Evaluation of anti-EGFR potential of quinazoline derivatives using molecular docking: An in silico approach.

Overexpression of epidermal growth factor receptor (EGFR) is commonly reported in epithelial malignancies such as oral squamous cell carcinoma. Inhibition of EGFR is, therefore, considered a potential therapeutic strategy. Among various anti-EGFR drugs, quinazoline-based tyrosine kinase inhibitors (TKIs) have gained increasing attention. Present study focused to investigate anti-EGFR potential of quinazoline-based compounds using in silico approach. Two widely used docking programs GOLD and AutoDock Vina were used for the study. Four drugs were docked on the X-ray crystallographic EGFR structure (1XKK). GOLD and AutoDock Vina produced results in terms of fitness score and binding affinity, respectively. GOLD prioritized varlitinib and AutoDock Vina preferred imatinib over other drugs. To reach the consensus from both software, all four drugs coupled with EGFR were studied rigorously. GOLD demonstrated varlitinib to be the best inhibitor with highest fitness score of 109, whereas AutoDock Vina revealed imatinib as the potent ligand with least binding energy of -10.9 kcal/mol. Most stable hydrogen bonds observed by GOLD and maximum number of hydrophobic contacts along with strong ionic interaction exhibited by varlitinib through both software have led us to conclude varlitinib as the most potent EGFR inhibitor in the studied group.

Prenatal diagnosis of maternal serum from mothers carrying ß-thalassemic fetus.

BACKGROUND: This study focuses on the discovery of protein biomarkers from the maternal serum of ß-thalassemic trait mothers carrying the normal fetus and ß-thalassemic major fetus. METHODS: Serum samples from ß-thalassemic trait mothers carrying major (N = 5) and normal fetuses (N = 5) were studied. The IVS1-5 thalassemia mutation was common among ß-thalassemic trait mothers who were carrying a homozygous ß-thalassemic fetus (IVS1-5/ IVS1-5 mutation) or a normal fetus (no mutation). We employed two-dimensional gel electrophoresis and mass spectrometry analysis to explore differentially expressed maternal serum proteins from thalassemia carrier couples with the same ß-thalassemia mutation. Western blotting was performed for one of the identified proteins to validate our data. RESULTS: Ten proteins were identified in the maternal serum of ß-thalassemic trait mothers carrying the ß-thalassemic major fetus and normal fetus. Among these, serotransferrin, haptoglobin, α-1 anti-trypsin, apo-lipoprotein A1, and the fibrinogen-ß chain were found to be upregulated in mothers carrying major fetuses and are known to be associated with pregnancy-related disorders. The expression of α-1 anti-trypsin was validated through western blotting. CONCLUSIONS: Proteins identified in the current study from maternal serum are reported to contribute to hereditary disorders. We suggest that these can serve as putative screening markers for non-invasive prenatal diagnosis in ß-thalassemic pregnancies.

Antioxidant Activity and Oxidative Stress Status in Maternal Serum of ß-thalassemic Mothers Carrying ß-thalassemic Major and Normal Fetuses.

Objectiveß-thalassemia is a genetic disorder characterized by reduction or absence of ß-globin chain with mutations in both copies (ß-thalassemia major) or in one copy (ß-thalassemia minor). Pregnancies in ß- thalassemic carrier women are considered symptom free but have risk of inheriting ß-thalassemic fetuses. Current study was designed to compare oxidative stress and antioxidants status in maternal serum from ß-thalassemic minor mothers having ß-thalassemic major and normal fetuses. Method: We investigated paraoxonase (PON1) and arylesterase (ARE) activities along with malondialdehyde (MDA) and reactive oxygen species (ROS) in maternal serum of ß-thalassemic carrier women. Results: PON1 and ARE activities were found to be significantly decreased, whereas the concentration of MDA and ROS were significantly increased in ß-thalassemic minor mothers with ß-thalassemic major fetuses. Conclusion: The study concludes that redox imbalance in ß-thalassemic trait mothers carrying thalassemic fetuses is higher than in mothers carrying normal fetuses.

