Practical Protocol Research Articles

Uptake and outcomes of implementing non-invasive prenatal testing (Prenatal Reflex DNA testing) into first-trimester contingent screening protocols for trisomy 21, 18, 13: A study protocol

Chromosomal anomalies (CAs), such as trisomies 21 (T21), 18 (T18), and 13 (T13), are significant contributors to birth defects and genetic conditions worldwide. Cell-free fetal DNA testing (cff-DNA) is the preferred method for detecting these trisomies from maternal blood samples. However, its adoption is hindered by high costs and complexities. To enhance efficiency and reduce financial strain, we propose a practical protocol. This approach involves a contingent test based on biochemical and ultrasound markers commonly used in clinical practice. We aim to leverage cost-effective contingent prenatal DNA screening for trisomies 21, 18, and 13. This is integrated into standard aneuploidy screening during the first trimester at prenatal centers. Screening participants are categorized into four pathways (1T Quad, 1T Combined, 1T Expanded Combined, and 1T Expanded Combined + Ductus Venosus Pulsatility Index) based on biochemical markers (PAPP-A, Free β-hCG, PlGF, AFP) and ultrasound indicators (NT, DV-PI) as per FMF guidelines. Pregnant subjects (Low/High/Moderate Risk) with a high chance of affected pregnancy (1:1000 ≤ Risk < 1:10) undergo the DNA reflex screening test using stored plasma samples.

Practical Machine Learning-Assisted Design Protocol for Protein Engineering: Transaminase Engineering for the Conversion of Bulky Substrates

Practical Machine Learning-Assisted Design Protocol for Protein Engineering: Transaminase Engineering for the Conversion of Bulky Substrates

Organic Base-Promoted C-N- and C-O-Coupled Domino Cyclization Strategy: Syntheses of Oxazine-6-ones and 4-Pyrimidinols.

Oxazine-6-one and 4-pyrimidinol are two important frameworks in pharmaceutical production. Herein, we disclosed a simple, efficient, inexpensive organic base-promoted and additive-stimulated protocol for the syntheses of variably functionalized oxazine-6-ones and 4-pyrimidinols employing acetonitrile solvent under conventional heating conditions using an oil bath through C-N and C-O coupled domino steps. This simple practicable productive protocol utilizes easily producible cheap precursors, namely, benzimidates or benzamidines, with differently substituted dicyano-olefins, and it comprises step economy, robustness, and moisture insensitive conditions affording high yield that avoids the use of transition-metal catalysts, multistep with multicomponent strategy, and harsh reaction conditions involving hazardous chemicals. This method is scalable into gram-scale production with good yield.

Musculoskeletal Health Systematic Review: Clinical and Pharmacological, Organizational and Legal, Administration and Pharmaceutical Management Aspects

This article examines current aspects of musculoskeletal health and pharmacotherapy of disorders of the human musculoskeletal system. An objective review of disorders of the musculoskeletal system was conducted in the areas of administration, clinical and pharmacological, organizational and legal, pharmaceutical management. The results of the systematic review show that the availability of diagnoses in the section "Orthopedics and traumatology" on the platform of the state expert center of the Ministry of Health of Ukraine is limited. It is necessary and urgent to expand the scientific base for practicing doctors. The study confirmed the importance of using new clinical protocols in medical and pharmaceutical practice for the implementation of evidence-based medicine and evidence-based pharmacy in Ukraine. Gradual improvement of medical and pharmaceutical practice allows to increase the quality, efficiency, safety, availability of pharmacotherapy for patients with disorders of the musculoskeletal system. The paper examines guidelines developed on the basis of health evidence provided by Duodecim Medical Publications. The study shows the availability of a wide range of treatment methods and helps to determine optimal therapy strategies in the field of orthopedics. It was found that non-steroidal anti-inflammatory drugs and analgesics are more often prescribed for the pharmacotherapy of disorders of the musculoskeletal system, which emphasizes the perspective of further clinical and pharmacological, organizational and legal researches in the pharmaceutical development and management of new drugs.

The Conflict between Regulatory Agencies over the 20,000-Fold Lowering of the Tolerable Daily Intake (TDI) for Bisphenol A (BPA) by the European Food Safety Authority (EFSA).

