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Abstract Objective The IPEC-J2 cell line is used as an in vitro small intestine model for swine, but it is also used as a model for the human intestine, presenting a relatively unique setting. By combining electric cell-substrate impedance sensing, with next-generation-sequencing technology, we showed that mRNA gene expression profiles and related pathways can depend on the growth phase of IPEC-J2 cells. Our investigative approach welcomes scientists to reproduce or modify our protocols and endorses putting their gene expression data in the context of the respective growth phase of the cells. Results Three time points are presented: (TP1) 1 h after medium change (= 6 h after seeding of cells), (TP2) the time point of the first derivative maximum of the cell growth curve, and a third point at the beginning of the plateau phase (TP3). Significantly outstanding at TP1 compared to TP2 was upregulated PLEKHN1, further FOSB and DEGS2 were significantly downregulated at TP2 compared to TP3. Any provided data can be used to improve next-generation experiments with IPEC-J2 cells.

Summary: Cellular crosstalk is an essential process influenced by numerous factors, including secreted vesicles that transfer nucleic acids, lipids, and proteins between cells. Extracellular vesicles (EVs) have been the center of many studies focusing on neurodegenerative disorders, but whether EVs display cell-type-specific features for cellular crosstalk during neurodevelopment is unknown. Here, using human-induced pluripotent stem cell-derived cerebral organoids, neural progenitors, neurons, and astrocytes, we identify heterogeneity in EV protein content and dynamics in a cell-type-specific and time-dependent manner. Our results support the trafficking of key molecules via EVs in neurodevelopment, such as the transcription factor YAP1, and their localization to differing cell compartments depending on the EV recipient cell type. This study sheds new light on the biology of EVs during human brain development.

For several years, the determination of a differential cell count of a raw milk sample has been proposed as a more accurate tool for monitoring the udder health of dairy cows compared with using the absolute somatic cell count. However, the required sample preparation and staining process can be labor- and cost-intensive. Therefore, the aim of our study was to demonstrate the feasibility of analyzing unlabeled blood and milk leukocytes from dairy cows by means of digital holographic microscopy (DHM). For this, we trained three different machine learning methods, i.e., k-Nearest Neighbor, Random Forests, and Support Vector Machine, on sorted leukocyte populations (granulocytes, lymphocytes, and monocytes/macrophages) isolated from blood and milk samples of three dairy cows by using fluorescence-activated cell sorting. Afterward, those classifiers were applied to differentiate unlabeled blood and milk samples analyzed by DHM. A total of 70 blood and 70 milk samples were used. Those samples were collected from five clinically healthy cows at 14-time points within a study period of 26 days. The outcome was compared with the results of the same samples analyzed by flow cytometry and (in the case of blood samples) also to routine analysis in an external laboratory. Moreover, a standard vaccination was used as an immune stimulus during the study to check for changes in cell morphology or cell counts. When applied to isolated leukocytes, Random Forests performed best, with a specificity of 0.93 for blood and 0.84 for milk cells and a sensitivity of 0.90 and 0.81, respectively. Although the results of the three analytical methods differed, it could be demonstrated that a DHM analysis is applicable for blood and milk leukocyte samples with high reliability. Compared with the flow cytometric results, Random Forests showed an MAE of 0.11 (SD = 0.04), an RMSE of 0.13 (SD = 0.14), and an MRE of 1.00 (SD = 1.11) for all blood leukocyte counts and an MAE of 0.20 (SD = 0.11), an RMSE of 0.21 (SD = 0.11) and an MRE of 1.95 (SD = 2.17) for all milk cell populations. Further studies with larger sample sizes and varying immune cell compositions are required to establish method-specific reference ranges.

BackgroundNewer 3D culturing approaches are a promising way to better mimic the in vivo tumor microenvironment and to study the interactions between the heterogeneous cell populations of glioblastoma multiforme. Like many other tumors, glioblastoma uses extracellular vesicles as an intercellular communication system to prepare surrounding tissue for invasive tumor growth. However, little is known about the effects of 3D culture on extracellular vesicles. The aim of this study was to comprehensively characterize extracellular vesicles in 3D organoid models and compare them to conventional 2D cell culture systems.MethodsPrimary glioblastoma cells were cultured as 2D and 3D organoid models. Extracellular vesicles were obtained by precipitation and immunoaffinity, with the latter allowing targeted isolation of the CD9/CD63/CD81 vesicle subpopulation. Comprehensive vesicle characterization was performed and miRNA expression profiles were generated by smallRNA-sequencing. In silico analysis of differentially regulated miRNAs was performed to identify mRNA targets and corresponding signaling pathways. The tumor cell media and extracellular vesicle proteome were analyzed by high-resolution mass spectrometry.ResultsWe observed an increased concentration of extracellular vesicles in 3D organoid cultures. Differential gene expression analysis further revealed the regulation of twelve miRNAs in 3D tumor organoid cultures (with nine miRNAs down and three miRNAs upregulated). MiR-23a-3p, known to be involved in glioblastoma invasion, was significantly increased in 3D. MiR-7-5p, which counteracts glioblastoma malignancy, was significantly decreased. Moreover, we identified four miRNAs (miR-323a-3p, miR-382-5p, miR-370-3p, miR-134-5p) located within the DLK1-DIO3 domain, a cancer-associated genomic region, suggesting a possible importance of this region in glioblastoma progression. Overrepresentation analysis identified alterations of extracellular vesicle cargo in 3D organoids, including representation of several miRNA targets and proteins primarily implicated in the immune response.ConclusionOur results show that 3D glioblastoma organoid models secrete extracellular vesicles with an altered cargo compared to corresponding conventional 2D cultures. Extracellular vesicles from 3D cultures were found to contain signaling molecules associated with the immune regulatory signaling pathways and as such could potentially change the surrounding microenvironment towards tumor progression and immunosuppressive conditions. These findings suggest the use of 3D glioblastoma models for further clinical biomarker studies as well as investigation of new therapeutic options.

BackgroundAtherosclerosis is a widespread disorder of the cardiovascular system. The early detection of plaques by circulating biomarkers is highly clinically relevant to prevent the occurrence of major complications such as stroke or heart attacks. It is known that extracellular vesicles (EVs) are important in intercellular communication in atherosclerotic disorders and carry many components of their cells of origin, including microRNAs (miRNAs). In this study, we test the assumption that miRNAs present in material acquired from plaques in patients undergoing surgery for atherosclerotic carotid artery stenosis are also expressed in circulating EVs obtained from the identical patients. This would allow the adoption of a liquid biopsy approach for the detection of plaques.MethodsWe studied 22 surgical patients with atherosclerotic carotid arterial stenosis and 28 healthy controls. EVs were isolated from serum by precipitation. miRNA expression profiles of serum-derived EVs were obtained by small RNA sequencing and in plaque material simultaneously acquired from patients. A comparative analysis was performed to identify circulating atherosclerosis-associated miRNAs that are also detectable in plaques.ResultsSeven miRNAs were found to be differentially regulated in patient serum compared with the serum of healthy controls. Of these, miR-193b-5p, miR-193a-5p, and miR-125a-3p were significantly upregulated in patients compared with that in healthy controls and present in both, circulating EVs and plaque material. An overrepresentation analysis of experimentally validated mRNA targets revealed an increased regulation of inflammation and vascular growth factors, key players in atherosclerosis and plaque formation.ConclusionOur findings suggest that circulating EVs reflect plaque development in patients with symptomatic carotid artery stenosis, which can serve as biomarker candidates for detecting the presence of atherosclerotic plaques.

