A cGAL-UAS bipartite expression toolkit for Caenorhabditis elegans sensory neurons.

Animals integrate sensory information from the environment and display various behaviors in response to external stimuli. In Caenorhabditis elegans hermaphrodites, 33 types of sensory neurons are responsible for chemosensation, olfaction, and mechanosensation. However, the functional roles of all sensory neurons have not been systematically studied due to the lack of facile genetic accessibility. A bipartite cGAL-UAS system has been previously developed to study tissue- or cell-specific functions in C. elegans. Here, we report a toolkit of new cGAL drivers that can facilitate the analysis of a vast majority of the 60 sensory neurons in C. elegans hermaphrodites. We generated 37 sensory neuronal cGAL drivers that drive cGAL expression by cell-specific regulatory sequences or intersection of two distinct regulatory regions with overlapping expression (split cGAL). Most cGAL-drivers exhibit expression in single types of cells. We also constructed 28 UAS effectors that allow expression of proteins to perturb or interrogate sensory neurons of choice. This cGAL-UAS sensory neuron toolkit provides a genetic platform to systematically study the functions of C. elegans sensory neurons.

Both entry to and exit from diapause arrest in Caenorhabditis elegans are regulated by a steroid hormone pathway.

Diapause arrest in animals such as Caenorhabditis elegans is tightly regulated so that animals make appropriate developmental decisions amidst environmental challenges. Fully understanding diapause requires mechanistic insight of both entry and exit from the arrested state. Although a steroid hormone pathway regulates the entry decision into C. elegans dauer diapause, its role in the exit decision is less clear. A complication to understanding steroid hormonal regulation of dauer has been the peculiar fact that steroid hormone mutants such as daf-9 form partial dauers under normal growth conditions. Here, we corroborate previous findings that daf-9 mutants remain capable of forming full dauers under unfavorable growth conditions and establish that the daf-9 partial dauer state is likely a partially exited dauer that has initiated but cannot complete the dauer exit process. We show that the steroid hormone pathway is both necessary for and promotes complete dauer exit, and that the spatiotemporal dynamics of steroid hormone regulation during dauer exit resembles that of dauer entry. Overall, dauer entry and dauer exit are distinct developmental decisions that are both controlled by steroid hormone signaling.

Structural and functional adaptation of Haloferax volcanii TFEα/ß.

The basal transcription factor TFE enhances transcription initiation by catalysing DNA strand-separation, a process that varies with temperature and ionic strength. Canonical TFE forms a heterodimeric complex whose integrity and function critically relies on a cubane iron-sulphur cluster residing in the TFEß subunit. Halophilic archaea such as Haloferax volcanii have highly divergent putative TFEß homologues with unknown properties. Here, we demonstrate that Haloferax TFEß lacks the prototypical iron-sulphur cluster yet still forms a stable complex with TFEα. A second metal cluster contained in the zinc ribbon domain in TFEα is highly degenerate but retains low binding affinity for zinc, which contributes to protein folding and stability. The deletion of the tfeB gene in H. volcanii results in the aberrant expression of approximately one third of all genes, consistent with its function as a basal transcription initiation factor. Interestingly, tfeB deletion particularly affects foreign genes including a prophage region. Our results reveal the loss of metal centres in Hvo transcription factors, and confirm the dual function of TFE as basal factor and regulator of transcription.

Transcription of cis Antisense Small RNA MtlS in Vibrio cholerae Is Regulated by Transcription of Its Target Gene, mtlA.

Vibrio cholerae, the facultative pathogen responsible for cholera disease, continues to pose a global health burden. Its persistence can be attributed to a flexible genetic tool kit that allows for adaptation to different environments with distinct carbon sources, including the six-carbon sugar alcohol mannitol. V. cholerae takes up mannitol through the transporter protein MtlA, whose production is downregulated at the posttranscriptional level by MtlS, a cis antisense small RNA (sRNA) whose promoter lies within the mtlA open reading frame. Though it is known that mtlS expression is robust under growth conditions lacking mannitol, it has remained elusive as to what factors govern the steady-state levels of MtlS. Here, we show that manipulating mtlA transcription is sufficient to drive inverse changes in MtlS levels, likely through transcriptional interference. This work has uncovered a cis-acting sRNA whose expression pattern is predominantly controlled by transcription of the sRNA's target gene.IMPORTANCEVibrio cholerae is a bacterial pathogen that relies on genetic tools, such as regulatory RNAs, to adapt to changing extracellular conditions. While many studies have focused on how these regulatory RNAs function, fewer have focused on how they are themselves modulated. V. cholerae expresses the noncoding RNA MtlS, which can regulate mannitol transport and use, and here we demonstrate that MtlS levels are controlled by the level of transcription occurring in the antisense direction. Our findings provide a model of regulation describing how bacteria like V. cholerae can modulate the levels of an important regulatory RNA. Our work contributes to knowledge of how bacteria deploy regulatory RNAs as an adaptive mechanism to buffer against environmental flux.

