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.

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.

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.

Intracranial pressure and haemodynamic changes during the tunnelling phase of ventriculoperitoneal shunt insertion.

BACKGROUND AND OBJECTIVE: The tunnelling phase of ventriculoperitoneal shunt insertion is the most painful part but patients are often given inadequate opioid analgesic for fear of post operative delayed recovery and/or respiratory depression. This may result in an increase in intracranial pressure. METHODS: Twenty adults scheduled to undergo ventriculoperitoneal shunt insertion were administered standard anaesthesia. Monitoring included heart rate, electrocardiogram, end-tidal carbon dioxide, invasive blood pressure, and oxygen saturation. Intracranial pressure was monitored by placing the ventricular end of shunt catheter in the dilated lateral ventricle. Five minutes before tunnelling, fentanyl 1 microg kg(-1) was administered. Mean arterial pressure, heart rate and intracranial pressure were recorded during tunnelling and subsequently at 1-min interval for 5 min. Data were analysed using t-test and repeated measured test. RESULTS: Tunnelling caused significant increase in mean arterial pressure (from 81.4 +/- 11.0 to 110.9 +/- 15.3 mmHg, P < 0.05), intracranial pressure (from 21.4 +/- 8.1 to 29.2 +/- 12.5 mmHg, P < 0.05) and heart rate (from 74.4 +/- 13.8 to 94.1 +/- 17.8 beats min(-1), P < 0.05). Whereas, the increase in haemodynamic parameters persisted for 3 min post-tunnelling, elevated intracranial pressure lasted for 2 min. CONCLUSION: Tunnelling significantly increases intracranial pressure and blood pressure despite prior fentanyl administration. This may be deleterious in the presence of intracranial pathology.

Comparative incidence of venous air embolism and associated hypotension in adults and children operated for neurosurgery in the sitting position.

BACKGROUND AND OBJECTIVE: Venous air embolism is a constant threat during neurosurgery performed in the sitting position. No large prospective study has compared the incidence of venous air embolism and associated hypotension between adults and children. METHODS: Four hundred and thirty patients (334 adults, 96 children) scheduled to undergo planned posterior fossa surgery in the sitting position (between January 1989 to December 1994) were studied with end-tidal carbon dioxide monitoring. Intraoperatively, a sudden and sustained decrease in end-tidal carbon dioxide tension of >0.7 kPa was presumed to be due to venous air embolism. Management during the episode was on the established guidelines. Hypotension (decrease in systolic arterial pressure of 20% or more from the previous level) was treated with crystalloids and/or a vasopressor. RESULTS: Capnometry detected a 28% incidence rate of air embolism in adults (93/334) and a 22% incidence rate in children (21/96) (P = 0.29). In both groups, the highest incidence rate of embolism took place during muscle handling (44% of adults versus 38% of children, P = 0.8). Embolic episodes were accompanied by hypotension in 37% of adults (34/93) and in 33% of children (7/21) (P = 0.98). To restore arterial pressure to pre-embolic levels, 53% of adults (18/34) and 43% of children (3/7) were administered vasopressors (P = 0.94). There was no intraoperative mortality. The surgical procedure on one adult was abandoned because of persistent hypotension following the embolic episode. CONCLUSION: The incidence of venous air embolism and consequent hypotension is similar in adults and children.

Reverse brain herniation during posterior fossa surgery.

Posterior fossa tumors are commonly associated with obstructive hydrocephalus. Relieving the raised intracranial pressure by draining the cerebrospinal fluid presents the possibility of reverse herniation of the brain. A 5-year-old male child with a diagnosis of posterior fossa space-occupying lesion and hydrocephalus was scheduled for craniectomy in the prone position. After craniectomy, the surgeons placed an intraventricular shunt catheter to drain out cerebrospinal fluid in an attempt to reduce the tense brain so as to facilitate easy dissection of the tumor. The patient had sudden and severe bradycardia followed by asystole. A diagnosis of reverse coning was made. Immediately, the surgeon injected 10-15 mL normal saline into the ventricles. There was a spontaneous return of the sinus rhythm and the rest of the course of surgery was uneventful. We present this case showing a rare phenomenon and its successful management.

ECG changes in pediatric patients with severe head injury.

Although ECG changes in subarachnoid hemorrhage and head injury have been described in adults, they have been rarely reported in children. We present 3 pediatric head-injured patients who developed severe ischemic changes on ECG. Three children (ages 9 months, 2.5 years, and 12 years) were admitted with severe head injury. All of them developed progressive ST segment depression of 4 to 7 mm during the surgical procedure. The first case, a 9-month-old child, also had bradycardia and cardiac arrest following ST depression. He was promptly resuscitated with simultaneous evacuation of extradural hematoma. In the other two cases, ST depression also gradually came up to baseline coinciding with surgical treatment of main pathology. All of the patients were ventilated postoperatively for 36 to 48 hours and discharged with no neurologic deficit. ECG changes and myocardial ischemia in head-injured patients have been attributed to extreme sympathetic stimulation and raised intracranial pressure in adults. But there has been no such systematic study in children. From our observations, we can conclude that ECG changes do occur in children with head injury, although the exact mechanism awaits further evaluation.

Neuropharmacological actions of some newly synthesized Mannich bases.

Benzamido (alkyl) methyl pyrrolidine Mannich bases were synthetized and subjected to certain neuropharmacological studies. All the bases reduced the pentobarbitone sleeping time and rota-rod grip of rats. The Mannich bases II, III and V raised the minimal electro-shock seizure threshold of rats. The TAB-induced pyrexia was not reduced by the bases I and III in rabbits. None of the bases showed any significant analgesic activity.