Rhodamine B functionalized silver nanoparticles paper discs as turn-on fluorescence sensor, coupled with a smartphone for the detection of microbial contamination in drinking water.

The precise detection of pathogenic microorganisms is crucial for the reduction of water-borne diseases. Herein, a filter-paper-based florescent chemosensor was fabricated for the detection of Escherichia coli and Staphylococcus aureus contamination exploiting protein-DNA interaction between the target and a specific probe. The sensing mechanism involved the self-assembly of Rhodamine B (RhB) on silver nanoparticles (AgNPs) surface that was labeled with a single-stranded DNA probe. This causes the fluorescence quenching of RhB by a distant-dependant process. The hybridization between pathogen-specific probe and bacterial surface protein causes the release of fluorescence of RhB, which was observed under UV light. For paper-based bio-surface preparation, the mixture comprising RhB-AgNP-ssDNA was drop-casted on filter paper discs. The conditions were optimized using isolated genomic DNA of the microbes. The method was applied for E.coli detection using an eae gene-based probe targeting intimin protein and S. aureus detection using tuf gene-based probe targeting EF-tuf protein on the microbe's surface. The chemosensor had a notable specificity and selectivity for E.coli, and S. aureus, with detection limits of 0.6 × 108 and 0.37 × 103 CFU/mL respectively. Moreover, the sensor was tested on real water samples, which presented excellent reproducibility of results (RSD ≤ 0.24%). Furthermore, the gradient change of fluorescence was captured by a smartphone, which allows direct detection of pathogens in a sensitive semi-quantitative way without the need for expensive instruments. The designed chemosensor can serve as a simple, inexpensive, and rapid method for the on-site detection of microbial contamination in drinking water.

Wire- and magnetic-seed-guided localization of impalpable breast lesions: iBRA-NET localisation study.

BACKGROUND: Wire localization is historically the most common method for guiding excision of non-palpable breast lesions, but there are limitations to the technique. Newer technologies such as magnetic seeds may allow some of these challenges to be overcome. The aim was to compare safety and effectiveness of wire and magnetic seed localization techniques. METHODS: Women undergoing standard wire or magnetic seed localization for non-palpable lesions between August 2018 and August 2020 were recruited prospectively to this IDEAL stage 2a/2b platform cohort study. The primary outcome was effectiveness defined as accurate localization and removal of the index lesion. Secondary endpoints included safety, specimen weight and reoperation rate for positive margins. RESULTS: Data were accrued from 2300 patients in 35 units; 2116 having unifocal, unilateral breast lesion localization. Identification of the index lesion in magnetic-seed-guided (946 patients) and wire-guided excisions (1170 patients) was 99.8 versus 99.1 per cent (P = 0.048). There was no difference in overall complication rate. For a subset of patients having a single lumpectomy only for lesions less than 50 mm (1746 patients), there was no difference in median closest margin (2 mm versus 2 mm, P = 0.342), re-excision rate (12 versus 13 per cent, P = 0.574) and specimen weight in relation to lesion size (0.15 g/mm2versus 0.138 g/mm2, P = 0.453). CONCLUSION: Magnetic seed localization demonstrated similar safety and effectiveness to those of wire localization. This study has established a robust platform for the comparative evaluation of new localization devices.

Impalpable breast lesion localisation, a logistical challenge: results of the UK iBRA-NET national practice questionnaire.

INTRODUCTION: Breast conserving surgery of impalpable breast lesions requires safe and effective localisation techniques. Wire localisation has traditionally been used, but has limitations. Newer techniques are now being introduced to mitigate this. The iBRA-NET group aims to robustly evaluate these new techniques in well-designed prospective studies. We report the first phase of this evaluation, a survey to establish current practice and service provision of breast localisation techniques in the UK. METHODS: A national practice questionnaire was designed using 'SurveyMonkey®' and was circulated to UK breast surgeons via the Association of Breast Surgery and the Mammary Fold. The questionnaire was live from 6th October 2018 to 6th April 2019. Only one response per unit was requested to reflect the unit's practice. RESULTS: Complete responses were received from 98 breast units across the UK. Wires were the mostly commonly used localisation technique (n = 82) with fewer units using Magseed® (n = 9), Radioguided Occult Lesion Localisation (n = 5) and Radioiodine Seed Localisation (n = 2). There was significant variation in practice and logistics involved. Frequent delays and theatre overruns were reported in 39 and 16 units, respectively. The median satisfaction score of the current technique was 7 out of 10. The main perceived limitation of existing localisation methods was logistics affecting theatre scheduling and the main barrier to introducing a new technique was cost. CONCLUSION: Wires are currently the most commonly used localisation technique but are associated with significant logistical issues. Newer techniques may offer a better solution but will need robust evaluation before they are adopted to ensure safety and efficacy.

