NiO/MnFe2O4 Nanocomposite Photoluminescence, Structural, Morphological, Magnetic, and Optical Properties: Photocatalytic Removal of Cresol Red under Visible Light Irradiation.

In this study, pure nickel oxide (NiO), manganese ferrite (MnFe2O4 or MFO), and binary nickel oxide/manganese ferrite (NiO/MFO1-4) nanocomposites (NCs) were synthesized using the Sol-Gel method. A comprehensive investigation into their photoluminescence, structural, morphological, magnetic, optical, and photocatalytic properties was conducted. Raman analysis, UV-Vis spectroscopy, Fourier-transform infrared spectroscopy, scanning electron microscopy, and X-ray diffraction techniques were used to characterize the materials. The synthesized samples exhibited superparamagnetic behavior, as revealed by our analysis of their magnetic properties. A lower recombination rate was shown by the photoluminescence analysis, which is helpful for raising photocatalytic activity. The photocatalytic activity was evaluated for the degradation of Cresol Red (CR) dye. 91.6% of CR dye was degraded by NiO/MFO-4 nanocomposite, and the NC dosage as well as solution pH affected the photocatalytic performance significantly. In four sequential photocatalytic cycles, the magnetically separable NCs were stable and recyclable. The enhanced photocatalytic activity and magnetic separability revealed the potential application of NiO/MFO-4 as an efficient photocatalyst for the removal of dyes from industrial wastewater under solar light irradiation.

Factors affecting the efficiency of equine embryo transfer (EET) in polo mares under subtropical conditions of Pakistan.

Equine embryo transfer (EET) is a prominent technology in the equine breeding industry, and its efficacy is affected by a number of factors. The current study aimed to determine the effects of the breed of donor/recipient mares, estrus/ovulation induction treatment, cooled transportation of embryos, and synchrony between donor and recipient mares on the efficiency of the EET under subtropical conditions of Pakistan. A total of eighty-four (n = 84) Polo-playing donor mares (Argentino-polo = 41 and Anglo-Arab = 43) and seventy (n = 70) recipient mares (light breed = 26 and heavy breed = 44) were used for EET. The donor mares exhibiting natural estrus (n = 28) were detected by teaser a stallion, and corpus luteum (CL) having mares (n = 56) were treated with prostaglandin (150 µg of Cloprostenol) for estrus induction. The mares' follicular growth was monitored through ultrasonography until the dominant follicle's size reached 35 mm or more with a moderate to obvious uterine edema score. Afterward, the mares were treated either with GnRH, i.e., 50 µg of Lecirelin acetate (n = 41) or Ovusyn, i.e., 1500 IU hCG (n = 43). Insemination with chilled semen was performed 24 hours later. The embryos were collected non-surgically, 7 or 8 days after ovulation, from the donor mares. The collected embryos were transferred into the well-synchronized recipient mares as fresh (n = 44) or chilled (n = 26) embryos. The pregnancy after ET was checked through ultrasonography. Statistical analysis revealed that the embryo recovery rate (ERR) remained significantly higher (P<0.05) for the Prostaglandin (PG) treated group of donors as compared to the natural heat group of donors. The breed of donor mares, type of ovulatory treatment given, and day of embryo collection did not significantly (P>0.05) affect the ERR. There was no significant effect of the type (fresh vs chilled), classification, and stage of development of embryo on pregnancy outcomes (P>0.05). ET pregnancy rate was significantly affected by the breed of recipient mares and ovulation synchrony between donor and recipient mares (P<0.05). In conclusion, under the subtropical conditions of Pakistan, PG-based estrus induction of donor mares, breed of recipient mares, and ovulation synchrony between the donor and recipient mares had a substantial effect on the efficiency of EET.

A comparative absorption study of sucrosomial® orodispersible vitamin D3 supplementation vs. a reference chewable tablet and soft gel capsule vitamin D3 in improving circulatory 25(OH)D levels in healthy adults with vitamin D deficiency-Results from a prospective randomized clinical trial.

