Paper-Based Flexible Nanoparticle Hybrid Substrate for Qualitative and Quantitative Analysis of Melamine in Powder Milk by SERS.

Melamine is a chemical compound that is added to dairy products to increase the apparent protein content for higher profit margins. However, extended consumption of melamine can cause health risks. The SERS technique has proven to be an important tool for detecting small compounds, such as melamine. Here, a paper-based flexible nanoparticles (NPs)-hybrid SERS substrate was designed by drop-casting pegylated gold nanoparticles (AuNPs) on the filter papers. In SERS characterization, this substrate exhibited an enhancement factor of 108 and a limit of detection (LOD) as low as 10-8 M for Rhodamine 6G dye. Furthermore, we successfully utilized these substrates to detect the melamine spiked milk sample with an LOD as low as 0.01 ppm. This hybrid SERS substrate offers a low-cost, biocompatible, and easy-to-use fabrication for large-scale production, which may be widely used in food safety applications.

Exploring structural antigens of yellow fever virus to design multi-epitope subunit vaccine candidate by utilizing an immuno-informatics approach.

BACKGROUND: Yellow fever is a mosquito-borne viral hemorrhagic disease transmitted by several species of virus-infected mosquitoes endemic to tropical regions of Central and South America and Africa. Earlier in the twentieth century, mass vaccination integrated with mosquito control was implemented to eradicate the yellow fever virus. However, regular outbreaks occur in these regions which pose a threat to travelers and residents of Africa and South America. There is no specific antiviral therapy, but there can be an effective peptide-based vaccine candidate to combat infection caused by the virus. Therefore, the study aims to design a multi-epitope-based subunit vaccine (MESV) construct against the yellow fever virus to reduce the time and cost using reverse vaccinology (RV) approach. METHODS: Yellow fever virus contains 10,233 nucleotides that encode for 10 proteins (C, prM, E, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) including 3 structural and 7 non-structural proteins. Structural proteins-precursor membrane protein (prM) and envelope protein (E)-were taken as a target for B cell and T cell epitope screening. Further, various immunoinformatics approaches were employed to FASTA sequences of structural proteins to retrieve B cell and T cell epitopes. MESV was constructed from these epitopes based on allergenicity, antigenicity and immunogenicity, toxicity, conservancy, and population coverage followed by structure prediction. The efficacy of the MESV construct to bind with human TLR-3, TLR-4, and TLR-8 were evaluated using molecular docking and simulation studies. Finally, in-silico cloning of vaccine construct was performed withpBR322 Escherichia coli expression system using codon optimization. RESULTS: Predicted epitopes evaluated and selected for MESV construction were found stable, non-allergenic, highly antigenic, and global population coverage of 68.03% according to in-silico analysis. However, this can be further tested in in-vitro and in-vivo investigations. Epitopes were sequentially merged to construct a MESV consisting of 393 amino acids using adjuvant and linkers. Molecular docking and simulation studies revealed stable and high-affinity interactions. Furthermore, in-silico immune response graphs showed effective immune response generation. Finally, higher CAI value ensured high gene expression of vaccine in the host cell. CONCLUSION: The designed MESV construct in the present in-silico study can be effective in generating an immune response against the yellow fever virus. Therefore, to prevent yellow fever, it can be an effective vaccine candidate. However, further downstream, in-vitro study is required.

Development of pharmacophore model to identify potential DNA gyrase inhibitors.

There is great concern in the medical community due to rapid increase in antibiotic resistance, causing 700,000 deaths annually worldwide. Therefore, there is paramount need to develop novel and innovative antibacterial agents active against resistant bacterial strains. DNA gyrase is a crucial enzyme in bacterial replication that is absent in eukaryotes, making it effective curative target for antibacterials. To identify potential DNA gyrase inhibitors by virtual screening of NCI database using a 3-step approach. A total of 271 compounds with known IC50 values against Escherichia coli DNA GyrA were selected to develop a pharmacophore model for dual screening approach to identify new potential hits from the NCI database. In the second step, the NCI database was also screened using in-house built NN-QSAR model. Molecular docking of common 5298 compounds screened from both methods were performed against E. coli DNA GyrA (PDB id- 6RKU), and 3004 compounds are reported to exhibit lower binding energies than ciprofloxacin (-6.77 Kcal/mol). The top three compounds (NCI371878, NCI371876 and NCI142159) reported with binding energy of -13.5, -13.19 and -13.03 Kcal/mol were further subjected to MD simulation studies for 100 ns supporting the stability of the docked complexes.

