The quality and safety of food are of major importance. Using contaminated animals' milk and meat may result in human disease. Among microorganisms, fungal toxins, especially aflatoxin B-1 (AFB1), are of special importance. Aflatoxin M-1 (AFM-1) is a metabolite that is produced by conversion and hydroxylation of AFB-1. Both toxins can cause acute and chronic mycotoxicosis mainly through ingestion of contaminated milk. Hence, it is critical to control and decrease these microorganisms. Despite cost-effective efforts, preventing foods contamination due to aflatoxins (AFs) is not only an expensive but also a difficult task. The best agricultural monitoring during preharvest and postharvest stages cannot eliminate the AFs, especially AFM-1 from milk and dairy products because of the high resistance of these toxins. There have been numerous studies investigating the methods of AF detoxification or reduction from infected milk. By focusing on advantages and disadvantages of preventative procedures using probiotics, antibodies, chemisorbents and even additives, one can choose one or several procedures to eliminate or reduce AFM-1 in milk and its byproducts efficiently.

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India is the world's second most populous country, accounting for a quarter of the world's annual incidence of tuberculosis (TB). Every year around 2 million people develop TB in India and 300,000 die due to the TB. The emergence of drug-resistant-TB (DR-TB) has become a major public health concern in India. This situation is worsened by the appearance of multi-drug-resistant (MDR) strains of Mycobacterium tuberculosis. We sought to determine the characteristics and relative frequency of transmission of MDR-TB in North India and their association with the clinical and epidemiological characteristics of TB-patients and to mitigate the impeding drug-resistant TB epidemic in the country; it will help to established TB surveillance system effectively in the country. Diagnosis of MDR-TB prompts an appropriate treatment for patients with presumptive MDR-TB or rifampicin resistance in TB patients who have failed treatment with first-line drugs. If left undiagnosed or poorly treated, MDR-TB patients suffer for months to years before succumbing to the disease; hence, transmission of MDR-TB continues MDR-TB patients were found to be significantly higher in previously treated patients in comparison to newly diagnosed patients. The emerging drug susceptibility testing patterns and enlisting the help of an expert in DR-TB should be sought sooner rather than later through more than 100 established DR-TB centers across the country. To control the primary transmission of MDR-TB in Northern India, we recommend that improving the social support, living standards, and medical security of each patient should become a priority.

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Background: This study aimed to compare the accuracy of Candida species identification by direct inoculation of positive blood culture specimens into CornMeal Tween^™ (CMT) 80 agar with the conventional methods. Methods: This study was performed in two phases: Phase 1: Inoculation of direct CMT from artificially spiked blood culture bottles with identified Candida species. Phase 2: Direct inoculation of smear-positive bottles on CMT agar for identification. Routine identification method was carried out using urease production, sensitivity to cycloheximide, CMT inoculation, and colony morphology on BiGGY agar. All isolates were also evaluated for sugar assimilation on the biochemical test panel Analytical profile index (API) 20C AUX (BioMerieux). Statistical Analysis Used: The correlation between both methods was determined as percentage agreement rate using kappa score. Results: In the first phase 50 artificially spiked blood culture bottles were included in the study. The sample set consisted of 24% C albicans, 38% C tropicalis, and 22% C glabrata isolates. There was 100% agreement between the directly inoculated and CMT inoculated from colonies at 48 hours. Phase 2: The overall agreement between directly inoculated CMT and conventional method of identification at 72 h was 92%. Conclusion: Direct inoculation of CMT agar can easily differentiate common Candida species microscopically on the basis of chlamydospores, blastopores, and arrangement of pseudo hyphae. However, identification of rare species from specimens containing two different species using direct method is challenging. Therefore, conventional methods including macroscopic/microscopic characteristics and biochemical profile with the aid of API with API 20 AUX would be a better choice for such cases.

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Tuberculosis is a highly infectious illness that has been considered a worldwide health danger. Existence inside macrophages is a main characteristic of Mycobacterium tuberculosis virulence and essential to make a perdurable infection in the human body. Recently, a pivotal cytotoxicity determinant of M. tuberculosis in macrophages was discovered and named channel protein with necrosis-inducing toxin (CpnT). CpnT includes an N-terminal channel domain and a C-terminal domain (CTD). The CTD is a secreted toxin and can induce a necrotizing process in the macrophages. The CTD has strong nicotinamide adenine dinucleotide (NAD⁺)-glycohydrolase activity that empties NAD⁺ reservoirs of cells, resulting in human cell necrosis. In this study, the structural and functional properties of the CTD were reviewed. Besides, to predict local similarity between the CTD and other protein sequences and infer the functional and evolutionary relationships, the Basic Local Alignment Search Tool was used. Several protein sequences of the Mycobacterium showed >50% similarity to the CTD, indicating species specificity of the CTD. However, some prokaryotic and eukaryotic sequences showed 20%–45% similarity to the CTD, indicating that the CTD belongs to an uncharacterized protein family including nonbacterial proteins.

