Back to Old Books Toward Affordable Research: Homemade Phenol-Based Reagent for Triphasic RNA Purification.

Covid-19 crisis did hit many socio-economic aspects in the whole world. In the scientific research, the problem is getting even worse, since most of materials and consumable are allocated to the health sector. Many research laboratories around the world have big delay in receiving their purchases to accomplish their research projects. In the developing countries, the situation is much more difficult, since most of the funding resources are directed to the Covid-19 crisis and there is a notable increase in reagents' prices. Therefore, the aim of the present study is to make a homemade reagents for RNA purification from eukaryotic cells/tissues. The homemade phenol-based RNA extraction reagents were prepared using saturated phenol pH 4.3 (adjusted by 0.5 M citrate buffer) and guanidine thiocyanate. To validate the phenol-based reagent, RNA was purified from different biological samples (cell line, tissues, and fungi) using homemade phenol-based versus a commercial one. Concentration of RNA samples extracted from the same number of cells were compared to assess the homemade preparation of phenol-based reagent. In conclusion, homemade phenol-based reagent is cost effective and comparable to the commercial one. Using homemade phenol-based, RNA extraction was successfully purified from different biological sources.

PTEN mutations prevalence in HER2-positive breast cancer patients / Prevalencia de mutaciones en pten en pacientes con cáncer de mama HER2-positivo

Introduction: HER2-positive tumors is one of the aggressive subtypes of breast cancer that indicate bad prognosis. Trastuzumab is one of the targeted therapy which inhibit HER2 receptors. Mutations/expression deregulations of the downstream of HER2 receptors could cause resistance to trastuzumab. PTEN is a tumor suppressor gene which directly regulates PI3K pathway which renders it one of the predictive markers of trastuzumab. Methods: In the present study, PTEN mutations were screened in 51 patients with HER2-positive breast cancer. Also, 16 patients were further analyzed for protein expression. Results: The mutations were detected in 3 out of 51 patients (5.9%). In addition, 56.3% of the 16 patients showed downregulation/loss of PTEN protein expression. The loss of PTEN was found in 75% of estrogen-receptor negative patients (p =0.130). Conclusions: The downregulation/loss of PTEN protein has the tendency to be associated with ER-negative reflecting its value as a treatment prediction marker. (AU)

Introducción: Los tumores HER2 positivos son uno de los subtipos agresivos de cáncer de mama que indican un mal pronóstico. Trastuzumab es una de las terapias dirigidas que inhiben los receptores HER2. Las mutaciones/desregulaciones de la expresión aguas abajo de los receptores HER2 podrían causar resistencia a trastuzumab. PTEN es un gen supresor de tumores que regula directamente la vía PI3K, lo que lo convierte en uno de los marcadores predictivos de trastuzumab. Metodos: En el presente estudio, las mutaciones de PTEN se examinaron en cincuenta y un pacientes con cáncer de mama positivo para HER2. Además, dieciséis pacientes fueron analizados más a fondo para determinar la expresión de proteínas. Resultados: Las mutaciones se detectaron en tres de 51 pacientes (5.9%). Además, el 56,3 % de los dieciséis pacientes mostró regulación baja/pérdida de la expresión de la proteína PTEN. También se encontró pérdida de PTEN en el 75% de los pacientes con receptores de estrógeno negativos. Conclusiones: La regulación baja/pérdida de la proteína PTEN tiende a asociarse con ER-negativo, lo que refleja su valor como marcador de predicción de tratamiento. (AU)

Assessment of correlation between asthenozoospermia and mitochondrial DNA mutations in Egyptian infertile men.

BACKGROUND: Asthenozoospermia is a chief reason for male seminal pathologies with an impression of around 19% of infertile patients. Spermatozoa mitochondrial DNA variations seem to link with low sperm motility. The objective of the study was to assess the relation between mitochondrial mutations and male sterility, especially in asthenozoospermia. The patient semen samples were investigated by studying the sperm physical characters; motility, viability, and morphological parameters were then classified into normozoospermia and asthenozoospermia. In addition, the level of malondialdehyde (MDA) as a bio-indicator of lipid peroxidation, seminal fructose, and total antioxidant capacity (TAC) were estimated. For molecular analysis, DNA from the semen samples was extracted using a DNA extraction kit. ND1, ND2, and ATPase6 genes were amplified by using a specific primer. After the purification procedure, each PCR product was sequenced to identify the single nucleotide polymorphisms (SNPs) in selected genes. RESULTS: A significant negative correlation between seminal plasma malondialdehyde levels and sperm motility was detected. Meanwhile, TAC analysis revealed significantly lower activity (p ≤ 0.05) in the sample of asthenozoospermic than in normozoospermic men. As regards the seminal plasma fructose, there was no significant difference in the fructose level of normozoospermia and asthenozoospermia cases. At the molecular level, 31 diverse nucleotide substitutions were recognized in mitochondrial DNA. Only ten (10) mutations led to amino acid transformation: four have deleterious effects, four are benign, and the other two have conflicting effectiveness. CONCLUSIONS: This study is the first in Egypt that is concerned with studying the relationship between the mitochondrial DNA mutations in human spermatozoa of asthenozoospermic patients and fertility. The results displayed scientific indications evidenced that there is an association between mitochondrial mutations and male infertility.

