Practical Protocol Research Articles

Practical Protocol and Parameter Optimizations For SBF-SEM Imaging – Tips & Tricks for Technically-Demanding Tasks

Practical Protocol and Parameter Optimizations For SBF-SEM Imaging – Tips & Tricks for Technically-Demanding Tasks

Practical issues related to immunocytochemistry on cytological smears: Tips and recommendations.

Immunocytochemistry (ICC) is essential for enhancing diagnostic accuracy and identifying markers for diagnosis, prognosis and targeted therapies. While cell blocks (CBs) are preferred for standardization and optimized staining, cytological smears are an alternative when CBs are unavailable. However, the literature on ICC protocols for smears is sparse. This review addresses preparation, fixation and protocols for nuclear and cytoplasmic antibodies on smears, drawing from our laboratory's experience. We reviewed procedures for ICC on cytological smears using existing literature and practical insights from our laboratory. Commercially available antibodies were found to be reliable for ICC on smears if specimens are properly prepared and fixed. Protocols developed in our laboratory maintained antigenicity and provided clear staining results. Although ICC on CBs is the gold standard for standardization, cytological smears are a viable alternative when CBs are unavailable. Success in ICC on smears depends on proper preparation and fixation. This review offers practical protocols and insights to help laboratories optimize ICC on cytological smears. Further research and standardization are necessary to enhance reproducibility and reliability of ICC on smears. The practical information provided is based on personal experience in our laboratory.

TP53 mutations in myeloid neoplasms: implications for accurate laboratory detection, diagnosis, and treatment.

Genetic alterations that affect the function of p53 tumor suppressor have been extensively investigated in myeloid neoplasms, revealing their significant impact on disease progression, treatment response, and patient outcomes. The identification and characterization of TP53 mutations play pivotal roles in subclassifying myeloid neoplasms and guiding treatment decisions. Starting with the presentation of a typical case, this review highlights the complicated nature of genetic alterations involving TP53 and provides a comprehensive analysis of TP53 mutations and other alterations in myeloid neoplasms. Currently available methods used in clinical laboratories to identify TP53 mutations are discussed, focusing on the importance of establishing a robust testing protocol within clinical laboratories to ensure the delivery of accurate and reliable results. The treatment implications of TP53 mutations in myeloid neoplasms and clinical trial options are reviewed. Ultimately, we hope that this review provides valuable insights into the patterns of TP53 alterations in myeloid neoplasms and offers guidance to establish practical laboratory testing protocols to support the best practices of precision oncology.

Oral Oncology: Two elective courses for advanced undergraduates - An Argentine teaching experience

The COVID-19 pandemic prompted significant adaptations in educational practices at Universidad Nacional de Córdoba, including the integration of e-learning and virtual education modalities. In response to an observed increase in advanced-stage oral cancer cases post-2020, we developed new educational strategies aimed at OC prevention and awareness. This study presents our experience with an innovative undergraduate program focusing on Oral Oncology and Oral Potentially Malignant Disorders. Two elective courses were integrated into the curriculum, featuring contributions from Oral Medicine and Pathology experts across several Latin American countries via virtual platforms. Each course provided comprehensive training, including clinical practice and telemedicine protocols for early lesion identification. A total of 107 students successfully completed the courses, highlighting the program's effectiveness in enhancing student engagement and competence in OC prevention. Recommendations from this initiative include reinforcing undergraduate curricula with OC-focused education and promoting international collaboration in higher education for comprehensive training. Future research should assess the long-term impact of such programs on early OC diagnosis and patient referral practices.

Hearing Screening for Older Adults With Cognitive Impairment: A Systematic Review.

: Hearing loss is the largest potentially modifiable risk factor for dementia. Early evaluation and intervention are crucial for older adults with cognitive impairment. However, pure-tone audiometry (PTA), the gold standard, may have limitations. This study reviewed auditory tests for detecting hearing loss in this population, comparing to PTA. Materials and Methods: Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, we conducted a systematic review in PubMed, Scopus, and Embase. Ten studies (1,071 participants) comparing auditory tests and PTA in patients with dementia and mild cognitive impairment were included. : Electrophysiological and behavioral tests demonstrated strong correlation and accurate hearing impairment detection compared to PTA. Conversely, self-reported assessment showed weaker correlations when aligned with hearing thresholds. : This study highlights the potential of electrophysiological and behavioral auditory tests in detecting hearing impairment in older adults with cognitive impairment, emphasizing the need for further research to develop practical screening protocols for this vulnerable population.

