International Journal of Molecular Sciences

Caffeine, the most widely consumed stimulant worldwide and primarily sourced from coffee, is well known for its central nervous system effects. Emerging evidence indicates that caffeine also modulates key cellular processes, including DNA repair. It inhibits the kinase activity of ATM and ATR-essential DNA damage response proteins, and impairs homologous recombination (HR)-mediated repair through multiple mechanisms. However, its effects on nonhomologous end joining (NHEJ), a major double-strand break (DSB) repair pathway, have been underexplored. In a recent study, we reported that caffeine inhibits NHEJ primarily by interfering with Ligase IV/XRCC4 complex, using in vitro and ex vivo model systems. Given coffees role as a primary dietary caffeine source, this study investigates the impact of Coffea arabica decoction on NHEJ-mediated DSB repair. High-performance liquid chromatography (HPLC) quantified caffeine levels in the decoction, followed by in vitro and ex vivo assays to evaluate NHEJ efficiency. Results demonstrate that coffee decoction inhibits end joining of both compatible and noncompatible DNA ends in cell-free systems derived from normal and cancer cells. Extrachromosomal repair assays confirmed impaired intracellular NHEJ, leading to accumulation of unrepaired DSBs in human cells. Kinetic analysis of {gamma}-H2AX foci formation and resolution revealed persistent DNA breaks and reduced repair kinetics. Reconstitution experiments verified that the decoction specifically targets the Ligase IV/XRCC4 complex. These findings, building on our previous work, establish coffee decoction as a potent NHEJ inhibitor, mirroring purified caffeines effects. This underscores caffeines interference with endogenous DNA repair, with profound implications for cancer therapy by sensitizing tumors to genotoxic treatments.

Purpose: To evaluate the efficacy and safety of oral L-ergothioneine (EGT) in alleviating pain and associated symptoms in women with primary dysmenorrhea (PD). Methods: In this randomized, double-blind, placebo-controlled trial, 40 women with PD (aged 18-30 years) were randomized (1:1) to receive EGT capsules (120 mg/day) or a matching placebo for 3 consecutive menstrual cycles. Outcomes evaluated at baseline and post-cycle included peak pain (Visual Analog Scale, VAS), Dysmenorrhea Symptom Score, and the COX Menstrual Symptom Scale (CMSS). Results: EGT significantly improved PD symptoms over 3 cycles. Mean VAS for peak pain decreased from 4.80 {+/-} 1.12 to 2.32 {+/-} 1.59 in the EGT group (p < 0.001), compared to a non-significant reduction (4.10 {+/-} 1.30 to 3.45 {+/-} 1.69) in the placebo group. The between-group difference at cycle 3 was significant (p < 0.01). A linear mixed-model confirmed a significant Time x Group interaction (p < 0.001), showing an accelerated decline in symptom severity for EGT. Furthermore, 84% of EGT-treated patients achieved [&ge;]50% VAS reduction versus 35% in the placebo group (p = 0.003). Serum inflammatory biomarkers showed no significant between-group differences or correlation with VAS improvements, suggesting EGT's analgesic effects likely operate via cytoprotective pathways independent of classical inflammatory cascades. No adverse events were reported. Conclusion: Oral EGT supplementation (120 mg/day) effectively and progressively mitigates menstrual pain and systemic symptoms in PD, offering a well-tolerated, non-pharmacological intervention. Trial Registration: ChiCTR2500112557; Retrospectively registered on 2025-11-17.

