title: Engineering Protein Activity into Off-the-Shelf DNA Devices

abstract: DNA-based devices are relatively straightforward to design by virtue of their predictable folding, but they lack biological activity. Conversely, protein-based devices offer a myriad of biological functions but are much more difficult to design due to their complex folding. This study bridges the fields of DNA engineering and protein engineering to generate a protein switch that is activated by a specific DNA sequence. A single protein switch, engineered from nanoluciferase using the alternate frame folding mechanism and herein called nLuc-AFF, is paired with different DNA technologies to create a biosensor for a DNA or RNA sequence of choice, sensors for serotonin and ATP, and a computational device that processes two DNA inputs. nLuc-AFF is a genetically-encoded, ratiometric, blue/green-luminescent biosensor whose output can be quantified by cell phone camera. nLuc-AFF is not falsely activated by decoy DNA and it retains full ratiometric readout in 100 % serum. The design approach can be applied to other proteins and enzymes to convert them into DNA-activated switches.


title: Long-term dynamic changes of NMDA receptors following an excitotoxic challenge

abstract: Excitotoxicity is a form of neuronal death characterized by the sustained activation of N-methyl-D-aspartate receptors (NMDARs) triggered by the excitatory neurotransmitter glutamate. NADPH-diaphorase neurons [also known as nNOS (+) neurons] are a subpopulation of aspiny interneurons, largely spared following excitotoxic challenges. Unlike nNOS (-) cells, nNOS (+) neurons fail to generate reactive oxygen species in response to NMDAR activation, a key divergent step in the excitotoxic cascade. However, additional mechanisms underlying the reduced vulnerability of nNOS (+) neurons to NMDAR-driven neuronal death have not been explored. Using functional, genetic, and molecular analysis in striatal cultures, we demonstrate that nNOS (+) neurons possess distinct NMDAR properties. These specific features are primarily driven by the peculiar redox milieu of this subpopulation. In addition, we found that nNOS (+) neurons exposed to a pharmacological maneuver set to mimic chronic excitotoxicity alter their responses to NMDAR-mediated challenges. These findings suggest the presence of mechanisms providing long-term dynamic regulation of NMDARs that can have critical implications in neurotoxic settings.

category: neuroscience


title: Essential regulatory functions of CaMKII T286 phosphorylation in LTP and two distinct forms of LTD

abstract: The Ca2+/calmodulin-dependent protein kinase II (CaMKII) mediates both long-term potentiation and depression (LTP and LTD) of excitatory synapses, two opposing forms of synaptic plasticity induced by strong versus weak stimulation of NMDA-type glutamate receptors (NMDARs). NMDAR-dependent LTD is prevalent in juvenile hippocampus, but in mature hippocampus, LTD is still readily induced by stimulating metabotropic glutamate receptors (mGluRs). Here we show that mGluR-dependent LTD also requires CaMKII and its T286 autophosphorylation that induces Ca2+-independent autonomous kinase activity. This autophosphorylation (i) accelerated CaMKII movement to excitatory synapses after LTP stimuli and (ii) was required for the movement to inhibitory synapses after NMDAR-LTD stimuli. Similar to NMDAR-LTD, the mGluR-LTD stimuli did not induce any CaMKII movement to excitatory synapses. However, in contrast to NMDAR-LTD, the mGluR-LTD did not involve CaMKII movement to inhibitory synapses and did not require additional T305/306 autophosphorylation. Taken together, even though CaMKII T286 autophosphorylation has a longstanding prominent role in LTP, it is also required for both major forms of LTD in hippocampal neurons, albeit with differential requirements for the heterosynaptic communication of excitatory signals to inhibitory synapses.


title: Integrated genomic analysis of AgRP neurons reveals that IRF3 regulates leptin's hunger-suppressing effects

abstract: AgRP neurons in the arcuate nucleus of the hypothalamus (ARC) coordinate homeostatic changes in appetite associated with fluctuations in food availability and leptin signaling. Identifying the relevant transcriptional regulatory pathways in these neurons has been a priority, yet such attempts have been stymied due to their low abundance and the rich cellular diversity of the ARC. Here we generated AgRP neuron-specific transcriptomic and chromatin accessibility profiles during opposing states of fasting-induced hunger and leptin-induced hunger suppression. Cis-regulatory analysis of these integrated datasets enabled the identification of 28 putative hunger-promoting and 29 putative hunger-suppressing transcriptional regulators in AgRP neurons, 16 of which were predicted to be transcriptional effectors of leptin. Within our dataset, Interferon regulatory factor 3 (IRF3) emerged as a leading candidate mediator of leptin-induced hunger-suppression. Gain- and loss-of-function experiments in vivo confirm the role of IRF3 in mediating the acute satiety-evoking effects of leptin in AgRP neurons, while live-cell imaging in vitro indicate that leptin can activate neuronal IRF3 in a cell autonomous manner. Finally, we employ CUT&RUN to uncover direct transcriptional targets of IRF3 in AgRP neurons in vivo. Thus, our findings identify AgRP neuron-expressed IRF3 as a key transcriptional effector of the hunger-suppressing effects of leptin.


title: SARS-CoV-2 entry sites are present in all structural elements of the human glossopharyngeal and vagal nerves: clinical implications

abstract: Severe acute respiratory syndrome coronavirus (SARS-CoV-2) infections result in the temporary loss of smell and taste (anosmia and dysgeusia) in about one third of confirmed cases. Several investigators have reported that the viral spike protein receptor is present in olfactory neurons. However, no study has been published to date showing the presence of viral entry sites angiotensin-converting enzyme 2 (ACE2), neuropilin1 (NRP1), and TMPRSS2, the serine protease necessary for priming the viral proteins, in human nerves that are responsible for taste sensation (cranial nerves: VII, IX and X). We used immunocytochemistry to examine three postmortem donor samples of the IXth (glossopharyngeal) and Xth (vagal) cranial nerves where they leave/join the medulla from three donors to confirm the presence of ACE2, NRP1 and TMPRSS2. Two samples were paraffin embedded; one was a frozen sample. In addition to staining sections from the latter, we isolated RNA from it, made cDNA, and performed PCR to confirm the presence of the mRNAs that encode the proteins visualized. All three of the proteins required for SARS-CoV-2 infections appear to be present in the human IXth and Xth nerves near the medulla. Direct infection of these nerves by the COVID-19 virus is likely to cause the loss of taste experienced by many patients. In addition, potential viral spread through these nerves into the adjacent brainstem respiratory centers might also aggravate the respiratory problems patients are experiencing.


title: Phosphoantigens are Molecular Glues that Promote Butyrophilin 3A1/2A1 Association Leading to Vγ9Vδ2 T Cell Activation

abstract:
Tumor cells and pathogen-infected cells are presented to human {gamma}{delta} T cells based on "inside-out" signaling in which metabolites called phosphoantigens (pAgs) inside target cells are recognized by the intracellular domain of a butyrophilin protein (BTN3A1), leading to an extracellular conformational change. Here, we report that pAgs function as molecular "glues" that initiate a heteromeric association between the intracellular domains of BTN3A1 and the structurally similar BTN2A1. Working with both exogenous and endogenous pAgs, we used x-ray crystallography, mutational studies, cellular assays, synthetic probe as well as molecular dynamics investigations to determine how pAgs glue intracellular BTN3A1 and BTN2A1 together for the "inside-out" signaling that triggers {gamma}{delta} T cell activation. This {gamma}{delta} T cell-specific mode of antigen sensing creates opportunities for the development of alternative immunotherapies against cancer and infectious diseases that do not involve {beta} T cells.

One Sentence SummaryThe responses of gamma-delta T cells to cancer cells or pathogens are initiated via the intracellular association of heteromeric butyrophilins that are glued together by isoprenoid metabolites.

category: immunology


title: Nonself Mutations in the Spike Protein Suggest an Increase in the Antigenicity and a Decrease in the Virulence of the Omicron Variant of SARS-CoV-2

abstract: Despite extensive worldwide vaccination, the current COVID-19 pandemic caused by SARS-CoV-2 continues. The Omicron variant is a recently emerged variant of concern and is now taking over the Delta variant. To characterize the potential antigenicity of the Omicron variant, we examined the distributions of SARS-CoV-2 nonself mutations (in reference to the human proteome) as 5 amino acid stretches of short constituent sequences (SCSs) in the Omicron and Delta proteomes. The number of nonself SCSs did not differ much throughout the Omicron, Delta, and Reference Sequence (RefSeq) proteomes but markedly increased in the receptor binding domain (RBD) of the Omicron spike protein compared to those of the Delta and RefSeq proteins. In contrast, the number of nonself SCSs decreased in non-RBD regions in the Omicron spike protein, compensating for the increase in the RBD. Several nonself SCSs were tandemly present in the RBD of the Omicron spike protein, likely as a result of selection for higher binding affinity to the ACE2 receptor (and hence higher infectivity and transmissibility) at the expense of increased antigenicity. Taken together, the present results suggest that the Omicron variant has evolved to have higher antigenicity and less virulence in humans despite increased infectivity and transmissibility.


title: RG203KR mutations in SARS-CoV-2 Nucleocapsid: Assessing the impact using Virus-like particle model system

abstract: The emergence and evolution of SARS-CoV-2 is characterized by the occurrence of diverse sets of mutations that affect virus characteristics, including transmissibility and antigenicity. Recent studies have focused mostly on Spike protein mutations; however, SARS-CoV-2 variants of interest (VoI) or concern (VoC) contain significant mutations in the nucleocapsid protein as well. To study the relevance of the mutations at the virion level, recombinant baculovirus expression system based VLPs were generated for the prototype Wuhan sequence along with Spike mutants like D614G, G1124V and the significant RG203KR mutation in Nucleocapsid. All the four structural proteins assembled in a particle wherein the morphology and size of the particle confirmed by TEM closely resembles the native virion. The VLP harbouring RG203KR mutations in nucleocapsid exhibited augmentation of humoral immune responses and enhanced neutralization by the immunized mice sera. Results demonstrate a non-infectious platform to quickly assess the implication of mutations in structural proteins of the emerging variant.

category: microbiology


title: Duplication and overexpression of the genes encoding a beta 1,3-glucan synthase confer the intrinsic resistance to echinocandin in Mucor circinelloides

abstract:
Procedures such as solid organ transplants and cancer treatments can leave many patients in an immunocompromised state resulting in an increased susceptibility to opportunistic diseases including fungal infections. Mucormycosis infections are continually emerging and pose a serious threat to immunocompromised patients. Currently there has been a sharp increase in mucormycosis cases as a secondary infection in patients battling SARS-CoV-2 infections. Mucorales fungi are notorious for presenting resistance to most antifungal drugs. The absence of effective means to treat these infections results in mortality rates approaching 100% in cases of disseminated infection. One of the most effective antifungal drug classes currently available are echinocandins. Echinocandins seem to be efficacious in treatment of many other fungal infections. Unfortunately, susceptibility testing has found that echinocandins have no to little effect on Mucorales. In this study, we found that the model Mucorales Mucor circinelloides genome carries three copies of the genes encoding for the echinocandin {beta}-(1,3)-D-glucan synthase (fksA, fksB, and fksC). Interestingly, we revealed that exposing M. circinelloides to micafungin significantly increased the expression of the fksA and fksB genes when compared to an untreated control. We further uncovered that the serine/threonine phosphatase calcineurin is responsible for the overexpression of fksA and fksB as deletion of calcineurin results in a decrease in expression of all three fks genes and a lower minimal inhibitory concentration (MIC) to micafungin. Taken together, this study demonstrates that the fks gene duplication and overexpression by calcineurin contribute to the intrinsic resistance to echinocandins in Mucor.