A Method Based on a Modified Fluorescence In Situ Hybridization (FISH) Approach for the Sensing of Staphylococcus aureus from Nasal Samples.

Staphylococcus aureus is a major source of bacteremia and develops several complications, causing high morbidity and mortality. Rapid identification and detection of these bacteria have become an important issue for biomedical applications. Herein, an optical method based on a modified fluorescence in situ hybridization (FISH) approach has been established using DNA hybridization technology for the swift detection of pathogenic S. aureus from clinical samples. The platform was constructed with single-stranded genomic DNA and microbial colony by directly immobilizing in agarose-polyvinyl alcohol (AG-PVA) hydrogel on the surface of a glass slide. The probe was based on an elongation factor encoding the tuf gene, which binds with equal affinity to single-stranded DNA targets as well as surface proteins on microbial cells. The probe was labeled with MFP488 fluorophore having excitation wavelength 501 nm. The hybridization of the labeled probe with the target DNA and surface proteins was carried out under optimal FISH conditions, and the detection of bacteria was based on temporary field excitation of the labeled probe under a fluorescence microscope. Positive hybridization signals were detected by high fluorescence intensity. In comparison to genomic DNA, robust signals were observed with microbial cells, perhaps due to the moonlighting effect of the elongation factor Tu (Ef-Tu) expressed on the surface of bacterial cells. The applicability of the developed platform was tested on pediatric nasal samples, and results were verified with real-time qPCR. The designed platform is stable and sensitive, and after detailed optimization, a portable structure for on-site detection of pathogenic bacteria from clinical samples can be produced.

Efficacy of various amendments for immobilization of potentially toxic elements in wastewater contaminated soils.

Farmers are using municipal wastewater either treated or untreated for irrigation because of limited fresh water resources. Wastewater extensively used for irrigation purposes is enriched with many nutrients. The reuse of wastewater is imposing a negative impact on human health and the ecosystem. It is a need of the day to identify and assess issues of the reuse of wastewater. In the current experiment, impact of organic/inorganic amendments was studied to mitigate the toxic effects of pollutants present in wastewater. Soil was brought from the site having consistent use of wastewater and different treatments were applied as per plan. The experiment has 28 treatments with 04 replications. Nine different amendments were used at 3 varying levels. Incubation time of 30 days was given after the addition of all treatments. The results of the study showed the application of FYM @ 5.0% w/w soil reduced soil pH (7.44), EC (2.16 dS m-1), SAR (8.14), lead (8.48 mg kg-1), cadmium (1.14 mg kg-1), nickel (10.55 mg kg-1) and arsenic (2.03 mg kg-1) when compared with control and other treatments. Usage of compost and horse waste followed FYM. On the basis of this study, it is recommended that wastewater can be used for irrigation purpose after treating with FYM preferably and compost in general.

Comparative expression analysis of sucrose phosphate synthase gene family in a low and high sucrose Pakistani sugarcane cultivars.

Sugarcane is the world's largest cultivated crop by biomass and is the main source of sugar and biofuel. Sucrose phosphate synthase (SPS) enzymes are directly involved in the synthesis of sucrose. Here, we analyzed and compared one of the important gene families involved in sucrose metabolism in a high and low sucrose sugarcane cultivar. A comprehensive in silico analysis of the SoSPS family displayed their phylogenetic relationship, gene and protein structure, miRNA targets, protein interaction network (PPI), gene ontology and collinearity. This was followed by a spatial expression analysis in two different sugarcane varieties. The phylogenetic reconstruction distributed AtSPS, ZmSPS, OsSPS, SoSPS and SbSPS into three main groups (A, B, C). The regulatory region of SoSPS genes carries ABRE, ARE, G-box, and MYC as the most dominant cis-regulatory elements. The PPI analysis predicted a total of 14 unique proteins interacting with SPS. The predominant expression of SPS in chloroplast clearly indicates that they are the most active in the organelle which is the hub of photosynthesis. Similarly, gene ontology attributed SPS to sucrose phosphate synthase and glucosyl transferase molecular functions, as well as sucrose biosynthetic and disaccharide biological processes. Overall, the expression of SPS in CPF252 (high sucrose variety) was higher in leaf and culm as compared to that of CPF 251 (low sucrose variety). In brief, this study adds to the present literature about sugarcane, sucrose metabolism and role of SPS in sucrose metabolism thereby opening up further avenues of research in crop improvement.

