Investigating the association of Angiotensin II Type I Receptor A1166C Polymorphism with Breast Cancer Risk in the Pakistani Population.

The polymorphisms of the Renin-Angiotensin System are related to many disorders like diabetes, cardiovascular disease, and different types of cancer. Among all the polymorphisms related to AGTR1, A1166C has been associated with several disorders, including cardiovascular diseases and breast cancer. This study was conducted to discover the association of AGTR1 polymorphism (A1166C) Renin-Angiotensin and its effect on the development and progression of breast cancer in the Pakistani population. One hundred forty participants, including seventy diagnosed breast cancer patients and seventy healthy individuals, were included in this study and genotyped with an allele-specific polymerase chain reaction. The most frequent genotype in healthy participants and breast cancer patients was CC. An insignificant (p value>0.05) risk of breast cancer was found with A1166C polymorphism in codominant (CC vs. AA OR=1.200 [0.256-5.631] and AC vs. AA 0.941 [OR=0.223-3.976]), dominant (OR=1.00 [0.240-4.167]), recessive (OR=1.230 [0.593-2.552]) and additive models (OR=1.028 [0.533-1.983]) of general population genotypes. Nonetheless, when the AA genotype was considered a reference group, a significant association was found between AC and CC genotypes and invasive ductal and ductal carcinoma development in breast cancer patients. In conclusion, this study demonstrated no significant association between AGTR1 (A1166C) polymorphism and breast cancer risk.

Death receptor pathway genes (Caspase and BID) expression analysis in cancerous cells in response to extracts of amla and turmeric.

Plant extracts antiproliferative effects were determined by using mammalian cells along the expression profile of Caspases 3, 8 and the BID gene of the death receptor-induced pathway. Two medicinal plants viz., Turmeric (Curcuma longa) and Amla (Emblica officinalis) extracts were examined for antiproliferative effect through Neutral Red-Dye uptake assay on Vero and MDA-MB 231 cell lines. A reverse transcriptase polymerase chain reaction was used to determine the expression of genes while GAPDH expression was used as an internal control. Expression of BID was up-regulated in methanolic turmeric extract-induced MDA-MB 231 cells while Caspases 3,8 expressions were the same in induced and uninduced MDA-MB 231 cells. Activated BID cleaved into tBID and activated the intrinsic pathway which caused death in methanolic turmeric extract-induced cancerous cells. Ethanolic extracts of turmeric exerted the strongest antiproliferative effects on Vero and methanolic extracts on MDA-MB 231 cells. The morphological studies of cell lines and gene expression analysis of turmeric methanolic extract-treated cells showed activation of apoptosis via converting BID into t-BID (intrinsic pathway) and activating Caspase-3 and Caspase-8 (extrinsic pathway). With the differential cytotoxicity and induction of apoptosis in induced cancer cells in comparison to uninduced cancerous cells, hence turmeric is a natural source of new anti-cancerous compounds.

MTHFR A1298C polymorphism: a predictor of reduced risk of preeclampsia in Punjab, Pakistan.

OBJECTIVES: This study aimed to investigate the genetic association between MTHFR (A1298C) SNP and preeclampsia (PE) in Punjab, Pakistan. METHODS: A sample of 80 pregnant women (40 healthy pregnant women and 40 with PE) was pooled for genotyping MTHFR A1298C polymorphism by using the tetra-primer amplification refractory mutation system (ARMS) PCR. The Genotypic and allelic assessments were performed using various statistical techniques. RESULTS: The AC genotype and C allele of MTHFR A1298C were found to be associated with decreased risk of PE (odds ratio [OR]: 0.31, risk ratio [RR]: 0.58, p = 0.01), and (odds ratio [OR]: 0.49, risk ratio [RR]: 0.61, p = 0.04), respectively. CONCLUSION: In conclusion, genetic polymorphism A1298C in MTHFR may pose a protective effect in the studied population.

Investigation of angiotensin-1 converting enzyme 2 gene (G8790A) polymorphism in patients of type 2 diabetes mellitus with diabetic nephropathy in Pakistani population.