MtlR negatively regulates mannitol utilization by Vibrio cholerae.

The phosphoenopyruvate:carbohydrate phosphotransferase system (PTS) enables Vibrio cholerae - and other bacteria - to recognize and transport exogenous carbon sources for energy, including the six-carbon sugar alcohol, mannitol. The mannitol-specific PTS transporter is encoded by mtlA and its expression is expected to be regulated by the putative repressor encoded by the mtlR gene. Here, we show that mtlR overexpression inhibits V. cholerae growth in medium supplied with mannitol as the sole carbon source and represses MtlA-mediated biofilm formation. We demonstrate that when V. cholerae is grown in non-mannitol medium, knocking out mtlR leads to both increased MtlA protein and mtlA mRNA levels, with these increases being especially pronounced in non-glucose sugars. We propose that in non-mannitol, non-glucose growth conditions, MtlR is a major regulator of mtlA transcription. Surprisingly, with regard to mtlR expression, transcript and protein levels are highest in mannitol medium, conditions where mtlA expression should not be repressed. We further show that MtlR levels increase during growth of the bacteria and linger in cells switched from mannitol to non-mannitol medium. Our data suggests an expression paradigm for mtlA where MtlR acts as a transcriptional repressor responsible for calibrating MtlA levels during environmental transitions.

Sensory integration of food availability and population density during the diapause exit decision involves insulin-like signaling in Caenorhabditis elegans.

Decisions made over long time scales, such as life cycle decisions, require coordinated interplay between sensory perception and sustained gene expression. The Caenorhabditis elegans dauer (or diapause) exit developmental decision requires sensory integration of population density and food availability to induce an all-or-nothing organismal-wide response, but the mechanism by which this occurs remains unknown. Here, we demonstrate how the ASJ chemosensory neurons, known to be critical for dauer exit, perform sensory integration at both the levels of gene expression and calcium activity. In response to favorable conditions, dauers rapidly produce and secrete the dauer exit-promoting insulin-like peptide INS-6. Expression of ins-6 in the ASJ neurons integrate population density and food level and can reflect decision commitment since dauers committed to exiting have higher ins-6 expression levels than those of non-committed dauers. Calcium imaging in dauers reveals that the ASJ neurons are activated by food, and this activity is suppressed by pheromone, indicating that sensory integration also occurs at the level of calcium transients. We find that ins-6 expression in the ASJ neurons depends on neuronal activity in the ASJs, cGMP signaling, a CaM-kinase pathway, and the pheromone components ascr#8 and ascr#2. We propose a model in which decision commitment to exit the dauer state involves an autoregulatory feedback loop in the ASJ neurons that promotes high INS-6 production and secretion. These results collectively demonstrate how insulin-like peptide signaling helps animals compute long-term decisions by bridging sensory perception to decision execution.

Fluorescence dynamics of lysosomal-related organelle flashing in the intestinal cells of Caenorhabditis elegans.

The biological roles of the autofluorescent lysosome-related organelles ("gut granules") in the intestinal cells of many nematodes, including Caenorhabditis elegans, have been shown to play an important role in metabolic and signaling processes, but they have not been fully characterized. We report here a previously undescribed phenomenon in which the autofluorescence of these granules increased and then decreased in a rapid and dynamic manner that may be associated with nutrient availability. We observed that two distinct types of fluorophores are likely present in the gut granules. One displays a "flashing" phenomenon, in which fluorescence decrease is preceded by a sharp increase in fluorescence intensity that expands into the surrounding area, while the other simply decreases in intensity. Gut granule flashing was observed in the different life stages of C. elegans and was also observed in Steinernema hermaphroditum, an evolutionarily distant nematode. We hypothesize that the "flashing" fluorophore is pH-sensitive, and the fluorescence intensity change results from the fluorophore being released from the lysosome-related organelles into the relatively higher pH environment of the cytosol. The visually spectacular dynamic fluorescence phenomenon we describe might provide a handle on the biochemistry and genetics of these lysosome-related organelles.