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Informatics studies represent the useful tool for development of the biologic-based therapeutics including proteins, antibodies, and vaccines. In this context, the bioinformatics studies are able to give more insights into the assessment of the effectiveness of the protein constructs, which have been developed as biologic-based therapeutics. Such investigations need a variety of data processing and analysis based on existing knowledge of gene functions and molecular interactions. Here, it is attempted to review some in silico approaches to assess physicochemical properties, posttranslational modification, hydrophobic behavior, solvent accessibility, allergenicity, and other properties of protein drugs and vaccines. Besides, in a part of this review, in silico strategies that are applicable during codon optimization of a recombinant protein are discussed.

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Background: Extrapulmonary tuberculosis (EPTB) constitutes about 15%–20% of all cases of TB. Patients with EPTB are more likely to have negative sputum smear results. It is often hard to diagnose because of difficulty of sampling and paucibacillary nature of samples. Methods: Smear negative extra pulmonary (EP) specimens were included in the study. An attempt was made to recover M. tuberculosis (MTB) from such EP specimens using rapid liquid culture (MGIT 960). Molecular Line probe assay (LPA) was used to determine the resistant for first line drugs, and second line drug resistant was determined using liquid culture. Results: Culture positivity was found in 21.3% (133/623) of specimens; of these 95.48% (127/133) were found to be MTB and 4.51% (6/133) of specimens were found to be non tubercular mycobacteria. Among MTB detected 18.9% of specimens were multi drug resistant, 3.90% were Rifampicin mono-resistant and 13.30% were Isoniazid resistant. Second line DST (N=29) was performed for Kanamycin and Ofloxacin; of which 3.4 % was found to be resistant to both drugs, 3.4 % was found to be resistant to Ofloxacin and 93.1% were sensitive to both drugs. Conclusion: Large percentage of drug resistance was observed in the study. Early recovery of MTB and determination of its drug resistance helped in early initiation of treatment and controlled further transmission of drug-resistant TB in the population.

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Background: Bacterial resistance is one of the most challenging and emerging public healthcare crisis in the modern era. Along with antibiotic degrading strategies, bacteria also evolved to produce extracellular polymers such as capsular polysaccharide (CPS) which not only provides immune protection but also act as a permeability barrier to antibiotics. The use of therapeutic enzymes alone and in combination with antibiotics has opened a new window for clinicians and researchers. Methods: In the present study, isolation of broad-spectrum capsular depolymerase bacterium was attempted from a number of environmental samples followed by 16SrRNA characterization. Optimization of capsular depolymerase production was performed by the one variable at time (OVAT) method and response surface methodology (RSM). Capsular depolymerase was partially purified using ammonium sulfate saturation method. Capsule stripping effects of depolymerase were analyzed using microscopic visualization of the capsule and antibiotic susceptibility test. Results: Thirty-two capsular depolymerase producing bacteria were isolated in this study and broad-spectrum depolymerase producing Isolate-30 was characterized as Bacillus siamensis SCVJ30 according to the 16srRNA sequencing. Depolymerase production was optimized using OVAT method and RSM. Relatively high yields (1.92 IU/ml) of capsular depolymerase were obtained in a medium containing 1 mg magnesium sulfate, 7 mg peptone and at 9 pH. A 115% increase in capsular depolymerase production was observed under optimal conditions than unoptimized conditions. Microscopic visualization of the capsule and antibiotic susceptibility testing postulates the positive effect of depolymerase on antibiotic effi cacy against Klebsiella pneumoniae. Conclusion: Further characterization of the enzyme will help in developing broad-spectrum depolymerase as a potent therapeutic agent against drug-resistant strains.