BACKGROUND: Type 2 diabetes mellitus is a multifactorial disease that escalates the risk of other associated complications such as diabetic neuropathy, retinopathy, and nephropathy. Diabetic nephropathy is a microvascular condition that leads to end-stage renal disease (ESRD). There are several genes involved in disease development and it is a challenging task to investigate all of these. Nonetheless, identifying individual gene roles can assist in evaluating the combinatorial effects with other genes. Angiotensin-1 converting enzyme 2 (ACE2), is the key regulator of blood pressure in the Renin-Angiotensin-Aldosterone System that hydrolyzes angiotensin II (vasoconstrictor) into angiotensin 1-7 (vasodilator). The association of different variants of the ACE2 with the risk of type 2 diabetes mellitus has been determined in various populations with susceptibility to other complications. This study was aimed to investigate the association of Angiotensin-1 converting enzyme 2 polymorphism, G8790A, with the increased risk of type 2 diabetes mellitus (T2DM) development with the complication of diabetic nephropathy (DN) in the Pakistani population. METHODS: In this case-control study, a total of 100 healthy controls and 100 patients of type 2 diabetes mellitus aged > 40 years, having disease duration ≥ 10 years were compared. The G8790A polymorphism in ACE2 was analyzed by allele-specific polymerase chain reaction (AS-PCR). The urinary albumin excretion (UAE), urinary creatinine, and albumin to creatinine ratios (ACR) were determined to assess renal function status. Pearson bivariate correlation coefficients were calculated to investigate the relationship among all the parameters. Crude and adjusted odds ratios were found to determine any risk association between ACE2 G8790A polymorphisms and disease development. The p-values < 0.05 were considered significant. RESULTS: A homogeneity was obtained regarding the distribution of data by sex, BMI, diastolic blood pressure, pulse rate and urinary creatinine levels between case and control groups. The ACR showed a significant correlation with UAE (r = 0.524, p = 0.001), urinary creatinine (r = -0.375, p = 0.001) and random blood sugar levels (r = 0.323, p = 0.005) with the complication of diabetic nephropathy in T2DM patient. Females with the AA genotype had a 10-fold increased risk for the development of type 2 Diabetes (OR = 9.5 [95% CI = 2.00-21.63] p<0.002). Males having A allele showed a significant association for susceptibility of type 2 Diabetes (OR = 3.807 [95% CI = 1.657-8.747] p<0.002). However, none of the genotypes or alleles revealed an association for diabetic nephropathy in male and female patients. Urinary ACR was also found to be positively correlated with UAE (r = 0.642 p = 0.001 & 0.524, p = 0.001) and random blood sugar levels (r = 0.302, p = 0.002 & r = 0.323, p = 0.005) in T2DM and T2DM+DN groups, respectively. CONCLUSION: The study finding indicated that female AG/AA genotype and male A genotype of G8790A polymorphism in the ACE2 gene were associated with type 2 diabetes mellitus as a genetic risk factor but are not associated with diabetic nephropathy in the Pakistani population.

A narrative review on the role of folate-mediated one-carbon metabolism and its associated gene polymorphisms in posing risk to preeclampsia.

Preeclampsia (PE) presents a major obstetrical problem for mother and fetus which is characterized by the onset of hypertension and proteinuria in formerly normotensive women. Altered folate-mediated one-carbon metabolism is one of the factors for PE development either due to nutritional insufficiencies such as folate deficiency or polymorphisms in genes that code for the key enzymes of the cycle. Commonly, there are four genes in the cycle whose polymorphisms have been described in relation to PE. These factors could cause elevation of homocysteine; the toxic metabolite, which subsequently leads to the development of PE. Sufficient levels of folate have been considered important during pregnancy and may reduce the risk of development of PE. This review aims at discussing genetic polymorphisms and nutritional deficiencies as probable predisposing factors and suggests considering fetal genotypes, varied ethnicities, and interaction of various other factors involved to render better conclusiveness to the present studies.

Extracellular microRNAs: key players to explore the outcomes of in vitro fertilization.