IntroductionBacteria inhabit the in- and outside of the human body, such as skin, gut or the oral cavity where they play an innoxious, beneficial or even pathogenic role. It is well known that bacteria can secrete membrane vesicles (MVs) like eukaryotic cells with extracellular vesicles (EVs). Several studies indicate that bacterial membrane vesicles (bMVs) play a crucial role in microbiome-host interactions. However, the composition of such bMVs and their functionality under different culture conditions are still largely unknown.MethodsTo gain a better insight into bMVs, we investigated the composition and functionality of E. coli (DSM 105380) bMVs from the culture media Lysogeny broth (LB) and RPMI 1640 throughout the different phases of growth (lag-, log- and stationary-phase). bMVs from three time points (8 h, 54 h, and 168 h) and two media (LB and RPMI 1640) were isolated by ultracentrifugation and analyzed using nanoparticle tracking analysis (NTA), cryogenic electron microscopy (Cryo-EM), conventional transmission electron microscopy (TEM) and mass spectrometry-based proteomics (LC–MS/MS). Furthermore, we examined pro-inflammatory cytokines IL-1β and IL-8 in the human monocyte cell line THP-1 upon bMV treatment.ResultsParticle numbers increased with inoculation periods. The bMV morphologies in Cryo-EM/TEM were similar at each time point and condition. Using proteomics, we identified 140 proteins, such as the common bMV markers OmpA and GroEL, present in bMVs isolated from both media and at all time points. Additionally, we were able to detect growth-condition-specific proteins. Treatment of THP-1 cells with bMVs of all six groups lead to significantly high IL-1β and IL-8 expressions.ConclusionOur study showed that the choice of medium and the duration of culturing significantly influence both E. coli bMV numbers and protein composition. Our TEM/Cryo-EM results demonstrated the presence of intact E. coli bMVs. Common E. coli proteins, including OmpA, GroEL, and ribosome proteins, can consistently be identified across all six tested growth conditions. Furthermore, our functional assays imply that bMVs isolated from the six groups retain their function and result in comparable cytokine induction.

The purpose of milking machines is to harvest milk at optimal quality and speed, while maintaining animal comfort and teat defense mechanisms against invading mastitis pathogens. Therefore, the milking machine is a very important piece of equipment on dairy farms to maintain a long healthy lactation by following the physiological conditions of the udder. The mechanical forces during long-term machine milking processes lead to changes in the teat tissue. This effect is related to the degree of adaptation of the milking machines to the physiological requirements of the individual udder anatomy and the physiological conditions of the lactating animals. If both, milking machine settings and liner design are not suitable for all teats and animals on the farm, some animals will not be fully milked, the teat condition will deteriorate over time and in the end, they may suffer from mastitis. Therefore, maintaining healthy udders and teats during milking is a central key component of an effective milking machine to produce good milk yield with higher quality by preventing mastitis and maintaining animal health and welfare. On large and thick teats, conventional liners often fit too tight, causing a massive mechanical stress load on the tissue. On small teats, however, they often do not adhere sufficiently close to the teat which can cause a considerable air admission and hence liner slips. The new liners, “Stimulor® StressLess” (Siliconform, Türkheim, Germany), have a wave-like lip construction and adapt well to the different teat sizes in a herd, thus ensuring consistent milking of lactating animals. A proper milking machine accommodates all teat sizes and forms, has a low vacuum to effectively open the teat and to stimulate physiological milk release and letdown. In addition, the right pulsation rate will maintain a stable vacuum on the teat area during milking. In conclusion, an ideal milking machine adapts to the morphological, anatomical, and physiological characteristics of the udder and teats of the lactating animals and it should achieve a physiologically ideal milking process that meets high animal welfare standards and increases milk production with a high quality standard.

Accuracy and transparency of scientific data are becoming more and more relevant with the increasing concern regarding the evaluation of data reproducibility in many research areas. This concern is also true for quantifying coding and noncoding RNAs, with the remarkable increase in publications reporting RNA profiling and sequencing studies. To address the problem, we propose the following recommendations: (a) accurate documentation of experimental procedures in Materials and methods (and not only in the supplementary information, as many journals have a strict mandate for making Materials and methods as visible as possible in the main text); (b) submission of RT‐qPCR raw data for all experiments reported; and (c) adoption of a unified, simple format for submitted RT‐qPCR raw data. The Real‐time PCR Data Essential Spreadsheet Format (RDES) was created for this purpose.

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease for which there is no cure. Accumulating research results suggest a role for extracellular vesicles (EVs) in the pathogenesis of COPD. This study aimed to uncover the involvement of EVs and their molecular cargo in the progression of COPD by identification of EV-associated protein and microRNA (miRNA) profiles. We isolated EVs from the bronchial alveolar lavage fluid (BALF) of 18 patients with COPD and 11 healthy controls using size-exclusion chromatography. EV isolates were characterized using nanoparticle tracking analysis and protein content. Proteomic analysis revealed a higher abundance of 284 proteins (log2FC > 1) and a lower abundance of 3 proteins (log2FC < −1) in EVs derived from patients with COPD. Ingenuity pathway analysis showed that proteins enriched in COPD-associated EVs trigger inflammatory responses, including neutrophil degranulation. Variances in surface receptors and ligands associated with COPD EVs suggest a preferential interaction with alveolar cells. Small RNAseq analysis identified a higher abundance of ten miRNAs and a lower abundance of one miRNA in EVs from COPD versus controls (Basemean > 100, FDR < 0.05). Our data indicate that the molecular composition of EVs in the BALF of patients with COPD is altered compared to healthy control EVs. Several components in COPD EVs were identified that may perpetuate inflammation and alveolar tissue destruction.

Extracellular vesicles (EVs) convey inter-organ communication in health and disease. Here, the authors report that adipocyte-derived EVs isolated from insulin-resistant obese but not lean male mice stimulate insulin secretion via the targeted transfer of insulinotropic proteins from adipose tissue to β-cells.

The aim of this review was to focus on the physiological aspects of milk somatic cell count (SCC) in small ruminants (SM). The SCC is an important component naturally present in milk and is generally used as an indicator of milk quality and udder health in milk producing ruminants. SCC contains the following cells: polymorphonuclear neutrophils (PMN), macrophages, lymphocytes, and many milk epithelial (MEC) cells, cell fragments, and cytoplasmic particles/vesicles. PMN (40–80%) represent the major cell type in milk in healthy uninfected goats, whereas the macrophages (45–88%) are the major cell type in sheep’s milk. However, dairy goats and sheep have an apocrine secretory system that produces cytoplasmic cellular particles/vesicles and large numbers of cell fragments, resulting in the physiological SCC limit being exceeded. It is obvious that the SCC level in milk of SM can be affected by various influencing factors, such as milk fraction, breed, stage of lactation, parity, type of birth, milking system, and others. An increase in the SCC above the physiological level not only indicates an udder or general health problem but reduces milk production, changes the milk composition, and hence affects milk processing. Moreover, the milking machine plays an important role in maintaining udder health in SM and stable SCC at physiological levels in the milk obtained. So far, there are no healthy or pathological physiological SCC levels defined in SM milk. Furthermore, a differential cell count (DCC) or even a high resolution DCC (HRDCC), which were recently developed for cattle milk, could also help in SM to gain deeper insight into the immunology of the mammary gland and find biomarkers to assess udder health. In conclusion, SCC is an indication of udder health or exposure of the udder to infectious agents or mechanical stress and should therefore always be considered a warning sign.

