Transcript-Level In Silico Analysis of Alzheimer's Disease-Related Gene Biomarkers and Their Evaluation with Bioactive Flavonoids to Explore Therapeutic Interactions.

Alzheimer's disease (AD) is a progressive brain disorder that can significantly affect the quality of life. We used a variety of in silico tools to investigate the transcript-level mutational impact of exonic missense rare variations (single nucleotide polymorphisms, SNPs) on protein function and to identify potential druggable protein cavities that correspond to potential therapeutic targets for the management of AD. According to the NIA-AA (National Institute on Aging-Alzheimer's Association) framework, we selected three AD biomarker genes (APP, NEFL, and MAPT). We systematically screened transcript-level exonic rare SNPs from these genes with a minor allele frequency of 1% in 1KGD (1000 Genomes Project Database) and gnomAD (Genome Aggregation Database). With downstream functional effect predictions, a single variation (rs182024939: K > N) of the MAPT gene with nine transcript SNPs was identified as the most pathogenic variation from the large dataset of mutations. The machine learning consensus classifier predictor categorized these transcript-level SNPs as the most deleterious variations, resulting in a large decrease in protein structural stability (ΔΔG kcal/mol). The bioactive flavonoid library was screened for drug-likeness and toxicity risk. Virtual screening of eligible flavonoids was performed using the MAPT protein. Identification of druggable protein-binding cavities showed VAL305, GLU655, and LYS657 as consensus-interacting residues present in the MAPT-docked top-ranked flavonoid compounds. The MM/PB(GB)SA analysis indicated hesperetin (-5.64 kcal/mol), eriodictyol (-5.63 kcal/mol), and sakuranetin (-5.60 kcal/mol) as the best docked flavonoids with the near-native binding pose. The findings of this study provide important insights into the potential of hesperetin as a promising flavonoid that can be utilized for further rational drug design and lead optimization to open new gateways in the field of AD therapeutics.

Comparison Of Potential Plasma Biomarkers In Alzheimer's Disease And Neurodegenerative Dementias In Pakistani Population.

This study aimed to compare the mean plasma levels of Amyloid ß42, Phosphorylated Tau and Neurofilament Light chain in patients diagnosed with Alzheimer's Clinical Syndrome (ACS), and other neurodegenerative dementias to find affordable and less-invasive means of diagnosing Alzheimer's disease (AD) early in its course. Blood samples of 36 subjects presenting with cognitive decline to the neurology OPDs of Dow and Civil hospitals, Karachi, were centrifuged, and plasma was stored at -80℃. Before analysis, it was thawed at 4℃ and protein levels were measured through ELISA. Two-thirds of the patients were females but age distribution across both the groups was not significantly different (p=0.21). No difference was observed in the mean plasma concentrations of Aß42, P-Tau, and NFL between the two groups (p-values 0.78 and 0.27 and 0.09 respectively). Our study suggests that despite being promising in CSF, Aß42, P-Tau, and NFL cannot differentiate between different neurodegenerative dementias when measured in plasma.

Plasma levels of phosphorylated tau and neurofilament light chain as potential biomarkers for Alzheimer's disease: A biochemical analysis in Pakistani population.

The National Institute on Aging-Alzheimer's Association's research framework in 2018 proposed a molecular construct for the diagnosis of Alzheimer's disease (AD). Nonetheless, the clinical exclusionary strategy is still the mainstay of AD diagnosis in Pakistan. We looked at the plasma levels of amyloid beta-42 (Aß-42), phosphorylated tau (P-tau), and neurofilament light (NFL) in patients with Alzheimer's clinical syndrome (ACS) and healthy controls (HC) from the Pakistani population to keep pace with the global efforts towards establishing accessible and affordable biochemical diagnostic markers for AD in Pakistan. Consultant neurologists screened patients who presented with cognitive impairment to three large tertiary care hospitals in Karachi, and after receiving informed consent, recruited participants with ACS and HC from the same facilities. We collected 5cc of blood in EDTA tubes along with demographic and lifestyle information of the subjects. Plasma aliquots were stored at -80°C after centrifugation. For analysis it was thawed at 4℃ and levels of the three proteins were measured through ELISA. Data from 28 ACS patients and 28 age matched healthy controls were evaluated. Among demographic factors, education and depression were related with health status (p = 0.03 and 0.003, respectively). NFL and P-tau mean values demonstrated a significant difference between the ACS and control groups (p = 0.003 and 0.006), however Aß42 did not (p = 0.114). ROC analysis showed that plasma P-tau and NFL, with AUCs of 0.717 and 0.735, respectively, could substantially distinguish ACS from the HC group (p = 0.007 and 0.003, respectively). Both plasma P-tau (r = -0.389; p = 0.004) and NFL (r = -0.424; p = 0.001) levels were significantly and negatively correlated with individuals' MMSE scores. NFL and plasma P-tau show promise in differentiating AD patients from healthy individuals. However, similar larger studies are needed to validate our findings.

