Probe Kit Research Articles

Quercetin promotes autophagy to alleviate cigarette smoke-related periodontitis.

Cigarette smoking has been reported as an independent risk factor for periodontitis. Tobacco toxins affect periodontal tissue not only locally but also systemically, leading to the deterioration and recurrence of periodontitis. However, the mechanism of cigarette smoke-related periodontitis (CSRP) is unclear and thus lacks targeted treatment strategies. Quercetin, a plant-derived polyphenolic flavonoid, has been reported to have therapeutic effects on periodontitis due to its documented antioxidant activity. This study aimed to evaluate the effects of quercetin on CSRP and elucidated the underlying mechanism. The cigarette smoke-related ligature-induced periodontitis mouse model was established by intraperitoneal injection of cigarette smoke extract (CSE) and silk ligation of bilateral maxillary second molars. Quercetin was adopted by gavage as a therapeutic strategy. Micro-computed tomography was used to evaluate the alveolar bone resorption. Immunohistochemistry detected the oxidative stress and autophagy markers in vivo. Cell viability was determined by Cell Counting Kit-8, and oxidative stress levels were tested by 2,7-dichlorodihydrofluorescein diacetate probe and lipid peroxidation malondialdehyde assay kit. Alkaline phosphatase and alizarin red staining were used to determine osteogenic differentiation. Network pharmacology analysis, molecular docking, and western blot were utilized to elucidate the underlying molecular mechanism. Alveolar bone resorption was exacerbated and oxidative stress products were accumulated during CSE exposure in vivo. Oxidative stress damage induced by CSE caused inhibition of osteogenic differentiation in vitro. Quercetin effectively protected the osteogenic differentiation of human periodontal ligament cells (hPDLCs) and periodontal tissue by upregulating the expression of Beclin-1 thus to promote autophagy and reduce oxidative stress damage. Our results established a role of oxidative stress damage and autophagy dysfunction in the mechanism of CSE-induced destruction of periodontal tissue and hPDLCs, and provided a potential application value of quercetin to ameliorate CSRP.

LoCoLotive: In silico mining for low-copy nuclear loci based on target capture probe sets and arbitrary reference genomes.

Universal target enrichment probe kits are used to circumvent the individual identification of loci suitable for phylogenetic studies in a given taxon. Under certain circumstances, however, target capture can be inefficient and costly, and lower numbers of marker loci may be sufficient. We therefore propose a computational pipeline that enables the easy identification of a subset of promising candidate loci for a taxon of interest. Target sequences used for probe design are filtered based on an assembled reference genome, resulting in presumably intron-containing single-copy loci as present in the reference taxon. The applicability of the proposed approach is demonstrated based on two probe kits (universal and family-specific) in combination with several publicly available reference genomes. Guided by commercial probe kits, LoCoLotive enables fast and cost-efficient marker mining. Its accuracy mainly depends on the quality of the reference genome and its relatedness to the taxa under study.

Evolutionary history, traits, and weediness inDigitaria(Poaceae: Panicoideae)

AbstractDigitaria is a large pantropical genus, which includes a number of economically problematic agricultural weeds. Difficulties in species identification and the circumscription of the genus have previously hindered progress in understanding its evolution and developing a stable classification. We investigate the evolutionary history of Digitaria by combining two approaches: (i) phylogenetic analyses of the currently available Sanger sequence data with previously unpublished sequences mainly from South East Asia, and (ii) newly generated genomic data from low-copy nuclear genes obtained using the Angiosperms353 probe kit, with new data mainly from Madagascar. Both methods gave congruent results. The combination of these results allowed us to confirm a new clade structure within Digitaria lineages, in partial agreement with the previous morphology-based classifications. We confirm that Digitaria as traditionally circumscribed is not monophyletic, comprising Anthephora, Baptorhachis, Chaetopoa, and Chlorocalymma. We present an inference of key character evolution with morphological support for clades with spikelets arranged in binate and ternate groups, with variable trichome morphology, spikelet length, and culm length. Geographical clade structure is also observed. No phylogenetic signal of weedy function was observed.

New Radio-Frequency Liquid Permittivity Measurement System Using Filter-Based Microfluidic Sensor

A new radio-frequency (RF) liquid permittivity measurement system using a filter-based microfluidic sensor is proposed in this paper. Integrating the filter-based sensor with an RF source, a power divider, and a gain/phase detector can form a new permittivity measurement system without using a bulky and expensive vector network analyzer (VNA) to acquire measured data. Since two coupled resonators with a strong coupling synthesize the filter-based sensor, it can provide a stronger electric-field concentration in the sensing area and improve the sensor’s sensitivity. The 8-μL water-ethanol mixtures with ethanol volume fractions (EVF) ranging from 10% - 90% in 10% increments are used as the test liquids to evaluate the proposed sensor and system. Compared with the measured results using the commercial dielectric probe kit, the maximum errors of ε′ <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">r</sub> and ε″ <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">r</sub> using the filter-based sensor with a VNA are -4.34% and 13.24%, respectively. At the same time, ε′ <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">r</sub> and ε′ <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">r</sub> errors are 8.28% and 14%, respectively, using the proposed permittivity measurement system. Since the proposed permittivity measurement system can flexibly synthesize the sensing area size, control the electric-field intensity, and get rid of a VNA, it makes it easier to satisfy the requirements of various microfluidic sensing scenarios.