Nanoplastics smaller than 1 µm accumulate as anthropogenic material in the food chain, but only little is known about their uptake and possible effects on potentially strongly exposed cells of the small intestine. The aim of the study was to observe the uptake of 100 nm polystyrene nanoplastics into a non-tumorigenic small intestine cell culture model (IPEC-J2 cells) and to monitor the effects on cell growth and gene regulation, compared to a 100 nm non-plastic silica nanoparticle reference. The intracellular uptake of both types of nanoparticles was proven via (confocal) fluorescence microscopy and complemented with transmission electron microscopy. Fluorescence microscopy showed a growth phase-dependent uptake of nanoparticles into the cells, hence further experiments included different time points related to epithelial closure, determined via electric cell substrate impedance sensing. No retardations in epithelial closure of cells after treatment with polystyrene nanoparticles could be found. In contrast, epithelial cell closure was partly negatively influenced by silica nanoparticles. An increased production of organic nanoparticles, like extracellular vesicles, was not measurable via nanoparticle tracking analysis. An assessment of messenger RNA by next generation sequencing and subsequent pathway analysis revealed that the TP53 pathway was influenced significantly by the polystyrene nanoparticle treatment. In both treatments, dysregulated mRNAs were highly enriched in the NOTCH signaling pathway compared to the non-particle control.

Donkey milk (DM) has been known in the world for 5000 years for its benefits for human nutrition and health. Nowadays, DM has become more and more attractive as a commercial product. DM contains several physiologically functional components, including high-quality whey proteins, vitamins, important minerals, unsaturated fatty acid and bioactive components. Therefore, it is not only consumed as food but also as a remedy. The average daily milk yield of a female donkey over the entire lactation season was 1.57 ± 1.12 kg/day and fluctuated between 0.20 and 6.00 kg/day. Average milk concentrations (±SD) of fat, protein, lactose, total solids and ash in DM were 0.63 ± 0.41%, 1.71 ± 0.24%, 6.34 ± 0.37%, 9.11 ± 0.95% and 0.39 ± 0.04%, respectively. Interestingly, DM is similar in composition to mare’s milk, and both are similar to mother’s milk. The anatomical and morphological properties of the mammary gland of the female donkey are special and can be compared with those of mare udders. However, the cistern cavity of the mammary gland of female donkeys is characterized by the presence of multiple pockets that open directly into the teat, instead of a single cistern cavity. Therefore, the mammary gland capacity in donkey mare is low and milking technique and routine are of most importance. So far there is no special milking machine for female donkeys and mares. The milking machines used nowadays were initially designed for smaller sheep and goat udders. The company Siliconform, Germany, has set itself the task of developing an optimized milking machine for donkey mares, which is adapted to the anatomical and morphological properties of the donkey mammary gland. Furthermore, it should achieve a physiologically ideal milking process meeting high animal welfare standards for increased milk production with high quality standards.

Restriction modification (RM) systems are known to provide a strong barrier to the exchange of DNA between and within bacterial species. Likewise, DNA methylation is known to have an important function in bacterial epigenetics regulating essential pathways such as DNA replication and the phase variable expression of prokaryotic phenotypes. To date, research on staphylococcal DNA methylation focused mainly on the two species Staphylococcus aureus and S. epidermidis. Less is known about other members of the genus such as S. xylosus, a coagulase-negative commensal of mammalian skin. The species is commonly used as starter organism in food fermentations but is also increasingly considered to have an as yet elusive function in bovine mastitis infections. We analyzed the methylomes of 14 S. xylosus strains using single-molecular, real-time (SMRT) sequencing. Subsequent in silico sequence analysis allowed identification of the RM systems and assignment of the respective enzymes to the discovered modification patterns. Hereby the presence of type I, II, III and IV RM systems in varying numbers and combinations among the different strains was revealed, clearly distinguishing the species from what is known for other members of the genus so far. In addition, the study characterizes a newly discovered type I RM system, encoded by S. xylosus but also by a variety of other staphylococcal species, with a hitherto unknown gene arrangement that involves two specificity units instead of one (hsdRSMS). Expression of different versions of the operon in E. coli showed proper base modification only when genes encoding both hsdS subunits were present. This study provides new insights into the general understanding of the versatility and function of RM systems as well as the distribution and variations in the genus Staphylococcus.

Abstract The determination of the somatic cell count of a milk sample is one of the most common methods to monitor udder health of a dairy cow. However, this procedure does not take into account the fact that cells in milk present a great variety of different cell types. The objective of our study was to establish a high-resolution differential cell count (HRDCC) by means of flow cytometry in blood and milk. We were able to detect ten subpopulations among the three main populations of immune cells and to determine their viability. Additionally, blood samples were analyzed for common laboratory biomarkers, i.e. differential blood counts, haptoglobin levels and several metabolic parameters. In this first feasibility study, we used three different vaccines to stimulate the immune system of five healthy cows each. Samples were collected shortly before, in between and after the vaccinations. Using multivariate statistical methods we saw a diagnostic benefit when HRDCCs were included compared to only the standard laboratory parameters. The impacts of all three vaccinations on the immune system were visible in blood HRDCCs as well as in milk HRDCCs. Cluster of Differentiation 8+ (CD8+) T cells, B cells and monocyte/macrophage subpopulations were among the most important and statistically relevant parameters for all treatments in both biofluids. Moreover, in one of the treatment groups intermediate monocytes showed a significant increase after both vaccinations. Although the use of HRDCC in blood or milk was shown to be highly relevant for early systemic diagnostic, to confirm these subpopulations further investigations in cows of different breed, lactation stage or health status are required.

BackgroundDegradation of the endothelial protective glycocalyx layer during COVID-19 infection leads to shedding of major glycocalyx components. These circulating proteins and their degradation products may feedback on immune and endothelial cells and activate molecular signaling cascades in COVID-19 associated microvascular injury. To test this hypothesis, we measured plasma glycocalyx components in patients with SARS-CoV-2 infection of variable disease severity and identified molecular signaling networks activated by glycocalyx components in immune and endothelial cells.MethodsWe studied patients with RT-PCR confirmed COVID-19 pneumonia, patients with COVID-19 Acute Respiratory Distress Syndrome (ARDS) and healthy controls (wildtype, n=20 in each group) and measured syndecan-1, heparan sulfate and hyaluronic acid. The in-silico construction of signaling networks was based on RNA sequencing (RNAseq) of mRNA transcripts derived from blood cells and of miRNAs isolated from extracellular vesicles from the identical cohort. Differentially regulated RNAs between groups were identified by gene expression analysis. Both RNAseq data sets were used for network construction of circulating glycosaminoglycans focusing on immune and endothelial cells.ResultsPlasma concentrations of glycocalyx components were highest in COVID-19 ARDS. Hyaluronic acid plasma levels in patients admitted with COVID-19 pneumonia who later developed ARDS during hospital treatment (n=8) were significantly higher at hospital admission than in patients with an early recovery. RNAseq identified hyaluronic acid as an upregulator of TLR4 in pneumonia and ARDS. In COVID-19 ARDS, syndecan-1 increased IL-6, which was significantly higher than in pneumonia. In ARDS, hyaluronic acid activated NRP1, a co-receptor of activated VEGFA, which is associated with pulmonary vascular hyperpermeability and interacted with VCAN (upregulated), a proteoglycan important for chemokine communication.ConclusionsCirculating glycocalyx components in COVID-19 have distinct biologic feedback effects on immune and endothelial cells and result in upregulation of key regulatory transcripts leading to further immune activation and more severe systemic inflammation. These consequences are most pronounced during the early hospital phase of COVID-19 before pulmonary failure develops. Elevated levels of circulating glycocalyx components may early identify patients at risk for microvascular injury and ARDS. The timely inhibition of glycocalyx degradation could provide a novel therapeutic approach to prevent the development of ARDS in COVID-19.

