Biological Research最新文献

Background: Selection for increased homogeneity of litter size has been proposed to improve resistance to stress and diseases in animals. Previous studies have shown that lines selected for decreased litter size variability (L lines) have higher litter sizes at parity than lines selected for increased litter size variability (H lines), possibly due to higher embryo survival in the oviduct. Thus, the study aimed at examining the effect of the line selection on the oviduct environment and its contribution to embryo development, particularly via extracellular vesicles (EVs). Oviductal EVs (oEVs) and their molecular cargo play essential roles in supporting early embryo development in different species, but little is so far known in rabbits.

Results: Oviductal fluid was collected by flushing oviducts from pregnant (with embryos at 72 h p.c., P) and control does (non-ovulated/non-pregnant, NO) from the two lines, resulting in 4 experimental groups: pregnant H line (H_P); pregnant L line (L_P); non-pregnant H line (H_NO); non-pregnant L line (L_NO). Oviductal EVs were isolated and characterized. RNA cargo of oEVs was analyzed by RNA-sequencing, revealing a high number of differential abundant (DA) genes between P vs. NO does in both lines (1223 DA genes in H line and 1519 in L line, FDR < 0.1%). Additionally, 27 and 25 miRNAs were found as DA between P vs. NO in H and L lines, respectively. Interestingly, functional enrichment analysis of DA genes and predicted target genes of identified miRNAs revealed biological terms such as embryo development, epithelium morphogenesis and differentiation, and cilium movement, which were only associated to L line for P and NO groups. Furthermore, the comparison between H and L lines identified 169 DA RNAs in NO does, but without significant differences in P does. For miRNAs, no differences were detected in H vs. L lines in P or NP does.

Conclusions: This is the first study unveiling the differential oEV RNA cargo between lines selected for low versus high variation in litter size, and in each line, between P and NO does. The changes in protein-coding RNA and miRNA cargo might reflect the different maternal support to the early embryo development in the different lines.

Background: Endometriosis (E) is multifactorial disease affecting around 10% of women worldwide. The association between E and infertility is clinically well recognized. For E patients to achieve a successful pregnancy, assisted reproductive technologies (ART) are considered as a treatment option. However, the impact of E on oocyte quality, its potential to be fertilized as well as pregnancy rates, is still under debate and with very few molecular clues explaining the clinical data. Alterations in protein-coding RNAs in cumulus cells (CCs), cells surrounding the oocytes and contributing to oocyte maturation, have been reported in E patients. But there is a lack of information regarding microRNAs (miRNAs), which control protein translation. Thus, we aimed: (1) to identify altered miRNA expression in CCs of E patients versus patients without the disease (control, C); and (2) to unveil if in E patients, CCs from fertilized oocytes display a different miRNA profile versus oocytes that failed fertilization. Small RNA-sequencing was performed on CCs from patients undergoing ART.

Results: A total of 85 differentially expressed (DE) miRNAs were identified in E versus C patients (FDR < 0.05). In E patients, 25 DE miRNAs were found between fertilized oocytes and oocytes that failed fertilization, while 13 DE miRNAs in C patients (FDR < 0.05). Comparisons among DE miRNAs highlighted three notable miRNA sets: Set (1) 35 DE miRNAs specific to E; Set (2) 27 DE miRNAs affected by both E and the potential to be fertilized; and Set (3) 6 DE miRNAs characteristic of a competent oocyte successfully fertilized despite the disease. Target gene analysis of DE miRNAs unveiled genes involved in oocyte meiosis, progesterone-mediated oocyte maturation pathway, embryo development, mitochondria and spindle alterations, calcium signaling, and oxidative stress.

Conclusion: This study identified for the first time an altered miRNA signature in CCs of E patients, pointing towards compromised oocyte competence. Besides, in E patients, a characteristic CCs miRNA footprint for oocytes that can be successfully fertilized despite the disease has been revealed. The study charts new territory for non-invasive diagnosis and personalized treatments based on miRNAs to improve oocyte competence in E patients under ART treatments.

