Green Chemistry Based Synthesis of Zinc Oxide Nanoparticles Using Plant Derivatives of Calotropis gigantea (Giant Milkweed) and Its Biological Applications against Various Bacterial and Fungal Pathogens.

Nanotechnology is a burning field of scientific interest for researchers in current era. Diverse plant materials are considered as potential tool in green chemistry based technologies for the synthesis of metal nanoparticles (NPs) to cope with the hazardous effects of synthetic chemicals, leading to severe abiotic climate change issues in today's agriculture. This study aimed to determine the synthesis and characterization of metal-based nanoparticles using extracts of the selected plant Calotropis gigantea and to evaluate the enzyme-inhibition activities and antibacterial and antifungal activity of extracts of metal-based zinc nanoparticles using C. gigantea extracts. The crystal structure and surface morphology were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM). C. gigantea was examined for antimicrobial activity against clinical isolates of bacteria and fungi. The water, ethanolic, and acetone extracts of C. gigantea were studied for their antagonistic action against bacterial strains (E. coli, S. aureus, P. multocida, and B. subtilis) and selected fungal strains (A. paracistic, F. solani, A. niger, S. ferrugenium, and R. nigricans). In vitro antimicrobial activity was determined by the disc diffusion method, where C. gigantea wastested for AChE and BChE inhibitory activity using Ellman's methodology. The kinetic analysis was performed by the proverbial Berthelot reaction for urease inhibition. The results showed that out of all the extracts tested, ethanolic and water extracts possessed zinc nanoparticles. These extracts showed the maximum zone of inhibition against F. solani and P. multocida and the lowest against S. ferrugenium and B. subtilis. A potential source of AChE inhibitors is certainly provided by the abundance of plants in nature. Numerous phyto-constituents, such as AChE and BChE inhibitors, have been reported in this communication. Water extract was active and has the potential for in vitro AChE and BChE inhibitory activity. The urease inhibition with flower extracts of C. gigantea revealed zinc nanoparticles in water extracts that competitively inhibited urease enzymes. In the case of cholinesterase enzymes, it was inferred that the water extract and zinc nanoparticles have more potential for inhibition of BChE than AChE and urease inhibition. Furthermore, zinc nanoparticles with water extract are active inthe inhibition of the bacterial strains E. coli, S. aureus, and P. multocida and the fungal strains A. paracistic, F. solani, and A. niger.

Evaluation of Bacterial Perpetuation Assays and Plant Biomolecules Antimicrobial Activity against Cotton Blight Bacterium Xanthomonas citri subsp. malvacearum; An Alternative Source for Food Production and Protection.

Cotton (Gossypium hirsutum) is a global cash crop which has gained importance in earning foreign exchange for each country. Bacterial blight caused by Xanthomonascitri subsp. malvacearum (Xcm) has been a seriousdisease in Pakistan's cotton belt on multiple occasions. Bacterium was isolated and identified through various biochemical and diagnostic tests wherehypersensitivity reaction, Gram staining, KOH (potassium hydroxide), catalase, starch hydrolysis, lecithinase and Tween 80 hydrolysis tests confirmed bacterium as Gram-negative and plant pathogenic. Xcm perpetuation assays wereevaluated on various cotton varieties under glasshouse conditions in completely randomized design by three different methods, wherein the scratch method proved to be the best upon CIM-496 and showed 83.33% disease incidence as compared with the other two methods, where Bt-3701 responded with 53.33% incidence via the spray gun method, and 50% with the water splash method on CIM-616, as compared with the control. Similarly, for disease severity percentage, Bt-3701 was pragmatic with 47.21% through scratch method, whereas, in the spray gun method, 45.51% disease severity was noted upon Bt-802, and 31.27% was calculated on Cyto-179 through the water splash method. Owing to the unique antibacterial properties of aqueous plant extracts, the poison food technique showed Aloe vera, Mentha piperita, Syzygiumcumini and Azadirachta indica with 17.77, 29.33, 18.33 and 20.22 bacterial colonies counted on nutrient agarmedium petri plate, respectively, as compared with the control. Measurement of the inhibition zone by disk diffusion technique showed Mentha piperita, Syzygiumcumini, Citrus limon, Moringa oleifera and Syzygium aromaticum to present the most promising results by calculating the maximum diameter of the inhibition zone, viz., 8.58, 8.55, 8.52, 8.49 and 8.41 (mm), respectively, at the highest tested concentration (75 ppm, parts per million) compared with the control. It is probable that the decoction's interaction with the pathogen population on the host plant will need to be considered in future experiments. However, at this moment, more research into the effective management of cotton bacterial blight by plant extracts in terms of concentration determination and development of biopesticides will provide future avenues to avoid environmental pollution.

K-29 linked ubiquitination of Arrdc4 regulates its function in extracellular vesicle biogenesis.