Introduction: MicroRNAs have a significant role in the regulation of the transcriptome. Several miRNAs have been proposed as potential biomarkers in different malignancies. However, contradictory results have been reported on the capability of miRNA biomarkers in cancer detection. The human biological clock involves molecular mechanisms that regulate several genes over time. Therefore, the sampling time becomes one of the significant factors in gene expression studies.Method: In the present study, we have tried to find miRNAs with minimum fluctuation in expression levels at different time points that could be more accurate candidates as diagnostic biomarkers. The small RNA-seq raw data of ten healthy individuals across nine-time points were analyzed to identify miRNAs with stable expression.Results: We have found five oscillation patterns. The stable miRNAs were investigated in 779 small-RNA-seq datasets of eleven cancer types. All miRNAs with the highest differential expression were selected for further analysis. The selected miRNAs were explored for functional pathways. The predominantly enriched pathways were miRNA in cancer and the P53-signaling pathway. Finally, we have found seven miRNAs, including miR-142-3p, miR-199a-5p, miR-223-5p, let-7d-5p, miR-148b-3p, miR-340-5p, and miR-421. These miRNAs showed minimum fluctuation in healthy blood and were dysregulated in the blood of eleven cancer types. Conclusion: We have found a signature of seven stable miRNAs which dysregulate in several cancer types and may serve as potential pan-cancer biomarkers.

The study aimed to evaluate the mRNA expression levels of various local novel adipokines, including vaspin, adiponectin, visfatin, and resistin, along with their associated receptors, heat shock 70 protein 5, adiponectin receptor 1, and adiponectin receptor 2, in the corpus luteum (CL) during luteal regression, also known as luteolysis, in dairy cows. We selected Fleckvieh cows in the mid-luteal phase (days 8–12, control group) and administered cloprostenol (PGF analog) to experimentally induce luteolysis. We collected CL samples at different time points following PGF application: before treatment (days 8–12, control group) and at 0.5, 2, 4, 12, 24, 48, and 64 h post-treatment (n = 5) per group. The mRNA expression was measured via real-time reverse transcription polymerase chain reaction (RT-qPCR). Vaspin was characterized by high mRNA levels at the beginning of the regression stage, followed by a significant decrease 48 h and 64 h after PGF treatment. Adiponectin mRNA levels were elevated 48 h after PGF. Resistin showed upregulation 4 h post PGF application. In summary, the alterations observed in the adipokine family within experimentally induced regressing CL tissue potentially play an integral role in the local regulatory processes governing the sequence of events culminating in functional luteolysis and subsequent structural changes in the bovine ovary.

Abstract Background Progranulin is a widely expressed pleiotropic growth factor with a central regulatory effect during the early immune response in sepsis. Progranulin signaling has not been systematically studied and compared between sepsis, community-acquired pneumonia (CAP), COVID-19 pneumonia and a sterile systemic inflammatory response (SIRS). We delineated molecular networks of progranulin signaling by next-generation sequencing (NGS), determined progranulin plasma concentrations and quantified the diagnostic performance of progranulin to differentiate between the above-mentioned disorders using the established biomarkers procalcitonin (PCT), interleukin-6 (IL-6) and C-reactive protein (CRP) for comparison. Methods The diagnostic performance of progranulin was operationalized by calculating AUC and ROC statistics for progranulin and established biomarkers in 241 patients with sepsis, 182 patients with SIRS, 53 patients with CAP, 22 patients with COVID-19 pneumonia and 53 healthy volunteers. miRNAs and mRNAs in blood cells from sepsis patients (n = 7) were characterized by NGS and validated by RT-qPCR in an independent cohort (n = 39) to identify canonical gene networks associated with upregulated progranulin at sepsis onset. Results Plasma concentrations of progranulin (ELISA) in patients with sepsis were 57.5 (42.8–84.9, Q25–Q75) ng/ml and significantly higher than in CAP (38.0, 33.5–41.0 ng/ml, p

This study aimed to determine the gene expression of different local novel adipokines, such as vaspin, adiponectin, visfatin, and resistin, and their known receptors, namely, heat shock 70 protein 5, adiponectin receptor 1, and adiponectin receptor 2, in the bovine corpus luteum (CL) during different phases of the estrous cycle (on days 1–2, 3–4, 5–7, 8–12, 13–18, >18) and pregnancy (at months 1–2, 3–4, 5–7, >7). The mRNA expression was measured by reverse transcription polymerase chain reaction (RT-qPCR). The mRNA expression levels were normalized to the geometric mean of all three constantly expressed reference genes (cyclophilin A, ubiquitin, ubiquitin C). Our findings suggest that adipokines are expressed and present in all investigated groups, and are specifically up- or downregulated during the estrus cycle and during pregnancy. Vaspin and adiponectin levels were upregulated in the middle and late cycle stages. Resistin was abundant during the CL regression stage and in the first months of pregnancy. The specific expression of adipokine receptors indicates their involvement in the local mechanisms that regulate CL function. Further investigations are required to elucidate the regulative mechanisms underlying the different local effects of adipokines on the ovarian physiology of cows.

Single-cell RNA-seq offers the opportunity to improve efficacy of T-cell based immunotherapy. Here the authors develop a plate-based method for cytotoxic T cell profiling. It captures a higher number of transcripts and detects gene with increased dynamic range in comparison to droplet-based methods.

Abstract Oncologic patients are regarded as the population most at risk of developing a severe course of COVID‐19 due to the fact that malignant diseases and chemotherapy often weaken the immune system. In the face of the ongoing SARS‐CoV‐2 pandemic, how particular patients deal with this infection remains an important question. In the period between the 15 and 26 April 2020, a total of 1227 patients were tested in one of seven oncologic outpatient clinics for SARS‐CoV‐2, regardless of symptoms, employing RT‐qPCR. Of 1227 patients, 78 (6.4%) were tested positive of SARS‐CoV‐2. Only one of the patients who tested positive developed a severe form of COVID‐19 with pneumonia (CURB‐65 score of 2), and two patients showed mild symptoms. Fourteen of 75 asymptomatic but positively tested patients received chemotherapy or chemo‐immunotherapy according to their regular therapy algorithm (±4 weeks of SARS‐CoV‐2 test), and 48 of 78 (61.5%) positive‐tested patients received glucocorticoids as co‐medication. None of the asymptomatic infected patients showed unexpected complications due to the SARS‐CoV‐2 infection during the cancer treatment. These data clearly contrast the view that patients with an oncologic disease are particularly vulnerable to SARS‐CoV‐2 and suggest that compromising therapies could be continued or started despite the ongoing pandemic. Moreover the relatively low appearance of symptoms due to COVID‐19 among patients on chemotherapy and other immunosuppressive co‐medication like glucocorticoids indicate that suppressing the response capacity of the immune system reduces disease severity.