A computational approach to discover antioxidant and anti-inflammatory attributes of silymarin derived from Silybum marianum by comparison with hydroxytyrosol.

Medicinal plants possess therapeutic potential for reducing reactive oxygen species (ROS)-mediated cellular damage. Hydroxytyrosol is one of the most potent antioxidants that served as control in the current study, including other synthetic antioxidants to computationally identify the antioxidant properties of Silymarin. The sequences of the receptors IκB kinase (IKK), Kelch-like ECH-associated protein 1 (Keap-1) and mitochondrial transcription factor A (Tfam) were retrieved from UniProtKB and homology modeling was performed using Swiss-Model server. Thereof the molecular docking and dynamic simulation studies were performed using Schrödinger's software version 11.5. From the current study, it was reported that on comparison of the binding energy of silymarin, hydroxytyrosol, α-tocopherol, ascorbic acid, butylated hydroxy anisole (BHA) and butylated hydroxytoluene (BHT), Silymarin exhibited better affinities with IKK receptor followed by Hydroxytyrosol suggesting it as the best or comparable of all other known antioxidants that could potentially suppress inflammation and other diseases. Also, Silymarin exhibited poorest binding affinity with Tfam promoting mitochondrial biogenesis, thereby scavenging ROS. However, with Keap-1, Silymarin is ranked 4th in the list, whereas hydroxytyrosol exhibited highest binding affinity to release oxidative stress. The stability of docked complexes made us conclude that Silymarin has comparable antioxidant properties to hydroxytyrosol, better anti-inflammatory potential and mitochondrial biogenesis enhancing properties to ultimately reduce oxidative stress. Now it can be tested further for in vitro or in vivo studies as potential drug against oxidative insult.Communicated by Ramaswamy H. Sarma.

Comparative genome analysis of Streptococcus strains to identify virulent genes causing neonatal meningitis.

AIM: To determine Streptococcus agalactiae genes responsible for causing neonatal meningitis. BACKGROUND: Streptococcus agalactiae strain 2603 V/R is causative agent of neonatal meningitis, maternal infection and sepsis in young children. World health organisation reported high burden of new born death caused by this bacterium. Streptococcus agalactiae colonizing epithelial cells of vagina and endothelial cells have high resistance to available antibiotic drugs which makes it essential to determine new drug targets. OBJECTIVES: To compare the genome of selected strain with the non-pathogenic strains of streptococcus and identify the virulent and antibiotic resistant genes for adaptation in host environment. METHOD: The whole genome of human pathogen Streptococcus agalactiae strain 2603 V/R was analysed and compared with Streptococcus dysgalactiae strains using visualization and annotation tools. Genomic islands, mobile genetic elements, virulent and resistant genes were studied. RESULTS: Genetically pathogenic strain is most similar to Streptococcus dysgalactiae subsp. equisimilis strain NCTC 7136. Comparative analysis revealed the importance of capsular polysaccharides and surface proteins responsible for avoiding immune system attachment to host epithelial cells and virulent behaviour. High number of genes coding for antibiotics resistance may provide a competitive advantage for survival of pathogenic Streptococcus agalactiae strain 2603 V/R in its niche. CONCLUSIONS: The comparative analysis of pathogenic strain Streptococcus agalactiae with non-pathogenic strains of Streptococcus dysgalactiae provided new insights in pathogenicity that could aid in recognization for new regions and genes for development of new drug development strategies considering presence of high number of resistance genes.

Discovery of Novel Inhibitors of Bacterial DNA Gyrase Using a QSAR-Based Approach.