Social Cohesion in Interactive Digital Heritage Experiences

Presently, social cohesion (SC) is a priority at different levels. Cultural heritage is an ideal context to promote SC through interactive digital technologies designed to engage groups of visitors. The purpose of the present study is to identify how to design digital heritage applications for SC and how to measure it. The results are based on the design of a cultural probe kit used to identify the design elements on top of which a collaborative and hybrid prototype, the Brancacci POV, was developed. Here, we analysed the results of this prototype, which included 107 visitors with respective groups of 5 participants and guided by an expert. From this analysis, the possibility of strengthening SC when collaborative tasks are included emerged. Additionally, it appeared to be possible to shorten the distance between citizens and cultural institutions if “mediated dialogue” approaches were adopted and if focus, motivation, trust and “in-group” perception of inclusion emerge when digital heritage experiences were set in intimate and quiet environments.

Klotho alleviates contrast-induced acute kidney injury by suppressing oxidative stress, inflammation, and NF-KappaB/NLRP3-mediated pyroptosis

Contrast-induced acute kidney injury (CI-AKI) is a common complication following percutaneous coronary intervention in coronary artery disease (CAD) patients with >30% incidence. Klotho is a multifunctional protein that inhibits oxidative stress and inflammation, but its role in CI-AKI is poorly understood. The present study aimed to explore the effects of klotho in CI-AKI. Six-week-old mice and HK-2 were divided into the control, contrast medium (CM), CM+klotho, and klotho groups. H&E staining evaluated kidney injury. Scr and BUN showed renal function. DHE probe and ELISA kit detected the levels of reactive oxygen species (ROS) in kidney tissue, superoxide dismutase (SOD), and malondialdehyde (MDA) in serum. Western blot detected the expressions of NF-κB and phosphorylated NF-κB (p-NF-κB) and pyroptosis-related protein levels of NLRP3, caspase-1, GSDMD, and cleaved-GSDMD in the kidney of CI-AKI mice. CCK-8 and lactate dehydrogenase (LDH) activity assays determined cell viability and damage. Fluorescent probe dichloro-dihydro-fluorescein diacetate (DCFH-DA) and enzyme-linked immunosorbent assay (ELISA) tested oxidative stress-related indicators. These included intracellular reactive oxygen species (ROS), superoxidase dismutase (SOD), and malondialdehyde (MDA). IL-6, TNF-α, IL-1β, and IL-18 in the cell supernatant were tested by ELISA assay and used to reflect inflammation responses. Propidium iodide (PI) staining showed the cell death of HK-2. The expressions of NF-κB, p-NF-κB and pyroptosis-related protein levels of NLRP3, caspase-1, GSDMD, and cleaved-GSDMD were detected by Western blot. Exogenous klotho administration reduced kidney histopathological alterations and improved renal function in vivo. The levels of reactive oxygen species (ROS) in renal tissue, superoxide dismutase (SOD), and malondialdehyde (MDA) in serum decreased after the klotho intervention. The expression levels of p-NF-κB and pyroptosis-related proteins, including NLRP3, caspase-1, GSDMD, and cleaved-GSDMD, were decreased in CI-AKI mice after the klotho intervention. In vitro, klotho significantly inhibited CM-induced oxidative stress and the production of IL-6 and TNF-α. Moreover, it was found that klotho inhibited the activation of p-NF-κB and down-regulated pyroptosis-related protein (NLRP3, caspase-1, GSDMD, and cleaved-GSDMD). Klotho has a protective effect on CI-AKI via suppressing oxidative stress, inflammation, and NF-κB/NLRP3-mediated pyroptosis that contributes to the potential therapy of CI-AKI.

Phylogenomics sheds new light on the drivers behind a long-lasting systematic riddle: the figwort family Scrophulariaceae.