Background: Vitamin D (Vit D) deficiency (VDD), associated with diverse health conditions, is commonly treated with Vit D3 supplements. However, the gastrointestinal (GI) absorption of Vit D3 in different formulations has not been well studied. Objective: We aimed to compare the absorption of an innovative phospholipids-sucrester matrix biodelivery vehicle-based (sucrosomial®) orodispersible Vit D3 preparation against a reference chewable tablet and soft gel capsule (SGC) Vit D3 formulations in Vit D-deficient healthy adults. Methods: In study 1, 25 subjects were randomized to receive a weekly single dose of 200,000 IU of sucrosomial® Vit D3 (n = 12) or chewable tablet Vit D3 (n = 13) for 3 weeks. In study 2, 20 subjects were randomized to receive a single dose of 200,000 IU every other week of sucrosomial® Vit D3 (n = 10) or SGC Vit D3 (n = 10) for 6 weeks. Circulatory 25-hydroxyvitamin D3 [25(OH)D] levels were reassessed after 2, 3, and 6 weeks in study 1 and after 4 and 6 weeks in study 2. Results: In study 1, after 2 weeks, circulatory 25(OH)D levels increased significantly in both Vit D3 treatment groups (p < 0.0001) but improved markedly in the sucrosomial® Vit D3 group, with no further considerable change after 3 and 6 weeks in both groups. Overall, at all three follow-ups, sucrosomial® Vit D3 treatment achieved significantly higher and sustained 25(OH)D levels (p < 0.001). In study 2, after 4 weeks, both Vit D3 treatment groups showed significant improvement in circulatory 25(OH)D levels (p < 0.0001) but substantially higher in the sucrosomial® group with statistically significant differences between the two treatment groups (p = 0.02). At the 6-week follow-up, only subjects in the sucrosomial® Vit D3 group showed a further increase in circulatory 25(OH)D levels (p = 0.049), but no further significant changes in the levels of the SGC Vit D3 group (p = 0.062), showing a statistically significant difference between the two treatment groups (p = 0.002). The Vit D3 treatment was well tolerated by all participants, and no treatment-emergent effects or serious adverse events were reported. Conclusion: Our results suggest that the sucrosomial® Vit D3 preparation absorbs efficiently in the GI system, achieving adequately higher and sustained circulatory Vit D levels in VDD, and thus can effectively contribute to the body protection against VDD-associated health conditions. Clinical trial registration: clinicaltrials.gov, identifier: NCT05706259.

Optical, Photocatalytic, Electrochemical, Magnetic, Dielectric, and Ferroelectric Properties of Cd- and Er-Doped BiFeO3 Prepared via a Facile Microemulsion Route.

A series of Cd- and Er-doped bismuth ferrites were synthesized using a simple microemulsion technique. The influence of Cd and Er doping on the structural, ferroelectric, photocatalytic, and dielectric properties of bismuth ferrite (BFO) was examined in this research. The prepared materials were examined by X-ray diffraction, Raman, scanning electron microscopy, and UV-vis techniques. The XRD patterns reflected the formation of a monophasic rhombohedral structure with the space group R3-c and an average crystallite size calculated to be in the range of 29 to 32 nm. The saturation polarization (Ps), coercivity (Hc), and retentivity (Pr) of the materials were investigated by a hysteresis loop (P-E), and it was perceived that increasing the dopant contents improved the Ps and Pr values, which may be due to the variation of metal cation valence states. In accordance with the photoluminescence (PL) spectra, a highly substituted material displayed lower recombination and increased charge separation rate (e--h+), which eventually contributed to a higher photocatalytic degradation performance of the prepared NMs. Furthermore, as the frequency and dopant concentration increased, the dielectric loss decreased, which could be due to different types of polarization. Bi1 - xCdxFe1 - yEryO3 showed well-saturated hysteresis loops (P-E) with enhanced saturation polarization near 9.7 × 10-4 µC·cm-2. The remnant polarization of the BFO and BFOCE NPs was 2.26 × 10-4 and 8.11 × 10-4 µC·cm-2, respectively, under a maximum electric field, which may be due to the variation of the metal cation valence states. The improved ferroelectric and dielectric properties of Bi1 - xCdxFe1 - yEryO3 NPs are attributed to the reduced concentration of defects, the different domain behavior, and the valence state of Cd and Er ions. The electrochemical (crystal violet (CV) and I-V) properties of Bi1 - xCdxFe1 - yEryO3 were all influenced by the dopant concentrations (Cd and Er). The synergistic effects of Cd and Er on the substituted material enhanced the specific capacitance in comparison to undoped BiFeO3. The photocatalytic activity to degrade CV under visible irradiation increased in BFOCE as the dopant (x,y) concentration increased from 0 to 0.25 by showing 84% dye degradation in comparison to pristine BiFeO3 (53% only) within 120 min under visible light. Moreover, the stability of these prepared nanoparticles was confirmed using recycling experiments, with the results indicating that the synthesized nanomaterials demonstrated promising stability and reusability.

Influence of endometritis on the follicular dynamics, recovery, quality, gene expression, nuclear maturation and in-vitro developmental competence of oocytes in Sahiwal cattle.

