Novel Biosynthesis of Copper Nanoparticles Using Zingiber and Allium sp. with Synergic Effect of Doxycycline for Anticancer and Bactericidal Activity.

Copper nanoparticles (CuNPs), due to their cost-effective synthesis, interesting properties, and a wide range of applications in conductive inks, cooling fluids, biomedical field, and catalysis, have attracted the attention of scientific community in recent years. The aim of the present study was to develop and characterize antibacterial and anticancer CuNPs synthesized via chemical and biological methods, and further synthesize CuNPs conjugated with doxycycline to study their synergic effect. During the chemical synthesis, ascorbic acid was used as a stabilizing agent, while Zingiber officinale and Allium sativum-derived extracts were used during the biological methods for synthesis of CuNPs. Characterization of CuNPs was performed by transmission electron microscopy (TEM), UV-visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), and X-ray crystallography (XRD). Antimicrobial evaluation of the nanomaterials against Pseudomonas aeruginosa and Escherichia coli was performed by using disk diffusion method, while anticancer behavior against HeLa and HepG2 cell lines was studied by MTT assay. TEM revealed spherical-shaped nanoparticles with mean size of 22.70 ± 5.67, 35.01 ± 5.84, and 19.02 ± 2.41 nm for CuNPs, Gin-CuNPs, and Gar-CuNPs, respectively, and surface plasmon resonance peaks were obtained at 570 nm, 575 nm, and 610 nm for CuNPs, Gar-CuNPs, and Gin-CuNPs, respectively. The results of FTIR confirmed the consumption of biomolecules from the plant extracts for the synthesis of CuNPs. XRD analysis also confirmed synthesis of CuNPs. Doxycycline-conjugated NPs exhibited more antibacterial effects than doxycycline or CuNPs alone. Copper nanoparticles prepared by biological synthesis are cost-effective and eco-friendly as compared to their chemical counterparts. The chemically synthesized nanoparticles displayed more significant antimicrobial activity when capped with doxycycline than Z. officinale and A. sativum-mediated CuNPs; however, green-synthesized nanoparticles showed greater anticancer activity than their chemical counterparts.

Matrix metalloprotease-9 polymorphism and its association with Atherosclerosis - A case-control study in Pakistani population.

OBJECTIVE: To determine the association of matrix metalloproteinase-9 gene with the onset of atherosclerosis in Pakistani population of Punjabi origin. METHODS: The case-control study was conducted from September 2015 to December 2016 at the Government College University, Lahore, Pakistan, and comprised atherosclerosis cases from the Punjab Institute of Cardiology, Lahore, as well as healthy controls. Single nucleotide polymorphismsrs3918242 (Sph1), rs17577 (Sty1) and rs2274756 (Taq1) were selected from the matrix metalloproteinase-9 gene for allelic and genotypic analysis. Direct sequencing and polymerase chain reaction-restriction fragment length polymorphism were performed for genotypic analysis. RESULTS: the 201 subjects, 100(49.8%) were controls and 101(50.2%) were cases. There were 75(75%) males among the controls and 70(69.3%) among the cases. Overall mean age of the controls was 47.3}13.1 years, and that of the cases was 59.2}10.2 years. Positive family history was a significant factor risk for atherosclerosis (p<0.05). Allele T and genotype CT and TT of rs3918242 were more frequent in the cases (p<0.05). Change in nucleotide at Sph1 site led towards -1562C >T polymorphism. The frequency of 'A' allele and 'GA' genotype for rs17577 was significantly higher in the cases (Sty1) (p<0.05). No association was detected between rs2274756 (Taq1) and atherosclerosis (p> 0.05). The co-expression of rs17577 and rs2274756 was significantly related with the onset of atherosclerosis (p<0.05). Haplotypes CAG, TAG and TGG were significantly involved in causing atherosclerosis (p<0.05) whereas CGG was protective against atherosclerosis in this population (p<0.05). CONCLUSIONS: Matrix metalloproteinase-9 gene was identified as a susceptible gene for the onset of atherosclerosis in Pakistani population of Punjabi origin.

