title: Structure insights, thermodynamic profiles, dsDNA melting activity, and liquid-liquid phase separation of the SARS-CoV-2 nucleocapsid N-terminal domain binding to DNA

abstract: The SARS-CoV-2 nucleocapsid protein (N) is a multifunctional promiscuous nucleic acid-binding protein, which plays a major role in nucleocapsid assembly and discontinuous RNA transcription, facilitating the template switch of transcriptional regulatory sequences (TRS). Here, we dissect the structural features of the N protein N-terminal domain (N-NTD), either with or without the SR-rich motif (SR), upon binding to single and double-stranded TRS DNA, as well as their activities for dsTRS melting and TRS-induced liquid-liquid phase separation (LLPS). Our study gives insights on specificity for N-NTD/N-NTD-SR interaction with TRS, including an unfavorable energetic contribution to binding along with hydrogen bonds between the triple-thymidine (TTT) motif in the dsTRS and {beta}-sheet II due to the defined position and orientation of the DNA duplex, a well-defined pattern ({Delta}H > 0 and {Delta}S > 0 for ssTRS, and {Delta}H < 0 and {Delta}S < 0 for dsTRS) for the thermodynamic profile of binding, and a preference for TRS in the formation of liquid condensates when compared to a non-specific sequence. Moreover, our results on DNA binding may serve as a starting point for the design of inhibitors, including aptamers, against N, a possible therapeutic target essential for the virus infectivity.

category: biophysics


title: Clinical and molecular features of acquired resistance to immunotherapy in non-small cell lung cancer

abstract: Although cancer immunotherapy with PD-(L)1 blockade is now routine treatment for patients with lung cancer, remarkably little is known about acquired resistance. We examined 1,201 patients with NSCLC treated with PD-(L)1 blockade to clinically characterize acquired resistance, finding it to be common (occurring in more than 60% of initial responders), with persistent but diminishing risk over time, and with distinct metastatic and survival patterns compared to primary resistance. To examine the molecular phenotype and potential mechanisms of acquired resistance, we performed whole transcriptome and exome tumor profiling in a subset of NSCLC patients (n=29) with acquired resistance. Systematic immunogenomic analysis revealed that tumors with acquired resistance generally had enriched signals of inflammation (including IFN{gamma} signaling and inferred CD8+ T cells) and could be separated into IFN{gamma} upregulated and stable subsets. IFN{gamma} upregulated tumors had putative routes of resistance with signatures of dysfunctional interferon signaling and mutations in antigen presentation genes. Transcriptomic profiling of cancer cells from a murine model of acquired resistance to PD-(L)1 blockade also showed evidence of dysfunctional interferon signaling and acquired insensitivity to in vitro interferon gamma treatment. In summary, we characterized clinical and molecular features of acquired resistance to PD-(L)1 blockade in NSCLC and found evidence of ongoing but dysfunctional IFN response. The persistently inflamed, rather than excluded or deserted, tumor microenvironment of acquired resistance informs therapeutic strategies to effectively reprogram and reverse acquired resistance.

category: cancer biology


title: Patterns of gene co-expression under water-deficit treatments and pan-genome occupancy in Brachypodium distachyon.

abstract: Natural populations are characterized by abundant genetic diversity driven by a range of different types of mutation. The tractability of sequence complete genomes has allowed new insights into the variable composition of genomes, summarized as a species pan-genome, which demonstrate that many genes are absent from the reference genomes whose analysis has dominated the initial years of the genomic era. Our field now turns towards understanding the functional consequence of these highly variable genomes. Here, we analyzed weighted gene co-expression networks from leaf transcriptome data for drought response in the purple false brome Brachypodium distachyon and investigated network topology and differential expression of genes putatively involved in adaptation to this stressor. We specifically asked whether genes with variable "occupancy" in the pan-genome - genes which are either present in all studied genotypes or missing in some genotypes - show different distributions among co-expression modules. Co-expression analysis united drought genes expressed in drought-stressed plants into 9 modules covering 343 hub genes (440 hub isoforms), and genes expressed under controlled water conditions into 13 modules, covering 724 hub genes (911 hub isoforms). We find that low occupancy pan-genes are under-represented among several modules, while other modules are over-enriched for low- occupancy pan-genes. We also provide new insight into the regulation of drought response in B. distachyon, specifically identifying one module with an apparent role in primary metabolism that is strongly responsive to drought. Our work shows the power of integrating pan-genomic analysis with transcriptomic data using factorial experiments to understand the functional genomics of environmental response.

category: plant biology


title: Optical manipulations reveal strong reciprocal inhibition but limited recurrent excitation within olfactory bulb glomeruli

abstract: The local circuitry within olfactory bulb glomeruli filters, transforms, and facilitates information transfer from olfactory sensory neurons to bulb output neurons. Two key elements of this circuit are glutamatergic tufted cells (TCs) and GABAergic periglomerular (PG) cells, both of which actively shape mitral cell activity and bulb output. A subtype of TCs, the external tufted cells (eTCs), can synaptically excite PG cells, but there are unresolved questions about other aspects of the glomerular connections, including the extent of connectivity between eTCs and the precise nature of reciprocal interactions between eTCs and PG cells. We combined patch-clamp recordings in OB slices and optophysiological tools to investigate local functional connections within glomeruli. When TCs were optically suppressed, we found a large decrease in excitatory post-synaptic currents (EPSCs) in "uniglomerular" PG cells that extend dendrites to one glomerulus, indicating that TC activation was required for most excitation of these PG cells. However, TC suppression had no effect on EPSCs in eTCs, arguing that TCs make few, if any, direct excitatory synaptic connections onto eTCs. The absence of synaptic connections between eTCs was also supported by recordings in eTC pairs. Lastly, we show using similar optical suppression methods that PG cells that express GAD65, mainly uniglomerular PG cells, provide strong inhibition onto eTCs. Our results indicate that the local network of TCs form potent reciprocal synaptic connections with GAD65-expressing uniglomerular PG cells but not other TCs. This configuration favors local inhibition over recurrent excitation within a glomerulus, limiting information transfer to downstream cortical regions.

category: neuroscience


title: Competent immune responses to SARS-CoV-2 variants in older adults following mRNA vaccination

abstract:
Aging is associated with a reduced magnitude of primary immune responses to vaccination and constriction of immune receptor repertoire diversity. Clinical trials demonstrated high efficacy of mRNA based SARS-CoV-2 vaccines in older adults but concerns about virus variant escape have not been well addressed. We have conducted an in-depth analysis of humoral and cellular immunity against an early-pandemic viral isolate and compared that to the P.1. (Gamma) and B.1.617.2 (Delta) variants in <50 and >55 age cohorts of mRNA vaccine recipients. We have further measured neutralizing antibody titers for B.1.617.1 (Kappa) and B.1.595; a SARS-CoV-2 isolate bearing Spike mutation E484Q. As reported, robust immunity required the second dose of vaccine. Older vaccinees manifested robust cellular immunity against early-pandemic SARS-CoV-2 and more recent variants, which remained statistically comparable to the adult group. The older cohort had lower neutralizing capacity at the first time point following the second dose, but at later time points immunity was indistinguishable between them. While the duration of these immune responses remains to be determined over longer periods of time, these results provide reasons for optimism regarding vaccine protection of older adults against SARS-CoV-2 variants and inform thinking about boost vaccination with variant vaccines.

eTOC summaryVaccine responses are often diminished with aging, but we found strong responses to SARS-CoV-2 in older adults following mRNA vaccination. T cell responses were not diminished when confronted by SARS-CoV-2 variants. Neutralizing Ab were reduced but not more than those in adults.

Graphical Abstract

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category: immunology


title: Democratizing Data-Independent Acquisition Proteomics Analysis on Public Cloud Infrastructures Via The Galaxy Framework

abstract: Data-independent acquisition (DIA) has become an important approach in global, mass spectrometric proteomic studies because it provides in-depth insights into the molecular variety of biological systems. However, DIA data analysis remains challenging due to the high complexity and large data and sample size, which require specialized software and large computing infrastructures. Most available open-source DIA software necessitate basic programming skills and cover only a fraction of the analysis steps, often yielding a complex of multiple software tools, severely limiting usability and reproducibility. To overcome this hurdle, we have integrated a suite of DIA tools in the Galaxy framework for reproducible and version-controlled data processing. The DIA suite includes OpenSwath, PyProphet, diapysef and swath2stats. We have compiled functional Galaxy pipelines for DIA processing, which provide a web-based graphical user interface to these pre-installed and pre-configured tools for their usage on freely accessible, powerful computational resources of the Galaxy framework. This approach also enables seamless sharing workflows with full configuration in addition to sharing raw data and results. We demonstrate usability of the all-in-one DIA pipeline in Galaxy by the analysis of a spike-in case study dataset. Additionally, extensive training material is provided, to further increase access for the proteomics community.

category: bioinformatics


title: NetSolP: predicting protein solubility in E. coli using language models

abstract:
Solubility and expression levels of proteins can be a limiting factor for large-scale studies and industrial production. By determining the solubility and expression directly from the protein sequence, the success rate of wet-lab experiments can be increased.

In this study, we focus on predicting the solubility and usability for purification of proteins expressed in Escherichia coli directly from the sequence. Our model NetSolP is based on deep learning protein language models called transformers and we show that it achieves state-of-the-art performance and improves extrapolation across datasets. As we find current methods are built on biased datasets, we curate existing datasets by using strict sequence-identity partitioning and ensure that there is minimal bias in the sequences.

