The adult mammalian heart exhibits limited regenerative capacity. Although the epicardium contributes to cardiac injury responses and remodeling, expandable adult human in vitro models for investigating epicardial activation and epithelial-to-mesenchymal (EMT)-associated cellular responses remain limited. Here, we isolated human epicardium-derived cells from the adult heart auricle, expressing WT1+/MSLN+/CRIP1+ and generated an expandable immortalized epicardium-derived cell (iEPDC) population, allowing the investigation of intercellular dynamics upon EMT activation. TGFβ signaling was modulated using SB431542 or TGFβ3. Morphological, immunocytochemical, transcriptomic and functional analyses were performed to investigate treatment-dependent responses. SB431542-treated iEPDCs displayed epithelial-like characteristics and elevated WT1, MSLN and CRIP1 expression, with CRIP1 detected at both transcript and protein levels. TGFβ3-treated cells expressed the mesenchymal markers VIM and CD105 and exhibited spindle-shaped morphology, increased migratory behavior and upregulation of mesenchymal- and remodeling-associated markers. Transcriptomic analyses revealed distinct treatment-dependent profiles, enrichment of ‘focal adhesion’, ‘ECM-receptor interaction’ and cytoskeleton-associated pathways in TGFβ3-treated iEPDCs and intermediate transcriptional characteristics in untreated cells. Together, these findings establish adult iEPDCs as an expandable in vitro model for investigating TGFβ-dependent epicardial activation and EMT-associated processes in the adult human heart. Furthermore, the integration of phenotypic, transcriptomic and functional findings revealed the treatment-responsive plasticity of adult human iEPDCs, supporting future studies of injury-associated epicardial activation.

Pseudoxanthoma elasticum (PXE) is an autosomal-recessive disorder caused by mutations in ATP-binding cassette subfamily C member 6 (ABCC6). In addition to the calcification and fragmentation of elastic fibers as the pathomechanistic cause of PXE, systemic and cellular oxidative stress have been reported. Human mesenchymal stem cells (hMSCs) with an ABCC6 knockdown were chosen to further investigate the oxidative stress associated with ABCC6 deficiency. The cells were treated with hydrogen peroxide to mimic external oxidative stress and the antioxidant Trolox to examine the cells' reaction to decreased oxidative stress. The level of different types of reactive species (RS) like nitric oxide and reactive oxygen species, the senescent phenotype, oxidative damage and mRNA expression of oxidative stress-related genes were evaluated. Knockdown of ABCC6 was shown to increase RS levels in hMSCs, induce a p53-dependent senescence-like phenotype and increase oxidative damage, while the mRNA expression of oxidative defense genes was elevated. The ABCC6-deficient cells exhibited an altered reaction to additional oxidative stress and the incubation with Trolox reversed these changes induced by ABCC6 knockdown. Our findings provide further evidence linking ABCC6-deficiency to oxidative stress and a senescence-like phenotype, while pointing towards antioxidants as part of a potential treatment for PXE.

The hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide. RNA viruses like HEV can establish viral populations with high intra-host variability, enabling them to rapidly adapt to changing immune responses, as observed in chronic infections. This study aimed to investigate how intra-host evolution shapes HEV populations during acute asymptomatic infection. Using a highly sensitive HEV amplicon sequencing approach, we characterized intra-host viral diversity and mutational signatures present in serum samples from a cohort of over 80 asymptomatic blood donors infected with acute HEV-3. The overall diversity within the host was constrained; however, several recurrent substitutions were identified, and four mutations in the polymerase region were found to be enriched among the donors. Despite being markedly replication-deficient in vitro, their replication defects could be rescued in an RNA-dependent RNA polymerase trans-complementation assay. Longitudinal sampling in a subset of donors revealed temporal shifts in variant frequencies, indicating ongoing early selection dynamics. In contrast to chronic HEV infection in immunosuppressed patients, acute-phase populations exhibited a significantly lower number of single-nucleotide variants (SNVs) and an absence of high-frequency variants. The presence of premature stop codons and other defective genomes was primarily detected during the acute phase of infection. This finding further supports the hypothesis that the early evolutionary landscape is highly dynamic, but constrained. Although HEV diversity is markedly restricted during acute infection, early evolutionary changes indicate that selective forces act even within the short window of acute, self-limiting, and asymptomatic disease. These findings offer mechanistic insights into early intra-host evolution, highlight conditions under which deleterious variants can transiently persist, and lay the groundwork for linking genotypic features to clinical outcomes and treatment responsiveness.IMPORTANCEThe hepatitis E virus is the leading cause of acute viral hepatitis globally. While studies of chronic infections have shown that HEV can evolve and adapt in ways that influence antiviral treatment responses, little is known about how the virus changes during the early, acute phase of infection. By analyzing viral populations in asymptomatic blood donors, this study demonstrates that HEV undergoes dynamic evolutionary changes even during short, self-limiting infections. Although overall viral diversity remains restricted, selective pressures still drive the emergence of new variants, including some with impaired replication capacity. These findings provide important mechanistic insight into early intra-host viral evolution and the conditions that allow defective variants to transiently persist. This work establishes a foundation for future studies linking viral genetic variation to disease progression, clinical outcomes, and treatment responsiveness.

