Probe Sets Research Articles

Joint Metric Learning-Based Class-Specific Representation for Image Set Classification.

With the rapid advances in digital imaging and communication technologies, recently image set classification has attracted significant attention and has been widely used in many real-world scenarios. As an effective technology, the class-specific representation theory-based methods have demonstrated their superior performances. However, this type of methods either only uses one gallery set to measure the gallery-to-probe set distance or ignores the inner connection between different metrics, leading to the learned distance metric lacking robustness, and is sensitive to the size of image sets. In this article, we propose a novel joint metric learning-based class-specific representation framework (JMLC), which can jointly learn the related and unrelated metrics. By iteratively modeling probe set and related or unrelated gallery sets as affine hull, we reconstruct this hull sparsely or collaboratively over another image set. With the obtained representation coefficients, the combined metric between the query set and the gallery set can then be calculated. In addition, we also derive the kernel extension of JMLC and propose two new unrelated set constituting strategies. Specifically, kernelized JMLC (KJMLC) embeds the gallery sets and probe sets into the high-dimensional Hilbert space, and in the kernel space, the data become approximately linear separable. Extensive experiments on seven benchmark databases show the superiority of the proposed methods to the state-of-the-art image set classifiers.

Development and evaluation of a multiplex real-time RT-PCR assay for simultaneous detection of H5, H7, and H9 subtype avian influenza viruses

H5, H7 and H9 are the major subtypes of avian influenza virus (AIV) that cause economic losses in the poultry industry and sporadic zoonotic infection. Early detection of AIV is essential for preventing disease spread. Therefore, molecular diagnosis and subtyping of AIV via real-time RT-PCR (rRT-PCR) is preferred over other classical diagnostic methods, such as egg inoculation, RT-PCR and HI test, due to its high sensitivity, specificity and convenience. The singleplex rRT-PCRs for the Matrix, H5 and H7 gene used for the national surveillance program in Korea have been developed in 2017; however, these methods were not designed for multiplexing, and does not reflect the sequences of currently circulating strains completely. In this study, the multiplex H5/7/9 rRT-PCR assay was developed with sets of primers and probe updated or newly designed to simultaneously detect the H5, H7 and H9 genes. Multiplex H5/7/9 rRT-PCR showed 100% specificity without cross-reactivity with other subtypes of AIVs and avian disease-causing viruses or bacteria, and the limit of detection was 1–10 EID50/0.1 ml (50% egg infectious dose). Artificial mixed infections with the three different subtypes could be detected accurately with high analytical sensitivity even under highly biased relative molecular ratios by balancing the reactivities of each subtype by modifying the concentration of the primers and probes. The multiplex H5/7/9 rRT-PCR assay developed in this study could be a useful tool for large-scale surveillance programs for viral detection as well as subtyping due to its high specificity, sensitivity and robustness in discriminating viruses in mixed infections, and this approach would greatly decrease the time, cost, effort and chance of cross-contamination compared to the conventional method of testing three subtypes by different singleplex rRT-PCR methods in parallel or in series.

Abstract B057: Oncogenic Foxl2 directs enhancer reprogramming and changes in 3D genome structure in ovarian granulosa cell tumors