Association Between Arsenic Toxicity, AS3MT Gene Polymorphism and Onset of Type 2 Diabetes.

Arsenic (As) exposure in drinking water has become a serious public health issue. AS3MT gene is involved in the metabolism of arsenic, so a single nucleotide polymorphism in this gene may lead to the development of type 2 diabetes in arsenic-exposed areas. This study aimed to evaluate the association of the AS3MT gene with the development of type 2 diabetes in highly arsenic-exposed areas of Punjab, Pakistan. Total 200 samples equal in number from high arsenic exposed-areas of Lahore (Nishtar) and Kasur (Mustafa Abad) were collected. rs11191439 was utilized as an influential variable to evaluate the association between arsenic metabolism and diabetes status to find a single nucleotide polymorphism in the AS3MT gene. We observed the arsenic level in drinking water of the arsenic-exposed selected areas 115.54 ± 1.23 µg/L and 96.88 ± 0.48 µg/L, respectively. The As level in the urine of diabetics (98.54 ± 2.63 µg/L and 56.38 ± 12.66 µg/L) was higher as compared to non-diabetics (77.58 ± 1.8 µg/L and 46.9 ± 8.95 µg/L) of both affected areas, respectively. Correspondingly, the As level in the blood of diabetics (6.48 ± 0.08 µg/L and 5.49 ± 1.43 µg/L) and non-diabetics (6.22 ± 0.12 µg/L and 5.26 ± 0.24 µg/L) in the affected areas. Genotyping showed significant differences in the frequencies of alleles among cases and controls. Nevertheless, notable disparities in genotype distribution were observed in SNPs rs11191439 (T/C) (P < 0.05) and when comparing T2D patients and non-diabetic control subjects. The AS3MT gene and clinical parameters show a significant association with the affected people with diabetes living in arsenic-exposed areas.

Green Synthesis and Characterization of Biologically Synthesized and Antibiotic-Conjugated Silver Nanoparticles followed by Post-Synthesis Assessment for Antibacterial and Antioxidant Applications.

The paper presents the antibacterial and antioxidant activities of silver nanoparticles (AgNPs) when conjugated with two antibiotics levofloxacin and ciprofloxacin as well as biologically synthesized nanoparticles from Moringa oleifera and Curcuma longa. Leaves of Moringa and powder of Curcuma were used in the green synthesis of silver nanoparticles. Ultraviolet-visible spectroscopy (UV), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM) were used for the characterization of the synthesized silver nanoparticles. Comparison of levofloxacin and ciprofloxacin and their conjugated AgNPs was also studied for antibacterial and antioxidant activity. The synthesis of Moringa-AgNPs, turmeric-AgNPs, levofloxacin-AgNPs, and ciprofloxacin-AgNPs was confirmed by UV spectroscopy. An absorption peak value of 400-450 nm was observed, and light to dark brown color indicated the synthesis of AgNPs. Moringa-AgNPs revealed high antioxidant activity (80.3 ± 3.14) among all of the synthesized AgNPs. Lev-AgNPs displayed the highest zone of inhibition for Staphylococcus aureus, while in Escherichia coli, Cip-AgNPs showed high antibacterial activity. Furthermore, AgNPs synthesized using green methods exhibit high and efficient antimicrobial activities against two food-borne pathogens. Biologically synthesized nanoparticles exhibited antibacterial activity against E. coli (13.73 ± 0.46 with Tur-AgNPs and 13.53 ± 0.32 with Mor-AgNPs) and S. aureus (14.16 ± 0.24 with Tur-AgNPs and 13.36 ± 0.77 with Mor-AgNPs) by using a well diffusion method with significant shrinkage and damage of the bacterial cell wall, whereas antibiotic-conjugated nanoparticles showed high antibacterial activity compared to biologically synthesized nanoparticles with 14.4 ± 0.37 for Cip-AgNPs and 13.93 ± 0.2 for Lev-AgNPs for E. coli and 13.3 ± 0.43 for Cip-AgNPs and 14.33 ± 0.12 for Lev-AgNPs for S. aureus. The enhanced efficiency of conjugated silver nanoparticles is attributed to their increased surface area compared to larger particles. Conjugation of different functional groups contributes to improved reactivity, creating active sites for catalytic reactions. Additionally, the precise control over the size and shape of green-synthesized nanoparticles further augments their catalytic and antibiotic activities.