Hepatitis E virus (HEV) is the leading cause of hepatitis globally and poses particular risks for immunocompromised individuals. Mandatory screening of blood donations for HEV RNA and retrospective individual testing of previous donations (lookback investigations) following a reactive result have been implemented in several countries to protect these patients. This includes Germany, where a sensitivity limit of 2000 IU/mL applies to index donations. In total, 334 HEV RNA-positive blood donations were detected at our blood donation service between 2018 and 2024. Lookback testing was applied in 211 cases, revealing previous HEV RNA-positive donations in 23.1% of donors (n = 48, 76 donations). Although 16 of these retrospectively tested HEV RNA-positive donations have already been transfused, no transfusion-transmitted HEV infection has been reported. The HEV RNA viral load in the lookback donation was below 50 IU/mL in 72.4% of cases. Routine testing effectively prevents highly viremic blood products entering the supply, significantly reducing the infection risk. While the administration of virus particles with low-viremic products cannot be ruled out, the remaining risk appears to be minimal and has been deemed so far acceptable for the safety of blood products. The lookback strategy further supports the screening strategy by retrospectively identifying blood products from low-viremic donations and enabling appropriate risk management.

Xylosyltransferase-II (XT-II), encoded by XYLT2, catalyzes the rate-limiting initial step of proteoglycan biosynthesis. Pathogenic mutations in XYLT2 cause spondylo-ocular syndrome (SOS), a rare disorder characterized by severe primary osteoporosis, skeletal dysplasia, and additional systemic manifestations. Although dysregulated macrophage polarization has been linked to bone remodeling disorders such as osteoporosis, which are associated with an imbalance toward pro-inflammatory and osteoclastogenic signaling, the role of XT-II in macrophage biology remains unexplored. In the present study, siRNA-mediated knockdown of XYLT2 was performed in human primary macrophages across three polarization states (M0, M1, M2) to investigate the impact of XT-II deficiency on macrophage polarization and inflammatory signaling. At the phenotypic level, XYLT2 deficiency destabilized M2 macrophage identity, as shown by significant reductions in M2-associated marker expression, including interleukin 1β, interleukin 6, CD163, and CD206, and impaired phagocytic capacity. A paradoxical reduction of pro-inflammatory markers alongside the induction of M2-associated features was observed in M1 macrophages, indicating a broader disruption of polarization boundary maintenance. The XYLT2 deficiency also promoted pro-inflammatory activation in unpolarized M0 macrophages. Cytokine profiling revealed a predominantly pro-inflammatory secretory shift, with exceptional induction of CXCL10 across all polarization states. At the signaling level, XYLT2 knockdown resulted in a reciprocal shift, characterized by suppression of NF-κB pathway components and nuclear p65 translocation alongside the constitutive activation of STAT1 and STAT3. Transcriptome-wide profiling by bulk mRNA sequencing confirmed a conserved interferon-associated gene expression program across all polarization states, with significant enrichment of innate immune sensing, JAK-STAT, and cytokine signaling pathways among upregulated genes. In summary, these findings demonstrate that XT-II fulfills an important role in maintaining macrophage polarization and that its deficiency induces a reprogramming of pro-inflammatory signaling, extracellular matrix remodeling, and cellular interactions in bone-relevant cell types. This is associated with transcriptional changes linked to osteoclast differentiation and altered bone-associated immune signaling, suggesting that XYLT2 deficiency may contribute to impaired bone homeostasis and providing new insights into the pathomechanisms of SOS.

