RT-PCR Sequencing Research Articles

A Novel Quali-Quantitative Defect of VWF

BackgroundVon Willebrand Factor (VWF) is a multimeric protein largely involved in both primary and secondary hemostasis. The diagnosis and classification of von Willebrand Disease (VWD) patients can be challenging. In this poster we explore the genetic defects and their structural consequences in a VWD patient with a disproportionately high bleeding phenotype for her VWD severity. A 31-year old female, initially diagnosed with VWD type 1, presented herself with a bleeding tendency (ISTH-BAT 13) disproportionate to the severity of her VWD with antigen levels of 36%. Additional analysis showed decreased FVIII-binding at 28%. The combination of both quantitative and functional defects of VWF was an indication for further genetic analysis in order to better define the subtype of VWD.AimsGenetic and structural analysis of VWF in a patient with a disproportionally high bleeding phenotype with regard to a mild decrease in VWF antigen.MethodsRoutine laboratory analysis for VWD was performed. Genetic screening was performed by exome sequencing of hemostasis related genes. VWF mRNA analysis was carried out by RT-PCR and Sanger sequencing. The X-ray structure of furin in complex with a peptide-based inhibitor (PDB ID: 6YD7) was used as a template to construct furin-VWF (759HR(R760S)SKRS764) complex. The derived structures (furin in complex with WT/R760S-VWF) were subjected to molecular dynamics (MD) simulations (200ns) and binding free energy (BFE) calculations by using standard parameters and protocols implemented in AMBER20 program.ResultsRoutine analysis showed PFA-ADP and PFA EPI >300 seconds, VWF:ACT of 37% with a VWF:AG of 36%. Collagen binding and FVIII-binding were 46% and 28% respectively. Genetic analysis of the VWF gene disclosed 2 heterozygous variants of unknown significance (VUS): c.2771 G>A (exon 21, p.Arg924Gln) has a 1-2.5% population prevalence and has been previously described in type 1 and 2N VWD. The other VUS (c.2278 C>A; exon 17) is a novel mutation predicting a major amino acid substitution (p.Arg760Ser) in the D2-domain of VWF. Sequencing of exons 17 and 21 in the patient's VWF mRNA revealed homozygosity for the mutated allele at both mutation sites, indicating that the two variants are in cis and that the ‘normal’ allele is not expressed at mRNA level. Molecular dynamics simulations of the novel c.2278 C>A mutation (Arg760Ser) predicts a markedly decreased binding of furin to its VWF binding site, possibly decreasing or preventing VWF pro-peptide cleavage. This in turn has been shown to lead to reduced FVIII-binding of VWF.ConclusionGenetic analysis shows one polymorphism (c.2771 G>A) and one variation of unknown significance (c.2278 C>A) in the patient's VWF-gene. The polymorphism is known to be of low pathogenicity. The c. 2278 C>A mutation was not known in any of the mutation databases and is a novel VWF mutation. Both mutations were shownto be present on the same allele. As the wild-type allele was not expressed on mRNA level, all of the patient's VWF protein includes both amino acid substitutions. Modeling and molecular dynamics simulations show a markedly decreased affinity of furin to its cleavage site on the VWF protein due to the Arg760Ser substitution, likely resulting in a persistent pro-peptide binding to the mature VWF protein. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

69 Immunophenotyping of TCR and BCR clonotypes

BackgroundT-cell receptor (TCR) and B-cell receptor (BCR) repertoire profiling holds great potential for understanding disease mechanisms and for the development of new therapeutics in infectious diseases, autoimmunity and in immuno-oncology. However, this potential could be greatly improved by combining information about receptor clonotypes with immuno-phenotypes of T and B cells.MethodsTo facilitate these studies, we developed a novel technology for combined profiling of all human TCR and BCR variable regions and phenotypic characterization of immune cells in bulk and at the single-cell level in PBMC and immune cell fraction samples. The developed TCR/BCR Immunophenotyping method involves multiplex RT-PCR amplification and sequencing of CDR3 regions of TCR and BCR genes and a set of the most informative T- and B-cell phenotyping genes. Bioinformatics analysis of NGS data allows profiling of TCR/BCR clonotypes, and identification of major immune cell subtypes and their activation status.ResultsData will be presented showing how combined TCR/BCR clonotype analysis combined with targeted expression profiling of immune cells can be applied for large-scale discovery of novel cell typing and activation biomarkers in several immune-responsive model systems.ConclusionsPreliminary studies demonstrate the assay has unparalleled throughput, sensitivity, and improved cost-effectiveness for high-throughput immunity biomarker discovery applications.