Extracellular vesicles (EVs) are important mediators of intercellular communication. However, EV biogenesis remains poorly understood. We previously defined a role for Arrdc4 (Arrestin domain containing protein 4), an adaptor for Nedd4 family ubiquitin ligases, in the biogenesis of EVs. Here we report that ubiquitination of Arrdc4 is critical for its role in EV secretion. We identified five potential ubiquitinated lysine residues in Arrdc4 using mass spectrometry. By analysing Arrdc4 lysine mutants we discovered that lysine 270 (K270) is critical for Arrdc4 function in EV biogenesis. Arrdc4K270R mutation caused a decrease in the number of EVs released by cells compared to Arrdc4WT , and a reduction in trafficking of divalent metal transporter (DMT1) into EVs. Furthermore, we also observed a decrease in DMT1 activity and an increase in its intracellular degradation in the presence of Arrdc4K270R . K270 was found to be ubiquitinated with K-29 polyubiquitin chains by the ubiquitin ligase Nedd4-2. Thus, our results uncover a novel role of K-29 polyubiquitin chains in Arrdc4-mediated EV biogenesis and protein trafficking.

Successful Outpatient Management of Children at a Secondary Care Hospital in Pakistan in a Dengue Fever Epidemic and Their Clinical Outcomes.

BACKGROUND: There is limited published literature on the feasibility of WHO 2009 guidelines for the management of dengue fever (DF) in Pakistani children. This study aimed to assess the outcome of children with DF who received outpatient treatment according to these guidelines during a DF epidemic. METHOD: This was a prospective cohort study conducted at Federal General Hospital, a secondary care hospital, Islamabad, Pakistan, from 1st August to 31st October 2019. Using WHO DF 2009 guidelines, children ≤13 years, diagnosed as confirmed DF (NS1 Ag +), were classified into the outpatient (DF) or the inpatient group (DF with warning signs or severe dengue (SD)). The inpatient group was admitted to the Pakistan Institute of Medical Sciences, a tertiary care hospital, and discharged on recovery. These children were followed for the primary outcome, i.e., recovery or hospitalization by day 14 of enrollment. Additionally, clinical and laboratory features (Hb, HCT, TLC, PLT, and ALT) of the patients in the outpatient who remained stable with those who progressed to inpatient care during follow-up were compared; also, time of recovery of blood counts was assessed. RESULTS: Of 93 children with DF, 87 (93.5%) received outpatient care at enrollment. Of these, 6 (7.8%) deteriorated by day 7 and were admitted to inpatient care. SD was present in 6/93 (6.4%) patients at presentation and were admitted. All children showed signs of recovery until day 14. Male gender (p=0.049), lower normal mean platelet (p=0.02), and high mean hematocrit (p=0.001) were associated with disease progression. CONCLUSION: The majority of children with confirmed DF who received outpatient treatment according to WHO 2009 guidelines were successfully managed. Additionally, confirmed DF with warning signs or SD were admitted and recovered. Regular follow-ups according to the guidelines are pertinent. Thrombocytopenia and high HCT were associated with disease progression.

Paediatric Contacts of Adult COVID-19 Patients: Clinical Parameters, Risk Factors, and Outcome.

BACKGROUND: There is insufficient data in Pakistan and in South Asia regarding paediatric COVID-19 demographics and related parameters. The main aim of this study was to assess the paediatric population exposed to SARS-CoV-2 infection, their clinical parameters, risk factors, and outcome. METHODS: This was a descriptive retrospective study conducted at the Pakistan Institute of Medical Sciences and Federal General Hospital Islamabad from 23rd July 2020 to 22nd August 2020. All paediatric contacts (≤13 years) of one hundred adult COVID-19 patients were included. Data of the index cases was taken from the medical records. Paediatric data was collected on the phone using a predesigned proforma. RESULTS: There were 137 paediatric contacts of 100 adult COVID-19 index cases. The index cases were predominantly males (67%) and belonged to the middle socioeconomic class (89%), and 14% succumbed to the disease. Females had more paediatric contacts. The mean age of contacts was 6.6 years, and the majority (80%) developed no symptoms. Among the symptomatic contacts, fever and cough were the most common symptoms. None of the contacts developed dyspnoea or required hospitalization. Majority of the contacts had been vaccinated with the BCG vaccine. Testing for COVID-19 was done in only 77 (56%) contacts, 25 (32%) by the government team, and 52 (67%) privately. A higher number of symptomatic contacts were positive (15/17 (88%)) as compared to that of the asymptomatic contacts (6/60 (10%)) (p = 0.002). Development of symptoms in the contacts was associated with the history of respiratory illnesses, recurrent infections, use of hematinics, a positive COVID-test result, and health professionals being index cases (p ≤ 0.01). Parents with higher education and in the health profession and the families of symptomatic contacts reported better compliance with quarantine regulations. CONCLUSION: A significant number of children were exposed to adult COVID-19 patients. Most paediatric contacts remained asymptomatic. Children with preexisting medical conditions and with parents in health profession were susceptible to infection.