Bringing resistance modulation to epidemic methicillin resistant S. aureus of dairy through antibiotics coupled metallic oxide nanoparticles.

The current study probed methicillin resistant S. aureus from milk of different dairy farms along with its response to multiple antibiotics, assessment of risk factors, and response to antibiotic coupled nanoparticle. XRD of Np was confirmed as miller indices (hkl) values i.e. (101), (100), (002), (110), (012) and (013) while STEM finally revealed 40-60 nm nanorods in aggregated form. Total of 6 preparations viz a viz gentamicin (G), chloramphenicol (C), zinc oxide nanoparticle (Np), gentamicin coupled Np (GNp), chloramphenicol coupled Np (CNp), and simultaneously coupling of gentamicin and chloramphenicol on Np (GCNp) were formulated for their potential to bring resistance modulation. Data analysis of this study revealed 24.59% MRSA from dairy milk appearing potentially associated (OR> 1, p < 0.05) with most of assumed risk factors. MRSA in response to various antibiotics showed highest resistance against amoxicillin (100%), penicillin (100%), vancomycin (100%), and linezolid (90%). Zone of inhibitions were increased by 249.76% (GNp), 184.86% (CNp), and 279.76% (GCNp) in case of coupled preparations. Significant reduced minimum inhibitory concentration was observed in case of GCNp (7.8125 ± 0.00 µg/mL) followed by GNp (15.00 ± 0.00 µg/mL) and CNp (41.67 ± 18.042 µg/mL) as compared to Np alone (125.00 ± 0.00 µg/mL). Minimum bactericidal concentrations for GCNp, GNp, and CNp, and Np were 31.125, 62.5, 125, and 500 µg/mL, respectively. The study thus concluded increased prevalence of MRSA while coupling of ZnO nanoparticles with antibiotics significantly brought resistance modulation to MRSA.

Transition-Metal-Free [4+3]-Cycloaddition of ortho-Quinone Methides and Isomünchnones: Catalytic and Diastereoselective Assembly of Oxa-bridged Oxazocine Scaffolds.

Cycloadditions are powerful processes to synthesize complex polycyclic scaffolds. Herein, we disclose a [4+3]-cycloaddition between an in situ generated ortho-quinone methide and an isomünchnone to yield oxa-bridged oxazocine cores, generating N2 and H2 O as the sole by-products. Using only catalytic amounts of camphorsulfonic acid, it is possible to generate both reactive intermediates in one step, eliminating the need for rhodium catalysts generally employed for isomünchnone formation. Spectroscopic data and X-ray crystallography indicate the formation of the syn diastereomer, with the main side-product arising from a hydrate participating in a competing [4+2]-cycloaddition pathway.

Isolation of buffalo poxvirus from clinical case and variations in the genetics of the B5R gene over fifty passages.

Outbreaks of buffalopox affect udder and teats, which may ultimately lead to mastitis in dairy buffalo and can significantly compromise the production. In this study, we report isolation of buffalo poxvirus and sequence analysis of the B5R gene collected from the buffalo clinically suspected to be poxvirus infected. The virus was isolated on BHK-21 cell line and was passaged for 50 times, B5R gene was amplified and sequenced using gene-specific primers, and analyzed at both nucleotide and amino acid levels. Phylogenetically, the isolate can be classified close to the previously reported Pakistani and Indian isolates with certain level of differential clustering patterns. Three significant putative mutations (I2K, N64D, and K111E) were observed in the B5R protein. The K111E was common with previous human isolate from Karachi, Pakistan in 2005. These mutations differed from poxviruses reported from the neighboring countries. Some deletion mutations were observed which were recovered in upcoming passages. The K111E mutation suggests potential to cause zoonotic infection in human all over the country.

Chitosan-based electrospun nanofibrous mats, hydrogels and cast films: novel anti-bacterial wound dressing matrices.

The development of highly efficient anti-bacterial wound dressings was carried out. For this purpose nanofibrous mats, hydrogels and films were synthesized from chitosan, poly(vinyl alcohol) and hydroxyapatite. The physical/chemical interactions of the synthesized materials were evaluated by FTIR. The morphology, structure; average diameter and pore size of the materials were investigated by scanning electron microscopy. The hydrogels showed a greater degree of swelling as compared to nanofibrous mats and films in phosphate buffer saline solution of pH 7.4. The in vitro drug release studies showed a burst release during the initial period of 4 h and then a sustained release profile was observed in the next upcoming 20 h. The lyophilized hydrogels showed a more slow release as compared to nanofibrous mats and films. Antibacterial potential of drug released solutions collected after 24 h of time interval was determined and all composite matrices showed good to moderate activity against Gram-positive and Gram-negative bacterial strains respectively. To determine the cytotoxicity, cell culture was performed for various cefixime loaded substrates by using neutral red dye uptake assay and all the matrices were found to be non-toxic.

