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Common pregnancy detection methods in dairy cattle have limits in sensitivity and timing. Thus, we aimed at finding new early pregnancy biomarkers in milk. A previous study in milk cells and skim milk suggested the possible use of several miRNAs for this purpose. In order to make the sampling procedure more convenient for daily use and to include the milk fat into analysis, here, a similar study was repeated using whole fresh milk of days 4, 12, 18 and 21 of pregnancy and non-pregnant day-matched controls, respectively. The microRNAs (miRNAs) were extracted from milk samples and sequenced on a high throughput sequencer (RNAseq). Promising miRNAs were validated via RT-qPCR. Several miRNAs were significantly regulated and six miRNAs (bta-miR-221, bta-miR-223, bta-miR-93, bta-miR-200c, bta-miR-125b and the closely related bta-miR-15b) showed a continuous trend, similar as in the milk cells of the previous study. However, pregnant and cyclic animals could not be separated completely and RT-qPCR validation of the RNAseq data was unsuccessful. Thus, miRNA of bovine whole fresh milk samples failed to show suitability for early pregnancy diagnosis.

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., Largely unexpected, only 78 of 1227 tested cancer patients showed a COVID‐19 infection of which only three patients were symptomatic, ranging from mild symptoms to pneumonia. These data clearly contrast the view that oncology patients are particularly vulnerable to SARS‐CoV‐2 and suggest that necessary chemotherapies could be continued or started despite the ongoing pandemic. One explanation could be the immunosuppressive effect of simultaneous systemic therapy which will be further investigated.

Porcine conceptuses synthesize estrogens between Day 11 and 12 as signal for maternal recognition of pregnancy. A preimplantational estrogen exposure to pregnant gilts has been associated with embryonic losses and changes in endometrial mRNA expression. MicroRNAs (miRNAs) play a key role in the mRNA regulation by modulating the expression. Effects of estrogens on endometrial miRNAs have not been investigated in this context so far. Thus, we studied the endometrial expression profile of miRNAs in the pig at gestational Day 10 after daily estradiol-17β (E2) application starting at fertilization using either 0, 0.05 (ADI-acceptable daily intake), 10 (NOEL-no-observed-effect level) and 1,000 (high dose) µg E2/kg body weight/day, respectively. In endometrial homogenates, E2 (p < 0.001) and total estrogen concentrations (p < 0.001) were significantly increased, namely 28- and 160-fold, respectively, in the high dose group as compared to the control. Additionally, total estrogens were sixfold elevated in the NOEL group. Interestingly, high-throughput sequencing of small non-coding RNA libraries did not indicate any differentially expressed miRNAs between the treatment groups and the control group. The expression of 12 potential E2 target miRNAs investigated by RT-qPCR were equally unaffected. Thus, preimplantational E2 exposure resulted in significantly higher endometrial estrogen concentrations, but did not perturb the expression profile of endometrial miRNAs.

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.

Septic shock is a common medical condition with a mortality approaching 50% where early diagnosis and treatment are of particular importance for patient survival. Novel biomarkers that serve as prompt indicators of sepsis are urgently needed. High‐throughput technologies assessing circulating microRNAs represent an important tool for biomarker identification, but the blood‐compartment specificity of these miRNAs has not yet been investigated. We characterized miRNA profiles from serum exosomes, total serum and blood cells (leukocytes, erythrocytes, platelets) of sepsis patients by next‐generation sequencing and RT‐qPCR (n = 3 × 22) and established differences in miRNA expression between blood compartments. In silico analysis was used to identify compartment‐specific signalling functions of differentially regulated miRNAs in sepsis‐relevant pathways. In septic shock, a total of 77 and 103 miRNAs were down‐ and up‐regulated, respectively. A majority of these regulated miRNAs (14 in serum, 32 in exosomes and 73 in blood cells) had not been previously associated with sepsis. We found a distinctly compartment‐specific regulation of miRNAs between sepsis patients and healthy volunteers. Blood cellular miR‐199b‐5p was identified as a potential early indicator for sepsis and septic shock. miR‐125b‐5p and miR‐26b‐5p were uniquely regulated in exosomes and serum, respectively, while one miRNA (miR‐27b‐3p) was present in all three compartments. The expression of sepsis‐associated miRNAs is compartment‐specific. Exosome‐derived miRNAs contribute significant information regarding sepsis diagnosis and survival prediction and could serve as newly identified targets for the development of novel sepsis biomarkers.

