Accurately Modeling RNA Stem-Loops in an Implicit Solvent Environment.

Ribonucleic acid (RNA) molecules can adopt a variety of secondary and tertiary structures in solution, with stem-loops being one of the more common motifs. Here, we present a systematic analysis of 15 RNA stem-loop sequences simulated with molecular dynamics simulations in an implicit solvent environment. Analysis of RNA cluster ensembles showed that the stem-loop structures can generally adopt the A-form RNA in the stem region. Loop structures are more sensitive, and experimental structures could only be reproduced with modification of CH···O interactions in the force field, combined with an implicit solvent nonpolar correction to better model base stacking interactions. Accurately modeling RNA with current atomistic physics-based models remains challenging, but the RNA systems studied herein may provide a useful benchmark set for testing other RNA modeling methods in the future.

The Structure and Function of the Bacterial Osmotically Inducible Protein Y.

The ability to adapt to osmotically diverse and fluctuating environments is critical to the survival and resilience of bacteria that colonize the human gut and urinary tract. Environmental stress often provides cross-protection against other challenges and increases antibiotic tolerance of bacteria. Thus, it is critical to understand how E. coli and other microbes survive and adapt to stress conditions. The osmotically inducible protein Y (OsmY) is significantly upregulated in response to hypertonicity. Yet its function remains unknown for decades. We determined the solution structure and dynamics of OsmY by nuclear magnetic resonance spectroscopy, which revealed that the two Bacterial OsmY and Nodulation (BON) domains of the protein are flexibly linked under low- and high-salinity conditions. In-cell solid-state NMR further indicates that there are no gross structural changes in OsmY as a function of osmotic stress. Using cryo-electron and super-resolution fluorescence microscopy, we show that OsmY attenuates plasmolysis-induced structural changes in E. coli and improves the time to growth resumption after osmotic upshift. Structure-guided mutational and functional studies demonstrate that exposed hydrophobic residues in the BON1 domain are critical for the function of OsmY. We find no evidence for membrane interaction of the BON domains of OsmY, contrary to current assumptions. Instead, at high ionic strength, we observe an interaction with the water channel, AqpZ. Thus, OsmY does not play a simple structural role in E. coli but may influence a cascade of osmoregulatory functions of the cell.

RNA language models predict mutations that improve RNA function.

Structured RNA lies at the heart of many central biological processes, from gene expression to catalysis. While advances in deep learning enable the prediction of accurate protein structural models, RNA structure prediction is not possible at present due to a lack of abundant high-quality reference data. Furthermore, available sequence data are generally not associated with organismal phenotypes that could inform RNA function. We created GARNET (Gtdb Acquired RNa with Environmental Temperatures), a new database for RNA structural and functional analysis anchored to the Genome Taxonomy Database (GTDB). GARNET links RNA sequences derived from GTDB genomes to experimental and predicted optimal growth temperatures of GTDB reference organisms. This enables construction of deep and diverse RNA sequence alignments to be used for machine learning. Using GARNET, we define the minimal requirements for a sequence- and structure-aware RNA generative model. We also develop a GPT-like language model for RNA in which triplet tokenization provides optimal encoding. Leveraging hyperthermophilic RNAs in GARNET and these RNA generative models, we identified mutations in ribosomal RNA that confer increased thermostability to the Escherichia coli ribosome. The GTDB-derived data and deep learning models presented here provide a foundation for understanding the connections between RNA sequence, structure, and function.

Mesoscopic mapping of hemodynamic responses and neuronal activity during pharmacologically induced interictal spikes in awake and anesthetized mice.

Imaging hemodynamic responses to interictal spikes holds promise for presurgical epilepsy evaluations. Understanding the hemodynamic response function is crucial for accurate interpretation. Prior interictal neurovascular coupling data primarily come from anesthetized animals, impacting reliability. We simultaneously monitored calcium fluctuations in excitatory neurons, hemodynamics, and local field potentials (LFP) during bicuculline-induced interictal events in both isoflurane-anesthetized and awake mice. Isoflurane significantly affected LFP amplitude but had little impact on the amplitude and area of the calcium signal. Anesthesia also dramatically blunted the amplitude and latency of the hemodynamic response, although not its area of spread. Cerebral blood volume change provided the best spatial estimation of excitatory neuronal activity in both states. Targeted silencing of the thalamus in awake mice failed to recapitulate the impact of anesthesia on hemodynamic responses suggesting that isoflurane's interruption of the thalamocortical loop did not contribute either to the dissociation between the LFP and the calcium signal nor to the alterations in interictal neurovascular coupling. The blood volume increase associated with interictal spikes represents a promising mapping signal in both the awake and anesthetized states.

