Effects of Drying Temperature and Solvents on In Vitro Diabetic Wound Healing Potential of Moringa oleifera Leaf Extracts.

The delayed healing of wounds among people with diabetes is a severe problem worldwide. Hyperglycemia and increased levels of free radicals are the major inhibiting factors of wound healing in diabetic patients. Plant extracts are a rich source of polyphenols, allowing them to be an effective agent for wound healing. Drying temperature and extraction solvent highly affect the stability of polyphenols in plant materials. However, there is a need to optimize the extraction protocol to ensure the efficacy of the final product. For this purpose, the effects of drying temperature and solvents on the polyphenolic composition and diabetic wound healing activity of Moringa oleifera leaves were examined in the present research. Fresh leaves were oven dried at different temperatures (10 °C, 30 °C, 50 °C, and 100 °C) and extracted in three solvents (acetone, ethanol, and methanol) to obtain twelve extracts in total. The extracts were assessed for free radical scavenging and antihyperglycemic effects using DPPH (2,2-diphenylpicrylhydrazyl) and α- glucosidase inhibition assays. Alongside this, a scratch assay was performed to evaluate the cell migration activity of M. oleifera on the human retinal pigment epithelial cell line. The cytotoxicity of the plant extracts was assessed on human retinal pigment epithelial (RPE) and hepatocellular carcinoma (Huh-7) cell lines. Using high-performance liquid chromatography, phenolic compounds in extracts of M. oleifera were identified. We found that an ethanol-based extract prepared by drying the leaves at 10 °C contained the highest amounts of identified polyphenols. Moringa oleifera extracts showed remarkable antioxidant, antidiabetic, and cell migration properties. The best results were obtained with leaves dried at 10 °C and 30 °C. Decreased activities were observed with drying temperatures of 50 °C and above. Moreover, M. oleifera extracts exhibited no toxicity on RPE cells, and the same extracts were cytotoxic for Huh-7 cells. This study revealed that M. oleifera leaves extracts can enhance wound healing in diabetic conditions due to their antihyperglycemic, antioxidant, and cell migration effects. The leaves of this plant can be an excellent therapeutic option when extracted at optimum conditions.

Ecofriendly phytosynthesized zirconium oxide nanoparticles as antibiofilm and quorum quenching agents against Acinetobacter baumannii.

The worldwide increase of multi-drug resistance has directed the researchers to focus on ecofriendly ways of nanoparticles synthesis with effective antivirulence properties. Here, we report the antibiofilm and quorum quenching (QQ) potential of zirconium oxide nanoparticles (ZrO2 NPs) synthesized from aqueous ginger extract against multi-drug resistant (MDR) Acinetobacter baumannii. The results indicated that ZrO2 NPs were of tetragonal shape with average diameter of 16 nm. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values for A. baumannii were 15.6 and 62.5 µg/ml, respectively, as revealed by broth microdilution assay. Exposure of bacterial cells to ZrO2 NPs resulted in reactive oxygen species (ROS) generation which in turn led to cellular membrane disruption as observed by an increase in leakage of cellular contents, such as proteins, sugars, and DNA. The antibiofilm activity was evaluated by microtiter plate assay and the results revealed that the percentage inhibition of biofilm was found to be 14.3-80.6%. ZrO2 NPs also obstructed the chemical composition of biofilms matrix by reducing the proteins and carbohydrate contents. Molecular docking studies of ZrO2 NPs with four proteins (2NAZ, 4HKG, 5D6H, and 5HM6) involved in biofilm formation of A. baumannii revealed the interaction of zirconium with target proteins. These findings suggested the in vitro efficacy of phytosynthesized ZrO2 NPs as antibiofilm and QQ agents that can be exploited in the development of alternative therapeutic options against MDR A. baumannii.

Biofabrication of ZnO nanoparticles using Acacia arabica leaf extract and their antibiofilm and antioxidant potential against foodborne pathogens.

