Simulating the Motion Underlying the Mechanism of Thioredoxin Reductase.

Thioredoxin reductase (TrxR) is an essential antioxidant in most cells; it reduces thioredoxin (Trx) and several more substrates, utilizing NADPH. However, the enzyme's internal active site is too small to accommodate the Trx substrate. Thus, TrxR evolved a disulfide shuttle that can carry reducing equivalents from the active site to the docking site of thioredoxin on the enzyme surface. Yet, in all available atomic structures of TrxR, access to the active site by the shuttle is sterically blocked. We find with computational dynamics that thermal motion at 37 °C allows the oxidized shuttle x to transiently access the active site. Once the shuttle is reduced, it becomes polar. Again, with molecular dynamics, we show that the polar shuttle will move outward toward the solution interface, whereas the oxidized, neutral shuttle will not. This work provides physical evidence for crucial steps in the enzyme mechanism that thus far were just conjectures. The total shuttle motion, from the active site toward the surface, is over 20 Å. TrxR may thus also be termed a molecular machine.

Factors Influencing COVID-19 Vaccine Confidence and Uptake in Australian Adults.

In January 2021, Australia initiated a national COVID-19 vaccine rollout strategy but faced setbacks, leading to negative press and media controversy, which may have diminished vaccine confidence. This study aimed to assess the factors influencing vaccine confidence in Australian adults (≥18 years of age) following the administration of a COVID-19 vaccine. Conducted at Blacktown Hospital, Sydney, a cross-sectional survey with 1053 respondents gauged vaccine confidence and influencing factors. The results showed overall high confidence (mean score 33/40). Trusted sources included the Australian Department of Health (77.8%), NSW Health (76.7%), and general practitioners (53.7%), while social media was distrusted (5.9%). The motivations for vaccination varied: university-educated individuals prioritised personal health (X2 = 17.81; p < 0.001), while religious and/or older respondents (≥50 years of age) emphasised community (X2 = 11.69; p < 0.001) and family protection (X2 = 17.314; p < 0.001). Multivariate logistic regression revealed use of the Australian Department of Health website as a trusted source of COVID-19 information as the strongest predictor of high confidence (>30; OR 1.43; p = 0.041), while exposure to fake news decreased confidence (OR 0.71; p = 0.025). The study underscores the importance of reliable health information sources in bolstering vaccine confidence and highlights the detrimental effects of misinformation. Promoting awareness of trustworthy health channels is crucial to combat vaccine hesitancy in Australia.

Global herpes zoster burden in adults with asthma: a systematic review and meta-analysis.

BACKGROUND: Asthma is a common respiratory disease, which may be associated with an increased risk of herpes zoster (HZ), often a debilitating disease associated with severe pain. This was the first systematic review with the objective of summarizing evidence on HZ burden in adults with asthma. METHODS: A global systematic literature review (SLR) and meta-analysis was conducted (Medline and Embase, 2003-2024), on HZ burden (incidence, risk, complications) in adults (≥18 years) with asthma. RESULTS: There were 19 studies included on HZ outcomes in adults with asthma. Pooled HZ incidence per 1000 person-years was 5.71 (95% confidence interval [CI] 4.68-6.96) in ≥18-year-olds (4.20 [3.09-5.70] in <60-year-olds versus 10.33 [9.17-11.64] in ≥60-year-olds). The pooled rate ratio for developing HZ was 1.23 [1.11-1.35] in ≥18-year-olds, and 1.36 [1.15-1.61] in ≥50-year-olds. The risk of HZ was higher in people with asthma using systemic corticosteroids; long-acting beta-agonists plus inhaled corticosteroids; and "add-on therapy". Asthma was also associated with an increased risk of post-herpetic neuralgia (odds ratio, OR 1.21 [1.06-1.37]) and HZ ophthalmicus (OR 1.9 [1.1-3.2]).Differences in study design, setting, case definitions, and follow-up durations led to heterogeneity. CONCLUSIONS: This SLR and meta-analysis found that adults with asthma have an increased risk of HZ, with higher risks in older age groups, and in those on certain treatments, such as oral corticosteroids. HZ vaccines are available for adults, including those with comorbidities such as asthma, and can be considered as part of integrated respiratory care.