Functional testing of cytotoxic lymphocytes is essential for research and quality control (QC), but most assays require freshly prepared target cells and extensive handling. A ready-to-thaw, no-wash, flow cytometry-based cytotoxicity assay was developed using pre-labeled K562 targets cryopreserved in STEM-CELLBANKER EX (SCB) as suitably sized aliquots. SCB tolerability was evaluated in K562, NK-92, and primary natural killer (NK) cells; post-cryopreservation label stability of CellTrace Violet (CTV) and carboxyfluorescein succinimidyl ester (CFSE) was assessed; freezing and thawing conditions were optimized; and wash versus no-wash workflows were compared using viability-based and absolute-count readouts, across effector-to-target (E:T) ratios with NK donors and NK-92 cells. Effector viability remained high at SCB concentrations up to 10%, and 5% SCB was selected for assay design. After cryopreservation, CTV labeling remained stable over the tested storage period, whereas CFSE showed substantial signal loss. Warm-medium thawing performed comparably to water-bath thawing, and the consolidated protocol (SCB plus fetal calf serum and thermal buffering) maintained high post-thaw target viability and recovery. In killing assays, lysis increased with increasing E:T ratios; omission of the post-thaw wash had minimal impact, and 5% SCB did not impair cytotoxic function. This ready-to-thaw workflow reduces hands-on time and sample manipulation, while improving standardization for reproducible results and enabling high-throughput functional testing and QC. ### Competing Interest Statement The authors have declared no competing interest. This work was funded by the University of Bielefeld and the Protestant Hospital of Bethel Foundation, University Hospital OWL of Bielefeld University. Neele Kusch and partly Jonathan Storm were financed and employed by the Protestant Hospital of Bethel Foundation.

Osteoporosis is characterized by reduced bone mineral density (BMD) and impaired bone microarchitecture, leading to increased fracture risk. Current bone turnover biomarkers reflect osteoclast and osteoblast activity but do not capture the status of bone extracellular matrix (ECM). Human xylosyltransferase (XT), which initiates glycosaminoglycan biosynthesis, is essential for ECM integrity and was recently implicated in skeletal disorders. To investigate its role in osteoporosis, serum activity of the two XT isoenzymes, XT-I and XT-II, were quantified in 40 postmenopausal women with osteoporosis and 40 age-matched healthy controls using a SPE-UPLC-MS/MS assay. Associations with BMD, fracture risk scores (FRAX) and markers of bone turnover were assessed using Pearson correlation analysis and principal component analysis (PCA). Serum XT-I activity was significantly reduced in postmenopausal osteoporosis patients, whereas XT-II activity remained unchanged. XT-I showed strong positive correlations with all BMD parameters and significant negative correlations with FRAX and bone turnover markers. PCA positioned XT-I activity alongside BMD parameters on the primary axis of disease-related variance, opposing bone resorption markers and fracture risk scores. These findings identify XT-I as a promising biomarker reflecting ECM remodeling in postmenopausal osteoporosis and support further investigation of its role in bone remodeling.

Hepatitis E virus (HEV) has attracted increasing attention in transfusion medicine in recent years. Mandatory testing regimes in Europe have resulted in not only ensuring the safety of blood products, but also providing information on the spread and immunology of HEV infections. We tracked a cohort of 497 donors identified as HEV RNA-positive during blood donation. Several follow-up samples were collected and serologically analyzed for 370 of them, up to five years after the index donation. In addition to the expected increase in immunoglobulins M (IgM) and G (IgG) titers at the beginning and the decrease over the years, we observed a proportion of 7.3% with positive anti-HEV IgM (long-term IgM-positive) and 9.1% with negative anti-HEV IgG (seroreversion) in five-year follow-ups, determined by serological tests from three different manufacturers. Both phenomena have an impact on the assessment of the correlation between incidence and seroprevalence. They are dependent on the sensitivity and specificity of serologic assays used and have a sex bias, which indicates a stronger, longer-lasting humoral immune response in women. These data offer new insights into the long-term development of immunity to HEV and thus complement short-term epidemiological data on the incidence and seroprevalence that have been obtained so far.

