Capture Protocol Research Articles

Understanding Wireless Debugging with Sniffers in Smart Home Environments

Wireless communication is becoming increasingly prevalent in smart home environments; the need for effective debugging methodologies has become paramount for system reliability and performance optimization. This comprehensive article analysis explores the implementation of wireless sniffers across multiple protocols, including Wi-Fi, Bluetooth, and Zigbee, with particular emphasis on their application in contemporary IoT devices. Through a detailed examination of chipset capabilities from major manufacturers, including Qualcomm Atheros, Texas Instruments, and Nordic Semiconductor, this article presents a structured approach to wireless debugging that encompasses packet capture mechanisms, signal analysis, and protocol verification. The investigation demonstrates how modern sniffer technologies can effectively diagnose common issues such as packet loss, signal interference, and protocol misconfigurations in smart home devices, including voice assistants, security systems, and environmental controls. By synthesizing practical implementation strategies with technical analysis, this article provides developers and system architects with actionable methodologies for improving wireless system reliability while highlighting the critical role of protocol analysis in ensuring robust IoT deployments.

Surface soil sampling underestimates soil carbon and nitrogen storage of long-term cover cropping

Cover cropping is a promising management practice for soil health and climate change mitigation by improving soil organic carbon (SOC) and total nitrogen (TN) stocks. However, limited studies focused on deeper soil layers (>30 cm depth) where soil C is more stable than that in surface soil (≤30 cm depth). Here, deep soil sampling was conducted in a 15-year cover cropping experiment, in a horticulture-grain system on sandy loam soil. The SOC and TN stocks were expressed on an equivalent soil mass basis using a cubic spline model. Overall, long-term cover cropping had significantly greater SOC and TN stocks by 22 % (95 %CI: 5–43 %) and 26 % (95 %CI: 6–49 %), respectively in the 0–120 cm depth, compared to no cover cropping. Additionally, the mean SOC and TN sequestration rate (0–30 cm depth) was 0.53 Mg C ha−1 yr−1 and 0.06 Mg N ha−1 yr−1, respectively. However, if only 0–15 cm depth was evaluated, long-term cover cropping did not significantly affect SOC and TN stocks. These results indicated that shallow sampling (<15 cm depth) may not provide comprehensive information on the effect of long-term cover cropping on soil C and N storage. To better understand the mechanism of bulk soil C and N storage, we investigated their distribution between particulate and mineral-associated organic matter pools (POM and MAOM). We found POM pool was the main store of bulk SOC and TN stocks in surface soils while it was the MAOM pool in deeper soil layers, without soil texture change with soil depth. These findings indicated that soil C and N sources for bulk SOC and TN accrual differed in surface and deeper soils. Our study demonstrated that long-term cover cropping can facilitate SOC accumulation in the soil below 15 cm deep, which calls into question carbon capture protocols that focus on shallow soil depths.

Fine mapping of RNA isoform diversity using an innovative targeted long-read RNA sequencing protocol with novel dedicated bioinformatics pipeline.

Solving the structure of mRNA transcripts is a major challenge for both research and molecular diagnostic purposes. Current approaches based on short-read RNA sequencing and RT-PCR techniques cannot fully explore the complexity of transcript structure. The emergence of third-generation long-read sequencing addresses this problem by solving this sequence directly. However, genes with low expression levels are difficult to study with the whole transcriptome sequencing approach. To fix this technical limitation, we propose a novel method to capture transcripts of a gene panel using a targeted enrichment approach suitable for Pacific Biosciences and Oxford Nanopore Technologies platforms. We designed a set of probes to capture transcripts of a panel of genes involved in hereditary breast and ovarian cancer syndrome. We present SOSTAR (iSofOrmS annoTAtoR), a versatile pipeline to assemble, quantify and annotate isoforms from long read sequencing using a new tool specially designed for this application. The significant enrichment of transcripts by our capture protocol, together with the SOSTAR annotation, allowed the identification of 1,231 unique transcripts within the gene panel from the eight patients sequenced. The structure of these transcripts was annotated with a resolution of one base relative to a reference transcript. All major alternative splicing events of the BRCA1 and BRCA2 genes described in the literature were found. Complex splicing events such as pseudoexons were correctly annotated. SOSTAR enabled the identification of abnormal transcripts in the positive controls. In addition, a case of unexplained inheritance in a family with a history of breast and ovarian cancer was solved by identifying an SVA retrotransposon in intron 13 of the BRCA1 gene. We have validated a new protocol for the enrichment of transcripts of interest using probes adapted to the ONT and PacBio platforms. This protocol allows a complete description of the alternative structures of transcripts, the estimation of their expression and the identification of aberrant transcripts in a single experiment. This proof-of-concept opens new possibilities for RNA structure exploration in both research and molecular diagnostics.

