Search

In Brief: PI3K-AKT signaling activates steroidogenesis by inducing estradiol and progesterone production, while MEK-ERK1/2 signaling regulates steroidogenesis by inhibiting estradiol and inducing progesterone production in granulosa cells (GCs). Both pathways are essential for glycolytic and mitochondrial metabolism in these cells., Abstract: The PI3K-AKT and MEK-ERK1/2 signaling pathways are integral to fundamental cellular processes, such as proliferation, viability and differentiation. In GCs, these pathways are activated by follicle-stimulating hormone (FSH) and IGF1 through respective receptors. We investigated the comparative transcriptome changes induced by the AKT and ERK (ERK1/2) pathways using corresponding inhibitors in GCs. GCs isolated from antral follicles showed positive signals for phospho-AKT and phospho-ERK proteins. Treatment of cultured GCs with FSH and IGF1 induced phospho-AKT and phospho-ERK levels. Transcriptome analysis revealed 1436 genes regulated by AKT and 654 genes regulated by the ERK pathway. Among these, 94 genes were commonly downregulated and 11 genes were commonly upregulated in both datasets, while 110 genes were oppositely regulated. Bioinformatics analysis revealed that the inhibition of the PI3K-AKT and MEK-ERK pathways downregulates key reproductive processes and upstream molecules. Notably, AKT inhibition affected FSH, ESRRG and HIF1 pathways, while ERK inhibition impacted CG, FOS, TGFβ, EGR1 and LH pathways. Transcriptome data showed that genes related to estradiol production were inhibited by ERK and induced by the AKT pathway. This was verified by radioimmunoassays, and mRNA and protein analysis of CYP19A1 and STAR genes. In addition, transcriptome data suggested the downregulation of glucose metabolism in GCs. Using validation experiments, we confirm that both pathways are essential for glucose uptake, lactate production and mitochondrial activity in GCs. These data provide a resource for informing future research for analyzing various novel candidate genes regulated by the AKT and ERK pathways in GCs and other cell types.

The current study presents the analysis of stromal cells obtained from an hyperplastic left-ovary of a Holstein cow. Cultured hyperplastic stromal cells displayed a fibroblast-like morphology and ceased proliferation after the 8th passage. The non-cancerous nature of stromal cells was confirmed by in vitro cell proliferation and migration assays. Negligible amounts of E2 were detected in the spent media of cultured stromal cells, which suggests that stromal cells were non-estradiol synthesizing cells. As revealed in immunofluorescence and gene expression analysis, the hyperplastic stromal cells explicitly expressed vimentin in their cytoskeleton. Upon hematoxylin staining, a highly dense population of stromal cells was observed in the stromal tissue of the hyperplastic ovary. To explore genome-wide alterations, mRNA microarray analysis was performed using Affymetrix Bovine Gene 1.0ST Arrays compared to normal ovarian derived stromal cells. The microarray identified 1396 differentially expressed genes, of which 733 were up- and 663 down-regulated in hyperplastic stromal cells. Importantly, asporin (ASPN) and vascular cell adhesion molecule 1 (VCAM1) were among the highly up-regulated genes. Higher expression of ASPN was also confirmed by immunohistochemistry and RT-qPCR analysis. Ingenuity pathway analysis (IPA) identified about 98 significantly enriched (-log (p value ≥ 1.3) canonical pathways, importantly of which the "Sirutin Signaling Pathway" and "Mitochondrial Dysfunction" were highly activated while "Oxidative phosphorylation" was inhibited. Additionally, higher proportion of hyperplastic stromal cells in the S-phase of cell cycle, could be attributed to higher expression levels of cell proliferation genes such as CCND2 and CDK6., (© 2024. The Author(s).)