Public health impact of herpes zoster vaccination on older adults in Singapore: a modeling study.

In Singapore, population aging and rising life expectancy are increasing herpes zoster (HZ) burden, which may be reduced by vaccination. The present study modeled the public health impact of HZ vaccination in Singapore using ZOster ecoNomic Analysis (ZONA) model adapted with Singapore-specific key model inputs, where available. Base case analysis was conducted in adults ≥ 50 years of age (YOA), exploring three vaccination strategies (no vaccination, recombinant zoster vaccine [RZV], zoster vaccine live [ZVL]) under mass vaccination setting (30% coverage). Scenario and sensitivity analyses were performed. Out of 1.51 million adults in 2021 (base case population), 406,513 (27.0%) cases of HZ, 68,264 (4.5%) cases of post-herpetic neuralgia (PHN), and 54,949 (3.6%) cases of other complications were projected without vaccination. RZV was estimated to avoid 73,129 cases of HZ, 11,094 cases of PHN, and 9,205 cases of other complications over the subjects' remaining lifetime; ZVL would avoid 17,565 cases of HZ, 2,781 cases of PHN, and 1,834 cases of other complications. The number needed to vaccinate to prevent one case of HZ/PHN was lower for RZV (7/41) than ZVL (26/163). Among all five age-stratified cohorts (50-59/60-64/65-69/70-79/≥80 YOA), RZV (versus no vaccination/ZVL) avoided the largest number of cases in the youngest cohort, 50-59 YOA. Results were robust under scenario and sensitivity analyses. Mass vaccination with RZV is expected to greatly reduce the public health burden of HZ among Singapore individuals ≥ 50 YOA. Findings support value assessment and decision-making regarding public health vaccination strategies for HZ prevention in Singapore.

Risk of shingles (herpes zoster) increases with age, especially from 50 years. Shingles is a major public health concern in Singapore, given its rapidly aging population. Vaccination can prevent shingles and reduce its public health burden. Two shingles vaccines are available in Singapore: recombinant zoster vaccine (RZV) since 2021, zoster vaccine live (ZVL) since 2008. To understand the value of preventing shingles via vaccination, this study assessed the public health impact of shingles vaccination. Three vaccination strategies (no vaccination, vaccination with RZV, vaccination with ZVL) were compared in 1.51 million Singapore adults aged 50 years and above. Without vaccination, public health burden of shingles would be high; an estimated 406,513 (27.0%) would have shingles, 68,264 (4.5%) would have shingles-related long-term nerve pain, 54,949 (3.6%) would have other shingles-related complications, and 17,762 (1.2%) would be hospitalized due to shingles. Shingles vaccination could reduce this public health burden: RZV avoided 73,129 cases of shingles, 11,094 cases of shingles-related long-term nerve pain, 9,205 cases of other shingles-related complications, and 2,827 hospitalizations due to shingles, which was 4­6 times that avoided with ZVL (shingles: 17,565; shingles-related long-term nerve pain: 2,781; other shingles-related complications: 1,834; hospitalizations due to shingles: 484). Shingles vaccination for adults aged 50 years and above, especially early vaccination from 50­59 years, could reduce its public health burden more than vaccination at later ages and contribute toward healthy aging, preventive care, and the Healthier SG initiative. Results support local public health value assessments and decision-making for shingles prevention.

Knowledge, attitude, and practice toward herpes zoster (HZ) and HZ vaccination: Concept elicitation findings from a multi-country study in the Asia Pacific.

