Inter-patient heterogeneity in the hepatic ischemia-reperfusion injury transcriptome: Implications for research and diagnostics.

Cellular responses induced by surgical procedure or ischemia-reperfusion injury (IRI) may severely alter transcriptome profiles and complicate molecular diagnostics. To investigate this effect, we characterized such pre-analytical effects in 143 non-malignant liver samples obtained from 30 patients at different time points of ischemia during surgery from two individual cohorts treated either with the Pringle manoeuvre or total vascular exclusion. Transcriptomics profiles were analyzed by Affymetrix microarrays and expression of selected mRNAs was validated by RT-PCR. We found 179 mutually deregulated genes which point to elevated cytokine signaling with NFκB as a dominant pathway in ischemia responses. In contrast to ischemia, reperfusion induced pro-apoptotic and pro-inflammatory cascades involving TNF, NFκB and MAPK pathways. FOS and JUN were down-regulated in steatosis compared to their up-regulation in normal livers. Surprisingly, molecular signatures of underlying primary and secondary cancers were present in non-tumor tissue. The reported inter-patient variability might reflect differences in individual stress responses and impact of underlying disease conditions. Furthermore, we provide a set of 230 pre-analytically highly robust genes identified from histologically normal livers (<2% covariation across both cohorts) that might serve as reference genes and could be particularly suited for future diagnostic applications.

Bei Mu Gua Lou San facilitates mucus expectoration by increasing surface area and hydration levels of airway mucus in an air-liquid-interface cell culture model of the respiratory epithelium.

BACKGROUND: Bei Mu Gua Lou San (BMGLS) is an ancient formulation known for its moisturizing and expectorant properties, but the underlying mechanisms remain unknown. We investigated concentration-dependent effects of BMGLS on its rehydrating and mucus-modulating properties using an air-liquid-interface (ALI) cell culture model of the Calu-3 human bronchial epithelial cell line and primary normal human bronchial epithelial cells (NHBE), and specifically focused on quantity and composition of the two major mucosal proteins MUC5AC and MUC5B. METHODS: ALI cultures were treated with BMGLS at different concentrations over three weeks and evaluated by means of histology, immunostaining and electron microscopy. MUC5AC and MUC5B mRNA levels were assessed and quantified on protein level using an automated image-based approach. Additionally, expression levels of the major mucus-stimulating enzyme 15-lipoxygenase (ALOX15) were evaluated. RESULTS: BMGLS induced concentration-dependent morphological changes in NHBE but not Calu-3 ALI cultures that resulted in increased surface area via the formation of herein termed intra-epithelial structures (IES). While cellular rates of proliferation, apoptosis or degeneration remained unaffected, BMGLS caused swelling of mucosal granules, increased the area of secreted mucus, decreased muco-glycoprotein density, and dispensed MUC5AC. Additionally, BMGLS reduced expression levels of MUC5AC, MUC5B and the mucus-stimulating enzyme 15-lipoxygenase (ALOX15). CONCLUSIONS: Our studies suggest that BMGLS rehydrates airway mucus while stimulating mucus secretion by increasing surface areas and regulating goblet cell differentiation through modulating major mucus-stimulating pathways.

Resilience and Protection of Health Care and Research Laboratory Workers During the SARS-CoV-2 Pandemic: Analysis and Case Study From an Austrian High Security Laboratory.

The SARS-CoV-2 pandemic has highlighted the interdependency of healthcare systems and research organizations on manufacturers and suppliers of personnel protective equipment (PPE) and the need for well-trained personnel who can react quickly to changing working conditions. Reports on challenges faced by research laboratory workers (RLWs) are rare in contrast to the lived experience of hospital health care workers. We report on experiences gained by RLWs (e.g., molecular scientists, pathologists, autopsy assistants) who significantly contributed to combating the pandemic under particularly challenging conditions due to increased workload, sickness and interrupted PPE supply chains. RLWs perform a broad spectrum of work with SARS-CoV-2 such as autopsies, establishment of virus cultures and infection models, development and verification of diagnostics, performance of virus inactivation assays to investigate various antiviral agents including vaccines and evaluation of decontamination technologies in high containment biological laboratories (HCBL). Performance of autopsies and laboratory work increased substantially during the pandemic and thus led to highly demanding working conditions with working shifts of more than eight hours working in PPE that stressed individual limits and also the ergonomic and safety limits of PPE. We provide detailed insights into the challenges of the stressful daily laboratory routine since the pandemic began, lessons learned, and suggest solutions for better safety based on a case study of a newly established HCBL (i.e., BSL-3 laboratory) designed for autopsies and research laboratory work. Reduced personal risk, increased resilience, and stress resistance can be achieved by improved PPE components, better training, redundant safety measures, inculcating a culture of safety, and excellent teamwork.

Quantification of increased MUC5AC expression in airway mucus of smoker using an automated image-based approach.

