Expression deregulation of matrix metalloproteinases and vasoconstriction related genes in Pakistani females with abnormal uterine bleeding.

BACKGROUND: Abnormal uterine bleeding (AUB) is irregular menstrual bleeding which has great impact on female health and life style. Various genetic factors are involved in etiology and pathology of AUB. Present study was designed to explore the association of PTGFR, MMP9, MMP2, TGFB3 and VEGFB with AUB. METHODS: Blood samples of 212 females with AUB were collected along with age-matched healthy control. Expression variation of targeted genes was evaluated using qPCR. Present study cohort was divided into different groups based on demographic parameters and all targeted genes were correlated with study demographics. RESULTS: Expression of targeted genes was significantly (P < 0.001) downregulated in females with AUB compared to control. Reduced (P < 0.01) expression of targeted genes was observed in all age groups (21-30, 31-40, 41-50 year) of AUB patients compared to respective control. Expression of VEGFB increased (P < 0.05) in AUB females with > 9 days bleeding compared to AUB patient had < 9 days bleeding. AUB women with miscarriage history showed upregulation in MMP2, TGFB3 (P < 0.05), and downregulation in MMP9 and VEGFB (P < 0.05) expression compared to AUB group with no miscarriage history. Expression of MMP2 increased (P < 0.05) in AUB females with > 60 kg body weigh compared to AUB patient with < 60 kg weight. CONCLUSION: Present study open a new window for diagnosis of AUB at early stages and suggested a possible involvement of PTGFR, MMP9, MMP2, TGFB3 and VEGFB as candidate biomarkers in AUB.

Probiotic potential of novel strains of Lactobacillus plantarum Lp -1: In vitro studies.

Five Lactobacillus strains isolated from vegetable and dairy products showed 99% similarity with Lactobacillus plantarum 1(Lp-1) using API -CHL 50 kit. Most of them proved to be sensitive to bacterial cell-wall inhibitors i.e. penicillin, ampicillin, amoxicillin and methicillin as studied by disc-diffusion method. These strains manifested profound tolerance to acidic-stress where Lp86 and Lp36 exhibited a good survival pattern at pH-2 for 4 hr retaining a survival count of 85% and 50%, respectively. A high survival of 85.7% was witnessed in Lp86 in presence of protease while Lp36 maintained 94.55% and 92.65% of population under the influence of enzyme pancreatin and pepsin. All the strains displayed marked tolerance against trypsin as the count did not drop below 77%. Absorbance and growth in terms of cfu/ml for bile-tolerance was examined for concentrations reflecting those in the GIT of humans, all the Lp-1 strains when grown with 1% bile showed a drop in the viable count by 1 log cycle i.e. from 1010 to 109cfu/ml. Fulfilling the above mentioned criteria these probiotic candidates displayed their capacity to reach the colon as viable metabolically active cells after successfully surviving under conditions similar to the gastrointestinal tract of humans. Upon examining the viability and stability of these probiotic candidates in most common foods serving as vehicle for probiotic delivery to the intestine, it was noticed that all the isolates tested sustained a probiotic approved number of 107 cfu/ml for effective function as recommended by WHO, after a maximum storage for one month. Hence, it could be justified that the selected probiotic candidates possess prominent probiotic potential. Therefore, L. plantarum 1 strains could prove to be an efficient probiotic after further in vivo studies to explore its safety in human subjects.

Optimization of physicochemical parameters for maximum amylase production by indigenously isolated Bacillus cereus AS2 strain.

Amylases are enzymes that catalyze the hydrolysis of starch into highly valuable products of economic significance. Amylases are used extensively in various industrial sectors. Microbial sources particularly Bacillus species are well known for the cost effective commercial production of amylase enzyme. Present study focuses on the enhancement of amylase enzyme production from an indigenously isolated Bacillus cereus AS2 strain via one variable at a time (OVAT) optimization of different physical and chemical factors. Purposely, eight parameters possibly affecting the amylase production including temperature, pH, incubation time, inoculum size, substrate concentration, metal ions, carbon and nitrogen sources were investigated. According to the results, amylase production was significantly boosted at maximum when the Bacillus cereus AS2 was grown at 45°C on pH 7.0 for 72 hours in the medium supplemented with 4% starch and 0.5% glycine. Among the different metal ions tested, CaCl2 (0.05%) was found significant to accelerate extracellular amylase production.

