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.

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.

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<0.001; ω = 0.31) and SP (χ2(3,N = 423) = 33.1; p<0.001; ω = 0.28). In total, only a small number of late maturing athletes were present (0.5-2.3%). Among relatively younger athletes, high percentages of early maturing athletes were found (43.1-43.3%). The findings indicate that relatively younger and less mature athletes are marginalized or totally excluded in alpine ski racing and soccer. Thus, selection criteria in both sports are effectively based on early biological development and relatively older age, both of which should be considered in future in the talent selection process. In this context, the easy feasible method of assessing the APHV can be used.

Evaluation and power of seroprevalence studies depend on the performed serological assays. The aim of this study was to assess four commercial serological tests from EUROIMMUN, DiaSorin, Abbott, and Roche as well as an in-house immunofluorescence and neutralization test for their capability to identify SARS-CoV-2 seropositive individuals in a high-prevalence setting. Therefore, 42 social and working contacts of a German super-spreader were tested. Consistent with a high-prevalence setting, 26 of 42 were SARS-CoV-2 seropositive by neutralization test (NT), and immunofluorescence test (IFT) confirmed 23 of these 26 positive test results (NT 61.9% and IFT 54.8% seroprevalence). Four commercial assays detected anti-SARS-CoV-2 antibodies in 33.3-40.5% individuals. Besides an overall discrepancy between the NT and the commercial assays regarding their sensitivity, this study revealed that commercial SARS-CoV-2 spike-based assays are better to predict the neutralization titer than nucleoprotein-based assays are.

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.

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.

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.

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 < dp < 100 μm size range. The MPFs ranged between 100 μm and 1000 μm in length. While most of the MPFs were of PET origin, the MPPs consisted mainly of PET, PP, PE and PS.

Transcription of the HIV-1 provirus generates a viral pre-mRNA, which is alternatively spliced into more than 50 HIV-1 mRNAs encoding all viral proteins. Regulation of viral alternative splice site usage includes the presence of splicing regulatory elements (SREs) which can dramatically impact RNA expression and HIV-1 replication when mutated. Recently, we were able to show that two viral SREs, GI3-2 and ESEtat, are important players in the generation of viral vif, vpr and tat mRNAs. Furthermore, we demonstrated that masking these SREs by transfected locked nucleic acid (LNA) mixmers affect the viral splicing pattern and viral particle production. With regard to the development of future therapeutic LNA mixmer-based antiretroviral approaches, we delivered the GI3-2 and the ESEtat LNA mixmers "nakedly", without the use of transfection reagents (gymnosis) into HIV-1 infected cells. Surprisingly, we observed that gymnotically-delivered LNA mixmers accumulated in the cytoplasm, and seemed to co-localize with GW bodies and induced degradation of mRNAs containing their LNA target sequence. The GI3-2 and the ESEtat LNA-mediated RNA degradation resulted in abrogation of viral replication in HIV-1 infected Jurkat and PM1 cells as well as in PBMCs.

The establishment of axon-dendrite polarity is fundamental for radial migration of neurons during cortex development of mammals. We demonstrate that the E3 ubiquitin ligases WW-Containing Proteins 1 and 2 (Wwp1 and Wwp2) are indispensable for proper polarization of developing neurons. We show that knockout of Wwp1 and Wwp2 results in defects in axon-dendrite polarity in pyramidal neurons, and their aberrant laminar cortical distribution. Knockout of miR-140, encoded in Wwp2 intron, engenders phenotypic changes analogous to those upon Wwp1 and Wwp2 deletion. Intriguingly, transcription of the Wwp1 and Wwp2/miR-140 loci in neurons is induced by the transcription factor Sox9. Finally, we provide evidence that miR-140 supervises the establishment of axon-dendrite polarity through repression of Fyn kinase mRNA. Our data delineate a novel regulatory pathway that involves Sox9-[Wwp1/Wwp2/miR-140]-Fyn required for axon specification, acquisition of pyramidal morphology, and proper laminar distribution of cortical neurons.