Herpes zoster (HZ) is a prevalent disease characterized by a painful rash. A multi­country study was conducted to elicit public and physician knowledge, attitude, and practice (KAP) toward HZ disease and vaccination for the assessment of local factors influencing HZ vaccine perceptions in four Asian-Pacific countries/territories One-to-one qualitative interviews were conducted in 2022, among the public (people aged ≥ 50 years, adults with parents aged ≥ 50 years, zoster vaccine live-vaccinated individuals aged ≥ 50 years in Republic of Korea, and HZ patients; n = 78) and physicians (general practitioners and specialists; n = 24). Themes surrounding KAP toward HZ and HZ vaccination were summarized using a thematic analysis. A substantial knowledge gap related to HZ was observed among the public, including its causes, long-term impacts, and the at-risk population. There was a low perceived risk of HZ and low general awareness of HZ vaccine availability, although country/territory-specific differences existed. Fear of HZ-associated pain contributed toward vaccination intent among HZ patients and adults with parents aged ≥ 50 years. HZ-naïve adults who were encouraged to receive the vaccine by others were not motivated to do so due to optimism bias. Physicians were perceived to be a reliable source of information. However, physicians did not always proactively discuss HZ vaccination due to time constraints and a perceived need to prioritize other vaccinations including influenza and pneumococcal vaccines. Initiatives are needed to improve public awareness of HZ and its complications, in terms of overall impact on individuals and society, and highlight the important role of physicians in recommending vaccination.

Identification of the novel HLA-DQA1*05:101 allele by next-generation sequencing in a family.

HLA-DQA1*05:101 differs from HLA-DQA1*05:01:01:02 by one nucleotide substitution at codon 221 (CGT>TGT) in exon 4.

GPR68-ATF4 signaling is a novel prosurvival pathway in glioblastoma activated by acidic extracellular microenvironment.

BACKGROUND: Glioblastoma multiforme (GBM) stands as a formidable challenge in oncology because of its aggressive nature and severely limited treatment options. Despite decades of research, the survival rates for GBM remain effectively stagnant. A defining hallmark of GBM is a highly acidic tumor microenvironment, which is thought to activate pro-tumorigenic pathways. This acidification is the result of altered tumor metabolism favoring aerobic glycolysis, a phenomenon known as the Warburg effect. Low extracellular pH confers radioresistant tumors to glial cells. Notably GPR68, an acid sensing GPCR, is upregulated in radioresistant GBM. Usage of Lorazepam, which has off target agonism of GPR68, is linked to worse clinical outcomes for a variety of cancers. However, the role of tumor microenvironment acidification in GPR68 activation has not been assessed in cancer. Here we interrogate the role of GPR68 specifically in GBM cells using a novel highly specific small molecule inhibitor of GPR68 named Ogremorphin (OGM) to induce the iron mediated cell death pathway: ferroptosis. METHOD: OGM was identified in a non-biased zebrafish embryonic development screen and validated with Morpholino and CRISPR based approaches. Next, A GPI-anchored pH reporter, pHluorin2, was stably expressed in U87 glioblastoma cells to probe extracellular acidification. Cell survival assays, via nuclei counting and cell titer glo, were used to demonstrate sensitivity to GPR68 inhibition in twelve immortalized and PDX GBM lines. To determine GPR68 inhibition's mechanism of cell death we use DAVID pathway analysis of RNAseq. Our major indication, ferroptosis, was then confirmed by western blotting and qRT-PCR of reporter genes including TFRC. This finding was further validated by transmission electron microscopy and liperfluo staining to assess lipid peroxidation. Lastly, we use siRNA and CRISPRi to demonstrate the critical role of ATF4 suppression via GPR68 for GBM survival. RESULTS: We used a pHLourin2 probe to demonstrate how glioblastoma cells acidify their microenvironment to activate the commonly over expressed acid sensing GPCR, GPR68. Using our small molecule inhibitor OGM and genetic means, we show that blocking GPR68 signaling results in robust cell death in all thirteen glioblastoma cell lines tested, irrespective of genetic and phenotypic heterogeneity, or resistance to the mainstay GBM chemotherapeutic temozolomide. We use U87 and U138 glioblastoma cell lines to show how selective induction of ferroptosis occurs in an ATF4-dependent manner. Importantly, OGM was not-acutely toxic to zebrafish and its inhibitory effects were found to spare non-malignant neural cells. CONCLUSION: These results indicate GPR68 emerges as a critical sensor for an autocrine pro-tumorigenic signaling cascade triggered by extracellular acidification in glioblastoma cells. In this context, GPR68 suppresses ATF4, inhibition of GPR68 increases expression of ATF4 which leads to ferroptotic cell death. These findings provide a promising therapeutic approach to selectively induce ferroptosis in glioblastoma cells while sparing healthy neural tissue.