This study aimed to investigate the dynamics of gene expression in pigs during heat stress (HS), focusing on both short-term (STHA) and long-term (LTHA) heat acclimation phases. A total of 12 castrated males were exposed to thermoneutral temperatures (24{degrees}C) for 14 days (TN) and then to a constant temperature of 30{degrees}C for 21 days. Rectal temperature measurements indicated a biphasic thermoregulatory response, with an initial peak followed by acclimation. Using whole blood transcriptome analysis at seven time points between day 5 before the initiation of HS challenge and day 13 post HS. A total of 525 genes were differentially expressed during the STHA (day 0-day 2) phase. A switch in the expression of most genes was observed around 20 hours after HS. Functional pathway enrichment analysis identified through shape-based clustering revealed the activation of the immune system, especially mediated through toll-like receptor signaling pathways. The LTHA phase (day 2-day 13) revealed 985 differentially expressed genes, with pathways associated with various metabolisms, including mitochondrial fatty acid beta-oxidation, and electron transport, ATP synthesis, and heat production by uncoupling proteins. Interestingly, oxidative phosphorylation was predicted to be activated during the LTHA, particularly in Complex V, whereas other complexes showed mixed regulation. Comparative pathway analysis indicated distinct metabolic adaptations between STHA and LTHA, with up-regulation of glucose and lipid metabolism in late STHA and down-regulation of lipid metabolism during LTHA. This study contributes to a better understanding of the time course of adaptation mechanisms in pigs to HS, underlying a coordinated regulation during STHA involving several stress-specific mechanisms (via the HSP) and metabolic variation to help pigs achieve homeothermy.

Purified proteins are routinely flash frozen for use in functional and structural studies, providing a convenient way to reproduce results across complex experiments. Rho guanine-nucleotide exchange factors (RhoGEFs) are no exception to this practice, yet the effects of freezing on their activity and stability remain largely uncharacterized. This gap potentially affects the characterization of these important enzymes and how results are interpreted with respect to their prospective use as therapeutic targets. Here, we tested the isolated DH/PH tandems of P-Rex1, P-Rex2, and PRG under different cryoprotectant conditions and monitored activity and thermostability over time after flash freezing. Our results show a clear divergence between the activity of fresh and frozen purified RhoGEF protein samples in as little as one week for some conditions. Specifically, the variability in data collected on frozen samples was greatly increased. Despite these differences, thermostability seems to be preserved for much longer timepoints across RhoGEFs. Moreover, despite eventual changes in both activity and thermostability with respect to freezing, there are no obvious changes in global conformation between fresh and frozen samples of the isolated P-Rex2 DH/PH tandem. From our data, there are few generalizable trends between the different RhoGEFs and no single cryoprotective agent tested was a silver bullet to preserve both activity and thermostability across RhoGEFs. Overall, our findings emphasize the unpredictable effects of freezing RhoGEFs. As such, RhoGEF freezing should be carefully characterized for each protein and critically viewed when comparing analyses between different studies.

NLRP3 inflammasome is a cytosolic multi-protein complex that plays a crucial role in the immune system, responding to various exogenous and endogenous stimuli by triggering protective inflammatory responses. However, aberrant NLRP3 inflammasome activation is implicated in numerous inflammatory diseases. Therefore, the NLRP3 inflammasome is an important pharmacological target for the treatment of multiple diseases. In this context, we screened various US-FDA-approved drugs for NLRP3 inflammasome inhibition. We found that among various drugs, minoxidil hydrochloride (MXL) effectively inhibits NLRP3 inflammasome, evidenced by reduced secretion of IL-1{beta} and IL-18 in J774A.1 cells treated with MXL. The IC50 values of MXL for inhibition of IL-1{beta} and IL-18 were calculated to be 1.2 and 1.06 {micro}M, respectively. MXL was found to prevent ASC oligomerization, thereby inhibiting the NLRP3 inflammasome and leading to CASP1 cleavage. Further investigation revealed that MXL also utilizes AMPK-mediated autophagy to modulate NLRP3 inflammasome activity. Using siAMPK and bafilomycin A1, an end-stage autophagy inhibitor, we elucidated crosstalk between the NLRP3 inflammasome and autophagic pathways, which was modulated by MXL. Furthermore, we demonstrated the efficacy of MXL in two different mouse models of inflammation, involving the NLRP3 inflammasome. MXL at doses of 10 and 20 mg/kg effectively inhibited the activation of NLRP3 inflammasome by monosodium urate in the air pouch model and by ATP in the peritoneal inflammation model, as evidenced by reduced secretion of 1{beta} and IL-18 in the lavage. Our study identifies MXL as a potent NLRP3 inflammasome inhibitor, warranting further investigation as a potential therapeutic agent for inflammatory diseases.