Background and PurposeIncreased levels of the chaperone protein GRP78 have been implicated in poorer outcomes of cancer therapy. We have therefore explored the functional connection between the expression of GRP78 and the development of radioresistance and metastatic behavior in HNSCC.Material and MethodsThe association between gene expression of GRP78 and survival in HNSCC patients was examined using the TCGA database. The influence of ionizing radiation on the GRP78 levels in HNSCC cell lines, their secreted extracellular vesicles (EV) and non-irradiated EV-recipient cells was investigated by Western Blot and FACS. The consequences of chemical inhibition or experimental overexpression of GRP78 on radioresistance and migration of HNSCC cells were analyzed by clonogenic survival and gap closure assays.ResultsElevated levels of GRP78 RNA in HNSCC correlated with poorer overall survival. Radiation increased GRP78 protein expression on the surface of HNSCC cell lines. Experimental overexpression of GRP78 increased both radioresistance and migratory potential. Chemical inhibition of GRP78 impaired cell migration. EVs were identified as a potential source of increased GRP78 content as elevated levels of surface GRP78 were found in EVs released by irradiated cells. These vesicles transferred GRP78 to non-irradiated recipient cells during co-cultivation.ConclusionsWe have identified the chaperone protein GRP78 as a potential driver of increased radioresistance and motility in HNSCC. The uptake of GRP78-rich EVs originating from irradiated cells may contribute to a poorer prognosis through bystander effects mediated by the transfer of GRP78 to non-irradiated cells. Therefore, we consider the chaperone protein GRP78 to be an attractive target for improving radiotherapy strategies.

BackgroundExtracellular vesicles (EVs) are mediators of cell-to-cell communication in inflammatory lung diseases. They function as carriers for miRNAs which regulate mRNA transcripts and signaling pathways after uptake into recipient cells. We investigated whether miRNAs associated with circulating EVs regulate immunologic processes in COVID-19.MethodsWe prospectively studied 20 symptomatic patients with COVID-19 pneumonia, 20 mechanically ventilated patients with severe COVID-19 (severe acute respiratory corona virus-2 syndrome, ARDS) and 20 healthy controls. EVs were isolated by precipitation, total RNA was extracted, profiled by small RNA sequencing and evaluated by differential gene expression analysis (DGE). Differentially regulated miRNAs between groups were bioinformatically analyzed, mRNA target transcripts identified and signaling networks constructed, thereby comparing COVID-19 pneumonia to the healthy state and pneumonia to severe COVID-19 ARDS.ResultsDGE revealed 43 significantly and differentially expressed miRNAs (25 downregulated) in COVID-19 pneumonia when compared to controls, and 20 miRNAs (15 downregulated) in COVID-19 ARDS patients in comparison to those with COVID-19 pneumonia. Network analysis for comparison of COVID-19 pneumonia to healthy controls showed upregulated miR-3168 (log2FC=2.28, padjusted

Atherosclerosis can occur throughout the arterial vascular system and lead to various diseases. Early diagnosis of atherosclerotic processes and of individual disease patterns would be more likely to be successful if targeted therapies were available. For this, it is important to find reliable biomarkers that are easily accessible and with little inconvenience for patients. There are many cell culture, animal model or tissue studies that found biomarkers at the microRNA (miRNA) and mRNA level describing atherosclerotic processes. However, little is known about their potential as circulating and liquid biopsy markers in patients. In this study, we examined serum-derived miRNA – profiles from 129 patients and 28 volunteers to identify potential biomarkers. The patients had four different atherosclerotic manifestations: abdominal aneurysm (n = 35), coronary heart disease (n = 34), carotid artery stenosis (n = 24) and peripheral arterial disease (n = 36). The samples were processed with an extracellular vesicle enrichment protocol, total-RNA extraction and small RNA-sequencing were performed. A differential expression analysis was performed bioinformatically to find potentially regulated miRNA biomarkers. Resulting miRNA candidates served as a starting point for an overrepresentation analysis in which relevant target mRNAs were identified. The Gene Ontology database revealed relevant biological functions in relation to atherosclerotic processes. In patients, expression of specific miRNAs changed significantly compared to healthy volunteers; 27 differentially expressed miRNAs were identified. We were able to detect a group-specific miRNA fingerprint: miR-122-5p, miR-2110 and miR-483-5p for abdominal aortic aneurysm, miR-370-3p and miR-409-3p for coronary heart disease, miR-335-3p, miR-381-3p, miR493-5p and miR654-3p for carotid artery stenosis, miR-199a-5p, miR-215-5p, miR-3168, miR-582-3p and miR-769-5p for peripheral arterial disease. The results of the study show that some of the identified miRNAs have already been associated with atherosclerosis in previous studies. Overrepresentation analysis on this data detected biological processes that are clearly relevant for atherosclerosis, its development and progression showing the potential of these miRNAs as biomarker candidates. In a next step, the relevance of these findings on the mRNA level is to be investigated and substantiated.

A key challenge of the 21st century will be to provide the growing world population with a sustainable and secure supply of food. Consequently, the dairy farming’s primary task is to lower milk losses and other inefficiencies associated with diseased cows. Moreover, a shift from curative to preventive health management would be desirable for mastitis and a wide variety of other infectious and non-infectious cattle diseases, some of which are known to have profound negative effects on the performance and well-being of cows. Differential cell counting (DCC), a procedure that aims to determine the proportions of different somatic cell types in raw milk samples, has not only the potential to optimize mastitis diagnostics, but it could furthermore serve as a diagnostic tool for monitoring the general and overall health status of dairy cows. Based on a broad search of the literature, the practical utility of various types of DCC is summarized and discussed in this review. Since it might be of advantage to interpret DCC with the aid of data from studies in humans, differences between the immune systems of humans and dairy cattle, with a special focus on surface marker expression profiles and γδ (gamma delta) T-cell characteristics, are also described.

Differential cell counts in milk offer a deeper insight into the immunology of the mammary gland and might even provide information about the systemic health status of a dairy cow. Consequently, their potential as a diagnostic method to identify biomarkers has been a subject of research for quite some time. The objective of our study was to closely monitor the immune status of eight healthy dairy cows throughout their whole lactation. For this, high-resolution differential cell counts in milk and blood were determined by means of flow cytometry, which included 10 subpopulations of the 3 main populations of immune cells and their viability. Milk and blood samples were taken twice a week in the first 100 days after calving and once a week during the remaining lactation period: in total, 55 (52–57) blood and 55 (52–57) milk samples per animal. In addition, six well-established routine laboratory biomarkers, i.e., haptoglobin, calcium, and different metabolic parameters (non-esterified fatty acids, β-hydroxybutyric acid, bilirubin, and glutamate dehydrogenase), were analyzed in all blood samples. Furthermore, a standard differential blood cell count was performed on all blood samples. We found substantial differences between cell count progressions in the blood and milk. The distribution of cell populations in the blood remained mostly stable throughout the lactation, albeit at different individual levels. Several cell populations in the milk showed a noticeable dynamic over time, which caused a separation of different lactation stages in clustering analyses. Gamma delta T cells and CD4+ T cells in the milk stood out as they showed characteristic fluctuations during the course of lactation as well as minor changes in the case of inflammation. The determination of a differential cell count has the potential to be a sensitive diagnostic and prognostic tool in bovine milk. Further studies need to show to what extent the method is suitable for routine diagnostics and how to deal with animal-specific differences.