A high number of SARS-CoV-2 infections are mild, often even asymptomatic. Because of high specificity and sensitivity, RT-PCR is considered the gold-standard for COVID-19 testing. The technology played a key role in detecting sources of infection at an early stage and therefore preventing larger outbreaks. This was especially important in case of critical infrastructure, such as hospitals. Until now, comprehensive studies concerning the impact of high-frequency PCR-testing in German tertiary care hospitals during the COVID-19 pandemic are lacking. We therefore analyzed about 285.000 oral swab probes of 3.421 healthcare-workers concerning SARS-CoV-2 RNA positivity between November 2020 and February 2023. Our data show that frequent PCR-testing is a useful tool concerning SARS-CoV-2 surveillance. Due to the longitudinal character of the study, we were able to observe SARS-CoV-2 variant-specific differences. For example, the omicron-variant led to high reinfection-rates as well as lower Ct-values. Nevertheless, reinfection rates in our hospital are much lower compared to other analyzed healthcare-worker cohorts described in the literature, which is again attributable to the frequent testing-regime implemented in the early phase of the pandemic. Our data further reveal a longer infection-duration in elderly compared to younger individuals.

Natural killer (NK) cells are promising candidates for adoptive immunotherapy, but their clinical application requires standardized expansion protocols that yield functional cells in sufficient numbers. This study examined how initial seeding density and donor-intrinsic variability affect NK cell proliferation and receptor phenotype during in vitro expansion in a G-Rex® 24-well plate under IL-2 stimulation. NK cells from healthy donors were analyzed longitudinally by flow cytometry, and targeted SNP sequencing of selected receptor genes (IL2RA, IL2RB, FCGR3A, NCR1, KLRK1, and ICAM-1) was performed to assess potential genetic contributions. A seeding density of 2.0 × 106 cells/cm2 promoted high expansion rates and favorable expression of activating receptors including CD16a, NKp46, and NKG2D. Nonetheless, marked inter-donor differences were observed. Some donors exhibited impaired proliferation and aberrant receptor expression, possibly associated with high-priority SNPs and distinct haplotype structures. Others showed robust proliferation despite the absence of identifiable genetic drivers, suggesting the involvement of variants in other genes or non-genetic mechanisms such as epigenetic priming or adaptive NK-cell differentiation. These results highlight the influence of both culture conditions and donor-intrinsic factors on NK-cell expansion outcomes. Integrating phenotypic and genetic analyses may improve the reproducibility and personalization of NK-cell-based manufacturing protocols for therapeutic use.

Xylosyltransferase-I (XT-I) plays a crucial role in skeletal development and cartilage integrity. An XT-I deficiency is linked to severe bone disorders, such as Desbuquois dysplasia type 2. While animal models have provided insights into XT-I’s role during skeletal development, its specific effects on adult bone homeostasis, particularly in human mesenchymal stem cell (hMSC) differentiation, remain unclear. This study investigates how XT-I deficiency impacts the differentiation of hMSCs into chondrocytes, osteoblasts, and adipocytes—key processes in bone formation and repair. The aim of this study was to elucidate for the first time the molecular mechanisms by which XT-I deficiency leads to impaired bone homeostasis. Using CRISPR-Cas9-mediated gene editing, we generated XYLT1 knockdown (KD) hMSCs to assess their differentiation potential. Our findings revealed significant disruption in the chondrogenic differentiation in KD hMSCs, characterized by the altered expression of regulatory factors and extracellular matrix components, suggesting premature chondrocyte hypertrophy. Despite the presence of perilipin-coated lipid droplets in the adipogenic pathway, the overall leptin mRNA and protein expression was reduced in KD hMSCs, indicating a compromised lipid metabolism. Conversely, osteogenic differentiation was largely unaffected, with KD and wild-type hMSCs exhibiting comparable mineralization processes, indicating that critical aspects of osteogenesis were preserved despite the XYLT1 deficiency. In summary, these results underscore XT-I’s pivotal role in regulating differentiation pathways within the bone marrow niche, influencing cellular functions critical for skeletal health. A deeper insight into bone biology may pave the way for the development of innovative therapeutic approaches to improve bone health and treat skeletal disorders.