He, we show that combined use of the EZH2 inhibitor GSK126 and the CDK4/6 inhibitor abemaciclib synergistically enhances antitumoral effects in preclinical GBM models. Dual blockade led to HIF1α upregulation and CalR translocation, accompanied by massive impairment of mitochondrial function. Basal oxygen consumption rate, ATP synthesis, and maximal mitochondrial respiration decreased, confirming disrupted endoplasmic reticulum-mitochondrial homeostasis. This was paralleled by mitochondrial depolarization and upregulation of the UPR sensors PERK, ATF6α, and IRE1α. Notably, dual EZH2/CDK4/6 blockade also reduced 3D-spheroid invasion, partially inhibited tumor growth in ovo, and led to impaired viability of patient-derived organoids. Mechanistically, this was due to transcriptional changes in genes involved in mitotic aberrations/spindle assembly (Rb, PLK1, RRM2, PRC1, CENPF, TPX2), histone modification (HIST1H1B, HIST1H3G), DNA damage/replication stress events (TOP2A, ATF4), immuno-oncology (DEPDC1), EMT-counterregulation (PCDH1) and a shift in the stemness profile towards a more differentiated state. We propose a dual EZH2/CDK4/6 blockade for further investigation., (© 2024. The Author(s).)

Background: Despite remarkable advances in the therapy of multiple sclerosis (MS), patients with MS may still experience relapses. High-dose short-term methylprednisolone (MP) remains the standard treatment in the acute management of MS relapses due to its potent anti-inflammatory and immunosuppressive properties. However, there is a lack of studies on the cell type-specific transcriptome changes that are induced by this synthetic glucocorticoid (GC). Moreover, it is not well understood why some patients do not benefit adequately from MP therapy., Methods: We collected peripheral blood from MS patients in relapse immediately before and after ∼3-5 days of therapy with MP at 4 study centers. CD19 + B cells and CD4 + T cells were then isolated for profiling the transcriptome with high-density arrays. The patients' improvement of neurological symptoms was evaluated after ∼2 weeks by the treating physicians. We finally analyzed the data to identify genes that were differentially expressed in response to the therapy and whose expression differed between clinical responders and non-responders., Results: After MP treatment, a total of 33 genes in B cells and 55 genes in T helper cells were significantly up- or downregulated. The gene lists overlap in 10 genes and contain genes that have already been described as GC-responsive genes in the literature on other cell types and diseases. Their differential expression points to a rapid and coordinated modulation of multiple signaling pathways that influence transcription. Genes that were previously suggested as potential prognostic biomarkers of the clinical response to MP therapy could not be confirmed in our data. However, a greater increase in the expression of genes encoding proteins with antimicrobial activity was detected in CD4 + T cells from non-responders compared to responders., Conclusion: Our study delved into the cell type-specific effects of MP at the transcriptional level. The data suggest a therapy-induced ectopic expression of some genes (e.g., AZU1, ELANE and MPO), especially in non-responders. The biological consequences of this remain to be explored in greater depth. A better understanding of the molecular mechanisms underlying clinical recovery from relapses in patients with MS will help to optimize future treatment decisions., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: MH received speaking fees and travel funds from Bayer HealthCare, Biogen, Merck Healthcare, Novartis and Teva. JK received personal compensation for consulting as well as speaker honoraria from Bayer, Biogen, Bristol Myers Squibb, Grifols, Janssen, Merck Serono, Novartis, Roche, Sanofi Genzyme, Takeda and Teva. MG received honoraria, speaking fees and research funding from Bayer, Biogen, Bristol Myers Squibb, Johnson & Johnson, Merck, Novartis, Roche, Sanofi and Teva as well as BMBF and DFG. AW received speaking fees and travel funds from Biogen, GlaxoSmithKline, Merck Serono, Novartis and Sanofi Genzyme. IL-P and KE are employees of Miltenyi Biotec, a biotechnology company that provides products and services for biomedical research as well as cell and gene therapy. UKZ received research support, speaking fees and travel funds from Alexion, Almirall, Bayer HealthCare, Biogen, Bristol Myers Squibb, Janssen, Merck Healthcare, Novartis, Roche, Sanofi Genzyme and Teva as well as the European Union, BMBF, BMWi and DFG. BF, DK, MS, SM and AD declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Thiazolopyridines are a highly relevant class of small molecules, which have previously shown a wide range of biological activities. Besides their anti-tubercular, anti-microbial and anti-viral activities, they also show anti-cancerogenic properties, and play a role as inhibitors of cancer-related proteins. Herein, the biological effects of the thiazolopyridine AV25R, a novel small molecule with unknown biological effects, were characterized. Screening of a set of lymphoma (SUP-T1, SU-DHL-4) and B- acute leukemia cell lines (RS4;11, SEM) revealed highly selective effects of AV25R. The selective anti-proliferative and metabolism-modulating effects were observed in vitro for the B-ALL cell line RS4;11. Further, we were able to detect severe morphological changes and the induction of apoptosis. Gene expression analysis identified a large number of differentially expressed genes after AV25R exposure and significant differentially regulated cancer-related signaling pathways, such as VEGFA-VEGFR2 signaling and the EGF/EGFR pathway. Structure-based pharmacophore screening approaches using in silico modeling identified potential biological AV25R targets. Our results indicate that AV25R binds with several proteins known to regulate cell proliferation and tumor progression, such as FECH, MAP11, EGFR, TGFBR1 and MDM2. The molecular docking analyses indicates that AV25R has a higher binding affinity compared to many of the experimentally validated small molecule inhibitors of these targets. Thus, here we present in vitro and in silico analyses which characterize, for the first time, the molecular acting mechanism of AV25R, including cellular and molecular biologic effects. Additionally, this predicted the target binding of the molecule, revealing a high affinity to cancer-related proteins and, thus, classified AVR25 for targeted intervention approaches.

Increased life expectancy in industrialized countries is causing an increased incidence of osteoporosis and the need for bioactive bone implants. The integration of implants can be improved physically, but mainly by chemical modifications of the material surface. It was recognized that amino-group-containing coatings improved cell attachment and intracellular signaling. The aim of this study was to determine the role of the amino group density in this positive cell behavior by developing controlled amino-rich nanolayers. This work used covalent grafting of polymer-based nanocoatings with different amino group densities. Titanium coated with the positively-charged trimethoxysilylpropyl modified poly(ethyleneimine) (Ti-TMS-PEI), which mostly improved cell area after 30 min, possessed the highest amino group density with an N/C of 32%. Interestingly, changes in adhesion-related genes on Ti-TMS-PEI could be seen after 4 h. The mRNA microarray data showed a premature transition of the MG-63 cells into the beginning differentiation phase after 24 h indicating Ti-TMS-PEI as a supportive factor for osseointegration. This amino-rich nanolayer also induced higher bovine serum albumin protein adsorption and caused the cells to migrate slower on the surface after a more extended period of cell settlement as an indication of a better surface anchorage. In conclusion, the cell spreading on amine-based nanocoatings correlated well with the amino group density (N/C).

