Molecular Cloning, Expression, Sequence Characterization and Structural Insight of Bubalus bubalis Growth Hormone-Receptor.

The current study focuses on molecular cloning, expression and structural characterization of growth hormone-receptor (GHR) and its extracellular domain as growth hormone binding protein (GHBP) from the liver of Nili-Ravi buffalo (Bubalus bubalis; Bb). RNA was isolated, genes were amplified by reverse transcriptase-polymerase chain reaction and sequence was characterized. The BbGHR sequence showed three amino acid variations in the extracellular domain when compared with Indian BbGHR. For the production of full length BbGHR and BbGHBP in Escherichia coli (E. coli) BL21 (RIPL) Codon Plus, expression plasmids were constructed under the control of T7lac promoter and isopropyl ß-D thiogalactopyranoside was used as an inducer. BbGHR and BbGHBP were expressed as inclusion bodies at ~ 40% and > 30% of the total E. coli proteins, respectively. The BbGHBP was solubilized and refolded by dilution method using cysteine-cystine redox potential. The recombinant BbGHBP was purified and biological activity was checked on HeLa cell lines showing increase cell proliferation in the presence of ovine GH (oGH), hence justifying the increase in the half-life of GH in the presence of BbGHBP. For the molecular interactions of oGH-BbGHBP multiple docking programs were employed to explore the subsequent interactions which showed high binding affinity and presence of large number of hydrogen bonds. Molecular Dynamics studies performed to examine the stability of proteins and exhibited stable structures along with favorable molecular interactions. This study has described the sequence characterization of BbGHR in Nili-Ravi buffaloes and hence provided the basis for the assessment of GH-GHR binding in other Bovidae species.

A computer aided drug discovery based discovery of lead-like compounds against KDM5A for cancers using pharmacophore modeling and high-throughput virtual screening.

KDM5A over-expression mediates cancer cell proliferation and promotes resistance toward chemotherapy through epigenetic modifications. As its complete mechanism of action is still unknown, there is no KDM5A specific drug available at clinical level. In the current study, lead compounds for KDM5A were determined through pharmacophore modeling and high-throughput virtual screening from Asinex libraries containing 0.5 million compounds. These virtual hits were further evaluated and filtered for ADMET properties. Finally, 726 compounds were used for docking analysis against KDM5A. On the basis of docking score, 10 top-ranked compounds were selected and further evaluated for non-central nervous system (CNS) and CNS drug-like properties. Among these compounds, N-{[(7-Methyl-4-oxo-1,2,3,4-tetrahydrocyclopenta [c] chromen-9-yl) oxy]acetyl}-l-phenylalanine (G-score: -11.363 kcal/mol) was estimated to exhibit non-CNS properties while 2-(3,4-Dimethoxy-phenyl)-7-methoxy-chromen-4-one (G-score: -7.977 kcal/mol) was evaluated as CNS compound. Docked complexes of both compounds were finally selected for molecular dynamic simulation to examine the stability. This study concluded that both these compounds can serve as lead compounds in the quest of finding therapeutic agents against KDM5A associated cancers.

A Computational Approach to Modeling an Antagonistic Angiogenic VEGFR1-IL2 Fusion Protein for Cancer Therapy.

In cancer treatment, immunotherapy has great potential for improving the prognosis of patients with hematologic and solid malignancies. In this study, various bioinformatics tools and servers were used to design an antiangiogenic fusion protein. After comprehensive evaluation, an antiangiogenic fusion protein was designed using a soluble extracellular domain of human vascular endothelial growth factor receptor 1 (sVEGFR-1) and human interleukin-2 (IL-2) joined by a flexible linker. The final construct was composed of 875 amino acids. The secondary structure of the fusion protein, obtained by CFSSP, PSIPRED, and SOPMA tools, consisted of 14.17% helices, 29.71% extended strands, 4.69% beta turns and 51.43% random coils. Tertiary structure prediction by Raptor X showed that the fusion protein comprises 3 domains with 875 modeled amino acids, out of which 26 positions (2%) were considered disordered. The Ramachandran plot revealed 89.3%, 7.1%, and 3.6% amino acid residues in favored, allowed, and outlier regions, respectively. Physical features of the Molecular Dynamic (MD) simulated system such as root mean square deviation, root mean square fluctuation, solvent-on hand surface region, and radius of gyration identified the fusion construct as a stable and compact protein with few fluctuations in its overall structure. Docking of the fusion protein showed that interaction between sVEGFR-1/VEGFA and IL-2/IL-2R still exists. In silico analysis revealed that the fusion protein comprising IL-2 and sVEGFR-1 has stable structure and the selected linker can efficiently separate the two domains. These observations may be helpful in determining protein stability prior to protein expression.

Role of serum VEGF-A biomarker for early diagnosis of ovarian cancer instead of CA-125.