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Background: Local drug delivery is well known for its advantages over systemic drug therapy. Among nonsteroidal anti-inflammatory drugs, diclofenac is most commonly used analgesic and anti-inflammatory agent. Its mouthwash preparation has gained attention; however, there has not been any cross-over study reported in literature. Methods: Ten patients, indicated for periodontal surgeries, randomly received diclofenac mouthwash for 1 min, twice daily for 4 days, or diclofenac tablet twice daily for 4 days. After at least 15 days, second surgery was performed and mode of therapy was switched. The patient was asked to score Numeric Rating Scale index either personally or telephonically for those 4 days. Collected data were tabulated and analyzed. Results: When unpaired t-test was used for intergroup comparison (mouthwash group and tablet Group) at 95% confidence interval, it showed P = 0.0852 considered not statistically significant. That means pain reduction in mouthwash group is as equal as tablet group. Conclusion: The effect of locally administered diclofenac sodium mouthwash is equivalent to systemically delivered diclofenac sodium tablets. Hence, it can be stated that topical formulation is sufficiently effective for pain relief after periodontal surgical procedures without subjecting the patients to systemic side effects.

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Background: Invasive pneumococcal disease (IPD) has high mortality among children. Pneumococcal bacteremia is an important marker of IPD but has received limited attention in predicting the outcomes of illness. The aim of this study was to evaluate clinical features, focus of infection, and comorbid of pneumococcal bacteremia in a pediatric cohort at a tertiary care center in Karachi, Pakistan. Methods: Children aged 0–14 years admitted to the Aga Khan University Hospital with pneumococcal bacteremia were identified through a laboratory database. Demographic, clinical, and outcome data were obtained from patients' medical records. Data entry and analysis were carried out in MS Excel and SPSS version 19.0. Results: Forty (n = 40) episodes of pneumococcal bacteremia in 39 children were identified. The median age of the cohort was 2.5 (interquartile range 4.15–1.1) years; 30% (n = 12) of these were infants aged 0–12 months. The primary focus of infection was identified as pneumonia in 47.5% (n = 19) and meningitis in 7.5% (n = 3). The prevalence of occult bacteremia was 27.5% (n = 11). Outcomes of infection at hospital discharge were assessed in 31 patients as nine patients were either transferred out or left against advice. Of 31 patients, 6 (19.4%) died and 25 (80.6%) were discharged home. Associations with mortality included sepsis (P = 0.017) and absence of a focus (occult bacteremia) (P = 0.007). Conclusion: Sepsis and occult bacteremia (without an underlying focus) were associated with mortality. No association was found between choice of antibiotics administered, penicillin minimum inhibitory concentration, and poor outcome at discharge. Other factors such as comorbidities, immune status, and focus of infection play an important role in predicting the outcomes of pneumococcal bacteremia.

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Background: Gas6 is a product of growth arrest-specific gene 6 and is mostly expressed by the growth-arrested organs and is implicated in thrombosis, various inflammation, and age-related diseases. However, its role in chronic obstructive pulmonary disease has not been investigated yet. Methods: We developed lipopolysaccharide-induced mouse model of chronic lung inflammation to study its involvement. Therefore, male C57BL/6 mice (20–25 g) were grouped into control and LPS group; each group having 15 animals each. LPS group was nebulized with LPS (1 mg/ml) for 45 min, once a week for 8 weeks to induce chronic inflammatory conditions in the lungs by use of nebulizer. The mice model was validated by assessing infiltration of various immune cells (T-cells, B-cells, and neutrophils) into the lungs and pro-inflammatory and anti-inflammatory cytokine profile by cytokine bead array kit by flow cytometry. Lung deterioration was assessed by lung histology. The level of gas6 protein from the lung homogenate was measured by quantikine enzyme-linked immunosorbent assay kit. Furthermore, lung homogenate was analyzed by Western blotting for expression profile of gas6 protein. Results: There was significant increase in the level of T-cells (19±8% vs 65±11%), B-cells (24±7% vs 64±10%), and neutrophils (22±9% vs 57±10%) and significant increase in the level of tumor necrosis factor (200 ± 17 pg/ml vs. 1222 ± 152 pg/ml) and IL-6 (106 ± 13 pg/ml vs. 448 ± 122 pg/ml). Lung deterioration was observed in LPS group. We observed significant increase in the level of gas6 protein in lung homogenate in LPS group (0.16 ± 0.1 ng/ml vs. 4.2 ± 0.1 ng/ml). Furthermore, we found significant increase in gas6 protein in the lung homogenate of LPS-treated group by Western blotting. Conclusion: The current study establishes the involvement gas6 protein in lung inflammation.

