Alpha Sequences Research Articles

First Report of Leaf Spot on Hydrangea macrophylla Caused by Boeremia exigua in Korea.

Hydragea (Hydrangea macrophylla) is a garden plant commonly used in parks and landscapes globally. In late May 2023, brown necrotic spots surrounded by chlorotic halos were observed on the leaves of H. macrophylla (cv. Renate Steiniger) at the Suncheonman National Garden in Suncheon (34°55'45.6" N 127°30'26.5" E), South Korea. The affected area covered approximately 20 m2, with the disease incidence ranging from 2% to 5%. Concentric rings with light brown centers measuring 0.5 to 6 cm were observed on the upper sides of the leaves. To isolate the pathogens, each infected leaf from three different plants was cut into 5 × 5 mm pieces, surface-sterilized with 70% ethanol for 1 min, and rinsed three times with sterile distilled water. The samples were cultured on 1.5% water agar amended with 100 μg/mL of streptomycin in the dark at 25 °C. Single-spore isolation from the grown hyphae was performed on the obtained isolates (SYP-1229 to 1231). The fungal colony on potato dextrose agar was olivaceous gray with floccose aerial mycelia, and black pycnidia developed on the medium after 14 days. The conidia observed were hyaline, cylindrical to ellipsoidal-oblong, and aseptate, measuring 4.9 to 9.1 μm × 1.9 to 4.2 μm (avg. 6.3 × 2.7 μm, n = 100) (length × width). The isolates were identified as Boeremia sp. based on their morphological characteristics (Boerema, 1976; Boerema, 2004). For molecular identification, the internal transcribed spacer (GenBank accession nos.: OR682193-OR682195), actin (OR689851- OR689853), β-tubulin (OR689854- OR689856) and translation elongation factor-1 alpha (OR689857- OR689859) sequences from the isolates SYP-1229 to 1231 exhibited 100% (558/558 bp), 100% (263/263), 100% (314/314), and 100% (316/316) similarity, respectively, with Boeremia exigua (EU167567, EU880846, OP611549, and KY484684, respectively) in GenBank BLAST search. Phylogenetics based on concatenated ITS, ACT, TUB2, and TEF sequences, using the maximum likelihood method of MEGA X (Kumar et al., 2018), showed that sequences of SYP-1229 to 1231 were clustered in the same clade as B. exigua. For pathogenicity tests conducted in pots, 10 mL conidial suspension (1 × 106 conidia/mL) derived from isolate SYP-1229, which had been cultured on PDA for 14 days, was sprayed onto two leaves of each of three independent healthy 1-year-old hydrangeas (cv. Renate Steiniger). The leaves of the control plants were inoculated with sterile distilled water, which was also applied to two leaves of each of the three independent healthy plants. The inoculated plants were placed in clear plastic boxes to maintain high humidity and incubated at 25 °C under 18 h of light. Brown spot lesions were observed on the inoculated two leaves seven days after inoculation, whereas the control were asymptomatic. Similar pathogenicity results were obtained for all three independent biological replicates. The fungus isolated from the lesion was identified as B. exigua through morphological characterization and ITS sequencing, thus fulfilling Koch's postulates. To the best of our knowledge, this is the first report of B. exigua causing brown-spot disease on H. macrophylla in South Korea. The pathogen has been previously reported H. macrophylla in Italy and the United States (Farr and Rossman 2017; Garibaldi et al. 2006), and H. paniculata in Italy (Garibaldi et al. 2018). This study will help in future detection and control to enhance the ornamental value of hydrangeas in Korea.

Morphological and phylogenetic analyses reveal four species of myxomycetes new to China.