The figwort family, Scrophulariaceae, comprises c.2000 species whose evolutionary relationships at the tribal level have proven difficult to resolve, hindering our ability to understand their origin and diversification. We designed a specific probe kit for Scrophulariaceae, targeting 849 nuclear loci and obtaining plastid regions as by-products. We sampled c.87% of the genera described in the family and use the nuclear dataset to estimate evolutionary relationships, timing of diversification, and biogeographic patterns. Ten tribes, including two new tribes, Androyeae and Camptolomeae, are supported, and the phylogenetic positions of Androya, Camptoloma, and Phygelius are unveiled. Our study reveals a major diversification at c.60 million yr ago in some Gondwanan landmasses, where two different lineages diversified, one of which gave rise to nearly 81% of extant species. ASouthern African origin is estimated for most modern-day tribes, with two exceptions, the American Leucophylleae, and the mainly Australian Myoporeae. The rapid mid-Eocene diversification is aligned with geographic expansion within southern Africa in most tribes, followed by range expansion to tropical Africa and multiple dispersals out of Africa. Our robust phylogeny provides a framework for future studies aimed at understanding the role of macroevolutionary patterns and processes that generated Scrophulariaceae diversity.

Abstract P2-09-03: Quantification of HER2 expression and spatial biology using computational pathology: A cross-assay validation study in breast cancer

Abstract Background Conventional pathologic scoring of HER2 by IHC is proven to distinguish potential responders to trastuzumab but has not been effective for next generation antibody drug conjugates (ADCs) such as trastuzumab deruxtecan (T-DXd), which is capable of bystander killing. Several alternative approaches have been deployed to measure HER2, including immunofluorescence and mRNA sequencing. We have developed a novel and fully automated computational pathology technique, Quantitative Continuous Scoring (QCS), to quantify the level and distribution of HER2 from digitized HER2 IHC slides in an objective, quantifiable, and reproducible manner on a per-cell basis [Gustavson et al., SABCS 2020]. To further validate this approach, we performed a systematic multi-omic comparison of QCS to orthogonal methods of HER2 quantitation on a cohort of primary and metastatic breast cancer cases (N=30). Methods HER2 was evaluated using three independent methods on serial tissue sections obtained from 30 archival FFPE breast cancer samples distributed over the full range of HER2 expression, from 0 to 3+. HER2-IHC staining (clone 4B5, Roche Tissue Diagnostics) was performed using standard methods and cases were scored by two pathologists using CAP/ASCO guidelines and H-scores were assigned. We performed FISH (HER2 IQFISH pharmDx [Dako]; PathVysion HER-2 DNA Probe Kit [Vysis]), mRNA quantification of ERBB2 transcript levels (Nano String), and immunofluorescence (IF; HER2 clone 29D8, CST). Imaged with Vectra (Akoya) and analyzed with Halo (Indica). QCS readouts were generated from the above-mentioned digital images of IHC slides by using a fully automated image analysis pipeline; readouts included per-cell staining intensity measurements of membranes and cytoplasmic sub-compartments in terms of optical density (OD) [Van der Laak, JQCS 2000], which were aggregated to a single slide-level score. Additionally, using the OD measurements and the cell locations, a Spatial Proximity Score (SPS) was computed, summing the percentage of cells with OD≥10 (corresponding to the limit of visual detection of IHC staining) as well as the percentage of cells with OD&amp;lt; 10 within a prespecified radius (25µm) of a neighboring cell with OD≥10. Results Our analysis demonstrated that QCS-based scoring correlates with orthogonal measurements used in this study. Comparing protein-based assays, the observed Pearson correlation was R=0.88 between QCS median membrane OD and IHC H-scores, R=0.86 with IF-based HER2 mean cell expression intensity, and R=0.85 with IF-based H-scores. Correlation with transcriptomic profiling was R=0.81 for OD vs. mRNA, however ERBB2 transcript levels did not distinguish between HER2 0, 1+, and 2+ FISH negative cases, while QCS was able to do so. Correlation between protein-based and nucleic-acid based assays were numerically worse, with R=0.64 for OD vs. FISH. All samples (including those with HER2 IHC scores of 0 and H-Scores &amp;lt; 10) had at least ~20% of cells with quantifiable HER2 expression by OD, the presence of which was confirmed using IF. For cases in the lowest quartile of HER2 expression by OD, SPS identified 20-50% additional HER2-null cells that were in close proximity to HER2-expressing cells that may be vulnerable to bystander killing. Conclusion QCS-based scoring is consistent with orthogonal protein-based measurements across the range of HER2 expression. Most importantly, QCS derived-spatial analysis features identify additional patients in the lower end of HER2 expression that might be highly relevant for ADC response prediction, particularly if a drug exerts bystander activity. Further clinical verification and validation on large cohorts is needed. Footnote: This study was approved by the IRB at MSKCC. Citation Format: Joshua Drago, Zonera Hassan, Jan Zaucha, Ansh Kapil, Fatemeh Derakhshan, Fresia Pareja, Shimulov Anatoliy, Fanni Ratzon, Travis J Hollman, Claire Myers, Jessica Chan, Andrea Spitzmuller, Mark Gustavson, Danielle Carroll, Dara Ross, Jorge Reis-Filho, Carl barrett, Sihem Khalifa, Schmidt Guenter, Hadassah Sade, Sarat Chandarlapaty. Quantification of HER2 expression and spatial biology using computational pathology: A cross-assay validation study in breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-09-03.