BACKGROUND: MicroRNAs (miRNAs) are small RNA molecules that modulate post-transcriptional gene regulation. They are often used as promising non-invasive biomarkers for the early diagnosis of cancer. However, their roles in assisted reproduction are still unknown. METHODS: This prospective study was designed to evaluate the expression profiles of seven extracellular miRNAs (miR-7-5p, miR-202-5p, miR-378-3p, miR-224, miR-320a, miR-212-3p, and miR-21-5p) in human follicular fluid (FF) to explore the outcomes of in vitro fertilization (IVF). Of 255 women, 145 were without polycystic ovary syndrome (PCOS), and their ovarian assets were normal (NOR), while 110 were with normo-androgenic PCOS. RESULTS: The combination of six FF miRNAs expression profile discriminated between PCOS and NOR women with a sensitivity of 79.2% and a specificity of 87.32% (AUC = 0.881 [0.61; 0.92], p = 0.001). MiR-202-5p significantly had a lower abundance level, and miR-378-3p had a high abundance level in pooled FF samples from patients treated with human menopausal gonadotropin (hMG) than those treated with recombinant follicle-stimulating hormone (rFSH) (p < 0.001). Our results showed that miRNA-320a was significantly different in top-quality embryos versus non-top-quality embryos on day 3 in NOR patients with a sensitivity of 80% and specificity of 71%, (AUC = [0.753 (0.651; 0.855)], p = 0.001). For clinical pregnancy outcome prediction, FF miRNA-21 exhibited high sensitivity (74.8%) and specificity (83.7%) with the AUC value of 0.774 (0.682; 0.865). CONCLUSION: Conclusively, our results provide evidence that miR-7-5p, miR-378-3p, miR-224, miR-212-3p were a differentially high expression in normo-androgenic PCOS patients than NOR patients. While miRNA-320a was significantly different in top-quality embryos versus non-top-quality embryos on day 3 (p = 0.001). The expression level of FF miR-212-3p was significantly related to the probability of embryos to develop into a high-quality blastocyst in patients with normal ovarian reserve.

Effects of plants extracts on the expression of major genes of JAK/STAT pathway.

This study analyzed the effects of the plant extracts (Citrus limon, Solanum lycopersicum, Zingiber officinale, Vitis vinifera and Allium sativum) on the growth of mammalian cells (Vero and MDA-MB-231) and evaluated the most effective plant extract for the expression of specific genes of the JAK/STAT pathway in human breast cancer cells. An antiproliferative bioassay involving neutral red-dye uptake was used to determine the anticancerous potential of plant extracts. In Vero cells, the ginger methanolic extract was least effective; whereas the lemon methanolic extract was more effective with 64 dilutions with IC50 51.42%. In MDA-MB-231 cells, the tomato and ginger methanolic, and grape water extracts were least effective, whereas lemon water extract was most effective with 32 dilutions with IC50 48.67%, by upregulating JAK1, JAK2, TYK2, IRF7 and IRF3 gene expressions of the JAK/STAT pathway. C. limon inhibited the growth of both Vero and MDA-MB 231 cells. It suggested that C. limon has anti-cancer potential by inducing the JAK/STAT pathway.

A Review: Molecular Chaperone-mediated Folding, Unfolding and Disaggregation of Expressed Recombinant Proteins.

The advancements in biotechnology over time have led to an increase in the demand of pure, soluble and functionally active proteins. Recombinant protein production has thus been employed to obtain high expression of purified proteins in bulk. E. coli is considered as the most desirable host for recombinant protein production due to its inexpensive and fast cultivation, simple nutritional requirements and known genetics. Despite all these benefits, recombinant protein production often comes with drawbacks, such as, the most common being the formation of inclusion bodies due to improper protein folding. Consequently, this can lead to the loss of the structure-function relationship of a protein. Apart from various strategies, one major strategy to resolve this issue is the use of molecular chaperones that act as folding modulators for proteins. Molecular chaperones assist newly synthesized, aggregated or misfolded proteins to fold into their native conformations. Chaperones have been widely used to improve the expression of various proteins which are otherwise difficult to produce in E. coli. Here, we discuss the structure, function, and role of major E. coli molecular chaperones in recombinant technology such as trigger factor, GroEL, DnaK and ClpB.

Gene cloning, expression enhancement in Escherichia coli and biochemical characterization of a highly thermostable amylomaltase from Pyrobaculum calidifontis.

Pcal_0768 gene encoding an amylomaltase, a 4-α-glucanatransferase belonging to family 77 of glycosyl hydrolases, from Pyrobaculum calidifontis was cloned and expressed in Escherichia coli. The recombinant protein was produced in E. coli in soluble and active form. However, the expression level was not very high. Analysis of the mRNA of initial seven codons at the 5'-end of the gene revealed the presence of a hair pin like secondary structure. This secondary structure was removed by site directed mutagenesis, without altering the amino acids, which resulted in enhanced expression of the cloned gene. Recombinant Pcal_0768 exhibited optimal amylomaltase activity at 80 °C and pH 6.9. Under these conditions, the specific activity was 690 U/ mg. Recombinant Pcal_0768 was highly thermostable with a half-life of 6 h at 100 °C. It exhibited the highest kcat value among the characterized glucanotransferases. No metal ions were required for activity or stability of the enzyme. Recombinant Pcal_0768 was successfully employed in the synthesis of modified starch for producing thermoreversible gel. To the best of our knowledge, till now this is the most thermostable enzyme among the characterized amylomaltases. High thermostability and starch modification potential make it a novel and distinct amylomaltase.