In this review, we discuss the development pipeline for transcriptional biomarkers in molecular diagnostics and stress the importance of a reliable gene transcript quantification strategy. Hence, a further focus is put on the MIQE guidelines and how to adapt them for biomarker discovery, from signature validation up to routine diagnostic applications. First, the advantages and pitfalls of the holistic RNA sequencing for biomarker development will be described to establish a candidate biomarker signature. Sequentially, the RT-qPCR confirmation process will be discussed to validate the discovered biomarker signature. Examples for the successful application of RT-qPCR as a fast and reproducible quantification method in routinemolecular diagnostics are provided. Based on the MIQE guidelines, the importance of “key steps” in RT-qPCR is accurately described, e.g., reverse transcription, proper reference gene selection and, finally, the application of automated RT-qPCR data analysis software. In conclusion, RT-qPCR proves to be a valuable tool in the establishment of a disease-specific transcriptional biomarker signature and will have a great future in molecular diagnostics or personalized medicine.

Abstract Background New treatment options are needed to prevent relapses following failed antibiotic therapies of Clostridium difficile infections (CDI) in humans. The concomitant therapy with an anti-C. difficile IgA containing whey protein concentrate can support the sustainable recovery of CDI patients. For 31 weeks, nine dairy cows were continuously vaccinated with several anti-C. difficile vaccines by certain routes of administration to produce anti-C. difficile IgA enriched milk. The study aimed at finding decisive differences between low responder (LR) and high responder (HR) cows (> 8.0 µg ml−1 total milk C. difficile specific IgA) concerning their immune response to vaccination on cellular and molecular biological levels. Results The results of total and differential cell counting (DCC) in blood and milk and the outcomes of the gene expression analysis of selected immune factors were assessed relating to the usage of two vaccine batches for injection (MucoCD-I batch A and B), marking two immunization (IM) periods, and compared to a control group (Ctr). The MucoCD-I batch A caused short-term leukopenia followed by leukocytosis in the blood of LR and HR. The total somatic cell counts in milk were not altered by the treatment. The DCC revealed that the leukocytes of the treated groups were partly impaired by the treatment. The gene expression analysis exposed cumulative and sustainable differences (p

The misuse of anabolic hormones or illegal drugs is a ubiquitous problem in animal husbandry and in food safety. The ban on growth promotants in food producing animals in the European Union is well controlled. However, application regimens that are difficult to detect persist, including newly designed anabolic drugs and complex hormone cocktails. Therefore identification of molecular endogenous biomarkers which are based on the physiological response after the illicit treatment has become a focus of detection methods. The analysis of the ‘transcriptome’ has been shown to have promise to discover the misuse of anabolic drugs, by indirect detection of their pharmacological action in organs or selected tissues. Various studies have measured gene expression changes after illegal drug or hormone application. So-called transcriptomic biomarkers were quantified at the mRNA and/or microRNA level by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) technology or by more modern ‘omics’ and high throughput technologies including RNA-sequencing (RNA-Seq). With the addition of advanced bioinformatical approaches such as hierarchical clustering analysis or dynamic principal components analysis, a valid ‘biomarker signature’ can be established to discriminate between treated and untreated individuals. It has been shown in numerous animal and cell culture studies, that identification of treated animals is possible via our transcriptional biomarker approach. The high throughput sequencing approach is also capable of discovering new biomarker candidates and, in combination with quantitative RT-qPCR, validation and confirmation of biomarkers has been possible. These results from animal production and food safety studies demonstrate that analysis of the transcriptome has high potential as a new screening method using transcriptional ‘biomarker signatures’ based on the physiological response triggered by illegal substances.

Abstract Objective Over the last decade, there has been a rapid expansion of the trendy water pipe smoking around the world especially among younger adults. The initial objective of this study was to identify the microbiota of the shisha, which may either be of no harm for the smoker or enhance the threat on his well-being. The total DNA for the metagenomics study was conducted on three different shishas from three different delivery shops in Jounieh, Lebanon. The microbiota in two solid parts of the shisha, shaft and hose, were analysed including the fresh tobacco and the water in the bowl. All samples were analysed using high-throughput sequencing of 16S rRNA gene amplicons. Results Overall, more than 40 bacterial genera were found in the three investigated shishas, some are commensal others are pathogenic. All three shishas showed similar microbial content regarding the bacteria inhabiting in water, shaft, or hose. From the results of this study it appears that a very large quantity of bacteria was found in the water pipes, some are harmful and others beneficial. We assume that the presence of gut dependent microbiota is related to the loose hygienic conditions in which the shisha is prepared.

The most common scoring system for critically ill patients is the Sequential Organ Failure Assessment (SOFA) score. Little is known about specific molecular signaling networks underlying the SOFA criteria. We characterized these networks and identified specific key regulatory molecules. We prospectively studied seven patients with sepsis and six controls with high-throughput RNA sequencing (RNAseq). Quantitative reverse transcription PCR (RT-qPCR) confirmation was performed in a second independent cohort. Differentially and significantly expressed miRNAs and their target mRNA transcripts were filtered for admission SOFA criteria and marker RNAs for the respective criteria identified. We bioinformatically constructed molecular signaling networks specifically reflecting these criteria followed by RT-qPCR confirmation of RNAs with important regulatory functions in the networks in the second cohort. RNAseq identified 82 miRNAs (45% upregulated) and 3254 mRNAs (50% upregulated) differentially expressed between sepsis patients and controls. Bioinformatic analysis characterized 6 miRNAs and 76 mRNA target transcripts specific for the SOFA criteria. RT-qPCR validated miRNA and mRNAs included IGFBP2 (respiratory system); MMP9 and PDE4B (nervous system); PPARG (cardiovascular system); AKR1B1, ANXA1, and LNC2/NGAL (acute kidney injury); GFER/ALR (liver); and miR-30c-3p (coagulopathy). There are specific canonical networks underlying the SOFA score. Key regulatory miRNA and mRNA transcripts support its biologic validity.

Ewing sarcoma (EwS) is an aggressive pediatric cancer of bone and soft tissues characterized by scant T cell infiltration and predominance of immunosuppressive myeloid cells. Given the important roles of extracellular vesicles (EVs) in cancer-host crosstalk, we hypothesized that EVs secreted by EwS tumors target myeloid cells and promote immunosuppressive phenotypes. Here, EVs were purified from EwS and fibroblast cell lines and exhibited characteristics of small EVs, including size (100–170 nm) and exosome markers CD63, CD81, and TSG101. Treatment of healthy donor-derived CD33+ and CD14+ myeloid cells with EwS EVs but not with fibroblast EVs induced pro-inflammatory cytokine release, including IL-6, IL-8, and TNF. Furthermore, EwS EVs impaired differentiation of these cells towards monocytic-derived dendritic cells (moDCs), as evidenced by reduced expression of co-stimulatory molecules CD80, CD86 and HLA-DR. Whole transcriptome analysis revealed activation of gene expression programs associated with immunosuppressive phenotypes and pro-inflammatory responses. Functionally, moDCs differentiated in the presence of EwS EVs inhibited CD4+ and CD8+ T cell proliferation as well as IFNγ release, while inducing secretion of IL-10 and IL-6. Therefore, EwS EVs may promote a local and systemic pro-inflammatory environment and weaken adaptive immunity by impairing the differentiation and function of antigen-presenting cells.