Uterine infections often lead to culling of valuable animals from a herd, resulting in genetic drain. The genetic potential of problematic females could be harvested by in-vitro embryo production. The objective of this study was to evaluate the effect of clinical endometritis on follicular dynamics, recovery, quality, gene expression, nuclear maturation and in-vitro developmental competence of oocytes in Sahiwal cattle. The B-mode ultrasonography was performed to examine the uterus for the presence of pus. Based on the history and reproductive examination of cows, a total of twelve (n = 12) Sahiwal cattle were selected for the experiment: (1) healthy group (n = 6) and (2) clinical endometritis group (n = 6). The 1st ovum pick-up (OPU) was conducted on day 165 postpartum. The collected cumulus oocytes complexes (COCs) were graded into A, B, C and D grades depending on the number of layers of cumulus cells and homogeneous nature of cytoplasm. Nuclear maturation was assessed by staining the oocytes with Hoechst 33,342. The results revealed that the number of medium-sized follicle (1.3 ± 0.1 versus 0.6 ± 0.1) and total number of follicles (9.1 ± 0.7 versus 6.6 ± 0.7) were higher (p < .05) in the healthy group as compared to clinical endometritis group, respectively. Similarly, the number of oocytes recovered (5.0 ± 0.4 versus 2.8 ± 0.4), oocytes with grade A, B and C (2.9 ± 0.3 versus 1.5 ± 0.3), proportion of oocytes with grade A or B (33 ± 0.0 versus 20 ± 0.1) and nuclear maturation (68 ± 0.1 versus 55 ± 0.1) were also higher (p < .05) in the healthy group as compared to clinical endometritis group, respectively. Perhaps, cleavage rate (55.1 ± 0.1 versus 46.2 ± 0.1) and blastocyst rate (29.7 ± 0.0 versus 26.3 ± 0.1) did not differ (p > .05) between the groups. Likewise, the expression level of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in immature oocytes did not differ (p > .05) between both the groups. In conclusion, clinical endometritis has a negative effect on follicular dynamics, oocyte recovery, oocyte quality and nuclear maturation; furthermore, the developmental competence of COCs is not compromised by it.

Effect of three schemes of ovum pick-up on the follicular dynamics, gene expression, and in-vitro developmental competence of oocytes in Sahiwal cattle.

This study aimed to compare the effect of three schemes of ovum pick-up (OPU) on follicular dynamics, oocytes recovery, oocytes quality, gene expression, nuclear maturation and in-vitro developmental competence of oocytes in Sahiwal cattle. Considering the follicle population, all the cows were divided equally in a 3 × 3 cross over design, and each cow received one of the three treatments: (a) twice weekly (TW; n = 6), (b) once weekly (OW; n = 6) and (c) bi-weekly OPU (BW; n = 6) in three periods, with the first OPU conducted on 4, 7 and 14 days after second dominant follicle puncture (DFP) in the TW, OW and BW OPU interval groups, respectively. The collected cumulus oocytes complexes (COCs) were graded into A, B, C and D grades depending on the number of layers of cumulus cells and homogeneous nature of cytoplasm. Nuclear maturation was assessed by staining the oocytes with Hoechst 33342. The growth rate (mm/day) of dominant follicle (DF) (F1) (0.49 ± 0.21 vs. 0.71 ± 0.26 vs. 1.30 ± 0.27) and first subordinate follicle (F2) (0.85 ± 0.27 vs. 0.71 ± 0.25 vs. 1.06 ± 0.29) did not differ (p > .05) among all the three groups. The proportion of animals bearing a corpus luteum (CL) in the BW OPU interval group (53.3%) was significantly higher (p < .05) as compared to TW (13.3%) and OW (18.3%) OPU interval groups. The number of medium-sized follicles and oocyte with grade A and B were significantly higher (p < .05) in the TW (1.16 ± 0.21 and 33.88 ± 0.03) OPU interval group as compared to the OW (0.88 ± 0.22 and 21.54 ± 0.03) and BW (0.55 ± 0.21 and 21.89 ± 0.02) OPU interval groups. However, the number of degenerated oocytes in BW (0.85 ± 0.16) OPU interval group was significantly higher (p < .05) as compared to the TW (0.16 ± 0.15) and OW (0.44 ± 0.16) OPU interval groups. Expression level of growth differentiation factor 9 in TW OPU interval group was significantly higher (p < .05) as compared to the OW and BW OPU interval groups. Likewise, expression level of bone morphogenetic protein 15 (BMP15) in the TW and BW OPU interval groups was significantly higher (p < .05) as compared to the OW OPU interval group. The nuclear maturation rate was significantly higher in the TW (63.64 ± 0.07) and BW (59.26 ± 0.08) OPU groups as compared to OW (51.43 ± 0.06) OPU interval group. However, the cleavage rate (59.30 ± 0.06 vs. 44.29 ± 0.06 vs. 56.67 ± 0.06) did not differ (p > .05) among the three groups. Whereas, the blastocyst rate tended to be higher (p = .06) in the TW (29.07 ± 0.05) and BW (28.33 ± 0.04) OPU interval groups as compared to OW (18.57 ± 0.05) OPU interval group. Taken together, it can be concluded that TW OPU interval scheme enhances the medium-sized follicles resulting in good quality oocytes, regulates the oocyte-derived paracrine factors, leading to higher nuclear maturation rates and improved embryonic development in-vitro.

