In-silico prediction of TGF-ß1 non-synonymous variants and their impact on binding affinity to Fresolimumab.

TGF-ß1 is a potent immunoregulatory cytokine that plays diverse roles in development, bone healing, fibrosis, and cancer. However, characterizing TGF-ß1 gene variants is challenging because the structural and functional consequences of these variants are still undetermined. In this study, we aimed to perform an in-silico analysis of TGF-ß1 non-synonymous variants and their pathogenic effects on the TGF-ß1 protein. A total of 10,252 TGF-ß1 SNPs were collected from the NCBI dbSNP database and in-silico tools (SIFT, PROVEAN, Mutation Taster, ClinVar, PolyPhen-2, CScape, MutPred, and ConSurf) were used. The in-silico predicted potential variants were further investigated for their binding to the TGF-ß1 targeting drug "Fresolimumab". Molecular docking was performed using HADDOCK and confirmed by PRODIGY and PDBsum. The in-silico analysis predicted four potential TGF-ß1 nsSNPs: E47G in the LAP domain of the propeptide and I22T, L28F, and E35D in the mature TGF-ß1 peptide. HADDOCK and molecular dynamics simulations revealed that the I22T and E35D variants have higher binding affinity for Fresolimumab as compared to the wild type and L28F variants. Molecular dynamics simulations (100 ns) and principal component analysis showed that TGF-ß1 variants influenced the protein structure and caused variations in the internal dynamics of protein complexes with the antibody. Among them, the E35D variant significantly destabilized the TGF-ß1 protein structure, resulting in rearrangement in the binding site and affecting the interactions with the Fresolimumab. This study identified four variants that can affect the TGF-ß1 protein structure and result in functional consequences such as impaired response to Fresolimumab.Communicated by Ramaswamy H. Sarma.

Systemic Diseases and Gastrointestinal Cancer Risk.

Importance: Gastrointestinal (GI) cancers are the second leading cause of cancer-related deaths worldwide. Observations: The global challenges GI cancers pose are high, especially in middle- and low-income countries. Patients with these cancers present with symptoms of poor appetite, weight loss, heartburn, abdominal pain, fatigue and anaemia. Several risk factors contribute to GI cancers, including age, gender, obesity, pathogenic infections, smoking cigarettes, alcohol consumption and dietary habits. Most of these cancers are sporadic. However, some patients are at high risk due to a family history of GI cancers. Systemic diseases affect multiple organs, and their chronic occurrence elicits inflammatory responses at various sites. These diseases also contribute to GI cancers. Conclusion and Relevance: In this review, we discuss that untreated systemic diseases, including diabetes, hepatitis, acquired immune deficiency syndrome, ulcers and hypertension, can potentially lead to GI cancers if they remain untreated for a longer period. Systemic diseases initiate oxidative stress, inflammatory pathways and genetic manipulations, which altogether confer risks to GI cancers. Here, we describe the association between systemic diseases and their underlying mechanisms leading to GI cancers.

miR-4482 and miR-3912 aim for 3'UTR of ERG mRNA in prostate cancer.

Ets-related gene (ERG) is overexpressed as a fusion protein in prostate cancer. During metastasis, the pathological role of ERG is associated with cell proliferation, invasion, and angiogenesis. Here, we hypothesized that miRNAs regulate ERG expression through its 3'UTR. Several bioinformatics tools were used to identify miRNAs and their binding sites on 3'UTR of ERG. The selected miRNAs expression was analyzed in prostate cancer samples by qPCR. The miRNAs overexpression was induced in prostate cancer cells (VCaP) to analyze ERG expression. Reporter gene assay was performed to evaluate the ERG activity in response to selected miRNAs. The expression of ERG downstream target genes was also investigated through qPCR after miRNAs overexpression. To observe the effects of selected miRNAs on cell proliferation and migration, scratch assay was performed to calculate the cell migration rate. miR-4482 and miR-3912 were selected from bioinformatics databases. miR-4482 and -3912 expression were decreased in prostate cancer samples, as compared to controls (p<0.05 and p<0.001), respectively. Overexpression of miR-4482 and miR-3912 significantly reduced ERG mRNA (p<0.001 and p<0.01), respectively) and protein (p<0.01) in prostate cancer cells. The transcriptional activity of ERG was significantly reduced (p<0.01) in response to miR-4482 and-3912. ERG angiogenic targets and cell migration rate was also reduced significantly (p<0.001) after miR-4482 and -3912 over-expression. This study indicates that miR-4482 and -3912 can suppress the ERG expression and its target genes, thereby, halt prostate cancer progression. These miRNAs may be employed as a potential therapeutic target for the miRNA-based therapy against prostate cancer.