This report identifies a bidirectional signaling axis connecting lipid metabolism to nuclear mechanotransduction, with the potential to control fatty acid/triglyceride metabolism. The sterol regulatory element-binding (SREBP) family of transcription factors control fatty acid, triglyceride and cholesterol synthesis and metabolism. The family consists of three members: SREBP1a, SREBP1c, and SREBP2, that are regulated by intracellular cholesterol levels and insulin signaling. The SREBP2-dependent control of the LDL receptor gene is a well-established target for cholesterol-lowering therapeutics and the activity of SREBP1c is an attractive target in metabolic disease. In the current report, we identify SYNE4 (nesprin-4), a component of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, as a direct target of the SREBP family of transcription factors, and show that nesprin-4 in turn supports SREBP1c function. We identify functional SREBP binding sites in the human SYNE4 promoter and demonstrate that these are required for the sterol- and SREBP-dependent regulation of the promoter. Furthermore, we show that the endogenous SYNE4 gene is also regulated by SREBP1/2 and intracellular sterol levels. Interestingly, SREBP2 is responsible for the sterol regulation of the SYNE4 gene in HepG2 cells, while SREBP1 is the major regulator in MCF7 cells, demonstrating that diberent cell types use diberent SREBP paralogs to regulate the same promoter/gene. Importantly, we find that nesprin-4 is a positive regulator of SREBP1c expression and function in HepG2 cells and during the diberentiation of human adipose-derived stem cells. In summary, the current report identifies a novel regulatory interaction between lipid metabolism and the LINC complex. Importantly, we demonstrate that this signaling axis is bidirectional, forming a closed loop that has the potential to control SREBP1c activity and thereby fatty acid and triglyceride synthesis/metabolism. Based on our data, we propose that the nesprin-4-dependent regulation of SREBP1c could represent a novel therapeutic target in metabolic disease.

Equine asthma (EA) is a highly prevalent, chronic, inflammatory disease of the lower airways ranging from mild-to-moderate to severe clinical presentations. Diagnosis currently relies on bronchoalveolar lavage fluid (BALF) cytology, an invasive method associated with interobserver variability, which highlights the need for more reproducible approaches. MicroRNAs (miRNAs) are small noncoding RNAs involved in post-transcriptional gene regulation. They are stable and readily detectable in body fluids and have shown promising results as biomarkers in human asthma. The aim of this study was to characterize miRNA abundance profiles in BALF and serum from horses with distinct EA endotypes to evaluate their biomarker potential and explore their involvement in disease pathogenesis. A total of 43 horses were included and classified as either EA (n=32) or controls (n=11), based on clinical examination and BALF cytology. The EA horses were further divided into three endotypes based on BALF inflammatory cell composition: neutrophilic asthma (n=10), mastocytic asthma (n=15), and mixed asthma (n=7). RNA was isolated from both serum and BALF samples and analyzed by quantitative real-time PCR (qPCR) targeting 103 miRNAs linked to asthma and pulmonary inflammation in humans. Differential miRNA abundance was analyzed across EA endotypes. The most significantly differentially abundant miRNAs were used for in silico target prediction and pathway enrichment analyses. Horses with mixed EA had significantly lower levels of eca-miR-125a-3p and eca-miR-125b-5p in BALF compared to controls. Additionally, eca-miR-146a-5p abundance was significantly increased in BALF from horses with neutrophilic EA compared to mastocytic EA. Target and pathway enrichment analyses for eca-miR-146a-5p identified immune-relevant pathways, such as MAPK and T-cell receptor signaling, supporting its involvement in inflammatory processes associated with asthma. This study identified three promising candidates, eca-miR-125a-3p, eca-miR-125b-5p, and eca-miR-146a-5p, as potential biomarkers associated with different EA endotypes. These miRNAs are interesting candidates for further investigation in an independent cohort.