A self-supervised deep Riemannian representation to classify parkinsonian fixational patterns

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder, and it remains incurable. Currently there is no definitive biomarker for detecting PD, measuring its severity, or monitoring of treatments. Recently, oculomotor fixation abnormalities have emerged as a sensitive biomarker to discriminate Parkinsonian patterns from a control population, even at early stages. For oculomotor analysis, current experimental setups use invasive and restrictive capture protocols that limit the transfer in clinical routine. Alternatively, computational approaches to support the PD diagnosis are strictly based on supervised strategies, depending of large labeled data, and introducing an inherent expert-bias. This work proposes a self-supervised architecture based on Riemannian deep representation to learn oculomotor fixation patterns from compact descriptors. Firstly, deep convolutional features are recovered from oculomotor fixation video slices, and then encoded in compact symmetric positive matrices (SPD) to summarize second-order relationships. Each SPD input matrix is projected onto a Riemannian encoder until obtain a SPD embedding. Then, a Riemannian decoder reconstructs SPD matrices while preserving the geometrical manifold structure. The proposed architecture successfully recovers geometric patterns in the embeddings without any label diagnosis supervision, and demonstrates the capability to be discriminative regarding PD patterns. In a retrospective study involving 13 healthy adults and 13 patients diagnosed with PD, the proposed Riemannian representation achieved an average accuracy of 95.6% and an AUC of 99% during a binary classification task using a Support Vector Machine.

Child and adolescent exposure to unhealthy food marketing across digital platforms in Canada

BackgroundChildren and adolescents are exposed to a high volume of unhealthy food marketing across digital media. No previous Canadian data has estimated child exposure to food marketing across digital media platforms. This study aimed to compare the frequency, healthfulness and power of food marketing viewed by children and adolescents across all digital platforms in Canada.MethodsFor this cross-sectional study, a quota sample of 100 youth aged 6–17 years old (50 children, 50 adolescents distributed equally by sex) were recruited online and in-person in Canada in 2022. Each participant completed the WHO screen capture protocol where they were recorded using their smartphone or tablet for 30-min in an online Zoom session. Research assistants identified all instances of food marketing in the captured video footage. A content analysis of each marketing instance was then completed to examine the use of marketing techniques. Nutritional data were collected on each product viewed and healthfulness was determined using Health Canada’s 2018 Nutrient Profile Model. Estimated daily and yearly exposure to food marketing was calculated using self-reported device usage data.Results51% of youth were exposed to food marketing. On average, we estimated that children are exposed to 1.96 marketing instances/child/30-min (4067 marketing instances/child/year) and adolescents are exposed to 2.56 marketing instances/adolescent/30-min (8301 marketing instances/adolescent/year). Both children and adolescents were most exposed on social media platforms (83%), followed by mobile games (13%). Both age groups were most exposed to fast food (22% of marketing instances) compared to other food categories. Nearly 90% of all marketing instances were considered less healthy according to Health Canada’s proposed 2018 Nutrient Profile Model, and youth-appealing marketing techniques such as graphic effects and music were used frequently.ConclusionsUsing the WHO screen capture protocol, we were able to determine that child and adolescent exposure to the marketing of unhealthy foods across digital media platforms is likely high. Government regulation to protect these vulnerable populations from the negative effects of this marketing is warranted.

Identification and in vitro characterization of UDP-GlcNAc-RNA cap-modifying and decapping enzymes.

In recent years, several noncanonical RNA caps derived from cofactors and metabolites have been identified. Purine-containing RNA caps have been extensively studied, with multiple decapping enzymes identified and efficient capture and sequencing protocols developed for nicotinamide adenine dinucleotide (NAD)-RNA, which allowed for a stepwise elucidation of capping functions. Despite being identified as an abundant noncanonical RNA-cap, UDP-sugar-capped RNA remains poorly understood, which is partly due to its complex in vitro preparation. Here, we describe a scalable synthesis of sugar-capped uridine-guanosine dinucleotides from readily available protected building blocks and their enzymatic conversion into several cell wall precursor-capped dinucleotides. We employed these capped dinucleotides in T7 RNA polymerase-catalyzed in vitro transcription reactions to efficiently generate RNAs capped with uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), its N-azidoacetyl derivative UDP-GlcNAz, and various cell wall precursors. We furthermore identified four enzymes capable of processing UDP-GlcNAc-capped RNA in vitro: MurA, MurBand MurC from Escherichia coli can sequentially modify the sugar-cap structure and were used to introduce a bioorthogonal, clickable moiety, and the human Nudix hydrolase Nudt5 was shown to efficiently decap UDP-GlcNAc-RNA. Our findings underscore the importance of efficient synthetic methods for capped model RNAs. Additionally, we provide useful enzymatic tools that could be utilized in the development and application of UDP-GlcNAc capture and sequencing protocols. Such protocols are essential for deepening our understanding of the widespread yet enigmatic GlcNAc modification of RNA and its physiological significance.