Transgenic expression of phytase in wheat endosperm increases bioavailability of iron and zinc in grains.

Phytate is a major constituent of wheat seeds and chelates metal ions, thus reducing their bioavailability and so the nutritional value of grains. Transgenic plants expressing heterologous phytase are expected to enhance degradation of phytic acid stored in seeds and are proposed to increase the in vitro bioavailability of mineral nutrients. Wheat transgenic plants expressing Aspergillus japonicus phytase gene (phyA) in wheat endosperm were developed till T3 generation. The transgenic lines exhibited 18-99 % increase in phytase activity and 12-76 % reduction of phytic acid content in seeds. The minimum phytic acid content was observed in chapatti (Asian bread) as compared to flour and dough. The transcript profiling of phyA mRNA indicated twofold to ninefold higher expression as compared to non transgenic controls. There was no significant difference in grain nutrient composition of transgenic and non-transgenic seeds. In vitro bioavailability assay for iron and zinc in dough and chapatti of transgenic lines revealed a significant increase in iron and zinc contents. The development of nutritionally enhanced cereals is a step forward to combat nutrition deficiency for iron and zinc in malnourished human population, especially women and children.

Efficacy of UV-C photolysis of bisphenol A on transcriptome alterations of genes in zebrafish embryos.

The purpose of this study was to investigate the efficacy of UV-C direct photolysis of bisphenol A (BPA) as a remediation method of BPA contamination. We used zebrafish embryos as a model organism to test the toxicity and residual biological activity by measuring cytochrome P4501A1 (CYP1A), aromatase B (Aro B) and heat shock proteins (HSP-70) transcript levels. The mRNA levels of CYP1A gene increased about two fold while exposure of zebrafish embryos at 72 hpf resulted in significant induction (P = 0.048) of Aro B at 100 µg/L of BPA. Exposure of zebrafish embryos at 72 hpf to increasing concentrations of BPA resulted in significant induction (P = 0.0031) of HSP-70 transcript level. UV treatment of BPA resulted in a significant reduction in toxicity by reducing mortality of zebrafish embryos. The results suggest that UV-C direct photolysis may be an effective method for remediation of BPA contamination. Further studies will be necessary for better understanding of the identity and relative activity of the UV degradation by-products.

Antimicrobial, antitumor and brine shrimp lethality assay of Ranunculus arvensis L. extracts.

To investigate the antitumor activity, brine shrimp lethality assay, antibacterial and antifungal activity of Methanol Extract (ME), Water Extract (WE), Acetone Extract (AE), Chloroform Extract (CE), Methanol-Water Extract (MWE), Methanol-Acetone Extract (MAE), Methanol-Chloroform Extract (MCE) of Ranunculus arvensis (L.). Antitumor activity was evaluated with Agrobacterium tumefaciens (At10) induced potato disc assay. Cytotoxicity was evaluated with brine shrimp lethality assay. Antibacterial activity was evaluated with six bacterial strains including Escherichia coli, Enterobacter aerogenes, Bordetella bronchiseptica, Klebsiella pneumoniae, Micrococcus luteus and Streptococcus anginosus and antifungal screening was done against five fungal strains including Aspergillus niger, A. flavus, A. fumigates, Fusarium solani and Mucor species by using disc diffusion method. Best antitumor activity was obtained with ME and WE, having highest IC50 values 20.27 ± 1.62 and 93.01 ± 1.33µg/disc. Brine shrimp lethality assay showed LC50 values of AE, MAE and ME were obtained as 384.66 ± 9.42µg/ml, 724.11 ± 8.01µg/ml and 978.7 ±8.01 µg/ml respectively. WE of R. arvensis revealed weak antimicrobial result against the tested microorganisms. On the other hand, the antifungal activity of the plant extracts was found to be insignificant. These findings demonstrate that extracts of R. arvensis possesses significant antitumor activity. Further extensive study is necessary to assess the therapeutic potential of the plant.

Evaluation of anthropogenic effects on water quality and bacterial diversity in Rawal Lake, Islamabad.

