In Vitro Biofilm-Mediated Biodegradation of Pesticides and Dye-Contaminated Effluents Using Bacterial Biofilms.

Overuse of pesticides in agricultural soil and dye-polluted effluents severely contaminates the environment and is toxic to animals and humans making their removal from the environment essential. The present study aimed to assess the biodegradation of pesticides (cypermethrin (CYP) and imidacloprid (IMI)), and dyes (malachite green (MG) and Congo red (CR)) using biofilms of bacteria isolated from pesticide-contaminated soil and dye effluents. Biofilms of indigenous bacteria, i.e., Bacillus thuringiensis 2A (OP554568), Enterobacter hormaechei 4A (OP723332), Bacillus sp. 5A (OP586601), and Bacillus cereus 6B (OP586602) individually and in mixed culture were tested against CYP and IMI. Biofilms of indigenous bacteria i.e., Lysinibacillus sphaericus AF1 (OP589134), Bacillus sp. CF3 (OP589135) and Bacillus sp. DF4 (OP589136) individually and in mixed culture were tested for their ability to degrade dyes. The biofilm of a mixed culture of B. thuringiensis + Bacillus sp. (P7) showed 46.2% degradation of CYP compared to the biofilm of a mixed culture of B. thuringiensis + E. hormaechei + Bacillus sp. + B. cereus (P11), which showed significantly high degradation (70.0%) of IMI. Regarding dye biodegradation, a mixed culture biofilm of Bacillus sp. + Bacillus sp. (D6) showed 86.76% degradation of MG, which was significantly high compared to a mixed culture biofilm of L. sphaericus + Bacillus sp. (D4) that degraded only 30.78% of CR. UV-VIS spectroscopy revealed major peaks at 224 nm, 263 nm, 581 nm and 436 nm for CYP, IMI, MG and CR, respectively, which completely disappeared after treatment with bacterial biofilms. Fourier transform infrared (FTIR) analysis showed the appearance of new peaks in degraded metabolites and disappearance of a peak in the control spectrum after biofilm treatment. Thin layer chromatography (TLC) analysis also confirmed the degradation of CYP, IMI, MG and CR into several metabolites compared to the control. The present study demonstrates the biodegradation potential of biofilm-forming bacteria isolated from pesticide-polluted soil and dye effluents against pesticides and dyes. This is the first report demonstrating biofilm-mediated bio-degradation of CYP, IMI, MG and CR utilizing soil and effluent bacterial flora from Multan and Sheikhupura, Punjab, Pakistan.

Antibacterial and Cytotoxic Effects of Biosynthesized Zinc Oxide and Titanium Dioxide Nanoparticles.

Nanotechnology is a rapidly developing field of research that studies materials having dimensions of less than 100 nanometers. It is applicable in many areas of life sciences and medicine including skin care and personal hygiene, as these materials are the essential components of various cosmetics and sunscreens. The aim of the present study was to synthesize Zinc oxide (ZnO) and Titanium dioxide (TiO2) nanoparticles (NPs) by using Calotropis procera (C. procera) leaf extract. Green synthesized NPs were characterized by UV spectroscopy, Fourier transform infrared (FTIR), X-ray diffraction (XRD), and Scanning Electron Microscopy (SEM) to investigate their structure, size, and physical properties. The antibacterial and synergistic effects of ZnO and TiO2 NPs along with antibiotics were also observed against bacterial isolates. The antioxidant activity of synthesized NPs was analyzed by their α-diphenyl-ß-picrylhydrazyl (DPPH) radical scavenging activity. In vivo toxic effects of the synthesized NPs were evaluated in albino mice at different doses (100, 200, and 300 mg/kg body weight) of ZnO and TiO2 NPs administered orally for 7, 14, and 21 days. The antibacterial results showed that the zone of inhibition (ZOI) was increased in a concentration-dependent manner. Among the bacterial strains, Staphylococcus aureus showed the highest ZOI, i.e., 17 and 14 mm against ZnO and TiO2 NPs, respectively, while Escherichia coli showed the lowest ZOI, i.e., 12 and 10 mm, respectively. Therefore, ZnO NPs are potent antibacterial agents compared to TiO2 NPs. Both NPs showed synergistic effects with antibiotics (ciprofloxacin and imipenem). Moreover, the DPPH activity showed that ZnO and TiO2 NPs have significantly (p > 0.05) higher antioxidant activity, i.e., 53% and 58.7%, respectively, which indicated that TiO2 has good antioxidant potential compared to ZnO NPs. However, the histological changes after exposure to different doses of ZnO and TiO2 NPs showed toxicity-related changes in the structure of the kidney compared to the control group. The current study provided valuable information about the antibacterial, antioxidant, and toxicity impacts of green synthesized ZnO and TiO2 NPs, which can be influential in the further study of their eco-toxicological effects.