Analytical Study Of Salivary Mmp-12 Expression In Oral Submucous Fibrosis.

BACKGROUND: Oral submucous fibrosis (OSF) is majorly a pathology pertaining to Asian population, reported most in Pakistan, India, Nepal, Bangladesh, and Taiwan. Equilibrium existing between Matrix metalloproteinases (MMP) & tissue inhibitors of matrix metalloproteinases is imperative for the normal integrity of connective tissues. However, this mechanism is disturbed in the presence of OSF, resulting in an increase in the extracellular matrix. METHODS: It is an analytical study including two groups with a total of 60 participants. The first group consists of 30 healthy participants and the other group consists of 30 patients presenting with oral submucous fibrosis. Collected samples of saliva were stored at -80 °C after centrifugation. For ELISA investigation, the procedure was performed as per manufacturer's instruction. Salivary matrix metalloproteinases- 12 concentration was estimated with the help of a standard curve. Data was analysed using SPSS 23. Mann Whitney test was applied to determine the difference existing in Matrix metalloproteinases- 12 levels between healthy and oral submucous fibrosis participants. p-value <0.05 was contemplated as significant. RESULTS: Statistical investigation indicated significant difference in Matrix metalloproteinases- 12 levels between Oral submucous fibrosis and healthy group (p<0.05). Saliva samples obtained from oral submucous fibrosis patients demonstrated raised concentrations of Matrix metalloproteinases- 12 as compared to healthy participants. CONCLUSIONS: Our study demonstrates significant upsurge in Matrix metalloproteinases- 12 expression in samples of saliva obtained from oral submucous fibrosis patients as compared to healthy individuals. Therefore, salivary Matrix metalloproteinases- 12 could serve as a useful diagnostic marker for OSF.

Diclofenac sodium enhances the antiepileptic effect of levetiracetam in pilocarpine induced epileptic mice model.

Epilepsy, a neuronal disorder has affected 1% of the world's population. Almost 35-40% of these patients get resistant to available anti-epileptic drugs (AEDs). Recent studies have shown the role of inflammation in the pathophysiology of epilepsy and a combination of anti-inflammatory and antiepileptic drugs could prove beneficial against epileptic seizures. Therefore, we aimed to examine the effect of levetiracetam (LEV) and diclofenac sodium (DFS) combination on pilocarpine (PLC) induced epileptic seizures in mice. Mice were divided into control and treatment groups. LEV alone and in combination with DFS was given for 3 days. On 3rd day after administering the required drugs, pilocarpine challenge was given intraperitoneally. Then, behavioral changes were observed for 90 minutes, including latency to first seizure, continuous seizures, duration of continuous seizures, and survival rate. Results showed significant improvement in the latencies to first (P<0.001) and continuous seizures (P<0.05), duration of the continuous seizure (p=0.001), and survival rate (P<0.01) in the combination treatment group as compared to the control or individual drug treatment groups. DFS enhances the efficacy of LEV, however, further mechanistic studies will be required to conclude if DFS can be given in combination with LEV for epilepsy treatment.

Association Of Subchorionic Hematoma With Preterm Labour In Patients With Threatened Abortion.

