infectivity and delayed entry kinetics of B.1.1.7 viruses were accompanied by inefficient proteolytic processing of spike. B.1.1.7 viruses failed to escape from neutralizing antibodies, but slightly dampened induction of innate immunity. The bronchial cell line NCI-H1299 supported 24- and 595-fold increased growth of B.1.1.7 and B.1.617.2 viruses, respectively, in the absence of detectable ACE2 expression and in a spike-determined fashion. Superior spread in NCI-H1299 cells suggests that VOCs employ a distinct set of cellular cofactors that may be unavailable in standard cell lines.


title: Global diversity and distribution of prophages is lineage-specific within the Ralstonia solanacearum plant pathogenic bacterium species complex

abstract: Ralstonia solanacearum is a destructive plant pathogenic bacterium and the causative agent of bacterial wilt disease, infecting over 200 plant species worldwide. In addition to chromosomal genes, its virulence is mediated by mobile genetic elements including integrated DNA of bacteriophages, i.e., prophages, which may carry fitness-associated auxiliary genes or modulate host gene expression. Although experimental studies have characterised several prophages that shape R. solanacearum virulence, the global diversity, distribution, and wider functional gene content of R. solanacearum prophages is unknown. In this study, prophages were identified in a diverse collection of 192 R. solanacearum draft genome assemblies originating from six continents. Prophages were identified bioinformatically and their diversity investigated using genetic distance measures, gene content, GC, and total length. Prophage distribution was characterised using metadata on R. solanacearum geographic origin and lineage classification (phylotypes), and their functional gene content was assessed by identifying putative prophage-encoded auxiliary genes. In total, 343 intact prophages were identified, forming ten genetically distinct clusters. These included five prophage clusters belonging to the Inoviridae, Myoviridae, and Siphoviridae phage families, and five uncharacterised clusters, possibly representing novel, previously undescribed phages. The prophages had broad geographical distribution being present across multiple continents. However, they were generally host phylogenetic lineage-specific, and overall, prophage diversity was proportional to the genetic diversity of their hosts. The prophages contained a myriad of auxiliary genes involved in metabolism and virulence of both phage and bacteria. Our results show that while R. solanacearum prophages are highly diverse globally, they make lineage-specific contributions to the R. solanacearum accessory genome, which could have resulted from shared coevolutionary history.


title: Global diversity and distribution of prophages are lineage-specific within the Ralstonia solanacearum plant pathogenic bacterium species complex


title: Deep mining of the Sequence Read Archive reveals bipartite coronavirus genomes and inter-family Spike glycoprotein recombination

abstract:
Genetic variation in RNA viruses is generated by point mutation and recombination as well as reassortment in the case of viruses with segmented genomes. While point mutation concerns only few sites per genome copy, recombination and reassortment can affect large genome regions, possibly facilitating the sudden emergence of novel traits. The contribution of recombination and reassortment to genomic plasticity and their rates remain poorly understood and might be underappreciated because of the lack of a comprehensive description of the virosphere.

Here we employed a computational approach that directly queries primary sequencing data in a highly parallelized way and involves a targeted viral genome assembly strategy. By screening more than 213,000 data sets from the Sequence Read Archive repository and using two metrics that quantitatively assess assembly quality we discovered 25 novel nidoviruses from a wide range of vertebrate hosts. These include eight fish coronaviruses with bipartite genomes, a giant 36.1 kilobase coronavirus genome with a duplicated Spike glycoprotein (S) gene, and 16 additional so far undescribed vertebrate nidoviruses. Some of these novel virus genomes encode protein domains that have not been described for nidoviruses. We provide evidence for a possible inter-family homologous recombination event involving S between ancestral bipartite coronaviruses and unsegmented tobaniviruses and report a case example of an individual fish simultaneously infected with members from both virus families. Our results shed light on the evolution and genomic plasticity of coronaviruses and identify recombinants with a possibly improved ability to cross species barriers, which might elevate their pandemic potential.


title: High-throughput identification of RNA localization elements reveals a regulatory role for A/G rich sequences

abstract: Hundreds of RNAs are enriched in the projections of neuronal cells. For the vast majority of them, though, the sequence elements within them that regulate their localization are unknown. To identify RNA elements capable of directing transcripts to neurites, we designed and deployed a massively parallel reporter assay that tested the localization regulatory ability of thousands of sequence fragments drawn from endogenous mouse 3' UTRs. We identified peaks of regulatory activity within several 3' UTRs and found that sequences derived from these peaks were both necessary and sufficient for RNA localization to neurites in mouse cells as well as active in human neurons. The active localization elements were enriched in adenosine and guanosine residues and were tens to hundreds of nucleotides long as shortening of the elements led to significantly reduced activity. Using RNA affinity and mass spectrometry, we found that the RNA-binding protein Unk was associated with the active localization elements. Depletion of Unk in cells reduced the neurite-enrichment of the elements, indicating a functional requirement for Unk in their trafficking. These results provide a framework for the unbiased, high-throughput identification of RNA elements and mechanisms that govern transcript localization.

category: molecular biology


title: Structural Basis for the Inhibition of IAPP Fibril Formation by the Hsp60 Co-Chaperonin Prefoldin

abstract: Chaperones, as modulators of protein conformational states, are key cellular actors to prevent the accumulation of fibrillar aggregates. Here, we integrated kinetic investigations with structural studies to elucidate how the ubiquitous co-chaperonin prefoldin (PFD) inhibits diabetes associated islet amyloid polypeptide (IAPP) fibril formation. We demonstrated that both human and archaeal PFD interfere similarly with the IAPP fibril elongation and secondary nucleation pathways. Using archaeal prefoldin model, we combined NMR spectroscopy with EM to establish that the inhibition of fibril formation is mediated by the binding of prefoldins coiled-coil helices to the flexible IAPP N-terminal segment accessible on the fibril surface and fibril ends. AFM demonstrates that binding of prefoldin to IAPP leads to the formation of lower amounts of aggregates, composed of shorter fibrils, clustered together. Linking structural models with observed fibrillation inhibition processes opens new perspectives for understanding the interference between natural chaperones and formation of disease-associated amyloids.

category: biophysics


title: DNA double-strand break end synapsis by DNA loop extrusion

abstract: DNA double-strand breaks (DSBs) occur every cell cycle and must be efficiently repaired. Non-homologous end joining (NHEJ) is the dominant pathway for DSB repair in G1-phase. The first step of NHEJ is to bring the two DSB ends back into proximity (synapsis). However, although synapsis is generally assumed to occur through passive diffusion, we show here that passive diffusion is unlikely to be consistent with the speed and efficiency of NHEJ observed in cells. Instead, we hypothesize that DNA loop extrusion facilitates synapsis. By combining experimentally constrained simulations and theory, we show that the simplest loop extrusion model only modestly facilitates synapsis. Instead, a loop extrusion model with targeted loading of loop extruding factors (LEFs), a small portion of long-lived LEFs as well as LEF stabilization by boundary elements and DSB ends achieves fast synapsis with near 100% efficiency. We propose that loop extrusion plays an underappreciated role in DSB repair.


title: Metastatic profiling of HER2-positive breast cancer cell lines in xenograft models

abstract:
Most studies on breast cancer metastasis have been performed using triple-negative breast cancer (TNBC) cells; thus, subtype-dependent metastatic ability of breast cancer is poorly understood. In this research, we performed intravenous injection (IVI) and intra-caudal arterial injections (CAI) using nine human epidermal growth factor receptor-2 (HER2)-positive breast cancer cell lines for evaluating their metastatic abilities. Our results showed that MDA-MB-453, UACC-893, and HCC-202 had strong bone metastatic abilities, whereas HCC-2218 and HCC-1419 did not show bone metastasis. HER2-positive cell lines could hardly metastasize to the lung through IVI. From the genomic analysis, gene signatures were extracted according to the breast cancer subtypes and their metastatic preferences. The UACC-893 cell line was identified as a useful model for the metastasis study of HER2-positive breast cancer. Combined with our previous result on brain proliferation ability, we provide a characteristic metastasis profile of HER2-positive breast cancer cell lines in this study.