The European Food Safety Authority (EFSA) recommended lowering their estimated tolerable daily intake (TDI) for bisphenol A (BPA) 20,000-fold to . BPA is an extensively studied high production volume endocrine disrupting chemical (EDC) associated with a vast array of diseases. Prior risk assessments of BPA by EFSA as well as the US Food and Drug Administration (FDA) have relied on industry-funded studies conducted under good laboratory practice protocols (GLP) requiring guideline end points and detailed record keeping, while also claiming to examine (but rejecting) thousands of published findings by academic scientists. Guideline protocols initially formalized in the mid-twentieth century are still used by many regulatory agencies. EFSA used a 21st century approach in its reassessment of BPA and conducted a transparent, but time-limited, systematic review that included both guideline and academic research. The German Federal Institute for Risk Assessment (BfR) opposed EFSA's revision of the TDI for BPA. We identify the flaws in the assumptions that the German BfR, as well as the FDA, have used to justify maintaining the TDI for BPA at levels above what a vast amount of academic research shows to cause harm. We argue that regulatory agencies need to incorporate 21st century science into chemical hazard identifications using the CLARITY-BPA (Consortium Linking Academic and Regulatory Insights on BPA Toxicity) nonguideline academic studies in a collaborative government-academic program model. We strongly endorse EFSA's revised TDI for BPA and support the European Commission's (EC) apparent acceptance of this updated BPA risk assessment. We discuss challenges to current chemical risk assessment assumptions about EDCs that need to be addressed by regulatory agencies to, in our opinion, become truly protective of public health. Addressing these challenges will hopefully result in BPA, and eventually other structurally similar bisphenols (called regrettable substitutions) for which there are known adverse effects, being eliminated from all food-related and many other uses in the EU and elsewhere. https://doi.org/10.1289/EHP13812.

Blood Pressure Variability in Acute Stroke: A Narrative Review.

The management of blood pressure variability (BPV) in acute stroke presents a complex challenge with profound implications for patient outcomes. This narrative review examines the role of BPV across various stages of acute stroke care, highlighting its impact on treatment strategies and prognostic considerations. In the prehospital setting, while guidelines lack specific recommendations for BP management, emerging evidence suggests a potential link between BPV and outcomes. Among ischaemic stroke patients who are ineligible for reperfusion therapies, BPV independently influences functional outcomes, emphasising the need for individualised approaches to BP control. During intravenous thrombolysis and endovascular therapy, the intricate interplay between BP levels, recanalisation status, and BPV is evident. Striking a balance between aggressive BP lowering and avoiding hypoperfusion-related complications is essential. Intracerebral haemorrhage management is further complicated by BPV, which emerges as a predictor of mortality and disability, necessitating nuanced BP management strategies. Finally, among patients with acute subarachnoid haemorrhage, increased BPV may be correlated with a rebleeding risk and worse outcomes, emphasizing the need for BPV monitoring in this population. Integration of BPV assessment into clinical practice and research protocols is crucial for refining treatment strategies that are tailored to individual patient needs. Future studies should explore novel interventions targeting BPV modulation to optimise stroke care outcomes.

Brucella canis: a clinical review and rationalising risk

Background: After a letter published by the chief veterinary officer in February 2021 highlighted an increase in the number of positive Brucella canis dogs, the frequency of B canis testing in the UK has increased substantially. To date, roughly 160 positive cases have been reported by the APHA. Amid growing unease, veterinary teams have implemented practice protocols to mitigate risk associated with this non‐endemic, zoonotic infection. Although well‐meaning, blanket policy and inappropriate application of serological tests can negatively impact client and patient welfare while fuelling health anxiety among staff. The combination of increased pet travel, increased popularity of importing dogs and a lack of centralised border requirements for dogs, means B canis is likely to be increasingly encountered and warrants regular, evidence‐based discussion to empower the profession and preserve canine welfare.Aim of the article: This article summarises the clinically relevant issues surrounding Brucella canis, focussing on epidemiology, transmission, human health and safety, clinical signs, diagnosis and treatment. Governmental recommendations and recent risk assessments by the Human Animal Infection and Risk Surveillance group are drawn on where appropriate. Moreover, the article aims to instil confidence in veterinary personnel when rationalising B canis infection risk, and help to ensure that canine and staff health are protected.