Abstract Background: Adult-type granulosa cell tumors (aGCTs) are a rare monogenic disease. Nearly all aGCTs carry a missense point mutation in the Forkhead domain containing FOXL2 (Foxl2 p.C134W) transcription factor, but the oncogenic mechanism of this mutation is unknown. Other Forkhead family transcription factors have well-described “pioneer” activity, binding to compacted, nucleosome-bound DNA and increasing accessibility for other regulatory proteins. Objectives: To investigate if oncogenic Foxl2-C134W has pioneering activity and if mutant Foxl2 operates its oncogenic activity through enhancer reprogramming. Methods: CRISPR/Cas9 editing was used to generate isogenic granulosa cell tumor cells lacking either the FOXL2 wild-type allele (single knock-out; SKO) or both the mutant and wild-type FOXL2 alleles (double knock-out; DKO). ATAC-Seq and endogenous Foxl2 ChIP-Seq were performed on these isogenic lines to determine the differential chromatin accessibility at Foxl2-bound regulatory regions across genotypes. Promoter capture HiC was performed using a bespoke genome-wide hybrid capture probe set, CHiCAGO was used to identify significant HiC interactions, and an activity-by-contact model was constructed to identify enhancer elements bound by endogenous Foxl2-C134W and determine their target promoter contact frequency (promoter capture HiC) and enhancer activity (geometric mean of H3K27ac and ATACseq signal. Results: Endogenous ChIP-seq of Foxl2-C134W from the SKO cell line identified 1147 high-confidence peaks. De novo motif discovery identified the canonical Foxl2 binding motif (TGTTTACATT, P = 1.4 × 10−7) in 44.9% of peaks, as well as a novel variant motif (TGTTTTGTCT, P = 6.7 × 10−15) in 68.5% of peaks. Median chromatin accessibility at Foxl2-C134W peak regions, as measured by ATAC-seq, was significantly decreased in the DKO cells compared to either SKO or parental cell lines (P < 2 × 10-16 for both comparisons). Decreased chromatin accessibility at Foxl2-C134W ChIP-seq peaks in the DKO cells was driven by peaks containing the novel variant Foxl2 binding motif. Promoter capture HiC through activity-by-contact model identified promoter-enhancer interactions lost in the DKO cell line involving several genes, including ADAMTS5, FOXF2, and PRICKLE1 (adjusted P < 0.05). Conclusion: Foxl2-C134W exhibits “pioneering” activity, increasing chromatin accessibility at key gene regulatory elements with associated re-programming of promoter-enhancer interactions. The oncogenic mechanism of Foxl2-C134W in granulosa cell tumors may involve changes in DNA binding specificity, re-directing this pioneering function to sites containing a novel variant Foxl2 binding motif. Citation Format: Veena K. Vuttaradhi, Eleonora Khlebus, Thomas Welte, Barrett Lawson, Robert Tyler Hillman. Oncogenic Foxl2 directs enhancer reprogramming and changes in 3D genome structure in ovarian granulosa cell tumors [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B057.

Short noncoding RNAs as predictive biomarkers for the development from inflammatory bowel disease unclassified to Crohn's disease or ulcerative colitis.

Numerous pathogenic processes are mediated by short noncoding RNAs (sncRNA). Twenty percent of inflammatory bowel disease (IBD) patients are labelled as IBD unclassified (IBDU) at disease onset. Most IBDU patients are reclassified as Crohn's disease (CD) or ulcerative colitis (UC) within few years. Since the therapeutic methods for CD and UC differ, biomarkers that can forecast the categorization of IBDU into CD or UC are highly desired. Here, we investigated whether sncRNAs can predict CD or UC among IBDU patients. 35 IBDU patients who were initially diagnosed with IBDU were included in this retrospective investigation; of them, 12, 15, and 8 were reclassified into CD (IBDU-CD), UC (IBDU-UC), or remained as IBDU (IBDU-IBDU), respectively. Eight IBD patients, were included as references. SncRNA profiling on RNA from mucosal biopsies were performed using Affymetrix miRNA 4.0 array. Selected probe sets were validated using RT-qPCR. Among all patients and only adults, 306 and 499 probe sets respectively were differentially expressed between IBDU-CD and IBDU-UC. Six of the probe sets were evaluated by RT-qPCR, of which miR-182-5p, miR-451a and ENSG00000239080 (snoU13) together with age and sex resulted in an AUC of 78.6% (95% CI: 60-97) in discriminating IBDU-CD from IBDU-UC. Based on the three sncRNAs profile it is possible to predict if IBDU patients within 3 years will be reclassified as CD or UC. We showed that the expression profile of IBDU patients differ from that of definite CD or UC, suggesting that a subgroup of IBDU patients may compose a third unique IBD subtype.

Perioperative and Anesthetic Considerations in Vascular Rings and Slings.

Vascular rings represent an increasingly prevalent and diverse set of congenital malformations in which the aortic arch and its primary branches encircle and constrict the esophagus and trachea. Perioperative management varies significantly based on the type of lesion, its associated comorbidities, and the compromise of adjacent structures. Multiple review articles have been published describing the scope of vascular rings and relevant concerns from a surgical perspective. This review seeks to discuss the perioperative implications and recommendations of such pathology from the perspective of an anesthesia provider.