Water quality and bacterial diversity in the surface water of Rawal Lake was investigated for a period of 8 months to evaluate the pollution load from anthropogenic effects of surrounding areas. Rawal Lake in Islamabad, Pakistan is an artificial reservoir that provides the water needs for the residents of Rawalpindi and Islamabad. Grabbed water samples were collected according to standard protocols from ten different locations of the lake and tributaries keeping in view the recharge points from adjacent areas. Temperature, pH, electrical conductivity, dissolved oxygen, total dissolved solids, hardness, alkalinity, and turbidity of water samples were determined to study the water quality characteristics. The physicochemical parameters showed higher values at the tributaries as compared to the sampling locations within the lake such as values of hardness and alkalinity were 298 and 244 mg/L, respectively, at the tributary of the Nurpur stream. Bacterial strains were isolated by streaking on differential and selective growth media by observing colony morphology and other biochemical tests such as Gram reaction, oxidase, and catalase test. Template DNA was prepared from pure cultivated bacteria and 16S rRNA gene analysis was performed using universal primers for bacteria. Sequencing was performed by using BigDye terminator cycle sequencing kit. Sequences of nearest relative microbial species were identified by using basic local alignment search tool and used as reference sequences for phylogenetic analysis. Phylogenetic trees were inferred using the neighbor-joining method. Sequencing and phylogenetic characterization of microbes showed various phylotypes, of which Firmicutes, Teobacteria, and Proteobacteria were predominant.

Hazardous or Advantageous: Uncovering the Roles of Heavy Metals and Humic Substances in Shilajit (Phyto-mineral) with Emphasis on Heavy Metals Toxicity and Their Detoxification Mechanisms.

Shilajit is a phyto-mineral diffusion and semi-solid matter used as traditional medicine with extraordinary health benefits. This study provides a comprehensive data on Shilajit with emphasis on heavy metal profile, associated toxicities, and metal detoxification mechanisms by humic substances present in Shilajit. Data was searched across papers and traditional books using Google Scholar, PubMed, Science Direct, Medline, SciELO, Web of Science, and Scopus as key scientific databases. Findings showed that Shilajit is distributed in almost 20 regions of the world with uses against 20 health problems as traditional medicine. With various humic substances, almost 11 biological activities were reported in Shilajit. This phyto-mineral diffusion possesses around 65 heavy metals including the toxic heavy metals like Cu, Al, Pb, As, Cd, and Hg. However, humic substances in Shilajit actively detoxify around 12 heavy metals. The recommended levels of heavy metals by WHO and FDA in herbal drugs is 0.20 and 0.30 ppm for Cd, 1 ppm for Hg, 10.00 ppm for As and Pb, 20 ppm for Cu, and 50 ppm for Zn. The levels of reported metals in Shilajit were found to be lower than the permissible limits set by WHO and FDA, except in few studies where exceeded levels were reported. Shilajit consumption without knowing permissible levels of metals is not safe and could pose serious health problems. Although the humic substances and few metals in Shilajit are beneficial in terms of chelating toxic heavy metals, the data on metal detoxification still needs to be clarified.

Exploring asymmetric influence of R&D expenditures on CO2 emissions in China: evidence from nonlinear ARDL model.

R&D spending upsurges technological advancement and innovation which results in lowering energy consumption and environmental degradation. The current study investigates the asymmetrical impact of R&D spending on CO2 emissions in China via employing annual data from 1980 to 2021 and the NARDL model for empirical analysis. The estimated results of the NARDL model confirmed that there are asymmetries in positive and negative coefficients of R&D spending in China. The results depict that the positive shock in R&D spending exerts a negative and statistically significant impact on CO2 emissions in both runs implying that an increase in R&D spending lowers CO2 emissions. However, the negative coefficient of R&D spending yields a positive and statistically significant impact on CO2 emissions revealing the fact that a negative shock in R&D spending results in the upsurge of CO2 emissions in China. According to these findings, the impact of positive and negative shocks in R&D spending on CO2 emissions is asymmetric. The findings also show that the impact of a negative shock in R&D spending is greater than the impact of a positive shock on CO2 emissions. In addition to the negative shock in R&D spending, increases in energy consumption, economic growth, and FDI inflows also contribute to an upsurge in CO2 emissions in China. The robustness of the estimated results is assessed using standard fully modified ordinary least square (FMOLS) and dynamic ordinary least square (DOLS) models. The FMOLS and DOLS results have been confirmed to be sound and consistent with the results of the NARDL model. The study suggests that the economic strategies should aim at investing in R&D spending to foster environment-friendly technological innovations and to lower environmental degradation in China.