The HORIZON III retrospective exploratory analysis: HER2 expression amplification in colorectal cancer.

194 Background: Although regularly used in breast and gastric cancer, HER2 testing is not routinely performed in colorectal cancer (CRC). Immunohistochemistry (IHC) and in situ hybridization (ISH) scoring have not been optimized for assessment of HER2 overexpression and amplification in CRC. Varying rates of HER2 overexpression (2%-11%) have been reported in previously untreated CRC (Wang World J Gastrointest Oncol 2019) and patients with advanced/metastatic CRC with HER2 amplification or overexpression respond poorly to current standard of care therapies (Sartore-Bianchi Oncologist 2019). We characterized HER2 prevalence in a retrospective cohort analysis of clinical trial patients. Methods: The HORIZON III trial (NCT00384176) evaluated FOLFOX + bevacizumab or cediranib (AZD2171) as first-line (1L) treatment in patients with metastatic CRC (Schmoll J Clin Oncol 2012). The primary objective of this analysis of a subset of HORIZON III samples was to characterize HER2 prevalence in 1L metastatic CRC. Secondary and exploratory objectives included correlating HER2 status to other molecular findings (eg, RAS/RAF mutations), clinicopathologic characteristics, and patient outcomes. IHC was performed using a monoclonal anti-HER2 antibody PATHWAY HER2 [4B5] Ventana on primary CRC tumor sections and scored according to ASCO/CAP guidelines for gastric cancer (Bartley Arch Pathol Lab Med 2016). H&amp;E staining was performed to determine the adequacy of tumor samples (ie, &gt; 100 viable tumor cells per specimen). All tumors with an IHC score of 2+ were analyzed for amplification by ISH (HER2 IQFISH pharmDx dual probe kit, Agilent Technologies K573111-5). Targeted mutation panel testing was used to determine other molecular alterations. Descriptive statistics were used to summarize baseline and clinical outcome data by HER2 status. The current analysis was approved by the AstraZeneca bioethics review board. Results: Of the 1614 patients in HORIZON III, 396 met the inclusion criteria of appropriate consent, sufficient tumor sample for analysis, and a unique identifier. HER2-positive tumors (IHC3+ or IHC2+/ISH+) were identified in 2.1% of samples; 1.3% were IHC3+ and 0.8% were IHC2+/ISH+. Compared with IHC3+, IHC2+ tumors were more heterogeneous with mixed components of cells weakly expressing HER2 and lacking HER2 expression. Conclusions: This exploratory analysis of the HORIZON III study provides insights into HER2 prevalence in 1L, unselected, advanced/metastatic CRC, finding that 2.1% of tumors were HER2 positive. The demographic characteristics of patients with analyzable samples were representative of the entire HORIZON III study population; however, further assessment of KRAS status and other clinicopathological characteristics is needed. These data may inform future clinical development and support selection of patients with CRC who are likely to benefit from HER2 targeting therapies. Clinical trial information: NCT00384176 .

Aquaporin5 Deficiency Aggravates ROS/NLRP3 Inflammasome-Mediated Pyroptosis in the Lacrimal Glands.

The pathogenesis of the lacrimal glands (LGs) is facilitated by inflammation mediated by the NACHT, LRR, and NLRP3 inflammasomes in dry eye disease. This research aimed to explore the protective effects of Aquaporin 5 (AQP5) on LGs by inhibiting reactive oxygen species (ROS) and the NLRP3 inflammasome. AQP5 knockout (AQP5-/-) mice were used to evaluate pathological changes in LGs. ROS generation was detected with a dichlorodihydro-fluorescein diacetate assay. Lipid metabolism was assessed by Oil Red O staining. The reversal of the mitochondrial membrane potential was detected using a JC-1 fluorescent probe kit. The effect of AQP5 on NLRP3/caspase-1/Gasdermin-D (GSDMD)-mediated pyroptosis was examined using pharmacological treatment of N-acetyl L-cysteine or MCC950. AQP5 loss significantly increased ROS generation, lipid metabolism disorders, TUNEL-positive cells, and reversal of the mitochondrial membrane potential in the AQP5-/- LGs. NLRP3 upregulation, increased caspase-1 and GSDMD activity, and enhanced IL-1β release were detected in the AQP5-/- mouse LGs and primary LG epithelial cells. MCC950 significantly suppressed NLRP3 inflammasome-related pyroptosis induced by AQP5 deficiency in LGs and primary LG epithelial cells. Furthermore, we discovered that prestimulating the AQP5-/- primary LG epithelial cells with N-acetyl L-cysteine decreased NLRP3 expression, caspase-1 and GSDMD activity levels, and IL-1β release. Our results revealed that AQP5 loss promoted NLRP3 inflammasome activation through ROS generation. Inhibiting the ROS or NLRP3 inflammasome significantly alleviated the damage and pyroptosis of AQP5-deficient LG epithelial cells, which could provide new insights into dry eye disease.