Microscopic analysis of mucus quantity and composition is crucial in research and diagnostics on muco-obstructive diseases. Currently used image-based methods are unable to extract concrete numeric values of mucosal proteins, especially on the expression of the key mucosal proteins MUC5AC and MUC5B. Since their levels increase under pathologic conditions such as extensive exposure to cigarette smoke, it is imperative to quantify them to improve treatment strategies of pulmonary diseases. This study presents a simple, image-based, and high-processing computational method that allows determining the ratio of MUC5AC and MUC5B within the overall airway mucus while providing information on their spatial distribution. The presented pipeline was optimized for automated downstream analysis using a combination of bright field and immunofluorescence imaging suitable for tracheal and bronchial tissue samples, and air-liquid interface (ALI) cell cultures. To validate our approach, we compared tracheal tissue and ALI cell cultures of isolated primary normal human bronchial epithelial cells derived from smokers and nonsmokers. Our data indicated 18-fold higher levels of MUC5AC in submucosal glands of smokers covering about 8% of mucosal areas compared to <1% in nonsmoking individuals, confirming results of previous studies. We further identified a subpopulation of nonsmokers with slightly elevated glandular MUC5AC levels suggesting moderate exposure to second-hand smoke or fine particulate air pollution. Overall, this study demonstrates a novel, user-friendly and freely available tool for digital pathology and the analysis of therapeutic interventions tested in ALI cell cultures.

Automated Quantitative Image Evaluation of Antigen Retrieval Methods for 17 Antibodies in Placentation and Implantation Diagnostic and Research.

Immunostaining in clinical routine and research highly depends on standardized staining methods and quantitative image analyses. We qualitatively and quantitatively compared antigen retrieval methods (no pretreatment, pretreatment with pepsin, and heat-induced pretreatment with pH 6 or pH 9) for 17 antibodies relevant for placenta and implantation diagnostics and research. Using our newly established, comprehensive automated quantitative image analysis approach, fluorescent signal intensities were evaluated. Automated quantitative image analysis found that 9 out of 17 antibodies needed antigen retrieval to show positive staining. Heat induction proved to be the most efficient form of antigen retrieval. Eight markers stained positive after pepsin digestion, with ß-hCG and vWF showing enhanced staining intensities. To avoid the misinterpretation of quantitative image data, the qualitative aspect should always be considered. Results from native placental tissue were compared with sections of a placental invasion model based on thermo-sensitive scaffolds. Immunostaining on placentas in vitro leads to new insights into fetal development and maternal pathophysiological pathways, as pregnant women are justifiably excluded from clinical studies. Thus, there is a clear need for the assessment of reliable immunofluorescent staining and pretreatment methods. Our evaluation offers a powerful tool for antibody and pretreatment selection in placental research providing objective and precise results.

Anti-Oxidative and Immune Regulatory Responses of THP-1 and PBMC to Pulsed EMF Are Field-Strength Dependent.

Innate immune cells react to electromagnetic fields (EMF) by generating reactive oxygen species (ROS), crucial intracellular messengers. Discrepancies in applied parameters of EMF studies, e.g., flux densities, complicate direct comparison of downstream anti-oxidative responses and immune regulatory signaling. We therefore compared the impact of different EMF flux densities in human leukemic THP1 cells and peripheral blood mononuclear cells (PBMC) of healthy donors to additionally consider a potential disparate receptivity based on medical origin. ROS levels increased in THP1 cells stimulated with lipopolysaccharide (LPS) after one hour of EMF exposure. Moreover, weak EMF mitigated the depletion of the reducing agent NAD(P)H in THP1. Neither of these effects occurred in PBMC. Landscaping transcriptional responses to varied EMF revealed elevation of the anti-oxidative enzymes PRDX6 (2-fold) and DHCR24 (6-fold) in THP1, implying involvement in lipid metabolism. Furthermore, our study confirmed anti-inflammatory effects of EMF by 6-fold increased expression of IL10. Strikingly, THP1 responded to weak EMF, while PBMC were primarily affected by strong EMF, yet with severe cellular stress and enhanced rates of apoptosis, indicated by HSP70 and caspase 3 (CASP3). Taken together, our results emphasize an altered susceptibility of immune cells of different origin and associate EMF-related effects with anti-inflammatory signaling and lipid metabolism.

Structural basis of STAT2 recognition by IRF9 reveals molecular insights into ISGF3 function.

Cytokine signaling through the JAK/STAT pathway controls multiple cellular responses including growth, survival, differentiation, and pathogen resistance. An expansion in the gene regulatory repertoire controlled by JAK/STAT signaling occurs through the interaction of STATs with IRF transcription factors to form ISGF3, a complex that contains STAT1, STAT2, and IRF9 and regulates expression of IFN-stimulated genes. ISGF3 function depends on selective interaction between IRF9, through its IRF-association domain (IAD), with the coiled-coil domain (CCD) of STAT2. Here, we report the crystal structures of the IRF9-IAD alone and in a complex with STAT2-CCD. Despite similarity in the overall structure among respective paralogs, the surface features of the IRF9-IAD and STAT2-CCD have diverged to enable specific interaction between these family members. We derive a model for the ISGF3 complex bound to an ISRE DNA element and demonstrate that the observed interface between STAT2 and IRF9 is required for ISGF3 function in cells.