Molecular epidemiology of dengue in North Kalimantan, a province with the highest incidence rates in Indonesia in 2019.

Dengue is endemic to Indonesia, a country that has largely varied geographical and demographic conditions across different regions. In 2019, dengue epidemic occurred in North Kalimantan province and recorded as the highest incidence rate in Indonesia. This study aims to investigate the molecular epidemiology of dengue during outbreak in the province and compare the epidemiological characteristics between two cities/towns in North Kalimantan, namely Malinau, an inland town surrounded by a dense rainforest, and Tarakan, an island city. A cross sectional study was conducted between September 2018 and July 2019. Dengue-like illness patients were recruited in hospitals and tested for dengue NS1 and IgG/IgM. Serological prevalence was measured using IgG ELISA, dengue virus (DENV) serotyping was conducted using RT-PCR and Envelope gene sequencing was performed to infer the virus origins and phylogeny. Clinical, demographical, and diagnostics data were also recorded and analyzed. We recruited 523 patients, 261 from Malinau and 262 from Tarakan. Among them, 349 patients were confirmed dengue. Cases in Malinau had a higher proportion of confirmed dengue (82.0%) compared to those in Tarakan (51.5%). Cases in Malinau were more likely to be dengue hemorrhagic fever with more severe hematological features compared to those in Tarakan. All four DENV serotypes were detected in both cities, the most prevalent serotype being DENV-2. The genetic characteristics of the viruses in the two towns was similar except for DENV-3. No sylvatic DENV was detected as well as alphaviruses and non-dengue flaviviruses during the outbreak. The molecular epidemiology of dengue in North Kalimantan revealed the similar virological characteristics but different clinical and demographic aspects in Malinau and Tarakan. The distinct dengue dynamics between different regions of Indonesia is prominent and this knowledge will be important for understanding future patterns of DENV transmission in the region.

Somatic hypermutation profiles in stereotyped IGHV4-34 receptors from South American chronic lymphocytic leukemia patients.

Chronic lymphocytic leukemia (CLL) is the most common mature B-cell neoplasm in the West. IGHV4-34 is one of the most frequently used genes in CLL patients, which usually display an indolent outcome. In this study, we explored the mutational profile of CLL patients expressing IGHV4-34 within different stereotypes and their association with prognostic factors and clinical outcome. A multi-institutional cohort of unselected 1444 CLL patients was analyzed by RT-PCR and bidirectional sequencing. Cytogenetics and molecular cytogenetics analyses were also performed. We identified 144 (10%) IGHV4-34 expressing cases, 119 mutated (M), 44 of them with stereotyped B-cell receptors. Subset #4 was the most frequent (56.8% of cases) followed by subsets #16 (13.6%), #29 (6.8%), and #201 (2.3%), with different distribution among countries. Analysis of somatic hypermutation profile showed significant differences among stereotyped subsets for G28>D/E, P45>S, E55>Q, and S64>I changes (p < 0.01) and high frequency of disruption of the glycosylation motif in the VH CDR2 region. All stereotyped IGHV4-34 cases showed normal karyotypes. Deletion 13q14 as a sole alteration was present in 42.8% of stereotyped cases with a different distribution among subsets. A shorter time to first treatment was found in non-stereotyped vs. stereotyped M-IGHV4-34 patients (p = 0.034). Our results add new information supporting the importance of recurrent amino acid changes at particular positions, contributing to refine the molecular characterization of South American CLL patients.