Background Extracellular vesicles (EVs) such as exosomes are key regulators of intercellular communication that can be found in almost all bio fluids. Although studies in the last decade have made great headway in discerning the role of EVs in many physiological and pathophysiological processes, the bioavailability and impact of dietary EVs and their cargo still remain to be elucidated. Due to its widespread consumption and high content of EV-associated microRNAs and proteins, a major focus in this field has been set on EVs in bovine milk and colostrum. Despite promising in vitro studies in recent years that show high resiliency of milk EVs to degradation and uptake of milk EV cargo in a variety of intestinal and blood cell types, in vivo experiments continue to be inconclusive and sometimes outright contradictive. Results To resolve this discrepancy, we assessed the potential postprandial transfer of colostral EVs to the circulation of newborn calves by analysing colostrum-specific protein and miRNAs, including specific isoforms (isomiRs) in cells, EV isolations and unfractionated samples from blood and colostrum. Our findings reveal distinct populations of EVs in colostrum and blood from cows that can be clearly separated by density, particle concentration and protein content (BTN1A1, MFGE8). Postprandial blood samples of calves show a time-dependent increase in EVs that share morphological and protein characteristics of colostral EVs. Analysis of miRNA expression profiles by Next-Generation Sequencing gave a different picture however. Although significant postprandial expression changes could only be detected for calf EV samples, expression profiles show very limited overlap with highly expressed miRNAs in colostral EVs or colostrum in general. Conclusions Taken together our results indicate a selective uptake of membrane-associated protein cargo but not luminal miRNAs from colostral EVs into the circulation of neonatal calves.

The low prevalence of asthma and allergies in farm children has partially been ascribed to the consumption of raw cow's milk. A literature search identified 12 publications on 8 pertinent studies. A meta-analysis corroborated the protective effect of raw milk consumption early in life (

This report summarises the presentations and activities of the ISEV Workshop on extracellular vesicle biomarkers held in Birmingham, UK during December 2017. Among the key messages was broad agreement about the importance of biospecimen science. Much greater attention needs to be paid towards the provenance of collected samples. The workshop also highlighted clear gaps in our knowledge about pre-analytical factors that alter extracellular vesicles (EVs). The future utility of certified standards for credentialing of instruments and software, to analyse EV and for tracking the influence of isolation steps on the structure and content of EVs were also discussed. Several example studies were presented, demonstrating the potential utility for EVs in disease diagnosis, prognosis, longitudinal serial testing and stratification of patients. The conclusion of the workshop was that more effort focused on pre-analytical issues and benchmarking of isolation methods is needed to strengthen collaborations and advance more effective biomarkers.

Molecular checkpoints that trigger the onset of islet autoimmunity or progression to human type 1 diabetes (T1D) are incompletely understood. Using T cells from children at an early stage of islet autoimmunity without clinical T1D, we find that a microRNA181a (miRNA181a)-mediated increase in signal strength of stimulation and costimulation links nuclear factor of activated T cells 5 (NFAT5) with impaired tolerance induction and autoimmune activation. We show that enhancing miRNA181a activity increases NFAT5 expression while inhibiting FOXP3+ regulatory T cell (Treg) induction in vitro. Accordingly, Treg induction is improved using T cells from NFAT5 knockout (NFAT5ko) animals, whereas altering miRNA181a activity does not affect Treg induction in NFAT5ko T cells. Moreover, high costimulatory signals result in phosphoinositide 3-kinase (PI3K)-mediated NFAT5, which interferes with FoxP3+ Treg induction. Blocking miRNA181a or NFAT5 increases Treg induction in murine and humanized models and reduces murine islet autoimmunity in vivo. These findings suggest targeting miRNA181a and/or NFAT5 signaling for the development of innovative personalized medicines to limit islet autoimmunity.

