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BackgroundThe emergence of novel SARS-CoV-2 variants challenges immunity, particularly among immunocompromised kidney transplant recipients (KTRs). To address this, vaccines have been adjusted to circulating variants. Despite intensive vaccination efforts, SARS-CoV-2 infections surged among KTRs during the Omicron wave, enabling a direct comparison of variant-specific immunity following-vaccination against Omicron BA.4/5 or Omicron infection in KTRs.Methods98 SARS-CoV-2 naïve KTRs who had received four vaccine doses were studied. Before and after a 5th antigen exposure, either via the bivalent vaccine composed of ancestral SARS-CoV-2 and Omicron BA.4/5 (29 KTRs) or via natural infection with Omicron (38 BA.4/5, 31 BA.1/2), spike-specific T cells were quantified using Elispot and serum pseudovirus neutralizing activity was assessed against the ancestral Wuhan strain, BA.5 and XBB.1.5.ResultsCompared to BA.4/5 vaccination, spike-specific T-cell responses and neutralization activity were higher up to six months post-Omicron infection and reached levels similar to healthy controls. Vaccinated KTRs showed modestly boosted neutralization activity against the Wuhan strain and BA.5, but not XBB.1.5. Baseline immunity correlated with immune responses three months post-vaccination and post-infection, indicating a predictive value for peak immune responses. Tixagevimab/Cilgavimab treatment was associated with robust neutralization of the Wuhan strain, but ineffective against XBB.1.5.ConclusionThe BA.4/5 vaccine improved neutralizing activity against the BA.4/5 variant, but not against the subsequently circulating XBB.1.5 variant in KTRs. Conversely, omicron infection boosted T cells and humoral responses more effectively, showing efficacy against XBB.1.5. These findings suggest that infection-induced immunity associates with greater protection than vaccination against future variants in KTRs.

Summary: The Hippo signaling pathway is an important regulator of organ growth and differentiation, and its deregulation contributes to the development of cancer. The activity of its downstream targets YAP/TAZ depends on adherens junctions. Plakophilin 4 (PKP4) is a cell-type specific adherens junction protein expressed in the proliferating cells of the epidermis. Here, we show that PKP4 diminishes proliferation as well as differentiation. Depletion of PKP4 increased proliferation but at the same time induced premature epidermal differentiation. PKP4 interacted with several Hippo pathway components, including the transcriptional co-activators YAP/TAZ, and promoted nuclear YAP localization and target gene expression. In differentiated keratinocytes, PKP4 recruited LATS and YAP to cell junctions where YAP is transcriptionally inactive. YAP depletion, on the other hand, reduced PKP4 levels and keratinocyte adhesion indicative of a feedback mechanism controlling adhesion, proliferation, and differentiation by balancing YAP functions.

Cross-sectional and longitudinal profiling of full sets of nucleic acids, peptides, or proteins as well as metabolites expressed in biospecimens acquired via a cardiovascular disease-oriented biobank may aid in the elucidation of the disease pathways and mechanisms underlying individual cardiovascular diseases, such as degenerative valvular heart disease. This may promote the development of novel and effective, personalized diagnostic and therapeutic strategies to efficiently reduce cardiovascular mortality and morbidity as well as its health and economic burden. This brief report aims to describe the unique, standardized, interdisciplinary, and interprofessional approach to cross-sectional and longitudinal cardiovascular biobanking and databasing at the University Hospital Augsburg. Moreover, we present the study protocol of a specific, well-defined, prospective, single-center research project involving cross-sectional and longitudinal cardiovascular biobanking. The aim of this project is to gain a better insight into the molecular mechanisms underlying aortic valve disease-induced cardiomyopathy and the long-term effect of surgical correction of the aortic valve pathology on the left ventricular myocardial molecule profile.

We present a novel small molecule antiviral chemotype that was identified by an unconventional cell-free protein synthesis and assembly-based phenotypic screen for modulation of viral capsid assembly. Activity of PAV-431, a representative compound from the series, has been validated against infectious viruses in multiple cell culture models for all six families of viruses causing most respiratory diseases in humans. In animals, this chemotype has been demonstrated efficacious for porcine epidemic diarrhoea virus (a coronavirus) and respiratory syncytial virus (a paramyxovirus). PAV-431 is shown to bind to the protein 14-3-3, a known allosteric modulator. However, it only appears to target the small subset of 14-3-3 which is present in a dynamic multi-protein complex whose components include proteins implicated in viral life cycles and in innate immunity. The composition of this target multi-protein complex appears to be modified upon viral infection and largely restored by PAV-431 treatment. An advanced analog, PAV-104, is shown to be selective for the virally modified target, thereby avoiding host toxicity. Our findings suggest a new paradigm for understanding, and drugging, the host–virus interface, which leads to a new clinical therapeutic strategy for treatment of respiratory viral disease.

Abstract Background As of today, the effect of coronavirus disease 2019 (COVID-19) on male fertility remains unclear. Studies published so far have partly contradictory results, likely due to very small sample sizes and heterogeneous populations. To gain a deeper understanding of the impact of COVID-19 on male fertility, we performed a prospective case–control study, in which we examined the ejaculate of 37 subjects, including 25 subjects in the acute phase of mild COVID-19 and 12 subjects who did not suffer from COVID-19. Determination of semen parameters, severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) qPCR, and infectivity analysis were performed in the acute phase of the disease and in series. Results Semen parameter values did not differ significantly between subjects with mild COVID-19 and the control group. The serial examination of semen parameters revealed no significant changes between 4, 18, and 82 days after the onset of symptoms. SARS-CoV-2 RNA or infectious particles could not be detected in any ejaculate. Conclusion Mild COVID-19 seems to have no detrimental effect on semen parameter values.

