Transient stabilization of human cardiovascular progenitor cells from hPSCs in vitro reflects stage-specific heart development in vivo.

AIM: Understanding the molecular identity of human pluripotent stem cell (hPSC)-derived cardiac progenitors and mechanisms controlling their proliferation and differentiation, is valuable for developmental biology and regenerative medicine. METHODS AND RESULTS: Here we show that chemical modulation of Histone Acetyl Transferases (HATs; by IQ-1) and WNT (by CHIR99021), synergistically enable the transient and reversible block of directed cardiac differentiation progression on hPSCs. The resulting stabilized cardiovascular progenitors (SCPs) are characterized by ISL1pos/KI-67pos/NKX2-5neg expression. In the presence of the chemical inhibitors, SCPs maintain a proliferation quiescent state. Upon small molecules removal SCPs resume proliferation and concomitant NKX2-5 upregulation triggers cell-autonomous differentiation into cardiomyocytes. Directed differentiation of SCPs into the endothelial and smooth muscle lineages confirms their full developmental potential typical of bona fide cardiovascular progenitors. Single-cell RNAseq-based transcriptional profiling of our in vitro generated human SCPs notably reflects the dynamic cellular composition of E8.25-E9.25 posterior second heart field (pSHF) of mouse hearts, hallmarked by NR2F2 expression. Investigating molecular mechanisms of SCP stabilization, we found that the cell-autonomously regulated Retinoic Acid (RA) and BMP signaling is governing SCPs transition from quiescence towards proliferation and cell-autonomous differentiation, reminiscent of a niche-like behavior. CONCLUSION: The chemically defined and reversible nature our stabilization approach provides an unprecedented opportunity to dissect mechanisms of cardiovascular progenitors' specification and reveal their cellular and molecular properties.

Comparison between beta-blockers and calcium channel blockers in patients with atrial fibrillation according to renal function.

BACKGROUND: Rate control is the most commonly employed first-line management strategy for atrial fibrillation (AF) in patients with chronic kidney disease (CKD). Principal agents used to control heart rate (HR) include beta-blockers (BB) and nondihydropyridine calcium channel blockers (ND-CCB). However, there is a paucity of published studies of the differences between those drugs in CKD patients. HYPOTHESIS: The present study aimed to investigate the differences, in terms of hospitalizations due to a poor HR control, in patients with AF under a rate-control strategy according to glomerular filtration rate (GFR). METHODS: The study cohort included 2804 AF patients under rate-control regime (BB or ND-CCB) between January 2014 and April 2020. The end point, determined by competing risk regression, was hospitalizations for AF with rapid ventricular response (RVR), slow ventricular response (SVR), and need for pacemaker. RESULTS: On multivariate analysis, there were no statistical differences between ND-CCB and BB for subjects with GFR > 60 mL/min/1.73 m2 (subdistribution heart rate [sHR] 0.850, 95% confidence interval [CI]: 0.61-1.19; p = .442) and GFR 30-59 mL/min/1.73 m2 (sHR 1.242, 95% CI: 0.80-1.63; p = .333), while in patients with GFR < 30 mL/min/1.73 m2, ND-CCB therapy was associated with increased hospitalizations due to poor HR control (sHR 4.53, 95% CI: 1.19-17.18; p = .026). CONCLUSION: In patients with GFR ≥ 30 mL/min/1.73 m2, the choice of ND-CCB or BB had no impact on hospitalizations due to poor HR control, while in GFR < 30 mL/min/1.73 m2, a possible association was detected. The effects of these drugs on GFR < 30 mL/min/1.73 m2 would require further investigation.

The formulation and in vitro evaluation of WS Biotin, a novel encapsulated form of D-Biotin with improved water solubility for hair and skin treatment applications.

