Quant-iT™ PicoGreen® dsDNA Quantification v2

The following protocol is intended for the quantification of double-stranded DNA using Quant-iT™PicoGreen® dsDNA Assay Kit (ThermoFisher). This protocol is a simplified and condensed version of the full protocol from the manufacturer. The procedure described here is for 96 reactions. If samples are run in duplicates, then this should allow quantifying 40 samples.

FPCount protocol - Full protocol v1

FPCount is a complete protocol for fluorescent protein calibration, consisting of: 1. FP expression/purification using Thermo's HisPur Cobalt Resin. 2. FP concentration determination in a microplate reader. 3. FP fluorescence quantification in a microplate reader. Results can be analysed with the corresponding R package, FPCountR. --- Summary 1. Expression 2. Harvesting/Washing 3. Lysis 4. Fractionation 5. Gel1: Verification of Expression/Fractions 6. Purification 7. Gel2: Verification of Purification 8. Protein concentration and buffer exchange 9. Quantification of FP concentration (part1) 10. Quantification of FP fluorescence 11. Quantification of FP concentration (part2) 12. Protein Storage 13. Calibration of Plate Reader

Assessing Fidelity to and Satisfaction with the “Adolescent Coping with Depression Course” (ACDC) Intervention in a Randomized Controlled Trial

This paper aims at describing the process for assessing the intervention fidelity of a randomized controlled trial (RCT) of an “Adolescent Coping with Depression Course” (ACDC) and to assess the participants’ satisfaction with the intervention. We applied the comprehensive fidelity model developed by the National Institutes of Health’s Behavior Change Consortium to examine how our intervention met the fidelity requirements under five categories. Data came from a two-arm parallel cluster RCT. Both qualitative and quantitative analyses of the ACDC intervention using the comprehensive fidelity model indicated that the level of fidelity in this study did not reach 100%. However, it was approaching a high level of treatment fidelity. Participants also expressed high levels of satisfaction (M = 3.65, SD = .95). This analysis is important to show how appropriately the intervention was implemented, areas for improvement to increase its fidelity, and to ensure the internal and external validity of the findings. Trial Registration: ISRCTN registry ISRCTN19700389. Registered 6 October 2015. https://doi.org/10.1186/ISRCTN19700389. Full Protocol: 10.1186/s12888-016-0954-y

IPAssess: A Protocol-Based Fingerprinting Model for Device Identification in the IoT

Internet of Things (IoT) are the most commonly used devices today, that provide services that have become widely prevalent. With their success and growing need, the number of threats and attacks against IoT devices and services have been increasing exponentially. With the increase in knowledge of IoT related threats and adequate monitoring technologies, the potential to detect these threats is becoming a reality. There have been various studies consisting of fingerprinting based approaches on device identification but none have taken into account the full protocol spectrum. IPAssess is a novel fingerprinting based model which takes a feature set based on the correlation between the device characteristics and the protocols and then applies various machine learning models to perform device identification and classification. We have also used aggregation and augmentation to enhance the algorithm. In our experimental study, IPAssess performs IoT device identification with a 99.6\% classification accuracy.

IPAssess: A Protocol-Based Fingerprinting Model for Device Identification in the IoT

Internet of Things (IoT) are the most commonly used devices today, that provide services that have become widely prevalent. With their success and growing need, the number of threats and attacks against IoT devices and services have been increasing exponentially. With the increase in knowledge of IoT related threats and adequate monitoring technologies, the potential to detect these threats is becoming a reality. There have been various studies consisting of fingerprinting based approaches on device identification but none have taken into account the full protocol spectrum. IPAssess is a novel fingerprinting based model which takes a feature set based on the correlation between the device characteristics and the protocols and then applies various machine learning models to perform device identification and classification. We have also used aggregation and augmentation to enhance the algorithm. In our experimental study, IPAssess performs IoT device identification with a 99.6\% classification accuracy.