The impact of net cleaning on fecal cortisol metabolites in farmed Atlantic Salmon

Stress assessment is crucial in aquaculture to ensure and improve the health and well-being of production animals including fish. The primary objective of this study was to utilize the assessment of fecal cortisol metabolites (FCMs) as a low-invasive stress determination method for farmed Atlantic salmon (Salmo salar L.) and to suggest an appropriate sampling timeframe for this measurement.Blood, mid-intestinal contents, and feces samples were collected before, during, and at 2 and 20 h after a stress-inducing event, namely, net cleaning. Plasma cortisol (PC) and its metabolite levels in the mid-intestinal contents (MICMs) and FCMs were assessed by an enzyme-linked immunosorbent assay (ELISA). The research reveals a distinctive temporal profile, with a sequential elevation in levels of PC, MICMs and FCMs in response to the effects of net cleaning. Peak FCMs levels were observed approximately 20 h after net cleaning and were significantly higher than peak PC levels (P < 0.05). The findings suggest that FCMs are a promising indicator for acute stress assessment. To enhance the understanding and improve the applicability of FCMs as stress indicators in fish farming practices the critical challenges regarding feces sampling and FCMs analyses are further discussed. Future research aimed at developing practical protocols and industry standards will emphasize method validation and include a greater number of sampling points as well as an expanded experimental period.This study signifies a crucial step toward understanding the role of FCMs as a reliable stress indicator in fish. It underscores the importance of accounting for the delayed and extended sampling patterns when assessing stress in aquatic settings. Moreover, it offers a less invasive and more labor-efficient approach for monitoring the overall health and welfare of salmonids than PC analysis, thereby promoting sustainability in this vital industry.

Imaging of the Liver and Pancreas: The Added Value of MRI.

MR is a powerful diagnostic tool in the diagnosis and management of most hepatic and pancreatic diseases. Thanks to its multiple sequences, the use of dedicated contrast media and special techniques, it allows a multiparametric approach able to provide both morphological and functional information for many pathological conditions. The knowledge of correct technique is fundamental in order to obtain a correct diagnosis. In this paper, different MR sequences will be illustrated in the evaluation of liver and pancreatic diseases, especially those sequences which provide information not otherwise obtainable with other imaging techniques. Practical MR protocols with the most common indications of MR in the study of the liver and pancreas are provided.

Abstract 6193: Exploring interplay between tumor intrinsic features and immune cell phenotypes in multi-tumor tissue microarrays by a single step 15-plex fluorescence immunohistochemistry and imaging on the Orion platform