Antimicrobial Efficacy of Biogenic Cobalt and Copper Nanoparticles against Pathogenic Isolates.

Biogenic synthesis of cobalt (Co) and copper (Cu) nanoparticles (NPs) was performed using the bacterial strains Escherichia coli and Bacillus subtilis. Prepared NPs were confirmed by a color change to maroon for CoNPs and green for CuNPs. The NPs characterization using FTIR showed the presence of functional groups, i.e., phenols, acids, protein, and aromatics present in the Co and CuNPs. UV-vis spectroscopy of E. coli and B. subtilis CuNPs showed peaks at 550 and 625 nm, respectively. For E. coli and B. subtilis CoNPs, peaks were observed at 300 nm and 350 nm, respectively. Antibacterial and antifungal activity of B. subtilis and E. coli Co and CuNPs was determined at 100 mg/mL concentration against two bacterial strains at 5, 2.5, and 1.5 mg/mL against fungal two strains F. oxysporum and T. viridi, respectively. B. subtilis CuNPs showed significantly higher inhibition zones (ZOI=25.7-29.7 mm) against E. coli and B. subtilis compared to other biogenic NPs. Likewise, B. Subtilis CuNPs showed lower MIC (4.3 ± 6.3) and MBC (5.3 mg/mL) values against both tested isolates. Antifungal activity of B. subtilis and E. coli CuNPs and CoNPs showed a concentration-dependent decrease in ZOI. Among all biogenic NPs, B. subtilis CoNPs showed the highest ZOI (25-30 mm) against F. oxysporum followed by E. coli CuNPs with maximum ZOI (20-27 mm) against T. viridi. Again, B. subtilis CoNPs and E. coli CuNPs showed lowest MIC and MFC values against both fungal isolates. In conclusion, the current study showed that biogenically synthesized B. subtilis Cu or CoNPs can be used as effective antimicrobial agents due to their potential antibacterial and antifungal potential.

In vitro Antimicrobial Activity of Biogenically Synthesized Nickel and Zinc Nanoparticles against Selected Pathogenic Bacterial Strains.