A new picorna-like virus identified in populations of the potato psyllid Bactericera cockerelli.

The potato/tomato psyllid Bactericera cockerelli (Hemiptera: Triozidae) is a pest of Solanaceae plants and a vector of the pathogenic bacterium 'Candidatus Liberibacter solanacearum', which is associated with zebra chip disease in potato. This disease is controlled through insecticide treatments, and more environmentally friendly management options are desirable. The objective of this study was to identify viruses present in potato psyllid populations that might be used as biocontrol agents for this insect pest. A new picorna-like virus, tentatively named "Bactericera cockerelli picorna-like virus" (BcPLV), was discovered in B. cockerelli populations maintained in greenhouses, through the use of high-throughput sequencing data and subsequent confirmation by RT-PCR and Sanger sequencing. BcPLV has a positive-sense 9,939-nt RNA genome encoding a single 2,947-aa polyprotein and is related to the Diaphorina citri picorna-like virus (DcPLV) found in Asian citrus psyllid Diaphorina citri populations. Based on their genome organization and the phylogeny of their RNA-dependent RNA polymerase domains, BcPLV and DcPLV together are proposed to comprise a new genus, provisionally named "Psylloidivirus", within the family Iflaviridae.

First Detection and Molecular Characterization of Apple Stem Grooving Virus, Apple Chlorotic Leaf Spot Virus, and Apple Hammerhead Viroid in Loquat in Spain.

Loquat (Eriobotrya japonica) is an important crop in Spain. To date, only one viral species, apple stem pitting virus (ASPV), has been detected in Spanish loquat orchards. In this study, the presence of additional viruses infecting this crop in Spain was investigated. RT-PCR and high-throughput sequencing (HTS) of symptomatic loquat plants led to first-time detection and characterization of apple stem grooving virus (ASGV), also known as citrus tatter leaf virus (CTLV), and apple chlorotic leaf spot virus (ACLSV) from Spain with description of nearly complete genomic sequences. The frequency of ACLSV infection was the highest, with over 30% of the samples testing positive and were also detected as coinfections with ASGV and ASPV, although most of the samples infected were symptomless. Studies on all the full-length sequences available in the databases were performed in order to establish the phylogenetic relationships of the Spanish isolates of these two viral species. Moreover, apple hammerhead viroid (AHVd) was also detected to infect loquat, the first host different from apple reported for this viroid to date.

Preliminary observations on ANGPT1 and ANGPT2 polymorphisms in systemic sclerosis: ANGPT2 rs2442598 and rs3739390 are associated with disease susceptibility and diffuse disease subtype.

Angiopoietin-1 (Ang-1) and -2 (Ang-2) concentrations were found to be associated with systemic sclerosis (SSc). We explored whether single nucleotide polymorphisms of Ang-1 (ANGPT1) and Ang-2 (ANGPT2) genes can predict SSc susceptibility in Polish Caucasian patients. Genotyping by reverse transcriptase–polymerase chain reaction and Sanger sequencing was performed in 48 patients with SSc and 38 controls. Individuals with the CC genotype of ANGPT2 rs2442598 were 3.29-fold more likely to develop SSc (odds ratio [OR], 3.288; 95% CI, 1.212–8.915; P = 0.02) compared with those carrying the CT variant. Subgroup analysis revealed that the G allele, CG, and CG+GG genotypes of ANGPT2 rs3739390 were associated with a 9-fold higher risk to develop a diffuse form of the disease compared with the C allele or CC genotype (OR, 9.00; 95% CI, 2.102–38.519; P = 0.002 and OR, 9.00; 95% CI, 1.112–72.824; P = 0.03, respectively) and patients carrying the CG variant presented with higher serum Ang-2 levels than those carrying the CC variant (P = 0.001). On the contrary, the likelihood of a diffuse disease subtype was 8.77-fold lower for the TT+AT than for the AA genotype of ANGPT1 rs2507800 (OR, 0.114; 95% CI, 0.014–0.932; P= 0.04). The C allele of ANGPT2 rs3739390 was associated with a 4.83-fold lower risk of digital ulcers (OR, 4.833; 95% CI, 1.089–21.437; P= 0.03). We concluded for the first time in the literature that the ANGPT2 rs2442598 polymorphism might represent a susceptibility locus for SSc, whereas the ANGPT2 rs3739390 and ANGPT1 rs2507800 variants may affect the disease profile.