Sequential rhodium/palladium catalysis: enantioselective formation of dihydroquinolinones in the presence of achiral and chiral ligands.

Compatible combinations of achiral and chiral ligands can be used in rhodium/palladium catalysis to achieve highly enantioselective domino reactions. The difference in rates of catalysis and minimal effects of ligand interference confer control in the domino sequence. The "all-in-one" 1,4-conjugate arylation and C-N cross-coupling through sequential Rh/Pd catalysis provides access to enantioenriched dihydroquinolinone building blocks.

Suzuki-Miyaura cross-coupling of unprotected ortho-bromoanilines with benzyl, alkyl, aryl, alkenyl and heteroaromatic boronic esters.

A Suzuki-Miyaura cross-coupling reaction was developed on unprotected ortho-bromoanilines. This operationally simple reaction was developed for the diversification of glucocorticoid receptor modulators (GRMs), showed compatibility to various boronic esters featuring unique functionalities, and was demonstrated on a gram scale.

Rate of Nutrition-Related Chronic Diseases Among a Multi-Ethnic Group of Uninsured Adults.

The prevalence of nutrition-related chronic diseases, such as obesity, cardiovascular disease, and type 2 diabetes, among adults in the U.S. is of increasing importance. These conditions adversely affect the overall public health, health care systems, and economy. Marginalized minority groups have been disproportionally affected by these conditions. Lack of or inadequate health insurance limits access to health care, which contributes to poor health outcomes among individuals with these conditions. South Florida is home to diverse racial/ethnic minority groups, many of whom are uninsured and do not have access to expert-delivered nutrition education services. It is imperative to thoroughly study the health needs of these underserved patient populations and examine the rate of nutrition-related conditions among them in order to develop medically and culturally tailored nutrition education programs for them. Therefore, the aim of this study was to assess the prevalence of nutrition-related diseases among multi-racial/ethnic uninsured individuals living in South Florida. A four-week electronic health record of adult patients (N=272) from a free clinic in South Florida was analyzed. Spearman`s correlation and binary regression models were used to assess the relationship between the variables. The sample included females (65%) and males (35%). The mean age was 49.08±14.56 years. Overall, 87% had at least one nutrition-related condition, with overweight/obesity being the most observed (75.2%), followed by hypertension (39%), dyslipidemia (27.2%), and diabetes (23.9%). BMI was a significant predictor of the prevalence of hypertension among Whites (p=0.008) and Blacks (p=0.002) but not Asians (p=0.536). Overall, a high rate of nutrition-related chronic diseases was found among uninsured adults in this study. This supports the need for increased medically, culturally, and economically tailored nutrition education programs in free clinic settings.

A protocol for identifying the binding sites of small molecules on the cystic fibrosis transmembrane conductance regulator (CFTR) protein.

We describe a protocol to identify the binding site(s) for a drug called ivacaftor that potentiates the CFTR chloride channel. We use photoaffinity probes-based on the structure of ivacaftor-to covalently modify the CFTR protein at the region that constitutes the drug binding site(s). We define the methods for photo-labeling CFTR, its membrane extraction, and enzymatic digestion using trypsin. We then describe the experimental methods to identify the modified peptides by using mass spectrometry. For complete details on the use and execution of this protocol, please refer to Laselva et al. (2021).

Helicobacter Pylori Eradication Therapy: Still a Challenge.