Type I interferons (IFN-Is) are pivotal in innate immunity against human immunodeficiency virus I (HIV-1) by eliciting the expression of IFN-stimulated genes (ISGs), which encompass potent host restriction factors. While ISGs restrict the viral replication within the host cell by targeting various stages of the viral life cycle, the lesser-known IFN-repressed genes (IRepGs), including RNA-binding proteins (RBPs), affect the viral replication by altering the expression of the host dependency factors that are essential for efficient HIV-1 gene expression. Both the host restriction and dependency factors determine the viral replication efficiency; however, the understanding of the IRepGs implicated in HIV-1 infection remains greatly limited at present. This review provides a comprehensive overview of the current understanding regarding the impact of the RNA-binding protein families, specifically the two families of splicing-associated proteins SRSF and hnRNP, on HIV-1 gene expression and viral replication. Since the recent findings show specifically that SRSF1 and hnRNP A0 are regulated by IFN-I in various cell lines and primary cells, including intestinal lamina propria mononuclear cells (LPMCs) and peripheral blood mononuclear cells (PBMCs), we particularly discuss their role in the context of the innate immunity affecting HIV-1 replication.

[This corrects the article DOI: 10.1371/journal.pone.0306306.].

Summary: Protein activities and interactions are determined by their subcellular localization. Elucidating the network of protein-protein interactions at a spatial resolution is essential for understanding the complexity of protein functions, their regulation, and cellular processes. Here, we present a protocol to determine the subcellular localization of protein interactions in non-transformed murine keratinocytes. We describe steps for nucleus/cytoplasm fractionation, immunoprecipitation from these fractions, and immunoblotting. We then detail binding quantification.For complete details on the use and execution of this protocol, please refer to Müller et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Abstract Background Pull-off forces of cement-retained zirconia reinforced lithium silicate (ZLS) in implant-supported single crowns on stock titanium abutments with respect to abutment height and implant cement were evaluated and compared. Methods Pull-off force of ZLS crowns on stock titanium abutments was evaluated concerning dental cement and abutment height. A total sample size of 64 stock abutments with heights of 3 mm (n = 32) and 5 mm (n = 32) was used. The ZLS crowns were cemented with four different types of cement (one temporary, two semi-permanent, and one permanent). After cementation, water storage, and thermocycling each sample was subjected to a pull-off test using a universal testing machine. Results The temporary cement showed the least pull-off force regardless of abutment height (3/5 mm: means 6 N/23 N), followed by the semi-permanent methacrylate-infiltrated zinc oxide cement (28 N/55 N), the semi-permanent methacrylate-based cement (103 N/163 N), and the permanent resin composite cement (238 N/820 N). Results of all types of cement differed statistically significantly from each other (p ≤ .012). The type of implant cement has an impact on the pull-off force of ZLS crowns and titanium abutments. Conclusions Permanent cements present higher retention than semi-permanent ones, and temporary cements present the lowest values. The abutment height had a subordinate impact.

Summary: Plakophilin 3 (PKP3) is a component of desmosomes and is frequently overexpressed in cancer. Using keratinocytes either lacking or overexpressing PKP3, we identify a signaling axis from ERK to the retinoblastoma (RB) protein and the E2F1 transcription factor that is controlled by PKP3. RB and E2F1 are key components controlling G1/S transition in the cell cycle. We show that PKP3 stimulates the activity of ERK and its target RSK1. This inhibits expression of the transcription factor RUNX3, a positive regulator of the CDK inhibitor CDKN1A/p21, which is also downregulated by PKP3. Elevated CDKN1A prevents RB phosphorylation and E2F1 target gene expression, leading to delayed S phase entry and reduced proliferation in PKP3-depleted cells. Elevated PKP3 expression not only increases ERK activity but also captures phosphorylated RB (phospho-RB) in the cytoplasm to promote E2F1 activity and cell-cycle progression. These data identify a mechanism by which PKP3 promotes proliferation and acts as an oncogene.

We provide follow-up data on the humoral immune response after COVID-19 vaccinations of two distinct cohorts aged below 60 and over 80 years to screen for age-related differences in the longevity and magnitude of the induction of the antibody responses post booster-vaccinations. While anti-SARS-CoV-2 spike-specific IgG and neutralization capacity waned rapidly after the initial vaccination schedule, additional boosters highly benefitted the humoral immune responses especially in the elderly cohort, including the neutralization of Omikron variants. Thus, adjusted COVID-19 booster vaccination schedules are an appropriate tool to overcome limitations in the success of vaccinations.