Background: Sickle cell disease (SCD) is characterised by chronic oxidative stress. However, there is limited information on how polymorphisms in cytokine genes influence oxidative stress in SCD patients. The study aimed to determine the effect of Interleukin gene (IL-10) and Tumor Necrosis Factor (TNF_αβ) polymorphisms on oxidative stress and cytokine levels in SCD patients from Mulago hospital.

Methods: A case control study with cross-sectional sample size of 163 SCD patients and 189 healthy controls was carried out. The extent of oxidative stress was quantified using Malondialdehyde (MDA) by spectrophotometry. Levels of IL-10 and TNF-α were measured using the Enzyme-Linked Immuno-Sorbent Assay (ELISA). The Amplification Refractory Mutation System polymerase chain reaction (ARMS-PCR) assay was used to genotype IL10-1082 A > G, (rs1800896), IL10-819 C > T (rs1800871) and TNF-α-308G > A (rs1800629) and TNF-β + 252 A > G (rs909253) gene polymorphisms.

Results: Samples showed significantly (P = 0.0063) higher median plasma levels of MDA in SCD patients (2.756µM) than healthy controls (2.364µM). A similar trend was observed with significantly (P < 0.0001) higher median plasma levels of IL-10 in SCD patients (20.37pg/ml) than healthy controls (7.5pg/ml). The most frequent genotype for IL-10 (-1082, rs1800896) gene polymorphism was heterozygous GA (62.6%). No significant association between IL10 (-1082G > A, rs1800896) gene polymorphisms and SCD was observed (OR = 1.08, 95% CI = 0.54-2.14, P = 0.87). Yet, IL10 homozygous GG (-1082, rs1800896) (22.12pg/ml) that was found to be significantly associated (P = 0.0234) with increased plasma levels of IL-10 as compared to heterozygous genotype (GA) (13.94pg/ml) in SCD patients. Similarly, higher levels of MDA were found to be significantly (P < 0.0001) associated with homozygous GG at IL-10 (-1082, rs1800896). The most frequent and only reported genotype for TNF-α/β gene polymorphisms were heterozygous GA, thus no associations were described.

Conclusion: In conclusion, our results suggest that the IL10 (-1082 G > A, rs1800896) gene polymorphism is associated with increased oxidative stress and IL-10 cytokine level in Ugandan SCD patients.

Background: In many types of tumors, the expression patterns of actin-binding proteins -fascin-1 and various isoforms of tropomyosin - are altered. Fascin-1 is an actin-bundling protein that promotes cancer cell motility, whereas tropomyosin functions as a tumor and metastasis suppressor. However, the mechanisms by which tropomyosin isoforms regulate fascin-1 remain poorly understood. This study aimed to investigate the reciprocal effects of fascin-1 and tropomyosin isoforms on their interactions with actin and on the formation of actin bundles.

Methods: Recombinant fascin-1 and the cytoskeletal tropomyosin isoforms encoded by TPM2 (Tpm2.1, Tpm2.3, and Tpm2.4) were expressed in BL21-DE3 cells and purified. High-speed centrifugation was employed to assess the actin affinities of fascin-1 and the Tpm2 isoforms. Actin filament bundling was analyzed using low-speed centrifugation and fluorescence microscopy. A pull-down assay was performed to examine direct interactions between fascin-1 and the Tpm2 isoforms. Confocal microscopy was used to analyze the localization of fascin-1 in the metastatic SAOS-2 LM5 cell line overexpressing Tpm2 isoforms.

Results: Among the three recombinant, acetylated Tpm2 isoforms, Tpm2.4 exhibited the highest affinity for F-actin. All Tpm2 isoforms strongly inhibited fascin-1-mediated actin bundling at low fascin-1 concentrations, with bundling restored only at substantially higher fascin-1 levels. The resulting actin bundles contained both Tpm2 and fascin-1; however, the number of filaments per bundle was reduced in the presence of any Tpm2 isoform. Fascin-1's affinity for actin was decreased in the presence of Tpm2 isoforms, and increased Tpm2 occupancy on actin filaments partially displaced fascin-1. In contrast, fascin-1 binding did not affect the affinity of Tpm2 isoforms for actin. Pull-down assays revealed that Tpm2 isoforms can directly interact with fascin-1, with Tpm2.4 showing the highest affinity. The inhibitory effect of Tpm2 on fascin-1-actin interactions was further supported by cellular data, which showed that overexpression of cytoplasmic Tpm2.1, Tpm2.3, or Tpm2.4 in SAOS-2 LM5 cells reduced fascin co-localization with actin.