Urinary miRNAs: Technical Updates.

Due to its non-invasive nature and easy accessibility, urine serves as a convenient bi-ological fluid for research purposes. Furthermore, urine samples are uncomplicated to preserve and relatively inexpensive. MicroRNA (miRNAs), small molecules that regulate gene expression post-transcriptionally, play vital roles in numerous cellular processes, including apoptosis, cell differentiation, development, and proliferation. Their dysregulated expression in urine has been proposed as a potential bi-omarker for various human diseases, including bladder cancer. To draw reliable conclusions about the roles of urinary miRNAs in human diseases, it is essential to have dependable and reproducible methods for miRNA extraction and profiling. In this review, we address the technical challenges associated with studying urinary miRNAs and provide an update on the current technologies used for urinary miRNA isolation, quality control assessment, and miRNA profiling, highlighting both their advantages and limitations.

Tracking the Variations in Trace Elements, Some Nutrients, Phenolics, and Anthocyanins in Grewia asiatica L. (Phalsa) at Different Fruit Development Stages.

Grewia asiatica L. (phalsa) is a very prevalent berry in Pakistan and is consumed extensively as raw or in the form of juice. Here, for the first time, we assessed phalsa from Pakistan in terms of variations in macro and micro minerals, nutrients, and bio-active phyto-constituents including total phenolic and anthocyanin contents at different fruit developmental stages. It was found that the sugars in phalsa increased from D1 (small at the initial fruit setting stage) to D6 development stage (fully ripened fruit) where sugars at D5 (near to fully ripe) and D6 stages were many times greater than at D1, D2 (unripe close to full-size completion), D3 (close to semi ripe), and D4 stage (semi ripened and full-size attainment). Total acidity of was declined in all developmental stages, where the D1 stage displayed maximum and D6 with the lowest acidity. Ascorbic acid was decreased from D1 to D2 and then increased gradually from D3 to D5 stages. At the D6 stage, again a steep decline in ascorbic acid was observed. The total phenolics (mg gallic acid equivalents/100g) at stage D6 were higher (136.02 ± 1.17), whereas D1 being the lowermost in total phenolic content (79.89 ± 1.72). For anthocyanins (mg/100g), an increasing pattern of changes was observed in all stages of phalsa fruit where the D1 stage showed lower (13.97 ± 4.84) anthocyanin contents which then increased gradually at stage D2 (67.79 ± 6.73), but increased sharply at D3 (199.66 ± 4.90), D4 (211.02 ± 18.85), D5 (328.41 ±14.96) and D6 (532.30 ± 8.51) stages. A total of four anthocyanins such as cyanidin, delphidine-3-glucoside, pelargonidin, and malvidin in phalsa were identified using HPLC procedures, and a significant > 90 % DPPH inhibition in phalsa was observed at the D5 and D6 development stages. The macro and micro minerals including Ni, Zn, Fe, Ca, Cu, Mg, Na, P, and K contents were decreased from initial (D1) stage to the final (D6) development stage, while only Fe displayed an increasing trend from the initial to final fruit development stages (D1-D6). Conclusively, these findings could be of great interest for patients who are intended to consume phalsa as adjuvant therapy against diabetes and metabolic syndromes and other diseases involving reactive oxygen species with minimum metal toxicity.

Mapping the landscape of medical research in the Arab world countries: A comprehensive bibliometric analysis.

OBJECTIVES: To describe the productivity, performance, and impact of medical research in the Arab world countries. METHODS: We carried out a bibliometric analysis using Clarivate Analytics databases from January 2017 to March 2023. We reported research productivity, national and international research collaboration patterns, impact of Arab medical research output compared to the global average, top medical journals publishing Arab-affiliated research, and performance of the most productive Arab institutions. RESULTS: The Arab world contributed 2.72% to global medical research publication, with a citation impact of 11.98 compared to the global impact of 12.02. Qatar, Lebanon, and Saudi Arabia led medical research publications per million population among Arab countries, ranking 26th, 36th, and 37th globally. Medical research publications increased by 87% annually from 2017-2022, with 70% of research originating from Saudi Arabia and Egypt. National collaborations accounted for 15% of Arab world publications, while international collaborations represented 66%. The median impact factor across the top 20 medical journals with Arab-affiliated authors was 5.14, with 50% being quartile one journals. The top 10 Arab-origin medical journals had a median impact factor of 3.13. Approximately 80% of the top 20 Arab institutions were academic, with a median publication count of 3,162.5 and a median citation impact of 14.5. CONCLUSION: The study provides insights into the state of medical research in the Arab countries, indicating room for improvement in the region's medical research.