Therapeutic Uses of HSP90 Inhibitors in Non-Small Cell Lung Carcinoma (NSCLC).

BACKGROUND: Non-Small Cell Lung Carcinoma is one of the major cause of morbidity and mortality worldwide with an incidence rate of 1.3 million cases per year. Heat shock protein 90 (HSP90) is a promising drug target in cancer treatment. HSP90 is required to activate numerous eukaryotic proto-oncogenic protein kinases, hence play a prominent role in cancer. METHOD: We reviewed fifty-five articles to highlight the importance of HSP90 in NSCLC and the recent developments of its inhibitors. RESULTS: This review showed that HSP90 inhibitors i.e. Ganestespib have shown great potential in the treatment of non-small cell lung carcinoma (NSCLC). Different HSP90 inhibitors has been designed till date that acts as effective drugs in tumour suppression. However, the utility of these drugs has been limited due to several drawbacks including hepato-toxicity, poor solubility, and poorly tolerated formulations. CONCLUSION: The goal of this review is to present the data in support of use of HSP90 inhibitors in NSCLC and to provide an overview of the on-going clinical trials involving new-generation HSP90 inhibitors.

Studies on the regioselectivity and kinetics of the action of trypsin on proinsulin and its derivatives using mass spectrometry.

Human M-proinsulin was cleaved by trypsin at the R(31)R(32)-E(33) and K(64)R(65)-G(66) bonds (B/C and C/A junctions), showing the same cleavage specificity as exhibited by prohormone convertases 1 and 2 respectively. Buffalo/bovine M-proinsulin was also cleaved by trypsin at the K(59)R(60)-G(61) bond but at the B/C junction cleavage occurred at the R(31)R(32)-E(33) as well as the R(31)-R(32)E(33) bond. Thus, the human isoform in the native state, with a 31 residue connecting C-peptide, seems to have a unique structure around the B/C and C/A junctions and cleavage at these sites is predominantly governed by the structure of the proinsulin itself. In the case of both the proinsulin species the cleavage at the B/C junction was preferred (65%) over that at the C/A junction (35%) supporting the earlier suggestion of the presence of some form of secondary structure at the C/A junction. Proinsulin and its derivatives, as natural substrates for trypsin, were used and mass spectrometric analysis showed that the k(cat.)/K(m) values for the cleavage were most favourable for the scission of the bonds at the two junctions (1.02±0.08×10(5)s(-1)M(-1)) and the cleavage of the K(29)-T(30) bond of M-insulin-RR (1.3±0.07×10(5)s(-1)M(-1)). However, the K(29)-T(30) bond in M-insulin, insulin as well as M-proinsulin was shielded from attack by trypsin (k(cat.)/K(m) values around 1000s(-1)M(-1)). Hence, as the biosynthetic path follows the sequence; proinsulin→insulin-RR→insulin, the K(29)-T(30) bond becomes shielded, exposed then shielded again respectively.

Inventory of 'slow exchanging' hydrogen atoms in human proinsulin and its derivatives: observations on the mass spectrometric analysis of deuterio-proteins in D(2)O.

Secondary structure elements of human proinsulin and of its tryptic products were compared by H/D exchange, in a single-pot, using mass spectrometry. Human proinsulin containing an N-terminal methionine, M-proinsulin, was engineered and converted into a perdeuterio derivative, which using an optimized mass spectrometric protocol and manual calculations gave a mass of 9669.6 (+/-1) Da showing the replacement, with deuterium of 146.4 from a total of 149 exchangeable hydrogen atoms (83 from amides and 66 from side-chains). Tryptic digestion of the perdeuterio-M-proinsulin, followed by the transfer of the digest from a deuterio- into a protio-medium showed, at the earliest time of analysis, that of the 27 (+/-1) D atoms retained in M-proinsulin, 24 (+/-1) were found in the insulin nucleus, M-insulin-RR, and 4.2 (+/-1) in the C-peptide-KR. A temporal analysis of the fate of D atoms in these species showed that whereas the C-peptide-KR rapidly exchanged its deuterium, losing all by 6 h, the loss of D atoms from M-proinsulin and M-insulin-RR was gradual and in each case, 12 deuterium atoms survived exchange for 72 h. At all time intervals the loss of D atoms from M-proinsulin mirrored that from M-insulin-RR plus the C-peptide-KR, suggesting that the secondary-structure elements of M-proinsulin are largely conserved in its two component parts.