Four species of myxomycetes (Arcyria pseudodenudata, Diderma europaeum, Lycogala irregulare, and Trichia armillata) new to China were observed via light microscope and scanning electron microscope, and detailed descriptions and illustrations are provided, along with comparisons with related species. Among them, A. pseudodenudata was discovered for the first time outside of the type locality, D. europaeum was discovered for the first time outside of Europe, and L. irregulare and T. armillata were reported again after being named. Phylogenetic analyses based on nuclear 18S rDNA and elongation factor-1 alpha sequences or nuclear 18S rDNA and cytochrome oxidase subunit I sequences was performed to provide a molecular basis for morphological identification. These specimens were deposited in the Herbarium of Fungi of Nanjing Normal University.

Characterization of Fusarium solani Associated with Tobacco (Nicotiana tabacum) Root Rot in Henan, China.

The aim of this study was to characterize the Fusarium solani species complex (FSSC) population obtained from tobacco roots with root rot symptoms by morphological characteristics, molecular tests, and assessment of pathogenicity. Cultures isolated from roots were white to cream with sparse mycelium on potato dextrose agar, with colony growth of 21.5 ± 0.5 to 29.5 ± 0.5 mm after 3 days. Sporodochia were cream on carnation leaf agar (CLA) and Spezieller Nährstoffarmer agar (SNA), and macroconidia formed in sporodochia were 3 to 6 septate and straight to slightly curved, with wide central cells, a slightly short blunt apical cell, and a straight to almost cylindrical basal cell with a distinct foot shape, ranging in size from 20.92 to 64.37 × 3.91 to 6.57 μm. Microconidia formed on CLA were reniform and fusiform, with 0 or 1 to occasionally 2 septa, that formed on long monophialidic conidiogenous cells, with a size range of 5.99 to 32.32 × 1.76 to 5.84 μm. Globose to oval chlamydospores were smooth- to rough-walled, 6.5 to 13.3 ± 0.37 μm in diameter, and terminal or intercalary and occurred singly, in pairs, or occasionally in short chains on SNA. Molecular tests consisted of sequencing and phylogenetic analysis of the translation elongation factor-1 alpha (EF-1α), RNA polymerase II largest subunit, and second largest subunit regions. All the obtained sequences revealed 98.14 to 100% identity to F. solani in both Fusarium ID and Fusarium MLST databases. Phylogenetic trees of the EF-1α gene and concatenated three-locus data showed that isolates from tobacco in Henan grouped in the proposed group 5, which is nested within FSSC clade 3 (FSSC 5). Twenty-seven of the 28 isolates caused root rot in artificially inoculated tobacco seedlings, with a disease severity index ranging from 15.00 ± 1.67 to 91.11 ± 2.22. Cross-pathogenicity tests showed that three representative isolates were virulent to six species of Solanaceae and two species of Poaceae, with disease severity indexes ranging from 6.12 ± 0.56 to 84.44 ± 0.00, indicating that these isolates have a wide host range. The results may inform the control of tobacco root rot through improved crop rotations.

Development of overcurrent relay based on wavelet transform for fault detection in transmission line

This study proposes a protection relay using a microcontroller to detect and classify faults in transmission lines based on the wavelet transform. An experimental model was constructed from an actual 115 kV transmission system prototype. The current signal was observed based on the fault type, phase, and position. Clark’s transform and the discrete wavelet transform (DWT) were applied to transform signals for analysis. Moreover, the performance of fault detection based on the output signals of Clark’s transform (alpha sequence, beta sequence, and zero sequence current) was compared to the performance of the alternative proposed fault detection method, which is based on the combining factor between alpha and beta sequence current. In addition, the influence of DWT level on fault analysis is also considered and is used to confirm the accuracy of fault detection. Results show that the proposed method is efficient for fault detection and classification. This finding allows the researcher to choose the appropriate analytical method. Moreover, it can also be used as the basis for overcurrent relay algorithm design in the effort to develop more advanced technologies.

First report of Fusarium ussurianum causing leaf spot on Hemerocallis citrina in Sichuan Basin of China.