Abstract Background Cancer metastases are the main cause of lethality. The five-year survival rate for patients diagnosed with advanced stage oral cancer is 30%. Hence, the identification of novel therapeutic targets is an urgent need. However, tumors are comprised of a heterogeneous collection of cells with distinct genetic and molecular profiles that can differentially promote metastasis making therapy development a challenging task. Here, we leveraged intratumoral heterogeneity in order to identify drivers of cancer cell motility that might be druggable targets for anti-metastasis therapy. Methods We used 2D migration and 3D matrigel-based invasion assays to characterize the invasive heterogeneity among and within four human oral cancer cell lines in vitro. Subsequently, we applied mRNA-sequencing to map the transcriptomes of poorly and strongly invasive subclones as well as primary tumors and matched metastasis. Results We identified SAS cells as a highly invasive oral cancer cell line. Clonal analysis of SAS yielded a panel of 20 subclones with different invasive capacities. Integrative gene expression analysis identified the Lymphocyte cell-specific protein-tyrosine kinase (LCK) as a druggable target gene associated with cancer cell invasion and metastasis. Inhibition of LCK using A-770041 or dasatinib blocked invasion of highly aggressive SAS cells. Interestingly, reduction of LCK activity increased the formation of adherens junctions and induced cell differentiation. Conclusion Analysis of invasive heterogeneity led to the discovery of LCK as an important regulator of motility in oral cancer cells. Hence, small molecule mediated inhibition of LCK could be a promising anti-metastasis therapy option for oral cancer patients.

Abstract Objectives Currently, there are no approved treatments for early disease stages of COVID-19 and few strategies to prevent disease progression after infection with SARS-CoV-2. The objective of this study is to evaluate the safety and efficacy of convalescent plasma (CP) or camostat mesylate administered within 72 h of diagnosis of SARS-CoV-2 infection in adult individuals with pre-existing risk factors at higher risk of getting seriously ill with COVID-19. Camostat mesylate acts as an inhibitor of the host cell serine protease TMPRSS2 and prevents the virus from entering the cell. CP represents another antiviral strategy in terms of passive immunization. The working hypothesis to be tested in the RES-Q-HR study is that the early use of CP or camostat mesylate reduces the likelihood of disease progression to (modified) WHO stages 4b-8 in SARS-CoV-2-positive adult patients at high risk of moderate or severe COVID-19 progression. Trial design This study is a 4-arm (parallel group), multicenter, randomized (2:2:1:1 ratio), partly double-blind, controlled trial to evaluate the safety and efficacy of convalescent plasma (CP) or camostat mesylate with control or placebo in adult patients diagnosed with SARS-CoV-2 infection and high risk for progression to moderate/severe COVID-19. Superiority of the intervention arms will be tested. Participants The trial is conducted at 10–15 tertiary care centers in Germany. Individuals aged 18 years or above with ability to provide written informed consent with SARS-CoV-2 infection, confirmed by PCR within 3 days or less before enrolment and the presence of at least one SARS-CoV-2 symptom (such as fever, cough, shortness of breath, sore throat, headache, fatigue, smell/and or taste disorder, diarrhea, abdominal symptoms, exanthema) and symptom duration of not more than 3 days. Further inclusion criteria comprise: Presence of at least one of the following criteria indicating increased risk for severe COVID-19: Age > 75 years Chronic obstructive pulmonary disease (COPD) and/or pulmonary fibrosis BMI > 40 kg/m2 Age > 65 years with at least one other risk factor (BMI > 35 kg/m2, coronary artery disease (CAD), chronic kidney disease (CKD) with GFR 35 kg/m2 with at least one other risk factor (CAD, CKD with GFR 3 days 10. SARS-CoV-2 PCR detection older than 3 days 11. SARS-CoV-2 associated clinical condition ≥ WHO stage 3 (patients hospitalized for other reasons than COVID-19 may be included if they fulfill all inclusion and none of the exclusion criteria) 12. Previously or currently hospitalized due to SARS-CoV-2 13. Previous antiviral therapy for SARS-CoV-2 14. ALT or AST > 5 x ULN at screening 15. Liver cirrhosis > Child A (patients with Child B/C cirrhosis are excluded from the trial) 16. Chronic kidney disease with GFR

Codon degeneracy of amino acid sequences permits an additional “mRNP code” layer underlying the genetic code that is related to RNA processing. In pre-mRNA splicing, splice site usage is determined by both intrinsic strength and sequence context providing RNA binding sites for splicing regulatory proteins. In this study, we systematically examined modification of splicing regulatory properties in the neighborhood of a GT site, i.e. potential splice site, without altering the encoded amino acids.We quantified the splicing regulatory properties of the neighborhood around a potential splice site by its Splice Site HEXplorer Weight (SSHW) based on the HEXplorer score algorithm. To systematically modify GT site neighborhoods, either minimizing or maximizing their SSHW, we designed the novel stochastic optimization algorithm ModCon that applies a genetic algorithm with stochastic crossover, insertion and random mutation elements supplemented by a heuristic sliding window approach.To assess the achievable range in SSHW in human splice donors without altering the encoded amino acids, we applied ModCon to a set of 1000 randomly selected Ensembl annotated human splice donor sites, achieving substantial and accurate changes in SSHW. Using ModCon optimization, we successfully switched splice donor usage in a splice site competition reporter containing coding sequences from FANCA, FANCB or BRCA2, while retaining their amino acid coding information.The ModCon algorithm and its R package implementation can assist in reporter design by either introducing novel splice sites, silencing accidental, undesired splice sites, and by generally modifying the entire mRNP code while maintaining the genetic code.