Dermoscopy can be safely and reliably used in ophthalmology

ObjectiveTo determine if dermoscopy, a technique widely utilized in dermatology for improved diagnosis of skin lesions, can be used comfortably for evaluating periorbital, eyelid, and conjunctival lesions. DesignProof-of-concept study in which a technique for performing dermoscopy near the eye was developed, related educational material was prepared, and a protocol for dermoscopic image capture was created. MethodsTechnicians used the developed materials to learn to take high-quality pictures with a 10x dermoscope attached to a standard cell phone camera. The images were assessed for diagnostic utility by an oculoplastic surgeon and two dermatologists. Participants115 patients recruited from ophthalmology clinics from July 2021 to April 2023 were photographed, yielding 129 lesions with high-quality dermoscopic images as assessed by an oculoplastic surgeon and two dermatologists. ResultsTechnicians reported a significant increase in confidence (measured on a 1–10 scale) with dermoscopy after training (pre-instruction mean = 1.72, median = 1, mode = 1, IQR = 1.25 vs mean = 7.69, median = 7.75, mode = 7 and 8, IQR = 1.5 post-instruction. Wilcoxon rank sum test with continuity correction, W = 0, p < 0.001, paired t = 13.95, p < 0.0001). Incorporating a contact plate with a 4 × 4mm reticule on the dermoscope aided in photographing ocular and periocular lesions. ConclusionMedical support staff in eye-care offices can be taught to use dermoscopes to capture high-quality images of periorbital, eyelid, and conjunctival lesions. Dermoscopy illuminates diagnostic features of lesions and thus offers a new avenue to improve decision-making in ophthalmology. Dermoscopy can be incorporated into telemedicine evaluations by ophthalmologists, oculoplastic surgeons, or affiliated dermatologists for triage of or rendering advice to patients and for planning of surgery if needed.

Stress-free single-cell transcriptomic profiling and functional genomics of murine eosinophils.

Eosinophils are a class of granulocytes with pleiotropic functions in homeostasis and various human diseases. Nevertheless, they are absent from conventional single-cell RNA sequencing atlases owing to technical difficulties preventing their transcriptomic interrogation. Consequently, eosinophil heterogeneity and the gene regulatory networks underpinning their diverse functions remain poorly understood. We have developed a stress-free protocol for single-cell RNA capture from murine tissue-resident eosinophils, which revealed distinct intestinal subsets and their roles in colitis. Here we describe in detail how to enrich eosinophils from multiple tissues of residence and how to capture high-quality single-cell transcriptomes by preventing transcript degradation. By combining magnetic eosinophil enrichment with microwell-based single-cell RNA capture (BD Rhapsody), our approach minimizes shear stress and processing time. Moreover, we report how to perform genome-wide CRISPR pooled genetic screening in ex vivo-conditioned bone marrow-derived eosinophils to functionally probe pathways required for their differentiation and intestinal maturation. These protocols can be performed by any researcher with basic skills in molecular biology and flow cytometry, and can be adapted to investigate other granulocytes, such as neutrophils and mast cells, thereby offering potential insights into their roles in both homeostasis and disease pathogenesis. Single-cell transcriptomics of eosinophils can be performed in 2-3 d, while functional genomics assays may require up to 1 month.

Comparison of Shoulder Range of Motion Quantified with Mobile Phone Video-Based Skeletal Tracking and 3D Motion Capture-Preliminary Study.

The accuracy of human pose tracking using smartphone camera (2D-pose) to quantify shoulder range of motion (RoM) is not determined. Twenty healthy individuals were recruited and performed shoulder abduction, adduction, flexion, or extension, captured simultaneously using a smartphone-based human pose estimation algorithm (Apple's vision framework) and using a skin marker-based 3D motion capture system. Validity was assessed by comparing the 2D-pose outcomes against a well-established 3D motion capture protocol. In addition, the impact of iPhone positioning was investigated using three smartphones in multiple vertical and horizontal positions. The relationship and validity were analysed using linear mixed models and Bland-Altman analysis. We found that 2D-pose-based shoulder RoM was consistent with 3D motion capture (linear mixed model: R2 > 0.93) but was somewhat overestimated by the smartphone. Differences were dependent on shoulder movement type and RoM amplitude, with adduction the worst performer among all tested movements. All motion types were described using linear equations. Correction methods are provided to correct potential out-of-plane shoulder movements. Shoulder RoM estimated using a smartphone camera is consistent with 3D motion-capture-derived RoM; however, differences between the systems were observed and are likely explained by differences in thoracic frame definitions.