A67 REDUCING INAPPROPRIATE GAMMA-GLUTAMYL TRANSFERASE TESTING FOR INPATIENTS: A QUALITY IMPROVEMENT INITIATIVE IN LAB WASTE REDUCTION APPLYING THE MODEL FOR CONTINUOUS IMPROVEMENT

Abstract Background Review of the literature identifies a rising trend in laboratory testing, with over 30% of tests estimated to be inappropriately repeated. Laboratory overutilization increases healthcare costs, and can lead to overdiagnosis, overtreatment and negative health outcomes. Indications for repeat Gamma Glutamyl Transferase (GGT) testing in adults are limited, particularly repeat testing within the same admission. Aims Our aim was to reduce the inappropriate ordering of repeat GGT testing by 25% for all inpatients at the London Health Sciences Centre (LHSC) over a one-year study period. Methods An interprofessional team was created to help engage relevant stakeholders, collect baseline data and reassess the indications for GGT testing. A combination of root cause analysis tools, specifically the Ishikawa diagram and Pareto chart, were employed to identify potential factors contributing to the overutilization of GGT testing. After prioritizing potential solutions, intervention bundles were developed, and Plan-Do-Study-Act (PDSA) cycles were created to target correctable factors. In PDSA cycle #1, the process started by eliminating GGT as a laboratory testing option in the three most commonly used admission order care sets. Considering the hierarchy of intervention effectiveness, PDSA cycle #2 involved implementing a computerized Clinical Decision Support (CDS) system to restrict the reordering of GGT tests within 72 hours of the same admission. Results Baseline data showed that in 2022, a total of 62,542 GGT tests were ordered, with an average of approximately 5,200 GGT tests ordered per month. Of these, 16.4% were ordered through the top 3 most prevalent admission order care sets, and around 25% of all GGT tests were repeats within 72 hours of admission. Referring to Figure 1, PDSA cycle #1 yielded no significant reduction in GGT testing. PDSA cycle #2 successfully reduced the proportion of repeat GGT tests ordered by 12% within two months of implementation, leading to an estimated annualized cost savings of approximately $37,440. Conclusions Our results establish the effectiveness of CDS systems in reducing laboratory testing overutilization, suggesting their superiority to individual care set targeting interventions, and emphasize the potential for cost-effective CDS development in contemporary healthcare. Funding Agencies None

Oligo-barcode illuminates holocentric karyotype evolution in Rhynchospora (Cyperaceae).

Holocentric karyotypes are assumed to rapidly evolve through chromosome fusions and fissions due to the diffuse nature of their centromeres. Here, we took advantage of the recent availability of a chromosome-scale reference genome for Rhynchospora breviuscula, a model species of this holocentric genus, and developed the first set of oligo-based barcode probes for a holocentric plant. These probes were applied to 13 additional species of the genus, aiming to investigate the evolutionary dynamics driving the karyotype evolution in Rhynchospora. The two sets of probes were composed of 27,392 (green) and 23,968 (magenta) oligonucleotides (45-nt long), and generated 15 distinct FISH signals as a unique barcode pattern for the identification of all five chromosome pairs of the R. breviuscula karyotype. Oligo-FISH comparative analyzes revealed different types of rearrangements, such as fusions, fissions, putative inversions and translocations, as well as genomic duplications among the analyzed species. Two rounds of whole genome duplication (WGD) were demonstrated in R. pubera, but both analyzed accessions differed in the complex chain of events that gave rise to its large, structurally diploidized karyotypes with 2n = 10 or 12. Considering the phylogenetic relationships and divergence time of the species, the specificity and synteny of the probes were maintained up to species with a divergence time of ~25 My. However, karyotype divergence in more distant species hindered chromosome mapping and the inference of specific events. This barcoding system is a powerful tool to study chromosomal variations and genomic evolution in holocentric chromosomes of Rhynchospora species.

Exploratory focused pharmacogenetic testing reveals novel markers associated with risperidone pharmacokinetics in Saudi children with autism.