Conclusion: Cytoplasmic Tpm2 isoforms regulate actin bundling activity of fascin-1 by organizing protein composition in the bundles, a mechanism that may contribute to the suppression of metastatic phenotype in cancer cells.

Rab11 and Rab35 have been implicated in large-scale intracellular membrane trafficking during the last phases of the cell cycle. Although both proteins are associated with cytokinetic abscission, they appear to perform distinct functions and give rise to different phenotypes in dividing cells. Despite a substantial body of research on each protein individually, no study to date has systematically compared Rab11 and Rab35 in the context of cancer cell division. As a result, the extent of their interrelationship and potential compensatory mechanisms remains unclear. Our data demonstrate that Rab11a, Rab11b and Rab35 expression levels are partially interrelated. We also show that Rab11 and Rab35 contribute to mitotic progression in different ways, particularly during specific stages of the M-phase. Notably, depletion of either Rab11 or Rab35 disrupts cytokinetic abscission and correlates with aberrant F-actin accumulation at the intercellular bridge. Furthermore, overexpression of related Rab proteins with overlapping functions does not rescue the cytokinetic defects caused by Rab11 or Rab35 downregulation in cancer cells. Therefore, this study aims to deepen our understanding of how Rab11 and Rab35 orchestrate the molecular events that drive the progression from late anaphase through the completion of cytokinesis.

Background: Recent studies have indicated the potential involvement of haptoglobin in a variety of events during mammalian reproduction, with previous research highlighting its efficacy in promoting porcine embryo development. The present study aims to provide a comprehensive investigation of haptoglobin expression and secretion in the bovine uterus, with a view to assessing its impact on bovine in vitro embryo production. A systematic study was conducted on cows in different oestrous stages, early follicular, late follicular, early luteal and late luteal stages. Relative haptoglobin mRNA abundance was quantified by RT-qPCR, and the expression of the protein was analysed by immunohistochemistry. The results were complemented by proteomic analyses of the uterine fluid. In vitro bovine fertilisation and embryo culture were carried out in the presence of haptoglobin.

Results: Haptoglobin mRNA expression in the bovine uterus is most abundant during the late luteal stage of the oestrous cycle. The presence of haptoglobin was demonstrated in the uterine epithelium and the uterine fluid in different stages of the oestrous cycle by immunohistochemistry and proteomic analyses. Furthermore, the addition of haptoglobin to in vitro culture improved the development of bovine embryos when the protein was present on the days corresponding to its passage through the uterus, with a higher blastocyst yield (P < 0.05) being observed in haptoglobin treatments compared with control groups. Haptoglobin appears to influence embryonic development by reducing mitochondrial activity and lipid content. Furthermore, transcripts associated with oxidative stress, lipid metabolism and cell cycle regulation were affected by the presence of haptoglobin.

Conclusions: The presence of haptoglobin protein in the female tract of cows during different stages of the oestrous cycle suggests that it plays a significant role in the reproductive process. The addition of haptoglobin during in vitro embryo production resulted in enhanced blastocyst rates and improved quality.

Background: Gallbladder cancer (GBC) is a highly lethal malignancy, often diagnosed at advanced stages when curative options are limited. Its rapid progression and high metastatic potential result in a 5-year survival rate below 5%. Recent evidence highlights the role of Endothelin-1 (ET1), a bioactive peptide, in promoting tumor aggressiveness through activation of its receptors (ETRs). However, therapeutic strategies have mainly focused on receptor inhibition, neglecting the modulation of ET1 availability. Therefore, this study aimed to evaluate the therapeutic potential of modulating ET1 levels through the application of recombinant Neprilysin (rNEP) to degrade ET1 or inhibition of Endothelin Converting Enzyme-1 (ECE1) to reduce its production in GBC cells.

