Expression of SPAG7 and its regulatory microRNAs in seminal plasma and seminal plasma-derived extracellular vesicles of patients with subfertility

Seminal plasma contains a variety of extracellular vesicles (EVs) that deliver RNAs including microRNAs (miRNAs) molecules. However, the roles of these EVs along with their delivered RNAs and their interactions with male infertility are not clear. Sperm-associated antigen 7 (SPAG 7) is expressed in male germ cells and plays a crucial role in several biological functions associated with sperm production and maturation. In this study, we aimed to identify the post-transcriptional regulation of SPAG7 in seminal plasma (SF-Native) and seminal plasma-derived extracellular vesicles (SF-EVs) collected from 87 men undergoing infertility treatment. Among the multiple binding sites for miRNAs within its 3’UTR of SPAG7, we identifed the binding of four miRNAs (miR-15b-5p, miR195-5p, miR-424-5p, and miR-497-5p) to the 3’UTR of SPAG7 by the dual luciferase assays. Analyzing sperm, we found reduced mRNA expression levels of SPAG7 in SF-EVs and SF-Native samples from oligoasthenozoospermic men. By contrast, two miRNAs (miR-424-5p and miR-497-5p) form the SF-Native samples, and four miRNAs (miR-195-5p, miR-424-5p, miR-497-5p, and miR-6838-5p) from the SF-EVs samples showed signifcantly higher expression levels in oligoasthenozoospermic men. The expression levels of miRNAs and SPAG7 were signifcantly correlated with basic semen parameters. These fndings contribute signifcantly to our understanding of regulatory pathways in male fertility by showing a direct link between upregulated miRNA, notably miR-424, and downregulated SPAG7 both in seminal plasma and in plasma-derived EVs likely contributing to oligoasthenozoospermia

Wrinkle in the plan: miR-34a-5p impacts chemokine signaling by modulating CXCL10/CXCL11/CXCR3-axis in CD4+, CD8+ T cells, and M1 macrophages

Background In 2016 the first-in-human phase I study of a miRNA-based cancer therapy with a liposomal mimic of microRNA-34a-5p (miR-34a-5p) was closed due to five immune related serious adverse events (SAEs) resulting in four patient deaths. For future applications of miRNA mimics in cancer therapy it is mandatory to unravel the miRNA effects both on the tumor tissue and on immune cells. Here, we set out to analyze the impact of miR-34a-5p over-expression on the CXCL10/CXCL11/CXCR3 axis, which is central for the development of an effective cancer control. Methods We performed a whole genome expression analysis of miR-34a-5p transfected M1 macrophages followed by an over-representation and a protein–protein network analysis. In-silico miRNA target prediction and dual luciferase assays were used for target identification and verification. Target genes involved in chemokine signaling were functionally analyzed in M1 macrophages, CD4+ and CD8+ T cells. Results A whole genome expression analysis of M1 macrophages with induced miR-34a-5p over-expression revealed an interaction network of downregulated target mRNAs including CXCL10 and CXCL11. In-silico target prediction in combination with dual luciferase assays identified direct binding of miR-34a-5p to the 3′UTRs of CXCL10 and CXCL11. Decreased CXCL10 and CXCL11 secretion was shown on the endogenous protein level and in the supernatant of miR-34a-5p transfected and activated M1 macrophages. To complete the analysis of the CXCL10/CXCL11/CXCR3 axis, we activated miR-34a-5p transfected CD4+ and CD8+ T cells by PMA/Ionomycin and found reduced levels of endogenous CXCR3 and CXCR3 on the cell surface. Conclusions MiR-34a-5p mimic administered by intravenous administration will likely not only be up-taken by the tumor cells but also by the immune cells. Our results indicate that miR-34a-5p over-expression leads in M1 macrophages to a reduced secretion of CXCL10 and CXCL11 chemokines and in CD4+ and CD8+ T cells to a reduced expression of CXCR3. As a result, less immune cells will be attracted to the tumor site. Furthermore, high levels of miR-34a-5p in naive CD4+ T cells can in turn hinder Th1 cell polarization through the downregulation of CXCR3 leading to a less pronounced activation of cytotoxic T lymphocytes, natural killer, and natural killer T cells and possibly contributing to lymphocytopenia

