Phylogenetic diversity and mutational analysis of New Delhi Metallo-β-lactamase (NDM) producing E. coli strains from pediatric patients in Pakistan

The evolution of NDM genes (bla_NDM) in E. coli is accounted for expansive multidrug resistance (MDR), causing severe infections and morbidities in the pediatric population. This study aimed to analyze the phylogeny and mutations in NDM variants of E. coli recovered from the pediatric population. Carbapenem-resistant clinical strains of E. coli were identified using microbiological phenotypic techniques. PCR technique used to amplify the bla_NDM genes, identified on agarose gel, and analyzed by DNA sequencing. The amino acid substitutions were examined for mutations after aligning with wild types. Mutational and phylogenetic analysis was performed using Lasergene, NCBI blastn, Clustal Omega, and MEGA software, whereas PHYRE2 software was used for the protein structure predictions. PCR amplification of the bla_NDM genes detected 113 clinical strains of E. coli with the contribution of bla_NDM-1 (46%), bla_NDM-4 (3.5%), and bla_NDM-5 (50%) variants. DNA sequencing of bla_NDM variants showed homology to the previously described bla_NDM-1, bla_NDM-4, and bla_NDM-5 genes available at GenBank and NCBI database. In addition, the mutational analysis revealed in frame substitutions of Pro60Ala and Pro59Ala in bla_NDM-4 and bla_NDM-5, respectively. The bla_NDM-1 was ortholog with related sequences of E. coli available at GenBank. The phylogenetic analysis indicated that the NDM gene variants resemble other microbes reported globally with some new mutational sites.     

Pilot scale production of functional foods using red palm olein: Antioxidant,vitamins’ stability and sensory quality during storage

The objective of this research work was to produce acceptable quality functional foods, namely, extruded snacks, digestive biscuits and pan bread, on a pilot scale, using vitamin E and β-carotene-rich red palm olein (RPOL) and red palm shortening (RPS). These products were evaluated for their chemical composition and sensory quality along with the antioxidants and vitamin contents during the six months of storage at room temperature (22 ± 1 °C). Extruded snacks and digestive biscuits prepared with RPOL and RPS were found to be good sources of these antioxidant vitamins. The average β-carotene content of the control and test snacks at the end of six months of storage ranged from 26.8 to 56.1 mg/kg fat, and from 430.9 to 468.9 mg/kg fat, respectively. The total vitamin E content in control and test snacks made in Plant No. 1 decreased after six months of storage from 786.1 to 704.4 mg/kg fat, and from 765.1 to 695.4 mg/kg fat, respectively. As expected, the total tocotrienol content was four to five times higher than the total tocopherols in control biscuits. The RPOL containing 600–750 ppm of carotenes (mainly α- and β-carotenes), 710–774 ppm of vitamin E, was found to be suitable for industrial application in producing acceptable quality pan bread, digestive biscuits and snacks. These functional foods contained significant amounts of β-carotene and total vitamin E, indicating the possibility of producing such foods rich in these two of the important antioxidant vitamins coming from a natural source. The research findings strongly indicate that good-quality pan bread, extruded snacks and digestive biscuits can successfully be produced to offer healthier eating choices to the consumers of this region, thereby promoting better health and productivity among the population.     

Mesenchymal stem cells and collagen patches for anterior cruciate ligament repair

AIM: To investigate collagen patches seeded with mesenchymal stem cells(MSCs) and/or tenocytes(TCs) with regards to their suitability for anterior cruciate ligament(ACL) repair. METHODS: Dynamic intraligamentary stabilization utilizes a dynamic screw system to keep ACL remnants in place and promote biological healing, supplemented by collagen patches. How these scaffolds interact with cells and what type of benefit they provide has not yet been investigated in detail. Primary ACL-derived TCs and human bone marrow derived MSCs were seeded onto two different types of 3D collagen scaffolds, Chondro-Gide?(CG) and Novocart?(NC). Cells were seeded onto the scaffolds and cultured for 7 d either as a pure populations or as "premix" containing a 1:1 ratio of TCs to MSCs. Additionally, as controls, cells were seeded in monolayers and in co-cultures on both sides of porous high-density membrane inserts(0.4 μm). We analyzed the patches by real time polymerase chain reaction, glycosaminoglycan(GAG), DNA and hydroxyproline(HYP) content. To determine cell spreading and adherence in the scaffolds microscopic imaging techniques, i.e., confocal laser scanning microscopy(c LSM) and scanning electron microscopy(SEM), were applied.RESULTS: CLSM and SEM imaging analysis confirmed cell adherence onto scaffolds. The metabolic cell activity revealed that patches promote adherence and proliferation of cells. The most dramatic increase in absolute metabolic cell activity was measured for CG samples seeded with tenocytes or a 1:1 cell premix. Analysis of DNA content and c LSM imaging also indicated MSCs were not proliferating as nicely as tenocytes on CG. The HYP to GAG ratio significantly changed for the premix group, resulting from a slightly lower GAG content, demonstrating that the cells are modifying the underlying matrix. Real-time quantitativepolymerase chain reaction data indicated that MSCs showed a trend of differentiation towards a more tenogenic-like phenotype after 7 d.CONCLUSION: CG and NC are both cyto-compatible with primary MSCs and TCs; TCs seemed to perform better on these collagen patches than MSCs.     

