Evaluation of the Anticancer Activity of Bioactive Fraction G Extracted from \u3cem\u3ePavetta crassipes\u3c/em\u3e in Malignant Brain Tumor Cell Lines

Objective: Natural products have served as sources of lead compounds that are commonly used in the treatment of human diseases including cancer. Pavetta crassipes has been widely demonstrated to have ethnopharmacological potential in the management of malaria, gastrointestinal conditions, central nervous system behavioral disorders, hypertension, and cancer. The goal of our study was to evaluate the biological and molecular effects of Fraction G, obtained from the plant Pavetta crassipes, on glioblastoma invasive growth and survival. Methodology: The antiproliferative effects of Fraction G, obtained from Pavetta crassipes, was evaluated using the trypan blue exclusion, (3-(4, 5-Dimethylthiazol- 2yl)-2, 5-Diphenyltetrazolium Bromide; MTT), and lactate dehydrogenase (LDH) assays. Flow cytometry and Western blotting analyses were carried out to examine the effects of Fraction G on cell cycle check-points and its effects on epidermal growth factor receptor-mediated signaling of AKT and MAPK pathways. Results: In this paper, we report that the Fraction G obtained from the plant Pavetta crassipes induced a reduction in glioma cell viability and proliferation as well as induced an increase in apoptosis as evidenced by cleaved PARP, increased caspase 3/7 activity, and cell cycle arrest in the G0/G1 check point. Furthermore, we report that Fraction G inhibited the phosphorylation of AKT and MAPK following EGF treatment. Conclusion: Taken together, our results demonstrate that Fraction G has potent inhibitory effects on pathways involved in glioblastoma proliferation and survival

Antimicrobial resistance pattern of methicillin-resistant Staphylococcus aureus isolated from sheep and humans in Veterinary Hospital Maiduguri, Nigeria

BACKGROUND AND AIM : Methicillin-resistant Staphylococcus aureus (MRSA), an important opportunistic pathogen, is a Gram-positive coccus known to be resistant to β-lactam antibiotics. Its virulence depends on a large range of factors, mainly extracellular proteins, such as enzymes and exotoxins, that contribute to causing a wide range of diseases in human and animal species. The major reasons for the success of this pathogen are its great variability, which enables it to occur and thrive at different periods and places with diverse clonal types and antibiotic resistance patterns within regions and countries. Infections caused by antibiotic-resistant S. aureus bring about serious problems in the general population (humans and animals). Infections with these pathogens can be devastating, particularly for the very young, adults and immunocompromised patients in both humans and animals. This study aimed to determine the presence of MRSA in both apparently healthy and sick sheep brought to the veterinary hospital as well as veterinary staff and students on clinical attachment in the hospital. MATERIALS AND METHODS : A total of 200 nasal swab samples were collected aseptically from sheep and humans (100 each) for the isolation of MRSA. The samples were processed by appropriately transporting them to the laboratory, then propagated in nutrient broth at 37°C for 24 h followed by subculturing on mannitol salt agar at 37°C for 24 h, to identify S. aureus. This was followed by biochemical tests (catalase and coagulase tests) and Gram staining. MRSA was isolated using Clinical Laboratory Standard Institute (CLSI) guideline and confirmed by plating onto Oxacillin (OX) Resistance Screening Agar Base agar. The antimicrobial susceptibility pattern of the MRSA isolates was determined using the disk diffusion method against 12 commonly used antimicrobial agents. RESULTS : The total rate of nasal carriage of S. aureus and MRSA was found to be 51% and 43% in sheep and humans, respectively. The MRSA prevalence in male and female sheep was 18% and 8%, while 9% and 8% were for male and female human samples, respectively. The antimicrobial susceptibility test showed 100% resistance to OX, cefoxitin, oxytetracycline, cephazolin, and penicillin-G (Pen) by MRSA isolates from humans. Conversely, there was 100% susceptibility to ciprofloxacin, imipenem, and gentamicin; for linezolid (LZD), it was 87.5%, norfloxacin (NOR) (71%), and erythromycin (ERY) (50%) susceptibility was recorded. The MRSA isolates from sheep recorded 100% resistance to the same set of drugs used for human MRSA isolates and were equally 100% susceptible to gentamicin, imipenem, LZD, ciprofloxacin, NOR (92%), and ERY (50%). CONCLUSION : This study determined the presence of MRSA in sheep and humans from the Veterinary Hospital, Maiduguri. It appears that certain drugs such as ciprofloxacin, imipenem, and gentamicin will continue to remain effective against MRSA associated with humans and sheep. Reasons for the observed patterns of resistance must be explored to reduce the burdens of MRSA resistance. Furthermore, the present study did not confirm the MRSA resistance genes such as mecA and spa typing to ascertain the polymorphism in the X-region using appropriate molecular techniques. Hence more studies need to be conducted to elucidate these findings using robust techniques.http://www.veterinaryworld.orgam2023Production Animal StudiesVeterinary Tropical Disease