Background: Nutrition has not only an impact on the general wellbeing of an animal but can also affect reproductive processes. In cattle, feeding regimes can influence the age of puberty onset and alter gonadal development. We analyzed effects of different milk replacer (MR) feeding regimes during rearing on ovarian physiology with specific emphasis on the numbers as well as gene expression characteristics of granulosa cells (GCs) at the age of puberty onset. Two groups of calves received either 10% or 20% of bodyweight MR per day during their first 8 weeks. After weaning, both groups were fed the same mixed ration ad libitum until slaughter at 8 months., Results: Animals of the 20% feeding group had a significantly higher body weight, but the proportion of animals having a corpus luteum at the time of slaughter was not different between groups, suggesting a similar onset of puberty. Calves of the 10% group showed a constant GC count regardless of the number of follicles (r = 0.23) whereas in the 20% group increasing numbers of GCs were detected with a higher follicle count (r = 0.71). As a first effort to find a possible molecular explanation for this unexpected limitation of GC numbers in the 10% group, we comparatively analyzed GC transcriptomes in both diet groups. The mRNA microarray analysis revealed a total of 557 differentially expressed genes comparing both groups (fold change > |1.5| and p < 0.05). OAS1X, MX2 and OAS1Z were among the top downregulated genes in the 20% vs. the 10% group, whereas top upregulated genes comprised BOLA and XCL1. All of these genes are known to be regulated by interferon. Subsequent signaling pathway analysis revealed the involvement of several immune response mechanisms in accordance with a number of interferons as upstream regulators., Conclusions: The results indicate that the plane of MR feeding in early life has an impact on the number and physiology of GCs later in life. This might influence the overall reproductive life initiated by the onset of puberty in cattle. In addition, the observed alterations in GCs of calves fed less MR might be a consequence of interferon regulated immunological pathways., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: The immune response is a crucial factor for mediating the benefit of cardiac cell therapies. Our previous research showed that cardiomyocyte transplantation alters the cardiac immune response and, when combined with short-term pharmacological CCR2 inhibition, resulted in diminished functional benefit. However, the specific role of innate immune cells, especially CCR2 macrophages on the outcome of cardiomyocyte transplantation, is unclear., Methods: We compared the cellular, molecular, and functional outcome following cardiomyocyte transplantation in wildtype and T cell- and B cell-deficient Rag2 del mice. The cardiac inflammatory response was assessed using flow cytometry. Gene expression profile was assessed using single-cell and bulk RNA sequencing. Cardiac function and morphology were determined using magnetic resonance tomography and immunohistochemistry respectively., Results: Compared to wildtype mice, Rag2 del mice show an increased innate immune response at steady state and disparate macrophage response after MI. Subsequent single-cell analyses after MI showed differences in macrophage development and a lower prevalence of CCR2 expressing macrophages. Cardiomyocyte transplantation increased NK cells and monocytes, while reducing CCR2 - MHC-II lo macrophages. Consequently, it led to increased mRNA levels of genes involved in extracellular remodelling, poor graft survival, and no functional improvement. Using machine learning-based feature selection, Mfge8 and Ccl7 were identified as the primary targets underlying these effects in the heart., Conclusions: Our results demonstrate that the improved functional outcome following cardiomyocyte transplantation is dependent on a specific CCR2 macrophage response. This work highlights the need to study the role of the immune response for cardiomyocyte cell therapy for successful clinical translation., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: Multiple sclerosis (MS) is a chronic, inflammatory and neurodegenerative disease that leads to irreversible damage to the brain and spinal cord. The goal of so-called "immune reconstitution therapies" (IRTs) is to achieve long-term disease remission by eliminating a pathogenic immune repertoire through intense short-term immune cell depletion. B cells are major targets for effective immunotherapy in MS., Objectives: The aim of this study was to analyze the gene expression pattern of B cells before and during IRT (i.e., before B-cell depletion and after B-cell repopulation) to better understand the therapeutic effects and to identify biomarker candidates of the clinical response to therapy., Methods: B cells were obtained from blood samples of patients with relapsing-remitting MS (n = 50), patients with primary progressive MS (n = 13) as well as healthy controls (n = 28). The patients with relapsing MS received either monthly infusions of natalizumab (n = 29) or a pulsed IRT with alemtuzumab (n = 15) or cladribine (n = 6). B-cell subpopulation frequencies were determined by flow cytometry, and transcriptome profiling was performed using Clariom D arrays. Differentially expressed genes (DEGs) between the patient groups and controls were examined with regard to their functions and interactions. We also tested for differences in gene expression between patients with and without relapse following alemtuzumab administration., Results: Patients treated with alemtuzumab or cladribine showed on average a > 20% lower proportion of memory B cells as compared to before IRT. This was paralleled by profound transcriptome shifts, with > 6000 significant DEGs after adjustment for multiple comparisons. The top DEGs were found to regulate apoptosis, cell adhesion and RNA processing, and the most highly connected nodes in the network of encoded proteins were ESR2, PHB and RC3H1. Higher mRNA levels of BCL2, IL13RA1 and SLC38A11 were seen in patients with relapse despite IRT, though these differences did not pass the false discovery rate correction., Conclusions: We show that B cells circulating in the blood of patients with MS undergoing IRT present a distinct gene expression signature, and we delineated the associated biological processes and gene interactions. Moreover, we identified genes whose expression may be an indicator of relapse risk, but further studies are needed to verify their potential value as biomarkers., (© 2023. The Author(s).)