Immunomodulation, Fish Health and Resistance to Staphylococcus aureus of Nile Tilapia (Oreochromis niloticus) Fed Diet Supplemented with Zinc Oxide Nanoparticles and Zinc Acetate.

Recently some metal-based nanoparticles have gained serious attention from aquaculture and the fish feed industry as feed supplements. Oral supplementation of zinc oxide nanoparticles (ZnO-NPs) in fish feed, replacing Zn acetate (conventionally used zinc), is suggested as a cost-effective and efficient approach. Our study assessed the response of Nile tilapia, Oreochromis niloticus, fingerlings after its diet supplemented with chemically synthesized ZnO-NPs and zinc acetate under controlled conditions. ZnO-NPs were chemically synthesized and characterized. Tilapia fingerlings with an average body weight of 09.12 ± 1.23 g were randomly distributed into five groups. An 8-week trial was set with control and four experimental groups. Basal diet (D1) was used as control, whereas D2, D3 and D4 comprising 20, 40, and 60 mgkg-1 ZnO-NPs supplementation were experimental diets. Additionally, D5 was composed of a basal diet supplemented with 40 mgkg-1 of conventionally used zinc acetate. Significant improvement (P < 0.05) was found in nanoparticles and Zn acetate supplemented groups as compared to control, while the 40 mgkg-1 Zn-NPs supplemented diet (D3) showed best performance in terms of health parameters, oxidative status and disease resistance. Antioxidant profiling was based on catalase, superoxide dismutase, glutathione's transferase, and malondialdehyde; hematology included Hb, WBCs, RBCs, HCT MCV, MCH and MCHC; immunological parameters comprised IgM, lysozyme activity, phagocytic activity, respiratory burst activity, cholesterol, aspartate aminotransferase, alanine aminotransferase, glucose content, and total serum proteins. We report that the D3 (40 mgkg-1 ZnO-NPs supplementation) significantly (P < 0.05) improved health-related parameters as compared to the other groups. Moreover, D3 also showed significantly decreased mortality percentage when challenged by Staphylococcus aureus, while the Zn acetate supplemented diet group showed better results as compared to control. Overall results suggest the basal diet supplemented with 40 mgkg-1 ZnO-NP for enhanced health parameters, oxidative status, immune response, and disease resistance. Hence, 40mgkg-1 ZnO-NP can be recommended to formulate the practical diet of fish to boost health improvement, immunomodulation, and resistance to bacterial disease.

Microplastics in water, sediments, and fish at Alpine River, originating from the Hindu Kush Mountain, Pakistan: implications for conservation.

