Genetic association of vitamin D receptor gene with female infertility.

BACKGROUND: Infertility is defined as failure to achieve a clinical pregnancy after 12 months of unprotected intercourse. It affects 15% of couples globally and 22% of couples within Pakistan. Female infertility can be caused by numerous genetic or environmental factors including hormone imbalances and exposure to chemicals or radiation. The prevalence of vitamin D deficiency among the adult population was reported to be 14-59% with a higher prevalence in Asian countries. Furthermore, the expression of Vitamin D receptor (VDR) can play a vital role in the reproductive organs of females. Hence, the aim of our present study was to check the association of VDR polymorphisms with infertile females. For this purpose, blood samples were collected for genotyping of four known VDR mutations [FokI (rs2228570), TaqI (rs731236), ApaI (rs7975232), and BsmI (rs1544410)] via PCR-based RFLP assay. RESULTS: Genotyping indicated that FokI, TaqI, and ApaI are associated with infertility (p = 0.004*, p = 0.013*, and p = 0.033*, respectively). However, BsmI did not show any significance. Multinomial regression analysis indicated that FokI heterozygous genotypes increase the risk of infertility by 2.5 times (hetero: OR = 2.5, 95%, p = 0.001*) as compared to wild type. Heterozygous genotypes of TaqI and ApaI were found to play a protective role and reduce the risk of infertility by 58 and 52%, respectively [TaqI: OR = 0.42, 95%, p = 0.004*, ApaI: OR = 0.48, 95%, p = 0.01*, respectively] as compared to wild type. Multinomial logistic regression analysis was also performed for allelic data as well. CONCLUSION: Thus, it could be summarized that among the studied polymorphisms of VDR, FokI SNP greatly increased the risk of infertility, while TaqI and ApaI genotypes protect from infertility. However, BsmI does not influence the risk of infertility in Pakistani females.

Novel quinoxaline-3-propanamides as VGFR-2 inhibitors and apoptosis inducers.

Vascular endothelial growth factor receptor-2 is a vital target for therapeutic mediation in various types of cancer. This study was aimed at exploring the cytotoxic activity of seventeen novel quinoxaline-3-propanamides against colon cancer (HCT-116) and breast cancer (MCF-7) using MTT assay. Results revealed that compounds 8, 9, and 14 elicited higher cytotoxicity than the reference drugs, doxorubicin (DOX) and sorafenib. Interestingly, they are more selective for HCT-116 (SI 11.98-19.97) and MCF-7 (SI 12.44-23.87) compared to DOX (SI HCT-116 0.72 and MCF-7 0.9). These compounds effectively reduced vascular endothelial growth factor receptor-2; among them, compound 14 displayed similar VEGFR-2 inhibitory activity to sorafenib (IC50 0.076 M). The ability of 14 to inhibit angiogenesis was demonstrated by a reduction in VEGF-A level compared to control. Furthermore, it induced a significant increase in the percentage of cells at pre-G1 phase by almost 1.38 folds (which could be indicative of apoptosis) and an increase in G2/M by 3.59 folds compared to the control experiment. A flow cytometry assay revealed that compound 14 triggered apoptosis via the programmed cell death and necrotic pathways. Besides, it caused a remarkable increase in apoptotic markers, i.e., caspase-3 p53 and BAX. When compared to the control, significant increase in the expression levels of caspase-3 from 47.88 to 423.10 and p53 from 22.19 to 345.83 pg per ml in MCF-7 cells. As well, it increased the proapoptotic protein BAX by 4.3 times while lowering the antiapoptotic marker BCL2 by 0.45 fold. Docking studies further supported the mechanism, where compound 14 showed good binding to the essential amino acids in the active site of VEGFR-2. Pharmacokinetic properties showed the privilege of these hits over sunitinib: they are not substrates of P-gp protein; this suggests that they have less chance to efflux out of the cell, committing maximum effect; and in addition, they do not allow permeation to the BBB.

Novel quinoxaline-based VEGFR-2 inhibitors to halt angiogenesis.

