Extracellular vesicle formation in Euryarchaeota is driven by a small GTPase.

Since their discovery, extracellular vesicles (EVs) have changed our view on how organisms interact with their extracellular world. EVs are able to traffic a diverse array of molecules across different species and even domains, facilitating numerous functions. In this study, we investigate EV production in Euryarchaeota, using the model organism Haloferax volcanii. We uncover that EVs enclose RNA, with specific transcripts preferentially enriched, including those with regulatory potential, and conclude that EVs can act as an RNA communication system between haloarchaea. We demonstrate the key role of an EV-associated small GTPase for EV formation in H. volcanii that is also present across other diverse evolutionary branches of Archaea. We propose the name, ArvA, for the identified family of archaeal vesiculating GTPases. Additionally, we show that two genes in the same operon with arvA (arvB and arvC) are also involved in EV formation. Both, arvB and arvC, are closely associated with arvA in the majority of other archaea encoding ArvA. Our work demonstrates that small GTPases involved in membrane deformation and vesiculation, ubiquitous in Eukaryotes, are also present in Archaea and are widely distributed across diverse archaeal phyla.

σ28-dependent small RNA regulation of flagella biosynthesis.

Flagella are important for bacterial motility as well as for pathogenesis. Synthesis of these structures is energy intensive and, while extensive transcriptional regulation has been described, little is known about the posttranscriptional regulation. Small RNAs (sRNAs) are widespread posttranscriptional regulators, most base pairing with mRNAs to affect their stability and/or translation. Here, we describe four UTR-derived sRNAs (UhpU, MotR, FliX and FlgO) whose expression is controlled by the flagella sigma factor σ28 (fliA) in Escherichia coli. Interestingly, the four sRNAs have varied effects on flagellin protein levels, flagella number and cell motility. UhpU, corresponding to the 3´ UTR of a metabolic gene, likely has hundreds of targets including a transcriptional regulator at the top flagella regulatory cascade connecting metabolism and flagella synthesis. Unlike most sRNAs, MotR and FliX base pair within the coding sequences of target mRNAs and act on ribosomal protein mRNAs connecting ribosome production and flagella synthesis. The study shows how sRNA-mediated regulation can overlay a complex network enabling nuanced control of flagella synthesis.

Evolutionary Landscape of SOX Genes to Inform Genotype-to-Phenotype Relationships.

The SOX transcription factor family is pivotal in controlling aspects of development. To identify genotype-phenotype relationships of SOX proteins, we performed a non-biased study of SOX using 1890 open-reading frame and 6667 amino acid sequences in combination with structural dynamics to interpret 3999 gnomAD, 485 ClinVar, 1174 Geno2MP, and 4313 COSMIC human variants. We identified, within the HMG (High Mobility Group)- box, twenty-seven amino acids with changes in multiple SOX proteins annotated to clinical pathologies. These sites were screened through Geno2MP medical phenotypes, revealing novel SOX15 R104G associated with musculature abnormality and SOX8 R159G with intellectual disability. Within gnomAD, SOX18 E137K (rs201931544), found within the HMG box of ~0.8% of Latinx individuals, is associated with seizures and neurological complications, potentially through blood-brain barrier alterations. A total of 56 highly conserved variants were found at sites outside the HMG-box, including several within the SOX2 HMG-box-flanking region with neurological associations, several in the SOX9 dimerization region associated with Campomelic Dysplasia, SOX14 K88R (rs199932938) flanking the HMG box associated with cardiovascular complications within European populations, and SOX7 A379V (rs143587868) within an SOXF conserved far C-terminal domain heterozygous in 0.716% of African individuals with associated eye phenotypes. This SOX data compilation builds a robust genotype-to-phenotype association for a gene family through more robust ortholog data integration.

Isolation, Purification, and Characterization of Membrane Vesicles from Haloarchaea.

Membrane vesicles (MVs), also described as extracellular vesicles (EVs), exosomes, or outer membrane vesicles (OMVs), are nano-sized (10-300 nm) spherical, membrane-bound structures deriving from the cell envelope. MVs have been studied extensively in both eukaryotic and prokaryotic systems, revealing a plethora of unique functions including cell-to-cell communication and protection of the cell. They are able to encapsulate specific cargos from nucleic acids to proteins, thereby concentrating cargo and providing protection from the extracellular environment. While MV production has been identified for all domains of life, with extensive investigation particularly for Bacteria and Eukaryota, it has only been studied in a few members of the archaeal domain, leaving a void of information concerning the role of MVs for the majority of Archaea. In addition, several discrepancies exist in the process of MV preparation and analysis between studies of MV production in different archaeal organisms. To further encourage the investigation of MVs in Archaea among the scientific community, we present a standardized method for the isolation, purification, and characterization of MVs based on the archaeal model organism, Haloferax volcanii. However, the described protocol can be applied to other Archaea with the appropriate modifications that are highlighted in Subheading 4.