The predictor is available at https://services.healthtech.dtu.dk/service.php?NetSolP-1.0


title: Automated imaging of duckweed growth and development

abstract: Duckweeds are some of the smallest angiosperms, possessing a simple body architecture and high rates of biomass accumulation. They can grow near-exponentially via clonal propagation. Understanding their reproductive biology, growth, and development is essential to unlock their potential for phytoremediation, carbon capture, and nutrition. However, there is a lack of non-laborious and convenient methods for spatially and temporally imaging an array of duckweed plants and growth conditions in the same experiment. We developed an automated microscopy approach to record time-lapse images of duckweed plants growing in 12-well cell culture plates. As a proof-of-concept experiment, we grew duckweed on semi-solid media with and without sucrose and monitored its effect on their growth over 3 days. Using the PlantCV toolkit, we quantified the thallus area of individual plantlets over time, and showed that L. minor grown on sucrose had an average growth rate four times higher than without sucrose. This method will serve as a blueprint to perform automated high-throughput growth assays for studying the development patterns of duckweeds from different species, genotypes, and conditions.


title: Gastrodia-Uncaria Water Extract Inhibits Endoplasmic Reticulum Stress and Matrix Metalloproteinase in Protecting against Cerebral Ischemia

abstract: Stroke is the second leading cause of death in worldwide, in which cerebral ischemia accounts for 87% of all cases. The building up of endoplasmic reticulum stress in cerebral ischemia contributes to the disruption of blood brain barrier and neuronal cell death. The only FDA-approved drug, recombinant tissue plasminogen activator, is still of limited use due to the narrow window period and lack of neuroprotective effect. Therefore, it is necessary to explore alternative treatment on cerebral ischemia. Tianma-Gouteng decoction is a traditional Chinese Medicine prescription used to treat brain diseases in China. In this study, we investigated the neuroprotective effect of a water extract consisting of Gastrodia elata and Uncaria rhynchophylla, which are the two main herbs in the decoction. Cerebral ischemia was induced in rats using middle cerebral artery occlusion. GUW-treated rats have significantly reduced infarction volume and recovered neurological functions. The number of protein aggregates and caspase-12 positive cells were significantly inhibited. In vitro oxygen-glucose deprivation / reoxygenation stroke model demonstrated that the unfolded protein response proteins GRP78 and PDI were upregulated by GUW. Less ubiquitin puncta and normalized ubiquitin distribution indicated the reduction in endoplasmic reticulum stress. Furthermore, a lower Evan blue signal and MMPsense signal was observed, suggesting that GUW may preserve the blood brain barrier integrity through inhibiting MMP activity. Taken together, this suggested that GUW protected ischemic neurons and the blood brain barrier through inhibiting endoplasmic reticulum stress.


title: Abnormal Recovery from Acute Stress in Huntingtons Disease Mice

abstract: Introduction: Comparisons of hypothalamic dysfunction between Huntingtons Disease (HD) patients and rodent models of HD have not always yielded similar results. Cortisol levels in HD patients have been contradictory, with reports ranging from hypo- to hypercorticoidism of morning measurements. Initial reports of major elevations in circulating corticosterone levels in the R6/2 mouse model of HD have only been followed up in one other closely related model, the R6/1 mouse, and the results were not perfectly congruent. To determine if abnormal stress hormones were a characteristic of disease, we examined diurnal and stress-induced corticosterone levels in multiple HD mouse models. Methods: We analyzed serum corticosterone levels from R6/2, Q140 and BACHD mice either diurnally (R6/2) or after restraint stress and subsequent recovery (all 3 models). Glucocorticoid secretion was also examined (R6/2) following dexamethasone suppression and ACTH stimulation. Blood samples were collected either by submandibular puncture or as trunk blood at sacrifice. Plasma corticosterone levels were assayed by RIA. Regional brain glucocorticoid receptor (GR) levels were visualized using immunofluorescence. Results: In the R6/2 mouse, basal levels of corticosterone, exhibited normal, sexually dimorphic diurnal rhythms but eventually became elevated towards end of life, after 13 weeks. The ability of the adrenal gland to produce corticosterone was not altered and the adrenal response to ACTH stimulation was only elevated in male R6/2 mice compared to littermates. Down regulation of GR receptors was only observed in the PVN at 16 weeks. R6/2 mice at both 6 and 12 weeks responded equivalently to littermates in the initial corticosterone response to restraint stress but serum corticosterone recovered from stress significantly more slowly. No differences were found in the initial stress response or in the recovery from stress for Q140 or BACHD mice, examined at 12 and 9 months, respectively. Conclusions: We did not find evidence of major disruption in glucocorticoid signaling in HD mouse models. A significantly slowed recovery from stress observed in the R6/2 mouse suggests such disruptions are context dependent. These results suggest that disruption of the HPA axis was subtle and occurred only at advanced stages of disease represented by comparisons among these HD mouse models.


title: CD8+ T cell signature in acute SARS-CoV-2 infection identifies memory precursors

abstract: Immunological memory is a hallmark of adaptive immunity and facilitates an accelerated and enhanced immune response upon re-infection with the same pathogen1,2. Since the outbreak of the ongoing coronavirus disease 19 (COVID-19) pandemic, a key question has focused on whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells stimulated during acute infection give rise to long-lived memory T cells3. Using spectral flow cytometry combined with cellular indexing of transcriptomes and T cell receptor (TCR) sequencing we longitudinally characterize individual SARS-CoV-2-specific CD8+ T cells of COVID-19 patients from acute infection to one year into recovery and find a distinct signature identifying long-lived memory CD8+ T cells. SARS-CoV-2-specific memory CD8+ T cells persisting one year after acute infection re-express CD45RA and interleukin-7 receptor  (CD127), upregulate T cell factor-1 (TCF1), and maintain low CCR7, thus resembling CD45RA+ effector-memory T (TEMRA) cells. Tracking individual clones of SARS-CoV-2-specific CD8+ T cells, we reveal that an interferon signature marks clones giving rise to long-lived cells, whereas prolonged proliferation and mammalian target of rapamycin (mTOR) signaling are associated with clone contraction and disappearance. Collectively, we identify a transcriptional signature differentiating short-from long-lived memory CD8+ T cells following an acute virus infection in humans.


title: Dichloroacetate improves systemic energy balance and feeding behavior during sepsis

abstract: Sepsis is a life-threatening organ dysfunction by dysregulated host response to an infection. The metabolic aberrations associated with sepsis underly an acute and organism wide hyper-inflammatory response and multiple organ dysfunction; however, crosstalk between systemic metabolomic alterations and metabolic reprograming at organ levels remains unknown. We analyzed substrate utilization by the respiratory exchange ratio, energy expenditure, metabolomic screening and transcriptional profiling in a cecal ligation and puncture (CLP) model, to show that sepsis increases circulating free fatty acids and acylcarnitines but decreases levels of amino acids and carbohydrates leading to a drastic shift in systemic fuel preference. Comparative analysis of previously published metabolomics from septic liver indicates a positive correlation with hepatic and plasma metabolites during sepsis. In particular, glycine deficiency was a common abnormality of both plasma and the liver during sepsis. Interrogation of the hepatic transcriptome in septic mice suggests that the septic liver may contribute to systemic glycine deficiency by downregulating genes involved in glycine synthesis. Interestingly, intraperitoneal injection of the pyruvate dehydrogenase kinase (PDK) inhibitor dichloroacetate (DCA) reverses sepsis-induced anorexia, energy imbalance, dyslipidemia, hypoglycemia, and glycine deficiency. Collectively, our data indicate that PDK inhibition rescues systemic energy imbalance and metabolic dysfunction in sepsis partly through restoration of hepatic fuel metabolism.


title: Impacts of Fluorescent Base Analogue Substitution on the Folding of a Riboswitch

abstract: Riboswitches are gene-regulating mRNA segments most commonly found in bacteria. A riboswitch contains an aptamer domain that binds to a ligand, causing a conformational change in a downstream expression platform. The aptamer domain of the Class I preQ1 riboswitch from Bacillus subtilis, which consists of a stem-loop structure and an adenine-rich single-stranded tail (L3), re-folds into a pseudoknot structure upon binding of its ligand, preQ1. To study the role of L3 in ligand recognition, we inserted 2-aminopurine (2-AP), a fluorescent base analogue of adenine (A), into the riboswitch at six different positions within L3. 2-AP differs from A in the relocation of its amino group from C6 to C2, allowing us to directly probe the significance of this specific functional group. We used circular dichroism spectroscopy and thermal denaturation experiments to study the structure and stability, respectively, of the riboswitch in the absence and presence of preQ1. At all labeling positions tested, 2-AP substitution inhibited the ability of preQ1 to stabilize the pseudoknot structure, with its location impacting the severity of the effect. Structural studies of the riboswitch suggest that at the most detrimental labeling sites, 2-AP substitution disrupts non-canonical base pairs. Our results show that these base pairs and tertiary interactions involving other residues in L3 play a critical role in ligand recognition by the preQ1 riboswitch, even at positions that are distal to the ligand binding pocket. They also highlight the importance of accounting for perturbations that fluorescent analogues like 2-AP may exert on the system being studied.

category: biochemistry


title: Proton-mediated gating mechanism of anion channelrhodopsin-1

abstract: Anion channelrhodopsin from Guillardia theta (GtACR1) has Asp234 (3.2 [A]) and Glu68 (5.3 [A]) near the protonated Schiff base. Here we investigate mutant GtACR1s (e.g., E68Q/D234N) expressed in HEK293 cells. The influence of the acidic residues on the absorption wavelengths were also analyzed, using a quantum mechanical/molecular mechanical approach. The calculated protonation pattern indicates that Asp234 is deprotonated and Glu68 is protonated in the original crystal structures. The D234E mutation and the E68Q/D234N mutation shortens and lengthens the measured and calculated absorption wavelengths, respectively, which suggests that Asp234 is deprotonated in the wild type GtACR1. Molecular dynamics simulations show that upon mutation of deprotonated Asp234 to asparagine, deprotonated Glu68 reorients towards the Schiff base and the calculated absorption wavelength remains unchanged. The formation of the proton transfer pathway via Asp234 toward Glu68 and the disconnection of the anion conducting channel are likely a basis of the gating mechanism.


title: Assessing target engagement using proteome-wide solvent shift assays

abstract: Recent advances in mass spectrometry (MS) have enabled quantitative proteomics to become a powerful tool in the field of drug discovery, especially when applied toward proteome-wide target engagement studies. Similar to temperature gradients, increasing concentrations of organic solvents stimulate unfolding and precipitation of the cellular proteome. This property can be influenced by physical association with ligands and other molecules, making individual proteins more or less susceptible to solvent-induced denaturation. Herein, we report the development of proteome-wide solvent shift assays by combining the principles of solvent-induced precipitation (Zhang et al., 2020) with modern quantitative proteomics. Using this approach, we developed solvent proteome profiling (SPP), which is capable of establishing target engagement through analysis of SPP denaturation curves. We readily identified the specific targets of compounds with known mechanisms of action. As a further efficiency boost, we applied the concept of area-under-the-curve analysis to develop solvent proteome integral solubility alteration (solvent-PISA) and demonstrate that this approach can serve as a reliable surrogate for SPP. We propose that by combining SPP with alternative methods, like thermal proteome profiling, it will be possible to increase the absolute number of high-quality melting curves that are attainable by either approach individually thereby increasing the fraction of the proteome that can be screened for evidence of ligand binding.