Hemerocallis citrina is a popular vegetable crop in China, due to abundant nutrients in its edible flower buds. In March 2021, serious symptoms of leaf spot were observed on nearly 90% cultivated H. citrina seedlings in the fields of Dazhou city (31°17'56″ N, 107°31'59″ E), Sichuan, China. Symptomatic leaves were collected from 15 seedlings in five different sampling sites (3 seedlings per site). Small pieces (5 × 3 mm) of lesion margin were excised, surface disinfected in 70% ethanol for 20 s and 1% sodium hypochlorite (NaClO) for 40 s, washed, dried, placed on potato dextrose agar (PDA) amended with streptomycin sulfate (50 mg/L) and incubated in dark at 25 ℃ for two days. Finally, eight purified isolates, HHC-FL22, HHC-FL23, HHC-FL25, HHC-FL26, HHC-FL27, HHC-FL28, HHC-FL29 and HHC-FL30, showing similar morphology were obtained through transferring hyphal tips to fresh PDA plates. On PDA plates, mycelia were initially white but gradually became light yellow, and scarlet diffusible pigments were also produced with time. On carnation leaf agar, our isolates produced slightly curved macroconidia with 4 to 8 septa that measured 3.1 to 5.7 × 36.8 to 69.3 µm (n = 30). Microconidia and chlamydospores were not observed. Our isolates were initially identified as Fusarium species based on morphological features (Leslie and Summerell 2006). To further confirm accurate identity, primers EF1/EF2 (O'Donnell et al. 2010), TRI1015B/TRI1013E (Hao et al. 2017), RPB1-F5/RPB1-G2R (O'Donnell et al. 2010), and fRPB2-5F/fRPB2-11aR and RPB2-5f2/RPB2-7cr (O'Donnell et al. 2012) were used to amplify gene sequences of translation elongation factor-1 alpha (TEF1), 3-O-acetyltransferase (Tri101), and DNA-directed RNA polymerase II largest (RPB1) and second largest subunit (RPB2), respectively. Our sequences were deposited in GenBank under accession numbers OQ860946 to OQ860953 (TEF1), OR393245 to OR393252 (Tri101), OP131893 to OP131900 (RPB1), and OQ860954 to OQ860961 and OP131885 to OP131892 (RPB2), respectively. BLASTN searches of our sequences showed 99 ~ 100% identity with TEF1 (FJ240301.1), Tri101 (FJ240345.1), RPB1 (MW233297.1) and RPB2 (KM361666.1) of F. ussurianum NRRL 45681, and 99.05 ~ 100% identity with TEF1 (FJ240305.1) and Tri101 (FJ240349.1) of F. ussurianum NRRL 45833, respectively. Two independent maximum-likelihood phylogenetic trees based on different combined datasets of TEF1, Tri101, RPB1 and RPB2 of Fusarium species confirmed that our isolates were F. ussurianum. To test pathogenicity, conidial suspension from HHC-FL23 (106 conidia / mL) were sprayed to seedlings of cultivar "chuanhuanghua No.1" (n = 3) and incubated in a greenhouse (25°C under 90% relative humidity, 16/8 h light/dark cycle). Controls were treated with ddH2O. Ten days post-inoculation, natural symptoms appeared on leaves inoculated with HHC-FL23, but control group seedlings remained disease-free. This experiment was repeated three times. All re-isolated pathogens from diseased leaves were molecularly and morphologically identified using methods described above. Consequently, the re-isolated fungi were identical to these inoculated. The leaf spot disease could cause foliar damage and even drastic yield loss of flower buds under severe conditions. To our knowledge, this is the first report of F. ussurianum causing leaf spot in H. citrina worldwide. Our study will assist in monitoring causal agent diversity of leaf spot and breeding new resistant varieties in H. citrina.

High-throughput RNA sequencing of the T cell receptor alpha and beta chains for simultaneous clonality and biological analyses in Sezary syndrome.