Bacterial resistance to already present antibiotics demands for new approaches in field of medicine. Scientists prefer nanoparticles (NPs) due to their promising potential in many applications. Two bacterial strains, Escherichia coli and Bacillus subtilis were used for biogenic synthesis of NPs. Characterization of prepared NPs was accomplished using UV-vis spectroscopy and fourier transform infrared spectroscopy (FTIR). The prepared NPs were confirmed by the color change from pale yellow to having white deposition for Zn NPs while from dark green to light green for Ni NPs. UV-vis spectroscopy of E. coli and B. subtilis based ZnNPs showed highest peak at 354nm and 362nm, respectively. Likewise, E. coli and B. subtilis NiNPs showed peaks at 246 nm and 238 nm, respectively. Antibacterial activity of B. subtilis based ZnNPs showed significant (p ≤ 0.05) zone of inhibition (ZOI; 27.3±0.6) against B. subtilis and 26.66±0.67 against E. coli at 100 mg/mL. Antibacterial activity of E. coli based ZnNPs showed 8.3±0.3 ZOI against B. subtilis and 6.6±0.3 ZOI against E. coli while NiNPs showed (25.0±0.0 mm) (ZOI) against B. subtilis and (25.0 ± 0.3 mm) against E. coli. Minimum inhibitory concentration (MIC) of E. coli ZnNPs showed values of 6.7±0.3 µg/mL for E. coli and 4.7±0.3 µg/mL for B. subtilis. MIC of B. subtilis ZnNPs showed 5.3±0.3 µg/mL for E. coli and 6.6±0.3 µg/mL for B. subtilis while NiNPs showed 33.0±1.0 µg/mL against E. coli and 24.0±1.0 µg/mL against B. subtilis as effective inhibitory concentrations. Minimum bactericidal concentration (MBC) of E. coli ZnNPs showed 7.3±0.3 µg/mL for E. coli and 8.3±0.3 µg/mL for B. subtilis. MBC of B. subtilis ZnNPs showed 7.6±0.3 µg/mL for E. coli and 8.6±0.3 µg/mL for B. subtilis while NiNPs showed 45.7±1.3 µg/mL against E. coli and 33.0±1.0 µg/mL against B. subtilis as effective inhibitory concentrations. It was concluded from the current study that biogenically synthesized ZnNPs and NiNPs are effective as promising antibacterial agents and have potential applications in biomedical fields.

Phenotypic and Molecular Characterization of CTX-M Type B-Lactamases in Gram Negative Bacterial Strains Isolated from Hospitals, Lahore, Pakistan.

One of the principal mechanisms that contribute resistance to antibiotics is the production of extended spectrum beta lactamase (ESBL) in Gram negative bacteria. In the present study, molecular methods were used to evaluate the prevalence of the extended-spectrum beta-lactamase (ESBL)-encoding CTX-M gene among Gram negative bacterial strains. In total, 148 clinical samples were collected from different tertiary care hospitals of Lahore, Pakistan. Disc synergy diffusion method was used to detect the presence of ESBL production. Moreover, antibiotic resistance patterns and molecular detection of bla CTX-M ESBLs, were also studied. The pathogens isolated from the 148 samples included Escherichia coli (43%) followed by Klebsiella sp. (28%), Proteus sp. (18%) and Pseudomonas sp. (11%). In all 148 strains, 95 (64%) were ESBL producers while 53 (36%) were non ESBL producers. The strains which were phenotypically ESBL producers, bla CTX-M were found in 46% E. coli strains, while 50% Klebsiella sp. were harboring the gene. A high resistance rate was observed against cephalosporins (cefopodoxime 67%, cefoperazone 73%, cephalexin 63% sparaxin 61%). Lower resistance was observed against meropenem among all isolated bacterial strains. Genotypic detection of bla CTX-M genes by PCR revealed 46% of E. coli and 50% of Klebsiella strains harbored bla CTX-M gene. The present study showed that ESBLs producers were resistant to commonly used antibiotics. Similarly, bla CTX-M ESBL production is more prevalent in our clinical isolates.

Population dynamics of pelagic rotifers in Marala Headworks (Pakistan).

Research work was designed to investigate the density and diversity of pelagic rotifers in a Lake near Marala Headworks. The physico-chemical parameters of water such as pH, dissolved oxygen, temperature, electrical conductivity, transparency and turbidity were evaluated. Correlation between rotifers and these parameters was also studied. Plankton sampling was done on monthly basis in order to check the population density of rotifers. In total, 18 species of rotifers were identified which belonged to 11 genera. The highest number of rotifers and their diversity was shown by genera namely Brachionus, Keratella, and Filinia. The Brachionus calyciflorus was dominant species in all the samples with mean population density (41%). Analysis of variance of physico-chemical parameters presented that the air and water temperature, electrical conductivity, transparency, dissolved oxygen and oxygen saturation were statistically significant in all the months. While pH was statistically non-significant (p≥0.05. Pearson correlation showed that oxygen and transparency were negatively correlated with rotifers density and diversity. Air and water temperature, concentration of hydrogen ions (pH), electrical conductivity and salinity showed positive relationship with density and diversity of rotifers.