title: CYP1B1 converts procarcinogens into genotoxins in Saccharomyces cerevisiae

abstract: CYP1B1 activates many chemical carcinogens into potent genotoxins, and allelic variants are risk factors in lung, breast, and prostate cancer. However, genetic instability phenotypes incurred by CYP1B1-activated metabolites have been investigated for only few compounds. In this study, we expressed human CYP1B1 in yeast strains that measure DNA damage-associated toxicity and frequencies of chromosomal translocations and mutations. DNA damage-associated toxicity was measured in a rad4 rad51 strain, defective in both DNA excision and recombinational repair. Frequencies of chromosomal translocations were measured in diploid yeast strains containing two his3 fragments, and mutation frequencies were measured by selecting for canavanine resistance (CanR) in haploid strains. These strains were exposed to benzo[a]pyrene dihydrodiol (BaP-DHD), aflatoxin B1 (AFB1), and the heterocyclic aromatic amines, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3-methylimidazo(4,5-f)quinoline (IQ). We observed that AFB1, BaP-DHD, IQ, and MeIQx conferred toxicity in the DNA repair mutant expressing CYP1B1. Translocation frequencies increased eight-fold and three-fold after exposure to 50 M AFB1 and 33 M BaP-DHD respectively. Only a two-fold increase in mutation frequency was observed after exposure to 50 M AFB1. However, a robust DNA damage response was observed after AFB1 exposure, as measured by the induction of the small subunit of ribonucleotide reductase, Rnr3. While CYP1B1-mediated activation of BaP-DHD and heterocyclic aromatic amines was expected, strong activation of AFB1 was not. These studies demonstrate that CYP1B1-mediated activation of carcinogens does not only activate compounds to become mutagens but also can convert compounds to become potent recombinagens.

category: pharmacology and toxicology


title: Gammaherpesvirus infection licenses age-associated B cells for pathogenicity in MS and EAE

abstract: While age-associated B cells (ABCs) are known to expand and persist following viral infection and during autoimmunity, their interactions are yet to be studied together in these contexts. Epstein-Barr virus (EBV) infection has long been implicated in multiple sclerosis (MS), and it is not known whether ABCs could play a role in mediating viral contribution to autoimmunity. Here, we show that the circulating ABC population is expanded in people with MS and that EBV infection and MS status differentially impact the circulating ABC phenotype. We then directly compared ABCs during viral infection and autoimmunity using mouse models of EBV, gammaherpesvirus 68 ({gamma}HV68), and MS, experimental autoimmune encephalomyelitis (EAE). We observed that splenic ABCs are expanded in a sex-biased manner during both latent virus infection and EAE, and each event drives the ABC population to opposing phenotypes. We have previously shown that latent{gamma} HV68 infection exacerbates EAE and here we show that mice lacking ABCs fail to display{gamma} HV68-enhanced disease. Collectively, these findings indicate that latent viral infection and central nervous system autoimmunity differentially impact the ABC population and suggests that viral infections such as EBV prime ABCs to contribute pathogenically in MS.


title: The Murine Neuronal Receptor NgR1 Is Dispensable for Reovirus Pathogenesis

abstract:
Engagement of host receptors is essential for viruses to enter target cells and initiate infection. Expression patterns of receptors in turn dictate host and tissue tropism and disease pathogenesis during infection. Mammalian orthoreovirus (reovirus) displays serotype-dependent patterns of tropism in the murine central nervous system (CNS) that are dictated by viral attachment protein {sigma}1. However, the receptor that mediates reovirus CNS tropism is unknown. Two proteinaceous receptors have been identified for reovirus, junctional adhesion molecule-A (JAM-A) and Nogo 66 receptor 1 (NgR1). Engagement of JAM-A is required for reovirus hematogenous dissemination but is dispensable for neural spread. To determine whether NgR1 functions in reovirus neuropathogenesis, we compared virus replication and disease following inoculation of wild-type (WT) and NgR1-/- mice. Genetic ablation of NgR1 did not alter replication of neurotropic reovirus strain T3SA- in the intestine and transmission to the brain following peroral inoculation. Viral titers in neural tissues following intramuscular inoculation, which provides access to neural dissemination routes, also were comparable in WT and NgR1-/- mice, suggesting that NgR1 is dispensable for reovirus neural spread to the CNS. The absence of both NgR1 and JAM-A also did not alter replication, neural tropism, and virulence of T3SA- following direct intracranial inoculation. In agreement with these findings, we found that the human but not the murine homolog of NgR1 functions as a receptor and confers efficient reovirus binding and infection of nonsusceptible cells in vitro. These results eliminate functions for JAM-A and NgR1 in shaping CNS tropism in mice and suggest that other receptors, yet to be identified, support this function.

IMPORTANCEThe CNS presents a range of barriers to pathogen invasion. Yet neurotropic viruses have evolved strategies to breach these barriers and establish infection by engagement of host factors that allow navigation to the CNS and neural cell entry. Human NgR1 was identified as a reovirus receptor in an RNA interference screen and is expressed in CNS neurons in a pattern overlapping with reovirus tropism. Using mice genetically lacking NgR1 expression, and following different routes of inoculation, we discovered that murine NgR1 is dispensable for reovirus dissemination to the CNS, tropism and replication in the brain, and resultant disease. Concordant with these results, expression of human but not murine NgR1 confers reovirus binding and infection of nonsusceptible cells in vitro. These results point to species-specific use of alternate receptors by reovirus. A detailed understanding of species- and tissue-specific factors that dictate viral tropism will inform development of interventions and targeted gene delivery and therapeutic viral vectors.

category: microbiology


title: Roles of proteins containing immunoglobulin-like domains in the conjugation of bacterial plasmids.

abstract: Horizontal transfer of bacterial plasmids generates genetic variability and contributes to the dissemination of the genes that enable bacterial cells to develop antimicrobial resistance (AMR). Several aspects of the conjugative process have long been known, namely, those related to the proteins that participate in the establishment of cell-to-cell contact and to the enzymatic processes associated with the processing of plasmid DNA and its transfer to the recipient cell. In this work, we describe the role of newly identified proteins that influence the conjugation of several plasmids. Genes encoding high-molecular-weight proteins that contain one or several immunoglobulin-like domains (Big) are located in the transfer regions of several plasmids that usually harbor AMR determinants. These Big proteins are exported to the external medium and target two extracellular organelles: the flagella and the conjugative pili. The plasmid-encoded Big proteins facilitate conjugation by reducing cell motility and facilitating cell-to-cell contact by binding both to the flagella and to the conjugative pilus. They use the same export machinery as that used by the conjugative pilus components. In the examples characterized in this paper, these proteins influence conjugation at environmental temperatures (i.e., 25{degrees}C). This suggests that they may play relevant roles in the dissemination of plasmids in natural environments. Taking into account that they interact with outer surface organelles, they could be targeted to control the dissemination of different bacterial plasmids carrying AMR determinants.


title: Quantification of archaea-driven freshwater nitrification: from single cell to ecosystem level

abstract: Deep oligotrophic lakes sustain large archaeal populations of the class Nitrososphaeria in their hypolimnion. They are thought to be the key ammonia oxidizers in these freshwater systems and as such responsible for the rate-limiting step in nitrification. However, the impact that planktonic Nitrososphaeria have on N cycling in lakes is severely understudied and yet to be quantified. Here, we followed this archaeal population in one of Central Europes largest lakes, Lake Constance, over two consecutive years using metagenomics and metatranscriptomics combined with stable isotope-based activity measurements. A single, highly abundant and transcriptionally active freshwater ecotype of Nitrososphaeria dominated the nitrifying community. Phylogenomic analysis of its metagenome-assembled genome showed that this ecotype represents a new lacustrine Nitrosopumilus species. Stable isotope probing revealed that Nitrososphaeria incorporated significantly more 15N-labeled ammonium than most other microorganisms at near-natural conditions and oxidized ammonia at an average rate of 0.22 {+/-} 0.11 fmol cell-1 d-1. This translates to 1.9 gigagram of ammonia oxidized per year, corresponding to 12% of the N-biomass produced annually by photosynthetic organisms in Lake Constance. Here, we show that ammonia-oxidizing archaea play an equally important role in the nitrogen cycle of deep oligotrophic lakes as their counterparts in marine ecosystems.


title: RTT109 AND Fun30 proteins mediate epigenetic regulation of the DNA damage response pathway in C. albicans

abstract: Fun30, an ATP-dependent chromatin remodeller, from S. cerevisiae mediates both regulation of gene expression as well as DNA damage response/repair. In this paper, we have characterized the biochemical and physiological function of Fun30 from the opportunistic fungi, C. albicans. Biochemically, the protein shows DNA-stimulated ATPase activity. Physiologically, the protein co-regulates transcription of RTT109, TEL1, MEC1, and SNF2-genes that encode for proteins involved in DNA damage response and repair pathway. The expression of FUN30, in turn, is regulated by histone H3 acetylation catalysed by Rtt109 encoded by RTT109. The RTT109Hz/FUN30Hz mutant strain shows sensitivity to oxidative stress and resistance to MMS as compared to the wild type strain. Quantitative PCR showed that the sensitivity to oxidative stress results from downregulation of MEC1, RAD9, MRC1 and RAD5 expression; ChIP experiments showed Fun30 but not H3ac regulates the expression of these genes in response to oxidative stress. In contrast, on treatment with MMS, the expression of RAD9 is upregulated and this upregulation is co-regulated by both Fun30 and H3 acetylation catalysed by Rtt109. Thus, Fun30 and H3 acetylation mediate the response of the fungal cell to genotoxic agents in C. albicans by regulating the expression of DNA damage response and repair pathway genes.

category: molecular biology


title: PICH, an ATP-dependent chromatin remodeling protein, transcriptionally co-regulates oxidative stress response.

abstract: Cells respond to oxidative stress by elevating the levels of antioxidants, signaling, and transcriptional regulation often implemented by chromatin remodeling proteins. The study presented in this paper shows that the expression of PICH, an ATP-dependent chromatin remodeler, is upregulated during oxidative stress in HeLa cells. We also show that PICH regulates the expression of Nrf2, a transcription factor regulating antioxidant response, both in the absence and presence of oxidative stress. In turn, Nrf2 regulates the expression of PICH in the presence of oxidative stress. Both PICH and Nrf2 together regulate the expression of antioxidant genes and this transcriptional regulation is dependent on the ATPase activity of PICH. In addition, H3K27ac modification also plays a role in activating transcription in the presence of oxidative stress. Co-immunoprecipitation experiments show that PICH and Nrf2 interact with H3K27ac in the presence of oxidative stress. Mechanistically, PICH recognizes ARE sequences present on its target genes and introduces a conformational change to the DNA sequences leading us to hypothesize that PICH regulates transcription by remodeling DNA. PICH ablation leads to reduced expression of Nrf2 and impaired antioxidant response leading to increased ROS content, thus, showing PICH is essential for the cell to respond to oxidative stress.