Microplastics (MP) pollution is an emerging threat to life and the environment. These particles are not restricted to human-inhabited lands but also found in different mountains and glaciers where the human population is relatively low. These MPs make their way to the river ecosystem from glaciers, rains, and municipal and industrial effluents. The current study was designed to highlight MPs' pollution in water, sediments, and fishes of the Swat River: originating from the Hindu Kush Mountain Range. These samples were collected from eight different sites across the river. An average concentration of MPs detected in water samples (305.79 ± 289.66 MPs/m3), fish (12.54 ± 8.02 MPs/individual), and sediments (588.29 ± 253.95 MPs/kg). The highest concentration was observed among water samples at Mingora city and the lowest at the confluence point of the rivers near Charsadda being 753.71 ± 330.08 MPs/m3 and 57.64 ± 31.98 MPs/m3, respectively. MP concentrations in the sediment samples were also the highest at Mingora city (834.0 ± 367.21 MPs/kg), and lowest at Chakdara (215.0 ± 20.0 MPs/kg). Among the fish samples, Schizothorax plagiostomus contained the highest while Wallago attu contained the lowest MP concentrations corresponding to 17.08 ± 8.27 MPs/individual and 5.0 ± 2.36 MPs/individual, respectively. Fibers were the most prevalent MPs in all the matrices representing 80%, 92%, and 85% of the total MP count in water, sediments, and fish samples. These findings highlighted that freshwater ecosystem are not free from MPs and are as much vulnerable to anthropogenic activities as marine ecosystem. Therefore, need attention not less than marine ecosystem awareness, education, ecotourism, sustainable reduction in plastic use, and strict rules and regulations could be helpful to prevent the anthropogenic menace.

Gold nanoparticles capped with L-glycine, L-cystine, and L-tyrosine: toxicity profiling and antioxidant potential.

Biomolecules-based surface modifications of nanomaterials may yield effective and biocompatible nanoconjugates. This study was designed to evaluate gold nanoconjugates (AuNCs) for their altered antioxidant potential. Gold nanoparticles (AuNPs) and their conjugates gave SPR peaks in the ranges of 512-525 nm, with red or blueshift for different conjugates. Cys-AuNCs demonstrated enhanced (p < 0.05) and Gly-AuNCs (p > 0.05) displayed reduced DPPH activity. Gly-AuNCs and Tyr-AuNCs displayed enhanced ferric-reducing power and hydrogen peroxide scavenging activity, respectively. Cadmium-intoxicated mice were exposed to gold nanomaterials, and the level of various endogenous parameters, i.e., CAT, GST, SOD, GSH, and MTs, was evaluated. GSH and MTs in liver tissues of the cadmium-exposed group (G2) were elevated (p < 0.05), while other groups showed nonsignificance deviations than the control group. It is concluded that these nanoconjugates might provide effective nanomaterials for biomedical applications. However, more detailed studies for their safety profiling are needed before their practical applications.

First Report on Microplastics Quantification in Poultry Chicken and Potential Human Health Risks in Pakistan.

Microplastics (MPs) are an emerging environmental health concern due to their widespread occurrence in food sources such as fish, meat, chicken, honey, sugar, salt, tea and drinking water, thereby posing possible risks to human health. This study aimed to observe the existence of MPs in the crop and gizzard of the farm chicken, a significant food source in Pakistan. Twenty-four chicken samples were taken from eight poultry farms across Punjab, Pakistan. A total of 1227 MP particles were found from 24 samples (crop and gizzards) originating from the 8 poultry farms. In all, 429 MP particles were found in 24 chicken crops, with a mean of 17.8 ± 12.1 MPs/crop. In contrast, 798 MP particles were found in 24 chicken gizzards, with a mean of 33.25 ± 17.8 MPs/gizzard. Comparatively larger particles, ranging between 300-500 µm, were more abundant (63%) than other considered sizes (300-150 µm [21%] and 150-50 µm [16%]). Additionally, fragments were the dominant type of shape in both sample types (crop [64%] and gizzard [53%]). The predominant colours of particles extracted from gizzards and crops were yellow (32%) and red (32%), respectively. Chemical characterisation of these particles detected four types of polymers: polyvinyl chloride (PVC) at 51.2%, followed by low-density polyethylene (LDPE) at 30.7%, polystyrene (PS) at 13.6% and polypropylene homopolymer (PPH) at 4.5%. In conclusion, we provide evidence for MPs in the gizzards and crops of farmed chickens which may originate from contaminated poultry feed. Only a few studies have been reported globally to assess MPs ingestion in chickens. The current study is the first report from Pakistan. It could be a valuable addition to support MPs literature to establish a relationship between MPs contamination and intake through the food chain.