Modeling the effect of climatic conditions and topography on malaria incidence using Poisson regression: a Retrospective study in Bannu, Khyber Pakhtunkhwa, Pakistan.

Background: Malaria has been identified as a crucial vector-borne disease around the globe. The primary aim of this study was to investigate the incidence of malaria in the district of Bannu and its relationship with climatic conditions such as temperature, rainfall, relative humidity, and topography. Methods: Secondary data were obtained from the metrological office and government hospitals across the district for 5 years (2013-2017). A Poisson regression model was applied for the statistical analysis. Results and discussion: The number of reported cases of malaria was 175,198. The regression analysis showed that temperature, relative humidity, and rainfall had a significant association (p < 0.05) with malaria incidence. In addition, the topographic variables were significantly associated (p < 0.05) with malaria incidence in the region. The percent variation in the odds ratio of incidence was 4% for every unit increase in temperature and 2% in humidity. In conclusion, this study indicated that the temperature, humidity, rainfall, and topographic variables were significantly associated with the incidence of malaria. Effective malaria control and interventions integrated with climatic factors must be considered to overcome the disease burden.

Rhinosinusitis and its impact on quality of life in children.

Rhinitis and rhinosinusitis are common conditions which have a significant burden on healthcare services and can lead to reduced productivity in patients across the entire age spectrum. Three disease-specific quality of life tools were analysed in hospital and community settings to explore prevalence, disease characteristics and financial costs to sufferers, and to compare these aspects between cohorts (young children, young adult and adults). These tools were Modified Sino-Nasal Outcome Test-20 (MSNOT-20) questionnaire for adults, MSNOT-20 Young Persons Questionnaire (MSYPQ) for 11-16-year-olds and Sami's Rhinosinusitis Diagnosis and Impact questionnaire for 5-10-year-olds. One of the top three symptoms in children aged 5-10 years was cough, which is a much less common symptom in adults. These validated, disease-specific, quality of life questionnaires provide a fuller illustration of the patient experience, allowing comprehensive comparative analysis across the ages.

Assessment of pesticide induced inhibition of Apis mellifera (honeybee) acetylcholinesterase by means of N-doped carbon dots/BSA nanocomposite modified electrochemical biosensor.

This work describes the development and optimization of an electrochemical method to evaluate pesticide induced inhibition of honey bee (Apis mellifera) acetylcholinesterase (AChE) by means of acetylcholinesterase biosensor. The inhibition assay was based on the detection of changes in electrochemical activity of the enzyme caused by pesticide. As transducer, nitrogen doped carbon dots BSA (N-CD/BSA) nanocomposite electrodeposited on pencil graphite electrode was used to covalently immobilize AChE. The as-synthesized nanocomposite and fabricated electrodes were characterized for the structural, functional and electrochemical properties. Nanocomposite promoted the electron transfer reaction to catalyze the electro-oxidation of thiocholine and a large current response was obtained by cyclic voltammetry at 0.77 V, indicating successful immobilization of AChE. The sensitivity of Diazinon, an OP insecticide, for honeybee AChE was tested under optimal conditions and a linear response ranging 10-250 nM was obtained with a detection limit of 8.9 nM, and sensitivity 9 uA/nM/cm2. The method showed a good operational reproducibility and selectivity of biosensor. Further, the molecular docking provided additional support to the experimental data suggesting irreversible nature and contact toxicity of the pesticide for honey bee AChE. The developed biosensor has proved useful for the diazinon detection in wheat samples with 99% recovery rate.

A novel construct of an electrochemical acetylcholinesterase biosensor for the investigation of malathion sensitivity to three different insect species using a NiCr2O4/g-C3N4 composite integrated pencil graphite electrode.