OBJECTIVE: To develop and evaluate the efficacy of WS Biotin, a novel water-soluble form of D-Biotin, for cosmetic use. METHODS: A new encapsulated form of D-Biotin was developed with the purpose of improving the water solubility of biotin. This novel form of encapsulated biotin was characterized by its physicochemical properties: particle size, D-Biotin content and solubility in water. Also, proliferation and gene expression in vitro tests in cell culture were performed to evaluate its effectiveness in promoting hair growth, an ELISA test was conducted for hair keratinization and skin lightening property was tested by analysing the intracellular melanin content. RESULTS: The developed WS Biotin microcapsules exhibit a particle size range of 2-30 µm with D-Biotin content of ~50% (w/w). The water solubility of WS Biotin was found to be 20-fold greater than free biotin. The obtained in vitro results indicated that WS Biotin enhances the expression of hair-related keratins in hair follicle keratinocytes, as well as the expression of hair growth-promoting genes in dermal papilla cells. Moreover, the melanin content in UVA-exposed epidermal melanocytes was reduced upon exposure to WS Biotin. CONCLUSION: In this work, a novel form of encapsulated biotin, WS Biotin, was developed in order to improve the water solubility of free biotin and was found to be effective for cosmetic use in both hair and skin applications.

OBJECTIF: Développer et évaluer l'efficacité de la WS Biotin, une nouvelle forme hydrosoluble de D-biotine, à usage cosmétique. MÉTHODES: Une nouveau format gélules de D-biotine a été développé dans le but d'améliorer la propriété d'hydrosolubilité de la biotine. Ce nouveau format de gélules de biotine a été caractérisé pour ses propriétés physicochimiques : taille des particules, teneur en D-biotine et solubilité dans l'eau. En outre, des tests in vitro de prolifération et d'expression génique en culture cellulaire ont été réalisés pour évaluer son efficacité à favoriser la croissance des cheveux, un test ELISA a été réalisé pour la kératinisation des cheveux et la propriété d'éclaircissement de la peau a été testée en analysant la teneur en mélanine intracellulaire. RÉSULTATS: Les microgélules de WS Biotin développées présentent une plage de tailles de particules de 2 à 30 micromètres avec une teneur en biotine D d'environ 50 % (p/p). L'hydrosolubilité de WS Biotin s'est avérée 20 fois plus élevée que celle de la biotine libre. Les résultats in vitro obtenus ont indiqué que WS Biotin améliorait l'expression des kératines capillaires dans les kératinocytes des follicules pileux, ainsi que l'expression des gènes favorisant la croissance dans les cellules papillaires dermiques. En outre, la teneur en mélanine dans les mélanocytes épidermiques exposés aux UVA a été réduite lors de l'exposition à WS Biotin. CONCLUSION: Dans ce travail, une nouvelle forme de biotine en gélule, WS Biotin, a été développée afin d'améliorer l'hydrosolubilité de la biotine libre et s'est avérée efficace pour une utilisation cosmétique dans les applications capillaires et cutanées.

Staged Treatment of Phlegmasia Cerulea Dolens in a Patient with Iliac Vein Compression Syndrome and Deep Vein Thrombosis: A Case Report.

Iliac vein compression syndrome (IVCS) is defined as extrinsic compression of the left common iliac vein (LCIV) between the overlying right common iliac artery and the lumbar vertebra. The most severe complication is phlegmasia cerulea dolens (PCD), a medical emergency that requires quick intervention to prevent irreversible limb ischemia. This article reports the case of a patient with PCD as the first manifestation of IVCS. The treatment included embolectomy and fasciotomy. Bilateral femoral iliac axis phlebography and cavography were performed 48 hours after the procedure. The IVCS was identified, and balloon predilatation of the lesions followed by implantation of self-expanding stents from the confluence of the LCIV with the inferior vena cava to the middle portion of the left external iliac vein was performed. Postprocedure phlebography demonstrated satisfactory final results, and a 12-month follow-up image showed patent stents and minimal intimal hyperplasia.

Depth camera based dataset of hand gestures.

The dataset contains RGB and depth version video frames of various hand movements captured with the Intel RealSense Depth Camera D435. The camera has two channels for collecting both RGB and depth frames at the same time. A large dataset is created for accurate classification of hand gestures under complex backgrounds. The dataset is made up of 29718 frames from RGB and depth versions corresponding to various hand gestures from different people collected at different time instances with complex backgrounds. Hand movements corresponding to scroll-right, scroll-left, scroll-up, scroll-down, zoom-in, and zoom-out are included in the data. Each sequence has data of 40 frames, and there is a total of 662 sequences corresponding to each gesture in the dataset. To capture all the variations in the dataset, the hand is oriented in various ways while capturing.