CCR5 and Biological Complexity: The Need for Data Integration and Educational Materials to Address Genetic/Biological Reductionism at the Interface of Ethical, Legal, and Social Implications.

In the age of genomics, public understanding of complex scientific knowledge is critical. To combat reductionistic views, it is necessary to generate and organize educational material and data that keep pace with advances in genomics. The view that CCR5 is solely the receptor for HIV gave rise to demand to remove the gene in patients to create host HIV resistance, underestimating the broader roles and complex genetic inheritance of CCR5. A program aimed at providing research projects to undergraduates, known as CODE, has been expanded to build educational material for genes such as CCR5 in a rapid approach, exposing students and trainees to large bioinformatics databases and previous experiments for broader data to challenge commitment to biological reductionism. Our students organize expression databases, query environmental responses, assess genetic factors, generate protein models/dynamics, and profile evolutionary insights into a protein such as CCR5. The knowledgebase generated in the initiative opens the door for public educational information and tools (molecular videos, 3D printed models, and handouts), classroom materials, and strategy for future genetic ideas that can be distributed in formal, semiformal, and informal educational environments. This work highlights that many factors are missing from the reductionist view of CCR5, including the role of missense variants or expression of CCR5 with neurological phenotypes and the role of CCR5 and the delta32 variant in complex critical care patients with sepsis. When connected to genomic stories in the news, these tools offer critically needed Ethical, Legal, and Social Implication (ELSI) education to combat biological reductionism.

High-Density Blood Transcriptomics Reveals Precision Immune Signatures of SARS-CoV-2 Infection in Hospitalized Individuals.

The immune response to COVID-19 infection is variable. How COVID-19 influences clinical outcomes in hospitalized patients needs to be understood through readily obtainable biological materials, such as blood. We hypothesized that a high-density analysis of host (and pathogen) blood RNA in hospitalized patients with SARS-CoV-2 would provide mechanistic insights into the heterogeneity of response amongst COVID-19 patients when combined with advanced multidimensional bioinformatics for RNA. We enrolled 36 hospitalized COVID-19 patients (11 died) and 15 controls, collecting 74 blood PAXgene RNA tubes at multiple timepoints, one early and in 23 patients after treatment with various therapies. Total RNAseq was performed at high-density, with >160 million paired-end, 150 base pair reads per sample, representing the most sequenced bases per sample for any publicly deposited blood PAXgene tube study. There are 770 genes significantly altered in the blood of COVID-19 patients associated with antiviral defense, mitotic cell cycle, type I interferon signaling, and severe viral infections. Immune genes activated include those associated with neutrophil mechanisms, secretory granules, and neutrophil extracellular traps (NETs), along with decreased gene expression in lymphocytes and clonal expansion of the acquired immune response. Therapies such as convalescent serum and dexamethasone reduced many of the blood expression signatures of COVID-19. Severely ill or deceased patients are marked by various secondary infections, unique gene patterns, dysregulated innate response, and peripheral organ damage not otherwise found in the cohort. High-density transcriptomic data offers shared gene expression signatures, providing unique insights into the immune system and individualized signatures of patients that could be used to understand the patient's clinical condition. Whole blood transcriptomics provides patient-level insights for immune activation, immune repertoire, and secondary infections that can further guide precision treatment.

Virus-induced genetics revealed by multidimensional precision medicine transcriptional workflow applicable to COVID-19.

Precision medicine requires the translation of basic biological understanding to medical insights, mainly applied to characterization of each unique patient. In many clinical settings, this requires tools that can be broadly used to identify pathology and risks. Patients often present to the intensive care unit with broad phenotypes, including multiple organ dysfunction syndrome (MODS) resulting from infection, trauma, or other disease processes. Etiology and outcomes are unique to individuals, making it difficult to cohort patients with MODS, but presenting a prime target for testing/developing tools for precision medicine. Using multitime point whole blood (cellular/acellular) total transcriptomics in 27 patients, we highlight the promise of simultaneously mapping viral/bacterial load, cell composition, tissue damage biomarkers, balance between syndromic biology versus environmental response, and unique biological insights in each patient using a single platform measurement. Integration of a transcriptome workflow yielded unexpected insights into the complex interplay between host genetics and viral/bacterial specific mechanisms, highlighted by a unique case of virally induced genetics (VIG) within one of these 27 patients. The power of RNA-Seq to study unique patient biology while investigating environmental contributions can be a critical tool moving forward for translational sciences applied to precision medicine.

