ASSOCIATION OF HAPTOGLOBIN GENOTYPES, BREAST CANCER AND MALARIA IN A POPULATION OF NIGERIAN WOMEN

Breast cancer is the leading cause of mortality among women with over a million cases recorded globally. Haptoglobin (Hp) is an acute phase glycoprotein whose major role is to remove free hemoglobin from circulation. The prevalence of Hp genotypes varies between populations from different countries and ethnic groups. Several studies have investigated the association of haptoglobin genotypes with breast cancer occurrence, but have reported conflicting results. However, only few studies have investigated the incidence of Hp genotypes, and their association to breast cancer occurrence and malaria in Nigeria. In this study, the association of haptoglobin genotypes, breast cancer and malaria was investigated among healthy women and clinically diagnosed breast cancer patients. Blood samples were collected from Lagos and Ogun States, and DNA was extracted using standard methods. Haptoglobin genotypes and malaria were detected by polymerase chain reaction (PCR) and agarose gel electrophoresis in breast cancer patients (n=75) and healthy controls (n=287). The percentage distribution of the 362 women that participated in the study was as follows: Hp 2-1 (39.8%) genotype had the highest prevalence, followed by Hp 1-1 (34.5%), and Hp 2-2 (25.7%). A highly significant increase in Hp 1-1 genotype (P<0.05) was observed among patients in the BC group when compared with the control group. Furthermore, a higher frequency of Hp 1 allele (54.4%) than Hp 2 allele (45.6%) was observed from both groups. However, no significant difference was observed in Hp 1 allele among the BC group when compared with the control group. A significant increase in Hp 1 allele (P<0.05) was observed among malaria-positive patients in the control group. An increase in Hp 1 allele was also observed in BC group, but it was not significant. The result of this study suggest an association between Hp 1-1 genotype and breast cancer occurrence, and an association between Hp 1 allele and increased risk of malaria infection

Determination of haptoglobin, hemoglobin genotypes and malaria incidence in Nigerian breast cancer patients

Breast cancer is the second leading cause of cancer morbidity and mortality globally. Cancer chemotherapy commonly result in hemolysis, which impacts patient overall health. There is a need to determine genetic factors associated with hemolysis in breast cancer patients. Haptoglobin (Hp), a polymorphic protein plays important role in hemoglobin clearance and disease predisposition, but has been reported to have no prognostic factor in breast cancer. However, understanding selection pressure that drives certain gene mutations in specific populations and how it confers protection or susceptibility to diseases is crucial. In Nigeria, breast cancer, malaria infection and sickle cell disease are prevalent and associated with hemolysis, but little is known of their association in breast cancer patients. This study aims to determine relationship between haptoglobin, hemoglobin genotypes and submicroscopic malaria co‐morbidity in clinically diagnosed breast cancer and healthy Nigerian women. DNA was extracted from blood using standard methods. Haptoglobin 2 and hemoglobin genotypes were detected by RFLP‐PCR, while Plasmodium falciparum infection was detected by primer specific amplification of plasmodium cytochrome oxidase III gene (cox III) in 75 clinically diagnosed breast cancer (BC) and 287 healthy women (control; HC). Proportions were determined and compared in the two groups and test of association was carried out with significance level set at P <0.05. In BC groups, 3 (4.1%) of 72 Hp 2‐2 phenotypes was detected compared to a significantly higher occurrence of 48 (16.7%) of 287 in HC group (p <0.05). Conversely, malaria infection was detected in 68 (94.4%) BC versus 255 (88.9%) in HC group. A similar proportion had Hp deletions (2 in BC and 8 in HC group). There was a low prevalence of hemoglobin S genotype in the entire population and relative risk for Hp 2‐2 polymorphism in hemoglobin genotypes was not significantly different. In conclusion, this study reports in breast cancer and healthy women an inverse correlation of haptoglobin (Hp2‐2) genotype with malaria incidence in southwest Nigeria. The results imply a possible protection against hemolysis and can play significant role in determining choice of cancer therapy for good patient treatment outcomes

ANTIDIABETIC ACTIVITY AND TOXICOLOGICAL EVALUATION OF THE METHANOL-DICHLOROMETHANE ROOT BARK EXTRACT OF NAUCLEA DIDERRICHII (DE WILD) MERR