Statements and DeclarationsO_ST_ABSFundingC_ST_ABSThis study was supported by JSPS KAKENHI (grant no. 18K16269: Grant-in-Aid for Early-Career Scientist to J.N.; grant no. 20J01794, Grant-in-Aid for JSPS fellows to J.N.; grant no. 20J23297, Grant-in-Aid for JSPS fellows to Y.H.) and partially supported by the grants for translational research programs from Fukushima Prefecture (S.W. and K.S.).

AuthorshipYH and KA performed the in vivo experiments and bioinformatical analyses. SW, and KS interpreted the data. YH, KA, and JN wrote the manuscript. JN conceived and designed the study. All the authors reviewed and edited the manuscript.

Competing InterestsThe authors declare that they have no competing interests.

Ethical approvalThe animal experiments were conducted under the approval of the ethics committee of Waseda University (2020-A067, 2021-A074).

category: cancer biology


title: A novel innate pathogen sensing strategy involving ubiquitination of bacterial surface proteins

abstract:
Sensing of pathogens by ubiquitination is critical for maintaining cytosolic sanctity. However, universal ubiquitination targets on bacteria, especially of proteinaceous origin, remain unidentified. Here, we unveil a novel strategy, involving recognition of degron-like motifs for identification of first protein-based ubiquitination substrates on phylogenetically distinct bacteria. Such motifs can form a new class of intra-cytosolic pathogen associated molecular patterns (PAMPs) as their incorporation enables identification of non-ubiquitin targets by host Ub-ligases. We find SCFFBW7 E3-ligase, supported by the regulatory kinase, GSK3{beta}, is crucial for effective pathogen detection and clearance. This may explain the enhanced risk of infections in Chronic Lymphocytic Leukaemia patients bearing FBXW7 mutations. We conclude that exploitation of such ubiquitous pathogen sensing strategy allows conservation of cellular resources and boost anti-microbial immunity.

One Sentence SummaryUbiquitination of bacterial surface proteins fosters sensing and clearance of diverse pathogens


title: CRISPR-Cas9 mediated knockout of SagD gene for overexpression of streptokinase in Streptococcus equisimilis

abstract:
Streptokinase is an enzyme that can break down the blood clots in some cases of myocardial infarction (Heart attack), pulmonary embolism, and arterial thromboembolism. Demand for streptokinase is high globally than the production due to increased incidences of various heart conditions. The main source of streptokinase is from various strains of Streptococcus. Expression of streptokinase in native strain Streptococcus equisimilis is limited due to the SagD inhibitor gene for production of streptokinase that needs to be knocked out in order to increase it expression. However, FasX is a small RNA (sRNA) present in group A Streptococcus species which is responsible for post-transcriptional regulation of streptokinase (ska) gene by binding at the 5 end of ska mRNA. S. equisimilis is a {beta}-hemolysin producing streptococcus bacterium (group C) containing the orthologue of FasX and natively expresses a clinically important thrombolytic streptokinase. In order to improve the stability of mRNA and increasing the expression of streptokinase which is inhibited by SagD. We used CRISPR-Cas9 to successfully knock-out of SagD gene and observed a 13.58-fold relative quantification of streptokinase expression in the mutant strain as compared to wild type. We have also demonstrated the successful target gene knockout using CRISPR-Cas9 in S. equisimilis that engineered strain can be used further for overexpression of streptokinase for therapeutic applications.

Graphical Abstract

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title: Human milk oligosaccharides reduce murine group B Streptococcus vaginal colonization with minimal impact on the vaginal microbiota

abstract:
Group B Streptococcus (GBS) colonizes the vaginal mucosa of a significant percentage of healthy women and is a leading cause of neonatal bacterial infections. Currently, pregnant women are screened in the last month of pregnancy and GBS-positive women are given antibiotics during parturition to prevent bacterial transmission to the neonate. Recently, human milk oligosaccharides (HMOs) isolated from breastmilk were found to inhibit GBS growth and biofilm formation in vitro, and women that make certain HMOs are less likely to be vaginally colonized with GBS. Using in vitro human vaginal epithelial cells and a murine vaginal colonization model, we tested the impact of HMO treatment on GBS burdens and the composition of the endogenous microbiota by 16S rRNA amplicon sequencing. HMO treatment reduced GBS vaginal burdens in vivo with minimal alterations to the vaginal microbiota. HMOs displayed potent inhibitory activity against GBS in vitro, but HMO pretreatment did not alter adherence of GBS or the probiotic Lactobacillus rhamnosus to human vaginal epithelial cells. Additionally, disruption of a putative GBS glycosyltransferase ({Delta}san_0913) rendered the bacterium largely resistant to HMO inhibition in vitro and in vivo but did not compromise its adherence, colonization, or biofilm formation in the absence of HMOs. We conclude that HMOs are a promising therapeutic bioactive to limit GBS vaginal colonization with minimal impacts on the vaginal microenvironment.

IMPORTANCEDuring pregnancy, GBS ascension into the uterus can cause fetal infection or preterm birth. Additionally, GBS exposure during labor creates a risk of serious disease in the vulnerable newborn and mother postpartum. Current recommended prophylaxis consists of administering broad-spectrum antibiotics to GBS-positive mothers during labor. Although antibiotics have significantly reduced GBS neonatal disease, there are several unintended consequences including altered neonatal gut bacteria and increased risk for other types of infection. Innovative preventions displaying more targeted antimicrobial activity, while leaving the maternal microbiota intact, are thus appealing. Using a mouse model, we found that human milk oligosaccharides (HMOs) reduce GBS burdens without perturbing the vaginal microbiota. We conclude that HMOs are a promising alternative to antibiotics to reduce GBS neonatal disease.


title: In vitro analysis of the effects of acetaminophen, ibuprofen and aspirin on motility of female adult Onchocerca volvulus worms

abstract: In view of the very ambitious global timelines for elimination of onchocerciasis in 2030, the search for alternative antifilarials cannot depend on drug development from scratch, and repurposed drugs offer cheaper and faster alternatives. Previous studies had demonstrated the presence and potential expression of amidase, an enzyme that can be targeted by repurposed analgesics, in Onchocerca volvulus worms. The aim of this study was to determine the effects of acetaminophen, ibuprofen and aspirin on the motility of adult O. volvulus worms. In total, thirty (30) female O. volvulus worms were exposed to acetaminophen, ibuprofen and aspirin in concentrations of either 5mg/ml, 2.5mg/ml, 1.25mg/ml or 0.63mg/ml control in duplicates. Worm motility was observed and recorded using the WormAssay software and a darkfield imaging apparatus starting on day 2 of incubation and ending on day 8. Acetaminophen, ibuprofen and aspirin inhibited O. volvulus motility by 2-fold compared to control in the first 24 hours of drug exposure. However, an extended exposure of the worms to these test drugs rather improved the motility of the worms. The study has demonstrated that a 24-hour exposure of O. volvulus worms to the analgesic drugs studied, results in significant inhibition in worm motility compared with the control group, but extended duration of exposure led to an enhancement in motility of the previously immobile worms. This finding supports the idea that aspirin and ibuprofen may have some longevity enhancing properties. Further research on the utility of these analgesics as possible anti-filarial drugs is thus warranted.


title: Nutritional dependence of sperm mitochondrial metabolism and small RNA biogenesis

abstract:
A wide spectrum of exogenous factors, including diet, environmental pollutants, stress, and seasonal changes have major impact on sperm quality and function. The molecular basis, however, that explains this susceptibility remains largely unknown. Using a combination of proteomics and small RNA (sRNA) sequencing, we show that Drosophila sperm display rapid molecular changes in response to dietary sugar, both in terms of metabolic/redox proteins and sRNA content, particularly miRNA and mitochondria derived sRNA (mt-sRNA). Thus, results from two independent omics point at the dynamics of mitochondria as the central aspect in rapid metabolic adjustments in sperm. Using specific stains and in vivo redox reporter flies, we show that diet indeed rapidly alters the production of mitochondrial derived reactive oxygen species (ROS). Quenching ROS via supplementation of N acetyl cysteine reduces diet-upregulated miRNA, but not mitochondrial-sRNA. Together, these results open new territories in our search for the mechanistic understanding of sperm health and disease.