Type II topoisomerases like DNA gyrase initiate ATP-dependent negative supercoils in bacterial DNA. It is critical in all of the bacteria but is missing from eukaryotes, making it a striking target for antibacterials. Ciprofloxacin is a clinically approved drug, but its clinical effectiveness is affected by the emergence of resistance in both Gram-positive and Gram-negative bacteria. Thus, it is vital to identify novel compounds that can efficiently inhibit DNA gyrase, and quantitative structure-activity relationship (QSAR) modeling is a quick and economical means to do so. A QSAR-based virtual screening approach was applied to identify new gyrase inhibitors using an in-house-generated combinatorial library of 29828 compounds from seven ciprofloxacin scaffold structures. QSAR was built using a data set of 271 compounds, which were identified as positive and negative inhibitors from existing data reported in in vitro studies. The best QSAR model was developed using the 5-fold cross-validation Neural Network in Orange, and it was based on five PaDEL descriptors with an accuracy and sensitivity of 83%. As a result of screening of an in-house-built combinatorial library with the best-developed QSAR model, 675 compounds were identified as potential inhibitors of DNA gyrase. These inhibitors were further docked with DNA gyrase using AutoDock to compare the binding mode and score of the selected/screened compounds, and 615 compounds exhibited a docking score comparable to or lower than that of ciprofloxacin. Out of these, the top five analogues 902b, 9699f, 4419f, 5538f, and 898b reported in our study have binding scores of -13.81, -12.95, -12.52, -12.43, and -12.41 kcal/mol, respectively. The MD simulations of these five analogues for 100 ns supported the interaction stability of analogues with Escherichia coli DNA gyrase. Ninety-one per cent of the analogues screened by the QSAR model displayed better binding energy than ciprofloxacin, demonstrating the efficacy of the generated model. The NN-QSAR model proposed in this manuscript can be downloaded from https://github.com/ritu225/NN-QSAR_model.git.

Co-expression analysis identifies networks of miRNAs implicated in biological ageing and modulated by short-term interval training.

Exercise training seems to promote healthy biological ageing partly by inducing telomere maintenance, yet the molecular mechanisms are not fully understood. Recent studies have emphasised the importance of microRNAs (miRNAs) in ageing and their ability to mirror pathophysiological alterations associated with age-related diseases. We examined the association between aerobic fitness and leukocyte telomere length before determining the influence of vigorous exercise training on the regulation of leukocyte miRNA networks. Telomere length was positively correlated to aerobic fitness (r = 0.32, p = 0.02). 104 miRNAs were differentially expressed after six weeks of thrice-weekly sprint interval training (SIT) in healthy men (q < 0.05). Gene co-expression analysis (WGCNA) detected biologically meaningful miRNA networks, five of which were significantly correlated with pre-SIT and post-SIT expression profiles (p < 0.001) and telomere length. Enrichment analysis revealed that the immune response, T cell differentiation and lipid metabolism associated miRNAs clusters were significantly down-regulated after SIT. Using data acquired from the Gene Expression Omnibus (GEO), we also identified two co-expressed miRNAs families that were modulated by exercise training in previous investigations. Collectively, our findings highlight the miRNA networks implicated in exercise adaptations and telomere regulation, and suggest that SIT may attenuate biological ageing through the control of the let-7 and miR-320 miRNA families.

Fibrolipomatous Hamartoma of Median Nerve - A Diagnostic Challenge.

Introduction: Fibrolipomatous hamartoma (FLH) is an uncommon slow-growing tumor of benign etiology, which predominantly affects the median nerve. Case Report: We report the case study of a 17-year-old male patient, who presented with complaints of a gradually increasing localized swelling over the volar aspect of left hand for 1 year. A contrast-enhanced Magnetic resonance (MR) scan of the left hand was performed which demonstrated characteristic findings. The patient was treated surgically and post-excision histopathological examination confirmed the diagnosis. Conclusion: The characteristic MR imaging features of coaxial cable-like appearance on axial section or spaghetti shaped enlarged nerve fascicles and fibrous tissue is confirmatory for a definitive diagnosis of FLH, which alleviates the need for any unnecessary biopsy.

Neuroimmune Mechanisms in Signaling of Pain During Acute Kidney Injury (AKI).

Acute kidney injury (AKI) is a significant global health concern. The primary causes of AKI include ischemia, sepsis and nephrotoxicity. The unraveled interface between nervous system and immune response with specific focus on pain pathways is generating a huge interest in reference to AKI. The nervous system though static executes functions by nerve fibers throughout the body. Neuronal peptides released by nerves effect the immune response to mediate the hemodynamic system critical to the functioning of kidney. Pain is the outcome of cellular cross talk between nervous and immune systems. The widespread release of neuropeptides, neurotransmitters and immune cells contribute to bidirectional neuroimmune cross talks for pain manifestation. Recently, we have reported pain pathway genes that may pave the way to better understand such processes during AKI. An auxiliary understanding of the functions and communications in these systems will lead to novel approaches in pain management and treatment through the pathological state, specifically during acute kidney injury.