Genetic Variation at 15 Autosomal STR Loci Among Seven Egyptian Populations.

Egypt is a transcontinental country containing substantial ethnic, cultural, and linguistic diversity among its people. This study was conducted to investigate the genetic variation at 15 AmpFlSTR Identifiler short tandem repeat (STR) loci, D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA, within and between seven Egyptian populations. Samples of 814 unrelated individuals from Northern Coast, Delta, Greater Cairo, Canal governorates, Northern Upper Egypt, Southern Upper Egypt, and Sinai were investigated. All loci were highly polymorphic in all sample populations. The data were analyzed to give information on allele frequencies and other population statistical parameters. After applying Bonferroni correction, the agreement with Hardy-Weinberg equilibrium (HWE) was confirmed for all loci (exact test), and for all loci with the exception of D3S1358, D19S433, and D18S51 (X2 test). The levels of genetic differentiation and the genetic relationships among populations were evaluated by coefficient of genetic differentiation (FST), AMOVA, and genetic distance of Nei. The most differentiated populations were found between Sinai and Southern Upper Egypt. These two populations showed the lowest within-population variation, whereas the population of Greater Cairo showed the highest within-population variation as indicated by the fixation index FIS. The varying levels of genetic relatedness among the populations in relation to their geographical distribution were analyzed using Mantel test. The results demonstrated that the effectiveness of STR markers enhances their value for identifying the genetic variation within and between Egyptian populations.

PIK3CA mutations in HER2-positive Breast Cancer Patients; Frequency and Clinicopathological Perspective in Egyptian Patients

Missense mutations in PIK3CA are common in breast cancers. They mostly involve exons 9 and 20 which encode kinase and helical domains of the protein and may result in its activation. PIK3CA activating mutations were previously shown to predict lower pathologic complete response (pCR) in HER2-positive breast cancer cases undergoing neoadjuvant human epidermal growth factor receptor 2-targeting therapy. Hence, the present work was conducted to estimate the mutation frequency in PIK3CA in 51 HER2-positive patients by direct sequencing. Our results showed 8 out of 51 (15.7%) to harbor PIK3CA mutations in either exon 9 or 20, or both. Three patients had mutations in both exons 9 and 20. Seven (13.7%) possess missense mutations in exon 20 which changed the amino acid sequence of the protein (H1047R, M1040I, and G1049G). Only four cases harbored mutations in exon 9, changing the codon sequences (E545K E545A, and R524K). Taking the clinicopathological data to account, the mutation frequency was greater in ductal than lobular carcinomas, in grade II rather than III and in lymph node positive lesions, with a higher HER2 score and which are ER/PR negative. However, none of the correlations proved statistically significant. In conclusion, to the best of our knowledge, the PIK3CA mutation frequency in this study is the first report regarding HER2-positive breast cancer patients in Egypt. Hereby, we highlight a moderate frequency which could be useful in the future as a predictive marker for anti-HER2 therapy.

Induction of P3NS1 Myeloma Cell Death and Cell Cycle Arrest by Simvastatin and/or γ-Radiation.

The present study was conducted to investigate the effect of γ-radiation alone or combined with a cytotoxic drug, simvastatin, on viability and cell cycling of a myeloma cell line. P3NS1 myeloma cells were treated with the selected dose of simvastatin (0.1 µM/l) 24 hours prior to γ-irradiation (0.25, 0.5 and 1 Gy). The cell viability, induction of apoptosis, cell death, cell cycling, generation of ROS, and expression of P53, Bax, Bcl2, caspase3, PARP1 and Fas genes were estimated. The results indicated that simvastatin (0.1 µM/l) treatment for 24 hours prior to γ- irradiation increased cell death to 37.5% as compared to 4.81% by radiation (0.5 Gy) alone. It was found that simvastatin treatment before irradiation caused arrest of cells in G0/G1 and G2/M phases as assessed using flow cytometry. Interestingly, simvastatin treatment of P3NS1 cells increased the intracellular ROS production and decreased antioxidant enzyme activity with increased P53, Bax and Caspase3 gene expression while that of Bcl2 was decreased. Consequently, our results indicated that pre-treatment with simvastatin increased radio sensitivity of myeloma tumor cells in addition to apoptotic effects through an intrinsic mitochondrial pathway.