CTCF: A novel fusion partner of ETO2 in a multiple relapsed acute myeloid leukemia patient

ETO2 is a nuclear co-repressor, which plays a critical role in the regulation of the cell cycle, self-renewal capacity, and differentiation of hematopoietic progenitor cells. We identified novel fusion transcripts involving ETO2 and CTCF by RNA-seq in a multiple relapsed AML case. The CTCF-ETO2 and ETO2-CTCF chimeric genes were validated by RT-PCR and Sanger sequencing. In addition, both transcripts apparently promoted cell proliferation via JAK/STAT3 pathway that is sensitive to STAT3 inhibitors. The novel fusions may have prognostic value and pathogenic mechanisms in acute myeloid leukemia.

Characterizing an Alternatively Spliced Variant of Chemokine Receptor 2 in Painful Diabetic Neuropathy

Prior research efforts have demonstrated a link between neuroinflammation and the progres-sion of Painful Diabetic Neuropathy (PDN), a chronic cascade of nerve damage that presents as tingling, numbness, hypersensitivity to touch, or intense pain. Current treatments are focused on pain manage-ment, serving to temporarily mask these symptoms without repressing or slowing nerve damage. The chemokine-receptor system has been closely stud-ied for its role in perpetuating neuropathic pain, alt-hough its precise mechanistic involvement remains unclear due to the network’s complexity. Because of its likely role in regulating neuropathic pain, target-ing CCR2 may be the key to effective treatment of PDN.Alternative splicing of CCR2 leads to two dis-tinct isoforms with different C-terminus sequences, CCR2A and CCR2B. The present study was intended to differentiate between these isoforms through spe-cific primer design, selection of optimized pairs, RT-PCR, and amplicon sequencing to verify the PCR products. However, the study has revealed a third, previously unreported isoform, CCR2C, due to evi-dence of alternative splicing and both the absence and insertion of parts of A and B. In the long term, we predict that the relationship between CCR2’s alternatively spliced transcript variants will lead to a distinct pattern of isoform prevalence in individuals suffering from PDN. Discerning the genetic profiles of patients with PDN and healthy individuals will clar-ify the complex mechanism driving CCR2’s intracel-lular interactions and offer more effective therapeu-tic options.

CircBAGE2 (hsa_circ_0061259) regulates CCND1 and PDCD10 expression by functioning as an miR-103a-3p ‘sponge’ to alter the proliferation and apoptosis of prostate cancer cells*

Abstract Objective The aim of the article is to explore the function of circBAGE2 (hsa_circ_0061259) in prostate cancer (PCa) cells. Methods Sequencing results of circBAGE2 were verified by quantitative RT PCR (qRT-PCR) and Sanger sequencing. Agarose gel electrophoresis was used to detect the resistance of GAPDH, BAGE2, and circBAGE2 to RNase R and their expression as cDNA and gDNAin 22RV1 cells. The biological functions of circBAGE2 were investigated by CCK8 assay and flow cytometry in 22RV1 cells transfected with siRNAs. Multiple databases were used to predict the target binding sites between circRNAs, miRNAs, and mRNAs. Western blotting was used to detect the expression of CCND1 and PDCD10. Results CircBAGE2 was significantly upregulated in PCa samples and PCa cells compared to that in matched normal tissues and normal cells, and CircBAGE2 knockdown inhibits cell proliferation and promotes apoptosis. Downregulation of circBAGE2 compromised the expression of CCND1 and PDCD10. The 3′ UTRs of CCND1 and PDCD10 were matched by miR-103a-3p, which shared binding sites with circBAGE2. Conclusion CircBAGE2 contributes to PCa progression by upregulating CCND1 and PDCD10 expression through its role as a ‘sponge’ of miR-103a-3p. CircBAGE2 may be a potential therapeutic target for PCa.