Single-protein Diffusion in the Periplasm of Escherichia coli.

The width of the periplasmic space of Gram-negative bacteria is only about 25-30 nm along the long axis of the cell, which affects free diffusion of (macro)molecules. We have performed single-particle displacement measurements and diffusion simulation studies to determine the impact of confinement on the apparent mobility of proteins in the periplasm of Escherichia coli. The diffusion of a reporter protein and of OsmY, an osmotically regulated periplasmic protein, is characterized by a fast and slow component regardless of the osmotic conditions. The diffusion coefficient of the fast fraction increases upon osmotic upshift, in agreement with a decrease in macromolecular crowding of the periplasm, but the mobility of the slow (immobile) fraction is not affected by the osmotic stress. We observe that the confinement created by the inner and outer membranes results in a lower apparent diffusion coefficient, but this can only partially explain the slow component of diffusion in the particle displacement measurements, suggesting that a fraction of the proteins is hindered in its mobility by large periplasmic structures. Using particle-based simulations, we have determined the confinement effect on the apparent diffusion coefficient of the particles for geometries akin the periplasmic space of Gram-negative bacteria.

Scalable noninvasive amplicon-based precision sequencing (SNAPseq) for genetic diagnosis and screening of ß-thalassemia and sickle cell disease using a next-generation sequencing platform.

ß-hemoglobinopathies such as ß-thalassemia (BT) and Sickle cell disease (SCD) are inherited monogenic blood disorders with significant global burden. Hence, early and affordable diagnosis can alleviate morbidity and reduce mortality given the lack of effective cure. Currently, Sanger sequencing is considered to be the gold standard genetic test for BT and SCD, but it has a very low throughput requiring multiple amplicons and more sequencing reactions to cover the entire HBB gene. To address this, we have demonstrated an extraction-free single amplicon-based approach for screening the entire ß-globin gene with clinical samples using Scalable noninvasive amplicon-based precision sequencing (SNAPseq) assay catalyzing with next-generation sequencing (NGS). We optimized the assay using noninvasive buccal swab samples and simple finger prick blood for direct amplification with crude lysates. SNAPseq demonstrates high sensitivity and specificity, having a 100% agreement with Sanger sequencing. Furthermore, to facilitate seamless reporting, we have created a much simpler automated pipeline with comprehensive resources for pathogenic mutations in BT and SCD through data integration after systematic classification of variants according to ACMG and AMP guidelines. To the best of our knowledge, this is the first report of the NGS-based high throughput SNAPseq approach for the detection of both BT and SCD in a single assay with high sensitivity in an automated pipeline.

Insights Into Clinical Outcomes of Acute Pancreatitis With Concomitant Acute Myocardial Infarction Using the National Inpatient Sample Database.

Acute pancreatitis (AP) and acute coronary syndrome (ACS) are common conditions, occasionally sharing overlapping symptoms, posing various clinical challenges. This study aims to investigate the demographics, outcomes, and risk factors of patients admitted with AP and ACS using the National Inpatient Sample database. The database from 2016 to 2019 was analyzed, identifying patients with a primary diagnosis of AP and dividing them into 2 groups: those with ACS and those without (non-ACS). Of the 112,874 patients with AP, 5,210 (0.46%) had ACS. The patients with AP with concomitant ACS were older, predominantly male, and had a higher prevalence of co-morbidities. Inpatient mortality was significantly higher in the AP with concomitant ACS cohort compared with the AP without ACS cohort (8.4% vs 0.5%, adjusted odds ratio 9.94, 95% confidence interval 7.79 to 12.67, p <0.05). In conclusion, patients with AP and ACS experienced worse clinical outcomes.

Super-resolving microscopy reveals the localizations and movement dynamics of stressosome proteins in Listeria monocytogenes.