Abstract Background The presence of nuclear mitochondrial DNA (numtDNA) has been reported within several nuclear genomes. Next to mitochondrial protein-coding genes, numtDNA sequences also encode for mitochondrial tRNA genes. However, the biological roles of numtDNA remain elusive. Results Employing in silico analysis, we identify 281 mitochondrial tRNA homologs in the human genome, which we term nimtRNAs (nuclear intronic mitochondrial-derived tRNAs), being contained within introns of 76 nuclear host genes. Despite base changes in nimtRNAs when compared to their mtRNA homologs, a canonical tRNA cloverleaf structure is maintained. To address potential functions of intronic nimtRNAs, we insert them into introns of constitutive and alternative splicing reporters and demonstrate that nimtRNAs promote pre-mRNA splicing, dependent on the number and positioning of nimtRNA genes and splice site recognition efficiency. A mutational analysis reveals that the nimtRNA cloverleaf structure is required for the observed splicing increase. Utilizing a CRISPR/Cas9 approach, we show that a partial deletion of a single endogenous nimtRNALys within intron 28 of the PPFIBP1 gene decreases inclusion of the downstream-located exon 29 of the PPFIBP1 mRNA. By employing a pull-down approach followed by mass spectrometry, a 3′-splice site-associated protein network is identified, including KHDRBS1, which we show directly interacts with nimtRNATyr by an electrophoretic mobility shift assay. Conclusions We propose that nimtRNAs, along with associated protein factors, can act as a novel class of intronic splicing regulatory elements in the human genome by participating in the regulation of splicing.

Abstract Background While the number of therapeutic options for treating inflammatory bowel diseases (IBD) is increasing, evidence for rational treatment decisions is scarce in many cases. In particular, appropriate biomarkers to predict the response to the anti-α4β7 integrin antibody vedolizumab are currently lacking. Methods We performed a cohort study with 21 patients suffering from ulcerative colitis (UC), in which first-time treatment with vedolizumab was initiated. CD4+ T cells were isolated from the peripheral blood and dynamic adhesion to recombinant mucosal vascular addressin cell adhesion molecule (MAdCAM-)1 in vitro as well as the effect of vedolizumab on such adhesion in vitro was determined. The expression of α4β1 integrin on peripheral blood CD4+ T cells was quantified by flow cytometry. Electronic patient records were reviewed to determine clinical response to vedolizumab. Results Dynamic adhesion of peripheral blood CD4+ T cells to MAdCAM-1 and the reduction of adhesion following vedolizumab treatment in vitro were higher and the change in α4β1 expression on CD4+ T cells was different in vedolizumab responders and non-responders. Responders could be identified with high specificity and positive-predictive value. Conclusions Determining dynamic adhesion of CD4+ T cells to MAdCAM-1 and the in vitro response to vedolizumab before treatment initiation or dynamic integrin regulation in the early course of treatment seem to be promising tools to predict the clinical response to vedolizumab therapy. Larger prospective studies are warranted.

The majority of COVID-19 patients experience mild to moderate disease course and recover within a few weeks. An increasing number of studies characterized the long-term changes in the specific anti-SARS-CoV-2 immune responses, but how COVID-19 shapes the innate and heterologous adaptive immune system after recovery is less well known. To comprehensively investigate the post-SARS-CoV-2 infection sequelae on the immune system, we performed a multi-omics study by integrating single-cell RNA-sequencing, single-cell ATAC-sequencing, genome-wide DNA methylation profiling, and functional validation experiments in 14 convalescent COVID-19 and 15 healthy individuals. We showed that immune responses generally recover without major sequelae after COVID-19. However, subtle differences persist at the transcriptomic level in monocytes, with downregulation of the interferon pathway, while DNA methylation also displays minor changes in convalescent COVID-19 individuals. However, these differences did not affect the cytokine production capacity of PBMCs upon different bacterial, viral, and fungal stimuli, although baseline release of IL-1Ra and IFN-γ was higher in convalescent individuals. In conclusion, we propose that despite minor differences in epigenetic and transcriptional programs, the immune system of convalescent COVID-19 patients largely recovers to the homeostatic level of healthy individuals.

Left ventricular (LV) reverse remodeling after aortic valve (AV) surgery is less predictable in chronic aortic regurgitation (AR) than in aortic stenosis (AS). We aimed to disclose specific LV myocardial protein signatures possibly contributing to differential disease progression. Global protein profiling of LV myocardial samples excised from the subaortic interventricular septum in patients with isolated AR or AS undergoing AV surgery was performed using liquid chromatography–electrospray ionization–tandem mass spectrometry. Based on label-free quantitation protein intensities, a logistic regression model was calculated and adjusted for age, sex and protein concentration. Web-based functional enrichment analyses of phenotype-associated proteins were performed utilizing g:Profiler and STRING. Data are available via ProteomeXchange with identifier PXD039662. Lysates from 38 patients, including 25 AR and 13 AS samples, were analyzed. AR patients presented with significantly larger LV diameters and volumes (end-diastolic diameter: 61 (12) vs. 48 (13) mm, p < 0.001; end-diastolic volume: 180.0 (74.6) vs. 92.3 (78.4), p = 0.001). A total of 171 proteins were associated with patient phenotype: 117 were positively associated with AR and the enrichment of intracellular compartment proteins (i.e., assigned to carbohydrate and nucleotide metabolism, protein biosynthesis and the proteasome) was detected. Additionally, 54 were positively associated with AS and the enrichment of extracellular compartment proteins (i.e., assigned to the immune and hematopoietic system) was observed. In summary, functional enrichment analysis revealed specific AR- and AS-associated signatures of LV myocardial proteins.