Persistent organic pollutants in Pakistan: Potential threat to ecological integrities in terms of genotoxicity and oxidative stress

As a consequence of both increasing population and industrialization in agro-economic sector, Pakistan has inevitably been confronted by multicomplex environmental challenges. Owing in part to poor regulatory framework, pollution due to persistent organic pollutants (POPs) has caused serious problems throughout the country. Resultantly, extensive use of POPs is causing vigorous deterioration of environment and human health. The current study addresses: (1) the general information on associated ecological effects and toxicity assessment by meta-analysis for local fauna and flora (2) their respective occurrence in living organisms; and (3) sources and distribution patterns of various POPs classes in environmental compartments of Pakistan. Based on the study, it can be concluded that the environment of Pakistan is highly contaminated with organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), dechlorane plus (DP), and polychlorinated naphthalenes (PCNs), which is further supported with the meta-analysis. Nevertheless, unavailability of environmental quality standards and food safety for POPs render it a forthcoming challenge of multicompartment toxicity exposure. Therefore, strategies must be planned for risk assessment of biologically active POPs, while the POP waste inventory should be elevated, along with the necessary measures to promote appropriate handling and treatment of POP as a matter of prime importance.     

Association of admission testosterone level with ST-segment resolution in male patients with ST-segment elevation myocardial infarction undergoing primary percutaneous coronary intervention

Background

Low level of testosterone may be associated with cardiovascular diseases in men, as some evidence suggests a protective role for testosterone in cardiovascular system. Little is known about the possible role of serum testosterone in response to reperfusion therapy in ST-elevation myocardial infarction (STEMI) and its relationship with ST-segment recovery. The present study was conducted to evaluate the association of serum testosterone levels with ST-segment resolution following primary percutaneous coronary intervention (PPCI) in male patients with acute STEMI.

Methods

Forty-eight men (mean age 54.55 ± 12.20) with STEMI undergoing PPCI were enrolled prospectively. Single-lead ST segment resolution in the lead with maximum baseline ST-elevation was measured and patients were divided into two groups according to the degree of ST-segment resolution: complete (> or =50%) or incomplete (<50%). The basic and demographic data of all patients, their left ventricular ejection fraction (LVEF) and laboratory findings including serum levels of free testosterone and cardiac enzymes were recorded along with angiographic finding and baseline TIMI (Thrombolysis in Myocardial Infarction) flow and also in-hospital complications and then these variables were compared between two groups.

Results

A complete ST-resolution (≥50%) was observed in 72.9% of the patients. The serum levels of free testosterone (P = 0.04), peak cardiac troponin (P = 0.03) were significantly higher and hs-CRP (P = 0.02) were lower in patients with complete ST-resolution compared to those with incomplete ST-resolution. In-hospital complications were observed in 31.2% of patients. The patients with a lower baseline TIMI flow (P = 0.03) and those who developed complications (P = 0.04) had lower levels of free testosterone. A significant positive correlation was observed between the left ventricular function and serum levels of free testosterone (P = 0.01 and r = +0.362).

Conclusion

This study suggests that in men with STEMI undergoing PPCI, higher serum levels of testosterone are associated with a better reperfusion response, fewer complications and a better left ventricular function.

     

Bioherbicidal activity of allelopathic bacteria against weeds associated with wheat and their effects on growth of wheat under axenic conditions

Allelopathic bacteria found to selectively inhibit weeds but not wheat in our earlier study were selected to evaluate their impact on three weeds and wheat under axenic conditions. Inoculated seeds of each species were sown in sand jars for 25 days. Results indicated that the applied strains variably inhibited germination of wild oat, little seed canary grass and broad leaved dock from 15.2 to 63.3, 18.5 to 58.7 and 18.4 to 60.5% and dry matter from 12.4 to 65, 22.8 to 81.4 and 21.7 to 71.3% than their controls, respectively. These effects were also evident in other growth parameters. Growth of wheat was significantly improved by four strains while others caused non-significant effects. Selectivity of these strains was also reflected in differential root colonization ability. These strains were characterized for various microbial and biochemical parameters. These strains may further be evaluated for their bioherbicidal activity under natural conditions.     

Properties of STAT1 and IRF1 enhancers and the influence of SNPs

Background

STAT1 and IRF1 collaborate to induce interferon-γ (IFNγ) stimulated genes (ISGs), but the extent to which they act alone or together is unclear. The effect of single nucleotide polymorphisms (SNPs) on in vivo binding is also largely unknown.