Patients affected by the rare disease pseudoxanthoma elasticum (PXE) exhibit the calcification of elastic fibers in ocular, dermal, and vascular tissues. These symptoms are triggered by mutations in the ATP-binding cassette transporter subfamily C member 6 (ABCC6), whose substrate remains unknown. Interestingly, ABCC6 is predominantly expressed in the liver tissue, leading to the hypothesis that PXE is a metabolic disorder. We developed a genome-editing system targeting ABCC6 in human immortalized hepatocytes (HepIms) for further investigations. The HepIms were transfected with an ABCC6-specific clustered regulatory interspaced short palindromic repeat (CRISPR-Cas9) genome-editing plasmid, resulting in the identification of a heterozygous (htABCC6HepIm) and a compound heterozygous (chtABCC6HepIm) clone. These clones were analyzed for key markers associated with the PXE pathobiochemistry. Hints of impaired lipid trafficking, defects in the extracellular matrix remodeling, the induction of calcification inhibitor expression, and the down regulation of senescence and inflammatory markers in ABCC6-deficienct HepIms were found. Our ABCC6 knock-out model of HepIms provides a valuable tool for studying the metabolic characteristics of PXE in vitro. The initial analysis of the clones mirrors various features of the PXE pathobiochemistry and provides an outlook on future research approaches.

The ageing phenotype is strongly driven by the exhaustion of adult stem cells (ASCs) and the accumulation of senescent cells. Cardiovascular diseases (CVDs) and heart failure (HF) are strongly linked to the ageing phenotype and are the leading cause of death. As the human heart is considered as an organ with low regenerative capacity, treatments targeting the rejuvenation of human cardiac stem cells (hCSCs) are of great interest. In this study, the beneficial effects of human blood serum on proliferation and senescence of hCSCs have been investigated at the molecular level. We show the induction of a proliferation-related gene expression response by human blood serum at the mRNA level. The concurrent differential expression of the TGFβ target and inhibitor genes indicates the participation of TGFβ signalling in this context. Surprisingly, the application of TGFβ1 as well as the inhibition of TGFβ type I and type II receptor (TGFβRI/II) signalling strongly increased the proliferation of hCSCs. Likewise, both human blood serum and TGFβ1 reduced the senescence in hCSCs. The protective effect of serum on senescence in hCSCs was enhanced by simultaneous TGFβRI/II inhibition. These results strongly indicate a dual role of TGFβ signalling in terms of the serum-mediated effects on hCSCs. Further analysis via RNA sequencing (RNA-Seq) revealed the participation of Ras-inactivating genes wherefore a prevention of hyperproliferation upon serum-treatment in hCSCs via TGFβ signalling and Ras-induced senescence is suggested. These insights may improve treatments of heart failure in the future.

Background: Awareness of transfusion -transmitted hepatitis E raised in recent years led to the mandatory testing of blood donations in some European countries for hepatitis E virus (HEV) RNA. However, little is known about the epidemiology of HEV infections. Aim: To and describe and analyse the epidemiology of HEV infections in blood donors in Germany. Methods: Data from routine testing of therapeutic blood products donated between January 2015 and December 2022 at the Uni. Blutspendedienst OWL were analysed at the Institute of Laboratory and Transfusion Medicine, Heart and Diabetes Centre North Rhine-Westphalia. A total of 731,630 allogenic blood donations from 119,610 individual blood donors were tested for HEV RNA in minipools of 96 samples. The HEV RNA -positive donations were analysed for the presence of anti -HE V IgM and IgG. The HEV strains were genotyped and various clinical liver -specific parameters were determined. Results: A total of 497 HEV-positive blood donations were identified, resulting in a yearly incidence of 1:1,474, from which 78.4% of the donations were RNA -only positive. Increased alanine aminotransferase activity was determined in 26.6% of HEV RNA -positive donors and was associated with the detection of IgG antibodies (1.2% anti -HE V IgM-positive, 11.9% anti -HE V IgM- and IgGpositive and 8.5% anti -HE V IgG-positive). An average incidence of 0.084-0.083% HEV RNA -positive donations in June and July in all years was observed, and a higher proportion of HEV RNA -positive men compared with women. All isolated HEV sequences corresponded to genotype 3. Conclusion :Our results underline the necessity of HEV RNA screening in blood donations.