Phenotypic characterization and genome analysis of a novel Salmonella Typhimurium phage having unique tail fiber genes.

Salmonella enterica serovar Typhimurium is a foodborne pathogen causing occasional outbreaks of enteric infections in humans. Salmonella has one of the largest pools of temperate phages in its genome that possess evolutionary significance for pathogen. In this study, we characterized a novel temperate phage Salmonella phage BIS20 (BIS20) with unique tail fiber genes. It belongs to the subfamily Peduovirinae genus Eganvirus and infects Salmonella Typhimurium strain (SE-BS17; Acc. NO MZ503545) of poultry origin. Phage BIS20 was viable only at biological pH and temperature ranges (pH7 and 37 °C). Despite being temperate BIS20 significantly slowed down the growth of host strain for 24 h as compared to control (P < 0.009). Phage BIS20 features 29,477-base pair (bp) linear DNA genome with 53% GC content and encodes for 37 putative ORFs. These ORFs have mosaic arrangement as indicated by its ORF similarity to various phages and prophages in NCBI. Genome analysis indicates its similarity to Salmonella enterica serovar Senftenberg prophage (SEStP) sequence (Nucleotide similarity 87.7%) and Escherichia virus 186 (~ 82.4% nucleotide similarity). Capsid genes were conserved however those associated with tail fiber formation and assembly were unique to all members of genus Eganvirus. We found strong evidence of recombination hotspot in tail fiber gene. Our study identifies BIS20 as a new species of genus Eganvirus temperate phages as its maximum nucleotide similarity is 82.4% with any phage in NCBI. Our findings may contribute to understanding of origin of new temperate phages.

Genome Analysis and Therapeutic Evaluation of a Novel Lytic Bacteriophage of Salmonella Typhimurium: Suggestive of a New Genus in the Subfamily Vequintavirinae.

Salmonella Typhimurium, a foodborne pathogen, is a major concern for food safety. Its MDR serovars of animal origin pose a serious threat to the human population. Phage therapy can be an alternative for the treatment of such MDR Salmonella serovars. In this study, we report on detailed genome analyses of a novel Salmonella phage (Salmonella-Phage-SSBI34) and evaluate its therapeutic potential. The phage was evaluated for latent time, burst size, host range, and bacterial growth reduction in liquid cultures. The phage stability was examined at various pH levels and temperatures. The genome analysis (141.095 Kb) indicated that its nucleotide sequence is novel, as it exhibited only 1-7% DNA coverage. The phage genome features 44% GC content, and 234 putative open reading frames were predicted. The genome was predicted to encode for 28 structural proteins and 40 enzymes related to nucleotide metabolism, DNA modification, and protein synthesis. Further, the genome features 11 tRNA genes for 10 different amino acids, indicating alternate codon usage, and hosts a unique hydrolase for bacterial lysis. This study provides new insights into the subfamily Vequintavirinae, of which SSBI34 may represent a new genus.

Emergence of plasmid-mediated mcr genes from Gram-negative bacteria at the human-animal interface.

BACKGROUND: The global emergence of plasmid-mediated colistin resistance (Col-R) conferred by mcr genes in gram-negative rods (GNRs) has jeopardized the last treatment option for multidrug-resistant bacterial infections in humans. This study aimed to assess the emergence of mcr gene-mediated Col-R in GNRs isolated from humans and animals in Pakistan. METHODS: Animal and clinical specimens collected from various sources were prospectively analysed using standard microbiological procedures. Pathogens were identified using the API 20E and API 20NE systems (bioMerieux). Minimum inhibitory concentration (MIC) against colistin was determined using the MIC detection methods, and multiplex polymerase chain reaction (PCR) was used to amplify the mcr-1 to mcr-5 genes. RESULTS: We isolated 126 (88.1%) animal and 17 (11.9%) human Col-R phenotypes, among which there was a significant association (P < 0.01) of Escherichia coli and Proteus mirabilis with animals and of Acinetobacter baumannii with humans. Animal strains exhibited statistically significant (P < 0.05) resistance to co-trimoxazole, chloramphenicol, and moxifloxacin, and the human pathogens exhibited statistically significant (P < 0.05) antibiotic resistance to cephalosporins, carbapenems, and piperacillin-tazobactam. For Col-R strains, MIC50 values were > 6 µg/mL and > 12 µg/mL for human and animal isolates, respectively. mcr genes were detected in 110 (76.9%) bacterial strains, of which 108 (98.2%) were mcr-1 and 2 (1.8%) were mcr-2. CONCLUSIONS: The detection of a considerable number of mcr-1 and mcr-2 genes in animals is worrisome, as they are now being detected in clinical pathogens. The acquisition of mcr genes by colistin-susceptible bacteria could leave us in a post-antibiotic era.