Dissection of TAF1 neuronal splicing and implications for neurodegeneration in X-linked dystonia-parkinsonism.

X-linked dystonia-parkinsonism (XDP) is a monogenic neurodegenerative disorder of the basal ganglia, which presents as a combination of hyperkinetic movements and parkinsonian features. The underlying genetic mechanism involves the insertion of a SINE-VNTR-Alu retrotransposon within the TAF1 gene. Interestingly, alterations of TAF1 have been involved in multiple neurological diseases. In XDP, the SINE-VNTR-Alu insertion in TAF1 has been proposed to result in alternative splicing defects, including the decreased incorporation of a neuron-specific microexon annotated as 34′. This mechanism has become controversial as recent studies failed to provide support. In order to resolve this conundrum, we examined the alternative splicing patterns of TAF1 mRNAs in XDP and control brains. The impact of the disease-associated SINE-VNTR-Alu on alternative splicing of microexon 34′ was further investigated in cellular assays. Subsequently, microexon 34′ incorporation was explored by RT-PCR and Nanopore long-read sequencing of TAF1 mRNAs from XDP and control brains tissues. Using cell-based splicing assays, we demonstrate that presence of the disease-associated SINE-VNTR-Alu does not affect the inclusion of microexon 34′. In addition, we show that (1) microexon 34′-containing TAF1 mRNAs are detected at similar levels in XDP as in controls and that (2) the architecture of TAF1 transcripts is remarkably similar between XDP and controls brains. These results indicate that microexon 34′ incorporation into TAF1 mRNA is not affected in XDP brains. Our findings shift the current paradigm of XDP by discounting alternative splicing of TAF1 microexon 34′ as the molecular basis for this disease.

Pneumonia Patients Caused by Co-infection With SARS-CoV-2 and Human Adenovirus in China.

Objectives: To data, no patients with obvious epidemiological relationship co-infected with SARS-CoV-2 and other pathogens have been reported. Here, we investigated 10 patients caused by co-infection with SARS-CoV-2 and human adenovirus (HAdV), resulting in third-generation transmission.Materials and Methods: From Jan 15, 2020, we enrolled 10 patients with pneumonia in Hunan Province, China. Epidemiological, clinical, and laboratory investigation results from these patients were analyzed. An epidemiological investigation was performed to assess whether patient infections were linked using conventional methods and metagenomic sequencing.Results: The presence of co-infection with SARS-CoV-2 and HAdV was determined via RT-PCR and metagenomic sequencing. Phylogenetic analysis revealed that SARS-CoV-2 and HAdV genomes clustered together, with similar genetic relationships. The first patient likely became co-infected during meetings or travel in Wuhan. The patient transmitted the virus via dinners and meetings, which resulted in four second-generation cases. Then, a second-generation case transmitted the virus to her family members or relatives via presymptomatic transmission.Conclusions: This study described an example of co-infection with SARS-CoV-2 and HAdV in pneumonia patients, which caused third-generation cases and inter-regional transmission via meetings, household interactions, and dinner parties. We also observed the persistent and presymptomatic transmission of co-infection, which has the potential to make the continued control of the COVID-19 pandemic challenging. Continuous surveillance is needed to monitor the prevalence, infectivity, transmissibility, and pathogenicity of SARS-CoV-2 co-infection with other pathogens to evaluate its real risk.

Identification and Molecular Characterization of a Novel Hordeivirus Associated With Yellow Mosaic Disease of Privet (Ligustrum vulgare) in Europe.