title: Characterization of early Alzheimer's-like pathological alterations in non-human primates with aging: a pilot study

abstract: Sporadic or late onset Alzheimers disease (LOAD) is a multifactorial neurodegenerative disease with aging the most known risk factor. Non-human primates (NHPs) may serve as an excellent model to study LOAD because of their close similarity to humans in many aspects including neuroanatomy and neurodevelopment. Recent studies reveal AD-like pathology in old NHPs. In this pilot study, we took advantage of brain samples from 6 Cynomolgus macaques that were divided into two groups: middle aged (average age 14.81 years) and older (average age 19.33 years). We found multiple AD-like pathological alteration in the prefrontal cortex (but not in the hippocampus) of the older NHPs including tau hyperphosphorylation, increased activity of AMP-activated protein kinase (AMPK), decreased expression of protein phosphatase 2A (PP2A), impairments in mitochondrial morphology and postsynaptic densities (PSDs) formation. These findings may provide insights into the factors contributing to the development of LOAD, particularly during the early stage transitioning from middle to old age. Future endeavors are warranted to elucidate mechanisms underlying the regional (and perhaps cellular) vulnerability with aging and the functional correlation of such pathological changes in NHPs.


title: Csp1, A Cold-Shock Protein Homolog in Xylella fastidiosa Influences Pili Formation, Stress Response, and Gene Expression

abstract:
Bacterial cold shock-domain proteins (CSPs) are conserved nucleic acid binding chaperones that play important roles in stress adaptation and pathogenesis. Csp1 is a temperature-independent cold shock protein homolog in Xylella fastidiosa, a bacterial plant pathogen of grapevine and other economically important crops. Csp1 contributes to stress tolerance and virulence in X. fastidiosa. However, besides general single stranded nucleic acid binding activity, little is known about the specific function(s) of this protein. To further investigate the role(s) of Csp1, we compared phenotypic differences between wild type and a csp1 deletion mutant ({Delta}csp1). We observed decreases in cellular aggregation and surface attachment with the {Delta}csp1 strain compared to the wild type. Transmission electron microscopy imaging revealed that {Delta}csp1 had reduced pili compared to the wild type and complemented strains. The {Delta}csp1 strain also showed reduced survival after long term growth, in vitro. Since Csp1 binds DNA and RNA, its influence on gene expression was also investigated. Long-read Nanopore RNA-Seq analysis of wild type and {Delta}csp1 revealed changes in expression of several genes important for attachment and biofilm formation in {Delta}csp1. One gene of intertest, pilA1, encodes a type IV pili subunit protein and was up regulated in {Delta}csp1. Deleting pilA1 increased surface attachment in vitro and reduced virulence in grapevines. X. fastidiosa virulence depends on bacterial attachment to host tissue and movement within and between xylem vessels. Our results show Csp1 may play a role in both virulence and stress tolerance by influencing expression of genes important for biofilm formation.

ImportanceXylella fastidiosa is a major threat to the worldwide agriculture industry (1, 2). Despite its global importance, many aspects of X. fastidiosa biology and pathogenicity are poorly understood. There are currently few effective solutions to suppress X. fastidiosa disease development or eliminate bacteria from infected plants(3). Recently, disease epidemics due to X. fastidiosa have greatly expanded(2, 4, 5), exacerbating the need for better disease prevention and control strategies. Our studies show that Csp1 is involved in X. fastidiosa virulence and stress tolerance. Understanding how Csp1 influences pathogenesis and bacteria survival can aide in developing novel pathogen and disease control strategies. We also streamlined a bioinformatics protocol to process and analyze long read Nanopore bacterial RNA-Seq data, which has previously not been reported for X. fastidiosa.


title: Systematic mapping of small nucleolar RNA targets in human cells

abstract: Altered expression of box C/D small nucleolar RNAs (snoRNAs) is implicated in human diseases, including cancer. Box C/D snoRNAs canonically direct site-specific, 2-O-methylation but the extent to which they participate in other functions remains unclear. To identify RNA targets of box C/D snoRNAs in human cells, we applied two techniques based on UV crosslinking, proximity ligation and sequencing of RNA hybrids (CLASH and FLASH). These identified hundreds of novel snoRNA interactions with rRNA, snoRNAs and mRNAs. We developed an informatic pipeline to rigorously call interactions predicted to direct methylation. Multiple snoRNA-rRNA interactions identified were not predicted to direct RNA methylation. These potentially modulate methylation efficiency and/or contribute to folding dynamics. snoRNA-mRNA hybrids included 1,300 interactions between 117 snoRNA families and 940 mRNAs. Human U3 is substantially more abundant than other snoRNAs and represented about 50% of snoRNA-mRNA hybrids. The distribution of U3 interactions across mRNAs also differed from other snoRNAs. Following U3 depletion, mRNAs showing altered abundance were strongly enriched for U3 CLASH targets. Most human snoRNAs are excised from pre-mRNA introns. Enrichment for snoRNA association with branch point regions of introns that contain snoRNA genes was common, suggesting widespread regulation of snoRNA maturation.

category: cell biology


title: E-cadherin regulates the stability and transcriptional activity of β-catenin in embryonic stem cells

abstract:
E-CADHERIN is abundantly expressed in embryonic stem cells (ESCs) and plays an important role in the maintenance of cell-cell adhesions. However, the exact function of this molecule beyond cell adhesion, in the context of cell fate decisions is largely unknown. Using mouse ESCs (mESCs), we demonstrate that E-CADHERIN and {beta}- CATENIN interact at the membrane and continue to do so upon internalization within the cell. Knockout of the gene encoding E-CADHERIN, Cdh1, in mESCs resulted in a failure to form tight colonies, accompanied by altered expression of differentiation markers, and retention of pluripotency factor expression during differentiation. Interestingly, Cdh1-/- mESCs showed a dramatic reduction in {beta}-CATENIN levels. Transcriptional profiling of Cdh1-/- mESCs displayed a significant alteration in the expression of a subset of {beta}-CATENIN targets, in a cell-state dependent manner. While treatment with a pharmacological inhibitor against GSK3{beta} could rescue levels of {beta}-CATENIN in Cdh1-/- mESCs, expression of downstream targets were altered in a context-dependent manner, indicating an additional layer of regulation within this subset. Together, our results reveal the existence of a cell-state-dependent regulation of {beta}-CATENIN and its transcriptional targets in an E-CADHERIN dependent manner. Our findings hint at hitherto unknown roles played by E- CADHERIN in regulating the activity of {beta}-CATENIN in ESCs.

Significance StatementAre cell adhesions only responsible for maintaining tissue architecture, or do they also regulate cell fate decisions during early embryonic stages by modulating the output of specific signalling pathways? In this study, we study the role of E- CADHERIN, a crucial component of cell-cell adhesions in the context of mouse embryonic stem cells (mESCs). We find that E-CADHERIN regulates the stability and activity of {beta}-CATENIN in mESCs through physical interactions. However, the loss of E-CADHERIN affected the expression of only a subset of downstream targets of {beta}-CATENIN in a cell-state dependent manner. This study highlights a critical cross-talk between molecules involved in cell-cell adhesion and the underlying signalling network critical for establishing cell fate during early mammalian development.


title: Conformation and membrane interaction studies of the potent antimicrobial and anticancer peptide palustrin-Ca

abstract:
Palustrin-Ca (GFLDIIKDTGKEFAVKILNNLKCKLAGGCPP) is a host defense peptide with potent antimicrobial and anticancer activities, first isolated from the skin of the American bullfrog Lithobates catesbeianus. The peptide is 31 amino acid residues long, cationic and amphipathic. Two-dimensional NMR spectroscopy was employed to characterise its three-dimensional structure in a 50/50% water/2,2,2-trifluoroethanol-d3 mixture. The structure is defined by an -helix that spans between Ile6-Ala26, and a cyclic disulphide bridged domain at the C-terminal end of the peptide sequence, between residues 23 and 29.

A molecular dynamics simulation was employed to model the peptides interactions with sodium dodecyl sulphate micelles, a widely used bacterial membrane-mimicking environment. Throughout the simulation, the peptide was found to maintain its -helical conformation between residues Ile6-Ala26, while adopting a position parallel to the surface to micelle, which is energetically-favourable due to many hydrophobic and electrostatic contacts with the micelle.


title: GM1 mediates the formation and maintenance of cytotoxic Aβ oligomers

abstract: The aggregation of amyloid beta (A{beta}) peptide is associated with Alzheimers disease (AD) pathogenesis. Cell membrane composition, especially monosialotetrahexosylganglioside (GM1), is known to promote the formation of A{beta} fibrils, yet little is known about the roles of GM1 in the early steps of A{beta} oligomer formation. Here, by using GM1-contained liposomes as a mimic of neuronal cell membrane, we demonstrate that GM1 is a critical trigger of A{beta} oligomerization and aggregation. We find that GM1 not only promotes the formation of A{beta} fibrils, but also facilitates the maintenance of A{beta} oligomers on liposome membranes. We structurally characterize the A{beta} oligomers formed on the membrane and find that GM1 captures A{beta} by binding to its arginine-5 residue. To interrogate the mechanism of A{beta} oligomer toxicity, we design a new liposome-based Ca2+-encapsulation assay and provide new evidence for the A{beta} ion channel hypothesis. Finally, we conduct cell viability assay to determine the toxicity of A{beta} oligomers formed on membranes. Overall, by uncovering the roles of GM1 in mediating early A{beta} oligomer formation and maintenance, our work provides a novel direction for pharmaceutical research for AD.