Herein, an electrochemical biosensor has been prepared to assess the sensitivity of an organophosphate insecticide, malathion, to acetylcholinesterase (AChE) enzyme of three insects including Apis mellifera (honeybee), Tribolium castaneum (red flour beetle), and Zootermopsis nevadensis (dampwood termite). A composite of nickel chromite (NiCr2O4) and graphitic carbon nitride (g-C3N4) was prepared and characterized for its morphological, chemical and electrical properties. The NiCr2O4/g-C3N4 composite integrated pencil graphite electrodes were used to covalently immobilize insect AChE enzymes and amperometric response of bioelectrodes was determined through cyclic voltammetry. The prepared bioelectrodes exhibited high enzyme immobilization efficiency and electro-catalytic performance. The integrated bioelectrodes could efficiently detect malathion induced inhibition of insects' AChEs. The linear ranges for malathion were found to be 0.1-1.6 µM, 1-40 nM and 2-100 nM, and LODs were 2 nM, 0.86 nM and 2.3 nM for A. mellifera, T. castaneum, and Z. nevadensis, respectively. Additionally, the biosensing platform developed using A. mellifera AChE was found highly sensitive and effective for malathion recoveries from spiked wheat flour samples with high recovery rates. Moreover, the proposed method was adequately reproducible and selective. The results revealed that A. mellifera AChE is less sensitive to inhibition by malathion as compared to T. castaneum, and Z. nevadensis AChE. The experimental results were validated through computational docking of malathion with insect AChEs and the results were in correspondence to experimental outcomes. The proposed method can be a plausible alternate to conventional analytical methods to assess the pesticide sensitivity and toxicity of various compounds against insect enzymes.

Diversity of Rotavirus Strains among Children with Acute Diarrhea in Karachi, Pakistan.

One of the common viral pathogens in infectious diarrhea is Rotavirus; in developing countries, it is a primary cause of deaths in children less than five years of age. This study was planned to find out the etiologic agents of acute watery diarrhea. In this study, 1465 stool samples were analyzed with the symptoms of acute diarrhea. Demographic data analysis showed no. of episodes of diarrhea, vomiting, and fever. All samples were checked by ELISA technique for the presence of Rotavirus circulating strains. More than 6% patients were found to be positive with Rotavirus. Common Rotavirus genotypes, including G2P4, G2P6, G3P4, G8P4, G8P6, G9P4, and G10P4, were detected in patients through RT-PCR. This study concluded that detection of rotavirus strain diversity and management of diarrheal patients may identify assortment of emerging strains and reduce emergence of antimicrobial resistance and repeated episodes of diarrhea, which may also help to avoid and manage the essential nutrients lost leading to malnutrition and stunted growth, as well as to reduce high mortality rate in young children less than five years.

Results of shared learning of a new magnetic seed localisation device - A UK iBRA-NET breast cancer localisation study.

INTRODUCTION: Shared learning is imperative in the assessment and safe implementation of new healthcare interventions. Magnetic seeds (Magseed®) potentially offer logistical benefit over wire localisation for non-palpable breast lesions but few data exist on outcomes comparing these techniques. A national registration study (iBRA-NET) was conducted to collate device outcomes. In order to share learning, thematic analysis was conducted to ascertain early clinical experiences of Magseed® and wire guided localisation and explore how learning events may be applied to improve clinical outcomes. METHODS: A qualitative study of 27 oncoplastic surgeons, radiologists and physicians was conducted in January 2020 to ascertain the feasibility and challenges associated with Magseed® versus wire breast localisation surgery. Four focus groups were asked to discuss experiences, concerns and shared learning outcomes which were tabulated and analysed thematically. RESULTS: Three key themes were identified comparing Magseed® and wire localisation of breast lesions relating to preoperative, intraoperative and postoperative learning outcomes. Percutaneous Magseed® detection, instrument interference and potential seed or wire dislodgement were the most common issues identified. Clinician experience suggested Magseed® index lesion identification was non-inferior to wire placement and improved the patient pathway in terms of scheduling and multi-site insertion. CONCLUSIONS: Prospective shared learning suggested Magseed® offered additional non-clinical benefits over wire localisation, improving the efficiency of the patient pathway. Recommendations for improving breast localisation technique, appropriate patient selection and clinical practice through shared learning are discussed that may aid other surgeons in the adoption of this relatively new technique.