Dielectric Characterization and Statistical Analysis of Ex-Vivo Burnt Human Skin Samples for Microwave Sensor Development

The dielectric properties of skin tissues in relation to different degrees of burn are a necessary prerequisite for designing non-invasive microwave sensing modalities. Due to the difficulties in obtaining human tissue samples, such databases are largely unavailable. To bridge the knowledge gap in this field, we attempt to create a dielectric database of various burn-degree skin samples and their statistical analysis in this work. This research is part of the European “Senseburn” project, which aims to create a non-invasive diagnostic tool that can measure the severity and depth of burns on humans in a clinical setting. In this work, several ex-vivo burnt samples were collected from the Uppsala University Hospital (Akademiska sjukhuset, Sweden). Out of that, eight samples with different degrees of burns in various human body locations were selected for the analysis. The dielectric characterization of the categorized samples was done using an Keysight N1501A dielectric open-end co-axial probe Kit. The dielectric characterization was made from 500 MHz to 10 GHz with 1001 points. The measurement was made systematically, and the clinician feedback forms were gathered and analyzed throughout the process. The measurement data followed the FASTCLUS procedure, which was initially analyzed using density plot, convergence, and cubic clustering criteria. For the statistical analysis, 11 frequency points were considered for eight samples. The results of the fundamental statistical analysis using the FASTCLUS procedure resulted in 88 data sets. Later, data sets were analyzed in sample-wise clusters. Every sample was made with two clusters, i.e., cluster 1, which consisted of healthy sectors, and cluster 2, which consisted of burnt sectors. We made the linear approximations for the sample-wise clusters and found the constant real permittivity difference. Furthermore, we found a pattern in the constant real permittivity differences of every sample that is proportional to the burn degrees. This information is needed in order to identify optimization parameters, i.e., the sensitivity with respect to dielectric difference for various burn degrees. For this purpose, extensive measurement campaigns across the microwave frequency band from 500 MHz – 10 GHz were conducted. Based on the analysis of dielectric data, each skin region of interest (ROI) has its own dielectric properties. Additionally, we developed a proof of concept non-invasive flexible microwave sensor based on the dielectric database collected from burnt ex-vivo human tissue samples. In this way, we could distinguish between phantoms with different dielectric properties in the burned human tissue sample range.

Application of fluorescence in situ hybridization in distinguishing acral melanoma in situ from acral junctional melanocytic nevus on the volar skin in Japanese patients.

Four-colored fluorescence in situ hybridization (FISH) is an ancillary diagnostic tool for melanoma. However, most studies that have investigated the usefulness of FISH primarily focused on advanced melanomas. The aim of the current study was to evaluate the effectiveness of FISH in distinguishing acral melanoma (AM) in situ from benign acral junctional nevus (AJN), two types of lesions that are difficult to differentiate via traditional clinical means. The authors investigated the usefulness of FISH in 91 acral melanocytic lesions, including 50 lesions with diagnostic discrepancies between dermoscopic and pathologic approaches or difficulty diagnosing between AM in situ and AJN, on the volar skin of Japanese patients. The authors classified the lesions based on the diagnosis of dermatologists and pathologists into four groups: (I) lesions with a unanimous diagnosis by dermatologists and pathologists as AM in situ or AJN (n=41); (II) lesions with a unanimous diagnosis by dermatologists only as AM in situ or AJN (n=21); (III) lesions with a unanimous diagnosis by pathologists only as AM in situ or AJN (n=15); and (IV) all other lesions (n=14). The dermatologists diagnosed the lesions by clinical and dermoscopic photographs alone, while the pathologists diagnosed the lesions by microscopy of hematoxylin and eosin-stained slides alone. In group I (AM in situ [n=20] and AJN [n=21]), four-colored FISH demonstrated 90% sensitivity and 81% specificity in distinguishing AM in situ from AJN. There was a significant correlation between the FISH results and the unanimous diagnoses by pathologists alone (p=0.03) in group III. However, FISH results were not significantly correlated with the unanimous diagnoses by dermatologists alone (p=0.33) in group II. In conclusion, the four-colored FISH probe kit was useful in distinguishing between AM in situ and AJN and may be an ancillary method when pathologists who are not experts of dermatopathology diagnose melanocytic lesions.

Effect of Coupling Medium on Penetration Depth in Microwave Medical Imaging.