A Low-Cost Luxometer Benchmark for Solar Illuminance Measurement System Based on the Internet of Things.

Natural illumination has an important place in home automation applications. Among other advantages, it contributes to better visual health, energy savings, and lower CO2 emissions. Therefore, it is important to measure illuminance in the most accurate and cost-effective way. This work compares several low-cost commercial sensors (VEML 7700, TSL2591, and OPT3001) with a professional one (ML-020S-O), all of them installed outdoors. In addition, a platform based on the Internet of Things technology was designed and deployed as a centralized point of data collection and processing. Summer months have been chosen for the comparison. This is the most adverse situation for low-cost sensors since they are designed for indoor use, and their operating range is lower than the maximum reached by sunlight. The solar illuminance was recorded every minute. As expected, the obtained bias depends on the solar height. This can reach 60% in the worst circumstances, although most of the time, its value stays below 40%. The positive side lies in the good precision of the recordings. This systematic deviation makes it susceptible to mathematical correction. Therefore, the incorporation of more sensors and data that can help the global improvement of the precision and accuracy of this low-cost system is left as a future line of improvement.

Nine actions to successfully restore tropical agroecosystems.

Well-designed approaches to ecological restoration can benefit nature and society. This is particularly the case in tropical agroecosystems, where restoration can provide substantial socioecological benefits at relatively low costs. To successfully restore tropical agroecosystems and maximise benefits, initiatives must begin by considering 'who' should be involved in and benefit from restoration, and 'what', 'where', and 'how' restoration should occur. Based on collective experience of restoring tropical agroecosystems worldwide, we present nine actions to guide future restoration of these systems, supported by case studies that demonstrate our actions being used successfully in practice and highlighting cases where poorly designed restoration has been damaging. We call for increased restoration activity in tropical agroecosystems during the current UN Decade on Ecosystem Restoration.

Unhealthy food advertising. A position paper by the AEP Committee on Nutrition and Breastfeeding.

INTRODUCTION: Some important factors influencing and maintaining unhealthy habits are food advertising and products accessibility. In order to develop and support recommendations, an analysis of the available evidence on the impact of food advertising on the health of children and adolescents has been carried out. METHODS: Literature review of systematic reviews and meta-analyses published up to January 2022 for the term "food advertising" that analyzed the impact of food advertising on weight, body mass index (BMI), adiposity, dietary intake, behavior toward the advertised product, its purchase or consumption in children and adolescents. RESULTS: Twenty-one systematic reviews fulfilled the inclusion criteria, including a total of 490 primary studies, 5 of which also contained a meta-analysis. The vast majority of the primary studies evaluate intermediate effects, related to the behavior of children and adolescents in relation to advertised products and their consumption. There is great variety in terms of the type of advertising and effects studied. Most of the studies agree that there is an association between food advertising and effect analyzed, being more evident in children under 12 years of age and in obese children. Most recent systematic reviews are focused on online advertising, noticing the negative effects especially in adolescents. CONCLUSIONS: Children and adolescents are a particularly vulnerable population to food advertising strategies. Despite the difficulty to demonstrate an independent effect, there is evidence of an association between food advertising and childhood and adolescents' behavior respect to the announced products, and the increase of consumption at short-term. In Spain unhealthy product advertising are still very common in the media and in the children and adolescent's online environment. The Nutrition and Breastfeeding Committee of the Spanish Association of Pediatrics supports the need for regulation and limitation of unhealthy food advertising, covering all media and marketing strategies.

TP53 Mutations Identified Using NGS Comprise the Overwhelming Majority of TP53 Disruptions in CLL: Results From a Multicentre Study.