Immobilisation efficacy of conducted electrical weapons on captive white-tailed deer

Context Wildlife emergencies (e.g. injured animals) often require capture or humane dispatch of animals to resolve the situation. Conducted electrical weapons (CEWs) have the potential to immobilise white-tailed deer (Odocoileus virginianus) for a short duration to avoid potential risks of extended immobilisation (e.g. chemical immobilisation) and increase safety for managers and the public during humane killing by gunshot. Aims To test rapid immobilisation with minimal long-term physiological consequences of CEW immobilisation of white-tailed deer. Methods We arranged five treatment groups of captive deer, including (1) chemical immobilisation with 5-s CEW exposure (n = 5), (2) chemical immobilisation with 15-s CEW exposure (n = 5), (3) 5-s CEW exposure with no chemical immobilisation (n = 10), (4) 15-s CEW exposure with no chemical immobilisation (n = 10) and (5) a control group with no chemical immobilisation or CEW exposure (n = 10). We conducted treatments 1 and 2 to determine short-term physiological effects of CEWs on deer, related to overall welfare. In treatment groups 1 and 2, we sedated deer to collect blood immediately before CEW exposure, and 2 days and 5 days post-exposure for serum biochemical analysis (to measure physiological markers of organ and tissue damage). We observed deer before, during and after treatments to evaluate potential behavioural changes. Key results All deer showed signs of muscle paralysis immediately after exposure to CEW and regained muscle control immediately after the exposure ended. Serum biochemistry results for treatment group 1 and 2 revealed significant increases in creatine kinase (CK) and aspartate aminotransferase (AST) 2 days post-treatment, before returning to pre-exposure concentrations by day 5-post-exposure, suggesting temporary myopathy. By 15-days post-exposure, deer that were only exposed to the CEW had 27 of 39 (69%) remotely fired probes still attached. No long-term behavioural or physiological changes were observed post-treatment. Conclusions Our findings suggest that for immobilisation of white-tailed deer, short-term exposure to a CEW to is a potential alternative to typical capture techniques and can provide sufficient immobilisation to approach and humanely kill deer by gunshot if necessary in wildlife emergencies. Implications As with all capture techniques, trainings and protocols should be developed to ensure the safety of personnel and animals during CEW exposures.

Strengthening the genomic surveillance of Francisella tularensis by using culture-free whole-genome sequencing from biological samples.

Francisella tularensis is a highly infectious bacterium that causes the zoonotic disease tularemia. The development of genotyping methods, especially those based on whole-genome sequencing (WGS), has recently increased the knowledge on the epidemiology of this disease. However, due to the difficulties associated with the growth and isolation of this fastidious pathogen in culture, the availability of strains and subsequently WGS data is still limited. To surpass these constraints, we aimed to implement a culture-free approach to capture and sequence F. tularensis genomes directly from complex samples. Biological samples obtained from 50 common voles and 13 Iberian hares collected in Spain were confirmed as positive for F. tularensis subsp. holarctica and subjected to a WGS target capture and enrichment protocol, using RNA oligonucleotide baits designed to cover F. tularensis genomic diversity. We obtained full genome sequences of F. tularensis from 13 animals (20.6%), two of which had mixed infections with distinct genotypes, and achieved a higher success rate when compared with culture-dependent WGS (only successful for two animals). The new genomes belonged to different clades commonly identified in Europe (B.49, B.51 and B.262) and subclades. Despite being phylogenetically closely related to other genomes from Spain, the detected clusters were often found in other countries. A comprehensive phylogenetic analysis, integrating 599 F. tularensis subsp. holarctica genomes, showed that most (sub)clades are found in both humans and animals and that closely related strains are found in different, and often geographically distant, countries. Overall, we show that the implemented culture-free WGS methodology yields timely, complete and high-quality genomic data of F. tularensis, being a highly valuable approach to promote and potentiate the genomic surveillance of F. tularensis and ultimately increase the knowledge on the genomics, ecology and epidemiology of this highly infectious pathogen.

High-resolution DNA methylation screening of the major histocompatibility complex in multiple sclerosis.