Introduction Helicobacter pylori (H. pylori) infection is prevalent worldwide. H. pylori therapies' adverse effects can contribute to noncompliance among patients. This study aimed to assess the association between compliance to H. pylori eradication therapy and adverse effects using various drug regimens. Method We conducted an observational study from September 2017 to February 2020 in two tertiary care hospitals in patients with dyspeptic symptoms. H. Pylori detection was done by histopathological examination of gastric mucosa during upper gastrointestinal endoscopy or stool for H. pylori antigen. Patients with positive results were randomly assigned one of the nine different regimens consisting of a combination of proton pump inhibitors along with at least two antibiotics. The antibiotics used in different combinations were amoxicillin, clarithromycin, metronidazole, doxycycline, levofloxacin, and bismuth sulfate. The treatment groups received standard triple therapy with and without probiotics, sequential, concomitant, levofloxacin-based triple therapy, or sequential and bismuth-based quadruple treatments. All treatments were given for two weeks. At the end of the treatment period, patients were interviewed about completing treatment and any adverse effects they may have experienced during therapy. Data were analyzed using IBM Statistical Package for the Social Sciences (SPSS) Statistics for Windows, Version 22.0 (Armonk, NY: IBM Corp.). Results A total of 250 patients were included in the study (62% males, 38% females) with a mean age of 37 years ± 13 years (range 12-84 years). Most patients completed the treatment regimen (80.4%), and 19.6% did not complete treatment because of adverse effects (p<0.005). The levofloxacin-based, concomitant, and standard triple regimen with probiotic treatments had the highest tolerance (≥85%). Common adverse effects were abdominal and epigastric pain (11%), alteration of taste, and diarrhea (6.5%). Conclusion H. pylori eradication therapy is always a challenge. Patient compliance to the treatment can only be ensured by medicines with fewer adverse effects. In our study, levofloxacin-based triple, concomitant, and standard triple regimens with probiotics are maximally acceptable treatments.

Identification of binding sites for ivacaftor on the cystic fibrosis transmembrane conductance regulator.

Ivacaftor (VX-770) was the first cystic fibrosis transmembrane conductance regulator (CFTR) modulatory drug approved for the treatment of patients with cystic fibrosis. Electron cryomicroscopy (cryo-EM) studies of detergent-solubilized CFTR indicated that VX-770 bound to a site at the interface between solvent and a hinge region in the CFTR protein conferred by transmembrane (tm) helices: tm4, tm5, and tm8. We re-evaluated VX-770 binding to CFTR in biological membranes using photoactivatable VX-770 probes. One such probe covalently labeled CFTR at two sites as determined following trypsin digestion and analysis by tandem-mass spectrometry. One labeled peptide resides in the cytosolic loop 4 of CFTR and the other is located in tm8, proximal to the site identified by cryo-EM. Complementary data from functional and molecular dynamic simulation studies support a model, where VX-770 mediates potentiation via multiple sites in the CFTR protein.

Epidemiology and Associated Risk Factors for Brucellosis in Small Ruminants Kept at Institutional Livestock Farms in Punjab, Pakistan.

Brucellosis is reportedly endemic in ruminants in Pakistan. Both Brucella abortus and B. melitensis infections have been decumented in domestic animals and humans in the country. This study aimed to identify the burden of anti-Brucella antibodies in small ruminants as well as associated potential risk factors with its occurrence at nine institutional livestock farms in Punjab, Pakistan. The sera collected from equal number of sheep and goats (500 from each species) were screened by indirect-ELISA for anti-smooth-Brucella antibodies followed by a serial detection by real-time PCR. Overall, 5.1% (51/1000) seropositivity was registered corresponding to 5% (25/500) prevalence in goats and 5.2% (26/500) in sheep. Brucella-DNA could not be detected in any of the tested sera by real-time PCR. Multiple logistic regression model indicated that farm location (OR 34.05), >4 years of age (OR 2.88), with history of reproductive disorders (OR 2.69), and with BCS of ≤ 3 (OR 12.37) were more likely to test positive for brucellosis at these farms. A routine screening, stringent biosecurity, and quarantine measures are warranted for monitoring and eradication of the infection. Similarly, isolation and molecular investigation of the etiologic agent(s) are needed to understand the relationship of epidemiology and out-breaks of brucellosis in the country.

Serological and Molecular Investigation of Coxiella burnetii in Small Ruminants and Ticks in Punjab, Pakistan.

Coxiellosis is a zoonotic disease caused by the obligate intracellular bacterium Coxiella burnetii affecting the productive and reproductive capabilities of animals. This study was conducted to gain insight into the seroprevalence of coxiellosis in small ruminants in seven farms of the Punjab, Pakistan. Potential risk factors were assessed. In total, 1000 serum samples (500 from sheep and 500 from goats) and 163 ticks were collected from the ruminants. All these 163 ticks were merged into 55 pools (29 pools for ticks from sheep and 26 pools for ticks from goat). Serum samples were investigated using an indirect ELISA and PCR. Coxiella burnetii DNA was detected in 29 pooled seropositive samples and 11 pooled ticks by real-time qPCR. Serological analysis revealed a prevalence of 15.6% and 15.0% in sheep and goats, respectively. A significant association was found between seropositivity and different variables like district, lactational status, reproductive status, body condition and reproductive disorders. Univariate analysis showed that detection of C. burnetii DNA in tick pools was significantly associated with the presence of ticks on sheep and goats. However, a non-significant association was found for the prevalence of C. burnetii DNA in serum pools. Hence, C. burnetii infection is prevalent in small ruminants and ticks maintained at livestock farms in Punjab, Pakistan.