IMPORTANCEThe recent emergence of mucormycosis cases in immunocompromised patients has become a more prevalent issue. The Mucorales fungi that cause these infections are known to be highly drug resistant, thus treatment options are limited and the mortalities of these types of infections remain unacceptably high. Echinocandins are one of the latest antifungal drugs developed to successfully treat fungal infections, but it remains unclear why Mucorales fungi are resistant to these treatments. In our study, we uncovered three copies of the genes (fks) encoding the drug target for echinocandins. Furthermore, we discovered that the serine-threonine phosphatase calcineurin is regulating the over-expression of these genes, which confers resistance. By inhibiting calcineurin we found that the expression of these drug targets decreases resulting in an increase in susceptibility to echinocandins both in vitro and in vivo


title: In planta transcriptomics reveals conflicts between pattern-triggered immunity and the AlgU sigma factor regulon

abstract: In previous work, we determined the transcriptomic impacts of flg22 pre-induced Pattern Triggered Immunity (PTI) in Arabidopsis thaliana on the pathogen Pseudomonas syringae pv. tomato DC3000 (Pto). During PTI exposure we observed expression patterns in Pto reminiscent of those previously observed in a Pto algU mutant. AlgU is a conserved extracytoplasmic function sigma factor which has been observed to regulate over 950 genes in Pto in vitro. We sought to identify the AlgU regulon in planta.and which PTI-regulated genes overlapped with AlgU-regulated genes. In this study, we analyzed transcriptomic data from RNA-sequencing to identify the AlgU in planta regulon and its relationship with PTI. Our results showed that approximately 224 genes are induced by AlgU, while another 154 genes are downregulated by AlgU in Arabidopsis during early infection. Both stress response and virulence-associated genes were induced by AlgU, while the flagellar motility genes are downregulated by AlgU. Under the pre-induced PTI condition, more than half of these AlgU-regulated genes have lost induction/suppression in contrast to naive plants, and almost all function groups regulated by AlgU were affected by PTI.


title: Understanding drivers of phylogenetic clustering and terminal branch lengths distribution in epidemics of Mycobacterium tuberculosis

abstract: Detecting factors associated with transmission is important to understand disease epidemics, and to design effective public health measures. Clustering and terminal branch lengths (TBL) analyses are commonly applied to genomic data sets of Mycobacterium tuberculosis (MTB) to identify sub-populations with increased transmission. Here, I used a simulation-based approach to investigate what epidemiological processes influence the results of clustering and TBL analyses, and whether difference in transmission can be detected with these methods. I simulated MTB epidemics with different dynamics (latency, infectious period, transmission rate, basic reproductive number R0, sampling proportion, and molecular clock), and found that all these factors, except the length of the infectious period and R0, affect the results of clustering and TBL distributions. I show that standard interpretations of this type of analyses ignore two main caveats: 1) clustering results and TBL depend on many factors that have nothing to do with transmission, 2) clustering results and TBL do not tell anything about whether the epidemic is stable, growing, or shrinking. An important consequence is that the optimal SNP threshold for clustering depends on the epidemiological conditions, and that sub-populations with different epidemiological characteristics should not be analyzed with the same threshold. Finally, these results suggest that different clustering rates and TBL distributions, that are found consistently between different MTB lineages, are probably due to intrinsic bacterial factors, and do not indicate necessarily differences in transmission or evolutionary success.


title: An in vitro and in vivo approach for the isolation of Omicron variant from human clinical specimens

abstract: Due to failure of virus isolation of Omicron variant in Vero CCL-81 from the clinical specimens of COVID-19 cases, we infected Syrian hamsters and then passage into Vero CCL-81 cells. The Omicron sequences were studied to assess if hamster could incorporate any mutation to changes its susceptibility. L212C mutation, Tyrosine 69 deletion, and C25000T nucleotide change in spike gene and absence of V17I mutation in E gene was observed in sequences of hamster passage unlike human clinical specimen and Vero CCL-81 passages. No change was observed in the furin cleavage site in any of the specimen sequence which suggests usefulness of these isolates in future studies.


title: Omicron variant escapes therapeutic mAbs contrary to eight prior main VOC

abstract: Monocolonal antibodies (mAbs) are currently used for active immunization of COVID-19 in immunocompromised patients. We herein show that in spite there are variations in susceptibility to available mAbs that are authorized for clinical use in France tested on the original B.1.1 virus and 9 variants of concern or of interest, the cocktail casirivimab/imdevimab (REGN-CoV-2) showed a major synergistic effect. However, none of the four mAbs either alone or in combination neutralized the new Omicron variant. Our data strongly warrant a reinforcement of protective measures against infection for immunocompromised patients.


title: In vitro evaluation of therapeutic antibodies against a SARS-CoV-2 Omicron B.1.1.529 isolate

abstract: The emergence and rapid spread of the Omicron variant of SARS-CoV-2, which has more than 30 substitutions in the spike glycoprotein, compromises the efficacy of currently available vaccines and therapeutic antibodies. Using a clinical strain of the Omicron variant, we analyzed the neutralizing power of eight currently used monoclonal antibodies compared to the ancestral B.1 BavPat1 D614G strain. We observed that six of these antibodies have lost their ability to neutralize the Omicron variant. Of the antibodies still having neutralizing activity, Sotrovimab/Vir-7831 shows the smallest reduction in activity, with a factor change of 3.1. Cilgavimab/AZD1061 alone shows a reduction in efficacy of 15.8, resulting in a significant loss of activity for the Evusheld cocktail (42.6 fold reduction) in which the other antibody, Tixagevimab, does not retain significant activity against Omicron. Our results suggest that the clinical efficacy of the initially proposed doses should be rapidly evaluated and the possible need to modify doses or propose combination therapies should be considered.


title: The SARS-CoV-2 variant, Omicron, shows rapid replication in human primary nasal epithelial cultures and efficiently uses the endosomal route of entry.

abstract: At the end of 2021 a new SARS-CoV-2 variant, Omicron, emerged and quickly spread across the world. It has been demonstrated that Omicrons high number of Spike mutations lead to partial immune evasion from even polyclonal antibody responses, allowing frequent re-infection and vaccine breakthroughs. However, it seems unlikely these antigenic differences alone explain its rapid growth; here we show Omicron replicates rapidly in human primary airway cultures, more so even than the previously dominant variant of concern, Delta. Omicron Spike continues to use human ACE2 as its primary receptor, to which it binds more strongly than other variants. Omicron Spike mediates enhanced entry into cells expressing several different animal ACE2s, including various domestic avian species, horseshoe bats and mice suggesting it has an increased propensity for reverse zoonosis and is more likely than previous variants to establish an animal reservoir of SARS-CoV-2. Unlike other SARS-CoV-2 variants, however, Omicron Spike has a diminished ability to induce syncytia formation. Furthermore, Omicron is capable of efficiently entering cells in a TMPRSS2-independent manner, via the endosomal route. We posit this enables Omicron to infect a greater number of cells in the respiratory epithelium, allowing it to be more infectious at lower exposure doses, and resulting in enhanced intrinsic transmissibility.


title: A dual-receptor mechanism between integrins and ACE2 widens SARS-CoV-2 tissue tropism

abstract: In addition to the ACE2 receptor, SARS-CoV-2 binds to integrins to gain host cell entry and trigger pro-inflammatory integrin-mediated signalling cascades. Integrins, therefore, are likely candidates for a dual-receptor mechanism with ACE2 to explain the increased infectivity seen in SARS-CoV-2 models. As integrins are primarily expressed in vasculature and persistent vasculopathy is seen in COVID-19, examining the role of endothelial integrin involvement is crucial in uncovering the pathophysiology of SARS-CoV-2.

category: cell biology


title: Cell culture model system utilizing engineered A549 cells to express high levels of ACE2 and TMPRSS2 for investigating SARS-CoV-2 infection and antivirals

abstract: Novel pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to pose an imminent global threat since its initial outbreak in December 2019. A simple in vitro model system using cell lines highly susceptible to SARS-CoV-2 infection are critical to facilitate the study of the virus cycle and to discover effective antivirals against the virus. Human lung alveolar A549 cells are regarded as a useful and valuable model for respiratory virus infection. However, SARS-CoV-2 uses the ACE2 as receptor for viral entry and the TMPRSS2 to prime the Spike protein, both of which are negligibly expressed in A549 cells. Here, we report the generation of a robust human lung epithelial cell-based model by transducing ACE2 and TMPRSS2 into A549 cells and show that the ACE2 enriched A549ACE2/TMPRSS2 cells (ACE2plus) and its single-cell-derived subclone (ACE2plusC3) are highly susceptible to SARS-CoV-2 infection. These engineered ACE2plus showed higher ACE2 and TMPRSS2 mRNA expression levels than currently used Calu3 and commercial A549ACE2/TMPRSS2 cells. ACE2 and TMPRSS2 proteins were also highly and ubiquitously expressed in ACE2plusC3 cells. Additionally, antiviral drugs like Camostat mesylate, EIDD-1931, and Remdesivir strongly inhibited SARS-CoV-2 replication. Notably, multinucleated syncytia, a clinical feature commonly observed in severe COVID-19 patients was induced in ACE2plusC3 cells either by virus infection or by overexpressing the Spike proteins of different variants of SARS-CoV-2. Syncytial process was effectively blocked by the furin protease inhibitor, Decanoyl-RVKR-CMK. Taken together, we have developed a robust human A549 lung epithelial cell-based model that can be applied to probe SARS-CoV-2 replication and to facilitate the discovery of SARS-CoV-2 inhibitors.