Measurement of cell surface coverage has become a common technique for the assessment of growth behavior of cells. As an indirect measurement method, this can be accomplished by monitoring changes in electrode impedance, which constitutes the basis of electric cell-substrate impedance sensing (ECIS). ECIS typically yields growth curves where impedance is plotted against time, and changes in single cell growth behavior or cell proliferation can be displayed without significantly impacting cell physiology. To provide better comparability of ECIS curves in different experimental settings, we developed a large toolset of R scripts for their transformation and quantification. They allow importing growth curves generated by ECIS systems, edit, transform, graph and analyze them while delivering quantitative data extracted from reference points on the curve. Quantification is implemented through three different curve fit algorithms (smoothing spline, logistic model, segmented regression). From the obtained models, curve reference points such as the first derivative maximum, segmentation knots and area under the curve are then extracted. The scripts were tested for general applicability in real-life cell culture experiments on partly anonymized cell lines, a calibration setup with a cell dilution series of impedance versus seeded cell number and finally IPEC-J2 cells treated with 1% and 5% ethanol.

Extracellular vesicles (EVs) play central physiological and pathophysiological roles in intercellular communication. Biomarker studies addressing disorders such as cardiovascular diseases often focus on circulating microRNAs (miRNAs) and may, depending on the type of disease and clinic routine, utilise patient specimens sampled from arterial or venous blood vessels. Thus, it is essential to test whether circulating miRNA profiles depend on the respective sampling site. We assessed potential differences in arterial and venous cell-free miRNA profiles in a cohort of 20 patients scheduled for cardiac surgery. Prior to surgery, blood was simultaneously sampled from the radial artery and the internal jugular vein. After precipitating crude EVs, we performed small RNA Sequencing, which failed to detect significantly regulated miRNAs using stringent filtering criteria for differential expression analysis. Filtering with less strict criteria, we detected four miRNAs slightly upregulated in arterial samples, one of which could be validated by reverse transcription real-time PCR. The applicability of these findings to purified arterial and venous EVs was subsequently tested in a subset of the initial study population. While an additional clean-up step using size-exclusion chromatography seemed to reduce overall miRNA yield compared to crude EV samples, no miRNAs with differential arteriovenous expression were detected. Unsupervised clustering approaches were unable to correctly classify samples drawn from arteries or veins based on miRNAs in either crude or purified preparations. Particle characterisation of crude preparations as well as characterisation of EV markers in purified EVs resulted in highly similar characteristics for arterial and venous samples. With the exception of specific pathologies (e.g. severe pulmonary disorders), arterial versus venous blood sampling should therefore not represent a likely confounder when studying differentially expressed circulating miRNAs. The use of either arterial or venous serum EV samples should result in highly similar data on miRNA expression profiles for the majority of biomarker studies. Abbreviations ACE inhibitors: Angiotensin-converting-enzyme inhibitors; ApoA1: Apolipoprotein A1; CNX: Calnexin; Cv: Coefficient of variation; cDNA: Complementary DNA; CABG: Coronary artery bypass graft; DGE: Differential gene expression; DPBS: Dulbecco’s Phosphate Buffered Saline; EVs: Extracellular vesicles; log2FC: Log2 fold change; baseMean: Mean miRNA expression; miRNA: MicroRNA; NTA: Nanoparticle Tracking Analysis; NGS: Next-Generation Sequencing; RT-qPCR: Reverse transcription quantitative real-time PCR; rRNA: Ribosomal RNA; RT: Room temperature; SEC: Size-exclusion chromatography; snoRNA: Small nucleolar RNA; snRNA: Small nuclear RNA; small RNA-Seq: Small RNA Sequencing; SD: Standard deviation; tRNA: Transfer RNA; TEM: Transmission electron microscopy; UA: Uranyl acetate.

The aim of this study was to characterize the regulation pattern of prostaglandin family members namely prostaglandin F2alpha (PTGF), prostaglandin E2 (PTGE), their receptors (PTGFR, PTGER2, PTGER4), cyclooxygenase 2 (COX-2), PTGF synthase (PTGFS), and PTGE synthase (PTGES) in the bovine follicles during preovulatory period and early corpus luteum (CL). Ovaries containing preovulatory follicles or CL were collected by transvaginal ovariectomy (n = 5 cows/group), and the follicles were classified: (I) before GnRH treatment; (II) 4 h after GnRH; (III) 10 h after GnRH; (IV) 20 h after GnRH; (V) 25 h after GnRH, and (VI) 60 h after GnRH (early CL). In these samples, the concentrations of progesterone (P4), estradiol (E2), PTGF and PTGE were investigated in the follicular fluid (FF) by validated EIA. Relative mRNA abundance of genes encoding for prostaglandin receptors (PTGFR, PTGER2, PTGER4), COX-2, PTGFS and PTGES were quantified by RT-qPCR. The localization of COX-2 and PTGES were investigated by established immunohistochemistry in fixed follicular and CL tissue samples. The high E2 concentration in the FF of the follicle group before GnRH treatment (495.8 ng/ml) and during luteinizing hormone (LH) surge (4 h after GnRH, 574.36 ng/ml), is followed by a significant (P

Clostridioides difficile infection (CDI) often develops after pretreatment with antibiotics, which can lead to damage of the intestinal microbiome. The approach of this study was to use specific polyclonal antibodies isolated from the milk of immunized cows to treat CDI, in contrast to the standard application of nonspecific antibiotics. To gain a deeper understanding of the role of the microbiome in the treatment of CDI with bovine antibodies, stool and intestinal fluid samples of hamsters were collected in large quantities from various treatments (>400 samples). The results show that the regeneration of the microbiome instantly begins with the start of the antibody treatment, in contrast to the Vancomycin-treated group where the diversity decreased significantly during the treatment duration. All antibody-treated hamsters that survived the initial phase also survived the entire study period. The results also show that the regeneration of the microbiome was not an antibody-induced regeneration, but a natural regeneration that occurred because no microbiota-inactivating substances were administered. In conclusion, the treatment with bovine antibodies is a functional therapy for both the acute treatment and the prevention of recurrence in hamsters and could meet the urgent need for CDI treatment alternatives in humans.