Exploring the prevalence and diversity of bovine ticks in five agro-ecological zones of Pakistan using phenetic and genetic tools.

Tick infestation is a leading cause of tick-worry and tick-borne diseases in livestock and associated economic losses in tropical and subtropical regions of the world. The cattle and buffalo populations in Pakistan are exposed to tick infestation throughout the year, but very little is known about the biology, diversity and distribution of tick species across different agro-ecological zones (AEZ) of the country. The present study aimed to investigate the prevalence (number of bovines infested with ticks out of the investigated population) and diversity of hard ticks infesting bovines in 30 villages located in five distinct AEZs (i.e. Arid, Indus delta, Northern irrigated plain, Sandy desert and Southern irrigated plain). We collected a total of 774 ticks (adult and nymphs) from cattle (n = 116) and water buffaloes (n = 88) on small-holder dairy farms (with <10 bovids per establishment) from September to November 2017. The overall tick prevalence was 46.1% (cattle: 47.9%; buffaloes: 44%), which varied significantly from 22.2% in the Indus delta to 70.5% in the Sandy desert. Tick prevalence was slightly higher in female (46.5%) than male animals (45%), and higher in calves (i.e. ≤ 1 year of age) (55%) than in young animals (i.e. up to 3 years of age) (39%) and adults (48%). Five tick species - Hyalomma anatolicum, Hyalomma hussaini, Hyalomma scupense, Rhipicephalus microplus and Rhipicephalus annulatus - were identified morphologically and then genetically. Genetic identification, achieved using the sequences of two mitochondrial (cytochrome c oxidase subunit 1 and 16S) and one nuclear ribosomal (second internal transcribed spacer) regions, was consistent with the morphological findings. Phylogenetic analyses of the DNA sequence data sets showed that the five species of tick identified here were closely related to the same species or closely related species from within and outside of Pakistan. Of five presently recognised taxa within the R. microplus complex, two were identified herein, including the R. microplus clade C and R. annulatus. This investigation provides the first genetic evidence of the occurrence of R. annulatus in Pakistan as well as Hy. hussaini and Hy. scupense in bovines specifically in the provinces of Sindh and Punjab, respectively. The present findings emphasise the importance of combining morphological and molecular approaches to study the diversity of ticks. Further longitudinal studies are required to establish seasonal variations in the prevalence and distribution of bovine ticks in different AEZs of Pakistan.

Effect of nabumetone on humoral immune responses in mice / Efeito da nabumetona nas respostas imunes humorais em camundongos

Nabumetone is used to reduce the pain and inflammation in rheumatoid arthritis. In the current study, immunomodulatory effect of Nabumetone is investigated in mice. The control group was administered normal saline orally as placebo. Nabumetone was administered orally via gavage in two treatment groups at 14mg/kg.b.w. doses and 28mg/kgb.w., respectively. Haemagglutination (HA) assay, Jerne hemolytic plaque and mice lethality assays were applied. In HA assay, the titer was significantly decreased in Nabumetone treatment groups (P< 0.001). In Jerne hemolytic plaque formation assay, there was a significant reduction (P< 0.001) in number of plaques in Nabumetone treated groups when compared with control. In mice lethality assay, there was a significant difference in mortality ratio of mice in control and Nabumetone treated groups (P< 0.001). Therefore, it is concluded that Nabumetone suppresses the humoral immune response in mice.(AU)

A nabumetona é usada na redução da dor e inflamação da artrite reumática. No presente estudo, o efeito imunomodulador é investigado em camundongos. O grupo de controle recebeu solução salina via oral como placebo. Nabumetona foi administrada oralmente via gavagem em dois grupos de tratamentos com doses de 14mg/kg.b.w. e 28mg/kgb.w., respectivamente. Foram realizados ensaios de hemaglutinação (HA), placa hemolítica de Jerne e letalidade dos camundongos. No ensaio HA, o grau foi significativamente menor nos grupos de tratamento com nabumetoma (P< 0.001). No ensaio de formação de placa hemolítica de Jerne houve redução significativa (P< 0.001) no número de placas em grupos tratados com nabumetoma comparado ao controle. No ensaio de letalidade dos camundongos houve diferença significativa no grau de mortalidade de camundongos no grupo de controle e grupos tratados com nabumetoma (P< 0.001). Portanto, conclui-se que a Nabumetoma suprime a resposta imune humoral em camundongos.(AU)

Effect of carboxylated poly l-Lysine as a cryoprotectant on post-thaw quality and in vivo fertility of Nili Ravi buffalo (Bubalus bubalis) bull semen.