Wild plants serve as a large reservoir of known and yet-unknown viruses and as a source of viral pathogens of cultivated plants. Yellow mosaic disease of forest shrub Ligustrum vulgare (privet) was recurrently observed in Europe for more than 100 years. Using a universal virus identification approach based on deep sequencing and de novo assembly of viral small interfering (si)RNAs we identified a causative agent of this disease in Switzerland and reconstructed its complete 3-segmented RNA genome. Notably, a short 3′-terminal common region (CR) attached to each segment via a ∼53–71 nucleotide poly(A) tract, as determined by RT-PCR sequencing, was initially identified as an orphan siRNA contig with conserved tRNA-like secondary structure. Phylogenomic analysis classified this virus as a novel member in the genus Hordeivirus of family Virgaviridae, which we named ligustrum mosaic virus (LigMV). Similar to other hordeiviruses, LigMV formed rod-shape virions (visualized by electron microscopy), was transmitted through seeds and could also be mechanically transmitted to herbaceous hosts Chenopodium quinoa and Nicotiana benthamiana. Blot hybridization analysis identified genomic and subgenomic RNAs, sharing the 3′-CR and likely serving as monocistronic mRNAs for seven evolutionarily-conserved viral proteins including two subunits of viral RNA-dependent RNA polymerase, coat protein, triple gene block proteins mediating viral movement and cysteine-rich suppressor of RNA silencing. Analysis of size, polarity, and hotspot profiles of viral siRNAs suggested that they are produced by the plant antiviral Dicer-like (DCL) proteins DCL2 and DCL4 processing double-stranded intermediates of genomic RNA replication. Whole genome sequencing of French and Austrian isolates of LigMV revealed its genetic stability over a wide geographic range (>99% nucleotide identity to Swiss isolates and each other), suggesting its persistence and spread in Europe via seed dispersal.

Clinical Implications of a Targeted RNA-Sequencing Panel in the Detection of Gene Fusions in Solid Tumors

The detection of recurrent gene fusions can help confirm diagnoses in solid tumors, particularly when the morphology and staining are unusual or nonspecific, and can guide therapeutic decisions. Although fluorescence in situ hybridization and PCR are often used to identify fusions, the rearrangement must be suspected, with only a few prioritized probes run. It was hypothesized that the Illumina TruSight RNA Fusion Panel, which detects fusions of 507 genes and their partners, would uncover fusions with greater sensitivity than other approaches, leading to changes in diagnosis, prognosis, or therapy. Targeted RNA sequencing was performed on formalin-fixed, paraffin-embedded sarcoma and carcinoma cases in which fluorescence in situ hybridization, RT-PCR, or DNA-based sequencing was conducted during the diagnostic workup. Of the 153 cases, 138 (90%) were sequenced with adequate quality control metrics. A total of 101 of 138 (73%) cases were concordant by RNA sequencing and the prior test method. RNA sequencing identified an additional 30 cases (22%) with fusions that were not detected by conventional methods. In seven cases (5%), the additional fusion information provided by RNA sequencing would have altered diagnosis and management. A total of 19 novel fusion pairs (not previously described in the literature) were discovered (14%). Overall, the findings show that a targeted RNA-sequencing method can detect gene fusions in formalin-fixed, paraffin-embedded specimens with high sensitivity.

Cloning and expression of circular transcript of mouse growth hormone receptor gene.

Based on reports in the literature and search results on the circBase database, 8 circular transcripts of the mouse growth hormone receptor (GHR) gene were identified. In order to confirm the existence of the circular transcripts of the GHR gene (circGHRs) and to explore their expression patterns, the Kunming mouse (Mus musculus) was used as a research animal. This study detected the existence of circGHRs by RT-PCR amplification and sequencing, one of which was selected as circGHR for detailed analysis. The circular structure of circGHR was confirmed by RNase R treatment and reverse transcription. The spatiotemporal expression of circGHR and GHR mRNA was analyzed by qRT-PCR. The results showed that the full length of mouse circGHR was 820 nt, which was formed by circularization of exons 2-8 of the transcript of the GHR gene. RNase R tolerance analysis shows that mouse circGHR has the general characteristics of circular molecules and is not easily degraded by RNase R. Compared with oligo-d(T)18 primers, random primers have higher reverse transcription efficiency for circGHR, which further shows that circGHR is a poly(A)-free cyclic structure molecule. Tissue expression profile results show that circGHR is highly expressed in the liver and kidney of 1 week-old and 7-week old Kunming mice, but is low in pectoral muscles and leg muscles. The time-series expression profile of circGHR does not show any significant difference between the liver and pectoral muscle tissue. The circGHR expression in the leg muscle was low before 5 weeks of age but increased after 7 weeks of age. This study confirmed the existence of a circular transcript circGHR of the mouse GHR gene, and initially revealed the expression pattern of circGHR. The results of the study laid a foundation for in-depth developmental studies on the biological functions of the mouse circGHR and its mechanism of action regarding the growth and development of mice.