title: A triad of kicknet sampling, eDNA metabarcoding, and predictive modeling to assess aquatic macroinvertebrate biodiversity

abstract: Monitoring freshwater biodiversity is essential to understand the impacts of human activities and for effective management of ecosystems. Thereby, biodiversity can be assessed through direct collection of targeted organisms, through indirect evidence of their presence (e.g. signs, environmental DNA, camera trap, etc.), or through extrapolations from species distribution models (SDM). Differences in approaches used in biodiversity assessment, however, may come with individual challenges and hinder cross-study comparability. In the context of rapidly developing techniques, we compared a triad of approaches in order to understand assessment of aquatic macroinvertebrate biodiversity. Specifically, we compared the community composition and species richness of three orders of aquatic macroinvertebrates (mayflies, stoneflies, and caddisflies, hereafter EPT) obtained via eDNA metabarcoding and via traditional in situ kicknet sampling to catchment-level based predictions of a species distribution model. We used kicknet data from 24 sites in Switzerland and compared taxonomic lists to those obtained using eDNA amplified with two different primer sets. Richness detected by these methods was compared to the independent predictions made by a statistical species distribution model using landscape-level features to estimate EPT diversity. Despite the ability of eDNA to consistently detect some EPT species found by traditional sampling, we found important discrepancies in community composition between the two approaches, particularly at local scale. Overall, the more specific set of primers, namely fwhF2/EPTDr2n, was most efficient for the detection of target species and for characterizing the diversity of EPT. Moreover, we found that the species richness measured by eDNA was poorly correlated to the richness measured by kicknet sampling and that the richness estimated by eDNA and kicknet were poorly correlated with the prediction of the statistical model. Overall, however, neither eDNA nor the traditional approach had strong links to the predictive models, indicating inherent limitations in upscaling species richness estimates. Future challenges include improving the accuracy and sensitivity of each approach individually yet also acknowledge their respective limitations, in order to best meet stakeholder demands addressing the biodiversity crisis we are facing.

category: ecology


title: Increasing adult density compromises anti-bacterial defense in Drosophila melanogaster

abstract: The density-dependent prophylaxis hypothesis predicts that risk of pathogen transmission increases with increase in population density, and in response to this, organisms mount a prophylactic immune response when exposed to high density. This prophylactic response is expected to help organisms improve their chances of survival when exposed to pathogens. Alternatively, organisms living at high densities can exhibit compromised defense against pathogens due to lack of resources and density associated physiological stress; the density stress hypothesis. We housed adult Drosophila melanogaster flies at different densities and measured the effect this has on their post-infection survival and resistance to starvation. We find that flies housed at higher densities show greater mortality after being infected with bacterial pathogens, while also exhibiting increased resistance to starvation. Our results are more in line with the density-stress hypothesis that postulates a compromised immune system when hosts are subjected to high densities.

category: evolutionary biology


title: Splicing QTL analysis focusing on coding sequences reveals pathogenicity of disease susceptibility loci.

abstract: Splicing QTL (sQTL) are one of the major causal mechanisms in GWAS loci, but their role in disease pathogenesis is poorly understood. One reason is the huge complexity of alternative splicing events producing many unknown isoforms. Here, we proposed two novel approaches, namely integration and selection, for this complexity by focusing on protein-structure of isoforms. First, we integrated isoforms with the same coding sequence (CDS) and identified 369-601 integrated-isoform ratio QTLs (i2-rQTLs), which altered protein-structure, in six immune subsets. Second, we selected CDS incomplete isoforms annotated in GENCODE and identified 175-337 isoform-ratio QTL (i-rQTL). By comprehensive long-read capture RNA-seq among these incomplete isoforms, we revealed 29 full-length isoforms with novel CDSs associated with GWAS traits. Furthermore, we have shown that disease-causal sQTL genes can be identified by evaluating their trans-eQTL effects. Our approaches highlight the understudied role of protein-altering sQTLs and are broadly applicable to other tissues and diseases.

category: genetics


title: Directed evolution of angiotensin-converting enzyme 2 (ACE2) variants with enhanced peptidase activity profiles for novel therapeutic applications

abstract: Angiotensin Converting-Enzyme 2 (ACE2) is currently being investigated for its ability to beneficially modulate the Angiotensin receptor (ATR) therapeutic axis to treat multiple human diseases, but its broad substrate scope and diverse physiological roles limit its potential as a therapeutic agent. In this work we engineer ACE2 variants with enhanced hydrolytic activity and specificity toward Angiotensin-II (Ang-II) that may lead to new therapeutic candidates with improved efficacy and reduced off-target side effects. We established a yeast display-based liquid chromatography screen that enabled use of directed evolution to discover ACE2 variants with improved Ang-II activity and specificity relative to the off-target peptide substrates Apelin-13 and Angiotensin-I (Ang-I). We screened ACE2 active site libraries to reveal three substitution-tolerant positions (M360, T371 and Y510) that can be mutated to enhance ACE2s activity profile and followed up on these hits with focused double mutant libraries to further improve the enzyme. Relative to wildtype ACE2, our top variant (T371L/Y510Ile) displayed a sevenfold increase in Ang-II turnover number (kcat), a sixfold diminished catalytic efficiency (kcat/Km) on Apelin-13, and an overall decreased activity on other ACE2 substrates that were not directly assayed in directed evolution screen. At physiologically relevant substrate concentrations, T371L/Y510Ile hydrolyzes more Ang-II than wildtype ACE2 with concomitant Ang-II:Apelin-13 specificity improvements reaching 30-fold. Our efforts have delivered ATR axis-acting therapeutic candidates with relevance to both established and unexplored ACE2 therapeutic applications and provide a foundation for further ACE2 engineering efforts.

category: biochemistry


title: Directed evolution of angiotensin-converting enzyme 2 (ACE2) peptidase activity profiles for therapeutic applications


title: Hydrodynamics of spike proteins dictate a transport-affinity competition for SARS-CoV-2 and other enveloped viruses

abstract: Many viruses, such as SARS-CoV-2 or Influenza, possess spike-decorated envelopes. Depending on the virus type, a large variability is present in spikes number, morphology and reactivity, which remains generally unexplained. Since viruses transmissibility depend on features beyond their genetic sequence, new tools are required to discern the effects of spikes functionality, interaction, and morphology. Here, we postulate the relevance of hydrodynamic interactions in the viral infectivity of enveloped viruses and propose micro-rheological characterization as a platform for viruses differentiation. To understand how the spikes affect virion mobility and infectivity, we investigate the diffusivity of spike-decorate structures using mesoscopic-hydrodynamic simulations. Furthermore, we explored the interplay between affinity and passive viral transport. Our results revealed that the diffusional mechanism of SARS-CoV-2 is strongly influenced by the size and distribution of its spikes. We propose and validate a universal mechanism to explain the link between optimal virion structure and maximal infectivity for many virus families.

category: biophysics


title: Serum metabolomic analysis of men on a low-carbohydrate diet for biochemically recurrent prostate cancer reveal the potential role of ketogenesis to slow tumor growth: A secondary analysis of the CAPS2 diet trial

abstract:
BackgroundSystemic treatments for prostate cancer (PC) have significant side effects. Thus, newer alternatives with fewer side effects are urgently needed. Animal and human studies suggest the therapeutic potential of low carbohydrate diet (LCD) for PC. To test this possibility, Carbohydrate and Prostate Study 2 (CAPS2) trial was conducted in PC patients with biochemical recurrence (BCR) after local treatment to determine the effect of a 6-month LCD intervention vs. usual care control on PC growth as measured by PSA doubling time (PSADT). We previously reported the LCD intervention led to significant weight loss, higher HDL, and lower triglycerides and HbA1c with a suggested longer PSADT. However, the metabolic basis of these effects are unknown.

MethodsTo identify the potential metabolic basis of effects of LCD on PSADT, serum metabolomic analysis was performed using baseline, month 3, and month 6 banked sera to identify the metabolites significantly altered by LCD and that correlated with varying PSADT.

ResultsLCD increased the serum levels of ketone bodies, glycine and hydroxyisocaproic acid. Reciprocally, LCD reduced the serum levels of alanine, cytidine, asymmetric dimethylarginine (ADMA) and 2-oxobutanoate. As high ADMA level is shown to inhibit nitric oxide (NO) signaling and contribute to various cardiovascular diseases, the ADMA repression under LCD may contribute to the LCD-associated health benefit. Regression analysis of the PSADT revealed a correlation between longer PSADT with higher level of 2-hydroxybutyric acids, ketone bodies, citrate and malate. Longer PSADT was also associated with LCD reduced nicotinamide, fructose-1, 6-biphosphate (FBP) and 2-oxobutanoate.