Buffalo bull sperm are more prone to cryo-injuries. Glycerol being the most common permeable cryoprotectant exerts cytotoxic effects on sperm which cause a reduction in fertility. Thus, the exploration of new cryoprotectant is needed. For this purpose, we investigated the effect of carboxylated poly l-Lysine (CPLL) as cryoprotectant used with different concentrations of glycerol on post-thaw sperm motility, kinematics, plasma membrane integrity, mitochondrial membrane potential (MMP), lipid peroxidation (LPO), catalase concentration and in vivo fertility of Nili Ravi buffalo bull semen. In experiment 1, semen samples (n = 15, bulls = 3) were diluted with Tris-citrate-egg yolk extender containing different concentration of CPLL [0% (C0), 0.25% (C0.25), 0.5% (C0.5), 0.75% (C0.75), 1% (C1)]. Each concentration of CPLL was added in extender containing either 7% (G7) or 5% (G5) glycerol. Diluted semen samples were cooled and cryopreserved using standard procedures. Post-thaw total and progressive motility, plasma membrane integrity, acrosome integrity, and MMP were found higher (P < 0.05) in group (G5C0.75) containing 0.75% CPLL and 5% glycerol as compared to the control group (G7C0) and other groups while LPO was recorded lower (P < 0.05) in the same group (G5C0.75). In experiment 2, in vivo fertility was compared between G5C0.75 (5% Glycerol+ 0.75% CPLL; depicted better post-thaw quality) and control group G7C0. Buffaloes were inseminated after 24 h of onset of estrus. Pregnancy diagnosis was performed per rectum at least 60 days post insemination. The fertility rates [56% (58/102) vs. 36% (37/103)] were higher (P < 0.05) in G5C0.75 as compared to the control group G7C0. Based upon these results, this study concludes that the addition of 0.75% CPLL in combination with 5% glycerol in freezing extender improves the post-thaw structure, function and in vivo fertility of Nili Ravi buffalo bull semen.

Isocratic High Performance Liquid Chromatography Assay for Quantification of Ceftiofur Hydrochloride in Bubaline Plasma.

We optimized and validated an isocratic high-performance liquid chromatography (HPLC) assay for quantification of ceftiofur hydrochloride in bubaline plasma. Ceftiofur, its metabolic products and protein-bound residues were cleaved, derivatized into desfuroylceftiofur acetamide and injected into HPLC system. The mobile phase comprising of sodium dihydrogen phosphate (0.025 M, pH 7) and acetonitrile (34:66, v/v), was driven at a flow rate of 1 mL/min, and separation was achieved using C18 column. Isocratic elution was performed with an injection volume of 45 µL and analyte was scanned at 310 nm. The linearity range, limit of detection and limit of quantification were 0.1-10 µg/mL, 0.03 µg/mL and 0.11 µg/mL respectively. Moreover, the accuracy, precision and recovery remained within the acceptable limits. The assay was effectively applied for determining the concentration of ceftiofur in plasma samples collected from ceftiofur-treated buffalo calves.

Avian Influenza A(H9N2) Virus in Poultry Worker, Pakistan, 2015.

Avian influenza A(H9N2) virus isolated from a poultry worker in Pakistan in 2015 was closely related to viruses detected in poultry farms. Observed mutations in the hemagglutinin related to receptor-binding affinity and antigenicity could affect cross-reactivity with prepandemic H9N2 vaccine strains.

Safety and efficacy of ketamine xylazine along with atropine anesthesia in BALB/c mice

Anesthetics are an indispensable prerequisite for surgical intervention and pharmacological animal studies. The objective of present study was to optimize the dose of ketamine (K) and xylazine (X) along with atropine sulfate (A) in order to achieve surgical tolerance in BALB/c mice. Several doses of ketamine (100, 150, 200 mg/kg) and xylazine (10, 15, 20 mg/kg) were mixed and combination of nine doses (K/X: 100/10, 100/15, 100/20, 150/10, 150/15, 150/20, 200/10,200/15,200/20) were evaluated (n=9 per combination). A constant dose of atropine (0.05 mg/kg) was also used to counter side effect. Time-related parameters were evaluated on the basis of reflexes. KX at dose 200/20 mg/kg produced surgical tolerance in all nine mice with duration 55.00±6.87 minutes. The induction time 0.97±0.09 minutes, sleeping time 90.67±5.81 minutes and immobilization time (102.23±6.83 minutes) were significantly higher than all combination. However, this combination was considered unsafe due to 11 % mortality. While, KX at dose 200/15 mg/kg results in none of the mortality, so was considered as safe. Moreover, this combination produces surgical tolerance in 89 % mice with duration (30.00±7.45 minutes). It was concluded that KX at dose 200/15 mg/kg along with atropine 0.05 mg/kg is safe for performing surgical interventions in BALB/c mice.