Estradiol and progesterone are the primary sex steroids produced by the ovary. Upon luteinizing hormone surge, estradiol-producing granulosa cells convert into progesterone-producing cells and eventually become large luteal cells of the corpus luteum. Signaling pathways and transcription factors involved in the cessation of estradiol and simultaneous stimulation of progesterone production in granulosa cells are not clearly understood. Here, we decipher that phosphorylated ERK1/2 regulates granulosa cell steroidogenesis by inhibiting estradiol and inducing progesterone production. Down-regulation of transcription factor FOXL2 and up-regulation of SOX9 by ERK underpin its differential steroidogenic function. Interestingly, the incidence of SOX9 is largely uncovered in ovarian cells and is found to regulate FOXL2 along with CYP19A1 and STAR genes, encoding rate-limiting enzymes of steroidogenesis, in cultured granulosa cells. We propose that the novel ERK1/2-SOX9/FOXL2 axis in granulosa cells is a critical regulator of ovarian steroidogenesis and may be considered when addressing pathophysiologies associated with inappropriate steroid production and infertility in humans and animals., (© 2023 Baddela et al.)

The myxozoan parasite Tetracapsuloides bryosalmonae is the causative agent of proliferative kidney disease (PKD)-a disease of salmonid fishes, notably of the commercially farmed rainbow trout Oncorhynchus mykiss . Both wild and farmed salmonids are threatened by this virulent/deadly disease, a chronic immunopathology characterized by massive lymphocyte proliferation and hyperplasia, which manifests as swollen kidneys in susceptible hosts. Studying the immune response towards the parasite helps us understand the causes and consequences of PKD. While examining the B cell population during a seasonal outbreak of PKD, we unexpectedly detected the B cell marker immunoglobulin M (IgM) on red blood cells (RBCs) of infected farmed rainbow trout. Here, we studied the nature of this IgM and this IgM + cell population. We verified the presence of surface IgM via parallel approaches: flow cytometry, microscopy, and mass spectrometry. The levels of surface IgM (allowing complete resolution of IgM - RBCs from IgM + RBCs) and frequency of IgM + RBCs (with up to 99% of RBCs being positive) have not been described before in healthy fishes nor those suffering from disease. To assess the influence of the disease on these cells, we profiled the transcriptomes of teleost RBCs in health and disease. Compared to RBCs originating from healthy fish, PKD fundamentally altered RBCs in their metabolism, adhesion, and innate immune response to inflammation. In summary, RBCs play a larger role in host immunity than previously appreciated. Specifically, our findings indicate that the nucleated RBCs of rainbow trout interact with host IgM and contribute to the immune response in PKD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Chan, Picard-Sánchez, Majstorović, Rebl, Koczan, Dyčka, Holzer and Korytář.)