Identification of the novel HLA-B*57:168 allele by next generation sequencing.

HLA-B*57:168 differs from HLA-B*57:01:01:01 by one nucleotide substitution at codon 325 (TGC > TCC) in exon 7.

Identification of the novel HLA-C*03:648 allele by next generation sequencing.

HLA-C*03:648 differs from HLA-C*03:04:02 by one nucleotide substitution at Codon -23 (CGG > CTG) in exon 1.

Association of New-Onset Atrial Fibrillation With All-Cause Mortality in COVID-19 Patients.

Background The COVID-19 pandemic has brought about unprecedented global health challenges, with its impact extending beyond respiratory manifestations to encompass cardiovascular complications, including arrhythmias. Dysrhythmias in COVID-19 are multifactorial, ranging from direct myocardial insult due to the cytokine storm to metabolic derangements. Objective In this study, we aim to examine the incidence of new-onset atrial fibrillation and to study its association with all-cause mortality of COVID-19. Methods A cross-sectional study was conducted at Cabell Huntington Hospital, West Virginia, utilizing electronic medical records of COVID-19 patients from 2020 to 2021. Inclusion criteria comprised patients aged >18 years with COVID-19 diagnosis and cardiac arrhythmias during hospitalization. Logistic regression analysis was employed to examine the relationship between demographic and clinical variables and in-hospital mortality. Results Of the 264 eligible patients, those aged >66 years had lower odds of in-hospital mortality (p < 0.001), while gender, ejection fraction, and diabetes mellitus did not significantly predict mortality. Atrial fibrillation (p = 0.011) and heart failure (p = 0.030) were associated with increased odds of mortality, while hypertension showed no significant predictive power (p = 0.791). Conclusion This study highlights the significance of atrial fibrillation and heart failure as predictors of in-hospital mortality in COVID-19 patients. Our findings underscore the importance of recognizing and managing arrhythmias in COVID-19 and call for further research on the mechanisms and long-term effects of these cardiac complications in the context of the pandemic. These insights can guide clinical practice and interventions to optimize patient outcomes.

The USP46 complex deubiquitylates LRP6 to promote Wnt/ß-catenin signaling.

The relative abundance of Wnt receptors plays a crucial role in controlling Wnt signaling in tissue homeostasis and human disease. While the ubiquitin ligases that ubiquitylate Wnt receptors are well-characterized, the deubiquitylase that reverses these reactions remains unclear. Herein, we identify USP46, UAF1, and WDR20 (USP46 complex) as positive regulators of Wnt signaling in cultured human cells. We find that the USP46 complex is similarly required for Wnt signaling in Xenopus and zebrafish embryos. We demonstrate that Wnt signaling promotes the association between the USP46 complex and cell surface Wnt coreceptor, LRP6. Knockdown of USP46 decreases steady-state levels of LRP6 and increases the level of ubiquitylated LRP6. In contrast, overexpression of the USP46 complex blocks ubiquitylation of LRP6 by the ubiquitin ligases RNF43 and ZNFR3. Size exclusion chromatography studies suggest that the size of the USP46 cytoplasmic complex increases upon Wnt stimulation. Finally, we show that USP46 is essential for Wnt-dependent intestinal organoid viability, likely via its role in LRP6 receptor homeostasis. We propose a model in which the USP46 complex increases the steady-state level of cell surface LRP6 and facilitates the assembly of LRP6 into signalosomes via a pruning mechanism that removes sterically hindering ubiquitin chains.