HighlightsO_LIDiet rapidly changes the proteomic and sRNA profiles in sperm
C_LIO_LIDiet sensitive sperm proteins are found in human infertility studies
C_LIO_LISperm mitochondrial ROS levels are modulated by diet
C_LIO_LIdme-miR-10 regulation is secondary to diet-induced ROS
C_LIO_LIDiet, but not diet-induced ROS, alters the expression of mitochondrial small RNA, especially tsRNA
C_LI


title: Limited specificity of molecular interactions incurs an environment-dependent fitness cost in bacteria

abstract: Reliable operation of cellular programs depends crucially on the specificity of biomolecular interactions. In gene regulatory networks, the appropriate expression of genes is determined through the specific binding of transcription factors (TFs) to their cognate DNA sequences. However, the large genomic background likely contains many DNA sequences showing similarity to TF target motifs, potentially allowing for substantial non-cognate TF binding with low specificity. Whether and how non-cognate TF binding impacts cellular function and fitness remains unclear. We show that increased expression of different transcriptional regulators in Escherichia coli and Salmonella enterica can significantly inhibit population growth across multiple environments. This effect depends upon (i) TF binding to a large number of DNA sequences with low specificity, (ii) TF cooperativity, and (iii) the ratio of TF to DNA. DNA binding due to the limited specificity of promiscuous or non-native TFs can thus severely impact fitness, giving rise to a fundamental biophysical constraint on gene regulatory design and evolution.


title: C/EBPα acts as an RNA binding protein essential for its chromatin recruitment and promotes Macrophage differentiation

abstract:
C/EBP has known to be a transcription factor that involved in Neutrophil differentiation for decades. However, exploring the Chromatin RNA Immunoprecipitation Sequencing (RIP), we discover that C/EBP is a RNA binding protein mainly interacts with RNA introns. Structure study and RNA electrophoretic mobility shift assay (REMSA) show that C/EBP interacts with RNA through two novel RNA binding domains distinct from its DNA binding domain. Mouse bone marrow transplantation and in vitro cytokine assay reveal that C/EBP RNA binding is critical for Macrophage differentiation but not Neutrophil differentiation. Mechanically, RNA binding domains control specific gene transcription. In particular, PU.1 intron 4 RNA interacts with C/EBP and recruit C/EBP to its enhancer site, which facilitate PU.1 expression. Taken together, C/EBP is demonstrated to be a RNA binding protein with unique function distinct from its DNA binding activity. Our finding transforms our knowledge of transcriptional regulation by transcription factor.

One-Sentence SummaryC/EBP interacts with RNA through novel RNA binding domains and regulates PU.1 expression during Macrophage differentiation.


title: Cholesterol biosynthetic pathway induces cellular senescence through ERRa

abstract: Cellular senescence is a cell program induced by various stresses that leads to a stable proliferation arrest and to a senescence-associated secretory phenotype. Accumulation of senescent cells during age-related diseases participates in these pathologies and regulates healthy lifespan. Recent evidences point out a global dysregulated intracellular metabolism associated to senescence phenotype. Nonetheless, the functional contribution of metabolic homeostasis in regulating senescence is barely understood. In this work, we describe how the mevalonate pathway, an anabolic pathway leading to the endogenous biosynthesis of poly-isoprenoids, such as cholesterol, acts as a positive regulator of cellular senescence in normal human cells. Mechanistically, this mevalonate-induced senescence is partly mediated by the downstream cholesterol biosynthetic pathway. This pathway promotes transcriptional activity of ERR leading to dysfunctional mitochondria, ROS production, DNA damage and a p53-dependent senescence. Supporting the relevance of these observations, increase of senescence in liver due to a high-fat diet regimen is abrogated in ERR knockout mouse. Overall, this work unravels the role of cholesterol biosynthesis in the induction of an ERR-dependent mitochondrial program leading to cellular senescence and related pathological alterations.


title: Lysosomes dock to omegasomes and ER-connected Phagophores During DNAJB12 and GABARAP-Dependent Selective-Autophagy of Misfolded P23H-Rhodopsin

abstract: We report on how the ER-associated-autophagy pathway (ERAA) delivers P23H-rhodopsin (P23H-R) to the lysosome. P23H-R accumulates in an ERAD-resistant conformation that is stabilized by DNAJB12 and Hsp70. P23H-R, DNAJB12, and FIP200 co-localize in discrete foci that punctuate the rim of omegasome rings coated by WIPI1. P23H-R tubules thread through the wall of WIPI1 rings into their central cavity. Transfer of P23H-R from ER-connected phagophores to lysosomes requires GABARAP, and is associated with the transient docking of lysosomes to WIPI1 rings. Instances of lysosomes docking to WIPI1 foci are constitutive, and increase 250% upon P23H-R expression. After departure from WIPI1 rings, new patches of P23H-R are seen in the membranes of lysosomes. The absence of GABARAP prevents transfer of P23H-R from phagophores to lysosomes without interfering with docking. These data identify lysosome docking to omegasomes as an important step in the DNAJB12 and GABARAP-dependent autophagic disposal of dominantly toxic P23H-R.


title: HiIDDD: A high-throughput imaging pipeline for the quantitative detection of DNA damage in primary human immune cells

abstract: DNA damage is a prominent biomarker for numerous diseases, including cancer and aging. Detection of DNA damage routinely relies on traditional microscopy or cytometric methods. However, these techniques are typically of limited throughput and are not ideally suited for large-scale longitudinal and population studies that require analysis of large sample sets. We have developed HiIDDD (High-throughput Immune cell DNA Damage Detection), a robust, semiquantitative and single-cell assay that measures DNA damage by high-throughput imaging using the two major DNA damage markers 53BP1 and {gamma}-H2AX. We demonstrate sensitive detection of DNA damage in a wide set of freshly isolated and cryopreserved primary human immune cells, including CD4+ and CD8+ T cells, B cells and monocytes with low inter-assay variability. As proof of principle, we demonstrate parallel batch processing of several immune cell types from multiple donors. We find common patterns of DNA damage in multiple immune cell types of donors of varying ages, suggesting that immune cell properties are specific to individuals. These results establish a novel high-throughput assay for the evaluation of DNA damage in large-scale studies.


title: The Polo kinase Cdc5 is regulated at multiple levels in the adaptation response to telomere dysfunction

abstract: Telomere dysfunction activates the DNA damage checkpoint to induce a cell cycle arrest. After an extended period of time, however, cells can bypass the arrest and undergo cell division despite the persistence of the initial damage, a process called adaptation to DNA damage. The Polo kinase Cdc5 in Saccharomyces cerevisiae is essential for adaptation and for many other cell-cycle processes. How the regulation of Cdc5 in response to telomere dysfunction relates to adaptation is not clear. Here, we report that Cdc5 protein level decreases after telomere dysfunction in a Mec1-, Rad53- and Ndd1-dependent manner. This regulation of Cdc5 is important to maintain long-term cell cycle arrest but not for the initial checkpoint arrest. We find that both Cdc5 and the adaptation-deficient mutant protein Cdc5-ad are heavily phosphorylated and several phosphorylation sites modulate adaptation efficiency. The PP2A phosphatases are involved in Cdc5-ad phosphorylation status and contribute to adaptation mechanisms. We finally propose that Cdc5 orchestrates multiple cell cycle pathways to promote adaptation.