Vascular endothelial growth factor receptor-2 is a dynamic target for therapeutic intervention in various types of cancer. This study was aimed at exploring the VEGFR-2 inhibitory activity of a novel library of quinoxalin-2-one derivatives such as 3-furoquinoxaline carboxamides, 3-pyrazolylquinoxalines, and 3-pyridopyrimidyl-quinoxalines. Among them, 6c, 7a, and 7d-f produced remarkable cytotoxicity against HCT-116 (IC50's 4.28-9.31 µM) and MCF-7 (IC50's 3.57-7.57 µM) cell lines using the MTT assay and doxorubicin (DOX) as a reference standard. Interestingly, results of cytotoxicity towards the human fibroblast cell line WI38 revealed that these hits demonstrated higher selectivity indices towards both HCT-116 (SI 8.69-23.19) and MCF-7 (SI 9.48-27.80) than DOX, SI 0.72 and 0.90, respectively. Then, these hits were subjected to a mechanistic study; they showed direct inhibition of VEGFR-2. Impressively, compound 7f displayed 1.2 times the VEGFR-2 inhibitory activity of sorafenib. The antiangiogenic potential of 7f was proved via lowering the level of VEGF-A, than that of control. It as well, exhibited scratch closure percent of 61.8%, compared with 74.5% of control at 48 hrs, indicating the potential anti-migratory effect of the compound 7f. It significantly increased the expression of tumor suppressor gene (p53) on MCF-7 cells by almost 18 folds and upregulated the caspase-3 level by 10.7 folds, compared to the control. Cell cycle analysis revealed cell cycle arrest at G2/M together with a PreG increase which indicated apoptosis induction potential. Annexin V-FITC apoptosis results proposed the two modes of cell death (apoptosis and necrosis) as an inherent mechanism of cytotoxicity of compound 7f. Molecular docking further supported the mechanism showing the affinity of target compounds for VEGFR-2 active site. Moreover, physicochemical and drug-like properties were assessed from the ADME properties.

A homozygous founder variant in PDE2A causes paroxysmal dyskinesia with intellectual disability.

Intellectual developmental disorder with paroxysmal dyskinesia or seizures (IDDPADS, OMIM#619150) is an ultra-rare childhood-onset autosomal recessive movement disorder manifesting paroxysmal dyskinesia, global developmental delay, impaired cognition, progressive psychomotor deterioration and/or drug-refractory seizures. We investigated three consanguineous Pakistani families with six affected individuals presenting overlapping phenotypes partially consistent with the reported characteristics of IDDPADS. Whole exome sequencing identified a novel missense variant in Phosphodiesterase 2A (PDE2A): NM_002599.4: c.1514T > C p.(Phe505Ser) that segregated with the disease status of individuals in these families. Retrospectively, we performed haplotype analysis that revealed a 3.16 Mb shared haplotype at 11q13.4 among three families suggesting a founder effect in this region. Moreover, we also observed abnormal mitochondrial morphology in patient fibroblasts compared to controls. Belonging to diverse age groups (13 years-60 years), patients presented paroxysmal dyskinesia, developmental delay, cognitive abnormalities, speech impairment, and drug-refractory seizures with variable onset of disease (as early as 3 months of age to 7 years). Together with the previous reports, we observed that intellectual disability, progressive psychomotor deterioration, and drug-refractory seizures are consistent outcomes of the disease. However, permanent choreodystonia showed variability. We also noticed that the later onset of paroxysmal dyskinesia manifests severe attacks in terms of duration. Being the first report from Pakistan, we add to the clinical and mutation spectrum of PDE2A-related recessive disease raising the total number of patients from six to 12 and variants from five to six. Together, with our findings, the role of PDE2A is strengthened in critical physio-neurological processes.

Study of the Side Effects of Pfizer and Oxford COVID-19 Vaccines in the Eastern Province of Saudi Arabia.

Background & Objective: The COVID-19 pandemic imposed global concern and became one of the deadliest pandemics of the twenty-first century. Several vaccines were developed against SARS-CoV-2 to counteract the effects of this virus. This study aims to determine the post-vaccination side effects of the most common COVID-19 vaccines used in the Eastern province of Saudi Arabia. Methods: This is a cross-sectional study using an online questionnaire distributed randomly through social media. Frequencies were calculated to determine participants' demographic information, vaccination details, and post-vaccination side effects. Univariate and multiple regression analysis were applied to test the association between individuals' willingness to receive a booster dose and different categorical variables. Results: A total of 1004 participants were included in the survey, of which 0.6%, 85.3% and 14.1% completed either one, two or three doses of the Pfizer mRNA vaccine and Oxford AstraZeneca vaccines, respectively. The similar common side effects between the first and the second doses were significantly associated with the type of vaccine received; these included fatigue (Pfizer 54.4%, Oxford 73.2%; p < 0.001), headache (Pfizer 33.2%, Oxford 44.7%; p = 0.002), and fever (Pfizer 25.1%, Oxford 57.6%; p < 0.001). Additionally, unusual side effects were also reported (palpitations and menstrual abnormalities). Getting SARS-CoV2 infection after vaccination was significantly associated with the type of vaccine received at the first dose (Chi-Square=5.496, p = 0.019). A statistically significant association was found between the individuals' willingness to receive a booster dose and their gender (Chi-Square = 39.493, p < 0.001), age (Chi-Square = 11.668, p = 0.02), presence of allergies (Chi-Square = 5.602, p = 0.018), and previous COVID-19 infection (Chi-Square = 9.495, p = 0.002). Conclusion: Despite the described side effects, further studies should be done to investigate the unusual and rare side effects to assess COVID-19 vaccines effectiveness and safety over longer period of time within a more diverse population.