BACKGROUND: One of the frequent obstetric complications in first trimester is vaginal bleeding. It has an incidence of 16%-25%. Subchorionic haemorrhage is the most common cause of first trimester miscarriage associated with vaginal bleeding. The objective of the study was to determine the association of subchorionic hematoma (SCH) with preterm labour in pregnant females with threatened abortion. METHODS: A Cohort Study was designed and conducted in the Obstetrics and Gynaecology Department, Zanana hospital, Dera Ismail Khan (D.I. Khan). A sample size of 418 subjects was equally divided into two equal groups. Non-probability consecutive sampling was used for collection of samples. Pregnant women presenting with sub chorionic hematoma were considered as cases while pregnant women without sub chorionic hematoma were considered as controls. The diagnosis of sub chorionic haemorrhage was established by ultrasound. Data was analysed by SPSS version 22.0 for description, i.e., frequencies and percentages were calculated for categorical variables and for continuous variables, mean±standard deviation (±SD) was calculated. To compare preterm labour in both groups Chi-square test was applied and p≤0.05 was taken as significant. Stratification was done with regard to age, gestational age, parity, history of hypertension, and weight to see the effect of these variables on preterm labour. Chi-square test was used for post stratification for both groups and p ≤ 0.05 was considered statistically significant and relative risk was calculated. RESULTS: The age range was from 18-40 years in group A with a mean age of 29.08±2.88 years while it was 28.41±2.94 years in group B. Mean gestational age was 13.99±3.15 weeks in group A and 11.42±3.37 weeks in group B. Mean parity was 0.91±1.14 in group A and 0.78±1.09 in group B respectively. Mean weight of subjects in group A was 68.31±10.27 and 67.55±10.09 Kg in group B. Majority of the patients were of 18-30 years in both groups (group A; 78.5% and group B; 78.9%). History of hypertension was noted in 33% in group A and 12.9% in group B. In group A, preterm labour was seen in 40 (19.1%) patients as compared to 61 (29.2%) patients in group B, (p=0.02, R.R=0.65). CONCLUSIONS: Subchorionic hematoma in patients with threatened abortion during the first half of the pregnancy may not increase the risk of preterm labour.

Estimation of salivary matrix metalloproteinases- 12 (MMP- 12) levels among patients presenting with oral submucous fibrosis and oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma is a global threat and accounts for approximately 90% of malignant oral lesions. The emergence of oral carcinoma is linked to precancerous lesions, which act as precursors of the disease. Matrix metalloproteinases appear to play a significant role in the pathogenesis of both precancerous conditions and oral malignancies due to their participation in remodeling of the extracellular matrix. METHODOLOGY: This is an analytical study conducted at Dow University of Health Sciences, Pakistan. Unstimulated saliva samples were collected from healthy, oral submucous fibrosis and oral squamous cell carcinoma patients. The level of MMP-12 was estimated using enzyme-linked immunosorbent assay. One-way Analysis of variance was run to determine if MMP-12 levels differ between the three groups, which was preceded by post hoc Tuckey test. MMP-12 cut off values were determined using Receiver operating characteristic curve. RESULTS: A significant difference in salivary MMP-12 expression was observed in OSF and OSCC (p < 0.001). The expression of salivary MMP-12 was higher in OSF and OSCC patients as compared to the healthy group (p < 0.001). The mean MMP-12 expression in OSCC appeared higher than in OSF cases (p < 0.05). MMP-12 value of [Formula: see text] 4.05 ng/ml and [Formula: see text] 4.20 ng/ml is predictive of OSF and OSCC respectively, with 100% sensitivity and specificity (p < 0.001). CONCLUSION: Increased expression of MMP-12 appears as the healthy patient advances to OSF and OSCC. The study results also demonstrate higher MMP-12 expression in OSCC patients as compared to OSF. Therefore, the estimation of salivary MMP-12 serves as a valuable non-invasive early diagnostic tool in diagnosing oral submucous fibrosis and oral squamous cell carcinoma.

Association Of Risk Factors With Spontaneous Preterm Labour With Intact Foetal Membranes.