The Role of Adult Vaccines as Part of Antimicrobial Stewardship: A Scoping Review.

BACKGROUND: Antimicrobial resistance (AMR) is a significant global health concern, causing an estimated 700,000 deaths annually. Although immunisation has been shown to significantly reduce AMR, the role of vaccines as part of antimicrobial stewardship (AMS) practices is often overlooked. OBJECTIVE: To identify and examine the available literature on the role of vaccines as part of AMS practices. METHOD: A scoping review was conducted in the following databases: MEDLINE, Embase, Scopus, CINAHL, CCRCT, IPA, and WoS, along with grey literature sources. The review was conducted using the JBI Methodology for Scoping Reviews and reported in line with the PRISMA-SCr checklist. RESULTS: Among the 1711 records identified, 34 met the inclusion criteria; 8 discussed only the concept, while 26 discussed both the concept and the vaccine implementation method in AMS practices. There were eight recommended and/or utilised types of AMS activities identified involving vaccines, under four key themes of vaccine-related AMS strategies: Education, Screening, Vaccination, and Monitoring. Influenza and pneumococcal vaccines had the most evidence for inclusion. CONCLUSION: Overall, the evidence supports the role of vaccines as part of AMS practices and the value of their inclusion in creating improved and comprehensive AMS strategies to further combat the development of AMR.

Scaling eukaryotic cell-free protein synthesis achieved with the versatile and high-yielding tobacco BY-2 cell lysate.

Eukaryotic cell-free protein synthesis (CFPS) can accelerate expression and high-throughput analysis of complex proteins with functionally relevant post-translational modifications (PTMs). However, low yields and difficulties scaling such systems have prevented their widespread adoption in protein research and manufacturing. Here, we provide detailed demonstrations for the capabilities of a CFPS system derived from Nicotiana tabacum BY-2 cell culture (BY-2 lysate; BYL). BYL is able to express diverse, functional proteins at high yields in 48 h, complete with native disulfide bonds and N-glycosylation. An optimized version of the technology is commercialized as ALiCE® and advances in scaling of BYL production methodologies now allow scaling of eukaryotic CFPS reactions. We show linear, lossless scale-up of batch mode protein expression from 100 µL microtiter plates to 10 and 100 mL volumes in Erlenmeyer flasks, culminating in preliminary data from a litre-scale reaction in a rocking-type bioreactor. Together, scaling across a 20,000x range is achieved without impacting product yields. Production of multimeric virus-like particles from the BYL cytosolic fraction were then shown, followed by functional expression of multiple classes of complex, difficult-to-express proteins using the native microsomes of the BYL CFPS. Specifically: a dimeric enzyme; a monoclonal antibody; the SARS-CoV-2 receptor-binding domain; a human growth factor; and a G protein-coupled receptor membrane protein. Functional binding and activity are demonstrated, together with in-depth PTM characterization of purified proteins through disulfide bond and N-glycan analysis. Taken together, BYL is a promising end-to-end R&D to manufacturing platform with the potential to significantly reduce the time-to-market for high value proteins and biologics.

Impaired Reorganization of Centrosome Structure Underlies Human Infantile Dilated Cardiomyopathy.