Prediction of Associations between microRNAs and Gene Expression in Glioma Biology

Despite progress in the determination of miR interactions, their regulatory role in cancer is only beginning to be unraveled. Utilizing gene expression data from 27 glioblastoma samples we found that the mere knowledge of physical interactions between specific mRNAs and miRs can be used to determine associated regulatory interactions, allowing us to identify 626 associated interactions, involving 128 miRs that putatively modulate the expression of 246 mRNAs. Experimentally determining the expression of miRs, we found an over-representation of over(under)-expressed miRs with various predicted mRNA target sequences. Such significantly associated miRs that putatively bind over-expressed genes strongly tend to have binding sites nearby the 3′UTR of the corresponding mRNAs, suggesting that the presence of the miRs near the translation stop site may be a factor in their regulatory ability. Our analysis predicted a significant association between miR-128 and the protein kinase WEE1, which we subsequently validated experimentally by showing that the over-expression of the naturally under-expressed miR-128 in glioma cells resulted in the inhibition of WEE1 in glioblastoma cells

Novel internal regulators and candidate miRNAs within miR-379/miR-656 miRNA cluster can alter cellular phenotype of human glioblastoma

Clustered miRNAs can affect functioning of downstream pathways due to possible coordinated function. We observed 78-88% of the miR-379/miR-656 cluster (C14MC) miRNAs were downregulated in three sub-types of diffuse gliomas, which was also corroborated with analysis from The Cancer Genome Atlas (TCGA) datasets. The miRNA expression levels decreased with increasing tumor grade, indicating this downregulation as an early event in gliomagenesis. Higher expression of the C14MC miRNAs significantly improved glioblastioma prognosis (Pearson’s r=0.62; p<3.08e-22). ENCODE meta-data analysis, followed by reporter assays validated existence of two novel internal regulators within C14MC. CRISPR activation of the most efficient internal regulator specifically induced members of the downstream miRNA sub-cluster and apoptosis in glioblastoma cells. Luciferase assays validated novel targets for miR-134 and miR-485-5p, two miRNAs from C14MC with the most number of target genes relevant for glioma. Overexpression of miR-134 and miR-485-5p in human glioblastoma cells suppressed invasion and proliferation, respectively. Furthermore, apoptosis was induced by both miRs, individually and in combination. The results emphasize the tumor suppressive role of C14MC in diffuse gliomas, and identifies two specific miRNAs with potential therapeutic value and towards better disease management and therapy

MicroRNA and Target Protein Patterns Reveal Physiopathological Features of Glioma Subtypes

Gliomas such as oligodendrogliomas (ODG) and glioblastomas (GBM) are brain tumours with different clinical outcomes. Histology-based classification of these tumour types is often difficult. Therefore the first aim of this study was to gain microRNA data that can be used as reliable signatures of oligodendrogliomas and glioblastomas. We investigated the levels of 282 microRNAs using membrane-array hybridisation and real-time PCR in ODG, GBM and control brain tissues. In comparison to these control tissues, 26 deregulated microRNAs were identified in tumours and the tissue levels of seven microRNAs (miR-21, miR-128, miR-132, miR-134, miR-155, miR-210 and miR-409-5p) appropriately discriminated oligodendrogliomas from glioblastomas. Genomic, epigenomic and host gene expression studies were conducted to investigate the mechanisms involved in these deregulations. Another aim of this study was to better understand glioma physiopathology looking for targets of deregulated microRNAs. We discovered that some targets of these microRNAs such as STAT3, PTBP1 or SIRT1 are differentially expressed in gliomas consistent with deregulation of microRNA expression. Moreover, MDH1, the target of several deregulated microRNAs, is repressed in glioblastomas, making an intramitochondrial-NAD reduction mediated by the mitochondrial aspartate-malate shuttle unlikely. Understanding the connections between microRNAs and bioenergetic pathways in gliomas may lead to identification of novel therapeutic targets