In microwave medical imaging, the human skin reflects most of microwave energy due to the impedance mismatch between the air and the body. As a result, only a small portion of the microwave energy can enter the body and work for medical purpose. One solution to tackle this issue is to use a coupling (or matching) medium, which can reduce unwanted reflections on the skin and meanwhile improve spatial imaging resolution. A few types of fluids were measured in this paper for their dielectric properties between 500 MHz and 13.5 GHz. Measurements were performed by a Keysight programmable network analyzer (PNA) with a dielectric probe kit, and dielectric constant and conductivity of the fluids were presented in this paper. Then, quantitative computations were exercised to present the attenuations due to the reflection on the skin and to the loss in each coupling medium, based on the measured liquid dielectric values. Finally, electromagnetic simulations verified that the coupling liquid can allow more microwave energy to enter the body to allow for a more efficient medical examination.

Phylogenomics and classification of Notropis and related shiners (Cypriniformes: Leuciscidae) and the utility of exon capture on lower taxonomic groups.

North American minnows of the Shiner Clade, within the family Leuciscidae, represent one of the most taxonomically complex clades of the order Cypriniformes due to the large number of taxa coupled with conserved morphologies. Species within this clade were moved between genera and subgenera until the community decided to lump many of the unclassified taxa with similar morphologies into one genus, Notropis, which has held up to 325 species. Despite phylogentic studies that began to re-elevate some genera merged into Notropis, such as Cyprinella, Luxilus, Lythrurus, and Pteronotropis, the large genus Notropis remained as a taxonomic repository for many shiners of uncertain placement. Recent molecular advances in sequencing technologies have provided the opportunity to re-examine the Shiner Clade using phylogenomic markers. Using a fish probe kit, we sequenced 90 specimens in 87 species representing 16 genera included in the Shiner Clade, with a resulting dataset of 1,004 loci and 286,455 base pairs. Despite the large dataset, only 32,349 bp (11.29%) were phylogenetically informative. In our maximum likelihood tree, 78% of nodes are 100% bootstrap supported demonstrating the utility of the phylogenomic markers at lower taxonomic levels. Unsurprisingly, species within Notropis as well as Hudsonius, Luxilus, and Alburnops are not resolved as monophyletic groups. Cyprinella is monophyletic if Cyprinella callistia is excluded, and Pteronotropis is monophyletic if it includes Hudsonius cummingsae. Taxonomic changes we propose are: restriction of species included in Alburnops and Notropis, elevation of the subgenus Hydrophlox, expansion of species included in Miniellus, movement of Hudsonius cummingsae to Pteronotropis, and resurrection of the genera Coccotis and Paranotropis. We additionally had two specimens of three species, Notropis atherinoides, Ericymba amplamala, and Pimephales vigilax and found signficant differences between the localities (1,086, 1,424, and 845 nucleotides respectively).

TaxonWorks as a Tool for Managing Large Biodiversity Projects

Large systematic revisionary projects incorporating data for hundreds or thousands of taxa require an integrative approach, with a strong biodiversity-informatics core for efficient data management to facilitate research on the group. Our original biodiversity informatics platform, 3i (Internet-accessible Interactive Identification) combined a customized MS Access database backend with ASP-based web interfaces to support revisionary syntheses of several large genera of leafhopers (Hemiptera: Auchenorrhyncha: Cicadellidae). More recently, for our National Science Foundation sponsored project, “GoLife: Collaborative Research: Integrative genealogy, ecology and phenomics of deltocephaline leafhoppers (Hemiptera: Cicadellidae), and their microbial associates”, we selected the new open-source platform TaxonWorks as the cyberinfrastructure. In the scope of the project, the original “3i World Auchenorrhyncha Database” was imported into TaxonWorks. At the present time, TaxonWorks has many tools to automatically import nomenclature, citations, and specimen based collection data. At the time of the initial migration of the 3i database, many of those tools were still under development, and complexity of the data in the database required a custom migration script, which is still probably the most efficient solution for importing datasets with long development history. At the moment, the World Auchenorrhyncha Database comprehensively covers nomenclature of the group and includes data on 70 valid families, 6,816 valid genera, 47,064 valid species as well as synonymy and subsequent combinations (Fig. 1). In addition, many taxon records include the original citation, bibliography, type information, etymology, etc. The bibliography of the group includes 37,579 sources, about 1/3 of which are associated with PDF files. Species have distribution records, either derived from individual specimens or as country and state level asserted distribution, as well as biological associations indicating host plants, predators, and parasitoids. Observation matrices in TaxonWorks are designed to handle morphological data associated with taxa or specimens. The matrices may be used to automatically generate interactive identification keys and taxon descriptions. They can also be downloaded to be imported, for example, into Lucid builder, or to perform phylogenetic analysis using an external application. At the moment there are 36 matrices associated with the project. The observation matrix from GoLife project covers 798 taxa by 210 descriptors (most of which are qualitative multi-state morphological descriptors) (Fig. 2). Illustrations are provided for 9,886 taxa and organized in the specialized image matrix and could be used as a pictorial key for determination of species and taxa of a higher rank. For the phylogenetic analysis, a dataset was constructed for 730 terminal taxa and &amp;gt;160,000 nucleotide positions obtained using anchored hybrid enrichment of genomic DNA for a sample of leafhoppers from the subfamily Deltocephalinae and outgroups. The probe kit targets leafhopper genes, as well as some bacterial genes (endosymbionts and plant pathogens transmitted by leafhoppers). The maximum likelihood analyses of concatenated nucleotide and amino acid sequences as well as coalescent gene tree analysis yielded well-resolved phylogenetic trees (Cao et al. 2022). Raw sequence data have been uploaded to the Sequence Read Archive on GenBank. Occurrence and morphological data, as well as diagnostic images, for voucher specimens have been incorporated into TaxonWorks. Data in TaxonWorks could be exported in raw format, get accessed via Application Programming Interface (API), or be shared with external data aggregators like Catalogue of Life, GBIF, iDigBio.