category: genetics


title: Genome-wide distribution of Rad26 and Rad1-Rad10 reveals their relationship with Mediator and RNA polymerase II

abstract: Transcription is coupled with DNA repair, especially within nucleotide excision repair (NER). Mediator is a conserved coregulator playing a key role in RNA polymerase (Pol) II transcription. Mediator also links transcription and NER via a direct contact with Rad2/XPG endonuclease. In this work, we analyzed the genome-wide distribution of Rad26/CSB and that of Rad1-Rad10/XPF-ERCC1, addressing the question on a potential interplay of these proteins with Mediator and Pol II in yeast Saccharomyces cerevisiae. Our genome-wide analyses show that Rad1-Rad10 and Rad26 are present on the yeast genome in the absence of genotoxic stress, especially on highly transcribed regions, with Rad26 binding strongly correlating with that of Pol II. Moreover, we revealed that Rad1-Rad10 and Rad26 colocalize with Mediator on intergenic regions and physically interact with this complex. Using kin28 TFIIH mutant, we showed that Mediator stabilization on core promoters lead to an increase in Rad1-Rad10 chromatin binding, whereas Rad26 occupancy is less impacted by Mediator and follows mainly a decrease in Pol II transcription. Combined with multivariate analyses, our results reveal the interplay between Rad1-Rad10, Rad26, Mediator and Pol II, modulated by the binding dynamics of Mediator and Pol II transcription. In conclusion, we extend the Mediator link to Rad1-Rad10 and Rad26 NER proteins and reveal important differences in Mediator relationships with Rad2, Rad1-Rad10 and Rad26. Our work thus contributes to new concepts of the functional interplay between transcription and DNA repair, relevant for human diseases including cancer and XP/CS syndromes.

category: genomics


title: Dynamic chromosomal interactions and control of heterochromatin positioning by Ki67

abstract: Ki67 forms a protective layer around mitotic chromosomes and is involved in genome positioning around nucleoli during interphase. However, a lack of genomic interaction maps has hampered a detailed understanding of the role of Ki67 in chromatin organization. We used pA-DamID to map genome-Ki67 interactions in three human cell lines and throughout the cell cycle. In mitosis, Ki67 shows a surprising preference for the distal 15-30 Mb of each chromosome. Early in interphase, when Ki67 is present in pre-nucleolar bodies, this is replaced by a pattern of large domains that overlaps with late-replicating DNA, which gradually shifts towards small chromosomes. Nucleolar perturbations indicate that these cell cycle dynamics correspond to nucleolar maturation during interphase. Furthermore, Ki67 competes with the nuclear lamina for interaction with late-replicating DNA, and controls replication timing at (peri)centromeric regions. Together, these results reveal a highly dynamic choreography of genome interactions and roles for Ki67 in genome organization.


title: A Tissue-Bioengineering Strategy for Modeling Rare Human Kidney Diseases In Vivo

abstract: The lack of animal models for certain human diseases precludes our understanding of disease mechanisms and our ability to test new therapies in vivo. Here we generated kidney organoids from Tuberous Sclerosis Complex (TSC) patient-derived-hiPSCs to recapitulate a rare kidney tumor called angiomylipoma (AML). Organoids derived from TSC2-/- hiPSCs but not from isogenic TSC2+/- or TSC2+/+ hiPSCs shared a common transcriptional signature and a myomelanocytic cell phenotype with kidney AMLs, and developed epithelial cysts, replicating two major TSC-associated kidney lesions driven by genetic mechanisms that cannot be robustly and consistently recapitulated with transgenic mice. Transplantation of multiple TSC2-/- kidney organoids into the kidneys of immunodeficient rats allowed us to recapitulate AML and cystic kidney disease in vivo, in a scalable fashion and with fidelity, and to test the efficiency of rapamycin-loaded nanoparticles as a novel approach to ablate AMLs by inducing apoptosis triggered by mTOR-inhibition. Collectively, these methods represent a novel tissue-bioengineering strategy for rare disease modeling in vivo.


title: Autophagy-dependent TOR reactivation drives fungal growth in living host rice cells

abstract: Eukaryotic filamentous plant pathogens with biotrophic growth stages like the devastating hemibiotrophic rice blast fungus Magnaporthe oryzae grow for extended periods in living host plant cells without eliciting defense responses. M. oryzae elaborates invasive hyphae (IH) that grow in and between living rice cells while separated from host cytoplasm by plant-derived membrane interfaces. However, although critical to the plant infection process, the molecular mechanisms and metabolic strategies underpinning this intracellular growth phase are poorly understood. Eukaryotic cell growth depends on activated target-of-rapamycin (TOR) kinase signaling, which inhibits autophagy. Here, using live-cell imaging coupled with plate growth tests and RNAseq, proteomic, quantitative phosphoproteomics and metabolic approaches, we show how cycles of autophagy in IH modulate TOR reactivation via -ketoglutarate to sustain biotrophic growth and maintain biotrophic interfacial membrane integrity in host rice cells. Deleting the M. oryzae serine-threonine protein kinase Rim15-encoding gene attenuated biotrophic growth, disrupted interfacial membrane integrity and abolished the in planta autophagic cycling we observe here for the first time in wild type.{Delta} rim15 was also impaired for glutaminolysis and depleted for -ketoglutarate. -ketoglutarate treatment of{Delta} rim15-infected leaf sheaths remediated{Delta} rim15 biotrophic growth. In WT, -ketoglutarate treatment suppressed autophagy. -ketoglutarate signaling is amino acid prototrophy- and GS-GOGAT cycle-dependent. We conclude that, following initial IH elaboration, cycles of Rim15- dependent autophagic flux liberate -ketoglutarate - via the GS-GOGAT cycle - as an amino acid-sufficiency signal to trigger TOR reactivation and promote fungal biotrophic growth in nutrient-restricted host rice cells.

category: plant biology


title: Dlk1-Dio3 cluster miRNAs regulate mitochondrial functions in Duchenne muscular dystrophy

abstract:
BackgroundDuchenne Muscular Dystrophy (DMD) is a severe muscle disease caused by impaired expression of dystrophin. While mitochondrial dysfunction is thought to play an important role in DMD, the mechanism of this dysfunction remains to be clarified. We recently identified in DMD and in other muscular dystrophies the upregulation of a large number of the Dlk1-Dio3 clustered miRNAs (DD-miRNAs), in both the muscle and the serum. The objective of the present study was to define the biological functions of DD-miRNAs in skeletal muscle, particularly in the context of muscular dystrophy.

MethodsDD-miRNAs expression pattern was characterized in vitro and in vivo, in normal and dystrophic situations. Epigenomic characterization was performed, to elucidate the molecular control of DD-miRNAs dysregulation. The biological effect of muscle DD-miRNAs dysregulation was investigated by an in vivo simultaneous overexpression of 14 DD-miRNAs in the wild-type muscle, together with CRISPR-Cas9-based knockdown of the entire DD-miRNA cluster in an iPS-derived myotubes. Omics data and bioinformatics tools were used for the prediction of DD-miRNAs biological functions, and functional characterization of mitochondrial pathways was performed.

ResultsWe found that DD-miRNAs dysregulation is not specific to DMD since observed in mouse models for other muscular dystrophies. We showed that DD-miRNAs expression in mdx, is reduced in satellite cells, but highly upregulated in regenerating myofibers, suggesting a myofibers origin of DD6miRNA upregulation in muscular dystrophy in both muscles and serum. We demonstrated that upregulation of DD-miRNAs in the dystrophic muscle is controlled epigenetically by DNA and histone methylation (p<0.0001 and p=0.001, respectively) at the Intergenic Differentially Methylated Region (IG-DMR) of Dlk1-Dio3 locus. Transcriptomic analysis revealed a substantial overlap between the dystrophic muscle of the mdx mouse and the normal muscle that overexpressed 14 DD-miRNAs. Bioinformatics analysis predicted that DD-miRNAs could regulate mitochondrial functions. The ectopic overexpression of 14 DD-miRNAs, in the healthy muscle, resulted in a drastic downregulation of mitochondrial oxidative phosphorylation (OxPhos) (NES=-2.8, p=8.7E-17), similarly to the level in dystrophic muscles of mdx mice and DMD patients (NES=-2.88, p=7.7E-28). Knocking down the entire DD-miRNA cluster in iPS-derived myotubes resulted in increased mitochondrial OxPhos expression and activities.

