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

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)

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

Power estimation for non-standardized multisite studies

AbstractA concern for researchers planning multisite studies is that scanner and T1-weighted sequence-related biases on regional volumes could overshadow true effects, especially for studies with a heterogeneous set of scanners and sequences. Current approaches attempt to harmonize data by standardizing hardware, pulse sequences, and protocols, or by calibrating across sites using phantom-based corrections to ensure the same raw image intensities. We propose to avoid harmonization and phantom-based correction entirely. We hypothesized that the bias of estimated regional volumes is scaled between sites due to the contrast and gradient distortion differences between scanners and sequences. Given this assumption, we provide a new statistical framework and derive a power equation to define inclusion criteria for a set of sites based on the variability of their scaling factors. We estimated the scaling factors of 20 scanners with heterogeneous hardware and sequence parameters by scanning a single set of 12 subjects at sites across the United States and Europe. Regional volumes and their scaling factors were estimated for each site using Freesurfer's segmentation algorithm and ordinary least squares, respectively. The scaling factors were validated by comparing the theoretical and simulated power curves, performing a leave-one-out calibration of regional volumes, and evaluating the absolute agreement of all regional volumes between sites before and after calibration. Using our derived power equation, we were able to define the conditions under which harmonization is not necessary to achieve 80% power. This approach can inform choice of processing pipelines and outcome metrics for multisite studies based on scaling factor variability across sites, enabling collaboration between clinical and research institutions

Overcoming a Conceptual Limitation of Industrial epsilon-Caprolactone Production via Chemoenzymatic Synthesis in Organic Medium

Bernhard LM, Gröger H. Overcoming a Conceptual Limitation of Industrial epsilon-Caprolactone Production via Chemoenzymatic Synthesis in Organic Medium. ChemSusChem . 2024.The multi-10.000 tons scale manufactured chemical epsilon-caprolactone attracts high industrial interest due to its favorable biodegradability properties. However, besides being of petrochemical origin yet, its industrial production has a conceptual limitation that is the difficult extraction of this highly water-soluble monomer from the water phase resulting from the aqueous solution of H2O2 applied as reagent. In this contribution, we report a chemoenzymatic cascade starting from bio-based phenol, which makes use of O2 instead of H2O2 and runs in pure organic medium, thus requiring only simply decantation and distillation as work-up. In a first step, phenol is hydrogenated quantitatively to cyclohexanol under solvent-free conditions with a Ru-catalyst. After simple removal of the heterogenous catalyst, cyclohexanol is converted to epsilon-caprolactone in a biocatalytic double oxidation with very high yields just requiring O2 as reagent. This biocatalytic process proceeds in pure organic medium, thus avoiding tedious extraction to isolate the highly water-soluble epsilon-caprolactone and enabling a dramatically simplified work-up by only centrifugal separation of lyophilized whole cells and solvent removal. This oxidation is accomplished using a tailor-made recombinant whole-cell catalyst containing an alcohol dehydrogenase and a cyclohexanone monooxygenase mutant. © 2024 Wiley‐VCH GmbH

Process Development of Enantioselective Imine Reductase- Catalyzed Syntheses of Pharmaceutically Relevant Pyrrolidines

Bernhard LM, McLachlan J, Gröger H. Process Development of Enantioselective Imine Reductase- Catalyzed Syntheses of Pharmaceutically Relevant Pyrrolidines. Organic Process Research & Development. 2022;26(7):2067–2074.Process development and optimization for an enzymatic reduction of 2-aryl-substituted pyrrolines to enantioselectively access the corresponding pyrrolidines have been carried out. Such chiral pyrrolidines are of high pharmaceutical interest and represent structural subunits in, e.g., larotrectinib and MSC2530818. This work enables access to the heterocyclic amine products with full conversion (>99%), excellent enantioselectivity (>99% ee), and good to high yield (up to 91%) in the presence of a readily available recombinant Escherichia coli-type whole-cell catalyst containing an imine reductase from Cupriavidus sp. HPC(L) and a glucose dehydrogenase in recombinant form. The developed processes utilizes cheap D-glucose as an economically favorable and nontoxic reducing agent and runs at a substrate loading of 18 g/L, which is also in an attractive range for larger-scale operations