The HLA-DRB1 gene in the major histocompatibility complex (MHC) region in chromosome 6p21 is the strongest genetic factor identified as influencing multiple sclerosis (MS) susceptibility. DNA methylation changes associated with MS have been consistently detected at the MHC region. However, understanding the full scope of epigenetic regulations of the MHC remains incomplete, due in part to the limited coverage of this region by standard whole genome bisulfite sequencing or array-based methods. We developed and validated an MHC capture protocol coupled with bisulfite sequencing and conducted a comprehensive analysis of the MHC methylation landscape in blood samples from 147 treatment naïve MS study participants and 129 healthy controls. We identified 132 differentially methylated region (DMRs) within MHC region associated with disease status. The DMRs overlapped with established MS risk loci. Integration of the MHC methylome with human leukocyte antigen (HLA) genetic data indicate that the methylation changes are significantly associated with HLA genotypes. Using DNA methylation quantitative trait loci (mQTL) mapping and the causal inference test (CIT), we identified 643 cis-mQTL-DMRs paired associations, including 71 DMRs possibly mediating causal relationships between 55 single nucleotide polymorphisms (SNPs) and MS risk. The results describe MS-associated methylation changes in MHC region and highlight the association between HLA genotypes and methylation changes. Results from the mQTL and CIT analyses provide evidence linking MHC region variations, methylation changes, and disease risk for MS.

Description of box trapping, immobilisation, anaesthesia monitoring and blood chemistry and serology in free-ranging European wildcats (Felis silvestris) in Southwest Germany

Specific questions in wildlife research and surveillance require safe and efficient capture, handling and anaesthesia protocols to enable sampling and transmitter placement in free-ranging individuals. For wild felids, various protocols are available, but detailed reports for European wildcats (Felis silvestris) are scarce. In particular, tools for anaesthesia monitoring under field conditions and reference values for heart rate, respiratory rate, oxygen saturation and body temperature are missing. In the present study, European wildcats were caught in box traps before being released into catch bags for manual restraint. Inside the bags, ketamine-xylazine anaesthesia was applied via intramuscular injection, adjusted to the animal’s body weight. During anaesthesia, samples were taken, and vital variables were monitored continuously. Haematology and blood chemistry parameters were obtained, along with serological markers for antibodies against feline immunodeficiency virus (FIV), feline coronavirus and antigens of feline leukaemia virus (FeLV). In total, 29 wildcats were captured, of which 21 were examined and marked with passive integrated transponders. Twelve wildcats were collared with GPS transmitters. Handling time under anaesthesia averaged 30 min (range 26–35 min). Heart rate ranged between 76 and 170 beats/min and respiratory rate between 20 and 52 breaths/min. Relative arterial oxygen saturation stayed mainly between 93 and 99%, and rectal temperature ranged between 36.2 and 40.2 °C. Further, FeLV antibodies were detected in 2/21 samples. The applied protocol facilitated safe and sufficient examination, sampling and transmitter placement, as well as the establishment of haematological and blood chemical values in free-ranging European wildcats for the first time.

The Presence of Somatic Mutation in Donor Does Not Affect Long-Term Outcomes in 339 Patients Following Allogeneic Hematopoietic Stem Cell Transplantation Using Uniform Fludarabine/Busulfan Myeloablative Conditioning and Cyclosporine/Methotrexate and ATG Gvhd Prophylaxis