An insect acetylcholinesterase biosensor utilizing WO3/g-C3N4 nanocomposite modified pencil graphite electrode for phosmet detection in stored grains.

This study was undertaken to assess the potential of Tribolium castaneum (Red flour beetle) acetylcholinesterase (Tc-AChE) based electrochemical biosensor integrating WO3/g-C3N4 nanocomposite modified Pencil graphite electrode to detect an organophosphate insecticide, Phosmet. The WO3/g-C3N4 nanocomposite provides a non-toxic, biocompatible surface for binding the enzyme on the electrode surface, attributed to its large surface area, high conductivity, and low ohmic resistance. The proposed biosensor shows a very good analytical performance with LOD 3.6 nM for Phosmet and effectively determined Phosmet in wheat with a 99% recovery rate. Furthermore, molecular docking deciphers the binding interactions of Phosmet with Tc-AChE using a modified AutoDock LGA algorithm and an AMBER03 force field in YASARA. The kinetic parameters strongly suggest the high potency of inhibitor with the enzyme. This study presents an adaptable, rapid, and straightforward approach that opens ways towards real progress in developing commercial biosensors for pesticide detection.

A Novel Mixed-Methods Platform Study Protocol for Investigating New Surgical Devices, with Embedded Shared Learning: Ibra-net Breast Lesion Localisation Study.

INTRODUCTION: New medical devices must have adequate research, such that outcomes are known, enabling patients to be consented with knowledge of the safety and efficacy of the device to be implanted. Device trials are challenging due to the learning curve and iterative assessment of best practice. This study is designed to pilot a national collaborative approach to medical device introduction by breast surgeons in the UK, using breast localisation devices as an exemplar. The aim is to develop an effective and transferable surgical device platform protocol design, with embedded shared learning. METHODS AND ANALYSIS: The iBRA-net localisation study is a UK based prospective, multi-centre platform study, comparing the safety and efficacy of novel localisation devices with wire-guided breast lesion localisation for wide local excision, using Magseed® as the pilot intervention group. Centres performing breast lesion localisation for wide local excision or excision biopsy will be eligible to participate if using one of the included devices. Further intervention arms will be added as new devices are CE marked. Outcomes will be collected via an online database. The primary outcome measure will be identification of the index lesion. Participating surgeons will be asked to record shared learning events via online questionnaires and focus group interviews to inform future study arms. ETHICS AND DISSEMINATION: The study will aim to collect data on 950 procedures for each intervention (Magseed® and wire localisation) from UK breast centres over an 18-month period. Shared learning will be prospectively evaluated via thematic analysis to refine breast localisation technique and to promote early identification of potential pitfalls and problems. Results will be presented at national and international conferences and published in peer reviewed journals. REGISTRATION: This is a UK national audit registered with Manchester University NHS Foundation Trust. HIGHLIGHTS: This protocol outlines a novel methodology for a collaborative national platform study to collate safety and efficacy data on new medical devices. Improved registration and audit of new medical devices is a major theme of the Cumberlege report of the Independent Medicines and Medical Devices Safety Review.We outline a protocol for a UK based multi-centre prospective audit to investigate the safety and efficacy of new surgical devices for breast lesion localisation. The study will run as a platform study using wire localisation as a control group and Magseed® as the first intervention arm.The protocol is designed for additional bolt-on intervention arms for other localisation devices, such as Hologic Localizer™ and Savi Scout®, when they become available to the European market. This will enable comparison of these devices to datasets already collected on wire and Magseed® localisation.The study includes a novel shared learning methodology using iterative online database reporting and surgical interviews to centrally distribute information on learning events, critical governance issues and recommended protocols for future use.

Formulation of novel chitosan guargum based hydrogels for sustained drug release of paracetamol.