OBJECTIVE: To assess the significance of serum vascular endothelial growth factor-A (VEGF-A) as a potential biomarker for the diagnosis of ovarian cancer instead of cancer antigen-125 (CA-125). METHODS: The case-control study was conducted at the obstetrics departments of Sir Ganga Ram Hospital and Jinnah Hospital, Lahore, Pakistan, from April to September 2018, and comprised cases of epithelial ovarian tumour and healthy female controls. Serum VEGF-A and CA-125 levels were evaluated using Luminex multi-analyte profiling technology and enzyme immunoassays technique. Age, stage, grading, metastasis and ascites of ovarian cancer patients were investigated and compared with serum VEGF-A and CA-125 levels. Data was analysed using SPSS 20. RESULTS: Of the 89 subjects, 44(49.4%) were cases and 45(49.6%) were controls. Among the cases, 13(29.5%) were benign and 31(70.5%) were malignant. The mean serum VEGF-A values were inversely proportional to the stages of ovarian cancer i.e. stage I, II, III and IV showed 762.2pg/ml, 267.3pg/ml, 233.1pg/ml and 125.5pg/ml VEGF-A levels respectively. A steady increase in the mean serum CA-125 values with the progression of the disease was observed i.e. in stage I, II, III and IV the levels of CA-125 were 146.2U/ml, 268.5U/ml and 477.2U/ml and 844.4U/ml respectively. CONCLUSIONS: The detection of high concentrations of serum VEGF-A level supported its use as one of the diagnostic parameters in the timely investigation of ovarian cancer.

Deleterious Variants in WNT10A, EDAR, and EDA Causing Isolated and Syndromic Tooth Agenesis: A Structural Perspective from Molecular Dynamics Simulations.

The dental abnormalities are the typical features of many ectodermal dysplasias along with congenital malformations of nails, skin, hair, and sweat glands. However, several reports of non-syndromic/isolated tooth agenesis have also been found in the literature. The characteristic features of hypohidrotic ectodermal dysplasia (HED) comprise of hypodontia/oligodontia, along with hypohidrosis/anhidrosis, and hypotrichosis. Pathogenic variants in EDA, EDAR, EDARADD, and TRAF6, cause the phenotypic expression of HED. Genetic alterations in EDA and WNT10A cause particularly non-syndromic/isolated oligodontia. In the current project, we recruited 57 patients of 17 genetic pedigrees (A-Q) from different geographic regions of the world, including Pakistan, Egypt, Saudi Arabia, and Syria. The molecular investigation of different syndromic and non-syndromic dental conditions, including hypodontia, oligodontia, generalized odontodysplasia, and dental crowding was carried out by using exome and Sanger sequencing. We have identified a novel missense variant (c.311G>A; p.Arg104His) in WNT10A in three oligodontia patients of family A, two novel sequence variants (c.207delinsTT, p.Gly70Trpfs*25 and c.1300T>G; p.Try434Gly) in EDAR in three patients of family B and four patients of family C, respectively. To better understand the structural and functional consequences of missense variants in WNT10A and EDAR on the stability of the proteins, we have performed extensive molecular dynamic (MD) simulations. We have also identified three previously reported pathogenic variants (c.1076T>C; p.Met359Thr), (c.1133C>T; p.Thr378Met) and (c.594_595insC; Gly201Argfs*39) in EDA in family D (four patients), E (two patients) and F (one patient), correspondingly. Presently, our data explain the genetic cause of 18 syndromic and non-syndromic tooth agenesis patients in six autosomal recessive and X-linked pedigrees (A-F), which expand the mutational spectrum of these unique clinical manifestations.

Effect of intravitreal bevacizumab on macular thickness: exploring serum and vitreous proangiogenic biomarkers in patients with diabetic macular edema

Background/aim: This study evaluates diabetic macular-edema (DME) patients for the effect of intravitreal bevacizumab (IVB) injection on macular thickness and proangiogenic biomarkers in serum and vitreous. Materials and methods: Forty DME patients were analyzed for macular thickness (MT). Twelve proangiogenic biomarkers in serum and vitreous were analyzed before and after IVB. Results: Significant decrease in MT with vitreal vascular endothelial growth factor-A (VEGF-A) was observed as expected after IVB, while serum VEGF-A did not follow a decreasing trend in contrast to VEGF-C, which decreased both in serum and vitreous. Other vitreal factors like bone morphogenetic protein-9 (BMP9) and fibroblast growth factor (FGF) were also significantly decreased, while endothelial growth factor (EGF) increased following IVB. Before IVB, significant negative correlations were vitreous BMP9 with serum FGF, vitreous human growth factor (HGF) and interleukin-8 (IL-8) with serum endothelin, and vitreous and serum FGF and serum placental growth factor (PLGF) with EGF. After IVB, negative correlations in serum vs. vitreous were found for both HGF and PLGF with BMP9, and angiopoietin with FGF. Cube average thickness was negatively correlated with serum FGF and positively correlated with vitreous PLGF and endothelin. Conclusion: Vascular endothelial growth factors are not the only factors that cause macular edema in diabetic patients. The effect of IVB on different proangiogenic biomarkers indicated a complex interplay of other factors in DME.