Biogenic Synthesis, Characterization and Antibacterial Properties of Silver Nanoparticles against Human Pathogens.

Biogenic synthesis of silver nanoparticles (AgNPs) is more eco-friendly and cost-effective approach as compared to the conventional chemical synthesis. Biologically synthesized AgNPs have been proved as therapeutically effective and valuable compounds. In this study, the four bacterial strains Escherichia coli (MT448673), Pseudomonas aeruginosa (MN900691), Bacillus subtilis (MN900684) and Bacillus licheniformis (MN900686) were used for the biogenic synthesis of AgNPs. Agar well diffusion assay revealed to determine the antibacterial activity of all biogenically synthesized AGNPs showed that P. aeruginosa AgNPs possessed significantly high (p < 0.05) antibacterial potential against all tested isolates. The one-way ANOVA test showed that that P. aeruginosa AgNPs showed significantly (p < 0.05) larger zones of inhibition (ZOI: 19 to 22 mm) compared to the positive control (rifampicin: 50 µg/mL) while no ZOI was observed against negative control (Dimethyl sulfoxide: DMSO). Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) concentration against four test strains also showed that among all biogenically synthesized NPs, P. aeruginosa AgNPs showed effective MIC (3.3-3.6 µg/mL) and MBC (4.3-4.6 µg/mL). Hence, P. aeruginosa AGNPs were characterized using visual UV vis-spectroscopy, X-ray diffractometer (XRD), fourier transform infrared (FTIR) and scanning electron microscopy (SEM). The formation of peak around 430 nm indicated the formation of AgNPs while the FTIR confirmed the involvement of biological molecules in the formation of nanoparticles (NPs). SEM revealed that the NPs were of approximately 40 nm. Overall, this study suggested that the biogenically synthesized nanoparticles could be utilized as effective antimicrobial agents for effective disease control.

Cloning and over Expression Studies of Ovine Somatotropin cDNA of Kajli (sheep breed) in a Prokaryotic System.

Genetic studies including the quest, cloning and expression of genes encoding proteins responsible for various vital physiological processes and beneficial characteristics of economic perspective have made the biotechnology research progressively auspicious. Due to its great zootechnical and industrial importance somatotropin gene have been cloned from various animal species. Current study was designed to clone mature ovine growth hormone complementary DNA (oGH cDNA) of a sheep breed, Kajli and carry out over expression studies of cloned GH cDNA in a suitable prokaryotic expression system. Sheep GH cDNA was cloned in T/A (thymine / adenine) vector with signal peptide and confirmed by nested polymerase chain reaction (PCR) and restriction digestion. The gene was then ligated in pLEX expression vector and restricted plasmids showed a fragment insert of ~ 600 bps. Restriction analysis confirmed positive clones, were induced for protein expression analysis. The pET vectors (plasmid for expression by T7 RNA polymerase) have an isopropylthio-ß-galactoside (IPTG) inducible strong T7 promoter and Escherichia coli expression strain of BL21 (DE3) pLysS contains DNA fragment from T7 phage which harbors RNA polymerase. Therefore, for expressing recombinant proteins, cells were induced with various IPTG concentrations to optimize expression levels. Cells were induced with different IPTG concentrations (0.1 to 0.8 mM) followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Results indicated maximum expression level of oGH at 5 hrs after induction of cells with 0.3 mM IPTG concentration with a molecular weight of 22 kDa. As for as cellular localization of protein is concerned accumulation of expressed oGH is observed in inclusion bodies. The successful expression of the cloned GH cDNA of sheep confirmed the functional viability of the clone. The above mentioned technique of genetic engineering has provided to boost the dairy industry by the production of large quantities of recombinant bovine somatotropin (rbST).

Recent Progress in Technetium-99m-Labeled Nanoparticles for Molecular Imaging and Cancer Therapy.