Microplastic Quantification in Aquatic Birds: Biomonitoring the Environmental Health of the Panjkora River Freshwater Ecosystem in Pakistan.

Microplastic pollution has become a global concern, with potential negative impacts on various ecosystems and wildlife species. Among these species, ducks (Anas platyrhynchos) are particularly vulnerable due to their feeding habits and proximity to aquatic environments contaminated with microplastics. The current study was designed to monitor microplastic (MP) pollutants in the freshwater ecosystem of the Panjkora River, Lower Dir, Pakistan. A total of twenty (20) duck samples were brought up for four months and 13 days on the banks of the river, with no food intake outside the river. When they reached an average weight of 2.41 ± 0.53 kg, all samples were sacrificed, dissected, and transported in an ice box to the laboratory for further analysis. After sample preparation, such as digestion with 10% potassium hydroxide (KOH), density separation, filtration, and identification, the MP content was counted. A total of 2033 MP particles were recovered from 20 ducks with a mean value of 44.6 ± 15.8 MPs/crop and 57.05 ± 18.7 MPs/gizzard. MPs detected in surface water were 31.2 ± 15.5 MPs/L. The major shape types of MPs recovered were fragments in crop (67%) and gizzard (58%) samples and fibers in surface water (56%). Other types of particles recovered were fibers, sheets, and foams. The majority of these detected MP particles were in the size range of 300-500 µm (63%) in crops, and 50-150 µm (55%) in gizzards, while in water samples the most detected particles were in the range of 150-300 µm (61%). Chemical characterization by FTIR found six types of polymers. Low-density polyethylene (LDPE) had the greatest polymer detection rate (39.2%), followed by polyvinyl chloride (PVC) (28.3%), high-density polyethylene (HDPE) (22.7%), polystyrene (6.6%), co-polymerized polypropylene (2.5%), and polypropylene homopolymer (0.7%). This study investigated the presence of microplastics in the crops and gizzards of ducks, as well as in river surface water. The results revealed the significant and pervasive occurrence of microplastics in both the avian digestive systems and the surrounding water environment. These findings highlight the potential threat of microplastic pollution to wildlife and ecosystems, emphasizing the need for further research and effective mitigation strategies to address this pressing environmental concern.

Colorimetric Detection of Mercury Ions in Water with Capped Silver Nanoprisms.

The emission of mercury (II) from coal combustion and other industrial processes may have impacts on water resources, and the detection with sensitive but rapid testing methods is desirable for environmental screening. Towards this end, silver nanoprisms were chemically synthesized resulting in a blue reagent solution that transitioned towards red and yellow solutions when exposed to Hg2+ ions at concentrations from 0.5 to 100 µM. A galvanic reduction of Hg2+ onto the surfaces is apparently responsible for a change in nanoprism shape towards spherical nanoparticles, leading to the change in solution color. There were no interferences by other tested mono- and divalent metal cations in solution and pH had minimal influence in the range of 6.5 to 9.8. The silver nanoprism reagent provided a detection limit of approximately 1.5 µM (300 µg/L) for mercury (II), which compared reasonably well with other reported nanoparticle-based techniques. Further optimization may reduce this detection limit, but matrix effects in realistic water samples require further investigation and amelioration.

Larval gut pH profile in pestiferous Chironomus crassicaudatus and Glyptotendipes paripes (Chironomidae: Diptera) in reference to the toxicity potential of Bacillus thuringiensis serovar israelensis.

Gut pH and pH optimum of amylase were measured in 4th-stage larvae of 2 pestiferous chironomid species, Chironomus crassicaudatus and Glyptotendipes paripes. The gut pH in both species was close to neutral, varying from 6.7 to 7.4 and 6.9 to 7.6 pH units for C. crassicaudatus and G. paripes, respectively. In both species, slightly alkaline pH values were measured in the anterior part of the mesenteron, whereas slightly acidic pH values were recorded in gastric caecae and the posterior part of mesenteron. Amylase pH optimum in both chironomid species remained between pH values of 5.5 and 7, being closer to pH value 7 than 5.5. This profile was consistent with the measured values of the gut pH. The recorded larval gut pH profile in the 2 species of chironomids in the present study is much lower than gut pH reported for mosquitoes or Lepidoptera larvae. This could be the reason for the relatively much lower susceptibility of chironomid larvae to Bacillus thuringiensis serovar israelensis toxin proteins than some other nematoceran Diptera, specifically mosquitoes.