BACKGROUND: During cardiomyocyte maturation, the centrosome, which functions as a microtubule organizing center in cardiomyocytes, undergoes dramatic structural reorganization where its components reorganize from being localized at the centriole to the nuclear envelope. This developmentally programmed process, referred to as centrosome reduction, has been previously associated with cell cycle exit. However, understanding of how this process influences cardiomyocyte cell biology, and whether its disruption results in human cardiac disease, remains unknown. We studied this phenomenon in an infant with a rare case of infantile dilated cardiomyopathy (iDCM) who presented with left ventricular ejection fraction of 18% and disrupted sarcomere and mitochondria structure. METHODS: We performed an analysis beginning with an infant who presented with a rare case of iDCM. We derived induced pluripotent stem cells from the patient to model iDCM in vitro. We performed whole exome sequencing on the patient and his parents for causal gene analysis. CRISPR/Cas9-mediated gene knockout and correction in vitro were used to confirm whole exome sequencing results. Zebrafish and Drosophila models were used for in vivo validation of the causal gene. Matrigel mattress technology and single-cell RNA sequencing were used to characterize iDCM cardiomyocytes further. RESULTS: Whole exome sequencing and CRISPR/Cas9 gene knockout/correction identified RTTN, the gene encoding the centrosomal protein RTTN (rotatin), as the causal gene underlying the patient's condition, representing the first time a centrosome defect has been implicated in a nonsyndromic dilated cardiomyopathy. Genetic knockdowns in zebrafish and Drosophila confirmed an evolutionarily conserved requirement of RTTN for cardiac structure and function. Single-cell RNA sequencing of iDCM cardiomyocytes showed impaired maturation of iDCM cardiomyocytes, which underlie the observed cardiomyocyte structural and functional deficits. We also observed persistent localization of the centrosome at the centriole, contrasting with expected programmed perinuclear reorganization, which led to subsequent global microtubule network defects. In addition, we identified a small molecule that restored centrosome reorganization and improved the structure and contractility of iDCM cardiomyocytes. CONCLUSIONS: This study is the first to demonstrate a case of human disease caused by a defect in centrosome reduction. We also uncovered a novel role for RTTN in perinatal cardiac development and identified a potential therapeutic strategy for centrosome-related iDCM. Future study aimed at identifying variants in centrosome components may uncover additional contributors to human cardiac disease.

Rapid screening and scaled manufacture of immunogenic virus-like particles in a tobacco BY-2 cell-free protein synthesis system.

Several vaccine platforms have been developed to fight pathogenic threats, with Virus-Like Particles (VLPs) representing a very promising alternative to traditional platforms. VLPs trigger strong and lasting humoral and cellular immune responses with fewer safety concerns and higher stability than other platforms. The use of extensively characterized carrier VLPs modified with heterologous antigens was proposed to circumvent the viral complexity of specific viruses that could lead to poor VLP assembly and yields. Although carrier VLPs have been successfully produced in a wide variety of cell-based systems, these are limited by low protein yields and protracted clone selection and optimization workflows that limit VLP screening approaches. In response, we have demonstrated the cell-free protein synthesis (CFPS) of several variants of the hepatitis B core (HBc) carrier VLP using a high-yielding tobacco BY-2 lysate (BYL). High VLP yields in the BYL system allowed in-depth characterization of HBc variants. Insertion of heterologous sequences at the spike region of the HBc monomer proved more structurally demanding than at the N-terminus but removal of the C-terminal domain allowed higher particle flexibility and insert acceptance, albeit at the expense of thermal and chemical stability. We also proved the possibility to scale the CFPS reaction up to 1L in batch mode to produce 0.45 grams of the native HBc VLP within a 48-hour reaction window. A maximum yield of 820 µg/ml of assembled VLP particles was observed at the 100µl scale and most remarkably the CFPS reaction was successfully scaled from 50µl to 1L without any reduction in protein yield across this 20,000-fold difference in reaction volumes. We subsequently proved the immunogenicity of BYL-derived VLPs, as flow cytometry and microscopy clearly showed prompt recognition and endocytosis of fluorescently labelled VLPs by human dendritic cells. Triggering of inflammatory cytokine production in human peripheral blood mononuclear cells was also quantitated using a multiplex assay. This research establishes BYL as a tool for rapid production and microscale screening of VLP variants with subsequent manufacturing possibilities across scales, thus accelerating discovery and implementation of new vaccine candidates using carrier VLPs.

Ultralow frictional healing explains recurring slow slip events.