Abstract Purpose: Deeper understanding of the intricacies between tumor microenvironments and immune cell infiltrates is critical for creation of next generation therapies. However, current immunohistochemistry protocols in clinical practice are restricted to one to six markers (cell types) and rely on multiple rounds of antigen retrieval and sequential staining over two days. This current workflow has high risk for tissue and signal loss. Hence, we explored utility of a single step staining workflow using 15 distinct antibodies conjugated novel flours (Argo Dye System) of formalin fixed paraffin embedded (FFPE) tumor microarrays and imaged on the Orion microscope (Rarecyte). Fidelity of the staining was assessed by comparing data from the Orion platform to internally developed clinical grade assays. The implementation of higher plex imaging will enable greater insight into immune surveillance, mechanisms of resistance and patient stratification while minimizing patient tissue required for analysis. Study Design: We utilized a custom panel developed by RareCyte (Hoescht, CD3e, CD4, CD8a, CD20, CD68, CD163, FOXP3, Ki67, Pan-CK, PD1, PDL1, CD45, Granzyme B, LAG3) to evaluate expression of immune markers on a pan-tumor and melanoma TMA including Hodgkin’s lymphoma, ovarian cancer, esophageal carcinoma, hepatic cell carcinoma, head and neck cancer. Serial sections were stained with 6-plex clinical grade assays, and correlation scores were determined between the 15-plex Orion and 6-plex assays utilizing TSA-based amplification. Results: In this study, the performance of Orion platform was assessed by quantitative and qualitative measures. Linear regression between the 6-plex clinical grade assays and 15-plex Orion workflow were calculated for a panel of biomarkers including CD3, CD4, CD8, CD68, CD163, CK, FOXP3, GZMB, Ki67, and PD1. Our results demonstrated that the Orion performed similarly to clinical grade assays both in terms of proportion of cells and fluorescence intensity (R2 ~ 0.7-0.98, Slope ~ 0.7-1.1). Conclusion: Our results suggest that the Orion platform offers a reliable and efficient solution for comprehensive biomarker analysis. Our results demonstrate that this platform can successfully capture the expression of 15 biomarkers on a single FFPE slide which offers workflow advantages over traditional TSA-based fIHC multiplexing that is limited to detection of 6 biomarkers on a single slide. The ability to analyze a wider range of biomarkers through a single platform has the potential to enhance the efficiency of biomarker analysis, leading to improved diagnostic and therapeutic outcomes in clinical practice. Citation Format: Richard Van Krieken, Jehovana O. Bender, Sophia Kekchidou, Michelle Burgin, Kristen Ruma, Naveen Dakappagari, Jennifer Bordeaux. Exploring interplay between tumor intrinsic features and immune cell phenotypes in multi-tumor tissue microarrays by a single step 15-plex fluorescence immunohistochemistry and imaging on the Orion platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6193.

Selective electrochemical thiocyanation/selenocyanation of C(sp3)-H and C(sp2)-H bonds

Herein, we report a general and eco-friendly electrochemical method for the selective thiocyanation/selenocyanation of C(sp3)-H and C(sp2)-H bonds in nitrogen-containing compounds under exogenous oxidant-free conditions. This is the first example of selective thiocyanation based on a nitrogen-containing compound with excellent regional principles and chemical selectivity for different reaction sites. This convenient and practical protocol has considerable potential for the modification of important structural backbones.

Validation and Implementation of OptiView and EnVision FLEX Detection Systems for Immunocytochemical Staining Protocols of the Ten Most Commonly Used Diagnostic Markers in Routine Cytopathological Practice.

The withdrawal of the iView detection system (iV) forced many cytopathology laboratories, including ours, to substitute immunocytochemical (ICC) staining protocols for routine practice with other detection systems. Our objective was to optimize, validate, and implement ICC protocols using OptiView (OV) and EnVision FLEX (EnV) detection systems, comparing the results with those obtained using iV. Residual cytologic samples with known diagnoses were used, testing antibodies for the ten most common markers in routine cytopathology diagnostics (calretinin, Ber-EP4, MOC-31, CKAE1/AE3, CK5/6, CD68, LCA, desmin, HBME-1, and WT1). Different staining parameters were tested using OV on BenchMark ULTRA and EnV on Dako Omnis immunostainer, respectively. Optimal staining protocols were then selected and validated on 10 positive and 10 negative cases. The staining results were compared with iV protocols through evaluation of UK NEQAS and internal scores. The optimal staining protocols with OV and EnV demonstrated similar or superior results compared to the existing iV protocols, with slightly stronger intensity regarding positive cells. We have successfully established and validated optimal ICC staining protocols for commonly used markers in routine cytopathology practice. These protocols may benefit other laboratories using similar staining platforms. However, the challenge regarding standardizing ICC protocols across different cytopathology laboratories remains unresolved.

Optimal individual and collective measurements for nonorthogonal quantum key distribution signals

We consider how the theory of optimal quantum measurements determines the maximum information available to the receiving party of a quantum key distribution (QKD) system employing linearly independent but nonorthogonal quantum states. Such a setting is characteristic of several practical QKD protocols. Due to nonorthogonality, the receiver is not able to discriminate unambiguously between the signals. To understand the fundamental limits that this imposes, the quantity of interest is the maximum mutual information between the transmitter (Alice) and the receiver, whether legitimate (Bob) or an eavesdropper (Eve). To find the optimal measurement—taken individually or collectively—we use a framework based on operator algebra and general results derived from singular-value decomposition, achieving optimal solutions for von Neumann measurements and positive operator-valued measures (POVMs). The formal proof and quantitative analysis elaborated for two signals allow us to conclude that optimal von Neumann measurements are uniquely defined and provide a higher information gain compared to POVMs. Interestingly, collective measurements not only do not provide additional information gain with respect to individual ones but also suffer from a gain reduction in the case of POVMs. Published by the American Physical Society 2024

Visible Light-Induced 1,2-Diphenyldisulfane-Mediated Defluoroborylation of Polyfluoroarenes.