Prevalence and Phylogenetics of H9n2 in Backyard and Commercial Poultry in Pakistan.

Surveillance of H9N2 is currently focused on areas central to the commercial poultry industry. This study determined the prevalence of H9N2 virus in commercial and backyard poultry flocks in Punjab Province, Pakistan. Oral and tracheal swabs were collected from commercial and backyard poultry from January 2015 through June 2016. Antisera against H5, H7, H9, and Newcastle disease viruses were used for virus identification. Molecular confirmation was made by reverse transcription PCR. Avian influenza virus subtypes H5 and H7 were not detected. The H9N2 virus was isolated in 5.7% of 905 tested flocks (5-10 birds/flock). Prevalence in commercial and backyard poultry was 6.7% of 687 flocks and 2.7% of 218 flocks, respectively. Hemagglutinin and neuraminidase-gene-based phylogenetic analysis of commercial and backyard poultry isolates showed 100% homology. Within sublineage B2 of Pakistan, identity among most recent isolates (2015) was 100%, compared to 75%-99% identity with previously isolated viruses (2010-12), indicating continued virus evolution. Most of the previously reported and currently studied viruses were isolated near the Pakistan-India border. Phylogenetic analysis showed that Pakistani and Indian isolates were closely related, indicating that avian influenza virus transmission may occur across this border.

Prevalencia y filogenia del virus de la influenza H9N2 en avicultura de traspatio y comercial en Pakistán. La vigilancia del virus de influenza H9N2 se centra actualmente en áreas centrales de la industria avícola comercial. Este estudio determinó la prevalencia del virus de influenza H9N2 en parvadas comerciales y de traspatio en la provincia de Punjab, Pakistán. Se recolectaron hisopos orales y traqueales de aves comerciales y de traspatio desde enero del 2015 hasta junio del 2016. Se usaron antisueros contra los virus de la enfermedad H5, H7, H9 y contra Newcastle para la identificación del virus. La confirmación molecular se realizó mediante transcripción reversa y PCR. No se detectaron los subtipos H5 y H7 del virus de la influenza aviar. El virus H9N2 se aisló en 5.7% de 905 parvadas analizadas (5-10 aves/parvada). La prevalencia en aves comerciales y domésticas fue del 6.7% de 687 bandadas y de 2.7% de 218 bandadas, respectivamente. El análisis filogenético basado en el gene de la hemaglutinina y de la neuraminidasa de los aislamientos de aves comerciales y de traspatio mostró 100% de identidad genética. Dentro del sublinaje B2 de Pakistán, la identidad entre los aislados más recientes (2015) fue del 100%, en comparación con el 75% al 99% de identidad con los virus aislados previamente (años 2010 al 2012), lo que indica la continua evolución del virus. La mayoría de los virus previamente reportados y estudiados actualmente se aislaron cerca de la frontera entre Pakistán e India. El análisis filogenético mostró que los aislamientos de Pakistán e India estaban estrechamente relacionados, lo que indica que la transmisión del virus de la influenza aviar puede ocurrir a través de esta frontera.

Immunomodulatory activities of gemifloxacin in mice.

OBJECTIVES: Gemifloxacin is a broad spectrum antibiotic and has shown excellent coverage against a wide variety of microorganisms. In this study, an attempt was made to evaluate the immunomodulatory potential of gemifloxacin in male swiss albino mice in vivo. MATERIALS AND METHODS: Three doses of gemifloxacin 25 mg/kg, 50 mg/kg and 75 mg/kg were used intraperitoneally (IP) for the evaluation of immune responses in mice. Delayed type hypersensitivity (DTH), heamagglutination assay, jerne hemolytic plaque formation assay and cyclophosphamide induced neutropenia assay were performed to evaluate the effect of gemifloxacin on immune responses. RESULTS: DTH assay has shown the significant immune suppressant potential of gemifloxacin at 25 mg/kg dose and 75mg/kg dose. Total leukocyte count (TLC) has shown decrease in leukocyte count (P<0.05) in drug treatment groups before cyclophosphamide administration and significant decrease (P<0.001) in leukocyte count after cyclophosphamide administration as compared to negative control group. Differential leukocyte count (DLC) has shown significant decrease (P<0.001) in percentage count of lymphocytes in 75 mg/kg treatment group in leukopenic mice while increase (P<0.01) in monocytes percentage in 50 mg/kg treatment group in leukopenic mice and increase in neutrophil percentage count (P<0.05) in all treatment groups was observed after cyclophosphamide administration. Humoral immune response is shown to be suppressed in dose dependent manner by both heamagglutination titre values (P<0.001) and jerne hemolytic plaque formation assay (P<0.001). CONCLUSION: The results of this work clearly demonstrate that gemifloxacin has significant immunomodulatory potential.