Prophylactic vaccination against SARS-CoV-2 is one of the most important measures to contain the COVID-19 pandemic. Recently, break-through infections following vaccination against this virus have been reported. Here, we describe the humoral immune response of break-through infections in fully vaccinated individuals of old age from an outbreak in a nursing home. In cooperation with the local health authority, blood samples from fully vaccinated and infected as well as fully vaccinated and uninfected residents of the nursing home were collected 4 weeks after the onset of the outbreak. The humoral immune response was determined in a neutralisation assay with replication-competent virus isolates and by a quantitative ELISA. In this outbreak a total of 23 residents and four health care workers were tested positive for SARS-CoV-2. Four residents were unvaccinated, including one with a severe course of disease who later severe disease course who later succumbed to infection. Despite their old age, all vaccinated residents showed no or only mild disease. Comparison of the humoral immune response revealed significantly higher antibody levels in fully vaccinated infected individuals compared to fully vaccinated uninfected individuals (p < 0.001). Notably, although only a minority of the vaccinated uninfected group showed neutralisation capacity against SARS-CoV-2, all vaccinated and infected individuals showed high-titre neutralisation of SARS-CoV-2 including the alpha and beta variant. Large SARS-CoV-2 outbreaks can occur in fully vaccinated populations, but seem to associate with mild disease. SARS-CoV-2 infection in fully vaccinated individuals is a strong booster of the humoral immune response providing enhanced neutralisation capacity against immune evasion variants.

Non-specific protective effects of certain vaccines have been reported, and long-term boosting of innate immunity, termed trained immunity, has been proposed as one of the mechanisms mediating these effects. Several epidemiological studies suggested cross-protection between influenza vaccination and COVID-19. In a large academic Dutch hospital, we found that SARS-CoV-2 infection was less common among employees who had received a previous influenza vaccination: relative risk reductions of 37% and 49% were observed following influenza vaccination during the first and second COVID-19 waves, respectively. The quadrivalent inactivated influenza vaccine induced a trained immunity program that boosted innate immune responses against various viral stimuli and fine-tuned the anti-SARS-CoV-2 response, which may result in better protection against COVID-19. Influenza vaccination led to transcriptional reprogramming of monocytes and reduced systemic inflammation. These epidemiological and immunological data argue for potential benefits of influenza vaccination against COVID-19, and future randomized trials are warranted to test this possibility. Author summary COVID-19, caused by the virus SARS-CoV-2, has claimed millions of lives and affected many more since its emergence. A number of studies have previously suggested that influenza vaccination can provide protection against COVID-19. Although multiple COVID-19 vaccines are currently available, emergence of new variants and inequity in vaccine distribution around the world make it crucial to identify alternative ways that can help in the fight against this pandemic. With this in mind, we investigated if seasonal influenza vaccination had any effect on COVID-19 incidence. Dutch healthcare workers who received the influenza vaccine in the previous flu season had 37% and 49% lower risk of SARS-CoV-2 infection in the first two waves of the pandemic, respectively. We also explored the mechanisms underlying this observation, using various techniques such as single-cell RNA sequencing, proteomics, and stimulation assays. The influenza vaccine reduced systemic inflammation and reprogrammed the immune cells to fine-tune the response against SARS-CoV-2. This study reveals influenza vaccination as a safe and helpful tool to decrease COVID-19 burden.

Desmosomes are intercellular junctions, which preserve tissue integrity during homeostatic and stress conditions. These functions rely on their unique structural properties, which enable them to respond to context-dependent signals and transmit them to change cell behavior. Desmosome composition and size vary depending on tissue specific expression and differentiation state. Their constituent proteins are highly regulated by posttranslational modifications that control their function in the desmosome itself and in addition regulate a multitude of desmosome-independent functions. This review will summarize our current knowledge how signaling pathways that control epithelial shape, polarity and function regulate desmosomes and how desmosomal proteins transduce these signals to modulate cell behavior.

We describe the unique disease course and cure of SARS-CoV-2 infection in a patient with SCID and graft failure. In absence of a humoral immune response, viral clearance was only achieved after transfusion of convalescent plasma. This observation underscores the necessity of the humoral immune response for SARS-CoV-2 clearance.

Objectives Mitral valve (MV) tenting parameters are indicators of left ventricular remodelling severity and may predict outcome in functional mitral regurgitation (FMR). We hypothesised that indexing of MV tenting area to body surface area (BSA), to mitral annulus diameter or gender-adjusted analysis of tenting parameters may improve their prognostic value.Methods We identified retrospectively 240 patients with consecutive FMR (mean age 68±10 years; men=135) from our institutional database who underwent isolated MV annuloplasty during a period of 7 years (2010–2016). Using preoperative two-dimensional transthoracic echocardiographic images, MV tenting parameters including tenting area, tenting height and annulus diameter were systematically assessed. Follow-up protocol consisted of chart review and structured clinical questionnaire. Primary study endpoint was the composite of death and adverse cardiac events (ie, MV reoperation, cardiac resynchronisation therapy implantation, ventricular assist device implantation or heart transplantation).Results BSA-indexed MV tenting area was identified as independent predictor of primary study endpoint (HR 1.9; 95% CI 1.1 to 3.5; p=0.02). After cut-off point analysis, BSA-indexed MV tenting area >1.35 cm2/m2 was significantly associated with primary study outcome (HR 2.3; 95% CI 1.3 to 4.0; p=0.003). Annulus-indexed MV tenting area showed only a tendency towards primary study endpoint prediction (HR 2.8; 95% CI 0.6 to 12.6; p=0.17). Between female and male patients, BSA-indexed MV tenting area was similar (1.42±0.4 cm2/m2 vs 1.45±0.4cm2/cm2; p=0.6) and gender was not associated with primary study outcome (HR 0.8; 95% CI 0.5 to 1.4; p=0.5).Conclusion In our FMR cohort, BSA-indexed MV tenting area showed the strongest association with negative outcomes following isolated MV annuloplasty. Patients with BSA-indexed MV tenting area >1.35cm2/m2 could potentially benefit from additional surgical maneuvers addressing left ventricular remodelling.