Previous investigations pointed out a role for antigen stimulation in Sezary syndrome (SS). High-throughput sequencing of the T cell receptor (TR) offers several applications beyond diagnostic purposes, including the study of T cell pathogenesis. We performed high-throughput RNA sequencing of the TR alpha (TRA) and beta (TRB) genes focusing on the complementarity-determining region 3 (CDR3) in 11 SS and one erythrodermic mycosis fungoides (MF) patients. Five psoriasis patients were employed as controls. Peripheral blood CD4+ cells were isolated and RNA sequenced (HiSeq2500). High-resolution HLA typing was performed in neoplastic patients. Highly expanded predominant TRA and TRB CDR3 were only found in SS patients (median frequency: 94.4% and 93.7%). No remarkable CDR3 expansions were observed in psoriasis patients (median frequency of predominant TRA and TRB CDR3: 0.87% and 0.69%, p < 0.001 compared to SS). CDR3 almost identical to the predominant were identified within each SS patient and were exponentially correlated with frequencies of the predominant CDR3 (R2 = 0.918, p < 0.001). Forty-six different CDR3 were shared between SS patients displaying HLA similarities, including predominant TRA and TRB CDR3 in one patient that were found in other three patients. Additionally, 351 antigen matches were detected (Cytomegalovirus, Epstein-Barr, Influenza virus, and self-antigens), and the predominant CDR3 of two different SS patients matched CDR3 with specificity for Influenza and Epstein-Barr viruses. Besides detecting clonality, these findings shed light on the nature of SS-related antigens, pointing to RNA sequencing as a useful tool for simultaneous clonality and biological analysis in SS.

Using combined single-cell gene expression, TCR sequencing and cell surface protein barcoding to characterize and track CD4+ T cell clones from murine tissues.

Single-cell gene expression analysis using sequencing (scRNA-seq) has gained increased attention in the past decades for studying cellular transcriptional programs and their heterogeneity in an unbiased manner, and novel protocols allow the simultaneous measurement of gene expression, T-cell receptor clonality and cell surface protein expression. In this article, we describe the methods to isolate scRNA/TCR-seq-compatible CD4+ T cells from murine tissues, such as skin, spleen, and lymph nodes. We describe the processing of cells and quality control parameters during library preparation, protocols for multiplexing of samples, and strategies for sequencing. Moreover, we describe a step-by-step bioinformatic analysis pipeline from sequencing data generated using these protocols. This includes quality control, preprocessing of sequencing data and demultiplexing of individual samples. We perform quantification of gene expression and extraction of T-cell receptor alpha and beta chain sequences, followed by quality control and doublet detection, and methods for harmonization and integration of datasets. Next, we describe the identification of highly variable genes and dimensionality reduction, clustering and pseudotemporal ordering of data, and we demonstrate how to visualize the results with interactive and reproducible dashboards. We will combine different analytic R-based frameworks such as Bioconductor and Seurat, illustrating how these can be interoperable to optimally analyze scRNA/TCR-seq data of CD4+ T cells from murine tissues.

Barcoding intracellular reverse transcription enables high-throughput phenotype-coupled T cell receptor analyses

Assays linking cellular phenotypes with Tcell or B cell antigen receptor sequences are crucial for characterizing adaptive immune responses. Existing methodologies are limited by low sample throughput and high cost. Here, we present INtraCEllular Reverse Transcription with Sorting and sequencing (INCERTS), an approach that combines molecular indexing of receptor repertoires within intact cells and fluorescence-activated cell sorting (FACS). We demonstrate that INCERTS enables efficient processing of millions of cells from pooled human peripheral blood mononuclear cell (PBMC) samples while retaining robust association between Tcell receptor (TCR) sequences and cellular phenotypes. We used INCERTS to discover antigen-specific TCRs from patients with cancer immunized with a novel mutant KRAS peptide vaccine. After exvivo stimulation, 28 uniquely barcoded samples were pooled prior to FACS into peptide-reactive and non-reactive CD4+ and CD8+ populations. Combining complementary patient-matched single-cell RNA sequencing (scRNA-seq) data enabled retrieval of full-length, paired TCR alpha and beta chain sequences for future validation of therapeutic utility.