Methods: The effects of rNEP and the ECE1 inhibitor SM19712 were evaluated in GBC cell lines by assessing ET1 levels, nuclear β-catenin localization, transcript levels of target genes, and changes in proliferation, migration, invasion, and stemness-associated markers in vitro.

Results: Both rNEP and SM19712 significantly reduced extracellular ET1 levels, nuclear β-catenin localization, and expression of genes such as CCND1, VEGFA, and BIRC5. Treatment also decreased the expression of EMT and stemness markers CD44 and Vimentin. Functionally, rNEP reduced cell migration, invasion, and colony formation, while SM19712 affected migration and colony formation. Isoform analysis revealed predominant expression of ECE1c, suggesting potential ET1-independent roles in invasion.

Conclusion: Modulating ET1 bioavailability through enzymatic degradation or inhibition of its synthesis reduces aggressiveness in GBC cells. These findings support the use of rNEP and ECE1 inhibition as promising strategies for GBC treatment, although further in vivo validation is required.

Objective: Sodium Thiosulfate (STS), a clinically approved agent for cyanide poisoning and vascular calcification, possesses antioxidant, anti-inflammatory, mitochondrial preservation, and metal chelation capabilities, rendering it a promising candidate for managing ischemia-reperfusion (IR) injury. The detrimental impact of high-fat diets (HD) on the outcomes of IR during renal surgeries is well-documented. However, the potential of STS to ameliorate renal IR injury in rat fed with high fat diet is not known.

Methods: Male Wistar rats were fed a standard diet (SD) or a high-fat diet (HD) for 16 weeks before undergoing an IR protocol (45 min of ischemia followed by 24 h of reperfusion). STS (10 mg/kg) was administered 30 min before IR.

Results: STS effectively mitigated IR-induced physiological decline and tissue damage in SD rats but was less effective in HD rats. To explore this difference, we measured renal mitochondrial quality. STS improved mitochondrial bioenergetics, balanced mitochondrial dynamics, and increased mitochondrial copy number in SD-IR rats more than in HD-IR rats. Additionally, STS significantly reduced oxidative stress and upregulated Pgc-1α, Polg, and Tfam genes in SD-IR rats but had a lesser effect in HD-IR rats. The 16-week HD significantly reduced renal mitochondrial quality at the basal level, hindering STS-mediated protection.

Conclusion: These findings highlight the efficacy of STS in managing renal IR and emphasize the need for nutritional support to restore mitochondrial function in high-fat diet subjects.

The yellowtail kingfish (Seriola lalandi) is a key species for the diversification of Chilean aquaculture. While controlled reproduction is essential for reliable fish production, the physiological and molecular bases underlying its reproductive cycle remain insufficiently explored. This study aimed to assess the expression patterns of brain-expressed neuroendocrine mRNAs involved in the activation of brain-pituitary-gonad (BPG) axis throughout different year seasons and to correlate them with ovarian maturation stages in S. lalandi females under captive conditions. Reproductive stages were determined by ovarian histology and gonadosomatic index (GSI) analysis. Expression levels of GnRH1 and GnRH2 genes (gnrh1 and gnrh2) as well as melatonin (mtn1ra), dopamine (drd2a), and kisspeptin (kiss1r) receptors were quantified by real time PCR in brain samples from adult individuals. Fish were maintained in temperature- and photoperiod-controlled tanks simulating the four seasons. Histological and GSI analyses identified four distinct reproductive stages. Gene expression peaked in winter and decreased in autumn, aligning with the seasonal progression of ovarian development. These results are consistent with the expected activation of the reproductive axis during the winter months in S. lalandi. Notably, the elevated expression of drd2a in winter suggests that modulation of GnRH action on pituitary function may not rely solely on dopaminergic inhibition. Taken together, our findings indicate that S. lalandi exhibits reproductive dynamics under captivity that mirror those of wild populations, supporting its use as a reliable model for studying reproductive physiology. Moreover, its responsiveness to environmental cues under controlled conditions enables the development of experimental strategies that would be logistically unfeasible in the wild.