The human pathogen Listeria monocytogenes can cope with severe environmental challenges, for which the high molecular weight stressosome complex acts as the sensing hub in a complicated signal transduction pathway. Here, we show the dynamics and functional roles of the stressosome protein RsbR1 and its paralogue, the blue-light receptor RsbL, using photo-activated localization microscopy combined with single-particle tracking and single-molecule displacement mapping and supported by physiological studies. In live cells, RsbR1 is present in multiple states: in protomers with RsbS, large clusters of stressosome complexes, and in connection with the plasma membrane via Prli42. RsbL diffuses freely in the cytoplasm but forms clusters upon exposure to light. The clustering of RsbL is independent of the presence of Prli42. Our work provides a comprehensive view of the spatial organization and intracellular dynamics of the stressosome proteins in L. monocytogenes, which paves the way towards uncovering the stress-sensing mechanism of this signal transduction pathway.

Association between inpatient palliative care encounter and 30-day all-cause readmissions after index hospitalization for chronic obstructive pulmonary disease.

BACKGROUND: Studies exist on the association between inpatient Palliative Care Encounter (iPCE) and 30-day rehospitalization among cancer and several non-cancer conditions but limited in persons with Chronic Obstructive Pulmonary Disease (COPD). OBJECTIVE: To assess the association between an iPCE with the risk of 30-day rehospitalization after an index hospitalization for COPD. METHODS: We conducted a cross-sectional analysis of the Nationwide Readmissions Database (2010-2014). Index hospitalizations were defined as persons ≥ 18 years of age, discharge destinations of either Home/Routine, Home with Home Care, or a Facility, and an index hospitalization with Diagnosis Related Group of COPD. The International Classification of Diseases, 9th revision codes were used to extract comorbidities and a Palliative Care Encounter (V66.7). RESULTS: There were 3,163,889 index hospitalizations and iPCE occurred in 21,330 (0.67%). There were 558,059 (17.63%) with a 30-day rehospitalization. An iPCE was associated with a significantly lower adjusted odds of 30-day readmission (Odds Ratio [OR], 0.50; 95% Confidence Interval [CI], 0.46 to 0.54).  By discharge destination, the odds of 30-day rehospitalization were for a discharged to a facility (OR, 0.37; 95% CI, 0.32 to 0.42), to home with home health (OR, 0.42; 95% CI, 0.37 to 0.47), and to home (OR, 0.98; 95% CI, 0.85 to 1.12) for those with relative to without iPCE. CONCLUSION: Inpatient PCE was associated with a 50% lower relative odds of 30-day rehospitalization after an index hospitalization for COPD. This association varied by discharge destination being statistically significant among those with a discharge destination of a facility (63%) and home with home care (58%).

How important is the N-terminal acetylation of alpha-synuclein for its function and aggregation into amyloids?

N-α-acetylation is a frequently occurring post-translational modification in eukaryotic proteins. It has manifold physiological consequences on the regulation and function of several proteins, with emerging studies suggesting that it is a global regulator of stress responses. For decades, in vitro biochemical investigations into the precise role of the intrinsically disordered protein alpha-synuclein (αS) in the etiology of Parkinson's disease (PD) were performed using non-acetylated αS. The N-terminus of α-synuclein is now unequivocally known to be acetylated in vivo, however, there are many aspects of this post-translational modifications that are not understood well. Is N-α-acetylation of αS a constitutive modification akin to most cellular proteins, or is it spatio-temporally regulated? Is N-α-acetylation of αS relevant to the as yet elusive function of αS? How does the N-α-acetylation of αS influence the aggregation of αS into amyloids? Here, we provide an overview of the current knowledge and discuss prevailing hypotheses on the impact of N-α-acetylation of αS on its conformational, oligomeric, and fibrillar states. The extent to which N-α-acetylation of αS is vital for its function, membrane binding, and aggregation into amyloids is also explored here. We further discuss the overall significance of N-α-acetylation of αS for its functional and pathogenic implications in Lewy body formation and synucleinopathies.