ConclusionThese results suggest a potential association of ketogenesis and TCA metabolites with slower PC growth and conversely glycolysis with faster PC growth. The link of high ketone bodies with longer PSADT supports future studies of ketogenic diets to slow PC growth.

category: cancer biology


title: Demonstration of a common DPhe7 to DNal(2')7 peptide ligand antagonist switch for the melanocortin-3 and melanocortin-4 receptors identifies systematic mischaracterization of the pharmacological properties of melanocortin peptides

abstract: Melanocortin peptides containing a D-naphthylalanine residue in position 7 (DNal(2)7), reported as melanocortin-3 receptor (MC3R) subtype-specific agonists in two separate publications, were found to lack significant MC3R agonist activity. The cell lines used at the University of Arizona for pharmacological characterization of these peptides, consisting of HEK293 cells stably transfected with human melanocortin receptor subtypes MC1R, MC3R, MC4R, or MC5R, were then obtained and characterized by quantitative PCR. While the MC1R cell line correctly expressed only the hMCR1, the three other cell lines were mischaracterized with regard to receptor subtype expression. Demonstration that a D-naphthylalanine residue in position 7, irrespective of the melanocortin peptide template, results primarily in antagonism of the MC3R and MC4R, then allowed us to search the published literature for additional errors. The erroneously characterized DNal(2)7-containing peptides date back to 2003; thus, our analysis suggests that systematic mischaracterization of the pharmacological properties of melanocortin peptides occurred.

category: pharmacology and toxicology


title: Cellular and gene expression patterns associated with root bifurcation in Selaginella

abstract:
The roots of lycophytes branch through dichotomy or bifurcation, which means that the root apex splits into two daughter roots. This is morphologically distinct from lateral root (LR) branching in the extant euphyllophytes, where LRs develop along the root axis at different distances from the apex. The process of root bifurcation is poorly understood, while such knowledge can be important, as it may represent an evolutionarily ancient strategy that roots recruited to form new stem cells or meristems. In this study, we examined root bifurcation in the lycophyte Selaginella moellendorffii. We characterized an in vitro developmental time-frame based on repetitive apex bifurcations, allowing us to sample different stages of dichotomous root branching and analyze the root meristem and root branching in S. moellendorffii at the microscopical and transcriptional level. Our results show that, in contrast to previous assumptions, initial cells in the root meristem are mostly not tetrahedral but rather show an irregular shape. Tracking down the early stages during root branching argues for the occurrence of a symmetric division of the single initial cell resulting in two apical stem cells allowing for root meristem bifurcation. Moreover, we generated a S. moellendorffii root branching transcriptome, which resulted in the delineation of a subset of core meristem genes. The occurrence of multiple meristem-related orthologues in this dataset, including inversely correlated expression profiles of a SCARECROW (SCR) versus a RETINOBLASTOMA-RELATED1 (RBR1) homologue suggests the presence of conserved pathways in the control of meristem and root stem cell establishment or maintenance.

One-sentence summaryThe root of the spike moss Selaginella moellendorffii bifurcates following a symmetric cell division of the single stem cell and involves conserved genetic modules known from angiosperm roots.

category: plant biology


title: Controlled exchange of protein and nucleic acid signals from and between synthetic minimal cells

abstract: Synthetic minimal cells are a class of small liposome bioreactors that have some, but not all functions of live cells. Here, we report a critical step towards the development of a bottom-up minimal cell: cellular export of functional protein and RNA products. We used cell penetrating peptide tags to translocate payloads across a synthetic cell vesicle membrane. We demonstrated efficient transport of active enzymes, and transport of nucleic acid payloads by RNA binding proteins. We investigated influence of a concentration gradient alongside other factors on the efficiency of the translocation, and we show a method to increase product accumulation in one location. We demonstrate the use of this technology to engineer molecular communication between different populations of synthetic cells, to exchange protein and nucleic acid signals. The synthetic minimal cell production and export of proteins or nucleic acids allows experimental designs that approach the complexity and relevancy of natural biological systems.

category: synthetic biology


title: Integrated Autolysis, DNA Hydrolysis and Precipitation Enables an Improved Bioprocess for Q-Griffithsin, a Broad-Spectrum Antiviral and Clinical-Stage anti-COVID-19 Candidate

abstract:
Across the biomanufacturing industry, innovations are needed to improve efficiency and flexibility, especially in the face of challenges such as the COVID-19 pandemic. Here we report an improved bioprocess for Q-Griffithsin, a broad-spectrum antiviral currently in clinical trials for COVID-19. Q-Griffithsin is produced at high titer in E. coli and purified to anticipated clinical grade without conventional chromatography or the need for any fixed downstream equipment. The process is thus both low-cost and highly flexible, facilitating low sales prices and agile modifications of production capacity, two key features for pandemic response. The simplicity of this process is enabled by a novel unit operation that integrates cellular autolysis, autohydrolysis of nucleic acids, and contaminant precipitation, giving essentially complete removal of host cell DNA as well as reducing host cell proteins and endotoxin by 3.6 and 2.4 log10 units, respectively. This unit operation can be performed rapidly and in the fermentation vessel, such that Q-GRFT is obtained with 100% yield and >99.9% purity immediately after fermentation and requires only a flow-through membrane chromatography step for further contaminant removal. Using this operation or variations of it may enable improved bioprocesses for a range of other high-value proteins in E. coli.

HighlightsO_LIIntegrating autolysis, DNA hydrolysis and precipitation enables process simplification
C_LIO_LIAutolysis reduces endotoxin release and burden to purification
C_LIO_LIQ-Griffithsin recovered from fermentation vessel at >99.9% purity and 100% yield
C_LIO_LIQ-Griffithsin purified to anticipated clinical grade without conventional chromatography
C_LIO_LIThe resulting bioprocess is 100% disposables-compatible, scalable, and low-cost
C_LI


title: Seed-competent tau monomer initiates pathology in PS19 tauopathy mice

abstract: Tau aggregation into ordered assemblies causes myriad neurodegenerative tauopathies. We previously reported that tau monomer exists in either inert (Mi) or seed-competent (Ms) conformational ensembles, and that Ms encodes strains, which are biologically active, self-propagating assemblies. We have previously isolated Ms from tauopathy brains, but it is unknown if disease begins with Ms formation followed by fibril assembly, or if Ms derives from fibrils and is an epiphenomenon. Consequently, we studied a tauopathy mouse model (PS19) that expresses full-length human (1N4R) tau containing a disease-associated mutation (P301S). Using tau repeat domain biosensor cells, we detected insoluble tau seeding activity at 2 months. We found insoluble tau protein assemblies by immunoblot at 3 months. We next immunopurified monomer from mice aged 1-6 weeks using size exclusion chromatography. We detected soluble seeding activity at 4 weeks, before insoluble material or larger assemblies, with assemblies ranging from n=1-3 tau units. By 5 and 6 weeks, large soluble assemblies had formed. This indicated the first detectable pathological forms of tau were Ms. We next tested for post-translational modifications of tau monomer from 1-6 weeks. We detected no phosphorylation unique to Ms in PS19 or Alzheimers disease brain. We conclude that tauopathy begins with formation of Ms monomer, whose activity is phosphorylation-independent. Ms self-assembles to form oligomers before it forms insoluble fibrils. The conversion of tau monomer from Mi to Ms thus constitutes the first detectable step in the initiation of tauopathy in this mouse model, with obvious implications for origins of disease in humans.


title: Volatile anesthetics inhibit presynaptic cGMP signaling to depress presynaptic excitability in rat hippocampal neurons

abstract: Volatile anesthetics alter presynaptic function including effects on Ca2+ influx and neurotransmitter release. These actions are proposed to play important roles in their pleiotropic neurophysiological effects including unconsciousness and amnesia. The nitric oxide and cyclic guanosine monophosphate (NO/cGMP) signaling pathway has been implicated in presynaptic mechanisms, and disruption of NO/cGMP signaling has been shown to alter sensitivity to volatile anesthetics in vivo. We investigated NO/cGMP signaling in relation to volatile anesthetic actions in cultured rat hippocampal neurons using pharmacological tools and genetically encoded biosensors of cGMP levels. Using the fluorescent biosensor cGull we found that electrical stmulation-evoked NMDA-type glutamate receptor-independent presynaptic cGMP transients were inhibited -33.2% by isoflurane (0.51 mM) and -23.8% by sevoflurane (0.57 mM) (p<0.0001) compared to a stimulation without anesthetic. Isoflurane and sevoflurane inhibition of stimulation-evoked increases in presynaptic Ca2+ concentration, measured with synaptophysin-GCaMP6f, and synaptic vesicle exocytosis, measured with synaptophysin-pHlourin, were reduced by in neurons expressing the cGMP scavenger sponGee. This reduction in anesthetic effect was recapitulated by inhibiting HCN channels, a cGMP-modulated effector that can facilitate glutamate release. We propose that volatile anesthetics depress presynaptic cGMP signaling and downstream effectors like HCN channels that are essential to presynaptic function and excitability. These findings identify a novel mechanism by which volatile anesthetics depress synaptic transmission via second messenger signaling involving the NO/cGMP pathway.


title: Cell type specific IL-27p28 (IL-30) deletion uncovers an unexpected pro-inflammatory property of IL-30 in autoimmune inflammation

abstract: IL-27 is an IL-12 family cytokine with potent immunoregulatory properties, capable of modulating inflammatory responses, including autoimmunity. While extensive studies have been performed to investigate the major target cells of IL-27 mediating its functions, the source of IL-27 especially during tissue specific autoimmune inflammation has not formally been tested. IL-27p28 subunit, also known as IL-30, was initially discovered as an IL-27-specific subunit, and its expression has thus been used as a surrogate for IL-27. However, there is emerging evidence that IL-27p28 can be secreted without Ebi3, a subunit that forms IL-27 with IL-27p28. Furthermore, IL-27p28 was also reported to act as a negative regulator antagonizing IL-27. In this study, we utilized various cell type specific IL-27p28-deficient mouse models and examined the major source of IL-27p28 in T cell mediated autoimmune neuroinflammation. We found that dendritic cell-derived IL-27p28 is dispensable for the disease development but that IL-27p28 expressed by infiltrating and CNS resident APC subsets, namely, infiltrating monocytes, microglia, and astrocytes, play an essential role in limiting inflammation. Unexpectedly, we observed that cell type specific IL-27p28 deficiency expressing severe disease phenotype is associated with dysregulated IL-27p28 expression in otherwise unaffected APC subsets, suggesting that disproportionate IL-27p28 expressed may increase disease susceptibility. Indeed, systemic recombinant IL-30 administration also induced severe disease. Taken together, our results uncover a pro-inflammatory property of IL-30 that supports encephalitogenic immunity in vivo.