CoolMPS: evaluation of antibody labeling based massively parallel non-coding RNA sequencing

Results of massive parallel sequencing-by-synthesis vary depending on the sequencing approach. CoolMPS™ is a new sequencing chemistry that incorporates bases by labeled antibodies. To evaluate the performance, we sequenced 240 human non-coding RNA samples (dementia patients and controls) with and without CoolMPS. The Q30 value as indicator of the per base sequencing quality increased from 91.8 to 94%. The higher quality was reached across the whole read length. Likewise, the percentage of reads mapping to the human genome increased from 84.9 to 86.2%. For both technologies, we computed similar distributions between different RNA classes (miRNA, piRNA, tRNA, snoRNA and yRNA) and within the classes. While standard sequencing-by-synthesis allowed to recover more annotated miRNAs, CoolMPS yielded more novel miRNAs. The correlation between the two methods was 0.97. Evaluating the diagnostic performance, we observed lower minimal P-values for CoolMPS (adjusted P-value of 0.0006 versus 0.0004) and larger effect sizes (Cohen's d of 0.878 versus 0.9). Validating 19 miRNAs resulted in a correlation of 0.852 between CoolMPS and reverse transcriptase-quantitative polymerase chain reaction. Comparison to data generated with Illumina technology confirmed a known shift in the overall RNA composition. With CoolMPS we evaluated a novel sequencing-by-synthesis technology showing high performance for the analysis of non-coding RNAs

miRNATissueAtlas2: an update to the human miRNA tissue atlas

Small non-coding RNAs (sncRNAs) are pervasive regulators of physiological and pathological processes. We previously developed the human miRNA Tissue Atlas, detailing the expression of miRNAs across organs in the human body. Here, we present an updated resource containing sequencing data of 188 tissue samples comprising 21 organ types retrieved from six humans. Sampling the organs from the same bodies minimizes intra-individual variability and facilitates the making of a precise highresolution body map of the non-coding transcriptome. The data allow shedding light on the organ- and organ system-specificity of piwi-interacting RNAs (piRNAs), transfer RNAs (tRNAs), microRNAs (miRNAs) and other non-coding RNAs. As use case of our resource, we describe the identification of highly specific ncRNAs in different organs. The update also contains 58 samples from six tissues of the Tabula Muris collection, allowing to check if the tissue specificity is evolutionary conserved between Homo sapiens and Mus musculus. The updated resource of 87 252 non-coding RNAs from nine noncoding RNA classes for all organs and organ systems is available online without any restrictions (https: //www.ccb.uni-saarland.de/tissueatlas2)

Chemoradiotherapy-induced increase in Th17 cell frequency in cervical cancer patients is associated with therapy resistance and early relapse

Cervical cancer therapy is still a major clinical challenge, as patients substantially differ in their response to standard treatments, including chemoradiotherapy (CRT). During cervical carcinogenesis, T-helper (Th)-17 cells accumulate in the peripheral blood and tumor tissues of cancer patients and are associated with poor prognosis. In this prospective study, we find increased Th17 frequencies in the blood of patients after chemoradiotherapy and a post-therapeutic ratio of Th17/CD4+ T cells > 8% was associated with early recurrence. Furthermore, Th17 cells promote resistance of cervical cancer cells toward CRT, which was dependent on the AKT signaling pathway. Consistently, patients with high Th17 frequencies in pretherapeutic biopsies exhibit lower response to primary CRT. This work reveals a key role of Th17 cells in CRT resistance and elevated Th17 frequencies in the blood after CRT correspond with early recurrence. Our results may help to explain individual treatment responses of cervical cancer patients and suggest evaluation of Th17 cells as a novel predictive biomarker for chemoradiotherapy responses and as a potential target for immunotherapy in cervical cancer