Structural and Functional Characterization of Novel Phosphotyrosine Phosphatase Protein from Drosophila melanogaster (Pupal Retina).

A novel pair of protein tyrosine phosphatases in Drosophila melanogaster (pupal retina) has been identified. Phosphotyrosyl protein phosphatases (PTPs) are structurally diverse enzymes increasingly recognized as having a fundamental role in cellular processes including effects on metabolism, cell proliferation, and differentiation. This study presents identification of novel sequences of PTPs and their comparative homology modeling from Drosophila melanogaster (Dr-PTPs) and complexation with the potent inhibitor HEPES. The 3D structure was predicted based on sequence homology with bovine heart low molecular weight PTPs (Bh-PTPs). The sequence homologies are approximately 50% identical to each other and to low molecular weight protein tyrosine phosphatases (PTPs) in other species. Comparison of the 3D structures of Bh-PTPs and Dr-PTPs (primo-2) reveals a remarkable similarity having a four stranded central parallel ß sheet with flanking α helices on both sides, showing two right handed ß-α-ß motifs. The inhibitor shows similar binding features as seen in other PTPs. The study also highlights the key catalytic residues important for target recognition and PTPs' activation. The structure guided studies of both proteins clearly reveal a common mechanism of action and inhibitor binding at the active site and will be expected to contribute toward the basic understanding of functional association of this enzyme with other molecules.

Eco-friendly textile desizing with indigenously produced amylase from Bacillus cereus AS2.

Starch is added to the fabric surface to secure weaving process. During finishing these sized particles are removed from the fabric and prepared it for printing and dyeing. Chemicals de-sizing agents damage fabric surfaces and reduce the quality of the product. An alternative to these conventional desizing agents is the use of biological molecules i.e. enzymes. The current study compares traditional de-sizing to bio-based de-sizing methods, as well as the optimization of fabric desizing settings using crude amylase. Amylase-producing Bacillus cereus AS2 was isolated from indigenous soil samples. The maximal fermentative de-sizing capability was discovered at 72 h, with no fabric surface degradation. Chemical desizing showed that the fabric lost all sizing agents to TEGEWA scale 9 within 1 h in presence of 5N HCl. Optimal studies for desizing showed that 1000 IU/ml of amylase resulted in maximum de-sizing within 15 h at 60 °C and 0.5% Triton-X. Water absorbance and weight loss, both parameters were used to check the desizing efficacy and it was found that de-sizing to same scale was occurred in the case of enzyme as well as commercially desized fabric. Enzyme desized cloth was found to be free of any starch particles in SEM micrographs, identical to industrially de-sized fabric, ensuring bioprocess efficacy.

Wild Edible Fruits as a Source of Food and Medicine: A Study among Tribal Communities of Southern Khyber Pakhtunkhwa.

Botanical surveys in all parts of Pakistan are mainly focused on ethnomedicinal uses of plants, and very little attention has been paid to documenting edible wild fruit species (EWFs). Multiple methodologies and tools were used for data collection. In a recent survey 74 EWF species belonging to 29 families were documented, including their medicinal uses for the treatment of various diseases. The most cited (23%) preparation method was raw, fresh parts. The UV and RFC of EWF species ranged from 0.08 to 0.4 and from 0.02 to 0.18, respectively. In terms of specific disease treatments and their consensus, the ICF ranged from 0 to 0.38. Sexual, gastrointestinal, and respiratory disorders had the highest use reports, and 11 species of plants had the highest FL of 100%. On the basis of uses reported by the inhabitants of seven districts of Southern Khyber Pakhtunkhwa Province, the CSI ranged from the lowest 1.3 to the highest 41. It is concluded that the traditional uses of EWF species depend mainly on socio-economic factors rather than climatic conditions or the number of species. However, there is a gradual loss of traditional knowledge among the younger generations. The present survey is the first baseline study about the socio-economic dimension of local communities regarding the use of EWF species for food as well as medicine.

Multisystem Inflammatory Syndrome in Children and Adolescents (MIS-C) under the Setting of COVID-19: A Review of Clinical Presentation, Workup and Management.