Limited data exists to show the correlation of (tumour protein 53) TP53 mutation detected by Next generation sequencing (NGS) and the presence/absence of deletions of 17p13 detected by FISH. The study which is the largest series to date includes 2332 CLL patients referred for analysis of del(17p) by FISH and TP53 mutations by NGS before treatment. Using a 10% variant allele frequency (VAF) threshold, cases were segregated into high burden mutations (≥10%) and low burden mutations (<10%). TP53 aberrations (17p [del(17p)] and/or TP53 mutation) were detected in 320/2332 patients (13.7%). Using NGS analysis, 429 TP53 mutations were identified in 303 patients (13%). Of these 238 (79%) and 65 (21%) were cases with high burden and low burden mutations respectively. In our cohort, 2012 cases did not demonstrate a TP53 aberration (86.3%). A total of 159 cases showed TP53 mutations in the absence of del(17p) (49/159 with low burden TP53 mutations) and 144 cases had both TP53 mutation and del(17p) (16/144 with low burden mutations). Only 17/2332 (0.7%) cases demonstrated del(17p) with no TP53 mutation. Validated NGS protocols should be used in clinical decision making to avoid missing low-burden TP53 mutations and can detect the vast majority of TP53 aberrations.

Effects of Chlorophyll Triplet States on the Kinetics of Spectral Hole Growth.

Spectral hole burning has been employed for decades to study various amorphous solids and proteins. Triplet states and respective transient holes were incorporated into theoretical models and software simulating nonphotochemical spectral hole burning (NPHB) and including all relevant distributions, in particular the distribution of the angle between the electric field of light E and transient dipole moment of the chromophore µ. The presence of a chlorophyll a triplet state with a lifetime of several milliseconds explains the slowdown of NPHB (on the depth vs illumination dose scale) with the increase of the light intensity, as well as larger hole depths observed in weak probe beam experiments, compared to those deduced from the hole growth kinetics (HGK) measurements (signal collected at a fixed wavelength while a stronger burning beam is on) in cytochrome b6f and chemically modified LH2. We also considered the solvent deuteration effects on triplet lifetime and concluded that both triplet states and local heating likely play a role in slowing down the HGK with increasing burn intensity.

Uses and Applications of Docosahexaenoic Acid (DHA) in Pediatric Gastroenterology: Current Evidence and New Perspective.

In this paper, we will review the dietary allowances of these fatty acids in the paediatric population, and also the indications in different pathologies within the field of pediatric gastroenterology. Finally, we will try to explain the reasons that may justify the difficulty in translating good results in experimental studies to the usual clinical practice. This "good results" may be too little to be detected or there may be other causes but misinterpreted as effects of DHA.

Outcomes of surgical treatment for isolated adrenal metastasis from non-small cell lung cancer.

OBJECTIVE: Long-term survival of patients who undergo surgical resection of isolated adrenal metastasis instead of nonsurgical treatment has shown higher values than those described for stage IVA. The primary endpoint was to evaluate overall survival (OS) of patients with single adrenal metastasis from non-small cell lung cancer (NSCLC), who underwent surgical treatment. The secondary endpoint was to evaluate and compare the OS and disease-free survival (DFS) according to: pathological lung tumour size, histology, lymph node involvement, type of metastasis at the time of diagnosis and laterality of the metastasis according to the primary lung tumour. METHODS: From August 2007 to March 2020, 13 patients with isolated adrenal gland metastasis were identified. We performed a descriptive observational study including patients with diagnosed single adrenal gland metastasis of resectable primary lung cancer and no history of other malignant disease. Clinical data obtained included patient demographics, metastases characteristics, laterality of the metastasis, time between surgeries, length of follow-up, survival status, pathological lung tumour size, histology and lymph node involvement. The variables analysed were OS and DFS. RESULTS: Median global OS was 31.9 months (interquartile range (IQR), 19.1-51.4). The 2- and 5-year OS estimated was 54% (95% CI: 29.5%-77.4%) and 36% (95% CI: 13.4%-68.1%), respectively. In patients with NSCLC without mediastinal lymph node involvement, we obtain a median OS of 40 months (IQR, 27.4-51.4) and a 2- and 5-year OS estimated of 75% (95% CI: 43.2%-92.2%) and 50% (95% CI: 18.7%-81.2%), respectively. Recurrence was detected in five patients with a median DFS of 11.9 months (IQR, 6-34.2). CONCLUSION: The resection of the adrenal metastasis should be considered if the primary lung cancer is resectable. Presence of mediastinal lymph node involvement should be ruled out through invasive staging of the mediastinum before performing adrenal and lung surgery. Proper selection of patients who would benefit from surgery is essential to obtain better survival results.