title: Unitary Structure of Palindromes in DNA

abstract: We investigate the quantum behavior encountered in palindromes within DNA structure. In particular, we reveal the unitary structure of usual palindromic sequences found in genomic DNAs of all living organisms, using the Schwingers approach. We clearly demonstrate the role played by palindromic configurations with special emphasis on physical symmetries, in particular subsymmetries of unitary structure. We unveil the prominence of unitary structure in palindromic sequences in the sense that vitally significant information endowed within DNA could be transformed unchangeably in the process of transcription. We introduce a new symmetry relation, namely purine-purine or pyrimidine-pyrimidine symmetries (p-symmetry) in addition to the already known symmetry relation of purine-pyrimidine symmetries (pp-symmetry) given by Chargaffs rule. Therefore, important vital functions of a living organisms are protected by means of these symmetric features. It is understood that higher order palindromic sequences could be generated in terms of the basis of the highest prime numbers that make up the palindrome sequence number. We propose that violation of this unitary structure of palindromic sequences by means of our proposed symmetries leads to a mutation in DNA, which could offer a new perspective in the scientific studies on the origin and cause of mutation.


title: GIV/Girdin binds BRCA1 and links trimeric G-proteins to DNA damage response

abstract:
Upon sensing DNA double-strand breaks (DSBs), eukaryotic cells either die or repair DSBs via one of two competing pathways, i.e., non-homologous end-joining (NHEJ) or homologous recombination (HR). We show that cell fate after DNA damage hinges on the guanine nucleotide-exchange modulator of heterotrimeric G-protein, Gi*{beta}{gamma}, GIV/Girdin. GIV suppresses HR by binding and sequestering BRCA1, a key coordinator of multiple steps within the HR pathway, away from DSBs; it does so using a C-terminal motif that binds BRCA1s BRCT-modules via both phospho-dependent and -independent mechanisms. GIV promotes NHEJ, and binds and activates Gi and enhances the  free G{beta}{gamma}[->]PI-3-kinase[->]Akt pathway, thus revealing the enigmatic origin of prosurvival Akt signals during dsDNA repair. Absence of GIV, or the loss of either of its two functions impaired DNA repair, and induced cell death when challenged with numerous cytotoxic agents. That GIV selectively binds few other BRCT-containing proteins suggests convergent signaling such that heterotrimeric G-proteins may finetune sensing, repair, and outcome after DNA damage.

GRAPHIC ABSTRACT

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org.highwire.dtl.DTLVardef@3d5202org.highwire.dtl.DTLVardef@1d32d89org.highwire.dtl.DTLVardef@152a85dorg.highwire.dtl.DTLVardef@1ab9953_HPS_FORMAT_FIGEXP  M_FIG C_FIG HIGHLIGHTSO_LINon-receptor G protein modulator, GIV/Girdin binds BRCA1
C_LIO_LIBinding occurs in both canonical and non-canonical modes
C_LIO_LIGIV sequesters BRCA1 away from dsDNA breaks, suppresses HR
C_LIO_LIActivation of Gi by GIV enhances Akt signals, favors NHEJ
C_LI

IN BRIEFIn this work, the authors show that heterotrimeric G protein signaling that is triggered by non-receptor GEF, GIV/Girdin, in response to double-stranded DNA breaks is critical for decisive signaling events which favor non-homologous end-joining (NHEJ) and inhibit homologous recombination (HR).


title: Regulation of DNA damage response by trimeric G-protein Signaling


title: Gestational insulin resistance is mediated by the gut microbiome-indoleamine 2,3-dioxygenase axis

abstract:
Background and aimsNormal gestation involves reprogramming of maternal gut microbiome (GM) that may contribute to maternal metabolic changes by unclear mechanisms. This study aimed to understand the mechanistic underpinnings of GM - maternal metabolism interaction.

MethodsThe GM and plasma metabolome of CD1, NIH-Swiss and C57BL/6J mice were analyzed using 16S rRNA sequencing and untargeted LC-MS throughout gestation and postpartum. Pharmacologic and genetic knockout mouse models were used to identify the role of indoleamine 2,3-dioxygenase (IDO1) in pregnancy-associated insulin resistance (IR). Involvement of gestational GM in the process was studied using fecal microbial transplants (FMT).

ResultsSignificant variation in gut microbial alpha diversity occurred throughout pregnancy. Enrichment in gut bacterial taxa was mouse strain and pregnancy time-point specific, with species enriched at gestation day 15/19 (G15/19), a point of heightened IR, distinct from those enriched pre- or post- pregnancy. Untargeted and targeted metabolomics revealed elevated plasma kynurenine at G15/19 in all three mouse strains. IDO1, the rate limiting enzyme for kynurenine production, had increased intestinal expression at G15, which was associated with mild systemic and gut inflammation. Pharmacologic and genetic inhibition of IDO1 inhibited kynurenine levels and reversed pregnancy-associated IR. FMT revealed that IDO1 induction and local kynurenine levels effects on IR derive from the GM in both mouse and human pregnancy.

ConclusionsGM changes accompanying pregnancy shift IDO1-dependent tryptophan metabolism toward kynurenine production, intestinal inflammation and gestational IR, a phenotype reversed by genetic deletion or inhibition of IDO1.

category: physiology


title: Fructose appetition in "taste-blind" P2X2/P2X3 double knockout mice

abstract: Inbred mouse strains differ in their postoral appetite stimulating response (appetition) to glucose and fructose. For example, C57BL/6J (B6) and FVB strains learn strong preferences for a flavor (CS+, e.g., cherry) paired with intragastric (IG) glucose infusions, but only FVB mice learned to prefer a CS+ paired with IG fructose infusions. Consistent with these findings, "tasteless" B6 knockout (KO) mice missing the taste signaling protein TRPM5 learn strong preferences for a CS+ added to glucose solution as well as for unflavored glucose but weak or no preferences for a fructose-paired CS+ or unflavored fructose. The present experiment reports that "tasteless" P2X2/P2X3 double-knockout (P2X2/3 DKO) mice, unlike TRPM5 KO mice, learned strong preferences for a CS+ mixed with fructose as well as for unflavored fructose. Whether differences in genetic backgrounds or other factors account for the fructose appetition displayed by P2X2/3 DKO mice but not TRPM5 KO mice remains to be determined.


title: Binocular Benefit Following Monocular Subretinal AAV Injection in a Mouse Model of Autosomal Dominant Retinitis Pigmentosa (adRP)

abstract: Autosomal dominant retinitis pigmentosa (adRP) is frequently caused by mutations in RHO, the gene for rhodopsin. In previous experiments in dogs with the T4R mutation in RHO, an AAV2/5 vector expressing both an shRNA directed to human and dog RHO mRNA and an shRNA-resistant human RHO cDNA (AAV-RHO820-shRNA820) prevented retinal degeneration for more than 8 months following injection. To confirm that this same vector could protect the retinas of a different species and bearing a different RHO mutation, we injected mice transgenic for human P23H RHO at postnatal day 30 in one eye. For nine months, we monitored their retinal structure using spectral- domain optical coherence tomography (SD-OCT) and retinal function using electroretinography (ERG). We compared these to P23H RHO transgenic mice injected with AAV-GFP. Though retinas continued to thin over time, compared to control injected eyes, AAV-RHO820-shRNA820 slowed the loss of photoreceptor cells and decreased ERG amplitudes in AAV-RHO820-shRNA820 eyes during the nine-month study period. Unexpectedly, we also observed preservation of retinal structure and function in the untreated contralateral eyes of AAV-RHO820-shRNA820 treated mice. PCR analysis and western blots provided evidence that a low amount of vector from injected eyes was present in uninjected eyes.


title: Transcriptional regulatory features associated with Coccidioides immitis phase transition

abstract: Coccidioidomycosis (Valley Fever) is an emerging endemic fungal infection with a rising incidence and an expanding geographic range. It is caused by Coccidiodes, which are thermally dimorphic fungi that grow as mycelia in soil but transition in the lung to form pathogenic spherules. The regulatory mechanisms underlying this transition are not understood. Exploiting capped small (cs)RNA-seq, which identifies actively initiated stable and unstable transcripts and thereby detects acute changes in gene regulation with remarkable sensitivity, here we report the changes in architectural organization and key sequence features underlying phase transition of this highly pathogenic fungus. Spherule transition was accompanied by large-scale transcriptional reprogramming, functional changes in transcript isoforms, and a massive increase in promoter-distal transcription of ncRNAs. Analysis of spherule-activated regulatory elements revealed a motif predicted to recruit a WOPR family transcription factor, which are known regulators of virulence in other fungi. We identify CIMG_02671 as a C. immitis WOPR homologue and show that it activates transcription in a WOPR motif-dependent manner, suggesting it is an important regulator of pathogenic phase transition. Collectively, this also highlights csRNA-seq as a powerful means to identify transcriptional mechanisms that control pathogenesis.


title: Cryo-EM structure of the NLRP3 decamer bound to the cytokine release inhibitory drug CRID3

abstract: NLRP3 is an intracellular sensor protein whose activation by a broad spectrum of exogenous and endogenous stimuli leads to inflammasome formation and pyroptosis. The mechanisms leading to NLRP3 activation and the way how antagonistic small molecules function remain poorly understood. Here we report the cryo-electron microscopy structures of full-length NLRP3 in its native form and complexed with the inhibitor CRID3 (also named MCC950). Inactive, ADP-bound NLRP3 is a decamer composed of homodimers of intertwined LRR domains that assemble back-to-back as pentamers with the NACHT domain located at the apical axis of this spherical structure. Molecular contacts between the concave sites of two opposing LRRs are mediated by an acidic loop extending from an LRR transition segment. Binding of CRID3 significantly stabilizes the NACHT and LRR domains relative to each other, allowing structural resolution of 3.9-4.2 [A]. CRID3 binds into a cleft, connecting four subdomains of the NACHT with the transition LRR. Its central sulfonylurea group interacts with the Walker A motif of the NLRP3 nucleotide-binding domain and is sandwiched between two arginines from opposing sites, explaining the specificity of NLRP3 for this chemical entity. With the determination of the binding site of this lead therapeutic, specific targeting of NLRP3 for the treatment of autoinflammatory and autoimmune diseases and rational drug optimization are within reach.