Therapeutic effect of hydrogen peroxide via altered expression of glutathione S-transferase and peroxiredoxin-2 in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) has a high incidence and mortality that epitomizes one of the prominent causes of cancer-related death globally. Novel therapeutic approaches are therefore required. Reactive oxygen species (ROS) are necessary for maintaining cell cycle. Although ROS is involved in HCC progression, hydrogen peroxide (H2O2) has anti-proliferative effect on HCC. METHOD: HCC Huh-7 cells were cultured and incubated with various concentrations of H2O2. Paraoxonase activity, levels of malondialdehyde, glutathione and protein oxidation were measured in treated and untreated Huh-7 cells. Furthermore, untreated and treated Huh-7 cells were subjected to two dimensional gel electrophoresis and identified protein spots which were differentially expressed by LC-MS/MS analysis. qRT-PCR was performed to validate the identified proteins. RESULTS: H2O2 depleted glutathione (GSH) with the concomitant up-regulation of GSTP1 and Prx2. H2O2 also increased malondialdehyde and protein oxidation, decreased the activity of paraoxonase in Huh-7 cells. CONCLUSION: H2O2 could be used as a novel therapeutic agent that might be beneficial in inducing cell cytotoxicity and hence suppress HCC proliferation.

Alendronate and FTI-277 combination as a possible therapeutic approach for hepatocellular carcinoma: An in vitro study.

BACKGROUND: An important product of mevalonate pathway is downstream synthesis of isoprenoid units that has long been implicated in development and progression of tumor. It has been speculated that inhibition of protein prenylation might be therapeutically beneficial. The objective of current study was to evaluate antitumor potential of a novel therapeutic combination of mevalonate pathway inhibitors, FTI-277 and alendronate. We also examined differentially expressed proteins in response to treatment using proteomics approach. METHODS: Huh-7 cells were incubated with different concentrations of FTI-277 alone and in combination with alendronate. Differential protein and gene expression was examined through two dimensional gel electrophoresis and real-time quantitative polymerase chain reaction (qPCR), respectively. Proteins were identified using tandem mass spectrometry analysis. RESULTS: Treatment of hepatocellular carcinoma (HCC) cell line with FTI-277 alone showed cell death in a time and dose dependent manner while in combination with alendronate, a synergistic apoptotic effect at 24 h was observed. Proteomic studies on the 20 µmol/L FTI-277 and 5 µmol/L alendronate +20 µmol/L FTI-277 treated cells revealed altered expression of different proteins including peroxiredoxin 2 (Prx2), glutathione S transferase 1 (GSTP1), Rho GTPase activating protein (RhoGAP), triosephosphate isomerase (TPI), and heat shock protein 60 (HSP60). Down-regulated expression of Prx2 and GSTP1 in treated cells was also confirmed by real-time qPCR analysis. CONCLUSIONS: Combined treatment of FTI-277 and alendronate on Huh-7 HCC cells showed cell death suggesting their anticancer potential. Such treatment approaches are likely to offer new therapeutic strategies.

Q192R paraoxonase1 polymorphism is a risk factor for cataract in Pakistani population.

Cataract, the lens opacity, is among major causes of blindness in Pakistani population. In recent past, oxidative stress is suggested to play crucial role in loss of transparency. Along with other antioxidants, Paraoxonase 1 (PON1) has also shown decreased activity in patients suffering from cataract. The aim of current study was to examine the possible association of PON polymorphism with predisposition of cataract formation in local population. The study was conducted on 51 cataract patients and 50 control subjects considering all ethical issues. DNA was extracted from whole blood and PON1 polymorphism was identified using tetra primer ARMS-PCR method for both positions L55M and Q192R. Tetra primer ARMS-PCR results revealed that association between L55M polymorphism and cataract was insignificant while 192R genotype PON1 frequency was higher among the people suffering from cataract (78.4%) as compared to control subjects (56%), (odds ratio=2.857, confidence interval=1.197-6.820). Hence, R allele is likely to be a risk factor for cataract with allele frequency (82.3%) and (odds ratio=4.552, confidence interval=1.716-12.073, p-value=0.002). PON1 Q192R polymorphism is likely to be a risk factor for cataract development in Pakistani population while PON1 L55M was not found to be associated with cataract.