The impact of SAMHD1 expression and mutation status in mantle cell lymphoma: An analysis of the MCL Younger and Elderly trial.

The sterile alpha motif and histidine-aspartic domain-containing protein 1 (SAMHD1) has been demonstrated to predict the response to high-dose cytarabine consolidation treatment in acute myeloid leukemia patients. Here, we evaluated SAMHD1 as potential biomarker for the response to high-dose cytarabine in mantle cell lymphoma (MCL) patients. We quantified SAMHD1 protein expression and determined the mutation status in patients of the MCL Younger and Elderly trials (n = 189), who had received high-dose cytarabine- or fludarabine-based polychemotherapy. Additionally, we quantified SAMHD1 expression in B cell lymphoma cell lines and exposed them to cytarabine, fludarabine, and clinically relevant combinations. Across both trials investigated, SAMHD1 mutations had a frequency of 7.1% (n = 13) and did not significantly affect the failure-free survival (FFS, P = .47). In patients treated with high-dose cytarabine- or fludarabine-containing regimes, SAMHD1 expression was not significantly associated with FFS or complete remission rate. SAMHD1 expression in B cell lymphoma cell lines, however, inversely correlated with their in vitro response to cytarabine as single agent (R = .65, P = .0065). This correlation could be reversed by combining cytarabine with other chemotherapeutics, such as oxaliplatin and vincristine, similar to the treatment regime of the MCL Younger trial. We conclude that this might explain why we did not observe a significant association between SAMHD1 protein expression and the outcome of MCL patients upon cytarabine-based treatment.

Genomic landscape of platinum resistant and sensitive testicular cancers.

While most testicular germ cell tumours (TGCTs) exhibit exquisite sensitivity to platinum chemotherapy, ~10% are platinum resistant. To gain insight into the underlying mechanisms, we undertake whole exome sequencing and copy number analysis in 40 tumours from 26 cases with platinum-resistant TGCT, and combine this with published genomic data on an additional 624 TGCTs. We integrate analyses for driver mutations, mutational burden, global, arm-level and focal copy number (CN) events, and SNV and CN signatures. Albeit preliminary and observational in nature, these analyses provide support for a possible mechanistic link between early driver mutations in RAS and KIT and the widespread copy number events by which TGCT is characterised.

Augmentation of Neurotoxicity of Anticancer Drugs by X-Ray Irradiation.

BACKGROUND: In order to investigate the combination effect of anticancer drugs and X-ray irradiation on neurotoxic side-effects (neurotoxicity), a method that provides homogeneously X-ray-irradiated cells was newly established. MATERIALS AND METHODS: PC12 cell suspension was irradiated by X-ray (0.5 Gy) in serum-supplemented medium, immediately inoculated into 96-microwell plates and incubated overnight. The medium was replaced with fresh serum-depleted medium containing 50 ng/ml nerve growth factor to induce differentiation toward nerve-like cells with characteristic neurites according to the overlay method without changing the medium. The differentiated cells were treated by anticancer drugs as well as antioxidants, oxaliplatin or bortezomib, and the viable cell number was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. RESULTS: Antioxidants and anticancer drugs were cytotoxic to differentiating PC12 cells. Combination of anticancer drugs and X-ray irradiation slightly reduced cell viability. CONCLUSION: The present 'population irradiation method' may be useful for the investigation of the combination effect of X-ray irradiation and any pharmaceutical drug.

Assessment of trauma scoring systems in patients subjected to exploratory laparotomy.