In our society, ageing, longevity, and neurodegenerative diseases are major concerns of public health. Recently, in Drosophila, we have identified a new cluster of three snoRNAs, including jouvence, and showed that each of them affect longevity and neurodegeneration. As these snoRNAs are required in the epithelium of the gut, these results point-out a causal relationship between the epithelium of the gut and the neurodegenerative lesions through the metabolic parameters, indicating a gut-brain axis. Here, we demonstrate that each snoRNA pseudouridylates a specific site on ribosomal-RNA, which consequently affects the amount of ribosomes as well as the translational efficacy. Moreover, using TRAP experiment assay, we also show that these lacks of pseudouridylations modify the translation of specific genes involved in lipid metabolism. Consequently, these lead to a chronic deregulation of trigycerides and sterols levels, whose correlate to an increase of neurogenerative lesions in old flies, as well as to a modification of longevity.

IP3R-Grp75-VDAC1 protein complex at the mitochondria-ER contact sites (MERCS) is involved in response to nutrients and control of glucose and energy metabolism, however, early alterations of the complex and MERCS in response to increased fat intake remain inconclusive. We investigated early effects of high-fat diet (HFD) on IP3R-Grp75-VDAC1 protein expression in correlation with ER-mitochondrial interaction in the liver of mice. Five-week-old mice were fed an HFD or a standard diet (SD) for 2 weeks (2W) or 8 weeks (8W). MERCS fractionation by a gradient ultracentrifugation, Western blot, transmission electron microscopy (TEM), Oroboros high-resolution respirometry were used to analyse liver tissues, while real-time PCR was used to profile genes responsive to HFD. No macroscopic morphological or functional alterations were observed in mice at 2W, while, expectedly, at 8W of HFD mice gained weight and glucose intolerance. Total IP3R protein was reduced at both 2W and 8W points by a post-transcriptional mechanism, while in MERCS, IP3R, VDAC1 and Grp75 were reduced at 8W time-point. TEM analysis revealed a significant reduction of mitochondrial coverage by MERCS, mitochondrial fragmentation and shortening of ER-mitochondria distance already at 2W time-point. Mitochondrial function and metabolism were largely spared. Markers of altered protein homeostasis such as Lmp2, Mecl-1 and Lmp7 showed an early upregulation. In conclusion, HFD induces early alterations in liver MERCS that precede gain of weight and glucose intolerance, suggesting their primary role in obesity and metabolic diseases and as potential therapeutic target.

Bisphenol A (BPA) and Bisphenol S (BPS) exposure disrupt ovarian function and granulosa cell (GC) steroidogenesis. Extracellular vesicles (EVs) and their miRNA cargo, as mediators of cellular response to environmental stimuli, might be involved in fertility and folliculogenesis. This study explored modulation of microRNA expression after 48h BPA or BPS exposure (10 {micro}M) in ovine primary GC and EVs from corresponding conditioned medium (CM EVs). Small RNA sequencing of control (0h) and 48h treated GC, CM EVs as well as follicular fluid EVs allowed identification of 533 ovine miRNAs, including 129 new sequences. BPA did not alter miRNA expression in GC, while BPS decreased cellular oar-24b miR. In contrast, BPA modified expression of 4 miRNAs in CM-EVs, including 3 new sequences, and two miRNAs were modified by BPS. Both compounds reduced expression of sequence homologous to miR-1306. Further studies are required to decipher their roles in bisphenol toxicity in GC.

Metabolic dysfunction-associated steatohepatitis (MASH) is associated with increased expression of peroxisome proliferator-activated receptor gamma (PPAR{gamma}, Pparg) and reduced expression of genes involved in methionine metabolism in the liver. The nuclear receptor PPAR{gamma} is activated by fatty acids, and the knockout of Pparg in hepatocytes (Pparg{Delta}Hep) reduced the negative effects of MASH on methionine metabolism. Here, we sought to determine whether hepatocyte Pparg is required for the transcriptional regulation of genes involved in hepatic methionine metabolism in conditions with altered fatty acid flux to the liver: fasting, refeeding, and high-fat diet (HFD)-induced obesity/steatosis. Fasting induced liver steatosis and increased the expression of key genes involved in the methionine metabolism in the liver, while 6h-refeeding reversed these effects and reduced the expression of phosphatidylethanolamine N-methyltransferase (Pemt) and cystathionine beta synthase (Cbs). Overall, fasting and refeeding did not alter hepatocyte Pparg expression nor Pparg{Delta}Hep affected fasting and refeeding-mediated regulation of methionine metabolism gene expression. Diet-induced steatosis reduced hepatic Pemt expression in control (Pparg-intact) mice, and the thiazolidinedione (TZD)-mediated activation of PPAR{gamma} in diet-induced obese control (Pparg-intact) mice reduced the expression of betaine homocysteine S-methyltransferase (Bhmt) and Cbs. However, diet-induced steatosis increased hepatocyte Pparg expression, and Pparg{Delta}Hep blocked the negative effects of HFD and TZD on hepatic methionine metabolism. The PPAR{gamma}-dependent reduction of hepatic Bhmt and Cbs expression was confirmed in mouse primary hepatocytes. Taken together, hepatocyte Pparg may serve as a negative regulator of hepatic methionine metabolism in diet-induced obese mice and these actions could contribute to promoting the onset of MASH.