Chloroacetamide fragment library screening identifies new scaffolds for covalent inhibition of the TEAD·YAP1 interaction.

Transcriptional enhanced associate domain (TEAD) binding to co-activator yes-associated protein (YAP1) leads to a transcription factor of the Hippo pathway. TEADs are regulated by S-palmitoylation of a conserved cysteine located in a deep well-defined hydrophobic pocket outside the TEAD·YAP1 interaction interface. Previously, we reported the discovery of a small molecule based on the structure of flufenamic acid that binds to the palmitate pocket, forms a covalent bond with the conserved cysteine, and inhibits TEAD4 binding to YAP1. Here, we screen a fragment library of chloroacetamide electrophiles to identify new scaffolds that bind to the palmitate pocket of TEADs and disrupt their interaction with YAP1. Time- and concentration-dependent studies with wild-type and mutant TEAD1-4 provided insight into their reaction rates and binding constants and established the compounds as covalent inhibitors of TEAD binding to YAP1. Binding pose hypotheses were generated by covalent docking revealing that the fragments and compounds engage lower, middle, and upper sub-sites of the palmitate pocket. Our fragments and compounds provide new scaffolds and starting points for the design of derivatives with improved inhibition potency of TEAD palmitoylation and binding to YAP1.

Genetic correlations between Alzheimer's disease and gut microbiome genera.

A growing body of evidence suggests that dysbiosis of the human gut microbiota is associated with neurodegenerative diseases like Alzheimer's disease (AD) via neuroinflammatory processes across the microbiota-gut-brain axis. The gut microbiota affects brain health through the secretion of toxins and short-chain fatty acids, which modulates gut permeability and numerous immune functions. Observational studies indicate that AD patients have reduced microbiome diversity, which could contribute to the pathogenesis of the disease. Uncovering the genetic basis of microbial abundance and its effect on AD could suggest lifestyle changes that may reduce an individual's risk for the disease. Using the largest genome-wide association study of gut microbiota genera from the MiBioGen consortium, we used polygenic risk score (PRS) analyses with the "best-fit" model implemented in PRSice-2 and determined the genetic correlation between 119 genera and AD in a discovery sample (ADc12 case/control: 1278/1293). To confirm the results from the discovery sample, we next repeated the PRS analysis in a replication sample (GenADA case/control: 799/778) and then performed a meta-analysis with the PRS results from both samples. Finally, we conducted a linear regression analysis to assess the correlation between the PRSs for the significant genera and the APOE genotypes. In the discovery sample, 20 gut microbiota genera were initially identified as genetically associated with AD case/control status. Of these 20, three genera (Eubacterium fissicatena as a protective factor, Collinsella, and Veillonella as a risk factor) were independently significant in the replication sample. Meta-analysis with discovery and replication samples confirmed that ten genera had a significant correlation with AD, four of which were significantly associated with the APOE rs429358 risk allele in a direction consistent with their protective/risk designation in AD association. Notably, the proinflammatory genus Collinsella, identified as a risk factor for AD, was positively correlated with the APOE rs429358 risk allele in both samples. Overall, the host genetic factors influencing the abundance of ten genera are significantly associated with AD, suggesting that these genera may serve as biomarkers and targets for AD treatment and intervention. Our results highlight that proinflammatory gut microbiota might promote AD development through interaction with APOE. Larger datasets and functional studies are required to understand their causal relationships.

Feasibility and Acceptability of a Text Message-Based Intervention to Reduce Overuse of Alcohol in Emergency Department Patients: Controlled Proof-of-Concept Trial.