Plate motion on shallow subduction megathrusts is accommodated by a spectrum of tectonic slip modes. However, the frictional properties and conditions that sustain these diverse slip behaviors remain enigmatic. Frictional healing is one such property, which describes the degree of fault restrengthening between earthquakes. We show that the frictional healing rate of materials entrained along the megathrust at the northern Hikurangi margin, which hosts well-characterized recurring shallow slow slip events (SSEs), is nearly zero (<0.0001 per decade). These low healing rates provide a mechanism for the low stress drops (<50 kilopascals) and short recurrence times (1 to 2 years) characteristic of shallow SSEs at Hikurangi and other subduction margins. We suggest that near-zero frictional healing rates, associated with weak phyllosilicates that are common in subduction zones, may promote frequent, small-stress-drop, slow ruptures near the trench.

ALiCE ® : A versatile, high yielding and scalable eukaryotic cell-free protein synthesis (CFPS) system.

Eukaryotic cell-free protein synthesis (CFPS) systems have the potential to simplify and speed up the expression and high-throughput analysis of complex proteins with functionally relevant post-translational modifications (PTMs). However, low yields and the inability to scale such systems have so far prevented their widespread adoption in protein research and manufacturing. Here, we present a detailed demonstration for the capabilities of a CFPS system derived from Nicotiana tabacum BY-2 cell culture (BY-2 lysate; BYL). BYL is able to express diverse, functional proteins at high yields in under 48 hours, complete with native disulfide bonds and N-glycosylation. An optimised version of the technology is commercialised as 'ALiCE ® ', engineered for high yields of up to 3 mg/mL. Recent advances in the scaling of BYL production methodologies have allowed scaling of the CFPS reaction. We show simple, linear scale-up of batch mode reporter proten expression from a 100 µL microtiter plate format to 10 mL and 100 mL volumes in standard Erlenmeyer flasks, culminating in preliminary data from 1 L reactions in a CELL-tainer® CT20 rocking motion bioreactor. As such, these works represent the first published example of a eukaryotic CFPS reaction scaled past the 10 mL level by several orders of magnitude. We show the ability of BYL to produce the simple reporter protein eYFP and large, multimeric virus-like particles directly in the cytosolic fraction. Complex proteins are processed using the native microsomes of BYL and functional expression of multiple classes of complex, difficult-to-express proteins is demonstrated, specifically: a dimeric, glycoprotein enzyme, glucose oxidase; the monoclonal antibody adalimumab; the SARS-Cov-2 receptor-binding domain; human epidermal growth factor; and a G protein-coupled receptor membrane protein, cannabinoid receptor type 2. Functional binding and activity are shown using a combination of surface plasmon resonance techniques, a serology-based ELISA method and a G protein activation assay. Finally, in-depth post-translational modification (PTM) characterisation of purified proteins through disulfide bond and N-glycan analysis is also revealed - previously difficult in the eukaryotic CFPS space due to limitations in reaction volumes and yields. Taken together, BYL provides a real opportunity for screening of complex proteins at the microscale with subsequent amplification to manufacturing-ready levels using off-the-shelf protocols. This end-to-end platform suggests the potential to significantly reduce cost and the time-to-market for high value proteins and biologics.

N-Glycans in Immortalized Mesenchymal Stromal Cell-Derived Extracellular Vesicles Are Critical for EV-Cell Interaction and Functional Activation of Endothelial Cells.

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EV) are widely considered as a cell-free therapeutic alternative to MSC cell administration, due to their immunomodulatory and regenerative properties. However, the interaction mechanisms between EV and target cells are not fully understood. The surface glycans could be key players in EV-cell communication, being specific molecular recognition patterns that are still little explored. In this study, we focused on the role of N-glycosylation of MSC-EV as mediators of MSC-EV and endothelial cells' interaction for subsequent EV uptake and the induction of cell migration and angiogenesis. For that, EV from immortalized Wharton's Jelly MSC (iWJ-MSC-EV) were isolated by size exclusion chromatography (SEC) and treated with the glycosidase PNGase-F in order to remove wild-type N-glycans. Then, CFSE-labelled iWJ-MSC-EV were tested in the context of in vitro capture, agarose-spot migration and matrigel-based tube formation assays, using HUVEC. As a result, we found that the N-glycosylation in iWJ-MSC-EV is critical for interaction with HUVEC cells. iWJ-MSC-EV were captured by HUVEC, stimulating their tube-like formation ability and promoting their recruitment. Conversely, the removal of N-glycans through PNGase-F treatment reduced all of these functional activities induced by native iWJ-MSC-EV. Finally, comparative lectin arrays of iWJ-MSC-EV and PNGase-F-treated iWJ-MSC-EV found marked differences in the surface glycosylation pattern, particularly in N-acetylglucosamine, mannose, and fucose-binding lectins. Taken together, our results highlight the importance of N-glycans in MSC-EV to permit EV-cell interactions and associated functions.