Silymarin protects the liver from α-naphthylisothiocyanate-induced cholestasis by modulating the expression of genes involved in bile acid homeostasis.

Cholestasis is characterized by impaired bile flow which results in inflammation, cirrhosis, and ultimately liver failure. The current study is aimed to evaluate the anti-cholestatic effect of silymarin against α-naphthylisothiocyanate (ANIT) induced cholestasis. Mice were gavaged with various doses of silymarin or ursodeoxycholic acid (UDCA) for 19 days. Then they were challenged with α-naphthylisothiocyanate (ANIT) and after 48 hours the animals were sacrificed to obtain blood and liver sections. Serum levels of bilirubin, aspartate transaminase (AST), alanine transaminase (ALP), and liver histology were analyzed. mRNA expression of selected transporters (Bile salt export pump (BSEP) and sodium taurocholate cotransporting polypeptide (NTCP)) and proteins (farnesoid x receptor (FXR) and Cytochrome P450 Family 7 Subfamily A Member 1 (Cyp7a1)) involved in bile acids biosynthesis, excretion and uptake were also evaluated by quantitative PCR. The results indicated that the serum levels of bilirubin, AST, and ALP were significantly higher in a cholestatic model group as compared to an untreated control group. However, in silymarin groups, the serum level of these parameters is significantly lower than in a cholestatic model group. Liver histology also showed that silymarin prevents ANIT-induced hepatic injury. mRNA expression of FXR, BSEP, and NTCP was downregulated and expression of Cyp7a1 was upregulated in a cholestatic model group as compared to an untreated control group. However, in silymarin treatment groups, the expression of FXR, BSEP and NTCP was upregulated and the expression of Cyp7a1 was downregulated as compared to the cholestatic model group. In conclusion, silymarin could alleviate hepatic injury by modulating the expression of genes involved in bile acid homeostasis.

Epidemiological Assessment of Oral Cancer Burden in Pakistan.

Oral cancer ranks first among males and is the primary cause of cancer-related deaths in Pakistan. We studied the epidemiology and risk factors associated with this cancer. The main risk factors in the Pakistani population include the usage of chewable and non-chewable tobacco, areca nut, betel leaf, poor dental hygiene practices, oncogenic viral infections, and genetic predispositions. The impact of socioeconomic status and the available health resources on the management of oral cancer is also discussed. It is concluded that being a low-middle economy efforts should be primarily focused on awareness for early screening, diagnosis, and prevention strategies.

In-Silico Analyses of Nonsynonymous Variants in the BRCA1 Gene.

BReast CAncer gene 1 (BRCA1)-a tumor suppressor gene plays an important role in the DNA repair mechanism. Several BRCA1 variants perturb its structure and function, including synonymous and nonsynonymous single nucleotide polymorphisms (SNPs). In the present study, we performed in-silico analyses of nonsynonymous SNPs (nsSNPs) of the BRCA1 gene. In total, 122 nsSNPs were retrieved from the NCBI SNP database and in-silico analyses were performed using computational prediction tools: SIFT, PROVEAN, Mutation Taster, PolyPhen-2, MutPred, and ConSurf. Of these tools, SIFT, PROVEAN, and Mutation Taster predicted 61 out of 122 nsSNPs as "damaging", based on structural homology analysis. PolyPhen-2 classified 22 nsSNPs as "probably damaging". These nsSNPs were further analyzed by MutPred to predict basic molecular mechanisms of amino acid alteration. ConSurf analysis predicted eleven conserved amino acid residues with structural and functional consequences. We identified five amino acid residues in the RING finger domain (L22, C39, H41, C44, and C47) and two in the BRCT domain (P1771 and I1707) with the potential to deter the BRCA1 protein function. This study provides insights into the effect of nsSNPs and amino acid substitutions in BRCA1.

SARS-COV-2 infection and lung tumor microenvironment.

Coronavirus Disease 2019 (COVID-19) is an acute respiratory syndrome, reported at the end of 2019 in China originally and immediately spread affecting over ten million world population to date. This pandemic is more lethal for the older population and those who previously suffered from other ailments such as cardiovascular diseases, respiratory disorders, and other immune system affecting abnormalities including cancers. Lung cancer is an important comorbidity of COVID-19. In this review, we emphasized the impact of lung tumor microenvironment (TME) on the possibility of enhanced severity of infection caused by the SARS-Co-V2. The compromised lung TME is further susceptible to the attack of viruses. The lung cells are also abundant in the virus entry receptors. Several SARS-Co-V2 proteins can modulate the lung TME by disrupting the fragile immune mechanisms contributing to cytokine storming and cellular metabolic variations. We also discussed the impact of medication used for lung cancer in the scenario of this infection. Since other respiratory infections can be a risk factor for lung cancer, COVID-19 recovered patients should be monitored for tumor development, especially if there is genetic susceptibility or it involves exposure to other risk factors.