OBJECTIVE: to assess the epidemiological profile of patients undergoing exploratory trauma laparotomy based on severity and prognosis criteria, and to determine the predictive accuracy of trauma scoring systems in terms of morbidity and mortality. METHODS: retrospective cohort study and review of medical records of patients undergoing exploratory laparotomy for blunt or penetrating trauma at the Hospital de Pronto Socorro de Porto Alegre, from November 2015 to November 2019. Demographic data, mechanism of injury, associated injuries, physiological (RTS and Shock Index), anatomical (ISS, NISS and ATI) and combined (TRISS and NTRISS) trauma scores, intraoperative findings, postoperative complications, length of stay and outcomes. RESULTS: 506 patients were included in the analysis. The mean age was 31 ± 13 years, with the majority being males (91.3%). Penetrating trauma was the most common mechanism of injury (86.2%), predominantly by firearms. The average RTS at hospital admission was 7.5 ± 0.7. The mean ISS and NISS was 16.5 ± 10.1 and 22.3 ± 13.6, respectively. The probability of survival estimated by TRISS was 95.5%, and by NTRISS 93%. The incidence of postoperative complications was 39.7% and the overall mortality was 12.8%. The most accurate score for predicting mortality was the NTRISS (88.5%), followed by TRISS, NISS and ISS. CONCLUSION: the study confirms the applicability of trauma scores in the studied population. The NTRISS seems to be the best predictor of morbidity and mortality.

Assessment of trauma scoring systems in patients subjected to exploratory laparotomy / Avaliação dos escores de trauma em pacientes submetidos à laparotomia exploradora

ABSTRACT Objective: to assess the epidemiological profile of patients undergoing exploratory trauma laparotomy based on severity and prognosis criteria, and to determine the predictive accuracy of trauma scoring systems in terms of morbidity and mortality. Methods: retrospective cohort study and review of medical records of patients undergoing exploratory laparotomy for blunt or penetrating trauma at the Hospital de Pronto Socorro de Porto Alegre, from November 2015 to November 2019. Demographic data, mechanism of injury, associated injuries, physiological (RTS and Shock Index), anatomical (ISS, NISS and ATI) and combined (TRISS and NTRISS) trauma scores, intraoperative findings, postoperative complications, length of stay and outcomes. Results: 506 patients were included in the analysis. The mean age was 31 ± 13 years, with the majority being males (91.3%). Penetrating trauma was the most common mechanism of injury (86.2%), predominantly by firearms. The average RTS at hospital admission was 7.5 ± 0.7. The mean ISS and NISS was 16.5 ± 10.1 and 22.3 ± 13.6, respectively. The probability of survival estimated by TRISS was 95.5%, and by NTRISS 93%. The incidence of postoperative complications was 39.7% and the overall mortality was 12.8%. The most accurate score for predicting mortality was the NTRISS (88.5%), followed by TRISS, NISS and ISS. Conclusion: the study confirms the applicability of trauma scores in the studied population. The NTRISS seems to be the best predictor of morbidity and mortality.

RESUMO Objetivo: analisar o perfil epidemiológico dos pacientes submetidos a laparotomia exploradora por trauma com base em critérios de gravidade e prognóstico, e determinar a acurácia preditiva dos escores de trauma em termos de morbimortalidade. Métodos: estudo de coorte retrospectiva e revisão de prontuários dos pacientes submetidos a laparotomia exploradora por trauma contuso ou penetrante no Hospital de Pronto Socorro de Porto Alegre no período de novembro de 2015 a novembro de 2019. Foram avaliados dados demográficos, mecanismo do trauma, lesões associadas, índices fisiológicos (RTS e Shock Index), anatômicos (ISS, NISS e ATI) e mistos (TRISS e NTRISS), achados intraoperatórios, complicações pós-operatórias, tempo de internação e desfecho. Resultados: foram incluídos 506 pacientes na análise. A idade média foi de 31 ± 13 anos, com predomínio do sexo masculino (91,3%). O trauma penetrante foi o mecanismo de lesão mais comum (86,2%), sendo a maioria por arma de fogo. A média do RTS na admissão hospitalar foi 7,5 ± 0,7. A média do ISS e do NISS foi 16,5 ± 10,1 e 22,3 ± 13,6, respectivamente. A probabilidade de sobrevida estimada pelo TRISS foi de 95,5%, e pelo NTRISS de 93%. A incidência de complicações pós-operatórias foi de 39,7% e a mortalidade geral de 12,8%. O escore com melhor acurácia preditiva foi o NTRISS (88,5%), seguido pelo TRISS, NISS e ISS. Conclusões: o estudo confirma a aplicabilidade dos escores de trauma na população em questão. O NTRISS parece ser o sistema com melhor acurácia preditiva de morbimortalidade.