Expression profiling of cancer-related galectins in acute myeloid leukemia.

Acute myeloid leukemia (AML) is the most common type of leukemia in adults with the lowest survival rate of all the leukemias. It is a heterogeneous disease in which a variety of cytogenetic and molecular alterations have been identified. Some galectins were previously reported to have important roles in cancer-like neoplastic transformation, tumor cell survival, angiogenesis, and tumor metastasis. Previous studies have showed that some galectin family members play a role in various types of leukemia. The present study aims at evaluating and clarifying the diagnostic and prognostic value of the expression of cancer-related galectins in relation to the clinicopathological characters of AML patients. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect expression profile of eight galectin family members (galectin-1, -2, -3, -4, -8, -9, -12, and -13) in 53 newly diagnosed de novo AML patients. The samples were collected from the inpatient clinic at National Cancer Institute (NCI), Cairo University (CU), diagnosed between July 2012 and May 2013. Our results show that patients with lower LGALS12 gene expression have a lower overall survival than those with higher expression (P value <0.026). Moreover, a statistically significant association between the LGALS4 gene expression and patient age is found. Hence, the higher expression of LGALS4 gene is associated with younger age (adjusted P value <0.001). In conclusion, galectin-12 may be a potential prognostic marker for AML.

The modifying effect of selenium and vitamins A, C, and E on the genotoxicity induced by sunset yellow in male mice.

The use of food additives in various products is growing up. It has attracted the attention towards the possible correlation between the mutagenic potential of food additives and various human diseases. This work evaluated the protective role of selenium and vitamins A, C and E (selenium ACE)(1) against the genotoxic effects induced by a synthetic food additive, sunset yellow, in mice. Six groups were studied including two control groups (negative and positive control), two groups are given single dose of sunset yellow (either 0.325, 0.65 or 1.3mg/kg body weight(2) alone or with selenium ACE) and two groups are given sunset yellow daily for 1, 2 or 3 weeks (0.325mg/kg b.wt./day alone or with selenium ACE), respectively. The study examined the induction of sister chromatid exchanges (SCE's)(3) in bone-marrow cells, chromosomal aberration in somatic (bone-marrow) and germ cells (spermatocytes) after single and repeated oral treatment, and the induction of morphological sperm abnormalities. The results showed that sunset yellow had genotoxic effects as indicated by increased frequency of SCE's, by chromosomal aberrations in both somatic and germ cells, and by increased morphological sperm abnormalities and DNA fragmentation. The results also indicated that the oral administration of selenium ACE significantly reduced the genotoxic effects of sunset yellow, a result that may support the use of antioxidants as chemopreventive agents in many applications.

Studies on the genotoxic effect of beryllium chloride and the possible protective role of selenium/vitamins A, C and E.

The genotoxic potential of beryllium chloride (BeCl2) was evaluated in vivo in mice using different endpoints. Chromosomal aberrations in bone marrow cells and in spermatocytes as well as sperm abnormalities were determined in the tested mice. The protective role of an orally administered drug consisting of selenium and vitamins A, C and E (selenium-ACE) was also studied. For analysis of chromosomal aberrations, both single and repeated oral treatments for a period of 3 weeks were performed. The doses used were 93.75, 187.50, 375, and 750 mg BeCl2/kg bw, which corresponds to 1/16, 1/8, 1/4, and 1/2 of the experimental LD50. BeCl2 induced a statistically significant increase in the percentage of chromosomal aberrations in both somatic and germ cells, with a dose- and time-response. The percentage of induced chromosomal aberrations was significantly reduced in all BeCl2-treated groups after oral administration of selenium-ACE. Beryllium chloride also induced a significant increase in the percentage of abnormal sperm. This percentage reached values of 9.62 +/- 0.32 and 5.56 +/- 0.31 in mice treated with the highest test dose of BeCl2 and with BeCl2+selenium-ACE, respectively, compared with 1.96 +/- 0.14 for the control. In conclusion, the results demonstrate the genotoxic effect of beryllium chloride and confirm the protective role of selenium-ACE against the genotoxicity of beryllium chloride.