Effects of 5'-azacytidine and alendronate on a hepatocellular carcinoma cell line: a proteomics perspective.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer related deaths around the world. Due to late diagnosis and development of drug resistance in patients suffering from HCC, development of more effective therapeutic strategies is inevitable. The aim of this study was to evaluate the combined apoptotic effect of 5'-Azacytidine (5'-AzaC) and alendronate (ALN) on Huh-7 HCC cell line and to explore differential expression at genomics and proteomics level. Incubation of HCC cell line with 5'-AzaC alone showed cell death in a time and dose dependent manner while in combination with ALN, increased cytotoxicity was observed. Up-regulation of CASP7(Caspase7) and LZTS1 (leucine zipper, putative tumor suppressor 1) and down-regulation of DNMT1(DNA (cytosine-5-)-methyltransferase 1) was noted in treated cells. Proteomic studies on the treated cells revealed altered expression of different proteins including peroxiredoxin 2 (Prx2), Annexin 5 (Anx5), Rho GTPase activating protein (RhoGAP), Nuclear factor-kappa B (NF-kB), tumor necrosis factor alpha-induced protein (TNF), triosephosphate isomerase (TPI), Glutathione S transferase (GSTP1) and Heat shock protein60 (HSP60). Our study demonstrated the cytotoxic effect of 5'-AzaC and ALN drug combination on Huh-7 HCC cells suggesting such combinations may be explored as a possible therapeutic approach. Current study revealed that Huh-7 HCC cells are sensitive to 5'-AzaC and ALN drug combination and such combination approaches could lead to the development of new therapeutic strategies. Furthermore, we also report the expression of Anx5 exclusively in untreated cancerous cell line indicating the possibility of being used as a potential therapeutic target and biomarker.

Docking studies of antidepressants against single crystal structure of tryptophan 2, 3-dioxygenase using Molegro Virtual Docker software.

Tryptophan 2, 3-dioxygenase (TDO) a heme containing enzyme found in mammalian liver is responsible for tryptophan (Trp) catabolism. Trp is an essential amino acid that is degraded in to N-formylkynurenine by the action of TDO. The protein ligand interaction plays a significant role in structural based drug designing. The current study illustrates the binding of established antidepressants (ADs) against TDO enzyme using in-silico docking studies. For this purpose, Fluoxetine, Paroxetine, Sertraline, Fluvoxamine, Seproxetine, Citalopram, Moclobamide, Hyperforin and Amoxepine were selected. In-silico docking studies were carried out using Molegro Virtual Docker (MVD) software. Docking results show that all ADs fit well in the active site of TDO moreover Hyperforin and Paroxetine exhibited high docking scores of -152.484k cal/mol and -139.706k cal/mol, respectively. It is concluded that Hyperforin and Paroxetine are possible lead molecules because of their high docking scores as compared to other ADs examined. Therefore, these two ADs stand as potent inhibitors of TDO enzyme.

Differential Protein Expression in Response to Varlitinib Treatment in Oral Cancer Cell Line: an In Vitro Therapeutic Approach.