Structure, microstructure and disorder in low temperature chemical vapor deposited SiC coatings

Chemical vapor deposited SiC coatings were investigated at different scales by X-Ray diffraction, Raman microspectroscopy and transmission electron microscopy. They were prepared under specific conditions explaining the various (micro)structures obtained. The deposits all have a columnar morphology with a preferential orientation and a faulted cubic structure. They differ in how disorder is incorporated in the structure. Fine XRD analyses and stacking fault density assessment by TEM revealed the one-dimensionally-disordered (ODD) polytype in the <111> textured coatings. The frequency and spatial distribution of stacking faults vary and sometimes locally generate periodic alpha sequences. A specific type of disorder was also identified where {111} planes are arranged parallel to the growth direction within the columns. These disorders, more energetic than stacking faults, induce multiple and particularly large Raman modes. Crystal distortions, such as dislocations, are localized at the ODD domain boundaries, which are frequently interrupted as they extend during the growth.

Characterization of a Novel Gja8 (Cx50) Mutation in a New Cataract Rat Model.

To describe a novel spontaneous cataract inbred strain isolated from large-scale breeding SD rats, identify the responsible gene mutation, and understand how this mutation affects lens function. Exome sequencing of 12 cataract-associated genes was performed in the affected and healthy relatives. Sequences of rat wild-type or mutant gap junction protein alpha 8 gene (Gja8) were transfected into cells. The expression level of protein was assayed by Western blot analysis. Subcellular localization of connexin 50 (Cx50) was analyzed in confocal fluorescent images. Wound-healing, 5-ethynyl-2'-deoxyuridine incorporation, and attachment assay were performed to characterize the cell migration, proliferation and adhesion. The abnormality was found to be inheritable in an autosomal semi-dominant pattern through different mating patterns. We found a G to T transversion at codon 655 in Gja8, leading to a substitution of valine by phenylalanine (p.V219F). Gja8V219F/+ heterozygotes expressed nuclear cataract while Gja8V219F/V219F homozygotes manifested microphthalmia in addition to cataract. Histology revealed fiber disorders and loss of organelle-free zone in the mutant lens. Cx50V219F altered its location in HeLa cells and inhibited the proliferation, migration and adhesion abilities of HLEB3 cells. The mutation also reduced the expression of focal adhesion kinase and its phosphorylation. The c.655G>T mutation (p.V219F) is a novel mutation in Gja8, inducing semi-dominant nuclear cataracts in a new spontaneous cataract rat model. The p.V219F mutation altered Cx50 distribution, inhibited lens epithelial cell proliferation, migration, and adhesion, and disrupted fiber cell differentiation. As a consequence, the nuclear cataract and small lens formed.

Using machine learning to detect coronaviruses potentially infectious to humans

Establishing the host range for novel viruses remains a challenge. Here, we address the challenge of identifying non-human animal coronaviruses that may infect humans by creating an artificial neural network model that learns from spike protein sequences of alpha and beta coronaviruses and their binding annotation to their host receptor. The proposed method produces a human-Binding Potential (h-BiP) score that distinguishes, with high accuracy, the binding potential among coronaviruses. Three viruses, previously unknown to bind human receptors, were identified: Bat coronavirus BtCoV/133/2005 and Pipistrellus abramus bat coronavirus HKU5-related (both MERS related viruses), and Rhinolophus affinis coronavirus isolate LYRa3 (a SARS related virus). We further analyze the binding properties of BtCoV/133/2005 and LYRa3 using molecular dynamics. To test whether this model can be used for surveillance of novel coronaviruses, we re-trained the model on a set that excludes SARS-CoV-2 and all viral sequences released after the SARS-CoV-2 was published. The results predict the binding of SARS-CoV-2 with a human receptor, indicating that machine learning methods are an excellent tool for the prediction of host expansion events.