Results

We show that IRF1 binds at proximal or distant ISG sites twice as often as STAT1, increasing to sixfold at the MHC class I locus. STAT1 almost always bound with IRF1, while most IRF1 binding events were isolated. Dual binding sites at remote or proximal enhancers distinguished ISGs that were responsive to IFNγ versus cell-specific resistant ISGs, which showed fewer and mainly single binding events. Surprisingly, inducibility in one cell type predicted ISG-responsiveness in other cells. Several dbSNPs overlapped with STAT1 and IRF1 binding motifs, and we developed methodology to rapidly assess their effects. We show that in silico prediction of SNP effects accurately reflects altered binding both in vitro and in vivo.

Conclusions

These data reveal broad cooperation between STAT1 and IRF1, explain cell type specific differences in ISG-responsiveness, and identify genetic variants that may participate in the pathogenesis of immune disorders.

     

Potato virus Y mRNA expression knockdown mediated by siRNAs in cultured mammalian cell line

RNA interference (RNAi) is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes. The goal of this research was to provide a highly robust and concise methodology for in-vitro screening of efficient siRNAs from a bulk to be used as a tool to protect potato plants against PVY invasion. In our study, a 480bp fragment of the capsid protein gene of potato virus Y (CP-PVY) was used as a target to downregulate PVY mRNA expression in-vitro, as the CP gene interferes with viral uncoating, translation and replication. A total of six siRNAs were designed and screened through transient transfection assay and knockdown in expression of CP-PVY mRNA was calculated in CHO-k cells. CP-PVY mRNA knockdown efficiency was analyzed by RT-PCR and real-time PCR of CHO-k cells co-transfected with a CP gene construct and siRNAs. Six biological replicates were performed in this study. In our findings, one CP gene specific siRNA out of a total of six was found to be the most effective for knockdown of CP-PVY mRNA in transfected CHO-k cells by up to 80%–90%.     

Therapeutic effects of Typha elephantina leave’s extract against paracetamol induced renal injury in rabbits

Present study focuses on ameliorative potential of Typha elephantina leave’s aqueous (TE.AQ) extract against Paracetamol (PCM) induced toxicity in rabbits. We fed the male rabbits with 300 mg PCM in alone and in combination with TE.AQ at different doses i.e. (100, 200 and 300 mg/kg body weight) or silymarin (100 mg/kg) daily for 21 days. PCM in alone significantly (P < 0.5) increased serum urea, uric acid, creatinine, total protein, albumin, globulin and blood urea nitrogen. Serum sodium, potassium and magnesium level were high. The glutathione, radical scavenging activity and Thiobarbituric acid reactive substances were significantly reduced. Treatment with TE.AQ at dose rate 300 mg/kg body weight and Silymarin significantly ameliorated all the parameters when compared with PCM administered group. The 100 and 200 mg of TE.AQ showed no significant effects. The histopathological examination confirmed the therapeutic potential of TE.AQ. These results established the presence of natural antioxidants in Typha elephantina leaves.     

Discovery of new 3-methylquinoxalines as potential anti-cancer agents and apoptosis inducers targeting VEGFR-2: design,synthesis, and in silico studies

There is an urgent need to design new anticancer agents that can prevent cancer cell proliferation even with minimal side effects. Accordingly, two new series of 3-methylquinoxalin-2(1H)-one and 3-methylquinoxaline-2-thiol derivatives were designed to act as VEGFR-2 inhibitors. The designed derivatives were synthesised and evaluated in vitro as cytotoxic agents against two human cancer cell lines namely, HepG-2 and MCF-7. Also, the synthesised derivatives were assessed for their VEGFR-2inhibitory effect. The most promising member 11e were further investigated to reach a valuable insight about its apoptotic effect through cell cycle and apoptosis analyses. Moreover, deep investigations were carried out for compound 11e using western-plot analyses to detect its effect against some apoptotic and apoptotic parameters including caspase-9, caspase-3, BAX, and Bcl-2. Many in silico investigations including docking, ADMET, toxicity studies were performed to predict binding affinity, pharmacokinetic, drug likeness, and toxicity of the synthesised compounds. The results revealed that compounds 11e, 11g, 12e, 12g, and 12k exhibited promising cytotoxic activities (IC₅₀ range is 2.1 − 9.8 µM), comparing to sorafenib (IC₅₀ = 3.4 and 2.2 µM against MCF-7 and HepG2, respectively). Moreover, 11b, 11f, 11g, 12e, 12f, 12g, and 12k showed the highest VEGFR-2 inhibitory activities (IC₅₀ range is 2.9 − 5.4 µM), comparing to sorafenib (IC₅₀ = 3.07 nM). Additionally, compound 11e had good potential to arrest the HepG2 cell growth at G2/M phase and to induce apoptosis by 49.14% compared to the control cells (9.71%). As well, such compound showed a significant increase in the level of caspase-3 (2.34-fold), caspase-9 (2.34-fold), and BAX (3.14-fold), and a significant decrease in Bcl-2 level (3.13-fold). For in silico studies, the synthesised compounds showed binding mode similar to that of the reference compound (sorafenib).