Epidermal growth factor receptor (EGFR) is the most frequently overexpressed receptor histologically exhibited by oral squamous cell carcinoma (OSCC) patients. Aberrated EGFR signaling may lead to recurrence and metastasis, thus laying the foundation of targeted therapy. Deactivating EGFR is likely to prevent downstream signaling thus resulting in apoptosis. Tyrosine kinase inhibitors (TKIs) have come into play to revert aggressiveness of OSCC. We exploited comparative proteomic analyses based on anti-EGFR potential of varlitinib, using cellular proteomes from treated and untreated groups of oral cancer cells to identify protein players functional during oral carcinogenesis. Following separation by two-dimensional electrophoresis, differentially expressed cellular proteins (varlitinib-treated and untreated cells) were analyzed and later identified using QTOF mass spectrometer. In silico analysis for protein-protein interaction was carried out using STRING. Six differentially expressed proteins were identified as binding immunoglobulin protein (BiP), heat shock protein 7 C (HSP7C), protein disulfide isomerase 1 A (PDIA1), vimentin (VIME), keratin type I cytoskeletal 14 (K1C14), and ß-Actin (ACTB). Relative expression of five proteins was found to be downregulated upon varlitinib treatment, whereas only K1C14 was upregulated in treated cells compared to control. Protein network analysis depicts the interaction between BiP, PDIA1, VIME, etc. indicating their role in oral carcinogenesis. Oral cancer cells show proteome shift based on varlitinib treatment compared to corresponding controls. Our data suggest candidature of varlitinib as a potent therapeutic agent and BiP, PDIA1, HSP7C, VIME, and ß-Actin as complementary/prognostic markers of OSCC.

Novel Synergistic Combination of Pamidronate and Temozolomide for Breast Cancer Therapeutics.

OBJECTIVE: Human breast cancer is among one major health concerns with high prevalence and mortality among women worldwide. Various cellular signaling pathways are implicated in carcinogenesis. One of the major pathways that affect the downstream cellular growth cascades is Mevalonate pathway (MVA). The inhibition of MVA is therapeutically beneficial for various cancers. Pamidronate (PAM) (MVA inhibitor), a nitrogen-containing bisphosphosphonate, is an antiresorptive FDAapproved drug. The objective of our study was to explore adjuvant therapy using a combination of PAM and an alkylating agent, Temozolomide (TMZ) against breast cancer. METHODS: We have examined the differential gene and protein expression in response to the combination treatment strategy. For gene expression analysis RT-qPCR and for proteomic study, twodimensional gel electrophoresis and mass spectrometry techniques were utilized. RESULTS: Combination treatment (PAM+TMZ) showed more pronounced cytotoxic effect as compared to single agent treatment. Our results indicate that MVA pathway regulatory genes (FDFT1, FDPS, KRAS) are significantly (p<0.05) downregulated in combination-treated breast cancer cells. The differential proteomic analysis showed lower expression of GFAP, PPA1 and TRIM68 proteins after synergistic treatment whereas, these proteins are found to be up-regulated in multiple cancers. CONCLUSION: The present study reveals that a combination of PAM and TMZ produces an effective anti-cancerous effect on breast cancer cells. Therefore, this novel therapeutic regimen is likely to provide a better treatment strategy for breast cancer.

Polymorphic Variants of ASS1 Gene Related to Arginine Metabolism and the Risk of HCC.

BACKGROUND: Hepatocellular carcinoma is a primary liver cancer and 6th most common cancer globally. Inefficient diagnostic strategies and the limited availability of treatments are the foremost reasons. Variable factors directly impact the disease burden, among them, molecular alterations have been found to play a significant role. In liver, argininosuccinate synthase-1 is a center of arginine metabolism and rate limiting enzyme of urea cycle. It also triggers multiple mechanisms that lead to HCC pathogenesis. OBJECTIVES: The aim of this study is to analyze the ASS1 gene expression, its polymorphic genotype and microsatellite instability among HCC patients from our Pakistani population. METHOD: Blood samples were collected from disease and healthy control individuals. Allele-Specific PCR was performed for SNP analysis. MSI of tri and tetra nucleotide repeats were analyzed by PCR. The differential expression of ASS1 gene was also investigated. Furthermore, the reactome database and STRING software were utilized for finding correlations between ASS1 gene with other associated gene/proteins. RESULTS: The GG wild-type genotype was more prevailed in the disease group as compared to the control. Significant downregulation in ASS1 and NOS2 genes was observed. Bioinformatics analysis reveals the correlation between ASS1 polymorphism and HCC development appears to be linked with the EMT pathway and polyamine production. Furthermore, MSI significantly resided in the disease group. Results were analyzed statistically to calculate the significance of obtained results. CONCLUSION: Study concludes that the insight of HCC mechanism through population-specific genetic mutations and altered gene expression of ASS1 might be helpful in early diagnostic and therapeutic purposes.