Pre-microRNA and Mature microRNA in Human Mitochondria

Chantier qualité GAInternational audienceBACKGROUND: Because of the central functions of the mitochondria in providing metabolic energy and initiating apoptosis on one hand and the role that microRNA (miRNA) play in gene expression, we hypothesized that some miRNA could be present in the mitochondria for post-transcriptomic regulation by RNA interference. We intend to identify miRNA localized in the mitochondria isolated from human skeletal primary muscular cells. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the potential origin of mitochondrial miRNA, we in-silico searched for microRNA candidates in the mtDNA. Twenty five human pre-miRNA and 33 miRNA aligments (E-value35) for the smallest RNA input concentration and 204 miRNA for the maximum RNA input concentration. In silico analysis predicted 80 putative miRNA target sites in the mitochondrial genome (E-value<0.05). CONCLUSIONS/SIGNIFICANCE: The present study experimentally demonstrated for the first time the presence of pre-miRNA and miRNA in the human mitochondria isolated from skeletal muscular cells. A set of miRNA were significantly detected in mitochondria fraction. The origin of these pre-miRNA and miRNA should be further investigate to determine if they are imported from the cytosol and/or if they are partially processed in the mitochondria

MiR-128 Inhibits Tumor Growth and Angiogenesis by Targeting p70S6K1

MicroRNAs are a class of small noncoding RNAs that function as critical gene regulators through targeting mRNAs for translational repression or degradation. In this study, we showed that miR-128 expression levels were decreased in glioma, and identified p70S6K1 as a novel direct target of miR-128. Overexpression of miR-128 suppressed p70S6K1 and its downstream signaling molecules such as HIF-1 and VEGF expression, and attenuated cell proliferation, tumor growth and angiogenesis. Forced expression of p70S6K1 can partly rescue the inhibitory effect of miR-128 in the cells. Taken together, these findings will shed light to the role and mechanism of miR-128 in regulating glioma tumor angiogenesis via miR-128/p70S6K1 axis, and miR-128 may serve as a potential therapeutic target in glioma in the future

NMDA Mediated Contextual Conditioning Changes miRNA Expression

We measured the expression of 187 miRNAs using quantitative real time PCR in the hippocampal CA1 region of contextually conditioned mice and cultured embryonic rat hippocampal neurons after neuronal stimulation with either NMDA or bicuculline. Many of the changes in miRNA expression after these three types of stimulation were similar. Surprisingly, the expression level of half of the 187 measured miRNAs was changed in response to contextual conditioning in an NMDA receptor-dependent manner. Genes that control miRNA biogenesis and components of the RISC also exhibited activity induced expression changes and are likely to contribute to the widespread changes in the miRNA profile. The widespread changes in miRNA expression are consistent with the finding that genes up-regulated by contextual conditioning have longer 3′ UTRs and more predicted binding sites for miRNAs. Among the miRNAs that changed their expression after contextual conditioning, several inhibit inhibitors of the mTOR pathway. These findings point to a role for miRNAs in learning and memory that includes mTOR-dependent modulation of protein synthesis

A Ten-microRNA Expression Signature Predicts Survival in Glioblastoma

Glioblastoma (GBM) is the most common and aggressive primary brain tumor with very poor patient median survival. To identify a microRNA (miRNA) expression signature that can predict GBM patient survival, we analyzed the miRNA expression data of GBM patients (n = 222) derived from The Cancer Genome Atlas (TCGA) dataset. We divided the patients randomly into training and testing sets with equal number in each group. We identified 10 significant miRNAs using Cox regression analysis on the training set and formulated a risk score based on the expression signature of these miRNAs that segregated the patients into high and low risk groups with significantly different survival times (hazard ratio [HR] = 2.4; 95% CI = 1.4–3.8; p<0.0001). Of these 10 miRNAs, 7 were found to be risky miRNAs and 3 were found to be protective. This signature was independently validated in the testing set (HR = 1.7; 95% CI = 1.1–2.8; p = 0.002). GBM patients with high risk scores had overall poor survival compared to the patients with low risk scores. Overall survival among the entire patient set was 35.0% at 2 years, 21.5% at 3 years, 18.5% at 4 years and 11.8% at 5 years in the low risk group, versus 11.0%, 5.5%, 0.0 and 0.0% respectively in the high risk group (HR = 2.0; 95% CI = 1.4–2.8; p<0.0001). Cox multivariate analysis with patient age as a covariate on the entire patient set identified risk score based on the 10 miRNA expression signature to be an independent predictor of patient survival (HR = 1.120; 95% CI = 1.04–1.20; p = 0.003). Thus we have identified a miRNA expression signature that can predict GBM patient survival. These findings may have implications in the understanding of gliomagenesis, development of targeted therapy and selection of high risk cancer patients for adjuvant therapy