Benchmarking long-read sequencing strategies for obtaining ASV-resolved rRNA operons from environmental microeukaryotes.

The use of short-read metabarcoding for classifying microeukaryotes is challenged by the lack of comprehensive 18S rRNA reference databases. While recent advances in high-throughput long-read sequencing provide the potential to greatly increase the phylogenetic coverage of these databases, the performance of different sequencing technologies and subsequent bioinformatics processing remain to be evaluated, primarily because of the absence of well-defined eukaryotic mock communities. To address this challenge, we created a eukaryotic rRNA operon clone-library and turned it into a precisely defined synthetic eukaryotic mock community. This mock community was then used to evaluate the performance of three long-read sequencing strategies (PacBio circular consensus sequencing and two Nanopore approaches using unique molecular identifiers) and three tools for resolving amplicons sequence variants (ASVs) (USEARCH, VSEARCH, and DADA2). We investigated the sensitivity of the sequencing techniques based on the number of detected mock taxa, and the accuracy of the different ASV-calling tools with a specific focus on the presence of chimera among the final rRNA operon ASVs. Based on our findings, we provide recommendations and best practice protocols for how to cost-effectively obtain essentially error-free rRNA operons in high-throughput. An agricultural soil sample was used to demonstrate that the sequencing and bioinformatic results from the mock community also translates to highly diverse natural samples, which enables us to identify previously undescribed microeukaryotic lineages.

Visible-Light-Driven Multicomponent Reactions for the Versatile Synthesis of Thioamides by Radical Thiocarbamoylation.

Thioamides are widely used structures in pharmaceuticals and agrochemicals, as well as important synthons for the construction of sulfur-containing heterocycles. This report presents a series of visible-light-driven multicomponent reactions of amines, carbon disulfide, and olefins for the mild and versatile synthesis of linear thioamides and cyclic thiolactams. The use of inexpensive and readily available carbon disulfide as the thiocarbonyl source in a radical pathway enables the facile assembly of structurally diverse amine moieties with non-nucleophilic carbon-based reaction partners. Radical thiocarbamoylative cyclization provides a practical protocol that complements traditional approaches to thiolactams relying on deoxythionation. Mechanistic studies reveal that direct photoexcitation of in situ formed dithiocarbamate anions as well as versatile photoinduced electron transfer with diverse electron acceptors are key to the reactions.

Visible‐Light‐Driven Multicomponent Reactions for the Versatile Synthesis of Thioamides by Radical Thiocarbamoylation

Thioamides are widely used structures in pharmaceuticals and agrochemicals, as well as important synthons for the construction of sulfur‐containing heterocycles. This report presents a series of visible‐light‐driven multicomponent reactions of amines, carbon disulfide, and olefins for the mild and versatile synthesis of linear thioamides and cyclic thiolactams. The use of inexpensive and readily available carbon disulfide as the thiocarbonyl source in a radical pathway enables the facile assembly of structurally diverse amine moieties with non‐nucleophilic carbon‐based reaction partners. Radical thiocarbamoylative cyclization provides a practical protocol that complements traditional approaches to thiolactams relying on deoxythionation. Mechanistic studies reveal that direct photoexcitation of in situ formed dithiocarbamate anions as well as versatile photoinduced electron transfer with diverse electron acceptors are key to the reactions.

Improving Clinical Communication: a qualitative study on the Informed Consent

In the context of the Patient-Centred Care paradigm (Epstein et al., 2005; Suojanen et al., 2012) and the shift toward the psychobiological model (Dean & Street, 2015; Muñoz & García-Izquierdo, 2020), there is a growing demand for the patient to be an active agent in the management of their health. Clinical communication should be conveyed accurately and empathetically (Bellés Fortuño & García-Izquierdo, forthcoming), especially in complex legal genres such as the Informed Consent (IC). The research carried out by the Gentt research group up-to-date has revealed that there is no specific monitoring with the use of IC protocols in clinical practice. In this paper, we present the results of a qualitative pilot study with a group of practitioners from the Valencian Community (Spain). A focus group was conducted where the articulation of communication with patients was analyzed. The study tries to define the practical insights of using the IC to draw conclusions that can improve clinical communication. Results show that MPs generally consider that the IC process needs improvement, especially when considering closeness with patients to enhance communication.