Nanotechnology has played a tremendous role in molecular imaging and cancer therapy. Over the last decade, scientists have worked exceptionally to translate nanomedicine into clinical practice. However, although several nanoparticle-based drugs are now clinically available, there is still a vast difference between preclinical products and clinically approved drugs. An efficient translation of preclinical results to clinical settings requires several critical studies, including a detailed, highly sensitive, pharmacokinetics and biodistribution study, and selective and efficient drug delivery to the target organ or tissue. In this context, technetium-99m (99mTc)-based radiolabeling of nanoparticles allows easy, economical, non-invasive, and whole-body in vivo tracking by the sensitive clinical imaging technique single-photon emission computed tomography (SPECT). Hence, a critical analysis of the radiolabeling strategies of potential drug delivery and therapeutic systems used to monitor results and therapeutic outcomes at the preclinical and clinical levels remains indispensable to provide maximum benefit to the patient. This review discusses up-to-date 99mTc radiolabeling strategies of a variety of important inorganic and organic nanoparticles and their application to preclinical imaging studies.

Solution-Processed PEDOT:PSS/MoS2 Nanocomposites as Efficient Hole-Transporting Layers for Organic Solar Cells.

An efficient hole-transporting layer (HTL) based on functionalized two-dimensional (2D) MoS2-poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) composites has been developed for use in organic solar cells (OSCs). Few-layer, oleylamine-functionalized MoS2 (FMoS2) nanosheets were prepared via a simple and cost-effective solution-phase exfoliation method; then, they were blended into PEDOT:PSS, a conducting conjugated polymer, and the resulting hybrid film (PEDOT:PSS/FMoS2) was tested as an HTL for poly(3-hexylthiophene):[6,6]-phenyl-C61-butyric acid methyl ester (P3HT:PCBM) OSCs. The devices using this hybrid film HTL showed power conversion efficiencies up to 3.74%, which is 15.08% higher than that of the reference ones having PEDOT:PSS as HTL. Atomic force microscopy and contact angle measurements confirmed the compatibility of the PEDOT:PSS/FMoS2 surface for active layer deposition on it. The electrical impedance spectroscopy analysis revealed that their use minimized the charge-transfer resistance of the OSCs, consequently improving their performance compared with the reference cells. Thus, the proposed fabrication of such HTLs incorporating 2D nanomaterials could be further expanded as a universal protocol for various high-performance optoelectronic devices.

Morphological diversity of Morus spp. (Mulberry accessions) grown in Muzaffarabad, Azad Jammu & Kashmir, Pakistan / La diversidad morfológica de Morus spp. (accesiones de mora) cultivados en Muzaffarabad, Azad Jammu y Cachemira, Pakistán

Genetic diversity of thirty mulberry accessions was determined by using the eleven different phenotypic characters. The study was conducted in field areas of Azad Jammu and Kashmir. The main objective of this study was to find out the diversity in morphological characters of Mulberry accessions found in Azad Jammu and Kashmir and Pakistan. The results showed that there is a significant difference in quantitative parameters among the thirty accessions (p≤0.001). The cluster analysis showed that the data is divided into two main groups at near 80 dissimilarity level. This study suggests that the Morus germplasm is quite diverse.

Se determinó la diversidad genética de treinta accesiones de mora utilizando once caracteres fenotípicos diferentes. El estudio se realizó en áreas de campo de Azad Jammu y Cachemira. El objetivo principal de este estudio fue conocer la diversidad en los caracteres morfológicos de lss accesiones de mora encontrados en Azad Jammu, Cachemira y Pakistán. Los resultados mostraron que hay una diferencia significativa en los parámetros cuantitativos entre las treinta accesiones de mora (p≤0.001). El análisis de conglomerados mostró que los datos se dividen en dos grupos principales a un nivel de disimilitud cercano a 80. Este estudio sugiere que el germoplasma de Morus es muy diverso.

Operative Versus Non-operative Treatment of Grade III Acromioclavicular Joint Dislocations and the Use of SurgiLig: a Retrospective Review.