Colorimetric enumeration of bacterial contamination in water based on ß-galactosidase gold nanoshell activity.

In this study we report a method for the rapid and sensitive estimation of bacterial cell concentration in solution based on a colorimetric enzyme/gold nanoshells conjugate system. The CTAB capped gold nanoshells are electrostatically attracted by both the bacterial surface and the enzyme ß-galactosidase. The preferential binding of cationic (CTAB)-functionalized gold nanoshells to the more negative bacterial surfaces leaves active ß-galactosidase in solution, providing an enzyme-amplified colorimetric response of the binding event. A progressive increase in the enzyme activity is evidenced by the conversion of the yellow-orange CPRG substrate into the red chromophore chlorophenol red, which can be correlated with increasing bacterial cell numbers. Using this strategy, the quantification of bacteria at concentrations as low as 10 bacteria/mL of solution has been achieved. The present method of bacterial cell load assessment offers a distinct potential advantage over other conventional methods such as plate counting in terms of ease of operation, rapidity, high sensitivity and quantitative detection of bacterial cells.

Poly-L-arginine Coated Silver Nanoprisms and Their Anti-Bacterial Properties.

The aim of this study was to test the effect of two different morphologies of silver nanoparticles, spheres, and prisms, on their antibacterial properties when coated with poly-L-arginine (poly-Arg) to enhance the interactions with cells. Silver nanoparticle solutions were characterized by UV-visible spectroscopy, transmission electron microscopy, dynamic light scattering, zeta potential, as well as antimicrobial tests. These ultimately showed that a prismatic morphology exhibited stronger antimicrobial effects against Escherichia coli, Pseudomonas aeruginosa and Salmonella enterica. The minimum bactericidal concentration was found to be 0.65 µg/mL in the case of a prismatic AgNP-poly-Arg-PVP (silver nanoparticle-poly-L-arginine-polyvinylpyrrolidone) nanocomposite. The anticancer cell activity of the silver nanoparticles was also studied, where the maximum effect against a HeLa cell line was 80% mortality with a prismatic AgNP-poly-Arg-PVP nanocomposite at a concentration of 11 µg/mL. The antimicrobial activity of these silver nanocomposites demonstrates the potential of such coated silver nanoparticles in the area of nano-medicine.

Genetic diversity in cytochrome c oxidase I gene of Anopheles mosquitoes.

Genetic diversity in cytochrome c oxidase I (coxI) among 7 species of Anopheles mosquitoes from Pakistan, and 37 species from different geographical regions of the world, was recorded. Automatic Barcode Gap Discovery (ABGD) analysis revealed a clear gap between intraspecific and interspecific distances of 7 species from Pakistan. However, genetic distances of 37 Anopheles species failed to adequately differentiate species in a global context. Intraspecific and interspecific divergences for 7 Anopheles species of Pakistan varied from 0.0% to 2.5% (mean = 0.49%) and 8% to 22.3% (mean = 12.77%), respectively. Similarly, intraspecific distances for 37 species from different parts of world ranged from 0.0% to 11.2% (mean = 0.65%) while values of interspecific divergences ranged from 3.4% to 35% (mean = 11.75%). Although phylogenetic tree revealed separate clades for 7 Anopheles species of Pakistan, it failed to produce separate clades for 37 species of the world. It is concluded that although standard barcode region is helpful for identifying Anopheles mosquitoes, combination of multi-locus approaches and morphology may be required to accurately identify species in this genus.