Objective: In southeastern Nigeria, Nauclea diderrichii (De Wild) Merr is used in the treatment of a wide range of ailments including diabetes mellitus (DM). This study evaluates the antidiabetic activity and toxicological profile of the methanol-dichloromethane root bark extract of N. diderrichii in normoglycemic and alloxan-induced diabetic models.Methods: Dried root barks of N. diderrichii were extracted using methanol and dichloromethane (1:1) to obtain N. diderrichii extract (NDE). The acute and sub-chronic toxicity tests were performed using standard procedures. The effect on alanine aminotransferase (ALT) aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), packed cell volume (PCV), hemoglobin concentration (HB) and total white blood cell (TWBC) count was determined in the rats after treatment. In order to elucidate its antidiabetic mode of action, an oral glucose tolerance test (OGTT) was performed using glucose (2 g/kg) as substrate and alloxan (100 mg/kg; i. v.) induced diabetic model. Glibenclamide (GLI 0.2 mg/kg) was used as the reference standard drug.Results: The results indicated that the LD50 of the extract is&gt;5000 mg/kg. ALT, AST, ALP PCV, HB and TWBC were insignificantly (p&gt;0.05) different compared with the control. No significant changes were observed in the organ weights compared with the control. In the acute and prolonged antidiabetic study, NDE (100, 200 and 400 mg/kg) significantly reduced the blood glucose level (BGL) by 14.66, 18.9, 25.80% and 75.11, 80.24, 83.74% respectively. In comparison, GLI, when administered, reduced BGL by 38.18 and 92.86% respectively.Conclusion: N. diderrichii possesses antidiabetic activity with good toxicological profile

A Review of Exosomal Isolation Methods: Is Size Exclusion Chromatography the Best Option?

Extracellular vesicles (EVs) are membranous vesicles secreted by both prokaryotic and eukaryotic cells and play a vital role in intercellular communication. EVs are classified into several subtypes based on their origin, physical characteristics, and biomolecular makeup. Exosomes, a subtype of EVs, are released by the fusion of multivesicular bodies (MVB) with the plasma membrane of the cell. Several methods have been described in literature to isolate exosomes from biofluids including blood, urine, milk, and cell culture media, among others. While differential ultracentrifugation (dUC) has been widely used to isolate exosomes, other techniques including ultrafiltration, precipitating agents such as poly-ethylene glycol (PEG), immunoaffinity capture, microfluidics, and size-exclusion chromatography (SEC) have emerged as credible alternatives with pros and cons associated with each. In this review, we provide a summary of commonly used exosomal isolation techniques with a focus on SEC as an ideal methodology. We evaluate the efficacy of SEC to isolate exosomes from an array of biological fluids, with a particular focus on its application to adipose tissue-derived exosomes. We argue that exosomes isolated via SEC are relatively pure and functional, and that this methodology is reproducible, scalable, inexpensive, and does not require specialized equipment or user expertise. However, it must be noted that while SEC is a good candidate method to isolate exosomes, direct comparative studies are required to support this conclusion

Effects of Stevioside on oxidative DNA damage in liver and kidney of High Fat Diet Induced type 2 diabetes in Rats

Type 2 diabetes mellitus (T2DM) is the most prevalent form of diabetes and it has been reported to be associated with oxidative stress-induced cellular dysfunction including diabetic nephropathy. Stevioside (STV), a natural non-caloric sweetener refined from the leaves of Stevia rebaudiana Bertoni, has been reported for its insulinotropic and antihyperlipemic effects. In order to investigate the influence of STV on oxidative stress and oxidative DNA damage, high fat-low streptozocin rat model of T2DM were treated orally with 0.125mg/Kg, 0.25mg/Kg and 0.50mg/Kg body weight of STV for 21days. The levels of plasma insulin and dipeptidyl peptidase-4 (DPP IV) were determined using enzyme-linked immunosorbent assay while other biomarkers of T2DM, organ function, oxidative stress and lipid profile were assayed spectrophotometrically. DNA damage in the liver and kidney was determined by assessing the internucleosomal DNA fragmentation pattern on agarose gel electrophoresis. STV treatment resulted in decrease in the levels of fasting plasma glucose, insulin and DPP IV as well as in the activities of plasma amylase and kidney angiotensin-converting enzyme. STV also significantly (p<0.05) improved plasma lipid profile and oxidative stress in the liver and kidney of the diabetic rats, with rats treated with 0.50mg/kg STV having the lowest levels of malondialdehyde and nitric oxide in liver and kidney. There was also a concomitant decrease in the fragmentation of genomic DNA in the liver and kidney of the diabetic rats. This ability of STV, administered orally, to prevent oxidative DNA damage in the liver and kidney of type 2 diabetic rats should contribute to its use in the management of T2DM