Triple-negative breast cancer (TNBC) is a particularly aggressive subtype of breast cancer with limited options for clinical intervention. As with many solid tumors, TNBC is known to promote invasiveness and metastasis by secreting extracellular vesicles (EVs) capable of modulating the behaviour of recipient cells. Recent investigations have demonstrated that high expression levels of glucocorticoid receptor (GR) in TNBC are linked to therapy resistance, higher recurrence rates and increased mortality. In addition to activating protein-coding genes, GR is also involved in the expression of short non-coding RNAs including microRNAs (miRNAs or miRs). The molecular mechanisms responsible for the oncogenic effects of GR on TNBC have yet to be fully elucidated; however, emerging evidence suggests that miRNAs may play a pivotal role in tumorigenesis and metastasis. Thus, the aim of this study was to identify GR-regulated cellular and vesicular miRNAs that might contribute to the particularly oncogenic phenotype of TNBC with a high GR expression. We analyzed miRNA profiles of three TNBC cell lines using an in vitro model of GR overexpression. Next-generation sequencing revealed minor, cell line-specific changes in cellular miRNA expression, whereas vesicular miRNAs were not significantly regulated by GR. Additionally, the analysis of predicted miRNA targets failed to establish a causal link between GR-induced miRNA expression and oncogenic signaling. On the whole, given that GR influences miRNA profiles to only a small degree, other mechanisms are more likely to be responsible for the increased mortality of patients with TNBC with a high GR expression.

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., Frontiers in Genetics, 8, ISSN:1664-8021

Aberrant immune activation mediated by T effector cell populations is pivotal in the onset of autoimmunity in type 1 diabetes (T1D). T follicular helper (TFH) cells are essential in the induction of high-affinity antibodies, and their precursor memory compartment circulates in the blood. The role of TFH precursors in the onset of islet autoimmunity and signaling pathways regulating their differentiation is incompletely understood. Here, we provide direct evidence that during onset of islet autoimmunity, the insulin-specific target T-cell population is enriched with a C-X-C chemokine receptor type 5 (CXCR5)(+)CD4(+) TFH precursor phenotype. During onset of islet autoimmunity, the frequency of TFH precursors was controlled by high expression of microRNA92a (miRNA92a). miRNA92a-mediated TFH precursor induction was regulated by phosphatase and tension homolog (PTEN) - phosphoinositol-3-kinase (PI3K) signaling involving PTEN and forkhead box protein O1 (Foxo1), supporting autoantibody generation and triggering the onset of islet autoimmunity. Moreover, we identify Krueppel-like factor 2 (KLF2) as a target of miRNA92a in regulating human TFH precursor induction. Importantly, a miRNA92a antagomir completely blocked induction of human TFH precursors in vitro. More importantly, in vivo application of a miRNA92a antagomir to nonobese diabetic (NOD) mice with ongoing islet autoimmunity resulted in a significant reduction of TFH precursors in peripheral blood and pancreatic lymph nodes. Moreover, miRNA92a antagomir application reduced immune infiltration and activation in pancreata of NOD mice as well as humanized NOD Scid IL2 receptor gamma chain knockout (NSG) human leucocyte antigen (HLA)-DQ8 transgenic animals. We therefore propose that miRNA92a and the PTEN-PI3K-KLF2 signaling network could function as targets for innovative precision medicines to reduce T1D islet autoimmunity.

In quantitative single-cell studies, the critical part is the low amount of nucleic acids present and the resulting experimental variations. In addition biological data obtained from heterogeneous tissue are not reflecting the expression behaviour of every single-cell. These variations can be derived from natural biological variance or can be introduced externally. Both have negative effects on the quantification result. The aim of this study is to make quantitative single-cell studies more transparent and reliable in order to fulfil the MIQE guidelines at the single-cell level. The technical variability introduced by RT, pre-amplification, evaporation, biological material and qPCR itself was evaluated by using RNA or DNA standards. Secondly, the biological expression variances of GAPDH, TNFα, IL-1β, TLR4 were measured by mRNA profiling experiment in single lymphocytes. The used quantification setup was sensitive enough to detect single standard copies and transcripts out of one solitary cell. Most variability was introduced by RT, followed by evaporation, and pre-amplification. The qPCR analysis and the biological matrix introduced only minor variability. Both conducted studies impressively demonstrate the heterogeneity of expression patterns in individual cells and showed clearly today's limitation in quantitative single-cell expression analysis.