title: Mito-FUNCAT-FACS reveals cellular heterogeneity in mitochondrial translation

abstract: Mitochondria possess their own genome that encodes components of oxidative phosphorylation (OXPHOS) complexes, and mitochondrial ribosomes within the organelle translate the mRNAs expressed from mitochondrial genome. Given the differential OXPHOS activity observed in diverse cell types, cell growth conditions, and other circumstances, cellular heterogeneity in mitochondrial translation can be expected. Although individual protein products translated in mitochondria have been monitored, the lack of techniques that address the variation in overall mitochondrial protein synthesis in cell populations poses analytic challenges. Here, we adapted mitochondrial-specific fluorescent noncanonical amino acid tagging (FUNCAT) for use with fluorescence-activated cell sorting (FACS) and developed mito-FUNCAT-FACS. The click chemistry-compatible methionine analog L-homopropargylglycine (HPG) enabled the metabolic labeling of newly synthesized proteins. In the presence of cytosolic translation inhibitors, HPG was selectively incorporated into mitochondrial nascent proteins and conjugated to fluorophores via the click reaction (mito-FUNCAT). The application of in situ mito-FUNCAT to flow cytometry allowed us to disentangle changes in net mitochondrial translation activity from those of the organelle mass and detect variations in mitochondrial translation in cancer cells. Our approach provides a useful methodology for examining mitochondrial protein synthesis in individual cells.

category: molecular biology


title: Wetland loss in the Neembucu Wetlands Complex, Paraguay, using remote sensing

abstract: South American wetlands are of global importance, yet limited delineation and monitoring restricts informed decision-making around the drivers of wetland loss. A growing human population and increasing demand for agricultural products has driven wetland loss and degradation in the Neotropics. Understanding of wetland dynamics and land use change can be gained through wetland monitoring. The Neembucu Wetlands Complex is the largest wetland in Paraguay, lying within the Paraguay-Parana-La Plata River system. This study aims to use remotely sensed data to map land cover between 2006 and 2021, quantify wetland change over the 15-year study period and thus identify land cover types vulnerable to change in the Neembucu Wetlands Complex. Forest, dryland vegetation, vegetated wetland and open water were identified using Random Forest supervised classifications trained on visual inspection data and field data. Annual change of -0.34, 4.95, -1.65, 0.40 was observed for forest, dryland, vegetated wetland and open water, respectively. Wetland and forest conversion is attributed to agricultural and urban expansion. With ongoing pressures on wetlands, monitoring will be a key tool for addressing change and advising decision-making around development and conservation of valuable ecosystem goods and services in the Neembucu Wetlands Complex.


title: Missense variants causing Wiedemann-Steiner syndrome preferentially occur in the KMT2A-CXXC domain and are accurately classified using AlphaFold2

abstract: Wiedemann-Steiner syndrome (WSS) is a neurodevelopmental disorder caused by de novo variants in KMT2A, which encodes a multi-domain histone methyltransferase. To gain insight into the currently unknown pathogenesis of WSS, we examined the spatial distribution of likely WSS-causing variants across the 15 different domains of KMT2A. Compared to variants in healthy controls, WSS variants exhibit a 64.1-fold overrepresentation within the CXXC domain - which mediates binding to unmethylated CpGs - suggesting a major role for this domain in mediating the phenotype. In contrast, we find no significant overrepresentation within the catalytic SET domain. Corroborating these results, we find that hippocampal neurons from Kmt2a-deficient mice demonstrate disrupted H3K4me1 preferentially at CpG-rich regions, but this has no systematic impact on gene expression. Motivated by these results, we combine accurate prediction of the CXXC domain structure by AlphaFold2 with prior biological knowledge to develop a classification scheme for missense variants in the CXXC domain. Our classifier achieved 96.0% positive and 92.3% negative predictive value on a hold-out test set. This classification performance enabled us to subsequently perform an in silico saturation mutagenesis and classify a total of 445 variants according to their functional effects. Our results yield a novel insight into the mechanistic basis of WSS and provide an example of how AlphaFold2 can contribute to the in silico characterization of variant effects with very high accuracy, establishing a paradigm potentially applicable to many other Mendelian disorders.


title: Lipoate protein ligase B primarily recognizes the C8-phosphopantetheine arm of its donor substrate and weakly binds the acyl carrier protein

abstract: Lipoic acid is a sulfur containing cofactor, indispensable for the function of several metabolic enzymes. In microorganisms, lipoic acid can be salvaged from the surroundings by Lipoate protein ligase A (LplA), an ATP-dependent enzyme. Alternatively, it can be synthesized by the sequential action of Lipoate protein ligase B (LipB) and Lipoyl synthase (LipA), in a two-step reaction. LipB uptakes octanoyl-chain from C8-acyl carrier protein (C8-ACP), a byproduct of the type II fatty acid synthesis pathway, and transfers it to a conserved lysine of the lipoyl domain of a dehydrogenase. The molecular basis of substrate recognition by LipB is still not fully understood. Using E. coli LipB as a prototype, we show that the enzyme mainly recognizes the 4-phosphopantetheine tethered acyl-chain of its donor substrate, and weakly binds the apo-acyl carrier protein. It can accept octanoate-from its own ACP, noncognate ACPs, as well as C8-CoA. Further, our NMR studies demonstrate the presence of an adenine and phosphate binding site in LipB, akin to LplA. A loop containing 71RGG73 sequence, analogous to the lipoate binding loop of LplA is also conserved. Collectively, our studies highlight commonalities between LipB and LplA in their mechanism of substrate recognition. This knowledge might be of significance in the treatment of mitochondrial fatty acid synthesis related disorders.


title: Melatonin drugs inhibit SARS-CoV-2 entry into the brain and virus-induced damage of cerebral small vessels

abstract: COVID-19 is a complex disease with short- and long-term respiratory, inflammatory and neurological symptoms that are triggered by the infection with SARS-CoV-2. Invasion of the brain by SARS-CoV-2 has been observed in humans and is postulated to be involved in post COVID condition. Brain infection is particularly pronounced in the K18-hACE2 mouse model of COVID-19. Here, we show that treatment of K18-hACE2 mice with melatonin and two melatonin-derived marketed drugs, agomelatine and ramelteon, prevent SARS-CoV-2 entry in the brain thereby reducing virus-induced damage of small cerebral vessels, immune cell infiltration and brain inflammation. Brain entry of SARS-CoV-2 through endothelial cells is prevented by melatonin through allosteric binding to human angiotensin-converting enzyme 2 (ACE2), which interferes with the cell entry receptor function of ACE2 for SARS-CoV-2. Our findings open new perspectives for the repurposing of melatonergic drugs in the prevention of brain infection by SARS-CoV-2 and COVID-19-related long-term neurological symptoms.


title: Akkermansia Muciniphila induces chronic extramedullary hematopoiesis through cooperative IL-1R and TLR signals

abstract:
Bacterial infections can activate and mobilize hematopoietic stem and progenitor cells (HSPCs) from the bone marrow (BM) to spleen, which is termed as extramedullary hematopoiesis (EMH). Recent studies suggest that commensal bacteria, particularly the microbiota, regulates not only the host immune system but also hematopoietic homeostasis. However, the impact of gut microbial species on hematopoietic pathology remains largely unknown. Here we found that systemic injection of Akkermansia muciniphila (A. m.), a mucin-degrading bacterium abundantly existing in the human gut rapidly activates BM myelopoiesis, and induces a slow but long-lasting hepato-splenomegaly, characterized by the expansion and differentiation of functional HSPCs, which we termed chronic EMH. Genetic deletion of Toll-like receptor-2 and -4 (TLR2/4) partially diminished A. m.-induced chronic EMH, while additional pharmacological inhibition of the interleukin-1 receptor (IL-1R) completely alleviated splenomegaly and EMH. Our results demonstrate that cooperative IL-1R- and TLR-mediated innate immune signals regulate commensal bacteria-driven EMH, which might be relevant for certain autoimmune disorders.

Article SummaryThe aim of our study is to understand how Akkermansia muciniphila (A.m.), one of the major mucin-degrading microbial species in the human gut activate the immune and hematopoietic systems in a mouse model. We found that a single injection of the A.m. membrane fraction can induce long-lasting hepatosplenomegaly with splenic EMH through cooperative IL-1R- and TLR-mediated innate immune signals.


title: Revealing genomic changes responsible for cannabinoid receptor loss in parrots: mechanism and functional effects

abstract: In vertebrates, an ancient duplication in the genes for cannabinoid receptors (CNRs) allowed the evolution of specialised endocannabinoid receptors expressed in the brain (CNR1) and the periphery (CNR2). While dominantly conserved throughout vertebrate phylogeny, our comparative genomic analysis suggests that certain taxa may have lost either the CNR1 regulator of neural processes or, more frequently, the CNR2 involved in immune regulation. Focussing on conspicuous CNR2 pseudogenization in parrots (Psittaciformes), a diversified crown lineage of cognitively-advanced birds, we highlight possible functional effects of such a loss. Parrots appear to have lost the CNR2 gene at at least two separate occasions due to chromosomal rearrangement. Using gene expression data from the brain and periphery of birds with experimentally-induced sterile inflammation, we compare CNR and inflammatory marker (interleukin 1 beta, IL1B) expression patterns in CNR2-deficient parrots (represented by the budgerigar, Melopsittacus undulatus and five other parrot species) with CNR2-intact passerines (represented by the zebra finch, Taeniopygia guttata). Though no significant changes in CNR expression were observed in either parrots or passerines during inflammation of the brain or periphery, we detected a significant up-regulation of IL1B expression in the brain after stimulation with lipopolysaccharide (LPS) only in parrots. As our analysis failed to show evidence for selection on altered CNR1 functionality in parrots, compared to other birds, CNR1 is unlikely to be involved in compensation for CNR2 loss in modulation of the neuroimmune interaction. Thus, our results provide evidence for the functional importance of CNR2 pseudogenization for regulation of neuroinflammation.