title: Limited accessibility of nitrogen supplied as amino acids, amides, and amines as energy sources for marine Thaumarchaeota

abstract: Genomic and physiological evidence from some strains of ammonia-oxidizing Thaumarchaeota demonstrate their additional ability to oxidize nitrogen (N) supplied as urea or cyanate, fueling conjecture about their ability to conserve energy by directly oxidizing reduced N from other dissolved organic nitrogen (DON) compounds. Similarly, field studies have shown rapid oxidation of polyamine-N in the ocean, but it is unclear whether Thaumarchaeota oxidize polyamine-N directly or whether heterotrophic DON remineralization is required. We tested growth of two marine Nitrosopumilus isolates on DON compounds including polyamines, amino acids, primary amines, and amides as their sole energy source. Though axenic cultures only consumed N supplied as ammonium or urea, there was rapid but inconsistent oxidation of N from the polyamine putrescine when cultures included a heterotrophic bacterium. Surprisingly, axenic cultures oxidized 15N-putrescine during growth on ammonia, suggesting co-metabolism or accelerated breakdown of putrescine by reactive metabolic byproducts. Nitric oxide, hydrogen peroxide, or peroxynitrite did not oxidize putrescine in sterile seawater. These data suggest that the N in common DON molecules is not directly accessible to marine Thaumarchaeota, with thaumarchaeal oxidation (and presumably assimilation) of DON-N requiring initial heterotrophic remineralization. However, reactive byproducts or enzymatic co-metabolism may facilitate limited thaumarchaeal DON-N oxidation.


title: Insertion sequences associated with antibiotic resistance genes in Enterococcus isolates from an inpatient with prolonged bacteremia.

abstract: Insertion sequences (ISs) and other transposable elements are associated with the mobilization of antibiotic resistance determinants and the modulation of pathogenic characteristics. In this work, we aimed to investigate the association between ISs and antibiotic resistance genes, and their role in dissemination and modification of antibiotic resistance phenotype. To that end, we leveraged fully resolved Enterococcus faecium and Enterococcus faecalis genomes of isolates collected over four days from an inpatient with prolonged bacteremia. Isolates from both species harbored similar IS family content but showed significant species-dependent differences in copy number and arrangements of ISs throughout their replicons. Here, we describe two inter-specific IS-mediated recombination events, and IS-medicated excision events in plasmids of E. faecium isolates. We also characterize a novel arrangement of the IS in a Tn1546-like transposon in E. faecalis isolates likely implicated in a vancomycin genotype-phenotype discrepancy. Furthermore, an extended analysis revealed a novel association between daptomycin resistance mutations in liaSR genes and a putative composite transposon in E. faecium offering a new paradigm for the study of daptomycin-resistance and novel insights into the route of daptomycin resistance dissemination. In conclusion, our study highlights the role ISs and other transposable elements play in rapid adaptation and response to clinically relevant stresses such as aggressive antibiotic treatment in enterococci.

category: genomics


title: Structure and lipid-mediated remodelling mechanism of the Na+/H+ exchanger NHA2

abstract: Na+/H+ exchangers catalyse an ion-exchange activity that is carried out in most, if not all cells. SLC9B2, also known as NHA2, correlates with the long-sought after sodium/lithium (Na+/Li+) exchanger linked to the pathogenesis of diabetes mellitus and essential hypertension in humans. Despite its functional importance, structural information and the molecular basis of its ion-exchange mechanism have been lacking. Here, we report the cryo EM structures of bison NHA2 in detergent and in nanodiscs at 3.0 and 3.5 [A] resolution, respectively. NHA2 shares closest structural similarity to the bacterial electrogenic Na+/H+ antiporter NapA, rather than other mammalian SLC9A members. Nevertheless, SSM-based electrophysiology results with NHA2 show the catalysis of electroneutral rather than electrogenic ion exchange, and the ion-binding site is quite distinctive, with a tryptophan-arginine- glutamate triad separated from the well-established ion-binding aspartates. These triad residues fine-tune ion binding specificity, as demonstrated by a salt-bridge swap mutant that converts NHA2 into a Li+-specific transporter. Strikingly, an additional N-terminal helix in NHA2 establishes a unique homodimer with a large [~] 25 [A] intracellular gap between protomers. In the presence of phosphatidylinositol lipids, the N-terminal helix rearranges and closes this gap. We confirm that dimerization of NHA2 is required for activity in vivo, and propose that the N- terminal helix has evolved as a lipid-mediated remodelling switch for regulation of transport activity.


title: Origin and evolutionary landscape of Nr2f transcription factors across Metazoa

abstract:
BackgroundNuclear Receptor Subfamily 2 Group F (Nr2f) orphan nuclear hormone transcription factors (TFs) are fundamental regulators of many developmental processes in invertebrates and vertebrates. Despite the importance of these TFs throughout metazoan development, previous work has not clearly outlined their evolutionary history.

ResultsWe integrated molecular phylogeny with comparisons of intron/exon structure, domain architecture, and syntenic conservation to define critical evolutionary events that distinguish the Nr2f gene family in Metazoa. Our data indicate that a single ancestral pre-metazoan Nr2f gene, we have termed Nr2f1/2/5/6, predated six main Bilateria subfamilies, which include a single Nr2f1/2/5 homolog that is present throughout protostomes and invertebrate deuterostomes, Nr2f1/2 homologs in agnathans, and Nr2f1, Nr2f2, Nr2f5, Nr2f6 orthologs that are found in gnathostomes. The three Nr2f1/2 members in agnathans are due to independent expansions not found in gnathostomes, while the vertebrate Nr2f1, Nr2f2, Nr2f5 members arose from whole-genome duplications (WGDs). However, Nr2f6 members are the most divergent subfamily, likely originating from an ancient duplication, and are only retained by gnathostomes. Interestingly, Nr2f5 TFs have been independently lost in both cartilaginous fish and amniotes, such as humans. Furthermore, our analysis shows there are differential expansions and losses of Nr2f genes in teleosts following their additional rounds of WGDs.

ConclusionOverall, our evolutionary genomic analysis of Nr2f proteins helps to reveal the origins and previously unrecognized relationships of this ancient transcription factor family, which may allow for greater insights into the conservation of Nr2f functions that shape Metazoan body plans.

category: evolutionary biology


title: The framework of plant regeneration in duckweed (Lemna turonifera) comprises genetic transcript regulation and cyclohexane release

abstract:
Regeneration is important for vegetative propagation of excellent variety, detoxification and the obtain of transgenic plant, but plant regeneration is time-consuming. Here, we found that duckweed regeneration could be enhanced by regenerating callus. Firstly, Genetic transcript regulation has been applied to study the molecular mechanism controlling regeneration. Auxin related genes have been significantly down-regulated in regenerating callus. Cytokinin signal pathway genes have been up-regulated in regenerating callus. Secondly, volatile organic compounds release has been analysised by gas chromatography/mass spectrum during the stage of plant regeneration, and 11 kinds of unique volatile organic compounds in the regenerating callus were increased. Among them, cyclohexane treatment enhanced duckweed regeneration by initiating root. Moreover, Auxin signal pathway genes were down-regulated in callus treated by cyclohexane. All together, these results provide novel mechanistic insights into how regenerating callus promotes duckweed regeneration.

Graphical abstract

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title: Optimal strategy for public good production is set by balancing intertemporal trade-off between population growth rate and carrying capacity

abstract:
Public goods are biomolecules that contribute to the community welfare. Their production can benefit populations in many ways, such as by providing access to previously unutilized resources. However, public good production has often been energetically costly, resulting in a reduction in the cellular growth rate. To reduce this cost, populations have evolved strategies to regulate biosynthesis of public good. Among these cell densities dependent regulation of public goods, as accomplished by quorum sensing, is a widely studied mechanism. Given that the fitness costs and benefits of public good production must be balanced, adoption of quorum sensing as a regulatory pathway by bacterial cells may have parallels with several economic principles that are used to study optimal investment decisions. Here, we explore the regulation of a public good, whose benefit is an increase in the carrying capacity, through experimental measurements of growth for engineered strains of Escherichia coli and analysis of those results using a modified logistic growth model. By varying the cell density at which the production of the public good was activated, we showed sharply-peaked optimum population fitness. Analysis further revealed that cell density associated with maximum public good benefits was determined by the trade-off between the cost of public good production, in terms of reduced growth rate, and benefits received from public good, in the form of increased carrying capacity. Moreover, our model showed that cells with luxRI quorum sensing seem to upregulate public good expression when the benefits from the production was immediate. These results demonstrate a case where a biological system apparently has evolved to optimize the timing of public good production to account for short-term costs and delays in reaping a future benefit.

Author summaryBacteria often cooperate by sharing resources, sometimes referred as public goods. The activity of public good molecules benefits all of the neighboring cells. Here we examine the public good amylase, an enzyme to break down starch into smaller pieces that can be eaten by cells. Amylase production benefits the entire population because it enables growth to a larger population size, but production of this enzyme also has a cost. Enzyme production requires cells to expend energy and cellular resources. To balance the cost and benefit of the production of amylase, cells have likely evolved a strategy to control the timing of the production of public goods. We tested the dependence of population fitness on the timing of amylase production, finding an optimal cell density for amylase production. Finding the balance means that cells have evolved to anticipate future events, such as when simple food sources are depleted. Similar balancing of immediate costs and future benefits has been observed and studied in economics. Our results revealed that cells which use the regulatory strategy quorum sensing to control amylase production have minimized the delay in obtaining a benefit from this public good.


title: Entrainment of Endangered Sturgeon by a Large Water Diversion: Rescue, Enumeration, and Conservation Opportunities

abstract: The Bonnet Carre Spillway diverts water from the Mississippi River through a floodway into Lake Pontchartrain to reduce river stages at New Orleans and prevent flood damages. Pallid Sturgeon, a federally listed species under the Endangered Species Act, and Shovelnose Sturgeon, listed under the Similarity of Appearance rule, are entrained through the Spillway structure and become trapped in the Spillway canals and other waterbodies. Five openings and corresponding rescue operations occurred between 2008 and 2019 after each Spillway closure. Operational parameters spanned a range of water temperatures and seasons with magnitude and duration of discharge varying across all openings. A total of 70 days with crew number ranging from 6 to 12 were expended to rescue 57 Pallid Sturgeon and 362 Shovelnose Sturgeon after the five openings that spanned 240 total days. More sturgeon were entrained at higher water temperatures, with greater numbers of bays opened, and for longer periods of time. Recovery of sturgeon is initially high but over time declines as sturgeon are depleted from the floodway, stranded in isolated waterbodies in the floodway, and/or displaced further downstream into Lake Pontchartrain during longer openings. Sturgeon that cannot find their way back to the floodway are unlikely to be rescued. Recent population studies indicate that less than 1% of the total population size in the Lower Mississippi River are entrained. However, this does not take into account those individuals entrained but not captured and the potential impacts of more frequent openings of the structure. Conservation recommendations are provided to increase catch efficiency and recovery of the endangered sturgeon.

category: ecology


title: The beneficial effect of chronic muscular exercise on muscle fragility is increased by Prox1 gene transfer in dystrophic mdx muscle

abstract:
PurposeGreater muscle fragility is thought to cause the exhaustion of the muscle stem cells during successive degeneration/repair cycles, leading to muscle wasting and weakness in Duchenne muscular dystrophy. Chronic voluntary exercise can partially reduce the susceptibility to contraction induced-muscle injury, i.e., muscle fragility, as shown by a reduced immediate maximal force drop following lengthening contractions, in the dystrophic mdx mice. Here, we studied the effect of Prospero-related homeobox factor 1 gene (Prox1) transfer (overexpression) on fragility in chronically exercised mdx mice, because Prox1 promotes slower type fibres in healthy mice and slower fibres are less fragile in mdx muscle.