Background: Multiple sclerosis (MS) is a chronic immune-mediated disease of the central nervous system to which a genetic predisposition contributes. Over 200 genetic regions have been associated with increased disease risk, but the disease-causing variants and their functional impact at the molecular level are mostly poorly defined. We hypothesized that single-nucleotide polymorphisms (SNPs) have an impact on pre-mRNA splicing in MS., Methods: Our study focused on 10 bioinformatically prioritized SNP-gene pairs, in which the SNP has a high potential to alter alternative splicing events (ASEs). We tested for differential gene expression and differential alternative splicing in B cells from MS patients and healthy controls. We further examined the impact of the SNP genotypes on ASEs and on splice isoform expression levels. Novel genotype-dependent effects on splicing were verified with splicing reporter minigene assays., Results: We were able to confirm previously described findings regarding the relation of MS-associated SNPs with the ASEs of the pre-mRNAs from GSDMB and SP140 . We also observed an increased IL7R exon 6 skipping when comparing relapsing and progressive MS patients to healthy subjects. Moreover, we found evidence that the MS risk alleles of the SNPs rs3851808 ( EFCAB13 ), rs1131123 ( HLA-C ), rs10783847 (TSFM), and rs2014886 ( TSFM ) may contribute to a differential splicing pattern. Of particular interest is the genotype-dependent exon skipping of TSFM due to the SNP rs2014886. The minor allele T creates a donor splice site, resulting in the expression of the exon 3 and 4 of a short TSFM transcript isoform, whereas in the presence of the MS risk allele C, this donor site is absent, and thus the short transcript isoform is not expressed., Conclusion: In summary, we found that genetic variants from MS risk loci affect pre-mRNA splicing. Our findings substantiate the role of ASEs with respect to the genetics of MS. Further studies on how disease-causing genetic variants may modify the interactions between splicing regulatory sequence elements and RNA-binding proteins can help to deepen our understanding of the genetic susceptibility to MS., Competing Interests: EP received travel funds from Novartis. MH received speaking fees and travel funds from Bayer HealthCare, Biogen, Merck, Novartis, and Teva. UKZ received research support as well as speaking fees and travel funds from Alexion, Almirall, Bayer HealthCare, Biogen, Bristol Myers Squibb, Janssen, Merck Serono, Novartis, Roche, Sanofi Genzyme, and Teva as well as EU, BMBF, BMWi, and DFG. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Putscher, Hecker, Fitzner, Boxberger, Schwartz, Koczan, Lorenz and Zettl.)

Background: Both bromodomain and extra-terminal domain (BET) proteins and spleen tyrosine kinase (SYK) represent promising targets in diffuse large B-cell (DLBCL) and Burkitt's lymphoma (BL). We evaluated the anti-lymphoma activity of the isoform-specific bivalent BET inhibitor AZD5153 (AZD) and the pan-BET inhibitor I-BET151 (I-BET) as single agents and in combination with SYK inhibitor Entospletinib (Ento) in vitro. Methods: The effect of the single agents on cell proliferation and metabolic activity was evaluated in two DLBCL and two BL cell lines. Proliferation, metabolic activity, apoptosis, cell cycle and morphology were further investigated after a combined treatment of AZD or I-BET and Ento. RNAseq profiling of combined AZD+Ento treatment was performed in SU-DHL-4 cells. Results: Both BET inhibitors reduced cell proliferation and metabolic activity in a dose- and time-dependent manner. Combined BET and SYK inhibition enhanced the anti-proliferative effect and induced a G0/G1 cell cycle arrest. SU-DHL-4 demonstrated a pronounced modulation of gene expression by AZD, which was markedly increased by additional SYK inhibition. Functional enrichment analyses identified combination-specific GO terms related to DNA replication and cell division. Genes such as ADGRA2 , MYB , TNFRSF11A , S100A10 , PLEKHH3 , DHRS2 and FOXP1-AS1 were identified as possible key regulators. Conclusion: Simultaneous inhibition of BET and SYK enhanced the anti-proliferative effects, and induced a combination-specific gene expression signature.