Comparative Analysis of Serum Proangiogenic Biomarkers between those with and without Diabetic Retinopathy.

OBJECTIVE: To compare the serum proangiogenic biomarkers in diabetic patients suffering from with and without diabetic retinopathy (DR). STUDY DESIGN: An observational study. PLACE AND DURATION OF STUDY: Arif Memorial Teaching Hospital, Lahore, Pakistan and Institute of Molecular Biology and Biotechnology/Centre for Research in Molecular Medicine, The University of Lahore, Lahore, Pakistan, from March to December 2017. METHODOLOGY: Forty patients with DR were included in group A and 15 patients without retinopathy (controls) were included in group B. Twelve serum pro-angiogenic biomarkers [Angiopoietin 2, Human Growth Factor (HGF), Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), Placental Growth Factor (PLGF), Vascular Endothelial Growth Factor-A and C (VEGF-A and VEGF-C), Bone Morphogenetic Protein 9 (BMP9), Follistatin, Leptin, Interleukin-8 (IL8), Endothelin (ET)] were analysed by xMAP flow cytometry technique, results were compared between the two groups and statistical analysis was done using Mann-Whitney U test. RESULTS: Serum ET, Follistatin and EGF were significantly raised in group A as compared to group B having p-values of 0.001, <0.001, and 0.033, respectively. Serum BMP9, Leptin, HGF, FGF and VEGF-C had p <0.001, 0.023, 0.020, and 0.009, respectively and were higher in group B than group A. CONCLUSION: Serum ET, Follistatin and EGF were significantly higher in DR patients as compared to those without DR and should be considered to be significant biomarkers of retinal complications in diabetes mellitus.

Soluble Production of Human Recombinant VEGF-A121 by Using SUMO Fusion Technology in Escherichia coli.

Human recombinant vascular endothelial growth factor-A121 (hrVEGF-A121) has applications in pharmaceutical industry especially in regenerative medicine. Here, we report the expression, purification, and characterization of hrVEGF-A121 in Escherichia coli expression system using human small ubiquitin-related modifier-3 (hSUMO3) fusion partner. Total RNA was isolated from healthy human gingival tissue, VEGF-A121 gene was RT-PCR amplified, and hSUMO3 gene was tagged at N-terminus. The fusion gene (SUMO3-VEGF-A121) was cloned in pET-22b(+) expression vector and transferred into E. coli strains; BL21 codon + and Rosetta-gami B(DE3). The hrVEGF-A121 expression was optimized for temperature, IPTG concentration, and time in Terrific Broth (TB). The positive transformants were sequenced and hrVEGF-A121 nucleotide sequence was submitted to Genbank (Accession No. KT581010). Approximately 40% of total cell protein expression was observed in soluble form on 15% SDS-PAGE. The hSUMO3 was cleaved from hrVEGF-A121 with SUMO protease and purified by Fast Protein Liquid Chromatography using anionic Hi-trap Resource Q column. From 100 ml TB, ~ 25.5% and ~ 6.8 mg of hrVEGF-A121 protein was recovered. The dimerized hrVEGF-A121 was characterized by Native PAGE and Western blot, using human anti-VEGF-A antibody and ESI-MS showed dimeric hrVEGF-A121 at 31,015 Da. The biological activity of hrVEGF-A121 was assessed in vitro by MTT and cell viability assay and observed to be bioactive.

Expression and rapid purification of recombinant biologically active ovine growth hormone with DsbA targeting to Escherichia coli inner membrane.

This study shows expression of recombinant ovine growth hormone (roGH) and targeting to the inner membrane using signal sequence, DsbA, in Escherichia coli (E. coli) cell. Factors such as temperature, IPTG induction, and expression conditions were studied and show diverse optical density with different media compositions. The optimum expression level of roGH in terrific broth medium was at 25 °C on induction with 20 µM IPTG in early logarithmic phase. SDS-PAGE analysis of expression and subcellular fractions of recombinant constructs revealed the translocation of roGH to the inner membrane of E. coli with DsbA signal sequence at the N terminus of roGH. The protein was easily solubilized by 40 % acetonitrile with ~90 % purity and was identified by Western blot, and analysis on MALDI-TOF/TOF confirmed a size of 21,059 Da. Relatively high soluble protein yield of 65.3 mg/L of roGH was obtained. The biological function of roGH was confirmed by HeLa cell line proliferation. This is the first study describing achievement of biologically active soluble roGH targeted to the inner membrane of E. coli and rapid purification with high yield.