Assessment of a HER-2 scoring system and its correlation of HER2-targeting antibody-drug conjugate therapy in urothelial carcinoma.

4572 Background: Human epidermal growth factor receptor 2 (HER2) overexpression is related to many tumor treatments. RC48-ADC, a novel humanized anti-HER2 antibody conjugated with monomethyl auristatin E, has shown a promising efficacy in patients with HER2-positive locally advanced or metastatic urothelial carcinoma (mUC). The characteristic expression and scoring systems of HER2 have existed in breast cancer, gastric cancer for many years, but not in UC. We aimed to explore the expression pattern of HER2 in UC and develop a validated HER2 scoring system. Methods: A total of 137 patients and 43 patients in two studies of cohort 1 and cohort 2 were enrolled, respectively. The patients of the cohort 2 were enrolled in the open-label, multicenter, phase II study of RC48-ADC. Formalin-fixed paraffin-embedded urothelial cancer samples were tested for HER2 status using the fluorescence in situ hybridization (FISH) PathVysion HER2 DNA probe kit (PathVysion, Abbott Molecular, USA). Immunohistochemistry (IHC) was performed using the Ventana Benchmark XT (Ventana Medical Systems, USA). The 2018 ASCO/CAP HER2 scoring system of breast cancer was adopted and modified to score HER2 expression level in UC. Results: The expression rate of HER2 (IHC 2+/3+) was 24.1% (33/137). In HER2 IHC status 3+ or 2+ patients, the HER2 amplified rate was 31% (13/42). The objective response rates in RC48-ADC treatment patients with IHC 3+, IHC 2+ and FISH +, IHC 2+ and FISH - were 58.8%, 66.7% and 40%, respectively. Heterogeneity of HER2 protein expression was 55.5% (15/27) and the objective response rate had no significant difference between patients with tumor heterogeneity and homogeneity. Conclusions: The modified HER2 testing scoring system could be applied to UC to determine which patient might benefit from anti-HER2-ADC treatment. There was a trend towards a better benefit for patients with HER2 amplification and the tumor heterogeneity did not influence the drug efficacy.

A novel fusion between CDC42BPB and ALK in a patient with quadruple wild‐type gastrointestinal stromal tumor

BackgroundGastrointestinal stromal tumors (GISTs) are the most common type of mesenchymal tumor in gastrointestinal tract, with striking features of morphology and immunohistochemistry. But GISTs in pregnancy could seldom be found. Pathogenic activating mutations of the proto‐oncogene KIT and PDGFRA are detected in majority GISTs, and adjuvant imatinib therapy targeting KIT and PDGFRA mutations is recommended for patients with high‐risk GIST. However, some rare subgroups with distinct molecular features remain uncovered and more therapeutic targets need to be revealed.MethodsThe DNA/RNA samples were detected using the NGS‐based YuanSu450 gene panel. After identifying the CDC42BPB‐ALK fusion by NGS, this novel fusion was further confirmed by Sanger sequencing. Subsequently, FISH analysis was performed using the Vysis ALK Break Apart FISH Probe kit to testify the ALK status. ALK protein expression was confirmed by IHC (D5F3 and 5A4).ResultsHerein, we reported the first case of quadruple wild‐type (WT) GIST with ALK‐CDC42BPB fusion and ALK (D5F3) overexpression. In this study, we described a 33‐year‐old pregnant patient in lactation who had a massive space occupying lesion (with the maximum diameter of 22 cm) in the stomach and was eventually diagnosed as quadruple WT GIST (KIT WT/PDGFRA WT/SDH WT/RAS‐P WT).ConclusionWe unexpectedly found that this GIST patient showed ALK (D5F3) overexpression and harbored a novel fusion CDC42BPB exon 24‐ALK in exon 20.