Purpose: We describe a diagnostic procedure suitable for scheduling (re-)vaccination against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) according to individual state of humoral immunization. Methods: To clarify the relation between quantitative antibody measurements and humoral ex vivo immune responsiveness, we monitored 124 individuals before, during and six months after vaccination with Spikevax (Moderna, Cambridge, MA, USA). Antibodies against SARS-CoV-2 spike (S1) protein receptor-binding domain (S1-AB) and against nucleocapsid antigens were measured by chemiluminescent immunoassay (Roche). Virus-neutralizing activities were determined by surrogate assays (NeutraLISA, Euroimmune; cPass, GenScript). Neutralization of SARS-CoV-2 in cell culture (full virus NT) served as an ex vivo correlate for humoral immune responsiveness. Results: Vaccination responses varied considerably. Six months after the second vaccination, participants still positive for the full virus NT were safely determined by S1-AB levels ≥1000 U/mL. The full virus NT-positive fraction of participants with S1-AB levels 70% as determined by surrogate assays (NeutraLISA or cPas). Participants that were full virus NT-negative and presumably insufficiently protected could thus be identified by a sensitivity of >83% and a specificity of >95%. Conclusion: The described diagnostic strategy possibly supports individualized (re-)vaccination schedules based on simple and rapid measurement of serum-based SARS-CoV-2 antibody levels. Our data apply only to WUHAN-type SARS-CoV-2 virus and the current version of the mRNA vaccine from Moderna (Cambridge, MA, USA). Adaptation to other vaccines and more recent SARS-CoV-2 strains will require modification of cut-offs and re-evaluation of sensitivity/specificity.

The C30 endopeptidase (3C-like protease; 3CLpro) is essential for the life cycle of SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2) since it plays a pivotal role in viral replication and transcription and, hence, is a promising drug target. Molecules isolated from animals, insects, plants, or microorganisms can serve as a scaffold for the design of novel biopharmaceutical products. Crotamine, a small cationic peptide from the venom of the rattlesnake Crotalus durissus terrificus, has been the focus of many studies since it exhibits activities such as analgesic, in vitro antibacterial, and hemolytic activities. The crotamine derivative L-peptides (L-CDP) that inhibit the 3CL protease in the low µM range were examined since they are susceptible to proteolytic degradation; we explored the utility of their D-enantiomers form. Comparative uptake inhibition analysis showed D-CDP as a promising prototype for a D-peptide-based drug. We also found that the D-peptides can impair SARS-CoV-2 replication in vivo, probably targeting the viral protease 3CLpro.

Concerns of possible transmission of SARS-CoV-2 from donors to patients by corneal transplantation have caused a decline in corneal transplantations. Graft culture media are routinely tested for infectious risks, but it is unclear whether this constitutes a viable means to avoid transmitting SARS-CoV-2 via keratoplasty. We found that SARS-CoV-2 RNA was not present in the medium after seven days of organ culture of corneas from donors (n = 4), who were SARS-CoV-2-positive upon tissue procurement. These medium samples showed no presence of viral RNA. To pursue this question under controlled conditions and further exclude the possibility of productive infection in corneal grafts, we inoculated corneoscleral discs from healthy donors (n = 8) with infectious SARS-CoV-2 and performed PCR testing of the culture medium at various time points. After seven days of culture, we also tested for SARS-CoV-2 RNA within the inoculated corneal tissue. The medium from tissue samples inoculated with SARS-CoV-2 showed no increase in viral RNA, which may indicate lack of viral replication in these corneal grafts. SARS-CoV-2-RNA was, however, detected on or in corneal tissue seven days after inoculation. Our data suggest that corneal grafts may not be permissive for replication of SARS-CoV-2 and demonstrates that PCR testing of culture media cannot safely exclude that tissue has been exposed to SARS-CoV-2. It also demonstrates the difficulty to differentiate between virus adherence and virus replication by PCR testing in SARS-CoV-2 exposed tissue.

In about half the cases of involuntary childlessness, a male infertility factor is involved. The PIWI-LIKE genes, a subclade of the Argonaute protein family, are involved in RNA silencing and transposon control in the germline. Knockout of murine Piwi-like 1 and 2 homologs results in complete infertility in males. The aim of this study was to analyze whether the mRNA expression of human PIWI-LIKE 1–4 genes is altered in ejaculated spermatozoa of men with impaired sperm characteristics. Ninety male participants were included in the study, among which 47 were with normozoospermia, 36 with impaired semen characteristics according to the World Health Organization (WHO) manual, 5th edition, and 7 with azoospermia serving as negative control for the PIWI-LIKE 1–4 mRNA expression in somatic cells in the ejaculate. PIWI-LIKE 1–4 mRNA expression in the ejaculated spermatozoa of the participants was measured by quantitative real-time PCR. In nonazoospermic men, PIWI-LIKE 1–4 mRNA was measurable in ejaculated spermatozoa in different proportions. PIWI-LIKE 1 (100.0%) and PIWI-LIKE 2 (49.4%) were more frequently expressed than PIWI-LIKE 3 (9.6%) and PIWI-LIKE 4 (15.7%). Furthermore, a decreased PIWI-LIKE 2 mRNA expression showed a significant correlation with a decreased sperm count (P = 0.022) and an increased PIWI-LIKE 1 mRNA expression with a decreased progressive motility (P = 0.048). PIWI-LIKE 1 and PIWI-LIKE 2 mRNA expression exhibited a significant association with impaired sperm characteristics and may be a useful candidate for the evaluation of the impact of PIWI-LIKE 1–4 mRNA expression on male infertility.