Methodsmdx mice received or not Prox1 transfer into the tibialis anterior muscle and performed voluntary running into a wheel during 1 month. We also performed Prox1 transfer in sedentary mdx mice. In situ maximal force production of the muscle in response to nerve stimulation was assessed before, during and after 10 lengthening contractions. Molecular and cellular parameters were also evaluated.

ResultsInterestingly, Prox1 transfer reduced the force drop following lengthening contractions in exercised mdx mice (p < 0.05 to 0.01), but not in sedentary mdx mice. It also increased the muscle expression of Myh7 (p < 0.001), MHC-2x (p < 0.01) and Trpc1 (p < 0.01), whereas it reduced that one of Myh4 (p < 0.001) and MHC-2b (p < 0.01) in exercised mdx mice. Moreover, Prox1 transfer decreased the maximal force (p < 0.01) before lengthening contraction in exercised mdx mice (p < 0.01), and reduced muscle weight (p < 0.0001) despite increased Mstn expression (p < 0.001).

ConclusionOur results indicate that the beneficial effect of Prox1 transfer on muscle fragility is only observed in chronically exercised mdx mice. Thus, Prox1 transfer combined to chronic exercise have the potential to substantially slow the progression of the dystrophic disease in the long term.


title: Reconstruction of a generic genome-scale metabolic network for chicken: investigating network connectivity and finding potential biomarkers

abstract: Chicken is the first sequenced avian that has a crucial role in human life for its meat and egg production. Because of various metabolic disorders, study the metabolism of chicken cell is important. Herein, the first genome-scale metabolic model of a chicken cell named iES1300, consists of 2427 reactions, 2569 metabolites, and 1300 genes, was reconstructed manually based on databases. Interactions of metabolic genes for growth were examined for E. coli, S. cerevisiae, human, and chicken metabolic models. The results indicated robustness to genetic manipulation for iES1300 similar to the results for human. iES1300 was integrated with transcriptomics data using algorithms and Principal Component Analysis was applied to compare context-specific models of the normal, tumor, lean and fat cell lines. It was found that the normal model has notable metabolic flexibility in the utilization of various metabolic pathways, especially in metabolic pathways of the carbohydrate metabolism, compared to the others. It was also concluded that the fat and tumor models have similar growth metabolisms and the lean chicken model has a more active lipid and carbohydrate metabolism.

category: systems biology


title: Numerical approaches for the rapid analysis of prophylactic efficacy against HIV with arbitrary drug-dosing schemes

abstract:
Pre-exposure prophylaxis (PrEP) is an important pillar to prevent HIV transmission. Because of experimental and clinical shortcomings, mathematical models that integrate pharmacological, viral- and host factors are frequently used to quantify clinical efficacy of PrEP. Stochastic simulations of these models provides sample statistics from which the clinical efficacy is approximated. However, many stochastic simulations are needed to reduce the associated sampling error. To remedy the shortcomings of stochastic simulation, we developed three numerical methods that allow predicting the efficacy of arbitrary prophylactic regimen directly from a viral dynamics model, without sampling. We apply the methods to various hypothetical dolutegravir (DTG) prophylaxis scenarios. The approaches are verified against one another, as well as state-of-the-art stochastic simulation. While the methods are more accurate than stochastic simulation, they are superior in terms of computational performance. For example, a continuous 6-month prophylactic profile is computed within a few seconds on a laptop computer. The methods computational performance, therefore, substantially expands the horizon of feasible analysis in the context of PrEP, and possibly other applications.

Author summaryPre-exposure prophylaxis (PrEP) is an important tool to prevent HIV transmission. However, experimental identification of parameters that determine prophylactic efficacy is extremely difficult. Clues about these parameters could prove essential for the design of next-generation PrEP compounds. Integrative mathematical models can fill this void: Based on stochastic simulation, a sample statistic can be generated, from which the prophylactic efficacy is estimated. However, for this sample statistic to be accurate, many simulations need to be performed.

Here, we introduce three numerical methods to directly compute the prophylactic efficacy from a viral dynamics model, without the need for sampling. Based on several examples with dolutegravir (DTG) -based short- and long-term PrEP, as well as post-exposure prophylaxis we demonstrate the correctness of the new methods and their outstanding computational performance. Due to the methods computational performance, a number of analysis, including formal sensitivity analysis are becoming feasible with the proposed methods.


title: A pipeline for the reconstruction and evaluation of context-specific human metabolic models at a large-scale

abstract:
Constraint-based (CB) metabolic models provide a mathematical framework and scaffold for in silico cell metabolism analysis and manipulation. In the past decade, significant efforts have been done to model human metabolism, enabled by the increased availability of multi-omics datasets and curated genome-scale reconstructions, as well as the development of several algorithms for context-specific model (CSM) reconstruction. Although CSM reconstruction has revealed insights on the deregulated metabolism of several pathologies, the process of reconstructing representative models of human tissues still lacks benchmarks and appropriate integrated software frameworks, since many tools required for this process are still disperse across various software platforms, some of which are proprietary.

In this work, we address this challenge by assembling a scalable CSM reconstruction pipeline capable of integrating transcriptomics data in CB models. We combined omics preprocessing methods inspired by previous efforts with in-house implementations of existing CSM algorithms and new model refinement and validation routines, all implemented in the Troppo Python-based open-source framework. The pipeline was validated with multi-omics datasets from the Cancer Cell Line Encyclopedia (CCLE), also including reference fluxomics measurements for the MCF7 cell line.

We reconstructed over 6000 models based on the Human-GEM template model for 733 cell lines featured in the CCLE, using MCF7 models as reference to find the best parameter combinations. These reference models outperform earlier studies using the same template by comparing gene essentiality and fluxomics experiments. We also analysed the heterogeneity of breast cancer cell lines, identifying key changes in metabolism related to cancer aggressiveness. Despite the many challenges in CB modelling, we demonstrate using our pipeline that combining transcriptomics data in metabolic models can be used to investigate key metabolic shifts. Significant limitations were found on these models ability for reliable quantitative flux prediction, thus motivating further work in genome-wide phenotype prediction.

Author summaryGenome-scale models of human metabolism are promising tools capable of contextualising large omics datasets within a framework that enables analysis and manipulation of metabolic phenotypes. Despite various successes in applying these methods to provide mechanistic hypotheses for deregulated metabolism in disease, there is no standardized workflow to extract these models using existing methods and the tools required to do so are mostly implemented using proprietary software.

We have assembled a generic pipeline to extract and validate context-specific metabolic models using multi-omics datasets and implemented it using the troppo framework. We first validate our pipeline using MCF7 cell line models and assess their ability to predict lethal gene knockouts as well as flux activity using multi-omics data. We also demonstrate how this approach can be generalized for large-scale transcriptomics datasets and used to generate insights on the metabolic heterogeneity of cancer and relevant features for other data mining approaches. The pipeline is available as part of an open-source framework that is generic for a variety of applications.


title: TMT-based proteomic analysis of the antibiotic effects of ShangKeHuangShui against Straphylococcus aureus.

abstract:
Post-traumatic infection is a serious orthopedic trauma complication commonly caused by Staphylococcus aureus (SAU). ShangKeHuangShui, also known as Yellow Aqueous Concentrate of Traumatology Herbs (YACTH), is prepared from six Chinese herbal medicines, which has been used for decades in our hospital to prevent post-traumatic infection.

In the present study, we investigated the in vitro antibacterial effects and underlying mechanism of YACTH against SAU. YACTH exhibited significant antibacterial activity against SAU with the minimum inhibitory concentration (MIC) of 8.625 mg/mL. Proteomic analysis based on Tandem mass tag (TMT) showed different protein expression levels in SAU under the YACTH and control conditions. Compared to the control group, the expression level of 490 proteins of YACTH treated group significantly changed (>1.2 fold, P <0.05). Biological informatics analysis showed that these differential proteins were widely involved in a variety of biological processes and multiple metabolic pathways. We then selected 26 target proteins to further conduct Parallel reaction monitoring (PRM). The results revealed that the 9 down-regulated proteins were mainly involved in RNA polymerase, oxidative phosphorylation, ABC transporter and glycolysis; whereas, the 17 up-regulated proteins are related to RNA degradation, mismatch repair and ribosome.

In conclusion, YACTH, which contains the water-soluble components of six Chinese herbs, is first reported to exhibit antibacterial activity against SAU in vitro. The results provide novel insights into the antibacterial mechanisms of YACTH on protein networks, which may help us find new potential antibiotic targets.


title: Evolution of Learning in Technology Adoption: The case of the U.S. Soybean Seed Industry.

abstract:
This paper examines how the evolution of learning affects technology adoption. We use a sequential adoption model that accounts for differences between forward-looking adopters, who consider future impacts of their learning, and myopic adopters, who only consider past learning. We apply the analysis to three panels of U.S. soybean farmers representing different stages of the genetically modified (GM) seed technology diffusion path. We show that uncertainty is considerably reduced over time due to increased learning efficiency. Our results indicate that a forward-looking model fits the early adopters and early majority stages better, while both models perform equally well in the laggard stage.

JEL classificationD83, Q31, Q33, Q16

category: scientific communication and education


title: Learning the rules of collective cell migration using deep attention networks

abstract:
Collective, coordinated cellular motions underpin key processes in all multicellular organisms, yet it has been difficult to express the  rules behind these motions in clear, interpretable forms. Here we apply deep attention networks to analyze several canonical living tissues systems and present the underlying collective migration rules for each tissue type using only cell migration trajectory data. We use these networks to learn the behaviors of key tissue types with distinct collective behaviors--epithelial, endothelial, and metastatic breast cancer cells--and show how the results complement traditional biophysical approaches. In particular, we present attention maps indicating the relative influence of neighboring cells to the learned turning decisions of a focal cell. These attention networks reveal distinct patterns of influence and attention unique to each model tissue. Endothelial cells exhibit tightly focused attention on their immediate forward-most neighbors, while cells in more expansile epithelial tissues are more broadly influenced by neighbors in a relatively large forward sector. Attention maps of ensembles of more mesenchymal, metastatic cells reveal completely symmetric attention patterns, indicating the lack of any particular coordination or direction of interest. Moreover, we show how attention networks are capable of detecting and learning how these rules changes based on biophysical context, such as location within the tissue and cellular crowding. That these results require only cellular trajectories and no modeling assumptions highlights the potential of attention networks for providing further biological insights into complex cellular systems.

