Validated assays for the quantification of C9orf72 human pathology.

A repeat expansion mutation in the C9orf72 gene is the leading known genetic cause of FTD and ALS. The C9orf72-ALS/FTD field has been plagued by a lack of reliable tools to monitor this genomic locus and its RNA and protein products. We have validated assays that quantify C9orf72 pathobiology at the DNA, RNA and protein levels using knock-out human iPSC lines as controls. Here we show that single-molecule sequencing can accurately measure the repeat expansion and faithfully report on changes to the C9orf72 locus in what has been a traditionally hard to sequence genomic region. This is of particular value to sizing and phasing the repeat expansion and determining changes to the gene locus after gene editing. We developed ddPCR assays to quantify two major C9orf72 transcript variants, which we validated by selective excision of their distinct transcriptional start sites. Using validated knock-out human iPSC lines, we validated 4 commercially available antibodies (of 9 tested) that were specific for C9orf72 protein quantification by Western blot, but none were specific for immunocytochemistry. We tested 15 combinations of antibodies against dipeptide repeat proteins (DPRs) across 66 concentrations using MSD immunoassay, and found two (against poly-GA and poly-GP) that yielded a 1.5-fold or greater signal increase in patient iPSC-motor neurons compared to knock-out control, and validated them in human postmortem and transgenic mouse brain tissue. Our validated DNA, RNA and protein assays are applicable to discovery research as well as clinical trials.

Natural flavonoid pectolinarin computationally targeted as a promising drug candidate against SARS-CoV-2.

Coronavirus disease-2019 (COVID-19) has become a global pandemic, necessitating the development of new medicines. In this investigation, we identified potential natural flavonoids and compared their inhibitory activity against spike glycoprotein, which is a target of SARS-CoV-2 and SARS-CoV. The target site for the interaction of new inhibitors for the treatment of SARS-CoV-2 has 82% sequence identity and the remaining 18% dissimilarities in RBD S1-subunit, S2-subunit, and 2.5% others. Molecular docking was employed to analyse the various binding processes used by each ligand in a library of 85 natural flavonoids that act as anti-viral medications and FDA authorised treatments for COVID-19. In the binding pocket of the target active site, remdesivir has less binding interaction than pectolinarin, according to the docking analysis. Pectolinarin is a natural flavonoid isolated from Cirsiumsetidensas that has anti-cancer, vasorelaxant, anti-inflammatory, hepatoprotective, anti-diabetic, anti-microbial, and anti-oxidant properties. The S-glycoprotein RBD region (330-583) is inhibited by kaempferol, rhoifolin, and herbacetin, but the S2 subunit (686-1270) is inhibited by pectolinarin, morin, and remdesivir. MD simulation analysis of S-glycoprotein of SARS-CoV-2 with pectolinarin complex at 100ns based on high dock-score. Finally, ADMET analysis was used to validate the proposed compounds with the highest binding energy.

STUDIES ON RADIATION SHIELDING PROPERTIES OF NEWLY DEVELOPED HIGH-DENSITY CONCRETE FOR ADVANCED RADIOTHERAPY FACILITIES.

The linear attenuation coefficients and tenth-value layers are determined experimentally for the newly developed Cement-based high-density Concrete and Fly-Ash-based Geopolymer high-density Concrete using Red-Mud-based synthetic aggregate made up from industrial waste. Linear attenuation coefficients were determined in narrow and broad beam conditions for five megavoltage X-ray photon beam energies, i.e. 6, 10, 15 MV, and 6 and 10 MV-FFF generated by Varian TrueBeam medical linear accelerator. These materials are found to be more effective in radiation shielding when compared with ordinary concrete and hematite ore-based high-density concrete making it a useful construction material for radiotherapy accelerator vaults. Similar values of linear attenuation coefficients are observed for all the above-mentioned X-ray beam energies when cement is replaced with fly-ash in ordinary concrete, hematite-based high-density concrete and red-mud-based high-density concrete, making it a good eco-friendly alternative of cement and useful for the construction of radiotherapy vaults.

Junior trainee group 'Buddy Scheme'.

The ethos of the junior trainees' group is to facilitate peer support, an important complement to pursuing our speciality of oral and maxillofacial surgery. Launched in September 2020, the Buddy Scheme enables peer-matching. Results demonstrate this scheme has successfully provided additional support for second-degree applicants. With further help from our senior colleagues, our ambition is to expand this scheme in 2021.

Surgical excision of non-melanoma skin cancer: no end in site?