Prevalence of RECQL germline variants in Pakistani early-onset and familial breast cancer patients.

BACKGROUND: The RecQ Like Helicase (RECQL) gene has previously been shown to predispose to breast cancer mainly in European populations, in particular to estrogen receptor (ER) and/or progesterone receptor (PR) positive tumor. Here, we investigated the contribution of pathogenic RECQL germline variants to hereditary breast cancer in early-onset and familial breast cancer patients from Pakistan. METHODS: Comprehensive RECQL variant analysis was performed in 302 BRCA1 and BRCA2 negative patients with ER and/or PR positive breast tumors using denaturing high-performance liquid chromatography followed by DNA sequencing. Novel variants were classified using Sherloc guidelines. RESULTS: One novel pathogenic protein-truncating variant (p.W75*) was identified in a 37-year-old familial breast cancer patient. The pathogenic variant frequencies were 0.3% (1/302) in early-onset and familial breast cancer patients and 0.8% (1/133) in familial patients. Further, three novel variants of unknown significance, p.I141F, p.S182S, and p.C475C, were identified in familial breast cancer patients at the age of 47, 68, and 47 respectively. All variants were absent in 250 controls. CONCLUSIONS: Our data suggest that the RECQL gene plays a negligible role in breast cancer predisposition in Pakistan.

Silybum marianum (milk thistle) improves vancomycin induced nephrotoxicity by downregulating apoptosis.

Increased use of vancomycin for treating infections, and the associated risk of causing nephrotoxicity lead to the present study. The antioxidant and anti-apoptotic potential of Silybum marianum is used along with vancomycin to reduce adverse effects on the kidney. Vero cells (monkey kidney cells) and mice were used to test S. marianum extract on vancomycin induced nephrotoxicity. Vero cells were treated with different concentrations of vancomycin and S. marianum for 24 h for determination of cytotoxic potential and mRNA levels of apoptotic genes p53 , p21, and cyt-c were measured. For in-vivo studies mice were divided into five groups; G1 control (untreated), G2 vehicle (olive oil), G3 vancomycin treated (300 mg/kg body weight), G4 (S. marianum; 400 mg/kg bodyweight and vancomycin 300 mg/kg bodyweight simultaneously) and G5 (S. marianum 400 mg/kg bodyweight and vancomycin 300 mg/kg bodyweight treatment started after day 4 of S. marianum treatment). After 10 days histopathological analysis of mice kidneys was performed, serum urea and creatinine were analysed and mRNA expression of p53 , p21, and cyt-c was evaluated. Expression of p53, p21, and cyt-c in Vero cells was elevated in response to vancomycin treatment, whereas after S. marianum administration expression of these genes reduced. Vancomycin showed apoptosis in cells at the concentration of 6 mg/ml (LC50). Urea and creatinine levels in mice were increased in response to vancomycin administration and kidney histology showed an abnormality in functional units. The apoptotic cells were very visible in kidney structure in vancomycin treated group. These symptoms were however relieved in groups where treatment of S. marianum extract was given. mRNA expression of p53 , p21, and cyt-c also reduced in S. marianum treated groups of mice. S. marianum extract has protective effects against renal damage from vancomycin induced oxidative stress and relieves symptoms may be by downregulating apoptotic genes.

Novel Biosynthesis of Copper Nanoparticles Using Zingiber and Allium sp. with Synergic Effect of Doxycycline for Anticancer and Bactericidal Activity.