The structural characterization and bioactivity assessment of nonspecific lipid transfer protein 1 (nsLTP1) from caraway (Carum carvi) seeds.

BACKGROUND: Carum carvi (caraway) of the Apiaceae family has been used in many cultures as a cooking spice and part of the folk medicine. Previous reports primarily focus on the medicinal properties of caraway seed essential oil and the whole seeds extract. However, no effort has been made to study caraway proteins and their potential pharmacological properties, including nonspecific lipid transfer protein (nsLTP), necessitating further research. The current study aimed to characterize nonspecific lipid transfer protein 1 (nsLTP1) from caraway seed, determine its three-dimensional structure, and analyze protein-ligand complex interactions through docking studies. We also evaluated nsLTP1 in vitro cytotoxic effect and antioxidant capacity. Additionally, nsLTP1 thermal- and pH- stability were investigated. METHODS: Caraway nsLTP1 was purified using two-dimensional chromatography. The complete amino acid sequence of nsLTP1 was achieved by intact protein sequence for the first 20 residues and the overlapping digested peptides. The three-dimensional structure was predicted using MODELLER. Autodock Vina software was employed for docking fatty acids against caraway nsLTP1. Assessment of nsLTP1 cytotoxic activity was achieved by MTS assay, and the Trolox equivalent antioxidant capacity (TAC) was determined. Thermal and pH stability of the nsLTP1 was examined by circular dichroism (CD) spectroscopy. RESULTS: Caraway nsLTP1 is composed of 91 residues and weighs 9652 Da. The three-dimensional structure of caraway nsLTP1 sequence was constructed based on searching known structures in the PDB. We chose nsLTP of Solanum melongena (PDB ID: 5TVI) as the modeling template with the highest identity among all other homologous proteins. Docking linolenic acid with caraway protein showed a maximum binding score of -3.6 kcal/mol. A preliminary screening of caraway nsLTP1 suppressed the proliferation of human breast cancer cell lines MDA-MB-231 and MCF-7 in a dose­dependent manner with an IC50 value of 52.93 and 44.76 µM, respectively. Also, nsLTP1 (41.4 µM) showed TAC up to 750.4 µM Trolox equivalent. Assessment of nsLTP1 demonstrated high thermal/pH stability. CONCLUSION: To the best of our knowledge, this is the first study carried out on nsLTP1 from caraway seeds. We hereby report the sequence of nsLTP1 from caraway seeds and its possible interaction with respective fatty acids using in silico approach. Our data indicated that the protein had anticancer and antioxidant activities and was thermally stable.

Chemical Hypoxic Preconditioning Improves Survival and Proliferation of Mesenchymal Stem Cells.