Nanotoxicity of Inert Materials: The Case of Gold, Silver and Iron.

Nanotechnology has opened a new horizon of research in various fields including applied physics, chemistry, electronics, optics, robotics, biotechnology and medicine. In the biomedical field, nanomaterials have shown remarkable potential as theranostic agents. Materials which are considered inert are often used in nanomedicine owning to their nontoxic profile. At nanoscale, these inert materials have shown unique properties that differ from bulk and dissolved counterparts. In the case of metals, this unique behavior not only imparts paramount advantages but also confers toxicity due to their unwanted interaction with different cellular processes. In the literature, the toxicity of nanoparticles made from inert materials has been investigated and many of these have revealed toxic potential under specific conditions. The surge to understand underlying mechanism of toxicity has increased and different means have been employed to overcome toxicity problems associated with these agents. In this review, we have focused nanoparticles of three inert metallic materials i.e. gold, silver and iron as these are regarded as biologically inert in the bulk and dissolved form. These materials have gained wider research interest and studies indicating the toxicity of these materials are also emerging. Oxidative stress, physical binding and interference with intracellular signaling are the major role player in nanotoxicity and their predominance is highly dependent upon size, surface coating and administered dose of nanoparticles. Current strategies to overcome toxicity have also been reviewed in the light of recent literature. The authors also suggested that uniform testing standards and well-designed studies are needed to evaluate nanotoxicity of these materials that are otherwise considered as inert. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Genetic diversity of Newcastle disease virus in Pakistan: a countrywide perspective.

BACKGROUND: Newcastle disease (ND) is one of the most deadly diseases of poultry around the globe. The disease is endemic in Pakistan and recurrent outbreaks are being reported regularly in wild captive, rural and commercial poultry flocks. Though, efforts have been made to characterize the causative agent in some of parts of the country, the genetic nature of strains circulating throughout Pakistan is currently lacking. MATERIAL AND METHODS: To ascertain the genetics of NDV, 452 blood samples were collected from 113 flocks, originating from all the provinces of Pakistan, showing high mortality (30-80%). The samples represented domesticated poultry (broiler, layer and rural) as well as wild captive birds (pigeons, turkeys, pheasants and peacock). Samples were screened with real-time PCR for both matrix and fusion genes (1792 bp), positive samples were subjected to amplification of full fusion gene and subsequent sequencing and phylogenetic analysis. RESULTS: The deduced amino acid sequence of the fusion protein cleavage site indicated the presence of motif (112RK/RQRR↓F117) typical for velogenic strains of NDV. Phylogenetic analysis of hypervariable region of the fusion gene indicated that all the isolates belong to lineage 5 of NDV except isolates collected from Khyber Pakhtunkhwa (KPK) province. A higher resolution of the phylogenetic analysis of lineage 5 showed the distribution of Pakistani NDV strains to 5b. However, the isolates from KPK belonged to lineage 4c; the first report of such lineage from this province. CONCLUSIONS: Taken together, data indicated the prevalence of multiple lineages of NDV in different poultry population including wild captive birds. Such understanding is crucial to underpin the nature of circulating strains of NDV, their potential for interspecies transmission and disease diagnosis and control strategies.

Role of PelF in pel polysaccharide biosynthesis in Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces three exopolysaccharides, Psl, Pel, and alginate, that play vital roles in biofilm formation. Pel is a glucose-rich, cellulose-like exopolysaccharide. The essential Pel biosynthesis proteins are encoded by seven genes, pelA to pelG. Bioinformatics analysis suggests that PelF is a cytosolic glycosyltransferase. Here, experimental evidence was provided to support this PelF function. A UDP-glucose dehydrogenase-based assay was developed to quantify UDP-glucose. UDP-glucose was proposed as the substrate for PelF. The isogenic pelF deletion mutant accumulated 1.8 times more UDP-glucose in its cytosol than the wild type. This suggested that PelF, which was found localized in the cystosol, uses UDP-glucose as substrate. Additionally, in vitro experiments confirmed that PelF uses UDP-glucose as substrate. To analyze the functional roles of conserved residues in PelF, site-directed mutagenesis was performed. The presence of the EX7E motif is characteristic for various glycosyltransferase families, and in PelF, E405/E413 are the conserved residues in this motif. Replacement of E405 with A resulted in a reduction of PelF activity to 30.35% ± 3.15% (mean ± standard deviation) of the wild-type level, whereas replacement of the second E, E413, with A did not produce a significant change in the activity of PelF. Moreover, replacement of both E residues did not result in a loss of PelF function, but replacement of the conserved R325 or K330 with A resulted in a complete loss of PelF activity. Overall, our data show that PelF is a soluble glycosyltransferase that uses UDP-glucose as the substrate for Pel synthesis and that conserved residues R325 and K330 are important for the activity of PelF.