I-KID study protocol: evaluation of efficacy, outcomes and safety of a new infant haemodialysis and ultrafiltration machine in clinical use: a randomised clinical investigation using a cluster stepped-wedge design

IntroductionThe I-KID study aims to determine the clinical efficacy, outcomes and safety of a novel non-CE-marked infant haemodialysis machine, the Newcastle Infant Dialysis Ultrafiltration System (NIDUS), compared with currently available therapy in the UK. NIDUS is specifically designed for renal replacement therapy in small babies between 0.8 and 8 kg.Methods and analysisThe clinical investigation is taking place in six UK centres. This is a randomised clinical investigation using a cluster stepped-wedge design. The study aims to recruit 95 babies requiring renal replacement therapy in paediatric intensive care units over 20 months.Ethics and disseminationThe study has high parent and public involvement at all stages in its design and parents will be involved in dissemination of results to parents and professionals via publications, conference proceedings and newsletters. The study has has ethics permissions from Tyne and Wear South Research Ethics Committee.Trial registration numbersIRAS ID number: 170 481MHRA Reference: CI/2017/0066ISRCT Number: 13 787 486CPMS ID number: 36 558NHS REC reference: 16/NE/0008Eudamed number: CIV-GB-18-02-023105Link to full protocol v6.0: https://fundingawards.nihr.ac.uk/award/14/23/26

Epidural morphine in COVID ARDS patients with high respiratory drive: a structured summary of a study protocol for a randomised controlled trial

Objectives We aim to study the effect of epidural morphine as a means to reduce high respiratory drive in COVID 19 patients on non-invasive ventilation (NIV)—primary end point—and to study its effect on respiratory parameters, subjective patient comfort, rates of endotracheal intubation, duration of mechanical ventilation and mortality. Trial design Parallel group, randomised, double blind, single centre placebo control trial. Allocation ratio 1:1, superiority trial Participants Trial site and population—COVID ICU patients in the All India Institute of Medical Sciences (AIIMS) Bhubaneswar, Odisha, India Inclusion and exclusion criteria Inclusion criteria Adult patients on NIV with COVID-19 Exclusion criteria Metabolic acidosis HCO3-< 16 and pH < 7.2. Severe hypoxemia warranting cessation of NIV and intubation, non-acceptance of NIV and proven sepsis. Technical difficulty for epidural catheterization, coagulation abnormalities, low respiratory drive and EOL orders. Sources or methods of recruitment—daily discussion at 8 am of new admissions to COVID ICU on NIV—consenting adult patients with COVID19 on NIV and high respiratory drive; not meeting exclusion criteria will be recruited for the trial and randomised. Intervention and comparator Patients of both groups will be turned to a lateral or sitting position (as comfortable), and an injection of local anaesthetic be given at lumbar 2–3/3–4 space. In the intervention group, an epidural catheter will be inserted using aseptic technique and fixed to the skin. The control group will have a sham catheter fixed exactly like in the intervention group, but not entering the epidural space. The intervention group will be administered injection morphine sulphate once every 18–24 h into the epidural space. The doses will be escalated daily (5–10 mg), titrated to effect: escalation limited by hypoventilation resulting in respiratory acidosis (pH < 7.2). The intervention will continue for a minimum of 2 doses and a maximum of 5 doses (96 h) of morphine. It will be stopped if the epidural catheter gets dislodged before the second dose or the patient is weaned off non-invasive ventilation to high flow mask for a continuous period of 24 h or requires endotracheal intubation. The patient will be followed up till death or 28 days after ICU discharge. Main outcomes Primary outcome—diaphragm thickening index fraction (average of minimum 3 readings) Secondary outcomes—ventilator parameters, sedation and pain scores, subjective comfort and dyspnoea scores, time to intubation, length of stay on NIV and 28-day mortality Timing of outcome assessment—every 8th hour assessment for 24 h after the last dose of epidural morphine or 120 h whichever is greater Randomisation A central random number list will be kept with the study research assistant. She will randomise according to the numbers available in the list using an allocation ratio of 1:1. An opaque sealed envelope concealing the allotted randomisation code will be dispatched to the ICU team. Blinding (masking) The assessor, patient, nurses and physicians will be blind to group allocation. One member of the team not involved in research will administer the intervention. Numbers to be randomised (sample size) Twenty-five patients per group; 50 patients total Trial status Protocol version 1. Not recruiting yet. Recruitment to begin by 24 July 2021 and end by 31 August 2022 Trial registration Central Trials Registry India CTRI CTRI/2021/07/035093. Registered on 23 July 2021. Prospectively registered Full protocol The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest of expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol

Prophylactic potential of honey and Nigella sativa L. against hospital and community-based SARS-CoV-2 spread: a structured summary of a study protocol for a randomised controlled trial

Objectives Considering the therapeutic potential of honey and Nigella sativa (HNS) in coronavirus disease 2019 (COVID-19) patients, the objective of the study is defined to evaluate the prophylactic role of HNS. Trial design The study is a randomized, placebo-controlled, adaptive clinical trial with parallel group design, superiority framework with an allocation ratio of 1:1 among experimental (HNS) and placebo group. An interim analysis will be done when half of the patients have been recruited to evaluate the need to adapt sample size, efficacy, and futility of the trial. Participants All asymptomatic patients with hospital or community based COVID-19 exposure will be screened if they have had 4 days exposure to a confirmed case. Non-pregnant adults with significant exposure level will be enrolled in the study High-risk exposure (<6 feet distance for >10min without face protection) Moderate exposure (<6 feet distance for >10min with face protection) Subjects with acute or chronic infection, COVID-19 vaccinated, and allergy to HNS will be excluded from the study. Recruitment will be done at Shaikh Zayed Post-Graduate Medical Institute, Ali Clinic and Doctors Lounge in Lahore (Pakistan). Intervention and comparator In this clinical study, patients will receive either raw natural honey (0.5 g) and encapsulated organic Nigella sativa seeds (40 mg) per kg body weight per day or empty capsule with and 30 ml of 5% dextrose water as a placebo for 14 days. Both the natural products will be certified for standardization by Government College University (Botany department). Furthermore, each patient will be given standard care therapy according to version 3.0 of the COVID-19 clinical management guidelines by the Ministry of National Health Services of Pakistan. Main outcomes Primary outcome will be Incidence of COVID-19 cases within 14 days of randomisation. Secondary endpoints include incidence of COVID-19-related symptoms, hospitalizations, and deaths along with the severity of COVID-19-related symptoms till 14th day of randomization. Randomisation Participants will be randomized into experimental and control groups (1:1 allocation ratio) via the lottery method. There will be stratification based on high risk and moderate risk exposure. Blinding (masking) Quadruple blinding will be ensured for the participants, care providers and outcome accessors. Data analysts will also be blinded to avoid conflict of interest. Site principal investigator will be responsible for ensuring masking. Numbers to be randomised (sample size) 1000 participants will be enrolled in the study with 1:1 allocation. Trial Status The final protocol version 1.4 was approved by institutional review board of Shaikh Zayed Post-Graduate Medical Complex on February 15, 2021. The trial recruitment was started on March 05, 2021, with a trial completion date of February 15, 2022. Trial registration Clinical trial was registered on February 23, 2021, www.clinicaltrials.gov with registration ID NCT04767087. Full protocol The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). With the intention of expediting dissemination of this trial, the conventional formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol. The study protocol has been reported in accordance with the Standard Protocol Items: Recommendations for Clinical Interventional Trials (SPIRIT) guidelines.