The genetically authenticated Finnish hop genotype LUKE 2541 obtained from wild was evaluated for antibacterial, anti-inflammatory, and anticancer activities. Water extracts from hop cones inhibited the Gram-positive bacteria Staphylococcus aureus and Bacillus cereus, with MIC values of 0.094- 0.188mg/mL, whereas Gram-negative strains showed limited sensitivity. In LPS-primed THP-1 cells, both IPA and IPA-Control extracts reduced reactive oxygen species formation in a dose-dependent manner, exhibiting similar IC50 values (50.41{micro}g/mL and 35.41{micro}g/mL). This hop genotype also displayed clear tissue- and solvent-dependent antiproliferative effects in human cancer cell lines. Bioactivity was strongly enriched in hop cones and predominantly associated with non-polar extracts, particularly hexane and dichloromethane fractions, which produced marked, dose-dependent reductions in cell viability. In contrast, aqueous and methanolic extracts were largely inactive, underscoring the critical importance of extraction chemistry and tissue selection. Sensitivity varied among cancer cell lines, with colorectal cells generally more responsive and leukemia cells less affected, highlighting cell-specific susceptibility. Further research is needed to elucidate underlying mechanisms, determine selectivity toward non-malignant cells, and identify the active compounds responsible for all in all investigated effects.

Milk microRNAs are believed to play gene regulatory functions in the consumers cells. Milk from different species is enriched in microRNAs predicted to influence immunity, metabolism, and intestinal homeostasis. For milk microRNAs to regulate gene expression in the consumer, they must survive digestion and be present at sufficient levels to influence intestinal cells and potentially beyond-intestinal cells. Milk microRNAs are proposed to be protected from degradation through their association with milk extracellular vesicles (EVs), which might also deliver them to cells. Studies on milk microRNA oral transfer and tissue bioavailability are limited by interspecies sequence homology, making it difficult to distinguish endogenous from exogenous microRNAs. Here, we used a transgenic (TG) cow model expressing four unique microRNA sequences (AmiRs) in its milk to study their association with milk EVs, their resistance to in vitro digestion, and AmiR uptake and regulatory activity in vitro. We confirmed the presence of the four milk EV populations in raw wild-type (WT) and TG cow milk, similar to those previously reported in commercial (pasteurized) cow milk, and confirmed their association with AmiRs and classical milk microRNAs. AmiRs showed differential resistance to simulated adult digestion. In vitro uptake studies showed a modest gene regulatory effect of AmiRs in Caco-2 cells incubated with TG milk EVs. The intent of using this model was to perform in vitro analysis which could lay the groundwork for later in vivo bioavailability studies, taking advantage of the uniqueness of the AmiRs sequences and bypassing the limitation of microRNA sequence homology. HighlightsO_LINew milk EV populations identified previously in commercial bovine milk were also identified in raw milk, indicating that they are not merely the result of processing C_LIO_LIThe routinely discarded EVs (12K and 35K) seem to be preferentially enriched with microRNAs in raw cow milk as was previously shown for commercial cow milk C_LIO_LIAmiRs in transgenic milk resist differentially to simulated digestion C_LI