Emergence of multidrug resistant pathogens is increasing globally at an alarming rate with a need to discover novel and effective methods to cope infections due to these pathogens. Green nanoparticles have gained attention to be used as efficient therapeutic agents because of their safety and reliability. In the present study, we prepared zinc oxide nanoparticles (ZnO NPs) from aqueous leaf extract of Acacia arabica. The nanoparticles produced were characterized through UV-Visible spectroscopy, scanning electron microscopy, and X-ray diffraction. In vitro antibacterial susceptibility testing against foodborne pathogens was done by agar well diffusion, growth kinetics and broth microdilution assays. Effect of ZnO NPs on biofilm formation (both qualitatively and quantitatively) and exopolysaccharide (EPS) production was also determined. Antioxidant potential of green synthesized nanoparticles was detected by DPPH radical scavenging assay. The cytotoxicity studies of nanoparticles were also performed against HeLa cell lines. The results revealed that diameter of zones of inhibition against foodborne pathogens was found to be 16-30 nm, whereas the values of MIC and MBC ranged between 31.25-62.5 µg/ml. Growth kinetics revealed nanoparticles bactericidal potential after 3 hours incubation at 2 × MIC for E. coli while for S. aureus and S. enterica reached after 2 hours of incubation at 2 × MIC, 4 × MIC, and 8 × MIC. 32.5-71.0% inhibition was observed for biofilm formation. Almost 50.6-65.1% (wet weight) and 44.6-57.8% (dry weight) of EPS production was decreased after treatment with sub-inhibitory concentrations of nanoparticles. Radical scavenging potential of nanoparticles increased in a dose dependent manner and value ranged from 19.25 to 73.15%. Whereas cytotoxicity studies revealed non-toxic nature of nanoparticles at the concentrations tested. The present study suggests that green synthesized ZnO NPs can substitute chemical drugs against antibiotic resistant foodborne pathogens.

BIN overlap confirms transcontinental distribution of pest aphids (Hemiptera: Aphididae).

DNA barcoding is highly effective for identifying specimens once a reference sequence library is available for the species assemblage targeted for analysis. Despite the great need for an improved capacity to identify the insect pests of crops, the use of DNA barcoding is constrained by the lack of a well-parameterized reference library. The current study begins to address this limitation by developing a DNA barcode reference library for the pest aphids of Pakistan. It also examines the affinities of these species with conspecific populations from other geographic regions based on both conventional taxonomy and Barcode Index Numbers (BINs). A total of 809 aphids were collected from a range of plant species at sites across Pakistan. Morphological study and DNA barcoding allowed 774 specimens to be identified to one of 42 species while the others were placed to a genus or subfamily. Sequences obtained from these specimens were assigned to 52 BINs whose monophyly were supported by neighbor-joining (NJ) clustering and Bayesian inference. The 42 species were assigned to 41 BINs with 38 showing BIN concordance. These species were represented on BOLD by 7,870 records from 69 countries. Combining these records with those from Pakistan produced 60 BINs with 12 species showing a BIN split and three a BIN merger. Geo-distance correlations showed that intraspecific divergence values for 49% of the species were not affected by the distance between populations. Forty four of the 52 BINs from Pakistan had counterparts in 73 countries across six continents, documenting the broad distributions of pest aphids.

Application of Organic Fertilizers Affect the Citrus Leafminer, Phyllocnistis citrella (Lepidoptera: Gracillariidae) Infestation and Citrus Canker Disease in Nursery Plantations.

Citrus leafminer (CLM), Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae), is one of the most important insect pests of Pakistan's citrus nursery stock and caused extensive damage to young flushes. The organic compost is a widespread technique used to manage insect pests and plant diseases. Different composts (biofert, tara root and vermicompost) at 0.5 and 0.25 kg/plant concentration in comparison to NPK fertilizer at 0.4 and 0.2 g/plant were evaluated for CLM infestation and the associated citrus canker disease in nursery plantations of Citrus reticulata Blanco. Application of biofert at 0.5 kg/plant reduced the CLM infestation up to 54.5% during Fall-2016 and 39.1% during Summer-2017 in comparison to control treatment. The CLM larval density was also found lower by the application of biofert followed by vermicompost during both seasons. Both concentrations of biofert followed by vermicompost at 0.5 kg/plant resulted in remarkable protection against citrus canker disease in both flushes. The incidence of canker associated with CLM infested leaves was also studied and found lower by the application of biofert and vermicompost compared with control treatment. Conclusively, the soil amendment using biofert and vermicompost affects the CLM population and canker infection in nursery plantations. These organic fertilizers can be used in future citrus IPM programs as a tool to suppress the CLM population and citrus canker disease.

Detection of Mycobacterium tuberculosis in AFB smear-negative sputum specimens through MTB culture and GeneXpert® MTB/RIF assay.