BACKGROUND: Acromioclavicular joint dislocations are common shoulder girdle injuries. The treatment of grade III acromioclavicular joint dislocations is controversial. Furthermore, the literature on the use of the Sur-giligTM synthetic ligament for reconstruction of dislocations is sparse. MATERIALS AND METHODS: This retrospective review aimed to establish whether operative treatment was superior to non-operative treatment in grade III acromioclavicular joint dislocations treated at our institute over a 5-year period. We also reviewed the effectiveness of reconstruction with SurgiligTM after acute and chronic dislocations across all grades of acromioclavicular joint dislocations. RESULTS: Twenty-five patients completed full follow-up with grade III dislocations. The mean follow-up in the operated group was 3.56 years and in the non-operated group this was 3.29 years. The mean Oxford Shoul-der Score (OSS) in the operated group was 39.8, whereas the mean OSS in the non-operated group was 45.9 (p=0.01). The mean pain score in the operated group was 2.2, and in the non-operated group this was 1.6. The mean satisfaction score in the operated group was 8.2 and that in the non-operated group was 7.8. There was no statistically significant difference in pain or satisfaction scores. In respect to the cohort treated with Surg-iligTM synthetic ligament, 22 patients across all grades of dislocations had this procedure performed. The mean post-operative Oxford Shoulder Score (OSS) was 40. CONCLUSIONS: 1. Non-operative treatment is not inferior to operative treatment for grade III acromioclavicular joint dislocations. The data from this study demonstrat-ed that the non-operated group had superior Ox-ford Shoul-der Scores that were statistically significant. 2. Additionally, the use of the SurgiligTM ligament appears to be effective in treating both chronic and acute acromioclavicular joint dislocations.

Exposure to sub-acute doses of fipronil and buprofezin in combination or alone induces biochemical, hematological, histopathological and genotoxic damage in common carp (Cyprinus carpio L.).

Use of pesticides or insecticides can be highly toxic to aquatic life forms due to leaching and agricultural runoff, rains or flood. Fipronil (FP) is a GABA receptor inhibitor, while buprofezin (BPFN) is an insect growth regulator. Presently, we exposed groups of aquaria acclimated carp fish (Cyprinus carpio) for 96h to sub-lethal concentrations of fipronil (400µgL(-1); 9.15×10(-7)molL(-1)) and buprofezin (BPFN, 100mgL(-1); 1.072×10(-6)molL(-1)) singly or in combination. The extent of damage was assessed at biochemical, hematological, molecular biological and histopathological level. Results obtained in treated fish were compared statistically with those of control non-treated fish and also among treatment groups. Significance level was p<0.05. Compared to control, serum total protein and globulin concentrations decreased significantly (p<0.0001) in fish treated with FP; while albumin concentration remained unaltered with all treatments. Glucose concentration decreased significantly (p<0.002) in fish treated with FP. In contrast, combined FP+BPFN treatment and BPFN treatment caused insignificant elevation of glucose concentration. Hematological assessment demonstrated significant decrease in red blood cell and thrombocyte counts, hemoglobin concentration and hematocrit percent; while white blood cell count showed an increase in all treatment groups (p<0.0001). Blood smears from pesticide treated fish revealed aberrant erythrocyte morphologies which included necrosis, micronuclear formation and hyperchromatosis. DNA laddering assay carried out on whole blood demonstrated excessive smear formation in combined FP+BPFN and BPFN treatment groups but no smear formation was noticeable in FP treated fish. Compared to control, whole blood DNA content increased significantly in the combined FP+BPFN and BPFN treatment groups (p<0.001 and p<0.009). With all treatments histopathological changes observed in the gills were: epithelial uplifting and necrosis of lamellae, lamellar atrophy, disruption of cartilaginous core, fusion and disorganization of lamellae and telangiectasia. In liver these were: karyorrhexis, hepatocellular hypertrophy, nuclear hypertrophy, melanomacrophage aggregates and central vein contraction, while in the kidney: deterioration of glomerulus and dilatation of Bowman's space, dilatation of renal tubules, thyroidisation, altered tubular lumen, nuclear hypertrophy, cellular atrophy, and cellular necrosis were the outcome. Our study revealed that FP and BPFN produce highly toxic effects on fish when given in combination or singly. To our knowledge, this is the first report on toxicity caused by FP and BPFN in single and combined state.