Earlier in its course, SARS-CoV-2 was primarily identified to cause an acute respiratory illness in adults, the elderly and immunocompromised, while children were known to be afflicted with milder symptoms. However, since mid-April of 2020, latent effects of the virus have begun emerging in children and adolescents, which is characterised by a multisystem hyperinflammatory state; thus, the term Multisystem Inflammatory Syndrome in Children (MIS-C) was introduced by the WHO and CDC. The syndrome manifests itself approximately 4 weeks after COVID-19 infection, with symptoms mimicking Kawasaki Disease and Kawasaki Disease Shock Syndrome. Demographically, MIS-C peaks in children aged 5 to 14 years, with clusters in Europe, North and Latin America seen, later followed by Asia. Although the exact pathophysiology behind the syndrome is unknown, recent studies have proposed a post-infectious immune aetiology, which explains the increased levels of immunoglobulins seen in affected patients. Patient presentation includes, but is not limited to, persistent fever, rash, gastrointestinal symptoms and cardiac complications including myocarditis. These patients also have raised inflammatory markers including C reactive protein, ferritin and interleukin-6. In poorly controlled patients, the syndrome can lead to multiorgan failure and death. The mainstay of treatment includes the use of intravenous immunoglobulins, steroids, immune modulators and aspirin. Adjunct therapy includes the use of low molecular weight heparin or warfarin for long term anticoagulation. Currently very little is known about the syndrome, highlighting the need for awareness amongst healthcare workers and parents. Moreover, with increased cases of COVID-19 as a result of the second wave, it is essential to keep MIS-C in mind when attending patients with a past history of COVID-19 exposure or infection. Additionally, once these patients have been identified and treated, strict follow-up must be done in order carry out long term studies, and to identify possible sequelae and complications.

Kinetic and thermodynamic studies of novel acid phosphates extracted from Cichorium intybus seedlings.

Herein for the first time a novel acid phosphatase from the seedlings of Cichorium intybus was purified to homogeneity by using various chromatographic techniques (salt precipitation, ion exchange, size exclusion and affinity chromatography) and thermodynamically characterized. The molecular mass of purified enzyme (66 kDa) was determined by SDS-PAGE under denaturing and non-denaturing conditions and by gel-filtration confirmed as dimer of molecular mass 130 kDa. The Michaelis-Menten (Km) constant for -p-NPP (0.3 mM) and (7.6 µmol/min/mg) Vmax. The enzyme was competitively inhibited by phosphate, molybdate and vanadate. Phenyl phosphate, ɑ and ß-glycero-phosphate and-p-NPP were found to be good substrate. When temperature increased from (55 °C to 75 °C), the deactivation rate constant (kd) was increased (0.1 to 4.6 min-1) and half- life was decreased from 630 min to 15 min. Various thermal denaturation parameters; change in enthalpy (ΔH°), change in entropy (ΔS°) and change in free energy (ΔG°) were found 121.93 KJ·mol-1, 72.45 KJ·mol-1 and 98.08 KJ·mol-1 respectively, confirming that acid phosphatase undergoes a significant process of unfolding during deactivation. The biochemical properties of acid phosphatase from C. intybus on the behalf of biological activity and its relationship to pH variations, thermal deactivation and kinetics parameters provide an insight into its novel features.

Multidrug resistant gram-negative bacteria in clinical isolates from Karachi.

A total of 54 gram-negative bacteria obtained from various pathological labs and hospitals of Karachi were screened for their resistance to ampicillin, chloramphenicol, gentamycin, kanamycin, neomycin, streptomycin and tetracycline antibiotics. Of the 54 bacteria, 50 were resistant to one or more antibiotics. Among the resistant bacteria, 13 out of 28 were found to transfer their resistances by conjugation. This indicates that at least 46% of clinical gram-negative bacteria in Karachi possess various types of transferable R plasmids, such as pAK5, pAK9, pAK10, pAK11, pAK12, pAK13, pAK14, pAK15, pAK16, pAK17, pAK18, pAK19, pAK20 and pAK21. The non-conjugative R plasmids included pMT14 and pZ26. Only pAK15 showed 26% segregation even after 20 consecutive transfers in plain broth (spontaneous segregation) whereas only pAK15 and pAK16 showed any significant loss of their markers in curing by acridine orange. The stability of R plasmids is more dangerous from clinical point of view.