Advice on chemical weapons sample stability and storage provided by the Scientific Advisory Board of the Organisation for the Prohibition of Chemical Weapons to increase investigative capabilities worldwide.

The Scientific Advisory Board (SAB) of the Organisation for the Prohibition of Chemical Weapons (OPCW) has provided advice on the long-term storage and stability of samples collected in the context of chemical weapons investigations. The information they compiled and reviewed is beneficial to all laboratories that carry out analysis of samples related to chemical warfare agents and is described herein. The preparation of this report was undertaken on request from the OPCW Director-General. The main degradation products for chemicals on the Schedules in the Annex on Chemicals of the Chemical Weapons Convention are tabulated. The expertise of the 25 scientists comprising the SAB, a review of the scientific literature on environmental and biomedical sample analysis, and answers to a questionnaire from chemists of nine OPCW Designated Laboratories, were drawn upon to provide the advice. Ten recommendations to ensure the long-term storage and stability of samples collected in relation to the potential use of chemical weapons were provided and are repeated here for the consideration of all laboratories worldwide.

KRAS and BRAF mutations in circulating tumour DNA from locally advanced rectal cancer.

There are limited data on circulating, cell-free, tumour (ct)DNA analysis in locally advanced rectal cancer (LARC). Digital droplet (dd)PCR was used to investigate KRAS/BRAF mutations in ctDNA from baseline blood samples of 97 LARC patients who were treated with CAPOX followed by chemoradiotherapy, surgery and adjuvant CAPOX ± cetuximab in a randomised phase II trial. KRAS mutation in G12D, G12V or G13D was detected in the ctDNA of 43% and 35% of patients with tumours that were mutant and wild-type for these hotspot mutations, respectively, according to standard PCR-based analyses on tissue. The detection rate in the ctDNA of 10 patients with less common mutations was 50%. In 26 cases ctDNA analysis revealed KRAS mutations that were not previously found in tissue. Twenty-two of these (84.6%) were detected following repeat tissue testing by ddPCR. Overall, the ctDNA detection rate in the KRAS mutant population was 66%. Detection of KRAS mutation in ctDNA failed to predict prognosis or refine patient selection for cetuximab. While this study confirms the feasibility of ctDNA analysis in LARC and the high sensitivity of ddPCR, larger series are needed to better address the role of ctDNA as a prognostic or predictive tool in this setting.

An evaluation of the challenges to developing tumor BRCA1 and BRCA2 testing methodologies for clinical practice.

Ovarian cancer patients with germline or somatic pathogenic variants benefit from treatment with poly ADP ribose polymerase (PARP) inhibitors. Tumor BRCA1/2 testing is more challenging than germline testing as the majority of samples are formalin-fixed paraffin embedded (FFPE), the tumor genome is complex, and the allelic fraction of somatic variants can be low. We collaborated with 10 laboratories testing BRCA1/2 in tumors to compare different approaches to identify clinically important variants within FFPE tumor DNA samples. This was not a proficiency study but an inter-laboratory comparison to identify common issues. Each laboratory received the same tumor DNA samples ranging in genotype, quantity, quality, and variant allele frequency (VAF). Each laboratory performed their preferred next-generation sequencing method to report on the variants. No false positive results were reported in this small study and the majority of methods detected the low VAF variants. A number of variants were not detected due to the bioinformatics analysis, variant classification, or insufficient DNA. The use of hybridization capture or short amplicon methods are recommended based on a bioinformatic assessment of the data. The study highlights the importance of establishing standards and standardization for tBRCA testing particularly when the test results dictate clinical decisions regarding life extending therapies.