BACKGROUND: Spontaneous preterm labour refers to the onset of uterine contractions of sufficient strength and frequency to effect progressive dilatation between 24 and 37 weeks of gestation. The objective of the study was to determine an association between risk factors and spontaneous preterm labour with intact foetal membranes. METHODS: A case control study was designed which was conducted in the Department of Obstetrics and Gynaecology, Ayub Teaching Hospital, Abbottabad. Sample size of 262 subjects was equally divided into two equal groups (131 cases and control each). Non-probability consecutive sampling was used collection of samples. Pregnant women presenting after 24 weeks of gestation with spontaneous preterm labour with intact membranes were considered as cases. While, pregnant women presenting after 37 complete weeks of gestation with normal pregnancy were considered as controls. The diagnosis of bacterial vaginosis was established by Amsel criteria. Asymptomatic bacteriuria was diagnosed by microscopy. Odds ratios were calculated as measure of association with calculation at 95% confidence interval. Level of 5% (p<0.05) was used to test for significant difference. RESULTS: Mean±SD age of cases was 26.97±7.072. Subjects with young maternal age (<20 years) were 27 (10.3%) from cases and 12 (4.6%) from controls (OR=2.575, 95% CI: 1.242, 5.338). The subjects with advanced maternal age (>35 years) were 26 (9.9%) from cases and 15 (5.7%) from controls (OR=0.522, 95% CI: 0.262, 1.039). 25 (19.1%) cases and 6 (4.6%) controls had bacterial vaginosis (OR=4.914, 95% CI: 1.943, 12.426). Asymptomatic bacteriuria was present in 03 (2.3%) subjects from cases and 2 (1.5%) from controls (OR=1.512, 95% CI: 0.248, 9.199). CONCLUSIONS: The study concluded that risk factors like young maternal age and bacterial vaginosis are significantly associated with spontaneous preterm labour with intact foetal membranes. While, association of other risk factors like advanced maternal age and asymptomatic bacteriuria with spontaneous preterm labour is still debatable.

Human papilloma virus 16/18: Fabricator of trouble in oral squamous cell carcinoma.

AIM: To find out the association between Human Papilloma Virus (HPV) genotypes 16/18 in Pakistani patients with oral squamous cell carcinoma (OSCC). METHODS: DNA from oral rinse of 300 subjects was taken. The subjects included 100 cases with OSCC and 200 controls. Samples were analyzed by both conventional and real time PCR using "HPV consensus Gp5+/Gp6+ and HPV 16, 18 specific primers". RESULTS: Out of 300 persons, 74/300 (25%) were found to be infected with HPV: "46/100(46%) from cases and 74/200(14%) from controls". The distribution was: HPV16, 6/300 (8%): 4/100 (9%) from OSCC group and 2/200 (8%) from controls while HPV 18 was 9/300(12%): 5/100(11%) from cases and 4/200(16%) from controls. Out of 300 subjects, 26(35%) were infected by "both HPV 16/18 (23(50%) from cases and 3(12%) from controls". Persons who were infected with HPV 16&18 had higher chances to develop OSCC as compared to those who didn't have HPV 16/18 (AOR: 21.4, 95% CI: 5.73 - 80.8). CONCLUSION: The exposure to high risk strains of Human papilloma virus (16/18) in combination can be fabricotor of trouble (p<0.001, Adjusted odds ratio; 21.42) in OSCC.

Protein Profiling of Non-model Plant Cuminum cyminum by Gel-Based Proteomic Approach.

INTRODUCTION: Cumin (Cuminum cyminum), a popular spice has been widely used in traditional medicine to cure various ailments. Despite the existence of scientific literature about its pharmacological properties, no successful proteome profiling has yet been attempted. OBJECTIVE: To optimise extraction of cumin proteins and analyse its profile by shotgun proteomics, using one-dimensional electrophoresis coupled with nano-ESI-LC-MS/MS. METHODOLOGY: As a first step, we have compared three extraction protocols for total proteins extraction from cumin. Extracted proteins were separated on one-dimensional gel and analysed by state-of-the-art linear ion trap (LTQ)-Orbitrap Velose and Q Exactive HF mass spectrometer. RESULTS: Evaluation of extraction method revealed significant differences in protein yield and proteome composition between the three extracts. LC-MS/MS allowed identification of several proteins with functional significance in various biological processes. CONCLUSION: This study provides identification of a large number of proteins and offers a molecular basis for future research on potential pharmacologically active cumin proteins. Copyright © 2017 John Wiley & Sons, Ltd.

Dithiothreitol (DTT) Acts as a Specific, UV-inducible Cross-linker in Elucidation of Protein-RNA Interactions.