Coordinated evolution at amino acid sites of SARS-CoV-2 spike.

SARS-CoV-2 has adapted in a stepwise manner, with multiple beneficial mutations accumulating in a rapid succession at origins of VOCs, and the reasons for this are unclear. Here, we searched for coordinated evolution of amino acid sites in the spike protein of SARS-CoV-2. Specifically, we searched for concordantly evolving site pairs (CSPs) for which changes at one site were rapidly followed by changes at the other site in the same lineage. We detected 46 sites which formed 45 CSP. Sites in CSP were closer to each other in the protein structure than random pairs, indicating that concordant evolution has a functional basis. Notably, site pairs carrying lineage defining mutations of the four VOCs that circulated before May 2021 are enriched in CSPs. For the Alpha VOC, the enrichment is detected even if Alpha sequences are removed from analysis, indicating that VOC origin could have been facilitated by positive epistasis. Additionally, we detected nine discordantly evolving pairs of sites where mutations at one site unexpectedly rarely occurred on the background of a specific allele at another site, for example on the background of wild-type D at site 614 (four pairs) or derived Y at site 501 (three pairs). Our findings hint that positive epistasis between accumulating mutations could have delayed the assembly of advantageous combinations of mutations comprising at least some of the VOCs.

Development of an ELISA Assay for the Determination of SARS-CoV-2 Protein Subunit Vaccine Antigen Content.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein subunit vaccine is one of the mainstream technology platforms for the development of COVID-19 vaccines, and most R&D units use the receptor-binding domain (RBD) or spike (S) protein as the main target antigen. The complexity of vaccine design, sequence, and expression systems makes it urgent to establish common antigen assays to facilitate vaccine development. In this study, we report the development of a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) to determine the antigen content of SARS-CoV-2 protein subunit vaccines based on the United States Pharmacopeia <1220> and ICH (international conference on harmonization) Q14 and Q2 (R2) requirements. A monoclonal antibody (mAb), 20D8, was identified as the detection antibody based on its high RBD binding activity (EC50 = 8.4 ng/mL), broad-spectrum anti-variant neutralizing activity (EC50: 2.7−9.8 ng/mL for pseudovirus and EC50: 9.6−127 ng/mL for authentic virus), good in vivo protection, and a recognized linear RBD epitope (369−379 aa). A porcine anti-RBD polyclonal antibody was selected as the coating antibody. Assay performance met the requirements of the analytical target profile with an accuracy and precision of ≥90% and adequate specificity. Within the specification range of 70−143%, the method capability index was >0.96; the misjudgment probability was <0.39%. The method successfully detected SARS-CoV-2 protein subunit vaccine antigens (RBD or S protein sequences in Alpha, Beta, Gamma, or Delta variants) obtained from five different manufacturers. Thus, we present a new robust, reliable, and general method for measuring the antigenic content of SARS-CoV-2 protein subunit vaccines. In addition to currently marketed and emergency vaccines, it is suitable for vaccines in development containing antigens derived from pre-Omicron mutant strains.

Surveillance, Diversity and Vegetative Compatibility Groups of Fusarium oxysporum f. sp. vasinfectum Collected in Cotton Fields in Australia (2017 to 2022)