Background: Schistosomiasis is a severe parasitic disease that is primarily driven by the host's immune response to schistosome eggs trapped in tissue and by the granulomatous inflammatory and fibrotic reaction they cause. Despite significant progress in understanding the complex immunological processes involved in the relationship between schistosomes and their host, neither an effective vaccine against the infection nor anti-fibrotic drugs currently exists, making the search for new targets for schistosome drugs and vaccine candidates even more important. In order to identify new molecular targets for defense against or elimination of the parasite, we investigate herein the interplay between the host and male or female schistosomes, clearly separating this from the action of the parasite eggs., Methods: For this purpose, we infected 6-8-week-old female NMRI mice with 100 male (M), female (F), or both (MF) S. mansoni cercariae and performed a comparative transcriptomic and flow cytometric analysis of their spleens., Results: Principal component analysis of a total of 22,207 transcripts showed a clear clustering of the experimental groups. We identified a total of 1,293 genes in group M, 512 genes in group F, and 4,062 genes in group MF that were differentially expressed compared to naive controls. The highest percentage of regulated genes (2,972; 65.9%) was found in group MF alone, but there was a large overlap between groups M and MF (798; 17.7%) and a small overlap between groups F and MF (91; 2.0%). Only 4.5% of genes (201) were revealed to be regulated in all experimental groups (M/F/MF). In addition, we were able to show that both worm sexes trigger immune responses in an egg-independent manner (non-polarized Th1 and Th2 response), with female worms exerting less regulatory influence than males., Conclusion: Our data show that adult schistosomes trigger sex-specific, egg-independent immune responses. The lists of genes regulated by adult female or male worms presented here may be useful in deciphering host-parasite interactions to identify targets for schistosome elimination., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Winkelmann, Rabes, Reinholdt, Koslowski, Koczan, Reisinger and Sombetzki.)

Constitutive activation of cyclin-dependent kinases (CDKs) or arginine auxotrophy are hallmarks of Glioblastoma multiforme (GBM). The latter metabolic defect renders tumor cells vulnerable to arginine-depleting substances, such as arginine deiminase from Streptococcus pyogenes (SpyADI). Previously, we confirmed the susceptibility of patient-derived GBM cells towards SpyADI as well as CDK inhibitors (CDKis). To improve therapeutic effects, we here applied a combined approach based on SpyADI and CDKis (dinaciclib, abemaciclib). Three arginine-auxotrophic patient-derived GBM lines with different molecular characteristics were cultured in 2D and 3D and effects of this combined SpyADI/CDKi approach were analyzed in-depth. All CDKi/SpyADI combinations yielded synergistic antitumoral effects, especially when given sequentially (SEQ), i.e., CDKi in first-line and most pronounced in the 3D models. SEQ application demonstrated impaired cell proliferation, invasiveness, and viability. Mitochondrial impairment was demonstrated by increasing mitochondrial membrane potential and decreasing oxygen consumption rate and extracellular acidification rate after SpyADI/abemaciclib monotherapy or its combination regimens. The combined treatment even induced autophagy in target cells (abemaciclib/SpyADI > dinaciclib/SpyADI). By contrast, the unfolded protein response and p53/p21 induced senescence played a minor role. Transmission electron microscopy confirmed damaged mitochondria and endoplasmic reticulum together with increased vacuolization under CDKi mono- and combination therapy. SEQ-abemaciclib/SpyADI treatment suppressed the DSB repair system via NHEJ and HR, whereas SEQ-dinaciclib/SpyADI treatment increased γ-H2AX accumulation and induced Rad51/Ku80. The latter combination also activated the stress sensor GADD45 and β-catenin antagonist AXIN2 and induced expression changes of genes involved in cellular/cytoskeletal integrity. This study highlights the strong antitumoral potential of a combined arginine deprivation and CDK inhibition approach via complex effects on mitochondrial dysfunction, invasiveness as well as DNA-damage response. This provides a good starting point for further in vitro and in vivo proof-of-concept studies to move forward with this strategy., (© 2022. The Author(s).)