Background: Autism spectrum disorders (ASDs) encompass a broad range of phenotypes characterized by diverse neurological alterations. Genomic studies have revealed considerable overlap between the molecular mechanisms implicated in the etiology of ASD and genes involved in the pharmacokinetic (PK) and pharmacodynamic (PD) pathways of antipsychotic drugs employed in ASD management. Given the conflicting data originating from candidate PK or PD gene association studies in diverse ethnogeographic ASD populations, dosage individualization based on "actionable" pharmacogenetic (PGx) markers has limited application in clinical practice. Additionally, off-label use of different antipsychotics is an ongoing practice, which is justified given the shortage of approved cures, despite the lack of satisfactory evidence for its safety according to precision medicine. This exploratory study aimed to identify PGx markers predictive of risperidone (RIS) exposure in autistic Saudi children. Methods: This prospective cohort study enrolled 89 Saudi children with ASD treated with RIS-based antipsychotic therapy. Plasma levels of RIS and 9-OH-RIS were measured using a liquid chromatography-tandem mass spectrometry system. To enable focused exploratory testing, genotyping was performed with the Axiom PharmacoFocus Array, which included a collection of probe sets targeting PK/PD genes. A total of 720 PGx markers were included in the association analysis. Results: A total of 27 PGx variants were found to have a prominent impact on various RIS PK parameters; most were not located within the genes involved in the classical RIS PK pathway. Specifically, 8 markers in 7 genes were identified as the PGx markers with the strongest impact on RIS levels (p < 0.01). Four PGx variants in 3 genes were strongly associated with 9-OH-RIS levels, while 5 markers in 5 different genes explained the interindividual variability in the total active moiety. Notably, 6 CYP2D6 variants exhibited strong linkage disequilibrium; however, they significantly influenced only the metabolic ratio and had no considerable effects on the individual estimates of RIS, 9-OH-RIS, or the total active moiety. After correction for multiple testing, rs78998153 in UGT2B17 (which is highly expressed in the brain) remained the most significant PGx marker positively adjusting the metabolic ratio. For the first time, certain human leukocyte antigen (HLA) markers were found to enhance various RIS exposure parameters, which reinforces the gut-brain axis theory of ASD etiology and its suggested inflammatory impacts on drug bioavailability through modulation of the brain, gastrointestinal tract and/or hepatic expression of metabolizing enzymes and transporters. Conclusion: Our hypothesis-generating approach identified a broad spectrum of PGx markers that interactively influence RIS exposure in ASD children, which indicated the need for further validation in population PK modeling studies to define polygenic scores for antipsychotic efficacy and safety, which could facilitate personalized therapeutic decision-making in this complex neurodevelopmental condition.

Long-insert sequence capture detects high copy numbers in a defence-related beta-glucosidase gene βglu-1 with large variations in white spruce but not Norway spruce

Conifers are long-lived and slow-evolving, thus requiring effective defences against their fast-evolving insect natural enemies. The copy number variation (CNV) of two key acetophenone biosynthesis genes Ugt5/Ugt5b and βglu-1 may provide a plausible mechanism underlying the constitutively variable defence in white spruce (Picea glauca) against its primary defoliator, spruce budworm. This study develops a long-insert sequence capture probe set (Picea_hung_p1.0) for quantifying copy number of βglu-1-like, Ugt5-like genes and single-copy genes on 38 Norway spruce (Picea abies) and 40 P. glauca individuals from eight and nine provenances across Europe and North America respectively. We developed local assemblies (Piabi_c1.0 and Pigla_c.1.0), full-length transcriptomes (PIAB_v1 and PIGL_v1), and gene models to characterise the diversity of βglu-1 and Ugt5 genes. We observed very large copy numbers of βglu-1, with up to 381 copies in a single P. glauca individual. We observed among-provenance CNV of βglu-1 in P. glauca but not P. abies. Ugt5b was predominantly single-copy in both species. This study generates critical hypotheses for testing the emergence and mechanism of extreme CNV, the dosage effect on phenotype, and the varying copy number of genes with the same pathway. We demonstrate new approaches to overcome experimental challenges in genomic research in conifer defences.