Copper nanoparticles (CuNPs), due to their cost-effective synthesis, interesting properties, and a wide range of applications in conductive inks, cooling fluids, biomedical field, and catalysis, have attracted the attention of scientific community in recent years. The aim of the present study was to develop and characterize antibacterial and anticancer CuNPs synthesized via chemical and biological methods, and further synthesize CuNPs conjugated with doxycycline to study their synergic effect. During the chemical synthesis, ascorbic acid was used as a stabilizing agent, while Zingiber officinale and Allium sativum-derived extracts were used during the biological methods for synthesis of CuNPs. Characterization of CuNPs was performed by transmission electron microscopy (TEM), UV-visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), and X-ray crystallography (XRD). Antimicrobial evaluation of the nanomaterials against Pseudomonas aeruginosa and Escherichia coli was performed by using disk diffusion method, while anticancer behavior against HeLa and HepG2 cell lines was studied by MTT assay. TEM revealed spherical-shaped nanoparticles with mean size of 22.70 ± 5.67, 35.01 ± 5.84, and 19.02 ± 2.41 nm for CuNPs, Gin-CuNPs, and Gar-CuNPs, respectively, and surface plasmon resonance peaks were obtained at 570 nm, 575 nm, and 610 nm for CuNPs, Gar-CuNPs, and Gin-CuNPs, respectively. The results of FTIR confirmed the consumption of biomolecules from the plant extracts for the synthesis of CuNPs. XRD analysis also confirmed synthesis of CuNPs. Doxycycline-conjugated NPs exhibited more antibacterial effects than doxycycline or CuNPs alone. Copper nanoparticles prepared by biological synthesis are cost-effective and eco-friendly as compared to their chemical counterparts. The chemically synthesized nanoparticles displayed more significant antimicrobial activity when capped with doxycycline than Z. officinale and A. sativum-mediated CuNPs; however, green-synthesized nanoparticles showed greater anticancer activity than their chemical counterparts.

Matrix metalloprotease-9 polymorphism and its association with Atherosclerosis - A case-control study in Pakistani population.

OBJECTIVE: To determine the association of matrix metalloproteinase-9 gene with the onset of atherosclerosis in Pakistani population of Punjabi origin. METHODS: The case-control study was conducted from September 2015 to December 2016 at the Government College University, Lahore, Pakistan, and comprised atherosclerosis cases from the Punjab Institute of Cardiology, Lahore, as well as healthy controls. Single nucleotide polymorphismsrs3918242 (Sph1), rs17577 (Sty1) and rs2274756 (Taq1) were selected from the matrix metalloproteinase-9 gene for allelic and genotypic analysis. Direct sequencing and polymerase chain reaction-restriction fragment length polymorphism were performed for genotypic analysis. RESULTS: the 201 subjects, 100(49.8%) were controls and 101(50.2%) were cases. There were 75(75%) males among the controls and 70(69.3%) among the cases. Overall mean age of the controls was 47.3}13.1 years, and that of the cases was 59.2}10.2 years. Positive family history was a significant factor risk for atherosclerosis (p<0.05). Allele T and genotype CT and TT of rs3918242 were more frequent in the cases (p<0.05). Change in nucleotide at Sph1 site led towards -1562C >T polymorphism. The frequency of 'A' allele and 'GA' genotype for rs17577 was significantly higher in the cases (Sty1) (p<0.05). No association was detected between rs2274756 (Taq1) and atherosclerosis (p> 0.05). The co-expression of rs17577 and rs2274756 was significantly related with the onset of atherosclerosis (p<0.05). Haplotypes CAG, TAG and TGG were significantly involved in causing atherosclerosis (p<0.05) whereas CGG was protective against atherosclerosis in this population (p<0.05). CONCLUSIONS: Matrix metalloproteinase-9 gene was identified as a susceptible gene for the onset of atherosclerosis in Pakistani population of Punjabi origin.

Evaluation of Serum Circulating Levels of ICAM-1 as Tuberculosis Risk-assessment Factor in Type 2 Diabetes Patients.

OBJECTIVE: Type 2 diabetes mellitus, due to its deteriorating effect on the immune system, makes a person susceptible to various other diseases, such as tuberculosis. The alarming increase in the number of diabetes mellitus cases in Pakistan may be a contributing factor to the increased tuberculosis incidence. The expression of cell adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) is important in determining cell permeability, and the latter's altered expression may ease the entry of infectious agents into the cell. Therefore, the present study evaluated the role of ICAM-1 in type 2 diabetes and tuberculosis patients so that a potential link between these 2 epidemics could be found. METHODS: To explore this hypothesis, the expression of ICAM-1 was measured tested in 3 groups of subjects: group I consisted of 100 healthy individuals (control), group II consisted of 100 type 2 diabetics, and group III consisted of 100 individuals with both type 2 diabetes and tuberculosis. Demographic information was obtained from all the participants and compared by group and ICAM-1 levels in the blood were determined by ELISA. RESULTS: The results revealed that, in comparison to group I, the individuals of group II had significantly (p ≤ 0.05) increased levels of ICAM-1, making them more prone to infection (by promoting the increased invasion of mycobacterium) and hence at increased risk of contracting tuberculosis. CONCLUSION: In conclusion, the present study demonstrated that elevated levels of ICAM-1 in patients with type 2 diabetes mellitus are likely associated with the development of tuberculosis.

Sequence-function correlations and dynamics of ERG isoforms. ERG8 is the black sheep of the family.