ConclusionsThe present study provides evidence for the modulation of mitochondrial activity in the dystrophic muscle by the upregulated DD-miRNAs and supports an updated model for mitochondrial dysfunction in DMD. The regulation of mitochondrial OxPhos by DD-miRNAs may have a broader impact beyond DMD in physiological and pathological situations of muscle adaptation and regeneration.

category: physiology


title: Locational memory of macrovessel vascular cells is transcriptionally imprinted

abstract: The locational predisposition of vascular pathologies illustrates the need for a better insight into vascular heterogeneity. To investigate the transcriptomic basis of angiodiversity, we isolated and analyzed transcriptomes from endothelial cells and vascular smooth muscle cells from nine different adult canine macrovessels: the aorta, coronary artery, vena cava, portal vein, femoral artery, femoral vein, saphenous vein, pulmonary vein, and pulmonary artery. We identified both reported and novel expression patterns defining specialized adult blood vessels. Our findings also show that adult vascular cells in culture express a remarkably high number of transcription factors crucial to organ development in the embryo. The persistent expression of these genes in culture indicates that these genes are not regulated by the flow or surrounding cell types but are rather fixed in the molecular memory. Therefore, our findings prompt the re-thinking of the extrapolation of results from single-origin endothelial cell systems.


title: The superior salinity tolerance of wheat cultivar Shanrong No. 3 cannot be attributed to elevated Ta-sro1 poly(ADP-ribose) polymerase activity

abstract: Saline soils limit the production of important staple crops such as wheat, particularly in arid and semiarid regions. Salt tolerance is a multi-gene trait and this complicates breeding of wheat varieties that deliver high yields under saline soil conditions. Notably, the elevated salinity tolerance of wheat cultivar Shanrong No. 3 (SR3) has been linked to a specific proteoform of the wheat SIMILAR TO RCD1 ONE (SRO1) protein that was created in an asymmetric genome hybridization with tall wheat grass. The two amino acid polymorphisms of the Ta-sro1 proteoform enhance the poly(ADP-ribose) polymerase (PARP) activity of the protein suggesting that altered poly-ADP-ribosylation of unknown substrate proteins or nucleic acids underlie the elevated salinity tolerance of cultivar SR3. To elucidate the molecular basis for the elevated PARP activity of the Ta-sro1 proteoform we solved a crystal structure of the catalytic PARP domain. Surprisingly, the structure revealed that the postulated binding site for the co-substrate NAD+ substantially differs from the structurally conserved NAD+ binding sites of canonical PARP enzymes. Consistently, we find that Ta-sro1 does not bind NAD+ and lacks ADP-ribosyltransferase activity. Therefore, although the structure revealed that one of the polymorphic amino acids is located close to the proposed active site, the elevated salinity tolerance of cultivar SR3 cannot be explained by altered ADP-ribosyltransferase activity of Ta-sro1.


title: Executive and Attentional Resting-State Hubs of the Human Cortical Connectome Revealed by a Causal Discovery Method for Data-Driven Effective Connectivity Analysis

abstract: We demonstrate a data-driven approach for calculating a "causal connectome" of directed connectivity from resting-state fMRI data using a greedy adjacency search and pairwise non-Gaussian edge orientations. We used this approach to construct n=442 causal connectomes. These connectomes were very sparse in comparison to typical Pearson correlation-based graphs (roughly 2.25% edge density) yet were fully connected in nearly all cases. Prominent highly connected hubs of the causal connectome were situated in attentional (dorsal attention) and executive (frontoparietal and cingulo-opercular) networks. These hub networks had distinctly different connectivity profiles: attentional networks shared incoming connections with sensory regions and outgoing connections with higher cognitive networks, while executive networks primarily connected to other higher cognitive networks and had a high degree of bidirected connectivity. Virtual lesion analyses accentuated these findings, demonstrating that attentional and executive hub networks are points of critical vulnerability in the human causal connectome. These data highlight the central role of attention and executive control networks in the human cortical connectome and set the stage for future applications of data-driven causal connectivity analysis in psychiatry.

category: neuroscience


title: Executive and Attentional Information Transfer Hubs of the Human Causal Connectome


title: The Resting-State Causal Human Connectome is Characterized by Hub Connectivity of Executive and Attentional Networks


title: Abstract representations emerge naturally in neural networks trained to perform multiple tasks

abstract: Humans and other animals demonstrate a remarkable ability to generalize knowledge across distinct contexts and objects during natural behavior. We posit that this ability depends on the geometry of the neural population representations of these objects and contexts. Specifically, abstract, or disentangled, neural representations - in which neural population activity is a linear function of the variables important for making a decision - are known to allow for this kind of generalization. Further, recent neurophysiological studies have shown that the brain has sufficiently abstract representations of some sensory and cognitive variables to enable generalization across distinct contexts. However, it is unknown how these abstract representations emerge. Here, using feedforward neural networks, we demonstrate a simple mechanism by which these abstract representations can be produced: The learning of multiple distinct classification tasks. We demonstrate that, despite heterogeneity in the task structure, abstract representations that enable reliable generalization can be produced from a variety of different inputs - including standard nonlinearly mixed inputs, inputs that mimic putative representations from early sensory areas, and even simple image inputs from a standard machine learning data set. Thus, we conclude that abstract representations of sensory and cognitive variables emerge from the multiple behaviors that animals exhibit in the natural world, and may be pervasive in high-level brain regions. We make several specific predictions about which variables will be represented abstractly as well as show how these representations can be detected.


title: Combination of structural and functional connectivity explains unique variation in specific domains of cognitive function.

abstract:
The relationship between structural and functional brain networks has been characterised as complex: the two networks mirror each other and show mutual influence but they also diverge in their organisation. This work explored whether a combination of structural and functional connectivity can improve predictive models of cognitive performance. Principal Component Analysis (PCA) was first applied to cognitive data from the Human Connectome Project to identify components reflecting five cognitive domains: Executive Function, Self-regulation, Language, Encoding and Sequence Processing. A Principal Component Regression (PCR) approach was then used to fit predictive models of each cognitive domain based on structural (SC), functional (FC) or combined structural-functional (CC) connectivity. Self-regulation, Encoding and Sequence Processing were best modelled by FC, whereas Executive Function and Language were best modelled by CC. The present study demonstrates that integrating structural and functional connectivity can help predict cognitive performance, but that the added explanatory value may be (cognitive) domain-specific. Implications of these results for studies of the brain basis of cognition in health and disease are discussed.