SHORT NONCODING RNAS AS PREDICTIVE BIOMARKERS FOR THE DEVELOPMENT FROM INFLAMMATORY BOWEL DISEASE UNCLASSIFIED TO CROHN’S DISEASE OR ULCERATIVE COLITIS

Abstract BACKGROUND Numerous pathogenic processes are mediated by short noncoding RNAs (ncRNA). Twenty percent of inflammatory bowel disease (IBD) patients are labelled as IBD unclassified (IBDU) at disease onset. Most IBDU patients are reclassified as Crohn's disease (CD) or ulcerative colitis (UC) within few years. Since the therapeutic methods for CD and UC differ, biomarkers that can forecast the categorization of IBDU into CD or UC are highly desired. Here, we investigated whether short ncRNAs can predict CD or UC among IBDU patients. METHODS Of 35 IBDU patients initially diagnosed with IBDU, 12, 15, and eight were reclassified into CD(IBDU-CD), UC(IBDU-UC), or remained as IBDU(IBDU-IBDU) respectively. Eight IBD patients, were included as references. Short ncRNA profiling on RNA from mucosal biopsies were performed using Affymetrix miRNA 4.0 array. Selected probe sets were validated using RT-qPCR. RESULTS Among all patients and only adults, 306 and 499 probe sets respectively were differentially expressed between IBDU-CD and IBDU-UC. Six of the probe sets were evaluated by RT-qPCR, of which miR-182-5p, miR-451a and ENSG00000239080 (snoU13) together with age and sex resulted in an AUC of 78.6% (95 % CI: 60 - 97) in discriminating IBDU-CD from IBDU-UC. CONCLUSIONS Based on the three-short ncRNAs profile it is possible to predict if IBDU patients within 3 years will be reclassified as CD or UC. We showed that the expression profile of IBDU patients differ from that of definite CD or UC, suggesting that a subgroup of IBDU patients may compose a third unique IBD subtype.

A Single Set of Well-Designed Aptamer Probes for Reliable On-site Qualitative and Ultra-Sensitive Quantitative Detection.

Aptamer-based probes are pivotal components in various sensing strategies, owing to their exceptional specificity and versatile programmable structure. Nevertheless, numerous aptamer-based probes usually offer only a single function, limiting their capacity to meet the diverse requirements of multi-faceted sensing systems. Here, we introduced supersandwich DNA probes (SSW-DNA), designed and modified on the outer surface of nanochannels with hydrophobic inner walls, enabling dual functionality: qualitative detection for on-site analysis and quantitative detection for precise analysis. The fragmented DNAs resulting from the target recognition, are subsequently identified through lateral flow assays, enabling robust on-site qualitative detection of microcystin-LR with an impressively low limit of detection (LOD) at 0.01 μg/L. Meanwhile, the nanochannels enable highly sensitive quantification of microcystin-LR through the current analysis, achieving an exceptionally low LOD at 2.5×10-7 μg/L, with a broad dynamic range spanning from 1×10-6 to 1×102 μg/L. Furthermore, the process of target recognition introduces just a single potential error propagation, which reduces the overall risk of errors during the entire qualitative and quantitative detection process. This sensing strategy broadens the scope of applications for aptamer-based composite probes, holding promising implications across diverse fields, such as medical diagnosis, food safety, and environmental protection.

The Interaction and Effect of a Small MitoBlock Library as Inhibitor of ALR Protein-Protein Interaction Pathway.

MIA40 and ALR of the MIA pathway mediate the import of protein precursors that form disulfides into the mitochondrial intermembrane space. This import pathway is suggested to be a linear pathway in which MIA40 first binds to the precursor via a disulfide linkage and oxidizes it. Subsequently, ALR re-oxidizes MIA40 and then ALR transfers electrons to terminal electron acceptors. However, the precise mechanism by which ALR and MIA40 coordinate translocation is unknown. With a collection of small molecule modulators (MB-5 to MB-9 and MB-13) that inhibit ALR activity, we characterized the import mechanism in mitochondria. NMR studies show that most of the compounds bind to a similar region in ALR. Mechanistic studies with small molecules demonstrate that treatment with compound MB-6 locks the precursor in a state bound to MIA40, blocking re-oxidation of MIA40 by ALR. Thus, small molecules that target a similar region in ALR alter the dynamics of the MIA import pathway differently, resulting in a set of probes that are useful for studying the catalysis of the redox-regulated import pathway in model systems.