Increasing evidence has demonstrated that mesenchymal stem cells (MSCs) have been linked to tissue regeneration both in vitro and in vivo. However, poor engraftment and low survival rate of transplanted MSCs are still a major concern. It has been found that the proliferation, survival, and migration of MSCs are all increased by hypoxic preconditioning. However, the molecular mechanism through which hypoxic preconditioning enhances these beneficial properties of MSCs remains to be fully investigated. Therefore, the present study is aimed to investigate the mechanism by which hypoxic preconditioning enhances the survival of MSCs. We used proteomic analysis to explore the molecules that may contribute to the survival and proliferation of hypoxic preconditioned (HP) MSCs. The analysis revealed a higher expression of prelamin A/C (Lmna), glutamate dehydrogenase 1(Glud1), Actin, cytoplasmic 1(Actb), Alpha-enolase (Eno1), Glucose-6-phosphate 1-dehydrogenase (G6pd), Protein disulfide-isomerase A3 (Pdia3), Malate dehydrogenase (Mdh1), Peroxiredoxin-6 (Prdx6), Superoxide dismutase (Sod1), and Annexin A2 (Anxa2) in HP-MSCs. These proteins are possibly involved in cellular survival and proliferation through various cellular pathways. This research could aid in understanding the processes involved in hypoxic preconditioning of MSCs and designing of cell-based therapeutic strategies for tissue regeneration.

Docking and molecular dynamics simulation studies on glycation-induced conformational changes of human paraoxonase 1.

Human paraoxonase 1 (huPON1) is a calcium-dependent esterase responsible for hydrolysis of a wide variety of substrates including organophosphates, esters, lactones, and paraoxon. Although its natural substrate is unknown, the action of PON as an antioxidant is well documented. Because recent reports have suggested glycation may induce reduced PON activity in diabetes, we investigated the structural features of huPON1 and its glycated mutant by template-based modeling, docking, and molecular dynamics (MD) simulations. Our results corroborated the importance of the His115-His134 dyad in both the lactonase and paraoxonase activity of huPON1. Structural alterations in the glycated model reflected weak interactions between the docked substrate and the active site cleft. We also used MD simulation to gain insight into glycation-induced conformational changes of huPON1 and the implication of this on depleted enzymatic activity. The catalytic calcium found on the surface interacts with the side chain oxygen of residues, including Asn224, Asn270, Asn168, Asp269, and Glu53, and this interaction with the respective residues undergoes minor displacement on glycation. The root-mean-square fluctuation had high motional flexibility in the non-glycated model whereas the conformation of the glycated structure was comparatively stable. Our findings emphasize the consequence of glycation-induced alterations and their effect on overall enzymatic activity.

Oxidative Stress Induced Cell Cycle Arrest: Potential Role of PRX-2 and GSTP-1 as Therapeutic Targets in Hepatocellular Carcinoma.

BACKGROUND: The increasing incidence and mortality rate of HCC is a major concern, especially for developing countries of the world. Hence, extensive research is being carried out in order to explore new approaches for developing successful therapeutic strategies for HCC. The controversial role of oxidative stress in the prognosis and treatment of various diseases such as cancer has become an area of great interest and intrigue for many scientists throughout the world. OBJECTIVE: We aim to investigate the role of induced oxidative stress on the suppression of HCC Huh-7 cancerous cells as a therapeutic approach. METHODS: Induction of oxidative stress via H2O2 treatment produced cell cytotoxicity in a dose dependent manner and also led to the overexpression of GSTP-1 and PRX-2. The expression of GSTP- 1 and PRX-2 was compared in HCC Huh-7 treated, untreated cells and normal hepatocytes using immunocytochemistry. Furthermore, the effects of oxidative stress on cell cycle arrest were also studied through flow cytometry. RESULTS: Our study demonstrated the inhibition of cancer cell proliferation as a result of H2O2 induction by arresting the cell cycle at the G2 phase. CONCLUSION: The induction of oxidative stress could be a potential therapeutic approach for treating HCC in the future. GSTP-1 and PRX-2 can serve as substantial therapeutic targets for the treatment of HCC.

Serum HSP90-Alpha and Oral Squamous Cell Carcinoma: A Prospective Biomarker.