Assessment of internal fit and micro leakage of conventionally fabricated ceramometallic restoration versus CAD wax and press veneering (in-vitro study).

STATEMENT OF PROBLEM: Accuracy of internal fit and microleakage for CAD-CAM systems used in metal coping fabrication and veneered with layering or pressing porcelain in ceramometallic restoration is unclear. MATERIAL AND METHODS: A master metal die was milled to resemble the right mandibular first molar preparation for coverage with ceramometallic restoration. Master die was duplicated to twenty-four resin specimen dies.They were divided into two groups according to metal coping construction technique using either conventional (C) or CAD (D) wax. Each group was subdivided into two subgroups (n = 6) according to the technique of porcelain veneering (layered or pressed) to fabricate ceramometallic restorations, where subgroup (CL, DL) were conventionally layered by porcelain and (CP, DP) were press veneered. A standardized thickness of metal and porcelain was performed in all specimens as per manufacturer's instructions for techniques ceramometallic restoration construction. Evaluation of internal fit was done with silicone replica technique using stereomicroscope at ×24 magnification where the thickness of silicon layer was measured at 20 reference points on each specimen. Then specimens were subjected to thermocycling. Sectioned specimens were assessed for microleakage using a stereomicroscope at ×12 magnification along die-cement interface with a five scale score. RESULTS: Mean internal gap values of veneering showed a statistically nonsignificant difference between specimens made with layering(L) and pressing(P). Different techniques of wax construction showed a non-significant difference in internal gap values between specimens made with conventional(C) and CAD(D) waxing. However, a significant difference was found in the internal gap at different sites. The highest internal gap was found at the occlusal surface, while the lowest gap was found at the finish line. The highest mean microleakage score was found with CAD wax and press veneering, while the lowest mean microleakage score was found with conventional wax and press veneering. CONCLUSION: Both construction techniques of ceramometallic restoration were considered reliable in restoration production within a clinically acceptable range regarding internal fit and microleakage. There is a strong positive correlation between internal fit and microleakage of ceramometallic restoration constructed.

Design and synthesis of novel tranilast analogs: Docking, antiproliferative evaluation and in-silico screening of TGFßR1 inhibitors.

The discovery of the antiproliferative potential of tranilast prompted additional studies directed at understanding the mechanisms of tranilast action. Its inhibitory effect on cell proliferation depends principally on the capacity of tranilast to interfere with transforming growth factor beta (TGFßR1) signaling. This work summarizes design, synthesis and biological evaluation of sixteen novel tranilast analogs on different tumors such as PC-3, HepG-2 and MCF-7 cell lines. The in vitro cytotoxicity was evaluated using MTT assay showed that, twelve compounds out of sixteen showed higher cytotoxic activities (IC50's 1.1-6.29 µM), than that of the reference standard, 5-FU (IC50 7.53 µM). The promising cytotoxic hits (4b, 7a, b and 14c-e), proved to be selective to cancer cells when their cytotoxicity's are examined on human normal cell line (WI-38). Then they are investigated for their possible mode of action as TGFßR1 inhibitors; remarkable inhibition of TGFßR1 by these hits was observed at the range of IC50 0.087-3.276 µM. The cell cycle analysis of the most potent TGFßR1 inhibitor, 4b revealed cell cycle arrest at G2/M phase on prostate cancer cells. Additionally, it is clearly indicated apoptosis induction at Pre-G1 phase, this is substantiated by significant increase in the expression on the tumor suppressor gene, p53 and up regulation the level of apoptosis mediator, caspase-3. In addition, in silico study was performed for validating the physicochemical and ADME properties which revealed that, all compounds are orally bioavailable with no side effects complying with Lipinski rule. The proposed mode of action can be further explored on the light of molecular modeling simulation of the most potent compounds, 4b and 14e which were docked into the active sites of TGFßR1 to predict their affinities toward the receptor.