Combining Ru-Catalyzed C-H Functionalization with Pd-Catalyzed Asymmetric Allylic Alkylation: Synthesis of 3-Allyl-3-aryl Oxindole Derivatives from Aryl α-Diazoamides.

Ruthenium-catalyzed C-H functionalization was successfully combined with palladium-catalyzed asymmetric allylic alkylation in one pot. The novel dual-metal-catalyzed reaction provides a variety of 3-allyl-3-aryl oxindoles from the corresponding α-diazoamides in up to 99% yield with up to 85% ee. The appropriate ligand choice is important to promote the sequential reaction, avoiding undesired metal interaction or ligand exchange.

Detection of Peste des Petits Ruminants Viral RNA in Fecal Samples of Goats after an Outbreak in Punjab Province of Pakistan: A Longitudinal Study.

Peste des petits ruminants (PPR) is a highly contagious viral disease of domestic and wild small ruminants and thus has serious socioeconomic implications. In Pakistan, during the year 2012-2013, estimated losses due to PPR were worth Rs. 31.51 billions. Close contact between infected and susceptible animals is an important route of transmission of PPR. Therefore, carrier animals play an important role in unnoticed transmission of PPR. The objective of the study was to investigate the detection of PPR virus in goats recovered from PPR. A suspected PPR outbreak was investigated and confirmed as PPR after analysing appropriate samples collected from infected animals using rRT-PCR. A longitudinal study was conducted over the period of 16 weeks to ascertain the detection of PPR virus (PPRV) in faecal samples of recovered goats. Ninety-six (96) faecal samples from each sampling were collected at 4, 8, 12, and 16 weeks after the outbreak. Faecal samples were analysed using rRT-PCR. Of 96 from each sampling a total of 46, 37, 29, and 25 samples were positive for PPR viral genome at 4, 8, 12, and 16 weeks, respectively, after recovery. Attempts were made for the isolation of PPR virus on Vero cells, but results were negative. These results indicated the detection of PPR viral RNA up to 16 weeks after infection. Therefore, these results may help in the future epidemiology of PPR virus shedding and possible role as source of silent infection for healthy animals especially when there is no history of any outbreak in nearby flock or area.

Synthesis of piroxicam loaded novel electrospun biodegradable nanocomposite scaffolds for periodontal regeneration.

Development of biodegradable composites having the ability to suppress or eliminate the pathogenic micro-biota or modulate the inflammatory response has attracted great interest in order to limit/repair periodontal tissue destruction. The present report includes the development of non-steroidal anti-inflammatory drug encapsulated novel biodegradable chitosan (CS)/poly(vinyl alcohol) (PVA)/hydroxyapatite (HA) electro-spun (e-spun) composite nanofibrous mats and films and study of the effect of heat treatment on fibers and films morphology. It also describes comparative in-vitro drug release profiles from heat treated and control (non-heat treated) nanofibrous mats and films containing varying concentrations of piroxicam (PX). Electrospinning was used to obtain drug loaded ultrafine fibrous mats. The physical/chemical interactions were evaluated by Fourier Transform Infrared (FT-IR) spectroscopy. The morphology, structure and pore size of the materials were investigated by scanning electron microscopy (SEM). The thermal behavior of the materials was investigated by thermal gravimetric analysis (TGA) and differential scanning calorimetry (DSC). Control (not heat treated) and heat treated e-spun fibers mats and films were tested for in vitro drug release studies at physiological pH7.4 and initially, as per requirement burst release patterns were observed from both fibers and films and later sustained release profiles were noted. In vitro cytocompatibility was performed using VERO cell line of epithelial cells and all the synthesized materials were found to be non-cytotoxic. The current observations suggested that these materials are potential candidates for periodontal regeneration.

Isolation and characterization of low pathogenic H9N2 avian influenza A viruses from a healthy flock and its comparison to other H9N2 isolates.