AIM: This study aims to perform differential protein expression analysis of serum samples from Oral Squamous Cell Carcinoma (OSCC) patients and healthy controls in search of potential diagnostic and/or prognostic biomarker(s). OBJECTIVE: OSCC is usually diagnosed late, which results in poor survival and high mortality. Identification of non-invasive prognostic biomarkers is of utmost importance for early diagnosis and proper management of the disease; hence we used a proteomic approach to identify potential biomarkers from serum. METHODS: Serum samples (OSCC n=45 and control n=30) were depleted, and proteins were separated using 2-D gel electrophoresis followed by identification by mass spectrometric analysis. Gene expression analysis of identified proteins in malignant and normal tissue was also performed to complement proteomics studies. RESULTS: Among differentially expressed proteins, up-regulation of heat shock protein alpha (HSP90α) from the serum of oral cancer patients was observed. We also observed elevated levels of Haptoglobin (HP) along with downregulation of Type II keratin cytoskeletal 1(KRT1) and serum albumin (ALB) in oral cancer patients. Gene expression studies on identified proteins in malignant and normal tissue revealed a similar pattern with the exception of KRT1. We believe that elevated levels of serum HSP90 alpha might be used as a potential biomarker. CONCLUSION: Our findings suggest a contribution of HSP90 alpha and other identified proteins in oral pathology as pro/anti-apoptotic modulators, thus considering their potential as predictive biomarkers.

Differential effects of memory enhancing and impairing doses of methylphenidate on serotonin metabolism and 5-HT1A, GABA, glutamate receptor expression in the rat prefrontal cortex.

Methylphenidate (MPD), a psychostimulant, is a prescription medicine for treating attention deficit hyperactivity disorder (ADHD). Previously we have shown that moderate doses of MPD enhanced learning and memory while higher doses impaired it. To understand neurochemical mechanisms and receptors involved in memory enhancing and impairing effects of MPD, the present study concerns the effects of these doses of MPD on serotonin, 5-HT1A, GABA, and NMDA receptor mRNA expression in the prefrontal cortex (PFC). We found that low doses (2.5 mg/kg) of MPD improved performance in the water-maze test but higher doses (5 mg/kg) impaired memory retention. Animals showing improved performance had high 5-HT metabolism in the PFC while these levels were not affected in the group treated with higher MPD doses and exhibiting impaired memory. There was downregulation of 5-HT1A receptors in the PFC of rats treated with higher dose MPD, which didn't occur in low dose of MPD treated animals. Further, a decrease in GABAAreceptor mRNA expression occurred in low doses of MPD treated animals and GluN2A expression was reduced in higher doses of MPD treated animals. The findings suggest that memory enhancing doses of MPD increase 5-HT and reduce GABAA receptor mRNA expression in the PFC to release excitatory glutamate neurons from the inhibitory influence of GABA. Conversely, higher dose of MPD downregulates 5-HT1A receptor mRNA expression to enhance inhibitory GABA influence on glutamate neurons and impair cognitive performance. The findings show an important role of 5-HT1A heteroreceptors in the PFC for improving therapeutic use of MPD and developing novel cognitive enhancers.

Structural characterization and in vitro lipid binding studies of non-specific lipid transfer protein 1 (nsLTP1) from fennel (Foeniculum vulgare) seeds.