category: genomics


title: An investigation into the spatiotemporal patterns of the Nymphalid butterfly Vagrans egista sinha (Kollar, )

abstract: Vagrans egista sinha (Kollar, [1844]), the Himalayan Vagrant is a subspecies of Nymphalid (Brush-footed) butterflies spread across Asia, whose western limit is in the north-west India. Observations of this subspecies have considerably increased over the past half-a-decade, with a spike in new sightings to the west of their previously known range. This has been considered as a range extension. The current study reports new records of this species from Bilaspur District, Himachal Pradesh, India (which are the first records for the district), through systematic and opportunistic sampling. This raises the question of whether the purported range extension towards the west could instead be a range shift or vagrancy, and whether there is any shift in elevational ranges in the populations across their known range. Questions pertaining to spatial differences in elevational ranges and seasonal variation, across their range, also piqued our curiosity. Using data from academic sources (such as published literature and museum collections), supplemented by data from public participation in scientific research and personal observations, these research questions are addressed. The accuracy of results when using citizen science data is also explored using the same dataset, focused on the impact of method of extraction of coordinates, and elevation derived from it under different scenarios. It was discovered that there has not been a range shift (either longitudinal or latitudinal) and observations dont suggest vagrancy but a case of range extension. Other results indicated that there was no climb of population to higher elevations, no spatial differences in elevational ranges in the populations, or seasonal variation in activities across their range. It was also discovered that the method of data collection by, and extraction from, citizen science databases, can influence the accuracy of the results. Some problems involved in collecting data are discussed, and remedial solutions are suggested.


title: Construct a variable-length fragment library for de novo protein structure prediction

abstract: Although remarkable achievements, such as AlphaFold2, have been made in end-to-end structure prediction, fragment libraries remain essential for de novo protein structure prediction, which can help explore and understand the protein-folding mechanism. In this work, we developed a variable-length fragment library (VFlib). In VFlib, a master structure database was first constructed from the Protein Data Bank through sequence clustering. The Hidden Markov Model (HMM) profile of each protein in the master structure database was generated by HHsuite, and the secondary structure of each protein was calculated by DSSP. For the query sequence, the HMM-profile was first constructed. Then, variable-length fragments were retrieved from the master structure database through dynamically variable-length profile-profile comparison. A complete method for chopping the query HMM-profile during this process was proposed to obtain fragments with increased diversity. Finally, secondary structure information was used to further screen the retrieved fragments to generate the final fragment library of specific query sequence. The experimental results obtained with a set of 120 nonredundant proteins showed that the global precision and coverage of the fragment library generated by VFlib were 55.04% and 94.95% at the RMSD cutoff of 1.5 [A], respectively. Compared to the benchmark method of NNMake, the global precision of our fragment library had increased by 62.89% with equivalent coverage. Furthermore, the fragments generated by VFlib and NNMake were used to predict structure models through fragment assembly. Controlled experimental results demonstrated that the average TM-score of VFlib was 16.00% higher than that of NNMake.

category: bioinformatics


title: Schild analysis of the interaction between parthenolide and cocaine on their effects onplanarian motility suggests an orthosteric relationship.

abstract: One of the best-characterized planarian behaviors induced by various compounds is the change in locomotor velocity. Previous work from our laboratory showed that the sesquiterpene lactone parthenolide and the local anesthetic cocaine, reduce planarian motility. Parthenolide reverses the cocaine-induced motility decrease and vice versa. However, the exact mechanism of the cocaine/parthenolide antagonism for this specific planarian behavior is still unknown. Here we report the results of a Schild analysis to determine whether the parthenolide/cocaine relationship is orthosteric or allosteric. Our results suggest an orthosteric relationship between these two compounds in the planarian Girardia tigrina. The simplest interpretation of our data is a shared binding site for cocaine and parthenolide. Still, we cannot rule out the possibility of distinct yet overlapping binding sites with the data available.


title: The rosy future paradox: Positive future thinking without task relevance enhances negative biases and anxiety for aversive events

abstract: Expectations have an important role in guiding behavior and the interpretation of novel information, but can contain negative biases such as is the case in anxiety disorders. Positive future thinking may provide an accessible way to attenuate these negatively biases. However, much is still unclear about the optimal form of such positive interventions, and it is unknown if the effects go beyond subjective experience. Here, we used a positive future thinking intervention to adapt the way a stressful event is experienced. Participants imagined either task-relevant (N = 21) or irrelevant (N = 21) positive future events before being subjected to the Trier Social Stress Task, or did not receive the intervention (N= 20). We recorded resting state EEG during the anticipation and recovery phases of the TSST to assess intervention and trait anxiety related differences in the level of frontal delta-beta coupling, which is considered a neurobiological substrate of emotion regulation. Results show that the intervention reduces event-related stress and anxiety, and increases social fixation behavior and task performance, but only if future thinking is task relevant. Paradoxically, task-irrelevant positive future thoughts enhance negative perceptual biases and stress reactivity. This increase in stress reactivity in the task-irrelevant group was corroborated by the elevated levels frontal delta-beta coupling during event anticipation, especially for those with high trait anxiety. This suggests an increased demand for emotion regulation following the task-irrelevant intervention, possibly due to the contextual incongruence between positive imagery and the stressor. Together, these results show that positive future thinking can mitigate the negative emotional, behavioral and neurobiological consequences of a stressful event, but that it should not be applied indiscriminately. Task-relevant positive future thinking can be an accessible way to boost efficacy of exposure therapy for pathological anxiety, and can help people deal with negative anticipation in daily life.


title: Continuous Daily Head and Trunk Acceleration Recording for the Assessment of Bone Stimulation in Astronauts

abstract: The acceleration of the head and hip along the x-, y-, and z-axis of 14 healthy subjects was recorded during two sessions of 12 consecutive hours. The magnitude, frequency content, and root mean square of the acceleration signals were used to determine the type of physical activity (sitting, standing, walking, etc.) during normal daily life on Earth. The acceleration signal slope (jerk) was also calculated to assess whether these activities were sufficient to maintain bone mineral density. These measurements indicated that the changes in vertical acceleration experienced by our subjects during normal daily life were presumably sufficient to maintain bone mineral density. However, these changes might not be sufficient for postmenopausal women and astronauts during long-term exposure to weightlessness during spaceflight.


title: A role for Lin28a in aging-associated decline of adult hippocampal neurogenesis

abstract: Hippocampal neurogenesis declines with aging. Wnt ligands and antagonists within the hippocampal neurogenic niche regulate the proliferation of neural progenitor cells and the development of new neurons, and the changes of their levels in the niche mediate aging-associated decline of neurogenesis. We found that RNA-binding protein Lin28a remained existent in neural progenitor cells and granule neurons in the adult hippocampus, and decreased with aging. Loss of Lin28a inhibited the responsiveness of neural progenitor cells to niche Wnt agonist and reduced neurogenesis, thus impairing pattern separation. Overexpression of Lin28a increased the proliferation of neural progenitor cells, promoted the functional integration of newborn neurons, restored neurogenesis in Wnt-deficient dentate gyrus, and rescued the impaired pattern separation in aging mice. Our data suggest that Lin28a regulates adult hippocampal neurogenesis as an intracellular mechanism by responding to niche Wnt signals, and its decrease is involved in aging-associated decline of hippocampal neurogenesis as well as related cognitive functions.


title: Phylogeographic analysis reveals an ancient East African origin of the human herpes simplexvirus 2 dispersal out-of-Africa.

abstract: Human herpes simplex virus 2 (HSV-2) is a globally ubiquitous, slow evolving DNA virus. HSV-2 genomic diversity can be divided into two main groups: an African lineage and worldwide lineage. Competing hypotheses have been put forth to explain the history of HSV-2. HSV-2 may have originated in Africa and then followed the first wave of human migration out of Africa between 50-100 kya. Alternatively, HSV-2 may have migrated out of Africa via the trans-Atlantic slave trade within the last 150-500 years. The lack of HSV-2 genomes from West and Central Africa, combined with a lack of molecular clock signal in HSV-2 has precluded robust testing of these competing hypotheses. Here, we expand the geographic sampling of HSV-2 genomes in order to resolve the geography and timing of divergence events within HSV-2. We analyze 65 newly sequenced HSV-2 genomes collected from primarily West and Central Africa along with 330 previously published genomes sampled over a 47-year period. Evolutionary simulations confirm that the molecular clock in HSV-2 is too slow to be detected using available data. However, phylogeographic analysis indicates that all biologically plausible evolutionary rates would place the ancestor of the worldwide lineage in East Africa, arguing against the trans-Atlantic slave trade as the source of worldwide diversity. The best supported evolutionary rates between 4.2x10-8 and 5.6x10-8 substitutions/site/year suggest a most recent common ancestor for HSV-2 around 90-120 kya and initial dispersal around 21.9-29.3 kya. These dates suggest HSV-2 left Africa during subsequent waves of human migration out of East Africa.


title: Explaining the microbial diversity conundrum: can microbiome composition be crucial for host fitness, while highly divergent among healthy individuals?

abstract: Recent empirical studies offer conflicting findings regarding the relation between host fitness and the composition of its microbiome, a conflict which we term the microbial {beta} - diversity conundrum: it has been shown that the microbiome is crucial for host wellbeing and survival. At the same time, different healthy individuals microbiome compositions, even in the same population, often differ dramatically, contrary to the notion that a vital trait should be highly conserved. Moreover, gnotobiotic individuals exhibit highly deleterious phenotypes, supporting the notion that the microbiome is paramount to host fitness. However, the introduction of almost arbitrarily selected microbiota into the system often achieves a significant rescue effect of the deleterious phenotypes, even microbiota from soil or phylogenetically distant host species, highlighting an apparent paradox. Here we suggest several solutions to the paradox using a computational framework, simulating the population dynamics of hosts and their microbiomes over multiple generations. The answers, relating to factors such as host population size, the specific mode of contribution of the microbes to host fitness, and the typical microbiome richness, offer solutions to the conundrum by creating scenarios where even when a hosts fitness is determined in full by its microbiome composition, this composition has little or no effect on the natural selection dynamics of the population.


title: The evolution of sexual dichromatism in a large radiation of landfowls: re-examining the female-biased selection in Wallace's model

abstract: Sexual dichromatism, the colour difference between males and females, has been particularly important for studying the interplay between sexual and natural selection. However, previous studies on the evolutionary forces of sexual dichromatism examing the Darwins and Wallaces model have produced mixed results. Phasianidae is a species-rich family with worldwide distribution, occupancy in nearly all terrestrial habitats, and a wide diversity of plumage patterns and colourations. Here, we use phylogenetic comparative methods to test the relationship between sexual dichromatism and colour complexity of males and females on both evolutionary direction and tempo including all species in Phasianidae. We show that the evolutionary direction of sexual dichromatism is negatively correlated with colour complexity in females but not males, and the evolutionary rates of sexual dichromatism are positively correlated with the evolutionary rates of colour complexity in both sexes. These results highlight the important role of female colour evolution in shaping sexual dichromatism in the pheasant family, and provide strong empirical supports for Wallaces hypothesis via a mosaic of sexual and natural selection in both sexes.