Cobalt-Catalyzed Borylative Reduction of Azobenzenes to Hydrazobenzenes via a Diborylated-Hydrazine Intermediate.

Cobalt-catalyzed borylative reduction of azobenzenes using pinacolborane is developed. The simple cobalt chloride catalyst and reaction conditions make this protocol attractive for hydrazobenzene synthesis. This borylative reduction shows good functional group compatibility and can be readily scaled up to the gram scale. Preliminary mechanistic studies clarified the proton source of the hydrazine products. This cobalt-catalyzed azobenzene borylative reaction provides a practical protocol to prepare synthetically useful diborylated hydrazines.

Development of best evidence-based practice protocols for central venous catheter placement and maintenance to reduce CLABSI.

Although evidence-based interventions can reduce the incidence of central line-associated bloodstream infection (CLABSI), there is a large gap between evidence-based interventions and the actual practice of central venous catheter (CVC) care. Evidence-based interventions are needed to reduce the incidence of CLABSI in intensive care units (ICU) in China. Professional association, guidelines, and database websites were searched for data relevant to CLABSI in the adult ICUs from inception to February 2020. Checklists were developed for both CVC placement and maintenance. Based on the Integrated Promoting Action on Research Implementation in Health Services framework, a questionnaire collected the cognition and practice of ICU nursing and medical staff on the CLABSI evidence-based prevention guidelines. From January 2018 to December 2021, ICU CLABSI rates were collected monthly. Ten clinical guidelines were included after the screening and evaluation process and used to develop the best evidence-based protocols for CVC placement and maintenance. The CLABSI rates in 2018, 2019, and 2020 were 2.98‰ (9/3021), 1.83‰ (6/3276), and 1.69‰ (4/2364), respectively. Notably, the CLABSI rate in 2021 was 0.38‰ (1/2607). In other words, the ICU CLABSI rate decreased from 1.69‰ to 0.38‰ after implementation of the new protocols. Additionally, our data suggested that the use of ultrasound-guidance for catheter insertion, chlorhexidine body wash, and the use of a checklist for CVC placement and maintenance were important measures for reducing the CLABSI rate. The evidence-based processes developed for CVC placement and maintenance were effective at reducing the CLABSI rate in the ICU.

Electrophotochemical metal-catalyzed synthesis of alkylnitriles from simple aliphatic carboxylic acids.

We report a practical and sustainable electrophotochemical metal-catalyzed protocol for decarboxylative cyanation of simple aliphatic carboxylic acids. This environmentally friendly method features easy availability of substrates, broad functional group compatibility, and directly converts a diverse range of aliphatic carboxylic acids including primary and tertiary alkyl acids into synthetically versatile alkylnitriles without using chemical oxidants or costly cyanating reagents under mild reaction conditions.

World Federation of Acupuncture-Moxibustion Societies Clinical Practice Guideline on Acupuncture-Moxibustion: Non-Specific Low Back Pain recommendation summaries: 世界针灸学会联合会《针灸临床实践指南:非特异性腰痛》推荐意见概述

Clinical Practice Guideline on Acupuncture and Moxibustion: Nonspecific low back pain was revised and released by the Standards Working Committee of World Federation of Acupuncture-Moxibustion Societies (WFAS) on October 9, 2023. This is the first clinical practice guideline (CPG) on acupuncture and moxibustion for nonspecific low back pain approved by an international academic organization, which provides the evidence-based recommendations and practical therapeutic protocols for international acupuncture practitioners. This CPG was developed by following Grades of Recommendation, Assessment, Development, and Evaluation (GRADE) methodology, and the Principles of the World Health Organization Handbook for Guideline Development. The guideline development group (GDG) from different countries, with different professions, played a critical role in the formulation of clinical questions, recommendations, and therapeutic protocols. Recommendations are the key content of a CPG and the direct answers to clinical questions. Hence, this article focuses on the recommendations of this CPG. The recommendations were formulated using the modified Delphi method and the GRADE grid rules, based on the updated systematic reviews of clinical evidence. A total of seven recommendations for ten clinical questions were formulated in this CPG, including one conditional recommendation for either the intervention or the comparison based on very low quality of evidence, and six conditional recommendations for the intervention based on very low quality of evidence.