Osteosarcoma (OS) is an aggressive bone cancer that most commonly affects children and young adults. OS exhibits a high degree of genomic complexity, as well as cellular plasticity, and dynamic transcriptional regulation is suggested to contribute to treatment resistance and metastasis. Cell lines are well characterized as models to advance our knowledge on OS biology. HOS and U2OS cells have increased invasiveness and higher migratory ability compared with MG63. In this study, we employed a tandem array of consensus transcription factor response elements (catTFREs) proteomic approach to characterize transcription factor (TF) regulatory networks related to OS aggressiveness. We mapped 7,594 proteins and enriched 352 transcription factors and coregulators. When we integrated proteomics with cell line specific gene expression and chromatin accessibility we classified the proteins into different OS cell line dependent sub-clusters and identified TFs and coregulators common for all cell lines and specific for individual cell lines. We demonstrate that RUNX2, MYBL2 and HMGA2 are specifically enriched in HOS and U2OS and may be linked to the cell aggressiveness. ETV5, JUNB, NFIX and ZEB1 were among TFs specific to MG63. Our analysis provides a more comprehensive understanding of the transcriptional drivers that shape OS regulatory landscapes and may have future therapeutic implications.

The RETINOBLASTOMA-RELATED (RBR) protein in plants functions as a cell-cycle inhibitor, regulating cell numbers in developing organs and establishing cellular quiescence during growth. Although the role of RBR counterparts in animals also involves regulating cell size, this potential function remains unexplored in plants. We investigated transgenic Arabidopsis plants with altered RBR levels and observed corresponding changes in cell size from embryogenesis through organ development. In addition, stomatal meristemoid cells with reduced RBR levels divided beyond the size threshold, whereas elevated RBR levels increased their size. RBR stimulated terminal differentiation in the stomatal lineage by inducing MUTE and CYCLIN D5;1 expression, whereas reduced RBR levels maintained asymmetric divisions through high SPEECHLESS and CYCLIN D3;1 expression. Interestingly, the cell proliferation-dependent phosphorylation of RBR at the conserved 911Ser site positively correlated with RBR protein levels in the transgenic lines and aligned with the effect of RBR on cell size. This study discusses the potential link between RBRs control of cell proliferation and cell size, providing new insights into the coordinated regulation of plant development.

Cryopreservation is essential for long-term storage of biological tissues. Yet, surprisingly, the precise molecular impact of cryopreservation on tissue transcriptomes remains poorly defined. This study provides the first resource of whole-genome transcriptomic changes following cryopreservation. This study used bulk RNA sequencing to examine how preservation method (snap freezing or vitrification) affects transcriptomes in mouse cerebral cortex and hippocampus. This allowed us to separate cryoprotectant-specific changes from cold induced-changes via snap freezing. In a subset of genes, tissues processed under vitrification conditions showed selective under-representation of a small but structurally coherent group of transcripts, with the hippocampus exhibiting greater vulnerability than the cortex. UniProt annotation revealed that affected transcripts were strongly enriched for proteins with membrane-associated, secretory-pathway, and multi-pass topologies, indicating that structurally complex membrane-integrated architectures are disproportionately sensitive to vitrification. Pathway-level analysis using iPANDA further showed that negative preservation scores in vitrified tissue clustered primarily within signal transduction and metabolic pathways, suggesting coordinated pathway-level disruption rather than global transcript loss. Together, these results demonstrate that vitrification conditions induce selective and structured molecular perturbations in neural tissue, defined by the under-recovery of transcripts associated with membrane and secretory pathway organization. This work highlights molecular vulnerability during vitrification and emphasizes the need for transcript-level evaluation when optimizing cryopreservation approaches for neural systems.