Extracellular vesicles (EVs) are intercellular communicators with key functions in physiological and pathological processes and have recently garnered interest because of their diagnostic and therapeutic potential. The past decade has brought about the development and commercialization of a wide array of methods to isolate EVs from serum. Which subpopulations of EVs are captured strongly depends on the isolation method, which in turn determines how suitable resulting samples are for various downstream applications. To help clinicians and scientists choose the most appropriate approach for their experiments, isolation methods need to be comparatively characterized. Few attempts have been made to comprehensively analyse vesicular microRNAs (miRNAs) in patient biofluids for biomarker studies. To address this discrepancy, we set out to benchmark the performance of several isolation principles for serum EVs in healthy individuals and critically ill patients. Here, we compared five different methods of EV isolation in combination with two RNA extraction methods regarding their suitability for biomarker discovery-focused miRNA sequencing as well as biological characteristics of captured vesicles. Our findings reveal striking method-specific differences in both the properties of isolated vesicles and the ability of associated miRNAs to serve in biomarker research. While isolation by precipitation and membrane affinity was highly suitable for miRNA-based biomarker discovery, methods based on size-exclusion chromatography failed to separate patients from healthy volunteers. Isolated vesicles differed in size, quantity, purity and composition, indicating that each method captured distinctive populations of EVs as well as additional contaminants. Even though the focus of this work was on transcriptomic profiling of EV-miRNAs, our insights also apply to additional areas of research. We provide guidance for navigating the multitude of EV isolation methods available today and help researchers and clinicians make an informed choice about which strategy to use for experiments involving critically ill patients.

The release of RNA-containing extracellular vesicles (EV) into the extracellular milieu has been demonstrated in a multitude of different in vitro cell systems and in a variety of body fluids. RNA-containing EV are in the limelight for their capacity to communicate genetically encoded messages to other cells, their suitability as candidate biomarkers for diseases, and their use as therapeutic agents. Although EV-RNA has attracted enormous interest from basic researchers, clinicians, and industry, we currently have limited knowledge on which mechanisms drive and regulate RNA incorporation into EV and on how RNA-encoded messages affect signalling processes in EV-targeted cells. Moreover, EV-RNA research faces various technical challenges, such as standardisation of EV isolation methods, optimisation of methodologies to isolate and characterise minute quantities of RNA found in EV, and development of approaches to demonstrate functional transfer of EV-RNA in vivo. These topics were discussed at the 2015 EV-RNA workshop of the International Society for Extracellular Vesicles. This position paper was written by the participants of the workshop not only to give an overview of the current state of knowledge in the field, but also to clarify that our incomplete knowledge – of the nature of EV(-RNA)s and of how to effectively and reliably study them – currently prohibits the implementation of gold standards in EV-RNA research. In addition, this paper creates awareness of possibilities and limitations of currently used strategies to investigate EV-RNA and calls for caution in interpretation of the obtained data.

In the past years, extracellular vesicles (EVs) have become an important field of research since EVs have been found to play a central role in biological processes. In pathogens, EVs are involved in several events during the host–pathogen interaction, including invasion, immunomodulation, and pathology as well as parasite–parasite communication. In this report, we summarised the role of EVs in infections caused by viruses, bacteria, fungi, protozoa, and helminths based on the talks and discussions carried out during the International Society for Extracellular Vesicles (ISEV) workshop held in São Paulo (November, 2016), Brazil, entitled Cross-organism Communication by Extracellular Vesicles: Hosts, Microbes and Parasites.

The formation, function, and subsequent regression of the ovarian corpus luteum (CL) are dynamic processes that enable ovary cyclical activity. Studies in whole ovary tissue have found microRNAs (miRNAs) to by critical for ovary function. However, relatively little is known about the role of miRNAs in the bovine CL. Utilizing small RNA next-generation sequencing we profiled miRNA transcriptome in bovine CL during the entire physiological estrous cycle, by sampling the CL on days: d 1–2, d 3–4, and d 5–7 (early CL, eCL), d 8–12 (mid CL, mCL), d 13–16 (late CL, lCL), and d > 18 (regressed CL, rCL). We characterized patterns of miRNAs abundance and identified 42 miRNAs that were consistent significantly different expressed (DE) in the eCL relative to their expression at each of the analyzed stages (mCL, lCL, and rCL). Out of these, bta-miR-210-3p, −2898, −96, −7-5p, −183-5p, −182, and −202 showed drastic up-regulation with a fold-change of ≥2.0 and adjusted P < 0.01 in the eCL, while bta-miR-146a was downregulated at lCL and rCL vs. the eCL. Another 24, 11, and 21 miRNAs were significantly DE only between individual comparisons, eCL vs. the mCL, lCL, and rCL, respectively. Irrespective of cycle stage two miRNAs, bta-miR-21-5p and bta-miR-143 were identified as the most abundant miRNAs species and show opposing expression abundance. Whilst bta-miR-21-5p peaked in number of reads in the eCL and was significantly downregulated in the mCL and lCL, bta-miR-143 reached its peak in the rCL and is significantly downregulated in the eCL. MiRNAs with significant DE in at least one cycle stage (CL class) were further grouped into eight distinct clusters by the self-organizing tree algorithm (SOTA). Half of the clusters contain miRNAs with low-expression, whilst the other half contain miRNAs with high-expression levels during eCL. Prediction analysis for significantly DE miRNAs resulted in target genes involved with CL formation, functionalization and CL regression. This study is the most comprehensive profiling of miRNA transcriptome in bovine CL covering the entire estrous cycle and provides a compact database for further functional validation and biomarker identification relevant for CL viability and fertility.

Toxin-induced Clostridium difficile infection (CDI) is a major disease characterized by severe diarrhea and high morbidity rates. The aim with this study was to develop an alternative drug for the treatment of CDI. Cows were repeatedly immunized to establish specific immunoglobulin G and A titers against toxins A (TcdA) and B (TcdB) and against C. difficile cells in mature milk or colostrum. The effect of three different concentrations of anti-C. difficile whey protein isolates (anti-CD-WPI) and the standard of care antibiotic vancomycin were investigated in an animal model of CD infected hamsters (6 groups, with 10 hamsters each). WPI obtained from the milk of exactly the same cows pre-immunization and a vehicle group served as negative controls. The survival of hamsters receiving anti-CD-WPI was 50, 80 and 100% compared to 10 and 0% for the control groups, respectively. Vancomycin suppressed the growth of C. difficile and thus protected the hamsters at the time of administration, but 90% of these hamsters nevertheless died shortly after discontinuation of treatment. In contrast, the surviving hamsters of the anti-CD-WPI groups survived the entire study period, although they were treated for only 75 h. The specific antibodies not only inactivated the toxins for initial suppression of CDI, but also provoked the inhibition of C. difficile growth after discontinuation, thus preventing recurrence. Oral administration of anti-CD-WPI is a functional therapy of CDI in infected hamsters for both primary treatment and prevention of recurrence. Thus, anti-CD-WPI could address the urgent unmet medical need for treating and preventing recurrent CDI in humans.

The molecular mechanisms underlying the selective neurodegeneration of motor neurons in amyotrophic lateral sclerosis (ALS) are inadequately understood. Recent breakthroughs have implicated impaired axonal transport, mediated by molecular motors, as a key element for disease onset and progression. The current work identifies the expression of 15 kinesin-like motors in healthy human motor cortex, including three novel isoforms. Our comprehensive quantitative mRNA analysis in control and sporadic ALS (SALS) motor cortex specimens detects SALS-specific down-regulation of KIF1Bβ and novel KIF3Aβ, two isoforms we show to be enriched in the brain, and also of SOD1, a key enzyme linked to familial ALS. This is accompanied by a marked reduction of KIF3Aβ protein levels. In the motor cortex KIF3Aβ localizes in cholinergic neurons, including upper motor neurons. No mutations causing splicing defects or altering protein-coding sequences were identified in the genes of the three proteins. The present study implicates two motor proteins as possible candidates in SALS pathology.