Predictions for the sPHENIX physics program

sPHENIX is a next-generation detector experiment at the Relativistic Heavy Ion Collider, designed for a broad set of jet and heavy-flavor probes of the Quark-Gluon Plasma created in heavy ion collisions. In anticipation of the commissioning and first data-taking of the detector in 2023, a RIKEN-BNL Research Center (RBRC) workshop was organized to collect theoretical input and identify compelling aspects of the physics program. This paper compiles theoretical predictions from the workshop participants for jet quenching, heavy flavor and quarkonia, cold QCD, and bulk physics measurements at sPHENIX.

An updated and extended version of the Melastomataceae probe set for target capture.

A probe set was previously designed to target 384 nuclear loci in the Melastomataceae family; however, when trying to use it, we encountered several practical and conceptual problems, such as the presence of sequences in reverse complement, intronic regions with stop codons, and other issues. This raised concerns regarding the use of this probe set for sequence recovery in Melastomataceae. In order to correct these issues, we cleaned the Melastomataceae probe set, extended it with additional sequences, and compared its performance with the original version. The final probe set targets 396 putative nuclear loci represented by 6009 template sequences. The probe set has been made available, along with details on the cleaning process, for reproducibility. We show that the new probe set performs better than the original version in terms of sequence recovery. This updated, extended, and cleaned probe set will improve the availability of phylogenomic resources across the Melastomataceae family. It is fully compatible with sequence recovery and extraction pipelines. The cleaning process can also be applied to any plant-targeting probe set that would need to be cleaned or updated if new genomic resources for the targeted taxa become available.

Compositae-ParaLoss-1272: A complementary sunflower-specific probe set reduces paralogs in phylogenomic analyses of complex systems.

A family-specific probe set for sunflowers, Compositae-1061, enables family-wide phylogenomic studies and investigations at lower taxonomic levels, but may lack resolution at genus to species levels, especially in groups complicated by polyploidy and hybridization. We developed a Hyb-Seq probe set, Compositae-ParaLoss-1272, that targets orthologous loci in Asteraceae. We tested its efficiency across the family by simulating target enrichment sequencing in silico. Additionally, we tested its effectiveness at lower taxonomic levels in the historically complex genus Packera. We performed Hyb-Seq with Compositae-ParaLoss-1272 for 19 Packera taxa that were previously studied using Compositae-1061. The resulting sequences from each probe set, plus a combination of both, were used to generate phylogenies, compare topologies, and assess node support. We report that Compositae-ParaLoss-1272 captured loci across all tested Asteraceae members, had less gene tree discordance, and retained longer loci than Compositae-1061. Most notably, Compositae-ParaLoss-1272 recovered substantially fewer paralogous sequences than Compositae-1061, with only ~5% of the recovered loci reporting as paralogous, compared to ~59% with Compositae-1061. Given the complexity of plant evolutionary histories, assigning orthology for phylogenomic analyses will continue to be challenging. However, we anticipate Compositae-ParaLoss-1272 will provide improved resolution and utility for studies of complex groups and lower taxonomic levels in the sunflower family.

CeGen: A Cost-Effective Probe Generation Scheme for Rule Verification in SDN

Rule verification is a prevalent way to troubleshoot network issues, which needs to generate a proper set of probes to exercise the rules. Existing probe generation schemes either determine a minimum number of probes using end-to-end probing, whose computation time is too long to handle rule dynamics, or generate and verify the rules one by one in a streaming way using per hop probing, which produces many more probes. In this letter, we propose CeGen, a cost-effective probe generation scheme that explores the possibility of generating a small number of probes leveraging per hop probing to strike a balance between the computation time and the number of probes. By modeling the network as a directed acyclic graph carrying state information, a small number of probes could be achieved by traversing the graph. We evaluate CeGen’s performance and compare with two solutions: ATPG and Monocle, on two data sets. The result shows CeGen could complete the probe generation in a short time while maintaining a small number of probes.