BACKGROUND:Despite combined anticoagulation therapy consisting of a vitamin K antagonist and an antiplatelet agent, thromboembolic complications often occur in patients with a left ventricular assist device (LVAD). In addition, bleeding events are also common. Resistance to antiplatelet drugs is a well-known phenomenon; however, the utilization of laboratory chemistry testing for the presence of such resistance, and then switching therapy, is controversial.METHODS:We tested 132 patients with LVAD (HeartWare n = 57, HeartMate II n = 22, HeartMate 3 n = 53) on acetylsalicylic acid (ASA) therapy for resistance and followed them for a maximum of 7 years regarding pump thrombosis. Light transmission aggregometry (LTA) and impedance aggregometry (IPA) were performed for testing platelet function.RESULTS:We could show that patients with ASA resistance displayed an increased risk of pump thrombosis, regardless of the test used (LTA: OR = 6.20, CI [1.86-20.64], p = 0.003; IPA: OR = 12.14, CI [3.00-49.07], p < 0.001). In patients with a HeartMate 3, we could not detect any pump thrombosis associated with aspirin resistance. Furthermore, there was no significant difference in bleeding events between patients with ASA resistance and ASA responders.CONCLUSION:Laboratory testing of ASA resistance seems to be a good tool to detect an increased risk of pump thrombosis, at least for patients with a HeartWare or HeartMate II. The extent to which these thromboses can be prevented with a change of medication has to be investigated in further studies. No pump thrombosis was detected in patients with a HeartMate 3, and the question should be asked as to what constellation of underlying and concomitant diseases must be present to justify ASA therapy for these patients.

Desbuquois dysplasia type 2 (DBQD2) and spondylo-ocular syndrome (SOS) are autosomal recessive disorders affecting the extracellular matrix (ECM) and categorized as glycosaminoglycan (GAG) linkeropathies. Linkeropathies result from mutations within glycosyltransferases involved in the synthesis of the tetrasaccharide linker, a linker between the core protein of proteoglycan (PG) and GAG. DBQD2 and SOS are caused by the isolated mutations of the xylosyltransferase (XT) isoforms. In this work, we successfully generated XYLT1- as well as XYLT2-deficient GAG linkeropathy model systems in human dermal fibroblasts using a ribonucleoprotein-based CRISPR/Cas9-system. Furthermore, it was possible to generate a complete XYLT-knockdown. Short- and long-term XT activity deficiency led to the mutual reduction in all linker transferase-encoding genes, suggesting a potential multienzyme complex with mutual regulation. Fibroblasts compensated for ECM misregulation initially by overexpressing ECM through the TGFβ1 signaling pathway, akin to myofibroblast differentiation patterns. The long-term reduction in one XT isoform induced a stress response, reducing ECM components. The isolated XYLT1-knockout exhibited α-smooth muscle actin overexpression, possibly partially compensated by unaltered XT-II activity. XYLT2-knockout leads to the reduction in both XT isoforms and a strong stress response with indications of oxidative stress, induced senescence and apoptotic cells. In conclusion, introducing XYLT-deficiency revealed temporal and isoform-specific regulatory differences.