Anti-COVID property of subcutaneous ivermectin in synergy with zinc among midlife moderately symptomatic patients: a structured summary of a study protocol for a randomised controlled trial

Objectives The study objective is to quantify the effectiveness of ivermectin (subcutaneous/oral IVM) in the presence or absence of zinc (Zn) for clinical and radiological improvement in coronavirus disease 2019 (COVID-19) patients with moderate severity. Trial design This quadruple-blinded, placebo-controlled randomized clinical trial will be a multiarmed multi-centered study with superiority framework. Participants Quinquagenarian and sexagenarian patients with moderate COVID-19 symptoms and positive severe respiratory syndrome coronavirus -2 (SARS-CoV-2) PCR will be included. Participants with co-morbidities and pregnant women will be excluded. Patient recruitment will be done in Shaikh Zayed Medical Complex, Doctors Lounge and Ali Clinic in Lahore (Pakistan). Intervention and comparator The registered patients will be allocated in 6 groups (30 participants each). Patients will be taking subcutaneous IVM at 200 μg/kg/48 h (Arm A) or subcutaneous IVM at 200 μg/kg/48 h and oral Zn 20mg/8 h (Arm B) or oral IVM at 0.2 mg/kg/day (Arm C) or oral IVM at 0.2 mg/kg/day and oral Zn 20mg/8 h (Arm D) or alone oral Zn 20mg/8 h (Arm E) or placebo alone (Arm X). Patients in all arms will receive standard care and respective placebo (empty capsule 8 hourly and/or subcutaneous normal saline 2ml/48 h). Main outcomes Primary endpoints will be duration of symptomatic phase and SARS-CoV-2 clearance along with high resolution CT (HRCT) chest score and clinical grade scale (CGS) on day 6. 30-day mortality will be documented as a secondary endpoint. SARS-CoV-2 clearance will be calculated by second PCR on day 7. HRCT chest score will be measured by the percentage and lung lobes involvement on day 6 with a maximum score of 25. CGS will be recorded on a seven-point scale; grade 1 (not hospitalized, no evidence of infection and resumption of normal activities), grade 2 (not hospitalized, but unable to resume normal activities), grade 3 (hospitalized, not requiring supplemental oxygen), grade 4 (hospitalized, requiring supplemental oxygen), grade 5 (hospitalized, requiring nasal high-flow oxygen therapy and/or noninvasive mechanical ventilation), grade 6 (hospitalized, requiring ECMO and/or invasive mechanical ventilation) and grade 7 (death). Randomisation A simple lottery method will be used to randomly allocate scrutinized patients in 1:1:1:1:1:1 ratio in 6 groups. Blinding (masking) Patients, primary care physicians, outcome assessors and the data collection team will be blinded. Numbers to be randomised (sample size) 180 participants will be randomized into six arms with five investigational and one placebo group. Trial Status Institutional Review Board Shaikh Zayed Post-Graduate Medical Complex, Lahore, Pakistan has approved the protocol (version 2.3) with ID SZMC/IRB/Internal0056/2020. The trial was approved on July 14, 2020, and enrolment started on July 30, 2020. The estimated completion date is October 30, 2021. Trial registration Clinical Trial has been retrospectively registered on www.clinicaltrials.gov with registration ID NCT04472585 dated July 16, 2020. Full protocol The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). With the intention of expediting dissemination of this trial, the conventional formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol. The study protocol has been reported in accordance with the Standard Protocol Items: Recommendations for Clinical Interventional Trials (SPIRIT) guidelines.

FindingNemo: A Toolkit of CoHex- and Glass Bead-based Protocols for Ultra-Long Sequencing on ONT Platforms v1

This collection of protocols is designed to enable ultra-long (UL) reads on Nanopore sequencers. It is split into five sections dealing with ultra-high molecular weight (UHMW) DNA: Extraction QC Library preparation Nemo clean-up using glass beads and Hexamminecobalt(III) Chloride, aka. CoHex. Flowcell priming and library loading We have tested and optimised the full protocol in human cell lines. Various options are available for each of the steps and we hope that the components here will be useful to the community and provide a long read toolkit.