Introduction: Clonal hematopoiesis with indeterminate potential (CHIP) constitutes a part of the biological aging process and comprises the acquisition of somatic mutations in hematopoietic stem cells (HSCs). In the context of allogeneic hematopoietic stem cell transplantation (HCT), the potential transfer of CHIP from donor to recipient may raise concerns on HCT-related complications. There are contradictory data on donor CHIP's impact on long-term transplant outcomes using very diverse conditioning regimen as well as various graft-versus-host disease (GVHD) prophylaxis. This study aims to evaluate the impact of donor CHIP on allogeneic HCT outcomes in a cohort of the patients transplanted using a uniform, myeloablative intensity conditioning of Fludarabine-Busulfan (4 days) as well as Cyclosporine/Methotrexate with 4.5 mg/kg rabbit antithymocyte globulin (ATG) over 3 days for GVHD prophylaxis. Patients and methods: Peripheral blood samples were obtained from recipients and their corresponding donors who underwent allogeneic HCT at Tom Baker Cancer Centre, Calgary, Canada. Samples were archived from 339 patients and 255 donors prior to HCT, and additional patient sampling was done on day 28 (D28), day 56 (D56), or day 84 post allogeneic HCT (D84). As a result, paired recipient-donor samples were available for 255 HCTs. This study was approved by the institutional review boards of Princess Margaret Cancer Centre, Toronto, and Tom Baker Cancer Centre, Calgary. For the detection of CHIP, we have applied a bar-coded error-corrected sequencing protocol, a single-molecule molecular inversion probe capture protocol (NAR Genom Bioinform, 2022), which was performed at Wiesmann Institute of Science (Rehovot, Israel). We designed probes targeting 34 genes covering CHIP mutations along with other AML related mutations. Bar-coded NGS library was generated and processed. All statistical analyses were performed using R statistical software 3.5.0 and EZR version 1.41. Results: Sixty-seven clonal mutations in 56 recipients (18.2%) and 22 clonal mutations in 18 donors (7.1%) were detected prior to HCT. For recipients, TET2, JAK2, RUNX1, PPM1D, and DNMT3A were detected in at least 2 patients or more. For donors, the most frequently mutated gene was TET2 (3.1%), and the seconds were DNMT3A and PTPN11 (1.2% for both). Baseline characteristics of 255 patient-donor pairs did not show any difference between D-CHIP and no D-CHIP groups except donor age. Donor CHIP status was distributed evenly throughout most of the demographics of recipients, type of disease, pre-transplant status, and transplant procedure. Most of the pts received peripheral blood stem cells (97%). With a median follow up duration 6.02 years, the 5-year OS rates tend to be higher in no D-CHIP group than D-CHIP group (63.1% vs 42.8%) with a HR of 1.73 (95% CI [0.90-3.35]), although it was not statistically significant (p=0.096). The cumulative incidence of relapse for both groups were not different in 5 years (28.1% vs. 28.4%, p=0.849). The cumulative incidence of NRM within 5 years in D-CHIP group was slightly higher than no D-CHIP group (28.4% vs 17.5%), but it was not statistically significant (p=0.250). Neutrophil engraftment by day 28 was 99.1% vs. 100%, without statistical difference between the two groups (p=0.785). The cumulative incidence of grade II-IV acute GVHD within day+100 seemed to be higher in D-CHIP group compared to no D-CHIP group, but it was not statistically significant (24.8% vs. 34.5%, p=0.148; Figure A). In terms of chronic GVHD, cumulative incidence of moderate to severe GVHD was significantly higher in 5 years after transplant (29.4% vs. 58.8%; HR 2.55 [1.331-4.96], p=0.006; Figure B). Even when we set the cut off level of donor CHIP positivity up to &amp;gt;1% or &amp;gt;10%, the association between donor CHIP and risk of chronic GVHD remained significant. The impact of donor CHIP on the cumulative incidence of moderate to severe GVHD was confirmed in multivariate analysis (HR 2.3 [1.3-4.1], p=0.003). Other factors associated with moderate to severe chronic GVHD were previous history of grade II-IV acute GVHD (p=0.008) and donor type (p=0.054). Conclusion: Under clinical setting of uniform conditioning regimen and GVHD prophylaxis incorporating ATG (4.5mg/kg), donor CHIP was found to be associated with higher risk of moderate to severe chronic GVHD, while it does not affect other allogeneic HCT outcomes.

Mapping Mammalian 3D Genome Interactions with Micro-C-XL.

Three-dimensional (3D) chromosome organization is a major factor in genome regulation and cell-type specification. For example, cis-regulatory elements, known as enhancers, are thought to regulate the activity of distal promoters via interaction in 3D space. Genome-wide chromosome conformation capture (3C)-technologies, such as Hi-C, have transformed our understanding of how genomes are organized in cells. The current understanding of 3D genome organization is limited by the resolution with which the topological organization of chromosomes in 3D space can be resolved. Micro-C-XL measures chromosome folding with resolution at the level of the nucleosome, the basic unit of chromatin, by utilizing micrococcal nuclease (MNase) to fragment genomes during the chromosome conformation capture protocol. This results in an improved signal-to-noise ratio in the measurements, thus facilitating the better detection of insulation sites and chromosome loops compared to other genome-wide 3D technologies. A visually supported, detailed, step-by-step protocol for preparing high-quality Micro-C-XL samples from mammalian cells is presented in this article.

Future Perspectives for the Identification and Sequencing of Nicotinamide Adenine Dinucleotide-Capped RNAs.