The relaxin-3/relaxin family peptide receptor 3 (RXFP3) neuropeptidergic system is emerging as a potential target for treating various neuropsychiatric diseases, particularly those involving dysregulated stress and arousal. RXFP3 is abundantly expressed in several hypothalamic nuclei, and in the zona incerta (ZI). These regions play a central role in the regulation of stress and arousal, however the function of relaxin-3/RXFP3 within these circuits is unknown. The purpose of this study was to begin characterising this function by describing the distribution and genetic signature of neurons that express RXFP3. We used RNAscope fluorescent in situ hybridisation to characterise the spatial expression pattern and neurochemical phenotype of cells expressing Rxfp3 mRNA throughout the mouse lateral hypothalamus (LH) and ZI. We found that Rxfp3 is expressed across the rostrocaudal extent of both the LH and ZI and follows a parabolic pattern of expression, peaking in more rostral areas of each nucleus. Neurochemical phenotyping of Rxfp3+ cells with Gad1, Slc17a6 (vGlut2), Pvalb, Th, and Sst showed that LH/ZI Rxfp3+ cells co-express each marker to varying extents, generally proportional to their overall abundance within each structure. Furthermore, LH/ZI Rxfp3+ cells overlapped with several known populations involved in various facets of fear learning and defensive behaviour, such as the dopaminergic A13 group, somatostatin-expressing rostral ZI neurons, and glutamatergic LH neurons. The neurochemical diversity of these neurons may reflect the overall role of both the LH and ZI as global regulators of behaviour and the role of relaxin-3/RXFP3 signalling in modulating high-vigilance states.

We present a novel integrative analysis of transposable elements (TEs) in 4 single cell RNA-seq (scRNA-seq) datasets of postmortem substantia nigra pars compacta samples of Parkinson Disease (PD) patients matched healthy controls, with the objective of building a cell-type specific trustworthy atlas of TEs that may clarify the role of TEs in sex differences in PD. We have used the soloTE tool to evaluate the TEs expression changes across all snRNA-seq studies identified in our previous systematic review, and then integrated the results using meta-analysis techniques. Finally, we evaluated the possible associations between TEs and protein coding genes by integrating our previous results in this matter with the information of TEs obtained, in order to propose the possible action mechanism by which some of the TEs contribute to PD.

Natural extracts could improve sperm storage and artificial insemination (AI). This study, for the first time, evaluates the suitability of a blueberry extract (Vaccinium corymbosum) obtained from pomace using a sustainable methodology as a supplement for bull semen extenders. Cryopreserved semen doses from eight bulls were combined in 9 pools (3 bulls/pool), supplemented with 0%, 1%, 5%, or 10% extract, and incubated up to 5 h at 38 {degrees}C. Motility was assessed hourly using OpenCASA, and the effects of treatment and time were evaluated using linear mixed-effects models. Motility was significantly better preserved with 1% extract (total and progressive motility, improved linear velocity and linearities, and decreased BCF and fractal dimension, related to hyperactivation). The effect of 5% was overall positive, but it was below 1%, whereas 10% mostly showed a negative effect. These results show that this natural extract could safely supplement bull semen extenders at least between 1% to 5%, and even help improve sperm motility. Therefore, this extract offers an opportunity to enhance cattle semen extenders using a sustainable approach, potentially improving reproductive outcomes.

The Alzheimers Amyloid Precursor Protein (APP) has determinant roles in neuronal development and function, both in its full-length conformation and as some of its proteolytic peptides, particularly secreted (s)APPa. Given that APP phosphorylation tightly regulates its trafficking, proteolysis, and protein-protein binding, it consequently affects several APP functions. The S655 residue, located in the basolateral sorting motif YTSI at APP C-terminus has been observed to be phosphorylated in mature full-length APP and its C-terminal fragments. Previously observed to modify APPs protein interactions, resulting in altered endolysosomal trafficking, andincreased half-life and sAPPa generation, phosphoS655 APP has potential to modulate APP-mediated neuronal differentiation. To study the phosphoS655 differential interactome relevant for neuronal differentiation, SH-SY5Y cells expressing Wt or S655 phosphomutants APP-GFP were differentiated at two time points. APP-GFP and their respective interacting partners were immunoprecipitated using GFP-trap, and interactors identified by mass spectrometry. Both dephospho and phosphoS655 interactomes were generally enriched in similar processes, primarily RNA processing and translation, as well as signal transduction, metabolism, and cytoskeleton remodeling. The smaller phosphoS655 interactome contributes for functional specialization via binding to e.g. FUBP3, ELAVL4, ATXN2, Tubulin, INA. Several of these specific binding partners are known to promote neurite outgrowth and likely underlie our experimental observation that phosphoS655 APP promotes neuritogenesis, particularly the formation of longer neuritic extensions. These results are not only important for the body of knowledge on this Alzheimers disease core protein, but may also aid in future therapies against this disease.