Non-melanoma skin cancer (NMSC) is the most common type of cancer in the UK and Ireland, accounting for approximately 20% of all new malignancies recorded, with evidence of increasing incidence. Surgical excision is regarded as the gold standard treatment for such lesions; however, incomplete excision incurs both physical and financial strain on patients and the healthcare service. Our aim is to identify which anatomical regions of the head and neck pose greater risk of incomplete excision and raise awareness for future surgical practice. Manual data collection was carried out on all patients admitted to the OMFS department for surgical excision of NMSCs between January 2016 and December 2017. Information collected included clinical notes and MDT outcomes. NMSCs excised via Mohs surgery. All tumours incompletely excised and requiring further treatment were noted with particular emphasis on the site of the lesion within the head and neck region, and the location of the positive margin on the specimen itself. A total of 506 NMSCs were excised (307 BCC, 199 SCC). Of these, 7.8% (n=24) and 6.0% (n=12), respectively, required further treatment due to incomplete excision. The incompletely exised lesions were predominately located on the nose (13.3%), temple (15.0%), and ear (12.8%). Our results demonstrate that our department is successfully excising NMSCs with minimal cases requiring further management. This study has identified anatomical profiles posing a higher risk of incomplete removal. Alongside the conventionally challenging sites of the nose and ear, we have highlighted additional risk within the temporal region. We therefore propose that Mohs surgery or a two-stage procedure should be considered as management for the temple at future MDTs.

COVID-19-associated mucormycosis presenting to the Emergency Department-an observational study of 70 patients.

BACKGROUND: Mucormycosis (MM) is a deadly opportunistic fungal infection and a large surge in COVID-19-associated mucormycosis (CAM) is occurring in India. AIM: Our aim was to delineate the clinico-epidemiological profile and identify risk factors of CAM patients presenting to the Emergency Department (ED). DESIGN: This was a retrospective, single-centre, observational study. METHODS: We included patients who presented with clinical features or diagnosed MM and who were previously treated for COVID-19 in last 3 months of presentation (recent COVID-19) or currently being treated for COVID-19 (active COVID-19). Information regarding clinical features of CAM, possible risk factors, examination findings, diagnostic workup including imaging and treatment details were collected. RESULTS: Seventy CAM patients (median age: 44.5 years, 60% males) with active (75.7%) or recent COVID-19 (24.3%) who presented to the ED in between 6 May 2021 and 1 June 2021, were included. A median duration of 20 days (interquartile range: 13.5-25) was present between the onset of COVID-19 symptoms and the onset of CAM symptoms. Ninety-three percent patients had at least one risk factor. Most common risk factors were diabetes mellitus (70%) and steroid use for COVID-19 disease (70%). After clinical, microbiological and radiological workup, final diagnosis of rhino-orbital CAM was made in most patients (68.6%). Systemic antifungals were started in the ED and urgent surgical debridement was planned. CONCLUSION: COVID-19 infection along with its medical management have increased patient susceptibility to MM.

Confidential image transfer: an ethico-legal dilemma.

Clinical photographs aid decision-making and represent important medicolegal records. Storage and transfer of images of the facial area must adhere to Caldicott Principles. Outside working hours, clinical photography services are often limited. Our Trust has introduced a Secure Clinical Image Transfer (SCIT) app allowing clinicians to take photographs on personal devices to be securely uploaded to the patient's electronic health record. To evaluate whether clinicians were taking clinical images in an insecure manner, clinicians completed an anonymous questionnaire before and after introduction of the SCIT app. The standard was 100% knowledge of, and adherence to, trust information governance guidelines. Response rate was 100% in both cycles. Introduction of the SCIT app reduced inappropriate clinical photography on personal devices. Our completed audit cycle shows that the SCIT app allows convenient, secure information capture on personal devices and automatic secure synchronisation to trust electronic health records.

Pilot project in rural western Madhya Pradesh, India, to assess the feasibility of using LED and solar-powered lanterns to remove kerosene lamps and related hazards from homes.