Ribonucleic acid (RNA) is composed primarily of four canonical building blocks. In addition, more than 170 modifications contribute to its stability and function. Metabolites like nicotinamide adenine dinucleotide (NAD) were found to function as 5'-cap structures of RNA, just like 7-methylguanosine (m7G). The identification of NAD-capped RNA sequences was first made possible by NAD captureSeq, a multistep protocol for the specific targeting, purification, and sequencing of NAD-capped RNAs, developed in the authors' laboratory in the year 2015. In recent years, a number of NAD-RNA identification protocols have been developed by researchers around the world. They have enabled the discovery and identification of NAD-RNAs in bacteria, archaea, yeast, plants, mice, and human cells, and they play a key role in studying the biological functions of NAD capping. We introduce the four parameters of yield, specificity, evaluability, and throughput and describe to the reader how an ideal NAD-RNA identification protocol would perform in each of these disciplines. These parameters are further used to describe and analyze existing protocols that follow two general methodologies: the capture approach and the decapping approach. Capture protocols introduce an exogenous moiety into the NAD-cap structure in order to either specifically purify or sequence NAD-capped RNAs. In decapping protocols, the NAD cap is digested to 5'-monophosphate RNA, which is then specifically targeted and sequenced. Both approaches, as well as the different protocols within them, have advantages and challenges that we evaluate based on the aforementioned parameters. In addition, we suggest improvements in order to meet the future needs of research on NAD-modified RNAs, which is beginning to emerge in the area of cell-type specific samples. A limiting factor of the capture approach is the need for large amounts of input RNA. Here we see a high potential for innovation within the key targeting step: The enzymatic modification reaction of the NAD-cap structure catalyzed by ADP-ribosyl cyclase (ADPRC) is a major contributor to the parameters of yield and specificity but has mostly seen minor changes since the pioneering protocol of NAD captureSeq and needs to be more stringently analyzed. The major challenge of the decapping approach remains the specificity of the decapping enzymes, many of which act on a variety of 5'-cap structures. Exploration of new decapping enzymes or engineering of already known enzymes could lead to improvements in NAD-specific protocols. The use of a curated set of decapping enzymes in a combinatorial approach could allow for the simultaneous detection of multiple 5'-caps. The throughput of both approaches could be greatly improved by early sample pooling. We propose that this could be achieved by introducing a barcode RNA sequence before or immediately after the NAD-RNA targeting steps. With increased processing capacity and a potential decrease in the cost per sample, protocols will gain the potential to analyze large numbers of samples from different growth conditions and treatments. This will support the search for biological roles of NAD-capped RNAs in all types of organisms.

Expression of the GCG gene and secretion of active glucagon-like peptide-1 varies along the length of intestinal tract in horses.

Active glucagon-like peptide-1 (aGLP-1) has been implicated in the pathogenesis of equine insulin dysregulation (ID), but its role is unclear. Cleavage of proglucagon (coded by the GCG gene) produces aGLP-1 in enteral L cells. The aim in vivo was to examine the sequence of the exons of GCG in horses with and without ID, where aGLP-1 was higher in the group with ID. The aims in vitro were to identify and quantify the expression of GCG in the equine intestine (as a marker of L cells) and determine intestinal secretion of aGLP-1. Genomic studies were case-control studies. Expression and secretion studies in vitro were cross-sectional. The GCG gene sequence of the exons was determined using a hybridisation capture protocol. Expression and quantification of GCG in samples of stomach duodenum, jejunum, ileum, caecum and ascending and descending colon was achieved with droplet digital PCR. For secretory studies tissue explants were incubated with 12 mM glucose and aGLP-1 secretion was measured with an ELISA. Although the median [IQR] post-prandial aGLP-1 concentrations were higher (p = 0.03) in animals with ID (10.2 [8.79-15.5]), compared with healthy animals (8.47 [6.12-11.7]), there was 100% pairwise identity of the exons of the GCG sequence for the cohort. The mRNA concentrations of GCG and secretion of aGLP-1 differed (p < 0.001) throughout the intestine. Only the exons of the GCG gene were sequenced and breeds were not compared. The horses used for the study in vitro were not assessed for ID and different horses were used for the small, and large, intestinal studies. Differences in post-prandial aGLP-1 concentration were not due to a variant in the exons of the GCG gene sequence in this cohort. Both the large and small intestine are sites of GLP-1 secretion.