Technical comparison of Abbott’s UroVysion® and Biocare’s CytoFISH urine fluorescence in situ hybridization (FISH) assays

BackgroundThis study aims to compare the technical performance of Abbott’s UroVysion and Biocare’s CytoFISH urine cytology probe panel and position the CytoFISH probe panel as an alternative to UroVysion. The CytoFISH probe panel was developed based on clinically sensitive chromosomes found to be amplified in bladder cancers, as well as a locus-specific probe also seen to be amplified in bladder tumors. After extensive testing comparing CytoFISH to UroVysion, we present here our findings for the two assays.Materials and methodsA total of 216 cases representing a mix of male (ages 36–99) and female (ages 46–91) patients were assayed with both probe sets. The CytoFISH and UroVysion probe panels were tested in accordance with the UroVysion procedure, as outlined in the manufacturer’s supplied package insert with the following exception: the probe volume used was 3µL for UroVysion and 5µL for CytoFISH.ResultsThe scoring used for the CytoFISH and UroVysion assays revealed a 95% concordance, suggesting that Biocare’s CytoFISH Test has at least the same clinical sensitivity and specificity as claimed by the Abbott UroVysion Kit. We found that the CytoFISH 5p15.2 locus-specific probe was easier to score than UroVysion’s 9p21 deletion.ConclusionThe high rate of concordance between the two assays suggests that Biocare’s CytoFISH assay is a robust alternative to Abbott’s UroVysion in the diagnosis and monitoring of bladder carcinoma.

Validation of digital droplet PCR assays for cell-associated HIV-1 DNA, HIV-1 2-LTR circle, and HIV-1 unspliced RNA for clinical studies in HIV-1 cure research

BackgroundCell-associated HIV-1 DNA, HIV-1 2-LTR circle, and HIV-1 unspliced RNA (usRNA) are important virological parameters for monitoring HIV-1 persistence and activation of latent HIV-1. Assays fully validated by CLIA and/or GCLP standards are needed for future clinical trials that seek to evaluate treatments directed towards HIV-1 cure. ObjectivesTo determine performance characteristics of sensitive, moderate-throughput, digital droplet PCR (ddPCR) assays for cell-associated HIV-1 DNA, HIV-1 2-LTR circle, and HIV-1 usRNA that can detect a broad range of HIV-1 M-group subtypes. Study DesignTo evaluate linearity, limit of detection, precision, and accuracy of each assay, contrived specimens were analyzed in a background of uninfected PBMC. Detection breadth was evaluated by in silico analysis of primer and probes sets and analysis of material harvested from PBMC infected in vitro with various HIV-1 subtypes. A cohort of clinical specimens from viremic and virologically suppressed individuals was analyzed to demonstrate applicability to clinical research. ResultsThe empirically determined limit of detection of these assays was 29, 7, and 60 copies per million PBMC for HIV-1 DNA, HIV-1 2-LTR circle, and HIV-1 usRNA, respectively. The assays detect a broad range of HIV-1 M-group subtypes. Finally, analysis of clinical specimens demonstrate that these assays can detect low levels of cell-associated HIV-1 DNA, HIV-1 usRNA, and HIV-1 2-LTR circle and correlate with clinical histories and viral loads of untreated and antiretroviral treated individuals. ConclusionsWe report the clinical validation of three HIV reservoir assays with broad HIV-1 coverage for future cure studies.

A phylogeny of Cereus (Cactaceae) and the placement and description of two new species

AbstractA new study of the phylogeny of Cereus investigates generic monophyly and previous subgeneric classification. A coalescent approach was used to assess phylogenetic relationships among Cereus species based on nuclear orthologous genes captured by the Cactaceae591 probe set. The analysis included two unusual new taxa from eastern and northern Brazil to ascertain their position within the genus. Amongst the additional taxa, one was found to be early derived to the Atlantic Forest and coastal Cereus species and is described as C. ingens. Its imposing habit, considerably broader flower, yellow fruits and large seeds differentiate it from other Cereus species, and its inland agreste forest habitat in Minas Gerais and Bahia leads it to be categorized as Endangered, as little remains of this vegetation type. A second new species, C. gerardi, is described from the state of Tocantins (northern Brazil). Vegetatively this taxon is remarkable for resembling members of Pilosocereus, but has typical Cereus flowers, matching those of the type of the genus, C. hexagonus. The definition and relationships of the genus within the tribe Cereeae were made more evident, partly recovering three of the four pre‐existing subgenera.