HighlightsO_LIWe assessed the relationship between cognitive domains and structural, functional and combined structural-functional connectivity.
C_LIO_LIWe found that Executive Function and Language components were best predicted by combined models of functional and structural connectivity.
C_LIO_LISelf-regulation, Encoding and Sequence Processing were best predicted by functional connectivity alone.
C_LIO_LIOur findings provide insight into separable contributions of functional, structural and combined connectivity to different cognitive domains.
C_LI


title: How development affects evolution

abstract: Natural selection acts on developmentally constructed phenotypes. Ever since the modern synthesis, many researchers have called for integration of development into evolution, but there has been a lack of general mathematical frameworks explicitly integrating the two. This has likely inhibited understanding of the evolutionary effects of development. Here we use a new mathematical framework that integrates development into evolution to analyse how development affects evolution. We show that, whilst selection pushes genetic and phenotypic evolution up the fitness landscape, development determines the admissible evolutionary pathway, such that evolutionary outcomes occur at path peaks rather than landscape peaks. Changes in development can generate path peaks, triggering adaptive radiations, even on constant, single-peak landscapes. Phenotypic plasticity, niche construction, extra-genetic inheritance, and developmental bias alter the evolutionary path and hence the outcome. Thus, extra-genetic inheritance can have permanent evolutionary effects by changing development and so the evolutionary path, even if extra-genetically acquired elements are not transmitted to future generations. Selective development, whereby phenotype construction points in the adaptive direction, may induce adaptive or maladaptive evolution depending on the developmental constraints. Moreover, developmental propagation of phenotypic effects over age enables the evolution of negative senescence. Overall, we find that development plays a major evolutionary role.

category: evolutionary biology


title: Long-range regulatory effects of Neandertal DNA in modern humans

abstract:
The admixture between modern humans and Neandertals has resulted in [~]2% of the genomes of present-day non-Africans being composed of Neandertal DNA. Association studies have shown that introgressed DNA significantly influences phenotypic variation in people today and that several of the phenotype-associated archaic variants had links to expression regulation as well. In general, introgressed DNA has been demonstrated to significantly affect the transcriptomic landscape in people today. However, little is known about how much of that impact is mediated through long-range regulatory effects that have been shown to explain [~]20% of expression variation.

Here we identified 60 transcription factors (TFs) with their top cis-eQTL SNP being of Neandertal ancestry in GTEx and predicted long-range Neandertal DNA-induced regulatory effects by screening for the predicted target genes of those TFs. We show that genes in regions devoid of Neandertal DNA are enriched among the target genes of some of these TFs. Furthermore, archaic cis-eQTLs for these TFs included multiple candidates for local adaptation and have associations with various immune traits, schizophrenia, blood cell type composition and anthropometric measures. Finally, we show that our results can be replicated in empirical trans-eQTLs with Neandertal variants.

Our results suggest that the regulatory reach of Neandertal DNA goes beyond the 40% of genomic sequence that it still covers in present-day non-Africans and that via this mechanism Neandertal DNA additionally influences the phenotypic variation in people today.


title: Adrenergic signaling controls early transcriptional programs during CD8+ T cell responses to viral infection

abstract: Viral infections drive the expansion and differentiation of responding CD8+ T cells into variegated populations of cytolytic effector and memory cells. While pro-inflammatory cytokines and cell surface immune receptors play a key role in guiding T cell responses to infection, T cells are also markedly influenced by neurotransmitters. Norepinephrine is a key sympathetic neurotransmitter, which acts to suppress CD8 + T cell cytokine secretion and lytic activity by signaling through the {beta}2-adrenergic receptor (ADRB2). Although ADRB2 signaling is considered generally immunosuppressive, its role in regulating differentiation of effector T cells in response to infection has not been investigated. Using an adoptive transfer approach, we compared the expansion and differentiation of wild type (WT) to Adrb2-/- CD8 + T cells throughout the primary response to vesicular stomatitis virus (VSV) infection in vivo. We measured the dynamic changes in transcriptome profiles of antigen-specific CD8 + T cells as they responded to VSV. Within the first 7 days of infection, WT cells out-paced the expansion of Adrb2-/- cells, which correlated with reduced expression of IL-2 and the IL-2R in the absence of ADRB2. RNASeq analysis identified over 300 differentially expressed genes that were both temporally regulated following infection and selectively regulated in WT vs Adrb2-/- cells. These genes contributed to major transcriptional pathways including cytokine receptor activation, signaling in cancer, immune deficiency, and neurotransmitter pathways. By parsing genes within groups that were either induced or repressed over time in response to infection, we identified three main branches of genes that were differentially regulated by the ADRB2. These gene sets were predicted to be regulated by specific transcription factors involved in effector T cell development, such as Tbx21 and Eomes. Collectively, these data demonstrate a significant role for ADRB2 signaling in regulating key transcriptional pathways during CD8 + T cells responses to infection that may dramatically impact their functional capabilities and downstream memory cell development.


title: MS2 RNA aptamer enhances prime editing in rice

abstract: Prime editing is a universal and very promising precise genome editing technology. However, optimization of prime editor (PE) from different aspects remains vital for its use as a routine tool in plant basic research and crop molecular breeding. In this report, we tested MS2-based prime editor (MS2PE). We fused the M-MLV reverse transcriptase (RT) gene variant to the MS2 RNA binding protein gene, MCP, and allowed the MCP-RT fusion gene to co-express with the SpCas9 nickase gene, SpCas9H840A, and various engineered pegRNAs harboring MS2 RNA (MS2pegR). Compared with control PEs, MS2PEs significantly enhanced editing efficiency at four of six targets in rice protoplasts, and achieved 1.2[~]10.1-fold increase in editing efficiency at five of six targets in transgenic rice lines. Furthermore, we tested total 22 different MS2pegR scaffolds, 3 RT variants or genes, 2 MCP variants, and various combinations of the Cas9 nickase, RT, and MCP modules. Our results demonstrated an alternative strategy for enhancing prime editing.


title: Downregulation of RyR and NCX in the neonatal rat ventricular myocyte modulates cytosolic

abstract: Calcium (Ca2+) is necessary for cardiac muscle contraction. RyR, NCX, and SERCA are key regulatory protein channels for cytosolic Ca2+ in cardiac myocytes. Expression levels of these proteins are a function of development, with protein expression shifting toward the adult phenotype over time. We investigated how downregulation by siRNAs of RyR and NCX affected expression levels of complimentary proteins and the corresponding intracellular Ca2+ transients. We compared experimentally observed Ca2+ transients to those predicted by mathematical models. Experiments show RyR downregulation decreased SERCA and increased NCX protein levels. The associated Ca2+ transient had a decreased amplitude, increased time-to-peak, 50%, and 90% Ca2+ removal with respect to the control cell. NCX downregulation increased SERCA production without significant changes in RyR expression levels. The corresponding [Ca2+] transient had increased amplitude, no change in time-to-peak and 50% Ca2+ removal, and increased 90% Ca2+ removal with respect to the control cell. Computational models that accurately predict the observed experimental data suggest compensatory changes occurring in the expression levels as well as biochemical activity of the regulatory proteins.


title: Regulatory variants active in iPSC-derived pancreatic progenitor cells are associated with Type 2 Diabetes in adults

abstract: Pancreatic progenitor cells (PPC) are an early developmental multipotent cell type that give rise to mature endocrine, exocrine, and ductal cells. To investigate the extent to which regulatory variants active in PPC contribute to pancreatic complex traits and disease in the adult, we derived PPC from induced pluripotent stem cells (iPSCs) of nine unrelated individuals and generated single cell profiles of chromatin accessibility (snATAC- seq) and transcriptome (scRNA-seq). While iPSC-PPC differentiation was asynchronous and included cell types from early to late developmental stages, we found that the predominant cell type consisted of NKX6-1+ progenitors. Genetic characterization using snATAC-seq identified 86,261 regulatory variants that either displayed chromatin allelic bias and/or were predicted to affect active transcription factor (TF) binding sites. Integration of these regulatory variants with 380 fine-mapped type 2 diabetes (T2D) risk loci identified regulatory variants in 209 of these loci that are functional in iPSC-PPC, either by affecting transcription factor binding or through association with allelic effects on chromatin accessibility. The PPC active regulatory variants in 65 of these loci showed strong evidence of causally underlying the association with T2D. Our study shows that studying the functional associations of regulatory variation in iPSC-PPC enables the identification and characterization of causal SNPs for adult Type 2 Diabetes.