Deployment of iron uptake machineries as targets against drug resistant strains of mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTB) requires a perpetual supply of iron for its sustenance. Iron scarcity and its limited availability in the host environment because of an encounter of various sites during the establishment of infection has led to the evolution of strategies for iron uptake, which includes biosynthesis of iron-chelating molecules called siderophores, Heme uptake pathways, recently discovered host iron transport protein receptors like glyceraldehyde-3-phosphate dehydrogenase and the development of machinery for proper storage of the acquired iron and its regulation. The components of the iron uptake machineries are viable targets in multidrug-resistant tuberculosis, some of which include the MmpL3 heme transfer protein, MbtA enzyme, and the ESX-3 system, while employment of approaches like the synthesis of siderophore drug conjugates, heme analogs, xenosiderophores as drug delivery agents, and the blockade of siderophore recycling are encouraged too. Thus, the mentioned discoveries stand as promising targets against various strains of MTB.

Prognostic Model of COVID-19 Severity and Survival among Hospitalized Patients Using Machine Learning Techniques.

We conducted a statistical study and developed a machine learning model to triage COVID-19 patients affected during the height of the COVID-19 pandemic in Hong Kong based on their medical records and test results (features) collected during their hospitalization. The correlation between the values of these features is studied against discharge status and disease severity as a preliminary step to identify those features with a more pronounced effect on the patient outcome. Once identified, they constitute the inputs of four machine learning models, Decision Tree, Random Forest, Gradient and RUSBoosting, which predict both the Mortality and Severity associated with the disease. We test the accuracy of the models when the number of input features is varied, demonstrating their stability; i.e., the models are already highly predictive when run over a core set of (6) features. We show that Random Forest and Gradient Boosting classifiers are highly accurate in predicting patients' Mortality (average accuracy ∼99%) as well as categorize patients (average accuracy ∼91%) into four distinct risk classes (Severity of COVID-19 infection). Our methodical and broad approach combines statistical insights with various machine learning models, which paves the way forward in the AI-assisted triage and prognosis of COVID-19 cases, which is potentially generalizable to other seasonal flus.

Acetazolamide-Challenged Arterial Spin Labeling Detects Augmented Cerebrovascular Reserve After Surgery for Moyamoya.

BACKGROUND: Cerebrovascular reserve (CVR) inversely correlates with stroke risk in children with Moyamoya disease and may be improved by revascularization surgery. We hypothesized that acetazolamide-challenged arterial spin labeling MR perfusion quantifies augmentation of CVR achieved by revascularization and correlates with currently accepted angiographic scoring criteria. METHODS: We retrospectively identified pediatric patients with Moyamoya disease or syndrome who received cerebral revascularization at ≤18 years of age between 2012 and 2019 at our institution. Using acetazolamide-challenged arterial spin labeling, we compared postoperative CVR to corresponding preoperative values and to postoperative perfusion outcomes classified by Matsushima grading. RESULTS: In this cohort, 32 patients (17 males) with Moyamoya underwent 29 direct and 16 indirect extracranial-intracranial bypasses at a median 9.7 years of age (interquartile range, 7.6-15.7). Following revascularization, median CVR increased within the ipsilateral middle cerebral artery territory (6.9 mL/100 g per minute preoperatively versus 16.5 mL/100 g per minute postoperatively, P<0.01). No differences were observed in the ipsilateral anterior cerebral artery (P=0.13) and posterior cerebral artery (P=0.48) territories. Postoperative CVR was higher in the ipsilateral middle cerebral artery territories of patients who achieved Matsushima grade A perfusion, in comparison to those with grades B or C (25.8 versus 17.5 mL, P=0.02). The method of bypass (direct or indirect) did not alter relative increases in CVR (8 versus 3.8 mL/100 g per minute, P=0.7). CONCLUSIONS: Acetazolamide-challenged arterial spin labeling noninvasively quantifies augmentation of CVR following surgery for Moyamoya disease and syndrome.

Amphibian behavioral diversity offers insights into evolutionary neurobiology.

Recent studies have served to emphasize the unique placement of amphibians, composed of more than 8000 species, in the evolution of the brain. We provide an overview of the three amphibian orders and their respective ecologies, behaviors, and brain anatomy. Studies have probed the origins of independently evolved parental care strategies in frogs and the biophysical principles driving species-specific differences in courtship vocalization patterns. Amphibians are also important models for studying the central control of movement, especially in the context of the vertebrate origin of limb-based locomotion. By highlighting the versatility of amphibians, we hope to see a further adoption of anurans, urodeles, and gymnophionans as model systems for the evolution and neural basis of behavior across vertebrates.