Flowrna in the Diagnosis of EBV-Related Lymphoproliferative Disease

Introduction Epstein-Barr (EBV) viraemia is frequently encountered in the context of haemophagocytic lymphohistiocytosis (HLH), systemic inflammation and lymphoproliferative disease (LPD). Although EBV typically infects B lymphocytes triggering both polyclonal proliferation and lymphoma, EBV can also infect T and NK cells. Understanding the phenotype of EBV-infected cells can help identify the underlying diagnosis and guide the use of appropriate targeted therapy. We have developed an EBV FlowRNA assay combining the detection of EBV-encoded small RNA (EBER) with multicolour flow cytometry (Collins, Blood, 2020). Here we present a larger case series illustrating how this assay can be used in diagnostic workup. Methods Anticoagulated blood samples were received from patients with EBV viraemia and either HLH or confirmed or suspected LPD within 36 hours of collection. Mononuclear cells were isolated by density centrifugation and one to three million cells were stained with a fixable viability marker and conjugated antibodies to cell surface markers for lymphocyte immunophenotyping. FlowRNA staining was then performed as per manufacturers' instructions (PrimeFlow RNA, Thermo Fisher), using target probe sets (Thermo Fisher) for EBER 1/2 and Beta-2-microglobulin (B2M). The sample was then acquired on a LSRFortessa X-20 cytometer and subsequent analysis performed in FlowJo version 10. Results Between February 2021 and June 2023, we received 23 adequate samples from 19 patients. A definitive EBV+ population was identified in single lymphocyte subsets from 15 samples. Significant EBV-infected subsets were identified; CD19+ B cells from 4 patients, CD56+ NK cells from 6 patients, CD4+ T cells from 1 patient and CD8+ T cells from 4 patients. All patients with EBV-infected T/NK cells had an underlying lymphoma. The frequency of infected cells detected ranged from 0.017% in a case of nodal CD8+ T cell lymphoma (figure A) to 22% in a patient with relapsed extra-nodal NK T lymphoma (ENKTL) with heavy marrow infiltration and an EBV viral load of 1x10 6 copies per ml (figure B). In 5 samples no EBER+ cells were identified despite EBV DNAemia of up 2x10 6 copies per ml. Although a small number of EBER+ events could be detected in a further two samples, the very low frequency of EBV+ cells made it impossible to confirm this was a genuine population. Discussion FlowRNA can be used to give useful clinical information with a short turnaround time. In two patients with suspected relapse of ENKTL, CD56+EBER+ cells could be detected in the peripheral blood despite no lesions being visible with imaging. The diagnosis was only later confirmed by bone marrow biopsy. Importantly, FlowRNA enables the distinction between EBV positivity in circulating tumour cells from bystander B cell reactivation. In one patient with known T cell lymphoma and EBV DNAemia, the B cells were EBER+ but tumour cells were negative confirming bystander infection. Conversely, in another patient with CD8+ nodal lymphoma, EBER+ T cells were identifiable pre and post-treatment. This technique can therefore be used to monitor therapy and has the potential to identify measurable residual disease (MRD). Clonality of EBER+ B cells can be confirmed by Kappa/Lambda staining; in two cases of EBV-related HLH, EBER detection in polyclonal B lymphocytes helped confirm EBV-related lymphoproliferation rather than underlying lymphoma. The assay can be highly sensitive, detecting very small lymphocyte populations. However, to achieve this at least 100,000 lymphocytes must be analysed. Measuring sufficient cell numbers can be challenging in the context of post-treatment lymphopenia, although using up to 50 ml of blood can increase the cell yield. Additionally, this technique can only detect EBER+ cells when present in the peripheral blood. We have noted negative FlowRNA with high EBV viral load in conditions previously associated with cell-free viral DNA including ENKTL and Hodgkin lymphoma, which represented 3/5 negative samples. FlowRNA has several significant strengths, it is highly sensitive, reproducible, has a short turnaround time and by its nature as a blood test, samples can be easily obtained. The ability to monitor low-frequency tumour cells in the blood gives potential for MRD monitoring. We are now working to validate this test for routine diagnostic use.