Cotton (Gossypium hirsutum) is a billion-dollar crop in regional New South Wales (NSW) and Queensland, Australia. Fusarium wilt (FW) caused by Fusarium oxysporum f. sp. vasinfectum (Fov) is an economically important disease. Initial disease losses of up to 90% when the disease was first detected resulted in fields being taken out of cotton production. The disease is now well-managed due to the adoption of highly resistant varieties. However, annual disease surveys recently revealed that the disease dynamic has changed in the past few seasons. With relatively mild and wet weather conditions during the 2021/22 growing season, FW was detected in eight surveyed valleys in NSW and Queensland, with the disease incidence as high as 44.5% and 98.5% in individual fields in early and late seasons, respectively. Fov is genetically distinct and evolved from local Fusarium oxysporum strains. Additionally, the pathogen was reported to evolve rapidly under continuous cotton cropping pressure. However, our knowledge of the genetic composition of the prevailing population is limited. Sequences of the translation elongation factor alpha 1 (TEF1) revealed that 94% of Fusarium isolates recovered from FW-infected cotton were clustered together with known Australian Fov and relatively distant related to overseas Fov races. All these isolates, except for nine, were further confirmed positive with a specific marker based on the Secreted in Xylem 6 (SIX6) effector gene. Vegetative compatibility group (VCG) analyses of 166 arbitrarily selected isolates revealed a predominance of VCG01111. There was only one detection of VCG01112 in the Border Rivers valley where it was first described. In this study, the exotic Californian Fov race 4 strain was not detected using a specific marker based on the unique Tfo1 insertion in the phosphate (PHO) gene. This study indicated that the prevalence and abundance of Fov across NSW and Queensland in the past five seasons was probably independent of its genetic diversity.

Relative infectivity of the SARS-CoV-2 Omicron variant in human alveolar cells.

With the continuous emergence of highly transmissible SARS-CoV-2 variants, the comparison of their infectivity has become a critical issue for public health. However, a direct assessment of the viral characteristic has been challenging because of the lack of appropriate experimental models and efficient methods. Here, we integrated human alveolar organoids and single-cell transcriptome sequencing to facilitate the evaluation. In a proof-of-concept study with four highly transmissible SARS-CoV-2 variants, including GR (B.1.1.119), Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (BA.1), a rapid evaluation of the relative infectivity was possible. Our system demonstrates that the Omicron variant is 5- to 7-fold more infectious to human alveolar cells than the other SARS-CoV-2 variants at the initial stage of infection. To our knowledge, for the first time, this study measures the relative infectivity of the Omicron variant under multiple virus co-infection and provides new experimental procedures that can be applied to monitor emerging viral variants.

Mass spectrometry synuclein detection in human plasma

AbstractBackgroundSynucleins (alpha, beta, and gamma) are potential biomarkers for Parkinson’s disease dementia (PDD), Dementia of Lewy body (DLB) and the Multi system atrophy (MSA). Their quantification with Mass spectrometry (MS) represents an interesting approach as it can precisely quantify synuclein proteoforms including mutations, alternative splicing, phosphorylation and other post‐translational modifications.MethodsWe developed an antibody free method starting with 95 µl of plasma. Patient cohort of 155 samples was analyzed with 57% PD, 22% LBD, 6% MSA, and 15% of control (CTRL) patient samples. Briefly, plasma proteins were differentially precipitated, clean‐up on AssayMap BRAVO (Agilent Technologies) and digest with trypsin/lys C. Liquid Chromatography‐Multiple Reaction Monitoring (LC‐MRM, Nexera Mikros – 8060, Shimadzu) method was developed to monitor 14 peptides originated from the alpha, beta and gamma synuclein sequence and one hemoglobin peptide. Method was validated following the EMA guidelines and compare to alpha synuclein immunodetection with Meso Scale Discovery platform (U‐plex human alpha synuclein kit).ResultsQuantitative assay based on LC‐MRM was validated on total alpha (140aa), beta (134aa) and gamma (127aa) synuclein peptides, and 4 alpha synuclein proteoforms (126, 112, 98 and 41) using 3 proteotypic peptides (one for 126/98, one for 112/98 and one for 41). After cohort quantification, alpha and beta synucleins were quantified in plasma with good performances and are able to statistically discriminate CTRL vs PD; CTRL vs LBD; CTRL vs MSA, and MSA vs PD. A slight correlation was found between the quantities obtain between MSD and MS assays.ConclusionMS based assay was successfully developed and validated to plasma synuclein proteoforms. Analysis of patient cohort indicate the ability of this assay to discriminate PD, DLB and MSA with a specificity of 81% and a sensitivity of 87%.