BACKGROUND: Emergency department (ED) patients have high rates of risky alcohol use, and an ED visit offers an opportunity to intervene. ED-based screening, brief intervention, and referral to treatment (SBIRT) reduces alcohol use and health care costs. Mobile health (mHealth) interventions may expand the impact of SBIRTs but are understudied in low-resource ED populations. OBJECTIVE: The objective of this study was to assess the feasibility of and patient satisfaction with a text-based mHealth extension of an ED screening program to reduce risky alcohol use in low-income, urban patients. METHODS: Research assistants screened a convenience sample of ED patients in person for risky alcohol use via the Alcohol Use Disorders Identification Test (AUDIT). Patients who reported AUDIT scores ≥8 and <20 were informed of their AUDIT score and risk. RAs invited patients with SMS text message-capable phones to receive mROAD (mobilizing to Reduce Overuse of Alcohol in the ED), an SMS text message-based extension of the ED screening program. mROAD is a 7-day program of twice-daily SMS text messages based on the National Institutes of Health's Rethinking Drinking campaign. Participants were allocated to a control group (daily sham text messages without specific guidance on behaviors, such as "Thanks for taking part!") or to the mROAD intervention group. Patients were interviewed at 30 days to assess acceptability, satisfaction, and changes in drinking behavior. Satisfaction was examined descriptively. Pre and post measurements of drinking behaviors and motivation were compared, as were differences in change scores between the intervention arms. RESULTS: Of 1028 patients screened, 95 (9.2%) exhibited risky alcohol use based on AUDIT, and 23/95 (24%) of those patients did not own an SMS text messaging-capable phone; this left 72/95 (76%) eligible patients. Among eligible participants, 48/72 (67%) agreed to enroll; 31/48 (65%) achieved follow-up (18/24 (75%) in the intervention group and 13/24 (55%) in the control group). Participants who completed follow-up reported high satisfaction. Changes in behavior were similar between the arms. Overall, the number of drinking days reported in the prior 30 days decreased by 5.0 (95% CI 1.7-8.3; P=.004), and the number of heavy drinking days decreased by 4.1 (95% CI 1.0 to 7.15, P=.01). Patients reported an 11-point increase (95% CI 2.6-20, P=.01, 10% overall increase) in motivation to change alcohol use via the Change Questionnaire. The were no statistical differences in drinking days, heavy drinking days, or motivation to change between the arms. CONCLUSIONS: The mROAD trial was feasible. Over three-quarters of ED patients with risky alcohol use owned a text message-capable phone, and two-thirds of these patients were willing to participate; only 1 patient opted out of the intervention. Although 35% of patients were lost to follow-up at 30 days, those patients who did follow up had favorable impressions of the program; more than 90% reported that SMS text messages were a "good way to teach," and 89% of intervention arm participants enjoyed the program and found that the messages were motivating. Both the mROAD and sham message groups showed promising changes in alcohol use and motivation to change. mROAD is a feasible intervention that may reduce rates of risky alcohol use in ED patients. TRIAL REGISTRATION: ClinicalTrials.gov NCT02158949; https://clinicaltrials.gov/ct2/show/NCT02158949.

IDseq-An open source cloud-based pipeline and analysis service for metagenomic pathogen detection and monitoring.

BACKGROUND: Metagenomic next-generation sequencing (mNGS) has enabled the rapid, unbiased detection and identification of microbes without pathogen-specific reagents, culturing, or a priori knowledge of the microbial landscape. mNGS data analysis requires a series of computationally intensive processing steps to accurately determine the microbial composition of a sample. Existing mNGS data analysis tools typically require bioinformatics expertise and access to local server-class hardware resources. For many research laboratories, this presents an obstacle, especially in resource-limited environments. FINDINGS: We present IDseq, an open source cloud-based metagenomics pipeline and service for global pathogen detection and monitoring (https://idseq.net). The IDseq Portal accepts raw mNGS data, performs host and quality filtration steps, then executes an assembly-based alignment pipeline, which results in the assignment of reads and contigs to taxonomic categories. The taxonomic relative abundances are reported and visualized in an easy-to-use web application to facilitate data interpretation and hypothesis generation. Furthermore, IDseq supports environmental background model generation and automatic internal spike-in control recognition, providing statistics that are critical for data interpretation. IDseq was designed with the specific intent of detecting novel pathogens. Here, we benchmark novel virus detection capability using both synthetically evolved viral sequences and real-world samples, including IDseq analysis of a nasopharyngeal swab sample acquired and processed locally in Cambodia from a tourist from Wuhan, China, infected with the recently emergent SARS-CoV-2. CONCLUSION: The IDseq Portal reduces the barrier to entry for mNGS data analysis and enables bench scientists, clinicians, and bioinformaticians to gain insight from mNGS datasets for both known and novel pathogens.