Usage of Direct Acting Oral Anticoagulants in Cirrhotic and Non-Cirrhotic Portal Vein Thrombosis: A Systematic Review.

Thrombosis of the portal vein (PVT) is generally seen in the setting of liver cirrhosis and to a lesser extent in the absence of cirrhosis. There is no clear guidance in relation to approaching treatment with anticoagulation in this condition. The professional societies and guidelines recommend treatment with traditional anticoagulation like low-molecular-weight heparin and vitamin-K antagonists in patients presenting with acute portal vein thrombosis. There is no clarity in relation to treatment in the setting of chronic PVT and in patients with cirrhosis. Also, the role of direct-acting oral anticoagulants (DOACs) that are becoming a preferred choice for anticoagulation for various other indications is not clear in the case of PVT. There are a very few studies in the medical literature that have investigated the role of DOACs in patients with PVT in different settings. Thus, we performed a systematic review of the literature to study the use of DOACs in PVT in patients with and without cirrhosis. The results of the available studies show that DOACS appears to be a promising choice for the treatment of patients with PVT. The availability of more data in the future along with better availability of the approved reversal agents for various DOACs is expected to make DOACS a preferred choice for the clinicians to treat patients with PVT.

Chemogenetic Tags with Probe Exchange for Live-Cell Fluorescence Microscopy.

Fluorogenic protein tagging systems have been less developed for prokaryotes than for eukaryotic cell systems. Here, we extend the concept of noncovalent fluorogenic protein tags in bacteria by introducing transcription factor-based tags, namely, LmrR and RamR, for probe binding and fluorescence readout under aerobic and anaerobic conditions. We developed two chemogenetic protein tags that impart fluorogenicity and a longer fluorescence lifetime to reversibly bound organic fluorophores, hence the name Chemogenetic Tags with Probe Exchange (CTPEs). We present an extensive characterization of 30 fluorophores reversibly interacting with the two different CTPEs and conclude that aromatic planar structures bind with high specificity to the hydrophobic pockets of these tags. The reversible binding of organic fluorophores to the CTPEs and the superior photophysical properties of organic fluorophores enable long-term fluorescence microscopy of living bacterial cells. Our protein tags provide a general tool for investigating (sub)cellular protein localization and dynamics, protein-protein interactions, and prolonged live-cell microscopy, even under oxygen-free conditions.

Measurement of Protein Mobility in Listeria monocytogenes Reveals a Unique Tolerance to Osmotic Stress and Temperature Dependence of Diffusion.

Protein mobility in the cytoplasm is essential for cellular functions, and slow diffusion may limit the rates of biochemical reactions in the living cell. Here, we determined the apparent lateral diffusion coefficient (D L ) of GFP in Listeria monocytogenes as a function of osmotic stress, temperature, and media composition. We find that D L is much less affected by hyperosmotic stress in L. monocytogenes than under similar conditions in Lactococcus lactis and Escherichia coli. We find a temperature optimum for protein diffusion in L. monocytogenes at 30°C, which deviates from predicted trends from the generalized Stokes-Einstein equation under dilute conditions and suggests that the structure of the cytoplasm and macromolecular crowding vary as a function of temperature. The turgor pressure of L. monocytogenes is comparable to other Gram-positive bacteria like Bacillus subtilis and L. lactis but higher in a knockout strain lacking the stress-inducible sigma factor SigB. We discuss these findings in the context of how L. monocytogenes survives during environmental transmission and interaction with the human host.

Case Report: Multiple Strokes and Digital Ischemia in a Young COVID-19 Patient.

COVID-19 is an infectious disease caused by SARS-CoV-2. This enveloped RNA coronavirus primarily has tropism for the respiratory tract. However, it has also been shown to have various extrapulmonary manifestations such as pulmonary embolism, ischemic strokes, deep venous thrombosis, or arterial thrombosis. We present a case of a 34-year-old woman who had severe COVID-19 infection with no respiratory symptoms and developed strokes in multiple vascular territories and digital ischemia due to thrombosis formation in the brachial circulation of her arm despite receiving therapeutic anticoagulation.