Samples from the secondary clarifier effluent of a waste water treatment plant (serving 98500 inhabitants) were analyzed to determine the microplastics (MP) emission.The samples were collected using a stainless steel centrifugal pump and filtered through a 10 μm stainless steel cartridge filter. Microplastics particles (MPPs) and microplastics fibers (MPFs) were recovered by chemical and physical sample purification. To remove natural organic matter, the samples were first subjected to oxidative treatment with H2O2 and NaClO. Inorganic materials were subsequently removed by density separation in ZnCl2 (ρ = 1.9 g/cm3) using a centrifuge. Special centrifuge tubes were developed for this purpose. Sample analysis was performed on a Si filter by Raman micro-spectroscopy. Particles with a diameter (dp) ≥ 10 μm were analyzed.The results were differentiated by dry and wet weather samples. On average, 5900 MPPs m−3 were identified in the effluent on wet weather days compared to 3000 MPPs m−3 on dry weather days. Most of the MPPs detected were in the 30 μm

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes COVID-19 and is responsible for the ongoing pandemic. Screening of potential antiviral drugs against SARS-CoV-2 depend on in vitro experiments, which are based on the quantification of the virus titer. Here, we used virus-induced cytopathic effects (CPE) in brightfield microscopy of SARS-CoV-2-infected monolayers to quantify the virus titer. Images were classified using deep transfer learning (DTL) that fine-tune the last layers of a pre-trained Resnet18 (ImageNet). To exclude toxic concentrations of potential drugs, the network was expanded to include a toxic score (TOX) that detected cell death (CPETOXnet). With this analytic tool, the inhibitory effects of chloroquine, hydroxychloroquine, remdesivir, and emetine were validated. Taken together we developed a simple method and provided open access implementation to quantify SARS-CoV-2 titers and drug toxicity in experimental settings, which may be adaptable to assays with other viruses. The quantification of virus titers from brightfield images could accelerate the experimental approach for antiviral testing.

Tourism has become a highly competitive sector, forcing destinations to reinvent and constantly enhance extraordinary experiences in accordance with their miscellaneous destination characteristics, whilst considering mountain biking as a growing phenomenon. This study, therefore, aims at providing a general profile of bike park visitors and at identifying key success factors for a bike park in the context of a destination. A quantitative survey (n = 325) was conducted in a cross-sectional design. The results suggest differing types of visitors with diversified perceptions of a bike park’s key success factors related to the overall perception of sojourn satisfaction. This research extends the literature by (1) shedding light on a booming phenomenon and revealing profiles of bike-park tourists, (2) pointing out key success factors of a bike park and (3) providing information about the perceptions of a bike park related to the overall tourism destination.

The relative age effect is a well-researched phenomenon, however there is still a dearth of understanding in track and field and female sport. This study investigated the role of relative age on selection for international competition of Spanish age group athletes between 2006-2014. Six hundred and forty two athletes competed for Spain at U20 or U18 age group international competition (n = 359 males; 283 females) across 9 years. The birthdates of these athletes were compared against the population of registered athletes at that time (14,502 males; 10,096 females). The results highlighted the influential role of relative age on selection to these opportunities. In line with previous research, this effect was mediated by age and gender, with stronger effects for both males and younger athletes (U18). The data best supported the 'maturation-selection' hypothesis as a mechanism for RAEs. These results highlight the need to carefully consider the role and need for international competitive opportunities at different age groups. A number of possible context relevant solutions are discussed, including correction adjustments techniques and competition structure within track and field.

Alpine ski racing is known to be a sport with a high risk of injuries. Because most studies have focused mainly on top-level athletes and on traumatic injuries, limited research exists about injury risk factors among youth ski racers. The aim of this study was to determine the intrinsic risk factors (anthropometrics, biological maturity, physical fitness, racing technique) for injury among youth alpine ski racers. Study participants were 81 youth ski racers attending a ski boarding school (50 males, 31 females; 9–14 years). A prospective longitudinal cohort design was used to monitor sports-related risk factors over two seasons and traumatic (TI) and overuse injuries (OI). At the beginning of the study, anthropometric characteristics (body height, body weight, sitting height, body mass index); biological maturity [status age at peak height velocity (APHV)]; physical performance parameters related to jump coordination, maximal leg and core strength, explosive and reactive strength, balance and endurance; and ski racing technique were assessed. Z score transformations normalized the age groups. Multivariate binary logistic regression (dependent variable: injury yes/no) and multivariate linear regression analyses (dependent variable: injury severity in total days of absence from training) were calculated. T-tests and multivariate analyses of variance were used to reveal differences between injured and non-injured athletes and between injury severity groups. The level of significance was set to p < 0.05. Relatively low rates of injuries were reported for both traumatic (0.63 TI/athlete) and overuse injuries (0.21 OI/athlete). Athletes with higher body weight, body height, and sitting height; lower APHV values; better core flexion strength; smaller core flexion:extension strength ratio; shorter drop jump contact time; and higher drop jump reactive strength index were at a lower injury risk or more vulnerable for fewer days of absence from training. However, significant differences between injured and non-injured athletes were only observed with respect to the drop jump reactive strength index. Regular documentation of anthropometric characteristics, biological maturity and physical fitness parameters is crucial to help to prevent injury in youth ski racing. The present findings suggest that neuromuscular training should be incorporated into the training regimen of youth ski racers to prevent injuries.