Tuberculosis (TB) is an important public health issue around the globe which is a chronic infectious disease and is still one of the major challenges for developing countries. The emergence of drug-resistant TB makes the condition worse and there is an urgent need of fast, highly sensitive diagnostic methods. This study was undertaken to evaluate the performance of GeneXpert® MTB/RIF assay and MTB culture for the detection of Mycobacterium tuberculosis (MTB) in sputum smear-negative pulmonary TB/drug-resistant tuberculosis (DR-TB) suspects. A total of 168 sputum smear-negative TB suspects were recruited for the study. Among the suspected TB cases, 52.98% were male and 47.02% were females with the mean age of 42 ± 17.6 years. All the sputum specimens collected from the study population were subjected to Ziehl-Neelsen (ZN) smear microscopy, GeneXpert MTB/RIF assay, and MTB culture. The results revealed that, out of 168 acid-fast bacilli (AFB)/ZN smear microscopy-negative sputum specimens, 48 (28.57%) and 58 (34.52%) were detected MTB positive by GeneXpert MTB/RIF assay and MTB culture, respectively, while 120 (71.43%) and 110 (65.48%) suspected TB cases were confirmed negative by GeneXpert MTB/RIF assay and MTB culture, respectively. The study concluded that GeneXpert assay was found to be a rapid and accurate tool for MTB detection in smear-negative sputum specimens. GeneXpert has advantage over ZN smear microscopy and MTB culture as it detects MTB and rifampicin resistance simultaneously within 2 h with minimal biohazards.

Inoculation of Nicotiana tabacum with recombinant potato virus X induces RNA interference in the solenopsis mealybug, Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae).

OBJECTIVES: The chitin synthase 1 (CHS1) gene in Phenacoccus solenopsis (PsCHS1) was evaluated as a potential target of RNA interference (RNAi) by using Potato virus X (PVX) as a vector (recombinant PVX) for expressing RNAi triggering elements in Nicotiana tabacum L. RESULTS: RT-PCR analysis confirmed the expression of PsCHS1 in N. tabacum inoculated with recombinant-PVX-PsCHS1 (treated). RT- and multiplex-PCR further showed a reduction in mRNA levels of the target gene in mealybugs feeding on treated plants. Mortality in parent adults and emerging nymphs (21 and 29%) exposed to the treated plants was significantly higher (P < 0.05) than those exposed to uninoculated (-ve control) or inoculated with non-recombinant PVX (PVX-control). The number of surviving adults and the combined number of adults and nymphs (47 and 60%) was significantly (P < 0.05) lower on the treated plants than the -ve (76%) or PVX (74%) control. The visual observations verified the physical deformities in mealybugs exposed to the treated plants. CONCLUSION: chitin synthase 1 is a potential RNAi target in P. solenopsis and the recombinant PVX can be used as a tool to evaluate candidate RNAi triggering elements in plants.

Use of recombinant tobacco mosaic virus to achieve RNA interference in plants against the citrus mealybug, Planococcus citri (Hemiptera: Pseudococcidae).

The citrus mealybug, Planococcus citri, is an important plant pest with a very broad plant host range. P. citri is a phloem feeder and loss of plant vigor and stunting are characteristic symptoms induced on a range of host plants, but P. citri also reduces fruit quality and causes fruit drop leading to significant yield reductions. Better strategies for managing this pest are greatly needed. RNA interference (RNAi) is an emerging tool for functional genomics studies and is being investigated as a practical tool for highly targeted insect control. Here we investigated whether RNAi effects can be induced in P. citri and whether candidate mRNAs could be identified as possible targets for RNAi-based P. citri control. RNAi effects were induced in P. citri, as demonstrated by specific target reductions of P. citri actin, chitin synthase 1 and V-ATPase mRNAs after injection of the corresponding specific double-stranded RNA inducers. We also used recombinant Tobacco mosaic virus (TMV) to express these RNAi effectors in Nicotiana benthamiana plants. We found that P. citri showed lower fecundity and pronounced death of crawlers after feeding on recombinant TMV-infected plants. Taken together, our data show that actin, chitin synthase 1 and V-ATPase mRNAs are potential targets for RNAi against P. citri, and that recombinant TMV is an effective tool for evaluating candidate RNAi effectors in plants.