MRNA analysis identifies deep intronic variants causing Alport syndrome and overcomes the problem of negative results of exome sequencing

Mutations in COL4A3, COL4A4 and COL4A5 genes lead to Alport syndrome (AS). However, pathogenic variants in some AS patients are not detected by exome sequencing. The aim of this study was to identify the underlying genetic causes of five unrelated AS probands with negative next-generation sequencing (NGS) test results. Urine COL4A3–5 mRNAs were analyzed in the probands with an uncertain inherited mode of AS, and COL4A5 mRNA of skin fibroblasts was analyzed in the probands with X-linked AS. RT-PCR and direct sequencing were performed to detect mRNA abnormalities. PCR and direct sequencing were used to analyze the exons with flanking intronic sequences corresponding to mRNA abnormalities. Six novel deep intronic splicing variants in COL4A4 and COL4A5 genes that cannot be captured by exome sequencing were identified in the four AS probands. Skipping of an exon was caused by an intronic variant, and retention of an intron fragment caused by five variants. In the remaining AS proband, COL4A5 variants c.2677 + 646 C > T and r.2678_r.2767del were detected at the DNA and RNA level, respectively, whereas it is unclear whether c.2677 + 646 C > T may not lead to r.2678_r.2767del. Our results reveal that mRNA analysis for AS genes from either urine or skin fibroblasts can resolve genetic diagnosis in AS patients with negative NGS results. We recommend analyzing COL4A3–5 mRNA from urine as the first choice for these patients because it is feasible and non-invasive.

Abstract P445: A Combinatorial Approach Comprehensively Improves The Maturation Of Human Induced Pluripotent Stem Cell Derived Atrial Cardiomyocytes

Introduction: The limited success of pharmacological approaches to atrial fibrillation ( AF ) is due to limitations of in vitro and in vivo models and inaccessibility of human atrial tissue. Patient-specific induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs) are a robust platform to model the heterogeneous myocardial substrate of AF, but their immaturity limits their fidelity. Objective: We hypothesized that a combinatorial approach of biochemical (triiodothyronine [ T3 ], insulin-like growth factor-1 [ IGF-1 ], and dexamethasone; collectively TID ), bioenergetic (fatty acids [ FA ]), and electrical stimulation ( ES ) will enhance electrophysiological ( EP ), structural, and metabolic maturity of iPSC- a CMs. Methods: We assessed maturation with whole cell patch clamping, calcium transients, immunofluorescence (IF), Seahorse Analyzer, contractility assay, RT-PCR, Western Blotting, and RNA sequencing (RNAseq). Using a time series with RNAseq we identified signaling pathways and transcriptional regulation that drive EP, structural, and metabolic atrial development and compared iPSC-aCM maturity with human aCMs (haCMs) obtained from the same patient. Results: TID+FA+ES significantly improved structural organization and cell morphology ( Fig. 1a ), enhanced membrane potential stability and improved depolarization ( Fig. 1b ), improved Ca 2+ kinetics with faster and increased Ca 2+ release from sarcoplasmic reticulum ( Fig. 1c ), and increased expression of Na + , Ca 2+ , and K + channels, markers of structural maturity, FA metabolism, and oxidative phosphorylation ( Fig. 1d ). There was no difference in each parameter between TID+FA+ES iPSC-aCMs and haCMs from the same patient. Conclusion: Our optimized, combinatorial TID+FA+ES approach markedly enhanced EP, structural, and metabolic maturity of human iPSC-aCMs, which will be useful for elucidating the genetic basis of AF developing precision drug therapies.