Protein-RNA cross-linking by UV irradiation at 254 nm wavelength has been established as an unbiased method to identify proteins in direct contact with RNA, and has been successfully applied to investigate the spatial arrangement of protein and RNA in large macromolecular assemblies, e.g. ribonucleoprotein-complex particles (RNPs). The mass spectrometric analysis of such peptide-RNA cross-links provides high resolution structural data to the point of mapping protein-RNA interactions to specific peptides or even amino acids. However, the approach suffers from the low yield of cross-linking products, which can be addressed by improving enrichment and analysis methods. In the present article, we introduce dithiothreitol (DTT) as a potent protein-RNA cross-linker. In order to evaluate the efficiency and specificity of DTT, we used two systems, a small synthetic peptide from smB protein incubated with U1 snRNA oligonucleotide and native ribonucleoprotein complexes from S. cerevisiae. Our results unambiguously show that DTT covalently participates in cysteine-uracil crosslinks, which is observable as a mass increment of 151.9966 Da (C(4)H(8)S(2)O(2)) upon mass spectrometric analysis. DTT presents advantages for cross-linking of cysteine containing regions of proteins. This is evidenced by comparison to experiments where (tris(2-carboxyethyl)phosphine) is used as reducing agent, and significantly less cross-links encompassing cysteine residues are found. We further propose insertion of DTT between the cysteine and uracil reactive sites as the most probable structure of the cross-linking products.

Isolation, purification and characterization of a nonspecific lipid transfer protein from Cuminum cyminum.

Cuminum cyminum, an aromatic plant from the family Umbelliferae, is used as a flavoring and seasoning agent in foods. This communication reports the characterization of a nonspecific lipid transfer protein nsLTP1 from its seeds. Plant nsLTPs are small basic proteins involved in transport of lipids between membranes. These proteins are known to participate in plant defense; however, the exact mechanism of their antimicrobial action against fungi or bacteria is still unclear. The cumin nsLTP1 has been purified using a combination of chromatographic procedures and further characterized using mass spectrometry, circular dichroism spectroscopy and Edman degradation. Amino acid sequence has been used to predict homology model of cumin nsLTP1 in complex with myristic acid, and lyso-myristoyl phosphatidyl choline (LMPC). Cumin nsLTP1 is a monomeric protein with a molecular weight of 9.7 kDa as estimated by SDS-PAGE and ESIMS. The protein shows an isoelectric point of 7.8 on 6% PAGE. The primary structure consists of 92 amino acids with eight conserved cysteine residues. The global fold of cumin nsLTP1 includes four alpha-helices stabilized by four disulfide bonds and a C-terminal tail. The role of internal hydrophobic cavity of the protein in lipid transfer is discussed.

Impeded electron transfer from a pathogenic FMN domain mutant of methionine synthase reductase and its responsiveness to flavin supplementation.

Methionine synthase reductase (MSR) is a diflavin oxidoreductase that transfers electrons from NADPH to oxidized cobalamin and plays a vital role in repairing inactive cobalamin-dependent methionine synthase. MSR deficiency is a recessive genetic disorder affecting folate and methionine metabolism and is characterized by elevated levels of plasma homocysteine. In this study, we have examined the molecular basis of MSR dysfunction associated with a patient mutation, A129T, which is housed in the FMN binding domain and is adjacent to a cluster of conserved acidic residues found in diflavin oxidoreductases. We show that the substitution of alanine with threonine destabilizes FMN binding without affecting the NADPH coenzyme specificity or affinity, indicating that the mutation's effects may be confined to the FMN module. The A129T MSR mutant transfers electrons to ferricyanide as efficiently as wild type MSR but the rate of cytochrome c, 2,6-dichloroindophenol, and menadione reduction is decreased 10-15 fold. The mutant is depleted in FMN and reactivates methionine synthase with 8% of the efficiency of wild type MSR. Reconstitution of A129T MSR with FMN partially restores its ability to reduce cytochrome c and to reactivate methionine synthase. Hydrogen-deuterium exchange mass spectrometric studies localize changes in backbone amide exchange rates to peptides in the FMN-binding domain. Together, our results reveal that the primary biochemical penalty associated with the A129T MSR mutant is its lower FMN content, provide insights into the distinct roles of the FAD and FMN centers in human MSR for delivering electrons to various electron acceptors, and suggest that patients harboring the A129T mutation may be responsive to riboflavin therapy.