Ageing-associated small RNA cargo of extracellular vesicles

Previous work on murine models and humans demonstrated global as well as tissue-specific molecular ageing trajectories of RNAs. Extracellular vesicles (EVs) are membrane vesicles mediating the horizontal transfer of genetic information between different tissues. We sequenced small regulatory RNAs (sncRNAs) in two mouse plasma fractions at five time points across the lifespan from 2–18 months: (1) sncRNAs that are free-circulating (fc-RNA) and (2) sncRNAs bound outside or inside EVs (EV-RNA). Different sncRNA classes exhibit unique ageing patterns that vary between the fcRNA and EV-RNA fractions. While tRNAs showed the highest correlation with ageing in both fractions, rRNAs exhibited inverse correlation trajectories between the EV- and fc-fractions. For miRNAs, the EV-RNA fraction was exceptionally strongly associated with ageing, especially the miR-29 family in adipose tissues. Sequencing of sncRNAs and coding genes in fat tissue of an independent cohort of aged mice up to 27 months highlighted the pivotal role of miR-29a-3p and miR-29b-3p in ageing-related gene regulation that we validated in a third cohort by RT-qPCR

Ageing-associated small RNA cargo of extracellular vesicles

Previous work on murine models and humans demonstrated global as well as tissue-specific molecular ageing trajectories of RNAs. Extracellular vesicles (EVs) are membrane vesicles mediating the horizontal transfer of genetic information between different tissues. We sequenced small regulatory RNAs (sncRNAs) in two mouse plasma fractions at five time points across the lifespan from 2–18 months: (1) sncRNAs that are free-circulating (fc-RNA) and (2) sncRNAs bound outside or inside EVs (EV-RNA). Different sncRNA classes exhibit unique ageing patterns that vary between the fcRNA and EV-RNA fractions. While tRNAs showed the highest correlation with ageing in both fractions, rRNAs exhibited inverse correlation trajectories between the EV- and fc-fractions. For miRNAs, the EV-RNA fraction was exceptionally strongly associated with ageing, especially the miR-29 family in adipose tissues. Sequencing of sncRNAs and coding genes in fat tissue of an independent cohort of aged mice up to 27 months highlighted the pivotal role of miR-29a-3p and miR-29b-3p in ageing-related gene regulation that we validated in a third cohort by RT-qPCR

The Quiescent Intracluster Medium in the Core of the Perseus Cluster

Clusters of galaxies are the most massive gravitationally-bound objects in the Universe and are still forming. They are thus important probes of cosmological parameters and a host of astrophysical processes. Knowledge of the dynamics of the pervasive hot gas, which dominates in mass over stars in a cluster, is a crucial missing ingredient. It can enable new insights into mechanical energy injection by the central supermassive black hole and the use of hydrostatic equilibrium for the determination of cluster masses. X-rays from the core of the Perseus cluster are emitted by the 50 million K diffuse hot plasma filling its gravitational potential well. The Active Galactic Nucleus of the central galaxy NGC1275 is pumping jetted energy into the surrounding intracluster medium, creating buoyant bubbles filled with relativistic plasma. These likely induce motions in the intracluster medium and heat the inner gas preventing runaway radiative cooling; a process known as Active Galactic Nucleus Feedback. Here we report on Hitomi X-ray observations of the Perseus cluster core, which reveal a remarkably quiescent atmosphere where the gas has a line-of-sight velocity dispersion of 164+/-10 km/s in a region 30-60 kpc from the central nucleus. A gradient in the line-of-sight velocity of 150+/-70 km/s is found across the 60 kpc image of the cluster core. Turbulent pressure support in the gas is 4% or less of the thermodynamic pressure, with large scale shear at most doubling that estimate. We infer that total cluster masses determined from hydrostatic equilibrium in the central regions need little correction for turbulent pressure.Comment: 31 pages, 11 Figs, published in Nature July