Generalizable FGFR prediction across tumor types using self supervised learning.

e15057 Background: Deep Learning models have demonstrated the ability to detect tumors, classify disease state, or infer genetic biomarkers from digital whole slide images (WSI) of Hematoxylin and Eosin (H&amp;E)-stained tissue. Such models can be deployed to improve clinical practice or efficiently recruit for clinical trials. However, the data to train these models is often very limited and image capture protocols vary between labs. We propose to use Self Supervised Learning (SSL) on large unlabeled histopathology data sets to improve generalizability and predictive performance of deep learning models on unseen data. Methods: We pre-trained a Convolutional Neural Network (CNN) using SSL on 25,120 unlabeled, digital WSIs from various sources (multiple scanners, hospital systems, labs, diseases, tissue sites). We then fine-tuned the pre-trained CNN to infer the presence or absence of select FGFR alterations in Muscle Invasive Bladder Cancer (MIBC) from the digital WSI. We applied this model (FGFRSSL) to WSIs of biopsies from other data sources and tumor types and compared with a model (FGFRTRAD) that was trained exclusively on MIBC samples without self-supervised learning. Results: The FGFRSSL model achieved an Area Under ROC Curve (AUC = 0.80) on MIBC WSIs and maintained performance on images from an independent lab (AUC=0.82). We further show that the model generalizes to Non-Muscle Invasive Bladder Cancer (NMIBC) samples with (AUC = 0.76) and Pan-Tumor WSIs (AUC: 0.83). The FGFRTRAD model trained without SSL-based pre-training achieved an Area Under ROC Curve (AUC: 0.76) on MIBC WSIs was less generalizable, showing lower performance for independent data (AUC = 0.72), NMIBC samples (AUC = 0.72), and Pan-Tumor samples (AUC = 0.64). Conclusions: We leveraged SSL pretraining to improve the reliability and generalizability of AI-based models across multiple data sources. We also demonstrate our model’s ability to infer FGFR status across multiple solid tumor types after training on only MIBC samples, suggesting the cell morphology conferred by FGFR alteration in cancer may be shared across diverse tumor types. These models represent a means for efficiently screening patients for actionable clinical biomarkers in a robust manner to guide clinical decisions and inform drug development efforts. [Table: see text]

Atrial stimulation protocols to test inducibility of atrial fibrillation

Abstract Funding Acknowledgements Type of funding sources: None. Background The electrophysiological inducibility of atrial fibrillation (AF) after pulmonary vein isolation (PVI) has been identified as a predictor of future AF recurrence. Stimulation protocols have not been sufficiently studied prospectively with respect to their reliability for inducing AF. Methods and results We conducted a prospective monocentric randomized clinical trial to evaluate different standardized electrical stimulation protocols on their potential to induce AF. Patients with symptomatic AF presenting in sinus rhythm scheduled for PVI were enrolled. The primary outcome was induction of AF. A total of 30 patients (age=62±2 years, 17% female, 13% persistent AF, left atrial systolic volume (LASV) 93±5 ml) were enrolled and randomized. To one of three different stimulation protocols performed at the beginning of PVI. Atrial fibrillation was induced in 4 out of 10 patients (40%) with fixed cycle length stimulation followed by up to 3 extrastimuli (protocol A), 6 out of 10 patients (60%) with burst stimulation (protocol B) and 9 out of 10 patients (90%) with fixed stimulation using the shortest cycle length with 1:1 atrial capture (protocol C), respectively. Time from start of stimulation to inducibility of AF was significantly lower in protocol C vs. protocol B and A (29±10 sec vs 40±14 sec vs 366±71 sec; p&amp;lt;0.0001). Isoprenaline was used in 10% in protocol A, 40% in protocol B and 80% protocol C. Overall, there were no significant differences concerning age (61±3 vs. 63±4 years; p=0.74), LASV (93±6 vs. 94±8 ml; p=0.98) or other parameters between patients with inducible and non-inducible AF. Conclusion With systematic stimulation protocols AF can be induced in up to 90%. Further studies are warranted to assess the specifity of these stimulations.

Breaking the tert-Butyllithium Contact Ion Pair: A Gateway to Alternate Selectivity in Lithiation Reactions.

The effects of Lewis basic phosphoramides on the aggregate structure of t-BuLi have been investigated in detail by NMR and DFT methods. It was determined that hexamethylphosphoramide (HMPA) can shift the equilibrium of t-BuLi to include the triple ion pair (t-Bu-Li-t-Bu)-/HMPA4Li+ which serves as a reservoir for the highly reactive separated ion pair t-Bu-/HMPA4Li+. Because the Li-atom's valences are saturated in this ion pair, the Lewis acidity is significantly decreased; in turn, the basicity is maximized which allowed for the typical directing effects within oxygen heterocycles to be overridden and for remote sp3 C-H bonds to be deprotonated. Furthermore, these newly accessed lithium aggregation states were leveraged to develop a simple γ-lithiation and capture protocol of chromane heterocycles with a variety of alkyl halide electrophiles in good yields.