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A 53-year-old male presented with multiple swellings over the left leg associated with sensory loss and weak dorsiflexion of the left foot for 4 months. Neurophysiological studies revealed absent sensory and motor action potentials in the left common peroneal nerve. Multiple hypoechoic areas were seen in the subcutaneous plane on ultrasound and were found to be nerve abscesses along the peroneal neurovascular bundle on magnetic resonance imaging of the left leg. Skin biopsy was suggestive of borderline tuberculoid leprosy. He was started on corticosteroids and paucibacillary multidrug therapy. The swelling subsided over the next 1 month, and the motor paralysis improved gradually with treatment.

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Background: Endothelial dysfunction has been associated for the cause of atherosclerosis or cardiovascular diseases (CVDs). Endothelial progenitor cells (EPCs) are exposed to oxidative stress during vascular injury as residents of blood vessel walls or as circulating cells homing to the sites of neovascularization. The current study was designed to analyze various subpopulations of EPCs and their DNA damage in CVDs. Methods: The study included 50 coronary artery disease (CAD) patients which was confirmed by angiography and 50 age-matched healthy controls without CAD. Flow cytometric analysis performed to measure subpopulations in EPCs in the peripheral blood using markers such as CD34, CD133, VEGFR2, and CD45. Oxidative DNA damage was analyzed in CD34+ cells. Mean EPC count was expressed as a percentage of total white blood cells. Three different subpopulations with CD45−/CD133+/VEGFR2+, CD45−/CD34+/VEGFR2+, and CD45−/CD34+/CD133+ coexpressions were measured with various percentages. Results: Subpopulation of CD45−/CD34+/VEGFR2+ cells had shown significant (P = 0.001) decrease in CAD patients in comparison with the healthy controls. There was no significant difference in the subpopulations of CD45−/CD34+/CD133+ cells (P = 0.005) and CD45−/CD133+/VEGFR2+ cells (P = 0.005) in CAD and healthy controls. The CD45−/CD34+/VEGFR2+ subpopulation EPC showed positive correlation with the severity of coronary stenosis (r = 0.35, P = 0.026), while other EPC subpopulation count did not show any correlation. Oxidative DNA damage was higher in CAD compared with controls. The number of EPC subpopulation CD45−/CD34+/VEGFR2+ was inversely correlated with oxidative DNA damage (P = 0.001), hypertension (P = 0.001), and diabetes mellitus (P = 0.004). Conclusion: We observed an association between CD45−/CD34+/VEGFR2 subpopulation EPCs and DNA damage in CAD condition. These findings support a cell biologist in searching the role of EPC populations in the pathophysiology or diagnosis of the disease by a clinician.

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Background: The present study was designed to evaluate the effects of vitamin E on phostoxin-induced changes in renal biochemical parameters in rats. Methods: Thirty disease free albino rats were selected to study the effect of acute aluminum phosphide poisoning (ALP poisoning) were further divided into 3 subgroups of ten each: A, B and C. Group A consists of rats given vehicle (Ginni Oil) only. Group B consists of rats given 5 ml “Celphos'mixture” (or 0.3mg/g body wt.). Group C consists of rats with acute Celphos poisoning along with Vitamin E (1.5 mg Vitamin E/g body weight of rat). Result: Mean serum creatinine concentration was significantly increased in both group B and group C as compared to group A. There is decrease in mean serum creatinine levels in acute ALP poisoning after vitamin E supplementation (group C) as compared to acute aluminium phosphide poisoning (group B), although this difference was not statistically significant. The rats were administered the doses via an infant feeding tube No. 8 and blood was obtained by Cardiac puncture one hour after feeding the dose. Serum Urea, serum creatinine and superoxide levels were estimated. The superoxide levels (nitroblue tetrazolium [NBT] reduction) were estimated. Mean serum urea concentration was significantly increased in both group B and group C as compared to group A. There is decrease in mean serum urea levels in acute ALP poisoning after vitamin E supplementation (group C) as compared to acute ALP poisoning (group B), although this difference was not statistically significant. NBT reduction was significantly increased in Group B as compared to Group A. Administration of Vitamin E to rats of Group C resulted in significant decrease of NBT reduction. Conclusion: Findings of the present study showed that Vitamin E via its antioxidant action and anti-inflamatory effects has protective effect on phosphine-induced toxicity in rats.

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