Modification of vaccination strategies is necessary to improve the immune response to SARS-CoV-2 vaccination in kidney transplant recipients (KTRs). This multicenter observational study analyzed the effects of the third SARS-CoV-2 vaccination in previously seronegative KTRs with the focus on temporary mycophenolate mofetil (MMF) dose reduction within propensity matched KTRs. 56 out of 174 (32%) previously seronegative KTRs became seropositive after the third vaccination with only three KTRs developing neutralizing antibodies against the omicron variant. Multivariate logistic regression revealed that initial antibody levels, graft function, time after transplantation and MMF trough levels had an influence on seroconversion (P < .05). After controlling for confounders, the effect of MMF dose reduction before the third vaccination was calculated using propensity score matching. KTRs with a dose reduction of ≥33% showed a significant decrease in MMF trough levels to 1.8 (1.2-2.5) μg/ml and were more likely to seroconvert than matched controls (P = .02). Therefore, a MMF dose reduction of 33% or more before vaccination is a promising approach to improve success of SARS-CoV-2 vaccination in KTRs.

Gene products linked to microcephaly have been studied foremost for their role in brain development, while their function in the development of other organs has been largely neglected. Here, we report the critical role of Cdk5rap2 in maintaining the germ cell pool during embryonic development. We highlight that infertility in Cdk5rap2 mutant mice is secondary to a lack of spermatogenic cells in adult mice as a result of an early developmental defect in the germ cells through mitotic delay, prolonged cell cycle, and apoptosis.

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.

We used enzyme-linked immunoassay methods to measure the prevalence and the levels of antibody responses to the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and four seasonal human coronaviruses (HCoV-OC43, HCoV-HKU1, HCoV 229E, and HCoV-NL63) in a cohort of 115 convalescent plasma donors infected with SARS-CoV-2 (1-61 days after symptom onset) compared to antibody levels in 114 individuals with no evidence of a recent infection with SARS-CoV-2. In the humoral response to the four seasonal coronaviruses, only HCoV-HKU1- and HCoV-229E-assays showed slightly elevated antibody levels in the COVID group compared to the control group. While in the COVID-group the levels of SARS-CoV-2 antibodies correlated significantly with disease severity, no association was found in the levels of antibodies against the seasonal coronaviruses. The most striking result in both groups was that the levels of antibodies against all tested coronaviruses, including the new SARS-CoV-2 showed a highly significant correlation with each other. There seems to be an individual predisposition to a weaker or stronger humoral immune response against all known seasonal human coronaviruses including the new SARS-CoV-2, which could lead to a definition of low and high responders against human coronaviruses with potential impact on the assessment of postinfection antibody levels and protection.

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.

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.

The underlying molecular mechanism and their general effect on the replication capacity of HIV 1 drug-resistance-associated mutations is often poorly understood. To elucidate the effect of two such mutations located in a region with a high density of spicing regulatory elements on the HIV-1-splicing outcome, bioinformatic predictions were combined with transfection and infection experiments. Results show that the previously described R263K drug-resistance-associated integrase mutation has additionally a severe effect on the ESE2b splicing regulatory element (SRE) in exon 2b, which causes loss of SD2b recognition. This was confirmed by an R263R silent mutation with a similar predicted effect on the exon 2b SRE. In contrast, a V260I mutation and its silent counterpart with a lower effect on ESS2b did not exhibit any differences in the splicing pattern. Since HIV-1 highly relies on a balanced splicing reaction, changes in the splicing outcome can contribute to changes in viral replication and might add to the effect of escape mutations toward antiviral drugs. Thus, a classification of mutations purely addressing proteins is insufficient.

A critical step in exon definition is the recognition of a proper splice donor (5΄ss) by the 5' end of U1 snRNA. In the selection of appropriate 5΄ss, cis-acting splicing regulatory elements (SREs) are indispensable. As a model for 5΄ss recognition, we investigated cryptic 5΄ss selection within the human fibrinogen Bβ-chain gene (FGB) exon 7, where we identified several exonic SREs that simultaneously acted on up- and downstream cryptic 5΄ss. In the FGB exon 7 model system, 5΄ss selection iteratively proceeded along an alternating sequence of U1 snRNA binding sites and interleaved SREs which in principle supported different 3' exon ends. Like in a relay race, SREs either suppressed a potential 5΄ss and passed the splicing baton on or splicing actually occurred. From RNA-Seq data, we systematically selected 19 genes containing exons with silent U1 snRNA binding sites competing with nearby highly used 5΄ss. Extensive SRE analysis by different algorithms found authentic 5΄ss significantly more supported by SREs than silent U1 snRNA binding sites, indicating that our concept may permit generalization to a model for 5΄ss selection and 3' exon end definition.

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.

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.