title: The last missing piece of the Triangle of U: the evolution of the tetraploid Brassica carinata genome

abstract: Ethiopian mustard (Brassica carinata) is an ancient crop with significant potential for expanded cultivation as a biodiesel feedstock. The remarkable stress resilience of B. carinata and desirable seed fatty acid profile addresses the ongoing food vs. fuel debate as the crop is productive on marginal lands otherwise not suitable for even closely related species. B. carinata is one of six key Brassica spp. that share three major genomes: three diploid species (AA, BB, CC) that spontaneously hybridized in a pairwise manner, forming three allotetraploid species (AABB, AACC, and BBCC). Each of these genomes has been researched extensively, except for that of B. carinata. In the present study, we report a high-quality, 1.31 Gbp genome with 156.9-fold sequencing coverage for B. carinata var. Gomenzer, completing and confirming the classic Triangle of U, a theory of the evolutionary relationships among these six species that arose almost a century ago. Our assembly provides insights into the genomic features that give rise to B. carinatas superior agronomic traits for developing more climate-resilient Brassica crops with excellent oil production. Notably, we identified an expansion of transcription factor networks and agronomically-important gene families. Completing the Triangle of U comparative genomics platform allowed us to examine the dynamics of polyploid evolution and the role of subgenome dominance in domestication and agronomical improvement.


title: Interpreting tree ensemble machine learning models with endoR

abstract: BackgroundTree ensemble machine learning models are increasingly used in microbiome science as they are compatible with the compositional, high-dimensional, and sparse structure of sequence-based microbiome data. While such models are often good at predicting phenotypes based on microbiome data, they only yield limited insights into how microbial taxa or genomic content may be associated. Results: We developed endoR, a method to interpret a fitted tree ensemble model. First, endoR simplifies the fitted model into a decision ensemble from which it then extracts information on the importance of individual features and their pairwise interactions and also visualizes these data as an interpretable network. Both the network and importance scores derived from endoR provide insights into how features, and interactions between them, contribute to the predictive performance of the fitted model. Adjustable regularization and bootstrapping help reduce the complexity and ensure that only essential parts of the model are retained. We assessed the performance of endoR on both simulated and real metagenomic data. We found endoR to infer true associations with more or comparable accuracy than other commonly used approaches while easing and enhancing model interpretation. Using endoR, we also confirmed published results on gut microbiome differences between cirrhotic and healthy individuals. Finally, we utilized endoR to gain insights into components of the microbiome that predict the presence of human gut methanogens, as these hydrogen-consumers are expected to interact with fermenting bacteria in a complex syntrophic network. Specifically, we analyzed a global metagenome dataset of 2203 individuals and confirmed the previously reported association between Methanobacteriaceae and Christensenellales. Additionally, we observed that Methanobacteriaceae are associated with a network of hydrogen-producing bacteria. Conclusion: Our method accurately captures how tree ensembles use features and interactions between them to predict a response. As demonstrated by our applications, the resultant visualizations and summary outputs facilitate model interpretation and enable the generation of novel hypotheses about complex systems. An implementation of endoR is available as an open-source R-package on GitHub (https://github.com/leylabmpi/endoR).


title: Molecular Dynamics of DNA Translocation by FtsK

abstract: The bacterial FtsK motor harvests energy from ATP to translocate double-stranded DNA during cell division. Here, we probe the molecular mechanisms underlying coordinated DNA translocation in FtsK by performing long timescale simulations of its hexameric assembly and individual subunits. From these simulations we predict signaling pathways that connect the ATPase active site to DNA-gripping residues, which allows the motor to coordinate its translocation activity with its ATPase activity. Additionally, we utilize well-tempered metadynamics simulations to compute free-energy landscapes that elucidate the extended-to-compact transition involved in force generation. We show that nucleotide binding promotes a compact conformation of a motor subunit, whereas the apo subunit is flexible. Together, our results support a mechanism whereby each ATP-bound subunit of the motor conforms to the helical pitch of DNA, and ATP hydrolysis/product release causes a subunit to lose grip of DNA. By ordinally engaging and disengaging with DNA, the FtsK motor unidirectionally translocates DNA.


title: An integrated approach to protein discovery and detection from complex biofluids

abstract:
Ovarian cancer, a leading cause of cancer related deaths among women, has been notoriously difficult to routinely screen for and diagnose early. Researchers and clinicians continue to seek routinely usable, non-invasive, screening methods as early detection significantly improves survival. Biomarker screening is ideal; however, currently available ovarian cancer biomarkers lack desirable sensitivity and specificity. Furthermore, the most fatal forms, high grade serous cancers often originate in the fallopian tube; therefore, sampling from the vaginal environment provides more proximal sources for tumor detection. To address these shortcomings and leverage proximal sampling, we developed an untargeted mass spectrometry microprotein profiling method and identified a signature of cystatin A, validated this protein in an animal model, and sought to overcome the limits of detection inherent to mass spectrometry by demonstrating that cystatin A is present at 100 pM concentrations using a label-free microtoroid resonator. The findings highlight the potential utility for early-stage detection where cystatin A levels would be low.

Significance StatementIt is now clear that high-grade serous ovarian cancer can originate in the fallopian tube epithelium. These tumors colonize the ovary and then metastasize throughout the peritoneum. This discovery has raised important, and yet unaddressed, questions how we might be able to detect and screen for this deadly disease for which there is no routine screening. We have leveraged vaginal lavages from a murine model of the disease as a complex biological fluid for untargeted discovery of microproteins using mass. We improved our limits of detection by conjugating a cystatin A antibody to the surface of a microtoroid resonator to allow us to specifically detect cystatin A from vaginal lavages at early time points across biological replicates.


title: Nearly maximal information gain due to time integration in central dogma reactions

abstract: Living cells process information about their environment through the central dogma processes of transcription and translation, which drive the cellular response to stimuli. Here, we study the transfer of information from environmental input to the transcript and protein expression levels. Evaluation of both experimental and analogous simulation data reveals that transcription and translation are not two simple information channels connected in series. Instead, we show that the central dogma reactions often create a time-integrating information channel, where the translation channel receives and integrates multiple outputs from the transcription channel. This information channel model of the central dogma provides new information-theoretic selection criteria for the central dogma rate constants. Using the data for four well-studied species we show that their central dogma rate constants achieve information gain due to time integration while also keeping the loss due to stochasticity in translation relatively low (< 0.5 bits).

category: systems biology


title: Regulation of PDF Receptor signaling controlling daily locomotor rhythms in Drosophila

abstract:
Each day and in conjunction with ambient daylight conditions, neuropeptide PDF regulates the phase and amplitude of locomotor activity rhythms in Drosophila through its receptor, PDFR, a Family B G protein-coupled receptor (GPCR). We studied the in vivo process by which PDFR signaling turns off, by converting as many as half of the 28 potential sites of phosphorylation in its C terminal tail to a non-phosphorylatable residue (alanine). We report that many such sites are conserved evolutionarily, and their conversion creates a specific behavioral syndrome opposite to loss-of-function phenotypes previously described for pdfr. That syndrome includes increases in the amplitudes of both Morning and Evening behavioral peaks, as well as multi-hour delays of the Evening phase. The precise behavioral effects were dependent on day-length, and most effects mapped to conversion of only a few, specific serine residues near the very end of the protein and specific to its A isoform. Behavioral phase delays under entraining conditions were often, but not always correlated with increases in circadian period. The behavioral phenotypes produced by the most severe PDFR variant were ligand-dependent in vivo, and not a consequence of changes to their pharmacological properties, nor of changes in their surface expression, as measured in vitro. The mechanisms underlying termination of PDFR signaling are complex, subject to regulation that is modified by season, and central to a better understanding of the peptidergic modulation of behavior.

AUTHOR SUMMARYIn multi-cellular organisms, circadian pacemakers create output as a series of phase markers across the 24 hour day to allow other cells to pattern diverse aspects of daily rhythmic physiology and behavior. Within circadian pacemaker circuits, neuropeptide signaling is essential to help promote coherent circadian outputs. In the fruit fly Drosophila 150 neurons are dedicated circadian clocks and they all tell the same time. In spite of such strong synchronization, they provide diverse phasic outputs in the form of their discrete, asynchronous neuronal activity patterns. Neuropeptide signaling breaks the clock-generated symmetry and drives many pacemakers away from their preferred activity period in the morning. Each day, neuropeptide PDF is released by Morning pacemakers and delays the phase of activity of specific other pacemakers to later parts of the day or night. When and how the PDF that is released in the morning stops acting is unknown. Furthermore, timing of signal termination is not fixed because day length changes each day, hence the modulatory delay exerted by PDF must itself be regulated. Here we test a canonical model of G protein-coupled receptor physiology to ask how PDF receptor signaling is normally de-activated. We use behavioral measures to define sequence elements of the receptor whose post-translational modifications (e.g., phosphorylation) may define the duration of receptor signaling.

category: neuroscience


title: Got Milk? Maternal immune activation during the mid-lactational period affects nutritional milk quality and adolescent offspring sensory processing in male and female rats

abstract: The neonatal environment requires a high level of maternal demand in terms of both breastfeeding and other forms of maternal care. Previous studies have underscored the importance of these maternal factors on offspring development and behavior. However, their contribution as dynamic variables in animal models of early life stress are often overlooked. In the present study, we show that lipopolysaccharide (LPS)-induced maternal immune activation (MIA) on postnatal day (P)10 elevated milk corticosterone concentrations, which recovered by P11. In contrast, both milk triglyceride and percent creamatocrit values demonstrated a prolonged decrease following MIA. Sustained changes to the nutritional quality of milk were also evidenced by an increase of microbial communities associated with inefficient energy and lipid metabolism. Nutritional deficits in early development have been associated with metabolic dysfunction later in life. Indeed, these nutritional milk changes were reflected by increased adolescent offspring bodyweights. While MIA did not decrease maternal care quality, there was a significant compensatory increase in maternal licking and grooming the day that followed the inflammatory challenge. However, this did not protect against disrupted neonatal huddling or later-life alterations in sensorimotor gating, mechanical allodynia, or hippocampal parvalbumin interneuron expression in MIA offspring. Since milk concentrations of LPS binding protein and interleukin-6 were not different between MIA and control dams, these offspring effects were likely driven by nutritional factors and not by a direct exposure to LPS or inflammatory molecules. Animal models of early life stress can impact both parents and their progeny; our data show that inflammatory stress affects maternal lactation quality which acts as a mechanism to confer multifaceted and compounding effects on offspring.