Linker histone variant H1t is closely associated with repressed repeat-element chromatin domains in pachytene spermatocytes.

BACKGROUND: H1t is the major linker histone variant in pachytene spermatocytes, where it constitutes 50-60% of total H1. This linker histone variant was previously reported to localize in the nucleolar rDNA element in mouse spermatocytes. Our main aim was to determine the extra-nucleolar localization of this linker histone variant in pachytene spermatocytes. RESULTS: We generated H1t-specific antibodies in rabbits and validated its specificity by multiple assays like ELISA, western blot, etc. Genome-wide occupancy studies, as determined by ChIP-sequencing in P20 mouse testicular cells revealed that H1t did not closely associate with active gene promoters and open chromatin regions. Annotation of H1t-bound genomic regions revealed that H1t is depleted from DSB hotspots and TSS, but are predominantly associated with retrotransposable repeat elements like LINE and LTR in pachytene spermatocytes. These chromatin domains are repressed based on co-association of H1t observed with methylated CpGs and repressive histone marks like H3K9me3 and H4K20me3 in vivo. Mass spectrometric analysis of proteins associated with H1t-containing oligonucleosomes identified piRNA-PIWI pathway proteins, repeat repression-associated proteins and heterochromatin proteins confirming the association with repressed repeat-element genomic regions. We validated the interaction of key proteins with H1t-containing oligonucleosomes by use of ChIP-western blot assays. On the other hand, we observe majority of H1t peaks to be associated with the intergenic spacer of the rDNA element, also in association with SINE elements of the rDNA element. Thus, we have identified the genomic and chromatin features of both nucleolar and extranucleolar localization patterns of linker histone H1t in the context of pachytene spermatocytes. CONCLUSIONS: H1t-containing repeat-element LINE and LTR chromatin domains are associated with repressive marks like methylated CpGs, histone modifications H3K9me3 and H4K20me3, and heterochromatin proteins like HP1ß, Trim28, PIWIL1, etc. Apart from localization of H1t at the rDNA element, we demonstrate the extranucleolar association of this linker histone variant at repeat-associated chromatin domains in pachytene spermatocytes. We hypothesize that H1t might induce local chromatin relaxation to recruit heterochromatin and repeat repression-associated protein factors necessary for TE (transposable element) repression, the final biological effect being formation of closed chromatin repressed structures.

Functional Outcomes After Revascularization Procedures in Patients With Hemorrhagic Moyamoya Disease.

BACKGROUND: Poor natural history of hemorrhagic Moyamoya disease (MMD) is related to high rehemorrhage rates between 32% and 61%. Postrevascularization, rehemorrhage rates reportedly decrease to 12% to 17%. OBJECTIVE: To evaluate long-term functional outcomes and rehemorrhage rates of hemorrhagic MMD patients treated with surgical revascularization and examine these in relation to clinical and radiological factors. METHODS: Patients treated surgically for hemorrhagic MMD over a 26-yr period were identified. Modified Rankin scale (mRS) was used to assess clinical status at presentation and functional outcomes. Multivariable regression analyses were performed to evaluate the risk factors associated with rehemorrhage rates and functional outcomes. RESULTS: A total of 104 patients (mean age: 38.04 yr) were identified. The mean mRS score at baseline was 1.3. Of 172 revascularized hemispheres, 157 (91.3%) were direct superficial temporal artery (STA)-middle cerebral artery (MCA) bypasses and the rest indirect. Over the mean follow-up of 61.4 mo, 8 of 104 patients (7.7%) experienced rehemorrhage with rehemorrhage rate per person-years of 1.9%. A total of 4 patients died with 1 related to rehemorrhage. At the last follow-up, mean mRS score improved to 1.1. No significant risk factors were identified in relation to the rehemorrhage rates (P < .05). The patients' initial mRS score was positively associated with mRS scores at the final follow-up (P < .001). STA-MCA direct bypass was associated with better performance status (P = .033). CONCLUSION: Rehemorrhage rate following surgical revascularization of the hemorrhagic MMD patients at 7.7% is lower compared with much higher natural history rates. Surgical revascularization improved patients' performance status. These outcomes support performing revascularization procedure with a preference for direct STA-MCA bypasses.