Background: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system with a well-established genetic contribution to susceptibility. Over 200 genetic regions have been linked to the inherited risk of developing MS, but the disease-causing variants and their functional effects at the molecular level are still largely unresolved. We hypothesised that MS-associated single-nucleotide polymorphisms (SNPs) affect the recognition and enzymatic cleavage of primary microRNAs (pri-miRNAs)., Methods: Our study focused on 11 pri-miRNAs (9 primate-specific) that are encoded in genetic risk loci for MS. The levels of mature miRNAs and potential isoforms (isomiRs) produced from those pri-miRNAs were measured in B cells obtained from the peripheral blood of 63 MS patients and 28 healthy controls. We tested for associations between SNP genotypes and miRNA expression in cis using quantitative trait locus (cis-miR-eQTL) analyses. Genetic effects on miRNA stem-loop processing efficiency were verified using luciferase reporter assays. Potential direct miRNA target genes were identified by transcriptome profiling and computational binding site assessment., Findings: Mature miRNAs and isomiRs from hsa-mir-26a-2, hsa-mir-199a-1, hsa-mir-4304, hsa-mir-4423, hsa-mir-4464 and hsa-mir-4492 could be detected in all B-cell samples. When MS patient subgroups were compared with healthy controls, a significant differential expression was observed for miRNAs from the 5' and 3' strands of hsa-mir-26a-2 and hsa-mir-199a-1. The cis-miR-eQTL analyses and reporter assays pointed to a slightly more efficient Drosha-mediated processing of hsa-mir-199a-1 when the MS risk allele T of SNP rs1005039 is present. On the other hand, the MS risk allele A of SNP rs817478, which substitutes the first C in a CNNC sequence motif, was found to cause a markedly lower efficiency in the processing of hsa-mir-4423. Overexpression of hsa-mir-199a-1 inhibited the expression of 60 protein-coding genes, including IRAK2, MIF, TNFRSF12A and TRAF1. The only target gene identified for hsa-mir-4423 was TMEM47., Interpretation: We found that MS-associated SNPs in sequence determinants of pri-miRNA processing can affect the expression of mature miRNAs. Our findings complement the existing literature on the dysregulation of miRNAs in MS. Further studies on the maturation and function of miRNAs in different cell types and tissues may help to gain a more detailed functional understanding of the genetic basis of MS., Funding: This study was funded by the Rostock University Medical Center (FORUN program, grant: 889002), Sanofi Genzyme (grant: GZ-2016-11560) and Merck Serono GmbH (Darmstadt, Germany, an affiliate of Merck KGaA, CrossRef Funder ID: 10.13039/100009945, grant: 4501860307). NB was supported by the Stiftung der Deutschen Wirtschaft (sdw) and the FAZIT foundation. EP was supported by the Landesgraduiertenförderung Mecklenburg-Vorpommern., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Aquaculture management involves regular handling procedures, but these can evoke stress responses in farmed fish. We compiled an extensive list of published parameters that indicate the most likely handling-induced physiological deviations from the norm. However, since these parameters are based almost exclusively on studies of rainbow trout and Atlantic salmon, we conducted a handling-challenge experiment with maraena whitefish ( Coregonus maraena ). This salmonid fish was sampled at either 3 or 24 h after a single 1-min handling or after 10 days of daily repeated 1-min handling. The cortisol levels were strongly elevated in some individuals at 3 h after the single handling challenge, but these elevations were not significantly different between the challenged and control cohorts. The phagocytic capacity of myeloid head-kidney cells stimulated with fluorophore-labeled, inactivated Aeromonas salmonicida was significantly decreased in maraena whitefish at 3 h after the handling challenge compared to control fish. Microarray analysis of head-kidney samples from the challenged and control fish revealed 12 differentially expressed genes at 3 h and 70 at 24 h after the single handling episode, but only 5 differentially expressed genes after 10 days of repeated daily handling. The identified genes were assigned to numerous stress- and immune-relevant functional pathways, including "glucocorticoid receptor signaling" (3 h post-challenge), "HIF1A signaling" (24 h post-challenge), or "complement system" (10 days of repeated challenge). Our data reveal the tight interconnection of immune and stress pathways in the head kidney of maraena whitefish and corroborate several parameters previously found regulated in other tissues of handling-stressed rainbow trout. These findings indicate that handling may compromise the health and welfare of maraena whitefish in aquaculture., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Martorell-Ribera, Koczan, Tindara Venuto, Viergutz, Brunner, Goldammer, Gimsa and Rebl.)