title: The structured flow on the brain's resting state manifold

abstract: AO_SCPLOWBSTRACTC_SCPLOWSpontaneously fluctuating brain activity patterns emerge at rest and relate to brain functional networks involved in task conditions. Despite detailed descriptions of the spatio-temporal brain patterns, our understanding of their generative mechanism is still incomplete. Using a combination of computational modeling and dynamical systems analysis we provide a complete mechanistic description in terms of the constituent entities and the productive relation of their causal activities leading to the formation of a resting state manifold via the network connectivity. We demonstrate that the symmetry breaking by the connectivity creates a characteristic flow on the manifold, which produces the major empirical data features including spontaneous high amplitude co-activations, neuronal cascades, spectral cortical gradients, multistability and characteristic functional connectivity dynamics. The understanding of the brains resting state manifold is fundamental for the construction of task-specific flows and manifolds used in theories of brain function such as predictive coding.


title: Keeping time and rhythm by replaying a sensory-motor engram

abstract: Imagine practicing a piece of music, or a speech, solely within the mind, without any sensory input or motor output. Our ability to implement dynamic internal representations is key for successful behavior, yet how the brain achieves this is not fully understood1-4. Here we trained primates to perceive, and internally maintain, rhythms of different tempos and performed large-scale recordings of neuronal activity across multiple areas spanning the sensory-motor processing hierarchy. Results show that perceiving and maintaining rhythms engage multiple brain areas, including visual, parietal, premotor, prefrontal, and hippocampal regions. Each area displayed oscillatory activity that reflected the temporal and spatial characteristics of an internal metronome which flexibly encoded fast, medium, and slow tempos on a trial-by-trial basis. The presence of widespread metronome-related activity across the brain, in the absence of stimuli and overt actions, is consistent with the idea that time and rhythm are maintained by a mechanism that internally replays the stimuli and actions that define well-timed behavior.


title: Low vision impairs implicit sensorimotor adaptation in response to small errors, but not large errors

abstract: Successful goal-directed actions require constant fine-tuning in response to errors introduced by changes in the body and environment. This implicit adaptive process has been assumed to operate in a statistically optimal fashion, reducing its sensitivity to errors when sensory uncertainty is high. However, recent work has shown that visual uncertainty attenuates implicit adaptation for small errors, but not large errors, a result that is at odds with an optimal integration hypothesis. This error size interaction has motivated a new hypothesis that sensory uncertainty impacts the distribution of the perceived error locations but not the systems sensitivity to errors. To examine these competing hypotheses, previous studies have experimentally manipulated uncertainty. But it is unknown which hypothesis best describes motor adaptation to sensory uncertainty experienced during daily life. To address this question, we recruited individuals with low vision due to diverse clinical conditions impacting visual uncertainty and matched controls. The groups were tested on visuomotor tasks designed to isolate implicit adaptation and maintain tight control over the error size. In two experiments, low vision was associated with attenuated implicit adaptation only for small errors, but not for large errors. Taken together with prior work in which visual uncertainty was experimentally manipulated, these results support the notion that increasing sensory uncertainty increases the likelihood that errors are mis-localized but does not affect error sensitivity, offering a novel account for the motor learning deficits seen in low vision.


title: Nested spatial and temporal modeling of environmental conditions associated with genetic markers of Vibrio parahaemolyticus in Washington state Pacific oysters

abstract: The Pacific Northwest (PNW) is one of the largest commercial harvesting areas for Pacific oysters (Crassostrea gigas) in the United States. Vibrio parahaemolyticus, a bacterium naturally present in estuarine waters, accumulates in shellfish and is a major cause of seafood-borne illness. Growers, consumers, and public-health officials have raised concerns about rising vibriosis cases in the region. V. parahaemolyticus genetic markers (tlh, tdh, trh) were estimated using an MPN-PCR technique in Washington State Pacific oysters regularly sampled between May and October from 2005 to 2019 (N=2,836); environmental conditions were also measured at each sampling event. Multilevel mixed-effects regression models were used to assess relationships between environmental measures and genetic markers as well as genetic marker ratios (trh:tlh, tdh:tlh, and tdh:trh), accounting for variation across space and time. Spatial and temporal dependence were also accounted for in the model structure. Model fit improved when including environmental measures from previous weeks (1-week lag for air temperature, 3-week lag for salinity). Positive associations were found between tlh and surface water temp, specifically between 15{degrees}C and 26{degrees}C, and between trh and surface water temperature up to 26{degrees}C. tlh and trh were negatively associated with 3-week lagged salinity in the most saline waters (> 27 ppt). There was also a positive relationship between tissue temperature and tdh, but only above 20{degrees}C. The tdh:tlh ratio displayed analogous inverted non-linear relationships as tlh. The non-linear associations found between the genetic targets and environmental measures demonstrate the complex habitat suitability of V. parahaemolyticus. Additional associations with both spatial and temporal variables also suggest there are influential unmeasured environmental conditions that could further explain bacterium variability. Overall, these findings confirm previous ecological risk factors for vibriosis in Washington State, while also identifying new associations between lagged temporal effects and pathogenic markers of V. parahaemolyticus.


title: Dual fates of exogenous tau seeds: lysosomal clearance vs. cytoplasmic amplification

abstract: Tau assembly propagation from the extracellular to intracellular space of a cell may underlie neurodegenerative tauopathies. The first step involves tau binding to heparan sulfate proteoglycans on the cell surface, followed by macropinocytosis. Pathological tau assemblies are thought to exit the vesicular compartment as "seeds" for replication in the cytoplasm. Tau uptake is highly efficient, but only [~]1-10% of cells that take up aggregates exhibit seeding. To investigate the basis for this observation, we used fluorescently tagged full-length (FL) tau fibrils added to native U2OS cells, and "biosensor" cells expressing FL tau or repeat domain fused to mClover (Clo). FL tau-Clo bound tubulin, but seeds triggered its aggregation in multiple locations simultaneously in the cytoplasm, generally independent of visible exogenous aggregates. Most exogenous tau trafficked to the lysosome, but imaging revealed a small percentage that slowly and steadily accumulated in the cytosol. Intracellular expression of Gal3-mRuby, which binds intravesicular galactosides and forms puncta upon vesicle rupture, revealed no evidence of vesicle damage following tau exposure. In fact, most seeded cells had no evidence of lysosome rupture. However, live cell imaging indicated that cells with pre-existing Gal3-positive puncta exhibited seeding at a slightly higher rate than the general population, indicating a potential role for vesicle instability as a predisposing factor. Clearance of tau seeds occurred rapidly in both vesicular and cytosolic fractions. Bafilomycin inhibited vesicular clearance, whereas MG132 inhibited cytosolic clearance. Tau seeds that enter the cell thus have at least two fates: lysosomal clearance that degrades most tau, and entry into the cytosol, where seeds replicate, and are cleared by the proteasome.


title: Modelling the distribution of rare invertebrates by correcting class imbalance and spatial bias

abstract:
AimSoil arthropods are important decomposers and nutrient cyclers, but are poorly represented on national and international conservation Red Lists. Opportunistic biological records for soil invertebrates are often sparse, and contain few observations of rare species but a relatively large number of non-detection observations (a problem known as class imbalance). Robinson et al. (2018) proposed a method for sub-sampling non-detection data using a spatial grid to improve class balance and spatial bias in bird data. For taxa that are less intensively sampled, datasets are smaller, which poses a challenge because under-sampling data removes information. We tested whether spatial under-sampling improved prediction performance of species distribution models for millipedes, for which large datasets are not available. We also tested whether using environmental predictor variables provided additional information beyond what is captured by spatial position for predicting species distributions.

LocationIsland of Ireland.

MethodsWe tested the spatial under-sampling method of Robinson et al. (2018) by using biological records to train species distribution models of rare millipedes.

ResultsUsing spatially under-sampled training data improved species distribution model sensitivity (true positive rate) but decreased model specificity (true negative rate). The decrease in specificity was minimal for rarer species and was accompanied by substantial increases in sensitivity. For common species, specificity decreased more, and sensitivity increased less, making spatial under-sampling most useful for rare species. Geographic coordinates were as good as or better than environmental variables for predicting distributions of two out of six species.

Main ConclusionsSpatial under-sampling improved prediction performance of species distribution models for rare soil arthropod species. Spatial under-sampling was most effective for rarer species. The good prediction performance of models using geographic coordinates is promising for modeling distributions of poorly studied species for which little is known about ecological or physiological determinants of occurrence.


title: Testing a theoretical framework for the environment-species abundance paradigm: a new approach to the Abundant Centre Hypothesis

abstract: The linkage between environment, a species fitness and its abundance is central to the theory of evolution. So far, all studies of this linkage have been heuristic and empirical due to an inability to determine fitness either experimentally (independent of abundance) or theoretically (from species-environment interaction). One category of such studies involves the Abundant Centre Hypothesis which posits that a species abundance rises to a maximum at the centre of its range. We argue that the confusing mix of results from ACH studies arises from ignoring the central premise that the abundance distribution cannot be independent of the environment. First, we employed a theoretical framework to identify an environmental context (an elevational transect; 200-2800 m in the eastern Himalayas) likely to favour ACH. We then improved upon some previously identified conceptual and methodological shortcomings of ACH studies. Using systematically collected bird data (245 species; 15867 records) from that transect we found that the community average abundance profile is symmetric, as expected by ACH. Notwithstanding which, the abundance profiles of individual species showed a small degree of asymmetry which was correlated with elevation. This elevational dependence may be due to the hard elevational limits at the lower and upper ends of the mountain, as expected from theoretical considerations. We also showed that the average abundance profile shape is close to gaussian, while ruling out uniform and inverted-quadratic shapes. This work demonstrates that selecting a particular category of environmental contexts can help in integrating theoretical tools into a field dominated by empirical studies. Such a union should spur the development of more detailed and testable theoretical models for better insights in an important field.