A green and practical protocol of defluoroborylation of polyfluoroarenes with stable and readily accessible NHC-borane was developed, using 1,2-diphenyldisulfane as a hydrogen atom transfer (HAT) and single electron transfer (SET) reagent precursor under visible-light irradiation, leading to the concise formation of value-added fluorinated organoboron scaffolds. Mechanism studies revealed the method underwent a boryl radical addition reaction with polyfluoroarene, followed by successive single electron transfer pathways and defluorination of the C-F bond to offer the targeted product. This unprecedented platform relies on 1,2-diphenyldisulfane and base without using expensive photocatalysts, highlighting the methodology has promising application value to prepare borylated polyfluoroarene compounds.

Pediatricians perception of stool color cards as a screening tool for biliary atresia: a cross-sectional survey in Sudan

ABSTRACT Background A stool color card has improved the prognosis of biliary atresia patients since it was first developed in Japan. This study investigated how Sudanese pediatric residents currently utilize stool color cards. Methods In January and February of 2022, 254 pediatric residents participated in this facility-based, cross-sectional study. The researchers developed a structured questionnaire, which was completed in Google form with a 100% response rate after it was pretested and validated. All the statistical analyses were performed with SPSS 25.0 (SPSS, Inc. Chicago, IL). The ethical committee of the Sudan Medical Specialization Board approved the study. All participants provided written informed consent. Results A total of 254 residents, ranging in age from 25 to 40 years, were enrolled in the study; 215 of whom were female (84.6%), and 39 were male (15.4%). Approximately 54.7% of the residents did not know about SCC. Of those who knew of SCC, 73% were proficient users. 71% of the participants properly identified the first three normal photographs of stool color out of the six photos in the SSC example. Nevertheless, only 13% of the participants correctly identified the final three abnormal images. The majority of residents (84.6%) were aware of when to send patients to gastroenterologists. At least one prereferral investigation was ordered by 83.8% of the residents. Conclusion The residents’ knowledge was unrelated to their practices. Educational interventions as well as practice protocols and guidelines are needed. More in-depth research with suitable designs and methodologies may offer additional clarity in the future.

Single-photon-based quantum secure protocol for the socialist millionaires’ problem

The socialist millionaires' problem, emanating from the millionaires’ problem, allows two millionaires to determine whether they happen to be equally rich while remaining their riches undisclosed to each other. Most of the current quantum solutions to the socialist millionaires’ problem have lower efficiency and are theoretically feasible. In this paper, we introduce a practical quantum secure protocol for the socialist millionaires’ problem based on single photons, which can be easily implemented and manipulated with current technology. Our protocol necessitates the involvement of a semi-honest third party (TP) responsible for preparing the single-photon sequences and transmitting them to Alice who performs Identity or Hadamard operations on the received quantum sequences via her private inputs and the secret keys, producing new quantum sequences that are subsequently sent to Bob. Similarly, Bob encodes his private inputs into the received quantum sequences to produce new quantum sequences, which are then sent to TP. By conducting single-particle measurements on the quantum sequences received from Bob, TP can ascertain the equality of private inputs between Alice and Bob, and subsequently communicate the comparison result to them. To assess the feasibility, the proposed protocol is simulated on IBM Quantum Cloud Platform. Furthermore, security analysis demonstrates that our protocol can withstand attacks from outsiders, such as eavesdroppers, and from insider participants attempting to grab the private input of another participant.