title: Immortalization and Functional Screening of Natively Paired Human T Cell Receptor Repertoires

abstract: Functional analyses of the T cell receptor (TCR) landscape can reveal critical information about protection from disease and molecular responses to vaccines. However, it has proven difficult to combine advanced next-generation sequencing technologies with methods to decode the peptide-major histocompatibility complex (pMHC) specificity of individual TCRs. Here we developed a new high-throughput approach to enable repertoire-scale functional evaluations of natively paired TCRs. In particular, we leveraged the immortalized nature of physically linked TCR:{beta} amplicon libraries to analyze binding against multiple recombinant pMHCs on a repertoire scale. To exemplify the utility of this approach, we also performed affinity-based functional mapping in conjunction with quantitative next-generation sequencing to track antigen-specific TCRs. These data successfully validated a new immortalization and screening platform to facilitate detailed molecular analyses of human TCRs against diverse antigen targets associated with health, vaccination, or disease.


title: CLUH controls astrin-1 expression to couple mitochondrial metabolism to cell cycle progression

abstract: Proliferating cells undergo metabolic changes in synchrony with cell cycle progression and cell division. Mitochondria provide fuel, metabolites, and ATP during different phases of the cell cycle, however it is not completely understood how mitochondrial function and the cell cycle are coordinated. CLUH is a post-transcriptional regulator of mRNAs encoding mitochondrial proteins involved in oxidative phosphorylation and several metabolic pathways. Here, we show a role of CLUH in regulating the expression of astrin, which is involved in metaphase to anaphase progression, centrosome integrity, and mTORC1 inhibition. We find that CLUH binds both the SPAG5 mRNA and its product astrin, and controls the synthesis and the stability of the full-length astrin-1 isoform. We show that CLUH interacts with astrin-1 specifically during interphase. Astrin-depleted cells show mTORC1 hyperactivation and enhanced anabolism. On the other hand, cells lacking CLUH show decreased astrin levels and increased mTORC1 signaling, but cannot sustain anaplerotic and anabolic pathways. In absence of CLUH, cells fail to grow during G1, and progress faster through the cell cycle, indicating dysregulated matching of growth, metabolism and cell cycling. Our data reveal a role of CLUH in coupling growth signaling pathways and mitochondrial metabolism with cell cycle progression.


title: Molecular pathology of a cystic fibrosis mutation is explained by loss of a hydrogen bond

abstract: The phosphorylation-activated anion channel CFTR is gated by an ATP hydrolysis cycle at its two cytosolic nucleotide binding domains, and is essential for epithelial salt-water transport. A large number of CFTR mutations cause cystic fibrosis. Since recent breakthrough in targeted pharmacotherapy, CFTR mutants with impaired gating are candidates for stimulation by potentiator drugs. Thus, understanding the molecular pathology of individual mutations has become important. The relatively common R117H mutation affects an extracellular loop, but nevertheless causes a strong gating defect. Here we identify a hydrogen bond between the side chain of arginine 117 and the backbone carbonyl group of glutamate 1124 in the cryo-electronmicroscopic structure of phosphorylated, ATP-bound CFTR. We address the functional relevance of that interaction for CFTR gating using macroscopic and microscopic inside-out patch-clamp recordings. Employing thermodynamic double-mutant cycles, we systematically track gating-state dependent changes in the strength of the R117-E1124 interaction. We find that the H-bond is formed only in the open state, but neither in the short-lived "flickery" nor in the long-lived "interburst" closed state. Loss of this H-bond explains the entire gating phenotype of the R117H mutant, including robustly shortened burst durations and strongly reduced intraburst open probability. The findings may help targeted potentiator design.


title: Global and context-specific transcriptional consequences of oncogenic Fbw7 mutations

abstract: Fbw7 is a ubiquitin ligase substrate receptor that targets proteins for proteasomal degradation. Most known Fbw7 substrates are transcription factors (TFs) and many are also oncoproteins (e.g., c-Myc, c-Jun, Notch). Fbw7 is an important tumor suppressor and FBXW7 mutations drive tumorigenesis through activation of oncogenic Fbw7 substrates. Defining the mechanisms of Fbw7-associated tumorigenesis is critical for developing targeted therapies. We thus determined the transcriptional consequences of oncogenic Fbw7 mutations by studying isogenic colorectal cancer cell lines with engineered FBXW7 null and heterozygous missense mutations. We used an integrated approach employing RNA-Seq and high-resolution mapping (CUT&RUN) of histone modifications and TF occupancy (c-Jun and c-Myc) to examine the combinatorial effects of mis-regulated Fbw7 substrates. Fbw7 mutations caused widespread transcriptional changes associated with active chromatin and altered TF occupancy at distal regulatory regions. Some regulatory changes were common to both FBXW7-mutant cell lines whereas others were FBXW7 mutation-specific. By comparing c-Jun and c-Myc binding sites, we also identified co-regulated elements, suggesting that Fbw7 substrates may have synergistic effects. One co-regulated gene was CIITA, a master regulator of MHC Class II gene expression, and Fbw7 loss increased CIITA and MHC Class II gene expression in colorectal cancer cells. Fbw7 mutations were also correlated with increased CIITA expression in TCGA colorectal tumors and cell lines, which may have immunologic implications for progression and treatment of Fbw7-associated cancers. This integrative analysis provides a framework for understanding normal and neoplastic context-specific Fbw7 functions.


title: Heterogeneous Tfh cell populations that develop during enteric helminth infection predict the quality of type 2 protective response

abstract:
T follicular helper (Tfh) cells are an important component of the germinal centre (GC)-mediated humoral immunity. Yet, how regulation of Tfh- GC responses impacts on effective responses to helminth infection are poorly understood. Here we show that chronic helminth Trichuris muris infection fails to induce Tfh-GC B cell responses, with Tfh cells expressing T-bet and IFN-{gamma}. In contrast, Tfh cells that express GATA-3 and IL-4 dominate responses to an acute, resolving infection. Accordingly, heightened expression and increased chromatin accessibility of Th1- and Th2 cell-associated genes is observed in chronic and acute induced Tfh cells, respectively. However, both acute and chronic Tfh cell populations retained the capacity to produce IL-21 in spite of the Th-biased response. Blockade of Tfh-GC interactions impaired type 2 immunity, highlighting the protective role of GC-dependent Th2-like Tfh cell responses against helminths. Collectively, these results provide new insights into the protective roles of Tfh-GC responses and identify distinct transcriptional and epigenetic features of Tfh cells that emerge during resolving or chronic helminth infections.

Author summaryAbout a quarter of the world population is afflicted with parasitic worm infection. Although deworming drugs can reduce the levels of the infection, they fail to prevent reinfections. Therefore, the most sustainable goal is to develop vaccines against human helminth parasites, which has been extremely challenging due to the lack of understanding of host-parasite interactions. While the protective roles of T helper 2 (Th2) cells are well established, the regulation of T follicular helper (Tfh) cells and their roles during helminth infection remain poorly defined. In this study, we describe the differential regulation of Tfh cell responses during chronic non-protective vs acute protective responses during helminth infection. We show that Tfh cells during chronic infection are rare and have strikingly different characteristics to acute-induced Tfh cells, which appear to be more like Th2 cells. Specifically, we show that blockade of Th2-like Tfh cell response during acute infection results in the host failing to expel the worms. Our study identifies that Tfh cell populations that emerge during chronic and acute infection are strikingly heterogeneous and critically important in mediating protective immune responses against helminths.


title: Phospho-regulation accommodates Type III secretion and assembly of a tether of ER-Chlamydia inclusion membrane contact sites

abstract: Membrane contact sites (MCS) are crucial for non-vesicular trafficking-based inter-organelle communication. ER-organelle tethering occurs in part through the interaction of the ER resident protein VAP with FFAT-motif containing proteins. FFAT motifs are characterized by a seven amino acidic core surrounded by acid tracks. We have previously shown that the human intracellular bacterial pathogen Chlamydia trachomatis establishes MCS between its vacuole (the inclusion) and the ER through expression of a bacterial tether, IncV, displaying molecular mimicry of eukaryotic FFAT motif cores. Here, we show that multiple layers of host cell kinase-mediated phosphorylation events govern the assembly of the IncV-VAP tethering complex. CK2-mediated phosphorylation of a C-terminal region of IncV enables IncV hyperphosphorylation of a phospho- FFAT motif core and serine-rich tracts immediately upstream of IncV FFAT motif cores. Phosphorylatable serine tracts, rather than genetically-encoded acidic tracts, accommodate Type III-mediated translocation of IncV to the inclusion membrane, while achieving full mimicry of FFAT motifs. Thus, regulatory components and post-translational modifications are integral to MCS biology, and intracellular pathogens such as C. trachomatis have evolved complex molecular mimicry of these eukaryotic features.