A Dynamic Adaptive Framework for Practical Byzantine Fault Tolerance Consensus Protocol in the Internet of Things

A Dynamic Adaptive Framework for Practical Byzantine Fault Tolerance Consensus Protocol in the Internet of Things

Rapid Glyco-Qualitative Assessment of Recombinant Proteins Using a Fully Automated System.

Protein glycosylation, a critical post-translational modification, influences the stability, efficacy, and immunogenicity of recombinant proteins, including biopharmaceuticals. Glycan structures exhibit significant heterogeneity, varying with production cell types, culture conditions, and purification methods. Consequently, monitoring and evaluating the glycan structures of recombinant proteins is vital, particularly in biopharmaceutical production. The lectin microarray, a technique complementary to mass spectrometry, boasts high sensitivity and ease of use. However, it typically requires more than a day to yield results. To adapt it to non-glycoscience research or drug product process development, an automated, high-throughput alternative is needed. Therefore, the world's first fully automated lectin-based glycan profiling system was developed, utilizing the "bead array in a single tip (BIST)" technology concept. This system allows for the preparation and storage of lectin-immobilized beads in units of 1,000, with customizable parallel insertion orders for various purposes. This article presents a practical protocol for research involving "glyco-qualified" recombinant proteins. After testing their reactivity against 12 polyacrylamide-glycan conjugates, 15 lectins were selected to increase the system's versatility. In addition, the sample labeling process was optimized by switching from Cy3 to biotin, reducing the overall processing time by 30 min. For immediate data qualification, lectin-binding signals are displayed as a dotcode on the top monitor. The system's reliability was confirmed through day-to-day reproducibility tests, repeatability tests, and long-term storage tests, with a coefficient of variation of <10%. This user-friendly and rapid glyco-analyzer has potential applications in the quality monitoring of endogenous glycoproteins for biomarker evaluation and validation. This method facilitates analysis for those new to glycoscience, thereby broadening its practical utility.

Photosynthetic Performance and Yield Losses of Winter Rapeseed (Brassica napus L. var. napus) Caused by Simulated Hail.

Winter oilseed rape (Brassica napus L.), Europe's foremost oilseed crop, is significantly impacted by hailstorms, leading to substantial yield reductions that are difficult to predict and measure using conventional methods. This research aimed to assess the effectiveness of photosynthetic efficiency analysis for predicting yield loss in winter rapeseed subjected to hail exposure. The aim was to pinpoint the chlorophyll fluorescence parameters most affected by hail stress and identify those that could act as non-invasive biomarkers of yield loss. The study was conducted in partially controlled conditions (greenhouse). Stress was induced in the plants by firing plastic balls with a 6 mm diameter at them using a pneumatic device, which launched the projectiles at speeds of several tens of meters per second. Measurements of both continuous-excitation and pulse-modulated-amplitude chlorophyll fluorescence were engaged to highlight the sensitivity of the induction curve and related parameters to hail stress. Our research uncovered that some parameters such as Fs, Fm', ΦPSII, ETR, Fo, Fv/Fm, and Fv/Fo measured eight days after the application of stress had a strong correlation with final yield, thus laying the groundwork for the creation of new practical protocols in agriculture and the insurance industry to accurately forecast damage to rapeseed crops due to hail stress.

Acquisition Efficiency and Technical Repeatability of Dual-Frequency 3D Vector MR Elastography of the Liver.

MR elastography (MRE) at 60 Hz is widely used for staging liver fibrosis. MRE with lower frequencies may provide inflammation biomarkers. To establish a practical simultaneous dual-frequency liver MRE protocol at both 30 Hz and 60 Hz during a single examination and validate the occurrence of second harmonic waves at 30 Hz. Retrospective. One hundred six patients (48 females, age: 50.0 ± 13.4 years) were divided as follows: Cohort One (15 patients with chronic liver disease [CLD] and 25 healthy volunteers) with simultaneous dual-frequency MRE. Cohort Two (66 patients with CLD) with second harmonic MRE. 3-T, single- or dual-frequency MRE at 30 Hz and 60 Hz. Liver stiffness (LS) in both cohorts was evaluated with manually placed volumetric ROIs by two independent analyzers. Image quality was assessed by three independent readers on a 4-point scale (0-3: none/failed, fair, moderate, excellent) based on the depth of wave propagation with 1/3 incremental penetration. The success rate was derived from the percentage of nonzero quality scores. Measurement agreement, bias, and repeatability of LS were assessed using intraclass correlation coefficients (ICCs), Bland-Altman plots, and repeatability coefficient (RC). Mann-Whitney U tests were used to evaluate the differences in image quality between different methods. A P-value <0.05 was considered statistically significant. Success rate was 97.5% in Cohort One and 91% success rate for the second harmonic MRE in Cohort Two. The second harmonic and conventional MRE showed excellent agreement in LS (all ICCs >0.90). The quality scores for the second harmonic wave images were lower than those from the conventional MRE (Z = -4.523). Compared with conventional and second harmonic methods, simultaneous dual-frequency had better image quality, high success rate and the advantage of intrinsic co-registration, while the second harmonic method can be an alternative if custom waveform is not available. 3 TECHNICAL EFFICACY: Stage 1.