High specificity and tight spatial restriction of self-biotinylation by DNA and RNA G-Quadruplexes complexed in vitro and in vivo with Heme.

Guanine-rich, single-stranded DNAs and RNAs that fold to G-quadruplexes (GQs) are able to complex tightly with heme and display strongly enhanced peroxidase activity. Phenolic compounds are particularly good substrates for these oxidative DNAzymes and ribozymes; we recently showed that the use of biotin-tyramide as substrate can lead to efficient GQ self-biotinylation. Such biotinylated GQs are amenable to polymerase chain reaction amplification and should be useful for a relatively non-perturbative investigation of GQs as well as GQ-heme complexes within living cells. Here, we report that in mixed solutions of GQ and duplex DNA in vitro, GQ biotinylation is specifically >104-fold that of the duplex, even in highly concentrated DNA gels; that a three-quartet GQ is tagged by up to four biotins, whose attachment occurs more or less uniformly along the GQ but doesn't extend significantly into a duplex appended to the GQ. This self-biotinylation can be modulated or even abolished in the presence of strong GQ ligands that compete with heme. Finally, we report strong evidence for the successful use of this methodology for labeling DNA and RNA within live, freshly dissected Drosophila larval salivary glands.

An Economical Method of Auricular Splinting in Management of Auricular Pseudocyst.

BACKGROUND: Pseudocyst of the auricle is a common benign disease. Many treatment modalities have been described for this benign condition ranging from simple aspiration to complex cutaneous surgeries involving skin de-roofing and debridement with diamond burr. the aim of treatment is to successfully resolve the seroma without damaging the underlying healthy cartilage, thus maintaining the normal contour of the auricle, and to prevent its recurrence. METHODS: In this study we describe incision and drainage of the pseudocyst with auricular splinting. RESULTS: Resolution was seen in 100.00 %, skin discolouration in 33.33%, skin thickening in 29.63% and deformity in 25.93% of the patients. CONCLUSION: The use of corrugated drain sheet splint is an ingenious method of aural pseudocyst management. This method is simple and can be performed by even less experienced surgeons and highly economical which prevents the recurrence and maintains the auricular aesthetics.

Nucleophosmin mutation analysis in acute myeloid leukaemia: Immunohistochemistry as a surrogate for molecular techniques.

BACKGROUND & OBJECTIVES: Mutation of nucleophosmin (NPM1) gene in the absence of FLT3-ITD (FMS related tyrosine kinase 3 - internal tandem duplications) mutation carries a good prognosis in cytogenetically normal acute myeloid leukaemia (AML). NPM1, a multifunctional nucleolar phosphoprotein that shuttles between nucleus and cytoplasm, gets trapped in the cytoplasm when mutated. Immunohistochemical (IHC) demonstration of its aberrant cytoplasmic location (NPMc+) has been suggested as a simple substitute for the standard screening molecular method. This study was aimed to assess the diagnostic utility of IHC on formalin fixed bone marrow biopsies in comparison with the reference molecular method (allele specific oligonucleotide - polymerase chain reaction; ASO-PCR) to predict NPM1 mutation status in AML patients. METHODS: NPM protein IHC was performed using mouse anti-NPM monoclonal antibody on 35 paraffin-embedded bone marrow biopsies of patients with primary AML of any French-American-British (FAB) subtype. Results of IHC were compared with those of ASO-PCR. RESULTS: Of the 35 AML patients, 21 (60%) were positive for NPM1 exon 12 gene mutation by ASO-PCR, 19 (90.47%) of these 21 were NPMc+. Thirteen of the 35 patients were negative by both the methods. One NPMc+ patient was not detected by ASO-PCR. IHC had a sensitivity and specificity of 90 and 93 per cent, respectively, compared to the molecular screening gold standard. INTERPRETATION & CONCLUSIONS: Mutation of NPM1 determined by the widely available and inexpensive IHC agrees closely with results of the standard molecular methods. Thus, technically and financially not well endowed laboratories can provide the prognostically and potentially therapeutically important information on NPM1 mutation using IHC.

Prevalence of common fusion transcripts in acute lymphoblastic leukemia: A report of 304 cases.