The Therapeutic Effect of Extracellular Vesicles on Asthma in Pre-Clinical Models: A Systematic Review Protocol

Asthma is the most common pediatric disease, characterized by chronic airway inflammation and airway hyperresponsiveness. There are several management options for asthma, but no specific treatment. Extracellular vesicles (EVs) are powerful cellular mediators of endocrine, autocrine and paracrine signalling, and can modulate biophysiological function in vitro and in vivo. A thorough investigation of therapeutic effects of EVs in asthma has not been conducted. Therefore, this systematic review is designed to synthesize recent literature on the therapeutic effects of EVs on physiological and biological outcomes of asthma in pre-clinical studies. An electronic search of Web of Science, EMBASE, MEDLINE, and Scopus will be conducted on manuscripts published in the last five years that adhere to standardized guidelines for EV research. Grey literature will also be included. Two reviewers will independently screen the selected studies for title and abstract, and full text based on the eligibility criteria. Data will be extracted, narratively synthesized and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. This systematic review will summarize the current knowledge from preclinical studies investigating the therapeutic effects of EVs on asthma. The results will delineate whether EVs can mitigate biological hallmarks of asthma, and if so, describe the underlying mechanisms involved in the process. This insight is crucial for identifying key pathways that can be targeted to alleviate the burden of asthma. The data will also reveal the origin, dosage and biophysical characteristics of beneficial EVs. Overall, our results will provide a scaffold for future intervention and translational studies on asthma treatment

Characterizing Extracellular Vesicles and Particles Derived from Skeletal Muscle Myoblasts and Myotubes and the Effect of Acute Contractile Activity

Extracellular vesicles (EVs), released from all cells, are essential to cellular communication and contain biomolecular cargo that can affect recipient cell function. Studies on the effects of contractile activity (exercise) on EVs usually rely on plasma/serum-based assessments, which contain EVs from many different cells. To specifically characterize skeletal muscle–derived vesicles and the effect of acute contractile activity, we used an in vitro model where C2C12 mouse myoblasts were differentiated to form myotubes. EVs were isolated from conditioned media from muscle cells at pre-differentiation (myoblasts) and post-differentiation (myotubes) and also from acutely stimulated myotubes (1 h @ 14 V, C-Pace EM, IonOptix, Westwood, MA, USA) using total exosome isolation reagent (TEI, ThermoFisher (Waltham, MA, USA), referred to as extracellular particles [EPs]) and differential ultracentrifugation (dUC; EVs). Myotube-EPs (~98 nm) were 41% smaller than myoblast-EPs (~167 nm, p n = 8–10). Two-way ANOVA showed a significant main effect for the size distribution of myotube vs. myoblast-EPs (p n = 10–13). In comparison, myoblast-EPs displayed a bimodal size distribution profile with peaks at p n = 6–9). Similar biophysical characteristics were observed when EVs were isolated using dUC: myotube-EVs (~195 nm) remained 41% smaller in average size than myoblast-EVs (~330 nm, p = 0.07, n = 4–6) and had comparable size distribution profiles to EPs isolated via TEI. Myotube-EVs also had 4.7-fold higher protein yield vs. myoblast EVs (p n = 4–6). Myotube-EPs exhibited significantly decreased expression of exosomal marker proteins TSG101, CD63, ALIX and CD81 compared with myoblast-EPs (p n = 7–12). Conversely, microvesicle marker ARF6 and lipoprotein marker APO-A1 were only found in the myotube-EPs (p n = 4–12). There was no effect of acute stimulation on myotube-EP biophysical characteristics (n = 7) or on the expression of TSG101, ARF6 or CD81 (n = 5–6). Myoblasts treated with control or acute stimulation–derived EPs (13 µg/well) for 48 h and 72 h showed no changes in mitochondrial mass (MitoTracker Red, ThermoFisher, Waltham, MA, USA), cell viability or cell count (n = 3–4). Myoblasts treated with EP-depleted media (72 h) exhibited ~90% lower cell counts (p n = 3). Our data show that EVs differed in size, distribution, protein yield and expression of subtype markers pre vs. post skeletal muscle–differentiation into myotubes. There was no effect of acute stimulation on biophysical profile or protein markers in EPs. Acute stimulation–derived EPs did not alter mitochondrial mass or cell count/viability. Further investigation into the effects of chronic contractile activity on the biophysical characteristics and cargo of skeletal muscle–specific EVs are warranted