Identifying genetic variants associated with the ICD10 (International Classification of Diseases10)-based diagnosis of cerebrovascular disease using a large-scale biomedical database.

OBJECTIVES: To utilize the UK Biobank to identify genetic variants associated with the ICD10 (International Classification of Diseases10)-based diagnosis of cerebrovascular disease (CeVD). BACKGROUND: Cerebrovascular disease occurs because of a complex interplay between vascular, environmental, and genetic factors. It is the second leading cause of disability worldwide. Understanding who may be genetically predisposed to cerebrovascular disease can help guide preventative efforts. Moreover, there is considerable interest in the use of real-world data, such as EHR (electronic health records) to better understand disease mechanisms and to discover new treatment strategies, but whether ICD10-based diagnosis can be used to study CeVD genetics is unknown. METHODS: Using the UK Biobank, we conducted a genome-wide association study (GWAS) where we analyzed the genomes of 11,155 cases and 122,705 controls who were sex, age and ancestry-matched in a 1:11 case: control design. Genetic variants were identified by Plink's firth logistic regression and assessed for association with the ICD10 codes corresponding to CeVD. RESULTS: We identified two groups of SNPs closely linked to PITX2 and LRRTM4 that were significantly associated with CeVD in this study (p < 5 x 10-8) and had a minor allele frequency of > 0.5%. DISCUSSION: Disease assignment based on ICD10 codes may underestimate prevalence; however, for CeVD, this does not appear to be the case. Compared to the age- and sex-matched control population, individuals with CeVD were more frequently diagnosed with comorbid conditions, such as hypertension, hyperlipidemia & atrial fibrillation or flutter, confirming their contribution to CeVD. The UK Biobank based ICD10 study identified 2 groups of variants that were associated with CeVD. The association between PITX2 and CeVD is likely explained by the increased rates of atrial fibrillation and flutter. While the mechanism explaining the relationship between LRRTM4 and CeVD is unclear, this has been documented in previous studies.

African Hydroclimate During the Early Eocene From the DeepMIP Simulations.

The early Eocene (∼56-48 Myr ago) is characterized by high CO2 estimates (1,200-2,500 ppmv) and elevated global temperatures (∼10°C-16°C higher than modern). However, the response of the hydrological cycle during the early Eocene is poorly constrained, especially in regions with sparse data coverage (e.g., Africa). Here, we present a study of African hydroclimate during the early Eocene, as simulated by an ensemble of state-of-the-art climate models in the Deep-time Model Intercomparison Project (DeepMIP). A comparison between the DeepMIP pre-industrial simulations and modern observations suggests that model biases are model- and geographically dependent, however, these biases are reduced in the model ensemble mean. A comparison between the Eocene simulations and the pre-industrial suggests that there is no obvious wetting or drying trend as the CO2 increases. The results suggest that changes to the land sea mask (relative to modern) in the models may be responsible for the simulated increases in precipitation to the north of Eocene Africa. There is an increase in precipitation over equatorial and West Africa and associated drying over northern Africa as CO2 rises. There are also important dynamical changes, with evidence that anticyclonic low-level circulation is replaced by increased south-westerly flow at high CO2 levels. Lastly, a model-data comparison using newly compiled quantitative climate estimates from paleobotanical proxy data suggests a marginally better fit with the reconstructions at lower levels of CO2.