Nano/Microfluidic Devices for Whole-Cell Biocatalyst Analysis by UV/VIS-Spectroscopy

Viefhues M, Schlüter L, Wedde S, Gröger H, Anselmetti D. Nano/Microfluidic Devices for Whole-Cell Biocatalyst Analysis by UV/VIS-Spectroscopy. In: Proceedings of the 20th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2016 (µTAS 2016). Vol 3. Dublin, Irland: The Chemical and Biological Microsystems Society (CBMS); 2017: 1635-1636

Enantioselective Synthesis of Secondary Amines by Combining Oxidative Rearrangement and Biocatalysis in a One-Pot Process

Bernhard LM, Zelenska K, Takashima M, Arisawa M, Murai K, Gröger H. Enantioselective Synthesis of Secondary Amines by Combining Oxidative Rearrangement and Biocatalysis in a One-Pot Process. Journal of Organic Chemistry. 2024.This contribution describes the development of chemoenzymatic one-pot processes, which combine an oxidative rearrangement and a biotransformation catalyzed by an imine reductase (IRED), for the synthesis of highly enantiomerically enriched secondary amines, such as an aryl-substituted pyrrolidine and a benzazepine. The benefits of this chemoenzymatic one-pot approach include high overall conversions (up to >99%), high enantiomeric excesses (up to >99% ee), and a straightforward synthetic approach toward secondary amines without the need to isolate the formed intermediate. For the initial chemical reaction, namely, the oxidative rearrangement, PhI(OAc)2 in methanol is used as a non-natural reagent, whereas the enzymatic step requires only stoichiometric amounts of d-glucose along with catalytic amounts of IRED, glucose dehydrogenase (GDH), and the cofactor NADPH. This methodology, demonstrating the compatibility of a "classic" organic synthesis using a non-natural, highly reactive reagent and a subsequent biocatalytic step, can be applied for different amines as substrates, thus making this concept a versatile tool in synthetic organic chemistry in general and for enantioselective synthesis of heterocyclic secondary amines in particular

Epstein-Barr Virus Infection of Cell Lines Derived from Diffuse Large B-Cell Lymphomas Alters MicroRNA Loading of the Ago2 Complex

Diffuse large B-cell lymphoma (DLBCL) is an aggressive lymphoid tumor which is occasionally Epstein-Barr virus (EBV)-positive and is further subtyped as activated B-cell (ABC) and germinal center B-cell (GCB) DLBCL, which has implications for prognosis and treatment. We performed Ago2-RNA immunoprecipitation followed by high throughput RNA sequencing (Ago2-RIP-Seq) to capture functionally active miRNAs in EBV-negative ABC-DLBCL and GC-DLBCL cell lines and their EBV-infected counterparts. In parallel, total miRNomes of these cells were sequenced to capture the cellular miRNA profile for comparison with the functionally active profile. Selected miRNAs with differential abundance were validated using RT-qPCR and Northern Blot. We found 6 miRNAs with differential abundance (2 upregulated and 4 downregulated miRNAs) between EBV-neg. and pos. ABC-DLBCL, and 12 miRNAs with differential abundance (3 upregulated and 9 downregulated miRNAs) between EBV-neg and -pos GC-DLBCL. Eight and twelve miRNAs were confirmed using RT-qPCR in ABC-DLBCL and GC-DLBCL, respectively. Selected miRNs were analyzed in additional type I/II vs. type III EBV latency DLBCL cell lines. Furthermore, up regulation of miR-221-3p and down regulation of let-7c-5p in ABC-DLBCL and up regulation of miR-363-3p and down regulation of 423-5p in GC-DLBCL was verified using RIP-Northern blot. Our comprehensive sequence analysis of the DLBCL miRNomes identified sets of deregulated miRNAs in the Ago2-RIP-seq. Our Ago2-IP-seq miRNomes profile could be considered as an important data set for detection of deregulated functionally active miRNAs in DLBCL and could possibly lead to identification of miRNAs as biomarkers for classification of DLBCL or even as targets for personalized targeted treatment