HIV-Resistant and HIV-Specific CAR-Modified CD4+ T Cells Mitigate HIV Disease Progression and Confer CD4+ T Cell Help In Vivo.

HIV infection preferentially depletes HIV-specific CD4+ T cells, thereby impairing antiviral immunity. In this study, we explored the therapeutic utility of adoptively transferred CD4+ T cells expressing an HIV-specific chimeric antigen receptor (CAR4) to restore CD4+ T cell function to the global HIV-specific immune response. We demonstrated that CAR4 T cells directly suppressed in vitro HIV replication and eliminated virus-infected cells. Notably, CAR4 T cells containing intracellular domains (ICDs) derived from the CD28 receptor family (ICOS and CD28) exhibited superior effector functions compared to the tumor necrosis factor receptor (TNFR) family ICDs (CD27, OX40, and 4-1BB). However, despite demonstrating limited in vitro efficacy, only HIV-resistant CAR4 T cells expressing the 4-1BBζ ICD exhibited profound expansion, concomitant with reduced rebound viremia after antiretroviral therapy (ART) cessation and protection of CD4+ T cells (CAR-) from HIV-induced depletion in humanized mice. Moreover, CAR4 T cells enhanced the in vivo persistence and efficacy of HIV-specific CAR-modified CD8+ T cells expressing the CD28ζ ICD, which alone exhibited poor survival. Collectively, these studies demonstrate that HIV-resistant CAR4 T cells can directly control HIV replication and augment the virus-specific CD8+ T cell response, highlighting the therapeutic potential of engineered CD4+ T cells to engender a functional HIV cure.

A randomised controlled trial of 'clockwise' ultrasound for low back pain.

AIMS: To examine how the choice of words explaining ultrasound (US) may influence the outcome of physiotherapy treatment for low back pain (LBP). METHODS: Sixty-seven patients with LBP < 3 months were randomly allocated to one of three groups - traditional education about US (control group [CG]), inflated education about US (experimental group [EG]) or extra-inflated education about US (extra-experimental group [EEG]). Each patient received the exact same application of US that has shown clinical efficacy for LBP (1.5 Watts/cm2 for 10 minutes at 1 Megahertz, pulsed 20% over a 20 cm2 area), but received different explanations (CG, EG or EEG). Before and immediately after US, measurements of LBP and leg pain (numeric rating scale), lumbar flexion (distance to floor) and straight leg raise (SLR) (inclinometer) were taken. Statistical analysis consisted of mixed-factorial analyses of variance and chi-square analyses to measure differences between the three groups, as well as meeting or exceeding minimal detectable changes (MDCs) for pain, lumbar flexion and SLR. RESULTS: Both EG and EEG groups showed a statistically significant improvement for SLR (p < 0.0001), while the CG did not. The EEG group participants were 4.4 times (95% confidence interval: 1.1 to 17.5) more likely to improve beyond the MDC than the CG. No significant differences were found between the groups for LBP, leg pain or lumbar flexion. CONCLUSION: The choice of words when applying a treatment in physiotherapy can alter the efficacy of the treatment.

Immediate effects of lumbar spine manipulation on the resting and contraction thickness of transversus abdominis in asymptomatic individuals.

STUDY DESIGN: Randomized, blinded, controlled crossover trial. OBJECTIVE: To determine if thrust joint manipulation (TJM) to the lumbar spine would result in changes to the resting and contraction thickness of transversus abdominis (TrA) in healthy individuals. BACKGROUND: Recent studies have demonstrated an immediate decrease in resting thickness and an increase in contraction thickness in TrA following lumbar TJM in patients with low back pain (LBP) who met a clinical prediction rule (CPR) for spinal manipulation. This observed phenomenon has not been investigated in healthy individuals. METHODS: Thirty-five healthy participants were randomly assigned to receive a TJM or sham manipulation treatment. All participants received instruction on how to produce an isolated concentric contraction of the TrA that involved visual ultrasound imaging biofeedback. Data were analyzed using ultrasound imaging to measure changes in thickness of the TrA at rest and during contraction, following the administration of each treatment. RESULTS: There were no interactions observed between treatment and time for TrA muscle thickness at rest (P = .351) and during the contracted state (P = .761). CONCLUSION: Our results indicate that TJM to the lumbar spine does not appear to affect the resting or contraction thickness of TrA in healthy individuals. These findings are in contrast to previous research in which patients with LBP who met a CPR demonstrated an immediate decrease in resting thickness and an increase in contraction thickness in TrA following lumbar TJM.