BACKGROUND: Globally, 300,000 deaths are estimated to occur annually and the incidence is far greater as a large majority of burns are small and go unreported. Ninety-five percent of the global burden of burns is found in low- and middle-income countries; however, there is relatively little in the literature regarding effective primary prevention in these areas. Flame is the most common cause of burn in Madhya Pradesh, the central state of India. The most common demographic among the burn unit inpatient of Choithram hospital Indore, is young women from 21 to 40 years of age, whose burns are primarily caused by kerosene lamps. A non electrical source of illumination is essential for every household in rural areas due to the infrequent and poor power supply. At the baseline, 23 kerosene lamp burns were reported by villagers in the past 5 years among the study population of this pilot project. METHOD: A pilot project to investigate the strategies for reducing the incidence of domestic burns in rural villages around the city of Indore was performed, by replacing kerosene lamps with safer and more sustainable alternatives, including solar-powered and light-emitting diode (LED) lamps. A total of 1042 households were randomly chosen from 18 villages within the Malwa region of Madhya Pradesh (population of 28,825) to receive the alternative light source (670 LED and 372 solar lamps). We investigated the efficacy of this strategy of reducing the incidence of burns, measured the social acceptance by villagers, and quantified the cost implications and availability of LED lamps in rural communities with a high incidence of burns. RESULTS: Replacing kerosene lamps with LED and solar alternatives was deemed socially acceptable by 99.34% of the participants and reduced the cost of lighting for impoverished rural villagers by 85% over 1 year. We successfully demonstrated a significant decrease in the use of kerosene lamps (p<0.01). More evidence is required to investigate the efficacy of this strategy in reducing burns. CONCLUSION: This pilot study highlights the viability of the approach of replacing kerosene lamps as an effective primary prevention strategy for reducing burns in rural areas. However, barriers remain to the wider adoption of these lamps, including accessibility and availability for the populations of rural India.

Maternal-neonatal transfusion compatibility irrespective of ABO mismatch--a prospective observational study.

OBJECTIVE: To determine transfusion compatibility of maternal RBCs for her neonate up to 4 weeks of age, irrespective of maternal-neonatal ABO mismatch. METHODS: This was a prospective observational study involving eligible mothers with their neonates delivered in participating site from 1 July 2012 till 31 December 2012. Mother's blood was collected before child birth. Neonatal blood sample was collected from placental end, shortly after birth. Blood Groups of mother-baby pair were individually tested for ABO and Rh-D groups. Pairs with negative Rh-D antigen/s or with same ABO blood groups were excluded. Thus, 28 pairs with both maternal and neonatal samples positive for Rh-D antigen and with different maternal neonatal ABO blood groups were included in the study. Blood samples were collected at birth and at 4 weeks. Cross matching was done at birth and at 4 weeks for each pair with standard blood bank protocols. RESULTS: All 28 pairs showed positive compatibility with standard blood bank cross-matching protocols at birth and at 4 weeks. CONCLUSIONS: Maternal blood irrespective of ABO compatibility might be a viable and potentially acceptable option for her new born baby in neonatal period. This may be especially important in developing world with limited blood bank resources.

Gold nanoparticles based dipstick immunoassay for the rapid detection of dichlorodiphenyltrichloroethane: an organochlorine pesticide.

Gold nanoparticles (GNPs) based dipstick competitive immunoassay was developed to detect organochlorine pesticide such as DDT at nanogram level (ppb). GNPs of definite size were synthesized and conjugated to anti-DDT antibodies (IgY), which served as the detecting reagent. DDA-BSA conjugate (antigen) was immobilized on to nitro cellulose (NC) membrane containing strip. GNPs conjugated anti-DDT antibodies were treated with different concentrations of free DDT ranging from 0.7 ng mL(-1) to 1000 ng mL(-1) to form an immunocomplex. This immunocomplex solution was further reacted with DDA-BSA conjugate immobilized NC membrane containing strips by dipping the strip in the immunocomplex solution. The free GNPs conjugated anti-DDT antibodies present in the immunocomplex solution were targeted for competitive binding with immobilized DDA-BSA on NC membrane containing strip. Depending on the concentration of free DDT in the sample the binding of GNPs conjugated anti-DDT antibodies to the immobilized DDA-BSA varied and was detected by the development of red color (due to gold nanoparticles) in the detection zone of NC membrane containing strips. The intensity of color development was inversely proportional to the DDT concentration with maximum intensity at zero DDT concentration. The lowest detection limit of DDT was determined to be 27 ng mL(-1) with the optimized conditions. The dipstick technique based on GNPs is suitable for the detection of several toxins in food and environmental samples and can be applied for rapid on-site testing of pesticides.

Maternal Factors for Low Birth Weight Babies.

BACKGROUND: Low birth weight is defined as the live births with less than 2.5 kg weight. It is a key determinant of infant survival, health and development. Low birth weight infants are at a greater risk of having a disability and for diseases such as cerebral palsy, visual problems, learning disabilities and respiratory problems. To reduce the low birth weight deliveries, we studied the maternal factors which adversely affect the fetus in utero and their impact on fetus. METHODS: A retrospective study was carried out on 40 low birth weight pregnancies out of 650 deliveries from July 2005 to Jun 2006. Maternal factors like age, parity, pre pregnancy body mass index, hemoglobin levels, bad obstetric history (history of stillbirth/neonatal death in previous pregnancies, three or more spontaneous consecutive abortions), pre eclampsia, fetal distress, mode of deliveries were studied. These results were compared with a random sample of 300 pregnant ladies taken from rest of the deliveries. Cases of multiple pregnancies and stillbirths were excluded. RESULT: We found that prepregnancy maternal body mass index (p<0.01 for BMI <20), unbooked status (p<0.01), pre eclampsia (p <0.01) and bad obstetric history (p<0.01) were the maternal factors which resulted in low birth weight babies in most of the cases. However in 10 (25%) cases, no contributory maternal factor was found. CONCLUSION: Prepregnancy maternal body mass index, unbooked status, pre eclampsia and bad obstetric history are significant maternal factors resulting in low birth weight babies.