Acute effects of different Tai Chi practice protocols on cardiac autonomic modulation

Tai Chi serves as an effective exercise modality for enhancing autonomic regulation. However, a majority of existing studies have employed the single routine (SR) protocol as the basis for health interventions. The extent to which the gong routine application (GRA) protocol achieves similar levels of exercise load stimulation as traditional single practice routines remains uncertain. Therefore, this study the distinct characteristics of autonomic load stimulation in these different protocols, thus providing a biological foundation to support the development of Tai Chi health promotion intervention programs. we recruited a cohort of forty-five university students to participate in the 15 min GRA protocol and SR protocol. We collected heart rate and heart rate variability indicators during periods of rest, GRA protocol, and SR protocol utilizing the Polar Scale. Additionally, we assessed the mental state of the participants using the BFS State of Mind Scale. In summary, the autonomic load is lower in the GRA protocol compared to the SR protocol, with lower sympathetic activity but higher parasympathetic activity in the former. Results are specific to college students, additional research is necessary to extend support for frail older adults. It is advised to incorporate GRA protocol alongside SR protocol in Tai Chi instruction. This approach is likely to enhance Tai Chi skills and yield greater health benefits.

Synthesis of N‐Difluoromethyl Carbonyl Compounds from N‐Difluoromethylcarbamoyl Fluorides

AbstractFluorinated small molecules are commonly used in functional small‐molecule chemistry, and N‐difluoromethyl (N‐CF2H) compounds are particularly intriguing due to their unique and unexplored physiochemical properties. However, despite limited progress, a general methodological approach to the synthesis of N‐CF2H compounds remains elusive. Here, guided by computation, we present a simple and practical protocol to access N‐CF2H amides and related carbonyl derivatives. The protocol involves a one‐pot conversion of thioformamides through desulfurization‐fluorination and acylation, providing N‐difluoromethylcarbamoyl fluoride building blocks that can be further diversified to a variety of unexplored N‐CF2H carbonyl compounds with rich functionality. Additionally, preliminary studies on their properties and stability showcased their potential application in pharmaceuticals and agrochemicals.

Synthesis of N-Difluoromethyl Carbonyl Compounds from N-Difluoromethylcarbamoyl Fluorides.

Fluorinated small molecules are commonly used in functional small-molecule chemistry, and N-difluoromethyl (N-CF2H) compounds are particularly intriguing due to their unique and unexplored physiochemical properties. However, despite limited progress, a general methodological approach to the synthesis of N-CF2H compounds remains elusive. Here, guided by computation, we present a simple and practical protocol to access N-CF2H amides and related carbonyl derivatives. The protocol involves a one-pot conversion of thioformamides through desulfurization-fluorination and acylation, providing N-difluoromethylcarbamoyl fluoride building blocks that can be further diversified to a variety of unexplored N-CF2H carbonyl compounds with rich functionality. Additionally, preliminary studies on their properties and stability showcased their potential application in pharmaceuticals and agrochemicals.

Neuroadaptive Output Formation Tracking for Heterogeneous Nonlinear Multiagent Systems With Multiple Nonidentical Leaders.

This article investigates the practical time-varying output formation tracking (TVOFT) problem for heterogeneous nonlinear multiagent systems (MASs) having multiple leaders, where agents herein could have heterogeneous dynamics and interact with each other under event-triggered communications. It is required that the outputs of followers not only track the predefined convex combination of multiple leaders but also achieve the desired time-varying formation simultaneously. The existing works on formation tracking problems for MASs with multiple leaders depend on the assumption that each follower is a well-informed or uninformed follower, where the well-informed follower is required to have all the leaders as its neighbor. To remove the limitation, a fully distributed observer-based formation tracking control protocol is developed and employed. First, an adaptive state observer with an edge-based event-triggered mechanism for estimating the states of multiple leaders is proposed based on the neighboring interactions, which eliminates the unexpected Zeno behavior. Second, a novel observer is constructed for each follower by exploiting the output information of the follower, in which the adaptive neural network (NN)-based approximation is exploited to compensate for the unknown nonlinearity. A practical TVOFT control protocol is then generated by the proposed observers, where the parameters are determined by an algorithm including five steps. With the help of Lyapunov stability theory and output regulation method, a practical TVOFT criterion for the considered closed-loop system is derived. Finally, the effectiveness of the proposed control scheme is illustrated by a numerical example.