Non-specific lipid transfer proteins (nsLTPs) are cationic proteins involved in intracellular lipid shuttling in growth and reproduction, as well as in defense against pathogenic microbes. Even though the primary and spatial structures of some nsLTPs from different plants indicate their similar features, they exhibit distinct lipid-binding specificities signifying their various biological roles that dictate further structural study. The present study determined the complete amino acid sequence, in silico 3D structure modeling, and the antiproliferative activity of nsLTP1 from fennel (Foeniculum vulgare) seeds. Fennel is a member of the family Umbelliferae (Apiaceae) native to southern Europe and the Mediterranean region. It is used as a spice medicine and fresh vegetable. Fennel nsLTP1 was purified using the combination of gel filtration and reverse-phase high-performance liquid chromatography (RP-HPLC). Its homogeneity was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. The purified nsLTP1 was treated with 4-vinyl pyridine, and the modified protein was then digested with trypsin. The complete amino acid sequence of nsLTP1 established by intact protein sequence up to 28 residues, overlapping tryptic peptides, and cyanogen bromide (CNBr) peptides. Hence, it is confirmed that fennel nsLTP1 is a 9433 Da single polypeptide chain consisting of 91 amino acids with eight conserved cysteines. Moreover, the 3D structure is predicted to have four α-helices interlinked by three loops and a long C-terminal tail. The lipid-binding property of fennel nsLTP1 is examined in vitro using fluorescent 2-p-toluidinonaphthalene-6-sulfonate (TNS) and validated using a molecular docking study with AutoDock Vina. Both of the binding studies confirmed the order of binding efficiency among the four studied fatty acids linoleic acid > linolenic acid > Stearic acid > Palmitic acid. A preliminary screening of fennel nsLTP1 suppressed the growth of MCF-7 human breast cancer cells in a dose-dependent manner with an IC50 value of 6.98 µM after 48 h treatment.

Identification of GIMAP7 and Rabl3 as Putative Biomarkers for Oral Squamous Cell Carcinoma Through Comparative Proteomic Approach.

Oral squamous cell carcinoma (OSCC) accounts for more than 90% of all oral cancers and has been listed as sixth most common human cancer. Due to late diagnosis and insufficient therapeutic response among patients, the survival rate remains very low accentuating the importance of early diagnostic markers. The study aimed to identify differentially expressed proteins in search for putative serum biomarkers and drug targets. Serum samples (n = 45) were depleted and resolved on two dimensional gel electrophoresis. Among differentially expressed proteins, two were identified using MALDI-TOF mass spectrometry. Gene expression levels of identified proteins were quantified in malignant and normal tissue using RT-qPCR. To validate serum Rabl3 expression, sandwich ELISA was performed. Proteomics analysis revealed two proteins which were found to be associated with oral cancer. The expression of GIMAP7 was found to be down regulated in serum of patients suffering from oral cancer while the expression of Rabl3 was found to be up-regulated. Gene expression analysis in malignant tissue and adjacent normal tissue revealed the same pattern. Quantitative ELISA was used to validate expression of Rabl3 in serum from oral cancer patients and healthy subjects which demonstrated significant up-regulation in cancer patients. Findings in current study demonstrate differential expression of novel putative biomarkers GIMAP7 and Rabl3 in oral cancer which suggests their potential role in oral cancer pathology and can be considered as predictive biomarkers.

Identification of Differentially Expressed Proteins from Smokeless Tobacco Addicted Patients Suffering from Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is the eight most common malignancy worldwide with an incidence rate of 40% in south-east Asia. Lack of effective diagnostic tools at early stage and disease recurrence despite extensive treatments are main reasons for high mortality and low survival rates. The aim of current study was to identify differentially expressed proteins to explore potential candidate biomarkers having diagnostic significance. We performed comparative proteomic analysis of paired protein samples (cancerous buccal mucosa and adjacent normal tissue) from OSCC patients using a combination of two dimensional gel electrophoresis and Mass spectrometric analysis. On the basis of spot intensity, seventeen proteins were found to be consistently differentially expressed among most of the samples which were identified through mass spectrometry. For validation of identified proteins, expression level of stratifin was determined using immuno-histochemistry and Western blot analysis. All identified proteins were analyzed by STRING to explore their interaction. Among uniquely identified proteins in this study, at least two candidate markers (Ig Kappa chain C region and Isoform 2 of fructose bisphosphate aldolase A) were found to be novel with respect to OSCC which can be explored further. Results presented in current study are likely to contribute in understanding the involvement of these molecules in carcinogenesis apart from their plausible role as diagnostic/prognostic markers.