Development of an evidence-based nursing practice program for preventing unplanned endotracheal extubations in the intensive care unit: A Delphi method study.

This study aims to develop an evidence-based nursing practice program to prevent unplanned endotracheal extubation (UEE) among adult patients in the intensive care unit (ICU). This study uses the Delphi method to develop an evidence-based nursing practice program. A comprehensive review of 18 databases and evidence-based websites was conducted to gather, assess and synthesize evidence on preventing UEEs in adult patients. Using this synthesized evidence, a questionnaire was formulated for further investigation. Subsequently, input was solicited from experts through Delphi surveys to establish an evidence-based nursing practice protocol for preventing UEEs in adult ICU patients. Consistency in consultation results guided subsequent rounds of consultation. The developed program comprised 43 evidence items categorized into nine dimensions, including risk assessment for unplanned extubation, sedation, analgesia, delirium, balloon management, psychosocial care, early extubation, catheter immobilization and protective restraints. Two rounds of expert inquiry yielded recovery rates of 94.7% and 100% for the first and second questionnaires, respectively. Kendall W values ranged from .224 to .353 (p < .001). This study developed an evidence-based nursing practice program to prevent UEE in adult ICU patients, employing evidence-based practices and Delphi expert consultation methods. However, further validation of the program's effectiveness is warranted. Findings were reported according to the Standards for Reporting Qualitative Research checklist. Nurses contributed to the study by participating in investigations. The program developed in this study offers an evidence-based framework for preventing unplanned extubation in hospitals, thereby reducing its incidence and enhancing the quality of nursing care.

Visible-Light-Induced Hydrodifluoromethylation of Unactivated Alkenes with Difluoroacetic Anhydride.

We herein described a practical and efficient protocol for hydrodifluoromethylation of unactivated alkenes using readily available difluoroacetic anhydride as a difluoromethyl source by merging photocatalysis and N-hydroxyphthalimide activation. This method features a wide substrate scope and excellent compatibility with various functional groups, as demonstrated by more than 50 examples, including bioactive molecules and pharmaceutical derivatives. Mechanism investigation indicated that N-hydroxyphthalimide may also serve as the hydrogen atom donor.

World Federation of Acupuncture-Moxibustion Societies (WFAS) Clinical Practice Guideline on Acupuncture and Moxibustion: Allergic Rhinitis recommendation summaries: 世界针灸学会联合会《针灸临床实践指南:过敏性鼻炎》推荐意见概述

Clinical Practice Guideline on Acupuncture and Moxibustion: Allergic Rhinitis was revised and released by the Standards Working Committee of World Federation of Acupuncture-Moxibustion Societies (WFAS) on October 9, 2023. This is the first clinical practice guideline (CPG) on acupuncture and moxibustion for allergic rhinitis approved by an international academic organization, which provides evidence-based recommendations and the practical therapeutic protocols for acupuncture practitioners globally. This CPG was developed by following Grades of Recommendation, Assessment, Development, and Evaluation (GRADE) methodology, and the principles of the World Health Organization Handbook for Guideline Development. The members in the guideline development group (GDG) were from different countries, with different professions, and they played a critical role in the development of clinical questions, recommendations, and therapeutic protocols. Recommendations are the core of a CPG and the direct answers to clinical questions, due to which this article focuses on the recommendations of this CPG. The recommendations were developed by using the modified Delphi method and the GRADE grid rules, based on the updated systematic reviews of clinical evidence. A total of 15 recommendations for 15 clinical questions were developed in this CPG, including one strong recommendation for intervention based on high-quality evidence, three conditional recommendations for either the intervention or the comparison based on very low quality of evidence, and 11 conditional recommendations for the intervention based on very low quality of evidence.