Several outbreaks of avian influenza (AI) caused by H9N2 subtype, have been reported in the poultry industry during 1990 around the globe. Currently, H9N2 are endemic in the large area of Middle and Far East, including Pakistan. Since H9N2 AI viruses are sporadically reported from humans, extensive incidence of H9N2 in poultry imposes a great risk for human health. In this context, continuous monitoring of the poultry and determining the genetic nature of these viruses are fundamental to predict any future threat. Thus gene sequences of one isolate of H9N2, isolated from commercial poultry flocks, were analyzed. The results of this investigation, based on hemagglutinin (HA), neuraminidase (NA) and non-structural genes, showed that Pakistani H9N2 isolates are closely related to each other and to other H9N2 isolates from the Middle East. However, several unusual substitutions with unknown functional consequences were observed in HA and NA proteins and thus warrant further investigations for their possible role in viral biology. In conclusion, these findings provide information regarding the genetic nature of H9N2 avian influenza viruses in Pakistani poultry and necessitate the sequencing of more H9N2 viruses from both naturally infected and vaccinated flocks.

Prevalence and molecular characterization of dengue viruses serotypes in 2010 epidemic.

During August to November 2010, a dengue (DEN) outbreak occurred, and several cities of Pakistan were affected. In this study, serum samples from 317 patients, based on clinical symptoms and blood test, were included from Lahore, Sheikhpura and Gujranwala cities for further diagnosis of different genotypes of DEN virus. The samples were categorized into 2 groups: group 1 containing patients with dengue fever (DF) without hemorrhagic manifestations (DF) (n=228) and group 2 involving patients having DF with hemorrhagic manifestations (dengue hemorrhagic fever) (n=89). The samples found positive for DEN in reverse transcriptase polymerase chain reaction (PCR) by targeting capsid and premembrane genes containing fragment of size ∼2.5 kb for groups 1 and 2 were found to be 83.33% and 91.01%, respectively; however, the detection limit was quite low by immunoglobulin M antibody capture enzyme linked immunosorbent assay in case of both groups and was found to be 34.70% and 68.53%, respectively. The 16.67% and 8.99% samples of groups 1 and 2, respectively, were found negative in reverse transcriptase PCR analysis. In case of group 1 and group 2, by adopting seminested multiplex PCR strategy, predominant genotype was DEN virus type 2 with prevalence rates of 45.17% and 65.16%, respectively. DEN virus type 1 was the second most prevailing genotype in the population with prevalence rates of 18.85% and 19.10% for groups 1 and 2, respectively.

Biological characterization and phylogenetic analysis of a novel genetic group of Newcastle disease virus isolated from outbreaks in commercial poultry and from backyard poultry flocks in Pakistan.

Newcastle disease (ND) is a contagious viral disease of many avian species particularly domestic poultry, and is responsible for devastating outbreaks in the poultry industries around the globe. In spite of its importance and endemicity in Southern Asia, data on the genetic nature of the viruses and epizootiological information of the disease is scarce. In this study, six isolates from an emerging wave of ND outbreaks in the north of Pakistan and two isolates from healthy poultry flocks were biologically and genetically characterized. Based on pathogenicity indices such as intracerebral pathogenicity index (ICPI), mean death time (MDT) and cleavage motifs in the fusion protein, all these isolates were classified as virulent. Phylogenetic analysis of the fusion (F), hemagglutinin-neuraminidase (HN) and matrix (M) genes indicated the emergence of a novel genetic group within lineage 5, distinct from isolates previously reported in the region. Several mutations in the neutralizing epitopes and functionally important motifs of the F and HN genes pose a need for re-evaluation of the currently used vaccine and vaccination practices. The characteristics of Newcastle disease virus (NDV) as virulent (F protein cleavage site, ICPI and MDT) in apparently healthy backyard poultry (BYP) explain that BYP can play crucial role in the epizootiology and spread of the disease. The present investigation provides essential information on the genetic nature of NDV circulating in Pakistan and its implication on disease diagnosis and control. Furthermore, these investigations emphasize the importance of continuous surveillance of ND in developing countries.

Rabies out-break in mules at Mansehra, Pakistan.

An unusual sickness in mules at Batrasi camp, District Mansehra, Pakistan, was reported. Twelve animals died with in 2-3 days after showing the clinical symptoms confusing with colic and nervous disorders. Animals did not respond to any treatment. A team of veterinary doctors/researchers from institute visited the place and collected the samples and information in all aspects related to any disease occurrence on epidemiological basis. Animals were also showing symptoms confusing with rabies. Brain samples were collected for rabies testing. Reverse transcriptase polymerase chain reaction (RT-PCR) by amplifying "N" region gene and mouse inoculation test (MIT) were performed and results showed that disease was nothing except rabies and RT-PCR is the rapid and sensitive method for diagnosis of rabies virus as compared to other conventional methods of diagnosis.