Solo Dining at Home in the Company of ICT Devices

The consumption of a solo meal is often subject to negative associations. Studies indicate that solo diners use information and communication technology (ICT) devices such as smartphones, to mitigate negative experiences such as boredom and loneliness, especially when dining in a public context. However, we know less about the motivation to use such devices and consequent meal experiences in a private context. For this exploratory qualitative study, we asked participants to fill out a cultural probe kit to capture their dining experience and use of ICT devices over a period of seven days. Once completed, the content was discussed with participants during a semi-structured interview. Data was analyzed using thematic analysis in a deductive and inductive form leading to four themes: (1) The experience of eating with others; (2) The use of electronic devices while eating; (3) The meaning of food; and (4) Relaxing features and influences. Participants indicated that eating alone can be a pleasurable experience that people enjoy and perceive as relaxing. ICT devices were named to play an essential part in the dining experience. The entertainment that devices provide can mitigate feelings of loneliness and uncomfortable silence when eating by oneself. We reflect on the findings and point out potential design avenues for future studies.

DOP79 Biomarkers for IBD using OLINK Proteomics inflammation panel: Preliminary results from the COLLIBRI consortium

Abstract Background Circulating serum proteins have provided insights into disease pathogenesis and are being used to identify prognostic, diagnostic and therapeutic biomarkers for chronic inflammatory diseases. With this pilot project, the Collaborative IBD Biomarker Research Initiative (COLLIBRI) consortium aimed to unravel disease heterogeneity in inflammatory bowel disease (IBD). Methods Serum samples were cross-sectionally obtained from 3,390 individuals (Crohn’s disease (CD), n=1815; ulcerative colitis (UC), n=1170; healthy, n=405) recruited at nine centres from Sweden and Belgium. Relative levels of 92 proteins were analysed using the Proseek Multiplex Inflammation I Probe kit 96x96 (Olink Proteomics, Uppsala, Sweden) and reported as arbitrary units, i.e., normalised protein expression on a log2 scale. Using a multivariate integrative approach, we identified protein signatures distinguishing CD and UC samples and attempted to identify clusters or subgroups within patients. Recruiting centre, cohort and batch information were considered for the integrative analysis. Optimisation was performed for identifying the number of components and features per component using 5-fold cross-validation and Leave-One-Group-Out-Cross-Validation, respectively. Information on transcriptional regulators was retrieved from the ReMap project using the orthogonal regulatory resource ChEA3. Results A panel of 8 proteins was identified which could segregate CD and UC patients (Figure 1). FGF19 exhibited a consistent trend of expression (downregulated in CD) across all batches of datasets. An integrated AUC of 72.5% was achieved across the different batches of samples used in the study with the highest AUC (79.2%, P-value 8.5e-07) being recorded for a single batch of samples (CD = 42, UC = 56). On a centre-specific dataset, the cross-centre integrated signature achieved an AUC of 75.1%. We identified three transcription factors (MEF2A, BATF, NFKB2), of which the two latter ones are known to modulate intestinal inflammation and which could potentially regulate the expression of at least half of the genes encoding the proteins in the predictive 8-protein panel. Conclusion We identified an integrated proteomic biomarker panel capable of separating CD and UC patients. Through further integration of confounder variables along with using other supervised and unsupervised approaches, subsequent analyses may further refine the molecular heterogeneity among CD and UC patients. Our results demonstrate the need for large datasets to identify relevant clusters of patients with IBD, since the diagnosis exhibits a high degree of clinical heterogeneity. *Equally contributed

Clinical Validation of the Xpert MTB/RIF Test for Identification of the Mycobacterium tuberculosis Complex in Acid-Fast Bacillus Smear-Positive MGIT Broth Cultures.

The identification of the Mycobacterium tuberculosis complex (MTBC) from smear-positive broth cultures can be achieved using several methods, including both lab-developed and commercially available molecular assays. In the United States, a commercially available probe-based assay has been used for over a decade by many laboratories for identification of MTBC directly from acid-fast bacilli (AFB) smear-positive broth cultures, including those recovered from the MGIT 960 system. However, recent difficulties in obtaining probe kits for identification resulted in mycobacteriology laboratories looking for alternative platforms to provide for rapid identification of MTBC and detection of rifampin resistance. The Xpert MTB/RIF test (Cepheid, Sunnyvale, CA) has shown high sensitivity for the diagnosis of MTBC from pulmonary specimens but is not often used for identification directly from smear-positive MGIT 960 broth cultures (Becton, Dickinson, Sparks, MD). We sought to validate the Xpert MTB/RIF test for use with AFB smear-positive MGIT 960 cultures in a clinical hospital setting. Overall, the assay showed a categorical agreement of 100% for identification of MTBC and detection of rifampin resistance. No false-positive results or cross-reactivity were noted. Findings indicate that the Xpert MTB/RIF test may be suitable as a rapid replacement for identification of MTBC and detection of rifampin resistance from AFB smear-positive MGIT 960 broth cultures.