Effect of Antenatal Expression of Breast Milk at Term in Reducing Breast Feeding Failures.

BACKGROUND: Though breast feeding is natural, during the first 2-3 days, when enough breast milk is not available with mother, she may introduce bottle feeding erroneously for improving nutrition to her baby. We studied the effect of antenatal expression of breast milk at term in reducing breast feeding failure as compared to conventional method of initiation of breast feeding. METHODS: A prospective study was carried out in 180 booked cases at term. Daily expression of breast milk at least once a day after 37 weeks of pregnancy was introduced in randomly selected 90 pregnant ladies. Prior examination was done to exclude any inverted or cracked nipples and appropriate treatment instituted. RESULT: The study group who expressed breast milk daily after 37 weeks did not find it difficult to initiate breast feeding after vaginal or cesarean delivery. Sufficient milk started flowing within half an hour of initiation of breast feeding in most 85 (94.4%) subjects of study group as compared to 63 (70%) patients of control group, which was statistically significant. There was no increase in any delivery complication. There were two partial breast feeding failures in control group but none in study group. CONCLUSION: Daily antenatal breast milk expression after 37 completed weeks of pregnancy significantly reduced the time for establishing full breast feeding and reduced breast feeding failures.

Thin-layer chromatography of aluminium: quantitative densitometric determination of Fe2+, Ni2+, Cu2+, and Si4+.

Thin-layer chromatography in combination with scanning densitometry is used as a tool for the quantitative determination of some impurity and additive elements in aluminium. Microgram levels of iron, silicon, copper, nickel, titanium, magnesium, manganese, and zinc present in a high concentration aluminium matrix is detected, and selective separations of some of these elements are achieved on silica gel H layers developed with a mobile phase containing aqueous sodium chloride solution. The quantitative determination of iron, silicon, nickel, and copper are obtained from the densitometric evaluation of chromatograms and are compared with the respective optical emission spectral analytical data.

Severe bradycardia and hypotension after connecting negative pressure to the subgaleal drain during craniotomy closure.

Negative pressure drainage systems are often used after craniotomy for evacuation of potential bleeding. There are several reports of haemodynamic disturbances with epidural negative pressure drainage, but such reports are very few for subgaleal drains placed over the bone flap. We report a case in which a patient developed severe cardiovascular disturbances after the vacuum drainage was connected to a subgaleal drain after craniotomy for aneurysm clipping. The patient had no significant cardiac history, had an uneventful intra-operative course and yet developed bradycardia and hypotension, which were reproducible and severe enough to require atropine administration. Anaesthetists must be aware of these effects, so that they can anticipate and treat such complications.

Detection of methyl parathion using immuno-chemiluminescence based image analysis using charge coupled device.

A novel method based on immuno-chemiluminescence and image analysis using charge coupled device (CCD) for the qualitative detection of methyl parathion (MP) with high sensitivity (up to 10 ppt) is described. MP antibodies raised in poultry were used as a biological sensing element for the recognition of MP present in the sample. The immuno-reactor column was prepared by packing in a glass capillary column (150 microl capacity) MP antibodies immobilized on Sepharose CL-4B through periodate oxidation method. Chemiluminescence principle was used for the detection of the pesticide. Light images generated during the chemiluminescence reaction were captured by a CCD camera and further processed for image intensity, which was correlated with pesticide concentrations. K(3)Fe(CN)(6) was used as a light enhancer to obtain detectable light images. Different parameters including concentrations of K(3)Fe(CN)(6), luminol, urea H(2)O(2), antibody, addition sequence of reactants and incubation time to obtain best images were optimized. The results obtained by image analysis method showed very good correlation with that of competitive ELISA for methyl parathion detection. Competitive ELISA method was used as a reference to compare the results obtained by CCD imaging.

Sudden asystole during surgery in the cerebellopontine angle.

We report a case of a 40-year-old lady undergoing surgery for a tumor in the cerebellopontine angle. Intraoperatively, patient had a sudden asystole without prior warning sign of bradycardia. It could have been the severe form of trigeminocardiac reflex. The cardiac rhythm returned spontaneously once the surgical manipulation stopped. The remainder of the operation was uneventful and no complication occurred afterwards. The possible mechanism of the event is discussed.