Molecular Detection of TEM, SHV and CTX-M Genes Among Gram-negative Klebsiella Isolates.

INTRODUCTION: In the past few years, the use of antimicrobial drugs against a wide range of pathogens has increased significantly. This extensive use of drugs has increased the resistance rate in microbial community widely. Molecular techniques for the detection of resistance are more reliable as compared to the conventional phenotypic method. BACKGROUND: The family Enterobacteriaceae is considered to be an important cause of nosocomial infections due to its predominantly active species such as E. coli, Klebsiella and Pseudomonas. These organisms are mainly involved in causing pneumonia, sepsis, post-surgical and urinary tract infections. Resistance against antimicrobial drugs among these isolates is increasing more rapidly all over the world. This study primarily focuses on the resistant isolates of Klebsiella species. The drug resistance in Klebsiella isolates is found to be associated with the production of resistance enzymes such as beta lactamase and extended spectrum beta lactamase (ESBLs) which confer resistance, most specifically against cephalosporins and extended spectrum cephalosporins. Since these enzymes are plasmid mediated, they can also produce resistance against several other antimicrobials. It has been found that among the genus Klebsiella, ESBLs are more prevalent in K. pneumoniae followed by K. oxytoca. METHODS: In this study, we estimated the distribution of ESBL producers among Klebsiella species and performed their genetic characterization. A total of 236 gram-negative isolates were collected from different microbiological laboratories, during the period January 2010 till January 2012. Among these gram-negative isolates, 125 were identified as Klebsiella species. After species identification, Kirby Bauer disk diffusion method was used for antimicrobial susceptibility profiling. Furthermore, the phenotypic detection of ESBL producers was performed by double disc synergy and combination disc methods. Resistance genes responsible for the production of beta lactamase and extended spectrum beta lactamase enzymes were detected by Polymerase chain reaction. DNA sequencing was performed by selecting an ESBL producing Klebsiella pneumoniae strain with a positive blaTEM gene. RESULTS: In this study, we found 48%, 43.2% and 2.4% of SHV, TEM, and CTX-M resistance genes respectively in Klebsiella isolates. The DNA sequence ESBL and blaTEM positive Klebsiella strain showed 94% similarity with Klebsiella pneumoniae KUN5033 blaTEM gene for class A beta-lactamase TEM-198 analyzed by the Basic Local Alignment Search Tool (BLAST). CONCLUSION: Since there is an intense need of research in the field of drug resistance for implementing strict antibiotic control policies in hospitals, health care centers, laboratories, etc., the present study is dedicatedly conducted to estimate the drug resistant Klebsiella isolates, specifically the Beta lactamase and Extended Spectrum Beta Lactamase producers, at molecular level.

Characterization of cyanidin- and quercetin-derived flavonoids and other phenolics in mature saskatoon fruits (Amelanchier alnifolia Nutt.).

In order to further characterize the anthocyanins, flavonols, and other phenolics present in mature saskatoon ( Amelanchier alnifolia Nutt.) fruit, extracts were characterized using high-performance liquid chromatography, gas chromatography, and liquid chromatography-mass spectrometry. Cyanidin 3-O-galactoside, cyanidin 3-O-glucoside, cyanidin 3-O-arabinoside, and cyanidin 3-O-xyloside were identified as the four major anthocyanins in the mature fruit. The quercetin-derived flavonols, quercetin 3-O-glucoside, quercetin 3-O-galactoside, quercetin 3-O-arabinoside, quercetin 3-O-xyloside, quercetin 3-O-arabinoglucoside, quercetin 3-O-robinobioside, and quercetin 3-O-rutinoside were also identified in mature fruit extracts. In addition, two chlorogenic acid isomers (hydroxycinnamates), 3-O-caffeoylquinic acid and 5-O-caffeoylquinic acid were detected. The total content of the anthocyanin-, flavonol-, and hydroxycinnamate-type phenolics detected in mature 'Smoky' saskatoon fruit was 140, 25, and 96 mg/100 g fresh weight, respectively. These data further our knowledge of the phenolic composition of mature saskatoon fruit, and as anthocyanins, flavonols, and hydroxycinnamates exhibit antioxidant activities, the presence and levels of these classes of phenolics will aid in the understanding of the potential health-beneficial effects of saskatoon fruits in the human diet.