category: microbiology


title: Signatures of Brain Network Alteration in Psychogenic Non-Epileptic Seizures: A Rest-EEG Study Based on Power Spectral Density and Phase Lag Index

abstract: The main challenge in the clinical assessment of Psychogenic Non-Epileptic Seizures (PNES) is the lack of an electroencephalographic marker in the electroencephalography (EEG) readout. Although decades of EEG studies have focused on detecting cortical brain function underlying PNES, the principle of PNES remains poorly understood. To address this problem, electric potentials generated by large populations of neurons were collected during the resting state to be processed after that by Power Spectrum Density (PSD) for possible analysis of PNES signatures. Additionally, the integration of distributed information of regular and synchronized multi-scale communication within and across inter-regional brain areas has been observed using functional connectivity tools like Phase Lag Index (PLI) and graph-derived metrics. A cohort study of 20 PNES and 19 Healthy Control subjects (HC) were enrolled. The major finding is that PNES patients exhibited significant differences in alpha-power spectrum in brain regions related to cognitive operations, attention, working memory, and movement regulation. Noticeably, we observed that there exists an altered oscillatory activity and a widespread inter-regional phase desynchronization. This indicates changes in global efficiency, node betweenness, shortest path length, and small worldness in the delta, theta, alpha, and beta frequency bands. Finally, our findings look into new evidence of the intrinsic organization of functional brain networks that reflects a dysfunctional level of integration of local activity across brain regions, which can provide new insights into the pathophysiological mechanisms of PNES.


title: Transcriptome profiling of the ventral pallidum reveals a role for pallido-thalamic neurons in cocaine reward

abstract: Psychostimulant exposure alters the activity of ventral pallidum (VP) projection-neurons. However, the molecular underpinnings of these circuit dysfunctions are unclear. Using RNA-sequencing followed by circuit-specific gene expression assays, we revealed a key role for the VP to mediodorsal thalamus (VP-MDT) projection neurons in cocaine-related behaviors in mice. Our analyses demonstrated that the transcription factor Nr4a1 bidirectionally modulated dendritic spine dynamics in VP-MDT neurons and positively regulated pathological drug use.


title: Natural variation further increases resilience of sorghum bred for chronically drought-prone environments

abstract: Climate-change-associated shifts in rainfall distribution together with a looming worldwide water crisis make drought resilience of central importance to food security. Even for relatively drought resilient crops such as sorghum, moisture stress is nonetheless one of the major constraints for production. Here, we explore the potential to use natural genetic variation to build on the inherent drought tolerance of an elite cultivar (Teshale) bred for Ethiopian conditions including chronic drought, evaluating a backcross nested-association mapping (BC-NAM) population using 12 diverse founder lines crossed with Teshale under three drought-prone environments in Ethiopia. All twelve populations averaged higher head exsertion and lower leaf senescence than the recurrent parent in the two highest-stress environments, reflecting new drought resilience mechanisms from the donors. A total of 154 QTLs were detected for eight drought responsive traits - the validity of these were supported in that 100 (64.9%) overlapped with QTLs previously detected for the same traits, concentrated in regions previously associated with  stay-green traits as well as the flowering regulator Ma6 and drought resistant gene P5CS2. Allele effects show that some favorable alleles are already present in the Ethiopian cultivar, however the exotic donors offer rich scope for increasing drought resilience. Using model-selected SNPs associated with eight traits in this study and three in a companion study, phenotypic prediction accuracies for grain yield were equivalent to genome-wide SNPs and were significantly better than random SNPs, indicating that these studied traits are predictive of sorghum grain yield. Rich scope for improving drought resilience even in cultivars bred for drought-prone regions, together with phenotypic prediction accuracy for grain yield, provides a foundation to enhance food security in drought-prone areas like the African Sahel.


title: Structural convergence for tubulin binding of CPAP and vinca domain microtubule inhibitors

abstract: Microtubule dynamics is regulated by various cellular proteins and perturbed by small molecule compounds. To what extent the mechanism of the former resembles that of the latter is an open question. We report here structures of tubulin bound to the PN2-3 domain of CPAP, a protein controlling the length of the centrioles. We show that an -helix of the PN2-3 N-terminal region binds and caps the longitudinal surface of the tubulin {beta} subunit. Moreover, a PN2-3 N-terminal stretch lies in a {beta}-tubulin site also targeted by fungal and bacterial peptide-like inhibitors of the vinca domain, sharing a very similar binding mode with these compounds. Therefore, our results identify several characteristic features of cellular partners that bind to this site and highlight a structural convergence of CPAP with small molecule inhibitors of microtubule assembly.


title: hyperTRIBER: a flexible R package for the analysis of differential RNA editing

abstract: AO_SCPLOWBSTRACTC_SCPLOWRNA editing by ADAR (adenosine deaminase acting on RNA) is gaining an increased interest in the field of post-transcriptional regulation. Fused to an RNA-binding protein (RBP) of interest, the catalytic activity of ADAR results in A-to-I RNA edits, whose identification will determine RBP-bound RNA transcripts. However, the computational tools available for their identification and differential RNA editing statistical analysis are limited or too specialised for general-purpose usage. Here we present hyperTRIBER, a flexible suite of tools, wrapped into a convenient R package, for the detection of differential RNA editing. hyperTRIBER is applicable to complex scenarios and experimental designs, and provides a robust statistical framework allowing for the control for coverage of reads at a given base, the total expression level and other co-variates. We demonstrate the capabilities of our approach on HyperTRIBE RNA-seq data for the detection of bound RNAs by the N6-methyladenosine (m6A) reader protein ECT2 in Arabidopsis roots. We show that hyperTRIBER finds edits with a high statistical power, even where editing proportions and RNA transcript expression levels are low, together demonstrating its usability and versatility for analysing differential RNA editing.

category: bioinformatics


title: A Network-based Deep Learning Framework Catalyzes GWAS and Multi-Omics Findings to Biology and Drug Repurposing for Alzheimer's Disease

abstract: Human genome sequencing studies have identified numerous loci associated with complex diseases, including Alzheimers disease (AD). Translating human genetic findings (i.e., genome-wide association studies [GWAS]) to pathobiology and therapeutic discovery, however, remains a major challenge. To address this critical problem, we present a network topology-based deep learning framework to identify disease-associated genes (NETTAG). NETTAG is capable of integrating multigenomics data along with the protein-protein interactome to infer putative risk genes and drug targets impacted by GWAS loci. Specifically, we leverage non-coding GWAS loci effects on expression quantitative trait loci (eQTLs), histone-QTLs, and transcription factor binding-QTLs, enhancers and CpG islands, promoter regions, open chromatin, and promoter flanking regions. The key premises of NETTAG are that the disease risk genes exhibit distinct functional characteristics compared to non-risk genes and therefore can be distinguished by their aggregated genomic features under the human protein interactome. Applying NETTAG to the latest AD GWAS data, we identified 156 putative AD-risk genes (i.e., APOE, BIN1, GSK3B, MARK4, and PICALM). We showed that predicted risk genes are: 1) significantly enriched in AD-related pathobiological pathways, 2) more likely to be differentially expressed regarding transcriptome and proteome of AD brains, and 3) enriched in druggable targets with approved medicines (i.e., choline and ibudilast). In summary, our findings suggest that understanding of human pathobiology and therapeutic development could benefit from a network-based deep learning methodology that utilizes GWAS findings under the multimodal genomic analyses.