Reverse transcription PCR-based detection of matrix and hemagglutinin-neuraminidase genes among Avian orthoavulavirus 1 clinical isolates in the Philippines, 1991-2017

Newcastle disease (ND) is a highly infectious disease that affects devastatingly the avian population worldwide. It is caused by Avian orthoavulavirus 1 (AOAV-1), or better known as Newcastle disease virus belonging to phylum Negarnaviricota, class Monjiviricetes, order Mononegavirales and family Paramyxoviridae. This virus consists of six principal structural proteins namely: the nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN) and the large protein RNA dependent RNA polymerase (L).. The present study aimed to molecularly detect the M and HN gene segments of the AOAV-1 field isolates from clinical cases in the Philippines from 1991 through 2017. RT-PCR amplification and sequence analyses using primers NDV-For4359 and NDV-Rev4788 which anneal to the matrix gene and primers NDV-For6369 and NDV-Rev6598 targeting the HN genes, identified all isolates to be AOAV-1. Determining the different genes of the virus would greatly help scientists and researchers to accurately identify the viral isolates in order to improve epidemiological studies and surveillance of the disease in the country.

Human astrovirus types 1, 4 and 5 circulating among children with acute gastroenteritis in a rural Brazilian state, 2010-2016.

This study combined conventional epidemiology of human astroviruses. From 2010 to 2016, 232 stool samples from children under 5 years of age were screened using NGS and conventional RT-PCR followed by genetic analysis in order to investigate the genotypic diversity of classical human astrovirus (HAstV) circulating in Tocantins State, Brazil. HAstV was detected in 16 cases (6.9%). Seven specimens (43.7%; 7/16) were positive according RT-PCR and next-generation sequencing (NGS) to investigate the molecular to both NGS and RT-PCR. NGS and RT-PCR individually revealed six (37.5%; 6/16) and three (18.8%; 3/16) additional positive samples, respectively. Sequencing of the HAstV-positive samples revealed HAstV-1a (9/16), HAstV-4c (3/16), and HAstV-5c (4/16) lineages.

The bile acid-activated retinoic acid response in dendritic cells is involved in food allergen sensitization.

Alteration of commensal microbiota is highly correlated with the prevalence of allergic reactions to food in the gastrointestinal tract. The mechanisms by which microbiota modulate food allergen sensitization in the mucosal site are not fully understood. We generate DCs specific knockout of retinoic acid receptor α (Rara) gene mice (DC KO Rara) to evaluate food sensitization. The bile acid-activated retinoic acid response was evaluated by flow cytometry, real-time RT-PCR and Illumina transcriptome sequencing. The global effect of Abx treatment on BA profiles in the mucosal lymph tissue mLN in mice was examined by UPLC-MS analysis. In this study, we demonstrate that depletion of commensal gut bacteria leads to enhanced retinoic acid (RA) signaling in mucosal dendritic cells (DCs). RA signaling in DCs is required for the production of food allergen-specific IgE and IgG1. Antibiotics induced an enlarged bile acid (BA) pool, and dysregulated BA profiles contributed to enhanced RA signaling in mucosal DCs. BA-activated RA signaling promoted DC upregulation of interferon I signature, RA signature, OX40L, and PDL2, which may lead to T helper 2 differentiation of CD4+ T cells. BA-activated RA signaling involved the farnesoid X receptor and RA receptor α (RARa) interaction. Depletion of bile acid reduces food allergen specific IgE and IgG1 levels in mice. Our research unveils a mechanism of food sensitization modulated by BA-RA signaling in DCs, which suggests a potential new approach for the intervention of food allergic reactions.