Author SummaryCollective behaviors are crucial to the function of multicellular life, with large-scale, coordinated cell migration enabling processes spanning organ formation to coordinated skin healing. However, we lack effective tools to discover and cleanly express collective rules at the level of an individual cell. Here, we employ a carefully structured neural network to extract collective information directly from cell trajectory data. The network is trained on data from various systems, including well-studied cell types (HUVEC and MDCK cells) which display visually distinct forms of collective motion, and metastatic cancer cells (MDA-MB-231) which are highly uncoordinated. Using these trained networks, we can produce attention maps for each system, which indicate how a cell within a tissue takes in information from its surrounding neighbors, as a function of weights assigned to those neighbors. Thus for a cell type in which cells tend to follow the path of the cell in front, the attention maps will display high weights for cells spatially forward of the focal cell. We present results in terms of additional metrics, such as accuracy plots and number of interacting cells, and encourage future development of improved metrics.


title: Prediction of fluorophore brightness in designed mini fluorescence activating proteins

abstract: The de novo computational design of proteins with predefined three-dimensional structure is becoming much more routine due to advancements both in force fields and algorithms. However, creating designs with functions beyond folding is more challenging. In that regard, the recent design of small beta barrel proteins that activate the fluorescence of an exogenous small molecule chromophore (DFHBI) is noteworthy. These proteins, termed mini Fluorescence Activating Proteins (mFAPs), have been shown increase the brightness of the chromophore more than 100-fold upon binding to the designed ligand pocket. The design process created a large library of variants with different brightness levels but gave no rational explanation for why one variant was brighter than another. Here we use quantum mechanics and molecular dynamics simulations to investigate how molecular flexibility in the ground and excited states influences brightness. We show that the ability of the protein to resist dihedral angle rotation of the chromophore is critical for predicting brightness. Our simulations suggest that the mFAP/DFHBI complex has a rough energy landscape, requiring extensive ground-state sampling to achieve converged predictions of excited-state kinetics. While computationally demanding, this roughness suggests that mFAP protein function can be enhanced by reshaping the energy landscape towards states that better resist DFHBI bond rotation.

category: biophysics


title: A newly identified group of P-like (PL) fimbriae from extra-intestinal pathogenic Escherichia coli (ExPEC) encode distinct adhesin subunits and mediate adherence to host cells

abstract:
Fimbrial adhesins play a critical role for bacterial adherence and biofilm formation. Sequencing of avian pathogenic Escherichia coli (APEC) strain QT598 identified a fimbrial gene cluster belonging to the {pi} group that we named PL (P-like) fimbriae, since genetic organization and sequence are similar to Pap and related fimbriae. Screening of genomic databases indicated that genes encoding PL fimbriae located on IncF plasmids are present in a diversity of E. coli isolates from poultry, human systemic infections, and other sources. As with P fimbriae, PL fimbriae exhibit sequence divergence in adhesin encoding genes, and strains could be divided into 5 classes based on differences in sequences of the PlfG adhesin protein. The plf genes from two predominant PlfG adhesin classes, PlfG-I and PlfG-II were cloned. PL fimbriae were visualized by electron microscopy, promoted biofilm formation, demonstrated distinct hemagglutination profiles and promoted adherence to human bladder and kidney epithelial cell lines. Hybrid fimbriae comprised of genes from plfQT598 wherein plfG was replaced by papG encoding adhesin genes were also shown to be functional and mediate adherence to epithelial cells, further indicating similarity and functional compatibility between these two types of fimbriae. Although deletion of plf genes did not significantly reduce colonization of the mouse urinary tract, plf gene expression was increased over 40-fold in the bladder compared to during in vitro culture. Overall, PL fimbriae represent a new group of fimbriae demonstrating both functional differences and similarities to P fimbriae and may contribute to adherence to cells and colonization of host tissues.

ImportanceFimbriae are important colonization factors in many bacterial species. The identification of a new type of fimbriae encoded on some IncF plasmids in E. coli was investigated. Genomic sequences demonstrated these fimbrial gene clusters have genetic diversity, particularly in the adhesin encoding PlfG gene. Functional studies demonstrated differences in hemagglutination specificity, although both types of Plf adhesin under study mediated adherence to human urinary epithelial cells. Such fimbriae may represent previously unrecognized adhesins that could contribute to host specificity and tissue tropism of some E. coli strains.


title: Chronic release of tailless phage particles from Lactococcus lactis

abstract: Lactococcus lactis strains residing in the microbial community of a complex dairy starter culture named "Ur" are hosts to prophages belonging to the family Siphoviridae. L. lactis strains (TIFN1 to TIFN7) showed detectable spontaneous phage production and release (109-1010 phage particles/mL) and up to 10-fold increases upon prophage induction, while in both cases we observed no obvious cell lysis, typically described for the lytic life cycle of Siphoviridae phages. Intrigued by this phenomenon, we investigated the host-phage interaction using strain TIFN1 (harboring prophage proPhi1) as a representative. We confirmed that during the massive phage release, all bacterial cells remain viable. Further, by monitoring phage replication in vivo, using a green fluorescence protein reporter combined with flow cytometry, we demonstrated that the majority of the bacterial population (over 80%) is actively producing phage particles when induced with mitomycin C. The released tailless phage particles were found to be engulfed in lipid membranes, as evidenced by electron microscopy and lipid staining combined with chemical lipid analysis. Based on the collective observations, we propose a model of phage-host interaction in L. lactis TIFN1, where the phage particles are engulfed in membranes upon release, thereby leaving the producing host intact. Moreover, we discuss possible mechanisms of chronic, or non-lytic release of LAB Siphoviridae phages and its impact on the bacterial host.


title: Manipulation of RNA Polymerase III by Herpes Simplex Virus-1

abstract: RNA Polymerase III (Pol III) transcribes noncoding RNA, including transfer RNA (tRNA), and acts as a pathogen sensor during the innate immune response. To promote enhanced proliferation, the Pol III machinery is commonly targeted during cancer and viral infection. Herein we employ DM-RNA-Seq, 4SU-Seq, ChIP-Seq, and ATAC-Seq to characterize how Herpes Simplex Virus-1 (HSV-1) perturbs the Pol III landscape. We find that HSV-1 stimulates tRNA expression 10-fold, with mature tRNAs exhibiting a 2-fold increase within 12 hours of infection. Perturbation of host tRNA synthesis requires nuclear viral entry, but not synthesis of specific viral transcripts, nascent viral genomes, or viral progeny. Host tRNA with a specific codon bias were not targeted--rather increased transcription was observed from euchromatic, actively transcribed loci. tRNA upregulation is linked to unique crosstalk between the Pol II and III transcriptional machinery. While viral infection is known to mediate host transcriptional shut off and lead to a depletion of Pol II on host mRNA promoters, we find that Pol II binding to tRNA loci increases. Finally, we report Pol III and associated factors bind the HSV genome, which suggests a previously unrecognized role in HSV-1 gene expression. These data provide insight into novel mechanisms by which HSV-1 alters the host nuclear environment, shifting key processes in favor of the pathogen.


title: De novo disease-associated mutations in KIF1A dominant negatively inhibit axonal transport of synaptic vesicle precursors

abstract:
KIF1A is a kinesin superfamily molecular motor that transports synaptic vesicle precursors in axons. Mutations in Kif1a lead to a group of neuronal diseases called KIF1A-associated neuronal disorder (KAND). KIF1A forms a homodimer and KAND mutations are mostly de novo and autosomal dominant; however, it is not known whether the function of wild-type KIF1A is inhibited by disease-associated KIF1A when they are dimerized. No reliable in vivo model systems to analyze the molecular and cellular biology of KAND caused by loss of function mutations have been developed; therefore, here, we established Caenorhabditis elegans models for KAND using CRISPR/cas9 technology and analyzed defects in axonal transport. In the C. elegans models, heterozygotes and homozygotes exhibited reduced axonal transport phenotypes. Suppressor screening using the disease model worm identified a mutation that recovers the motor activity of disease-associated human KIF1A. In addition, we developed in vitro assays to analyze the motility of single heterodimers composed of wild-type KIF1A and disease-associated KIF1A. Disease-associated KIF1A significantly inhibited the motility of wild-type KIF1A when heterodimers were formed. These data indicate the molecular mechanism underlying the dominant nature of de novo KAND mutations.

Significance StatementKIF1A is a molecular motor that transports synaptic vesicle precursors in axons. Recent studies have identified many KIF1A mutations in congenital neuropathy patients; however, the molecular mechanism of pathogenesis remains largely elusive. This study established loss of function models for KIF1A-associated neuronal disorder (KAND) in Caenorhabditis elegans to analyze the molecular and cell biology of the disease in vivo. Genetic screening using the disease model could find a mutation that recovers the motor activity of disease-associated KIF1A. This study also established in vitro single-molecule assays to quantitatively analyze the effect of KAND mutations when mutant KIF1A forms heterodimers with wild-type KIF1A. Our findings provide a foundation for future genetic screening and for drug screening to search for KAND treatments.


title: Matrix elasticity gradients guide neuronal polarity by controlling microtubule network mobility

abstract: Neuronal polarization and axon specification depend on extracellular cues, intracellular signaling, cytoskeletal rearrangements and polarized transport, but the interplay between these processes has remained unresolved. The polarized transport of kinesin-1 into a specific neurite is an early marker for axon identity, but the mechanisms that govern neurite selection and polarized transport are unknown. We show that extracellular elasticity gradients control polarized transport and axon specification, mediated by Rho-GTPases whose local activation is necessary and sufficient for polarized transport. Selective Kinesin-1 accumulation furthermore depends on differences in microtubule network mobility between neurites and local control over this mobility is necessary and sufficient for proper polarization, as shown using optogenetic anchoring of microtubules. Together, these results explain how mechanical cues can instruct polarized transport and axon specification.