Recent in vitro and in vivo studies on anthocyanins confirmed numerous health-promoting effects in humans. Daily anthocyanin intake can be estimated via food databases, but the amount absorbed by the organism still remains uncertain because anthocyanin bioavailability is yet to be elucidated in its entirety. For this purpose, suitable and validated methods of sample preparation and analysis are required. Therefore, a sample preparation method for anthocyanin metabolite analysis in plasma was successfully established and validated. The validation yielded acceptable results for the anthocyanins in terms of recovery (54−108%) and precision (coefficient of variation (CV) < 15%). The UHPLC-MS method used in the consecutive reaction monitoring (CRM) mode was sufficiently sensitive, resulting in limits of detection

The sensorimotor system (SMS) plays an important role in sports and in every day movement. Several tools for assessment and training have been designed. Many of them are directed to specific populations, and have major shortcomings due to the training effect or safety. The aim of the present study was to design and assess a dynamic sensorimotor test and training device that can be adjusted for all levels of performance. The novel pneumatic-driven mechatronic device can guide the trainee, allow independent movements or disrupt the individual with unpredicted perturbations while standing on a platform. The test-reliability was evaluated using intraclass correlation coefficient (ICC). Subjects were required to balance their center of pressure (COP) in a target circle (TITC). The time in TITC and the COP error (COPe) were recorded for analysis. The results of 22 males and 14 females (23.7 ± 2.6 years) showed good to excellent test–retest reliability. The newly designed Active Balance System (ABS) was then compared with the Biodex Balance System SD® (BBS). The results of 15 females, 14 males (23.4 ± 1.6 years) showed modest correlation in static and acceptable correlation in dynamic conditions, suggesting that ABS could be a reliable and comparable tool for dynamic balance assessments.

Since the relative age effect (RAE) characterizes a problem in all age categories of alpine ski racing and soccer and the fact that, yet, to date the underlying factors have not been well investigated, the aim of the present study was to assess the influence of the biological maturity status on the RAE among youth alpine ski racers (YSR) and soccer players (SP). In total, 183 male and female YSR selected for national final races and 423 male SP selected for Elite Youth Development Centres were investigated. Additionally, a comparison group of 413 non-athletes was evaluated. The birth months were split into four relative age quarters. The biological maturity status was assessed by the age at peak height velocity (APHV) method; according to the M±SD of the comparison group, the athletes were divided into normal, early and late maturing. Chi2-tests indicated a significant RAE among YSR (χ2(3,N = 183) = 18.0; p

The relative age effect (RAE) is a well-documented phenomenon in youth sports. This effect exists when the relative age quarter distribution of selected athletes shows a biased distribution with an over-representation of relatively older athletes. In alpine ski racing, it exists in all age categories (national youth levels up to World Cup). Studies so far could demonstrate that selected ski racers are relatively older, taller and heavier. It could be hypothesized that relatively younger athletes nearly only have a chance for selection if they are early maturing. However, surprisingly this influence of the biological maturity status on the RAE could not be proven, yet. Therefore, the aim of the present study was to investigate the influence of the biological maturity status on the RAE in dependence of the level of competition. The study investigated 372 elite youth ski racers: 234 provincial ski racers (P-SR; high level of competition) and 137 national ski racers (N-SR; very high level of competition). Anthropometric characteristics were measured to calculate the age at peak height velocity (APHV) as an indicator of the biological maturity status. A significant RAE was present among both P-SR and N-SR, with a larger effect size among the latter group. The N-SR significantly differed in APHV from the P-SR. The distribution of normal, early and late maturing athletes significantly differed from the expected normal distribution among the N-SR, not among the P-SR. Hardly any late maturing N-SR were present; 41.7% of the male and 34% of the female N-SR of the last relative age quarter were early maturing. These findings clearly demonstrate the significant influence of the biological maturity status on the selection process of youth alpine ski racing in dependence of the level of competition. Relatively younger athletes seem to have a chance of selection only if they are early maturing.

The relative age effect (RAE), which refers to an over-representation of selected athletes born early in the selection year, was proven to be present in alpine ski racing in all age categories at both national and international levels. However, the influential factors on, or the causal mechanisms of, the RAE are still unknown. Therefore, the aim of the present study was to examine three possible influential factors on the relative age effect in alpine skiing: physical performance, anthropometric characteristics and biological maturational status. The study included the investigation of 282 elite Austrian youth ski racers and 413 non-athletes (comparison group) of the same age (10-13 years) and region. Six physical performance tests were performed, body mass and height were assessed, and the age at peak height velocity (APHV) was calculated. A significant RAE was present in the ski racers. No differences were shown in the physical performance characteristics or in the calculated APHV between the relative age quarters. These results suggest that ski racers born in the last quarter can counteract the relative age disadvantages if they already present the same level of physical performance and maturational status as those born at the beginning of the year. The height and weight of ski racers born at the beginning of the year were significantly higher compared to the non-athletes, and ski racers born in relative age quarter 1 were taller and heavier compared to the ski racers of the other quarters. This indicates that the anthropometric characteristics influence the selection process in alpine ski racing, and that relatively older athletes are more likely to be selected if they exhibit advanced anthropometric characteristics.