The collagen superfamily, as the major structural component of the extracellular matrix, encompasses 28 distinct subtypes, with type-I and -III forming fibrils crucial for the matrix scaffold. During collagen biogenesis, trimers of type-I and -III procollagen are secreted into the extracellular matrix. The N- and C-terminal propeptides of these trimers are proteolytically cleaved from procollagen during secretion, initiating collagen fibril formation. The propeptides are released into extracellular space and, therefore, have been used to quantify collagen biogenesis. But high-throughput methods for the quantification of these biomarkers are still lacking. This study presents a state-of-the-art multiplexed approach for the simultaneous quantification of PINP, PICP, PIIINP and PIIICP from cell culture supernatants. The ability of targeted proteomics to quantify these propeptides from cell culture samples was assessed in this study. Using tryptic digestion and solid phase extraction, we were able to accurately quantify precollagen propeptides in a range of 3 to 1000 ng/mL. The assay showed an average inter-assay variance of 6.86% with an overall recovery ranging from 92 to 98%. The assay was validated using recombinant protein standards diluted in surrogate matrix and tested using transforming growth factor β1 mediated induction of normal human dermal fibroblasts. In summary, the assay presented in this paper offers a novel, robust, and precise high-throughput method for measuring human collagen propeptides in cell culture supernatants, empowering researchers to assess collagen biogenesis effectively in in vitro experiments.

Xylosyltransferase-I and-II (XT-I,-II) possess a central role during the glycosylation of proteoglycans (PGs). They catalyze the formation of an O-glycosidic bond between the xylosyl residue of uridinediphosphate-xylose and the core protein of a PG. Thereafter, three following glycosyltransferases lead to the generation of a tetrasaccharide linker, which connects the PG core protein to the respective glycosaminoglycan.The selective quantification of XT-I and XT-II activity is of biological and clinical interest due to their association with fibrotic processes and skeletal dysplasia. There is no assay available to date that simultaneously determines the activity of the two XT isoforms. Although an XT-I selective UPLC MS/MS-based assay was published by Fischer et al., in 2021, the determination of XT-II activity can only be performed simultaneously by the improved assay presented here. To establish the assay, two synthetic peptides, selectively xylosylated by the respective isoform, were identified and the associated mea-surement parameters for the mass spectrometer were optimized. In addition, the quantitative range of the xylosylated peptides were validated, and the incubation time of the enzyme reaction was optimized for cell culture samples and human sera. The specific enzyme kinetics (KM and Vmax) of the respective XT isoform for the two peptides were also determined. Subsequently, a mathematical model was developed, allowing the simultaneous determination of XT-I and XT-II activity from the chromatographic results.Summarized, a mass spectrometric method suitable for the simultaneous analysis of XT-I and XT-II activity in cell culture lysates, supernatants and human sera was successfully developed.(c) 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Cardiovascular diseases (CVDs) are often linked to ageing and are the major cause of death worldwide. The declined proliferation of adult stem cells in the heart often impedes its regenerative potential. Thus, an investigation of the proliferative potential of adult human cardiac stem cells (hCSCs) might be of great interest for improving cell-based treatments of cardiovascular diseases. The application of human blood serum was already shown to enhance hCSC proliferation and reduce senescence. Here, the underlying signalling pathways of serum-mediated hCSC proliferation were studied. We are the first to demonstrate the involvement of the transcription factor NF-κB in the serum-mediated proliferative response of hCSCs by utilizing the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC). RNA-Sequencing (RNA-Seq) revealed ATF6B, COX5B, and TNFRSF14 as potential targets of NF-κB that are involved in serum-induced hCSC proliferation.

Natural killer (NK) cells hold promise in cancer treatment due to their ability to spontaneously lyse cancer cells. For clinical use, high quantities of pure, functional NK cells are necessary. Combining adherence-based isolation with specialized media showed the unreliability of the isolation method, but demonstrated the superiority of the NK MACS® medium, particularly in suboptimal conditions. Neither human pooled serum, fetal calf serum (FCS), human platelet lysate, nor chemically defined serum replacement could substitute human AB serum. Interleukin (IL-)2, IL-15, IL-21, and combined CD2/NKp46 stimulation were assessed. IL-21 and CD2/NKp46 stimulation increased cytotoxicity, but reduced NK cell proliferation. IL-15 stimulation alone achieved the highest proliferation, but the more affordable IL-2 performed similarly. The RosetteSep™ human NK cell enrichment kit was effective for isolation, but the presence of peripheral blood mononuclear cells (PBMCs) in the culture enhanced NK cell proliferation, despite similar expression levels of CD16, NKp46, NKG2D, and ICAM-1. In line with this, purified NK cells cultured in NK MACS® medium with human AB serum and IL-2 demonstrated high cytotoxicity against primary glioblastoma stem cells.