AIM: Information about fusion transcripts in acute lymphoblastic leukemia (ALL) is used to risk-stratify patients, decide on the treatment and to detect minimal residual disease. This study was conducted to determine the frequency of common fusion transcripts BCR-ABL, TEL-AML1, MLL-AF4 and E2A-PBX1 for B-ALL and SIL-TAL1 for T-ALL as seen at a tertiary care center in India. METHODS: Up to 304 new cases of ALL (271 B-ALL and 33 T-ALL) diagnosed on morphology, cytochemistry and immunophenotyping were studied. All were screened for the common fusion transcripts by RT-PCR. RESULTS: Both our B- (218/271; 80.4%) and T-ALL (26/33; 78.8%) patients were largely children. In the B-ALL children, BCR-ABL was detected in 26/218 (11.9%), E2A-PBX1 in 13/218 (5.9%), TEL-AML1 in 16/218 (7.3%) and MLL-AF4 in 3/218 (1.4%) patients. Adult B-ALL cases had BCR-ABL in 15/53 (28.3%) and E2A-PBX in 2/53 (3.8%); however, no other fusion transcript was detected. SIL-TAL1 was found in four of 26 pediatric (15%) and zero of 7 adult T-ALL cases. CONCLUSION: The higher incidence of BCR-ABL and lower incidence of TEL-AML1 in our ALL patients, both in children and adults as compared with the West, suggests that patients in India may be biologically different. This difference may explain at least in part the higher relapse rate and poorer outcome in our B-ALL cases.

The restrained expression of NF-kB in renal tissue ameliorates folic acid induced acute kidney injury in mice.

The Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) represent family of structurally-related eukaryotic transcription factors which regulate diverse array of cellular processes including immunological responses, inflammation, apoptosis, growth & development. Increased expression of NF-kB has often been seen in many diverse diseases, suggesting the importance of genomic deregulation to disease pathophysiology. In the present study we focused on acute kidney injury (AKI), which remains one of the major risk factor showing a high rate of mortality and morbidity. The pathology associated with it, however, remains incompletely known though inflammation has been reported to be one of the major risk factor in the disease pathophysiology. The role of NF-kB thus seemed pertinent. In the present study we show that high dose of folic acid (FA) induced acute kidney injury (AKI) characterized by elevation in levels of blood urea nitrogen & serum creatinine together with extensive tubular necrosis, loss of brush border and marked reduction in mitochondria. One of the salient observations of this study was a coupled increase in the expression of renal, relA, NF-kB2, and p53 genes and proteins during folic acid induced AKI (FA AKI). Treatment of mice with NF-kB inhibitor, pyrrolidine dithio-carbamate ammonium (PDTC) lowered the expression of these transcription factors and ameliorated the aberrant renal function by decreasing serum creatinine levels. In conclusion, our results suggested that NF-kB plays a pivotal role in maintaining renal function that also involved regulating p53 levels during FA AKI.

A Time-motion Comparison of Itemized Treatment Costs in First and Second In Vitro Fertilization Attempts: A United Kingdom Fertility Centre Experience.

Objective: To assess the difference in cost between initial and second in vitro fertilization (IVF) cycles in the United Kingdom. Methods: This prospective time-motion analysis captured data on average time spent on 31 representative components of the IVF sequence as provided by clinical team members in seven categories. Audits of consumables and observations on personnel costs were made from total of 120 fertility patients undergoing initial or second IVF cycles (n=736) between 1 January 2002 and 31 December 2002 at a UK assisted fertility unit. Results: Patients spent an average of 16.71±4.3 hrs with staff during an initial IVF cycle, resulting in direct personnel costs of £577.05±151.01. When consumables were included, each initial cycle cost the clinic approximately £2246.57±151.01. For second IVF cycles, patients spent significantly less time with staff compared to their first IVF cycle (6.94±2.44 hrs; p<0.05), corresponding to £257.53±90.77 in personnel cost. Conclusions: This is the first economic appraisal of the IVF treatment sequence in the UK using a timemotion analysis model. Our study found that when combined with consumables, total institutional costs for second IVF cycles were significantly reduced when compared to initial cycles (£1813.12±90.77; p<0.05). Aggregating data from all IVF cycles performed within the fertility centre during the study interval, initial cycles were found to be front-loaded, resulting in £252,420 more in institutional costs as compared with subsequent IVF cycles. While these observations were registered in 2003, an inflation adjustment using recent European Commission Eurostat data for healthcare finds the difference between initial and subsequent fresh IVF cycles in present currency to be approximately £579.14 per cycle. Time-motion analysis can identify episodes of care that can be streamlined to improve outcomes and reduce cost.