The report presents the formulation of hydrogel based on biopolymers chitosan and guar gum after cross-linking for sustained release of a commonly used orally prescribed analgesic Paracetamol. The oral ingestion of Paracetamol is associated with complications of the gastric tract and liver metabolism that can be effectually avoided by using transdermal drug delivery systems. The formulated transdermal patch was characterized for physicochemical properties including swelling, bonding pattern (using FTIR Fourier Transform Infra-Red and Scanning Electron Microscopy SEM) and antimicrobial activity. Biocompatibility and cytotoxicity was examined in vitro using cell culture in HeLa cell lines. After characterizing the novel formulated hydrogel were employed for the preparation of drug encapsulated in alginate beads as a transdermal patch. After formulation of the transdermal patch, the drug release was studied using an avian skin model. The results followed zero order kinetics and Non-Fickian law for diffusion. Paracetamol due to its small molecular mass (151.163g/mol) released in a sustained manner. The released drug successfully retained its biological effects including anti-inflammatory and anti-protease activity, indicating no interaction between the drug and the formulated hydrogel. It was shown that the formulated hydrogels could be safely used as a dermal patch for the sustained drug release of Paracetamol.

Structure-function relation of somatotropin with reference to molecular modeling.

Somatotropin, commonly known as growth hormone (GH) is a polypeptide chain containing about 190 amino acid residues, produced by the pituitary gland in mammals and is responsible for a number of anabolic processes. It has two disulphide bridges, with 4 alpha helices arranged in anti-parallel distinctive manner. GH molecule binds with two receptor molecules to exhibit its full biological activity. In this review, the information regarding characterization, structure and function is updated. A number of human growth hormone variants (naturally occurring and produced by recombinant DNA- technology) are visualised, and structure related functions are revealed. 1) The di-sulphide bridges are not essential for the biological activity of the molecule. The two chain variants of GH are able to show full biological activity. 2) The different domains of GH could be related to its functions 3) N-terminus of the molecule is involved in the galactopoietic activity of the molecule. 4) A single amino acid residue at a particular position could determine the magnitude of hormone receptor binding. 5) Role of Trp 86 is critical in packing of the apha helices bundles of the molecule. 6) Hydrophobic cores are essential for the stability of GH molecule 7) Salt bridges and hydrogen bonds are also important for the binding of the molecule with its receptors. 8) GH molecule has two binding sites for receptor molecules, Site I and Site II which are sterically coupled. The placental growth hormone has also been discussed and compared with the pituitary derived GH for its structure and function.

Comparison in effect of different metal ions, pH and reducing agent on the protease activity in human hyper mature and mature cataract.

This study was undertaken to isolate and characterize the protease activity of human eye lens sample of mature and hyper mature cataract. Samples were collected just after surgery of the cataract lens and were stored at -20 degrees C. The total protein extract was isolated from 5 samples in each case (mature and hyper mature cataract) and clear supernatant obtained after centrifugation was used as an enzyme source. The optimum pH for the proteases of mature cataract was 7.5 while the proteases of hyper mature cataract were recorded for maximum activity at pH 5.5 and 7.5. The optimum temperature for both enzyme sources was 50 degrees C. Effect of different metal ions such as potassium, lead, silver, zinc and borate was studied. In each case protease activity was increased. Reducing agent e.g. beta mercaptoethanol also caused an increase in activity indicating the involvement of sulfhydryl groups. Protease activity was also located on agar plates.

Identification of novel catalytic features of endo-beta-1,4-glucanase produced by mulberry longicorn beetle Apriona germari.

Mulberry longicorn beetle, Apriona germari, has been reported to produce two endo-beta-1,4-glucanases or AgEGases (accession Nos. Q6SS52 and Q5XQD1). AgEGase sequence contains catalytic motif (amino acid residues 37-48), which is the characteristic of family Glycohydrolase 45 and is identified as the substrate binding site. The application of bioinformatics approaches includes sequence analysis, structural modeling and inhibitor docking to relate the structure and function of AgEGases. We have dissected the sequence and structure of AgEGase catalytic motif and compared it with crystal structure of Humicola insolens endoglucanases V. The results show an involvement of sulfur containing amino acid residues in the active site of the enzyme. Cys residues and position of disulfide bonds are highly conserved between the two structures of endoglucanases of A. germari. Surface calculation of AgEGase structure in the absence of Cys residues reveals greater accessibility of the catalytic site to the substrate involving Asp42, a highly conserved residue. For the inhibition study, tannin-based structure was docked into the catalytic site of AgEGase using ArgusLab 4.0 and it resulted in a stable complex formation. It is suggested that the inhibition could occur through formation of a stable transition state analog-enzyme complex with the tannin-based inhibitor, as observed with other insect cellulases in our laboratory.