Engineering Riboswitches in Vivo Using Dual Genetic Selection and Fluorescence-Activated Cell Sorting.

Riboswitches, noncoding RNAs that bind a small molecule effector to control gene expression at the level of transcription or translation, are uniquely suited to meet challenges in synthetic biology. To expand the limited set of existing riboswitches, we developed a riboswitch discovery platform that couples dual genetic selection and fluorescence-activated cell sorting to identify novel riboswitches from a 108 random-sequence library in which the aptamer domain of the ThiM#2 riboswitch was replaced with an N40 sequence. In a proof-of-principle validation, we identified novel riboswitches for the small molecule theophylline. Our best riboswitch (Hit 3-5) displays 2.3-fold activation of downstream gene expression in the presence of theophylline. Random mutagenesis of Hit 3-5, coupled with selections and screens, afforded improved riboswitches displaying nearly 3-fold activation. To the best of our knowledge, this is the first report of in vivo directed evolution of an aptamer domain to generate a functional riboswitch.

Tai chi for health benefits in patients with multiple sclerosis: A systematic review.

The aim of this systematic review was to evaluate the existing evidence on the effectiveness and safety of Tai chi, which is critical to provide guidelines for clinicians to improve symptomatic management in patients with multiple sclerosis (MS). After performing electronic and manual searches of many sources, ten relevant peer-reviewed studies that met the inclusion criteria were retrieved. The existing evidence supports the effectiveness of Tai chi on improving quality of life (QOL) and functional balance in MS patients. A small number of these studies also reported the positive effect of Tai chi on flexibility, leg strength, gait, and pain. The effect of Tai chi on fatigue is inconsistent across studies. Although the findings demonstrate beneficial effects on improving outcome measures, especially for functional balance and QOL improvements, a conclusive claim should be made carefully for reasons such as methodological flaws, small sample size, lack of specific-disease instruments, unclear description of Tai chi protocol, unreported safety of Tai chi, and insufficient follow-up as documented by the existing literature. Future research should recruit a larger number of participants and utilize the experimental design with a long-term follow-up to ascertain the benefits of Tai chi for MS patients.

A Systematic Review and Meta-Analysis Baduanjin Qigong for Health Benefits: Randomized Controlled Trials.

Objective. To investigate the effects of practicing Baduanjin Qigong on different health outcomes. Methods. Six electronic databases were used for literature search through entering the following key words: Baduanjin Qigong, quality of life, sleep quality, and health-related outcomes. Results. Nineteen randomized controlled trials were used for meta-analysis. The aggregated results from this systematic review have shown significant benefits in favour of Baduanjin Qigong on quality of life (SMD, -0.75; 95% CI -1.26 to -0.24; P = 0.004), sleep quality (SMD, -0.55; 95% CI -0.97 to -0.12; P = 0.01), balance (SMD, -0.94; 95% CI -1.59 to 0.30; P = 0.004), handgrip strength (SMD, -0.69; 95% CI -1.2 to -0.19; P = 0.007), trunk flexibility (SMD, -0.66; 95% CI -1.13 to -0.19; P = 0.006), systolic (SMD, -0.60; 95% CI -0.94 to -0.27; P = 0.0004) and diastolic blood pressure (SMD, -0.46; 95% CI -0.73 to -0.20; P = 0.0005), and resting heart rate (SMD, -0.87; 95% CI -1.47 to -0.27; P = 0.005). The aggregated results of meta-analyses examining the effect of Baduanjin Qigong on leg power, cardiopulmonary endurance, and pulmonary function remain unclear because of a small number of studies. Conclusions. The aggregated results from this systematic review show that Baduanjin Qigong practice is beneficial for quality of life, sleep quality, balance, handgrip strength, trunk flexibility, systolic and diastolic blood pressure, and resting heart rate. Further studies are necessary to confirm the effects of Baduanjin Qigong on leg power, cardiopulmonary endurance, and pulmonary function (e.g., vital capacity), while considering a long-term follow-up. Registration Number. This trial is registered with International Prospective Register of Systematic Reviews (PROSPERO): CRD42016036966.