The transcription factor ERG is known to have divergent roles. On one hand, it acts as differentiation factor of endothelial cells. On the other hand, it has pathological roles in various cancers. Genomic analyses of the ERG gene show that it gives rise to several isoforms. However, functional differences between these isoforms, representing potential reasons for distinct effects in diverse cell types have not been addressed in detail so far. We set out to investigate the major protein isoforms and found that ERG8 contains a unique C-terminus. This isoform, when expressed as GFP-fusion protein, localized mainly to the cytosol, whereas the other major isoforms (ERG1-4) were predominantly nuclear. Using site directed mutagenesis and laser scanning microscopy of live cells, we could identify nuclear localization (NLS) and nuclear export sequences (NES). These analyses indicated that ERG8 lacks a classical NLS and the DNA-binding domain, but holds an additional NES within its distinctive C-terminus. All the tested isoforms were shuttling between nucleus and cytosol and showed a high degree of mobility. ERG's 1 to 4 were transcriptionally active on ERG-promoter elements whereas ERG8 was inactive, which is in line with the absence of a DNA-binding domain. Fluorescence resonance energy transfer (FRET) microscopy revealed that ERG8 can bind to the transcriptionally active ERG's. Knockdown of ERG8 in endothelial cells resulted in upregulation of endogenous ERG-transcriptional activity implying ERG8 as an inhibitor of the active ERG isoforms. Quantitative PCR revealed a different ratio of active ERG's to ERG8 in cancer- versus non-transformed cells.

Mechanistic insights into PTS2-mediated peroxisomal protein import: the co-receptor PEX5L drastically increases the interaction strength between the cargo protein and the receptor PEX7.

The destination of peroxisomal matrix proteins is encoded by short peptide sequences, which have been characterized as peroxisomal targeting signals (PTS) residing either at the C terminus (PTS1) or close to the N terminus (PTS2). PTS2-carrying proteins interact with their cognate receptor protein PEX7 that mediates their transport to peroxisomes by a concerted action with a co-receptor protein, which in mammals is the PTS1 receptor PEX5L. Using a modified version of the mammalian two-hybrid assay, we demonstrate that the interaction strength between cargo and PEX7 is drastically increased in the presence of the co-receptor PEX5L. In addition, cargo binding is a prerequisite for the interaction between PEX7 and PEX5L and ectopic overexpression of PTS2-carrying cargo protein drastically increases the formation of PEX7-PEX5L complexes in this assay. Consistently, we find that the peroxisomal transfer of PEX7 depends on cargo binding and that ectopic overexpression of cargo protein stimulates this process. Thus, the sequential formation of a highly stable trimeric complex involving cargo protein, PEX7 and PEX5L stabilizes cargo binding and is a prerequisite for PTS2-mediated peroxisomal import.

Some secrets of fluorescent proteins: distinct bleaching in various mounting fluids and photoactivation of cyan fluorescent proteins at YFP-excitation.

BACKGROUND: The use of spectrally distinct variants of green fluorescent protein (GFP) such as cyan or yellow mutants (CFP and YFP, respectively) is very common in all different fields of life sciences, e.g. for marking specific proteins or cells or to determine protein interactions. In the latter case, the quantum physical phenomenon of fluorescence resonance energy transfer (FRET) is exploited by specific microscopy techniques to visualize proximity of proteins. METHODOLOGY/PRINCIPAL FINDINGS: When we applied a commonly used FRET microscopy technique--the increase in donor (CFP)-fluorescence after bleaching of acceptor fluorophores (YFP), we obtained good signals in live cells, but very weak signals for the same samples after fixation and mounting in commercial microscopy mounting fluids. This observation could be traced back to much faster bleaching of CFP in these mounting media. Strikingly, the opposite effect of the mounting fluid was observed for YFP and also for other proteins such as Cerulean, TFP or Venus. The changes in photostability of CFP and YFP were not caused by the fixation but directly dependent on the mounting fluid. Furthermore we made the interesting observation that the CFP-fluorescence intensity increases by about 10-15% after illumination at the YFP-excitation wavelength--a phenomenon, which was also observed for Cerulean. This photoactivation of cyan fluorescent proteins at the YFP-excitation can cause false-positive signals in the FRET-microscopy technique that is based on bleaching of a yellow FRET acceptor. CONCLUSIONS/SIGNIFICANCE: Our results show that photostability of fluorescent proteins differs significantly for various media and that CFP bleaches significantly faster in commercial mounting fluids, while the opposite is observed for YFP and some other proteins. Moreover, we show that the FRET microscopy technique that is based on bleaching of the YFP is prone to artifacts due to photoactivation of cyan fluorescent proteins under these conditions.