Socio-economic impact of ecological agriculture at the territorial level

This deliverable investigates the socio-economic effects of ecological approaches to farming through implementing two participatory approaches, namely Delphi exercise and Q-method, at the level of a case study area (CSA). The focus is on how people and other productive assets are employed and remunerated by ecological approaches to agriculture, particularly those aspects that can influence employment, and drive the prosperity and vitality of local communities and some rural businesses. It is based on the collaborative research on Task 4.2 ‘Socio-economic impact of ecological agriculture at the territorial level’ of the LIFT project between UNIKENT (United Kingdom-UK) (Task Leader), BOKU (Austria), INRAE (France), VetAgro Sup (France), DEMETER (Greece), MTA KRTK (Hungary), UNIBO (Italy), IRWiR PAN (Poland), IAE-AR (Romania), SLU (Sweden), SRUC (UK). Beginning with the Delphi exercise, this deliverable presents qualitative information extracted from stakeholders in the following four steps. First, the researchers build a presentation of differences between ecological and conventional farming approaches in each CSA. Second, stakeholders elaborate on how they understand ecological farming approaches to exist in each CSA. Third, stakeholders develop a scenario of adoption of ecological approaches to farming depending on two factors: pattern (ecological farms forming clusters or randomly spread within the territory) and rate of adoption 10 years in the future. After establishing this scenario across two rounds, the stakeholders explore the socio-economic effects of their adoption scenario. The Q-methodology then presents a Q-set of statements that the Delphi has developed and, through factor analysis,studies the key stakeholder perspectives of the socio-economic effects of the perceived adoption of ecological practices in 10 years in the future. Four key results can be derived from the Delphi exercise and the Q-methodology. First, a higher adoption of ecological farming approaches, especially so at a 50% adoption rate, is mostly thought by stakeholders in the Delphi Exercise to lead to an increase in skill level and quality of life in on-farm employment. This is as a result of an increased diversity of farming enterprises on farms using ecological farming approaches, the interest generated from this, the knowledge of natural processes and biology required, engagement with nature and change in machinery that is coming into the industry. Strongly related to this need for skills is a predicted increase in the number of advisers and civil servants to deal with more complicated farms and incentives as well as monitoring of ecological effects on farm. An increase in required skill level is repeated across all Q-studies. Second, especially where farms are clustered together, Delphi Exercise respondents predict an increase in the trade of inputs such as manure and compost replacing synthetic fertiliser, as well as more sharing of capital and labour. Q-methodology highlights that these clusters may support a stronger social movement, more consumers buying local food and increase collaboration between farmers. Supply chains are expected to become shorter as farmers sell more directly and there are fewer intermediaries upstream of the farming sector. As farmers collaborate more with each other on environmental objectives, trading inputs and sharing best practices, farmer relationships should improve in rural communities. Third, Delphi exercise finds that contracting, machinery purchasers, and machinery traders and dealers could increase, decrease or display no change – the anticipated effects are mixed. Stakeholders are in no doubt that machinery use will change and therefore new skills will need to be learnt, but the wider effect on machinery purchase is uncertain. However, stakeholders conclude that a greater specialisation in machinery will occur leading to changes in farm management as well as the suppliers of this machinery. Q-methodology highlights that ecological practices will not mean the end of machinery and a lot more labour – often machinery will be useful in weeding and reducing physical labour as technology has significantly improved and skills are improving too in order to use these technologies. Fourth, Delphi respondents argued that although rural populations might be little affected by ecological farming, a shift in people moving from urban to rural settlements, and thereby a higher rural population density, seeking a more attractive rural environment, might contribute to higher local consumer demand. The Q-methodology highlights that where there is high adoption, rural areas are expected to become more attractive, as landscapes will have a much greater variety of crops instead of fields of monocrops. This variety of crops may include agroforestry (farmers interested in ecological approaches to farming may also be interested in agroforestry as a way of boosting their yields and protecting crops and livestock from the elements) as well as intercropping