CTIM-31. IMMUNOLOGIC MONITORING AFTER ADOPTIVE CELL THERAPY IN PEDIATRIC PATIENTS WITH RECURRENT MEDULLOBLASTOMA USING IMMUNOGENOMICS APPROACH

Abstract BACKGROUND Adoptive cellular therapy (ACT) using transfer of tumor-specific lymphocytes has emerged as a potent strategy for treatment of advanced and refractory malignancies. We have employed the use of total tumor RNA (TTRNA)-pulsed DCs in the ex vivo expansion of tumor-specific lymphocytes for use in adoptive cellular therapy targeting pediatric malignant brain tumors. METHODS Total RNA was isolated from patient PBMC samples collected prior to adoptive cellular therapy and after 2-weeks and 4-weeks following immunotherapy treatment from 18 patients (Re-MATCH protocol, FDA IND BB-14058). We applied bulk RNA-Seq and high-throughput T cell receptor sequencing to monitor changes in blood draws after ACT. RESULTS We observed an increase of expression level of genes responsible for T cell activation in PBMC after adoptive cell therapy. We found that these genes were involved in T cell signaling pathways, proliferative signals, and cytokine production. The data revealed that certain T-cells were clonally expanded after ACT, with an increasing number of “hyper-expanded” TCR clones (comprising &amp;gt; 1% of all TCR beta or alpha sequences) after adoptive cellular therapy. These TCR clones established a new balance in the peripheral blood that was stable over the month following the completion of cellular immunotherapy. Hyper-expanded TCR clones and increased TCR diversity following ACT was associated with radiographic response to ACT and prolonged progression-free and overall survival. Limited TCR expansion and diversity following ACT was observed in patients with short overall and progression-free survival. CONCLUSIONS These results emphasize the importance of gene expression profiling to achieve higher resolution in monitoring immune responses in patients receiving immunotherapeutic treatment. Also, these findings support further study of the use of TCR sequencing to monitor responses to adoptive cellular therapy and suggest that TCR clonal expansion and increasing TCR diversity following treatment may be associated with positive clinical responses.

Reactive Centre Loop Mutagenesis of SerpinB3 to Target TMPRSS2 and Furin: Inhibition of SARS-CoV-2 Cell Entry and Replication.

The SARS-CoV-2 virus can utilize host cell proteases to facilitate cell entry, whereby the Spike (S) protein is cleaved at two specific sites to enable membrane fusion. Furin, transmembrane protease serine 2 (TMPRSS2), and cathepsin L (CatL) are the major proteases implicated, and are thus targets for anti-viral therapy. The human serpin (serine protease inhibitor) alpha-1 antitrypsin (A1AT) shows inhibitory activity for TMPRSS2, and has previously been found to suppress cell infection with SARS-CoV-2. Here, we have generated modified serpin inhibitors with increased specificity for these cellular proteases. Using SerpinB3 (SCCA-1), a cross-class inhibitor of CatL, as a scaffold, we have designed and produced reactive centre loop (RCL) variants to more specifically target both furin and TMPRSS2. Two further variants were generated by substituting the RCL P7–P1 with the spike protein S1/S2 cleavage site from either SARS-CoV-2 alpha or delta (P681R) sequences. Altered inhibitory specificity of purified recombinant proteins was verified in protease assays, with attenuated CatL inhibition and gain of furin or TMPRSS2 inhibition, as predicted, and modified serpins were shown to block S protein cleavage in vitro. Furthermore, the serpin variants were able to inhibit S-pseudoparticle entry into A549-ACE2-TMPRSS2 cells and suppress SARS-CoV-2 replication in Vero E6 cells expressing TMPRSS2. The construct designed to inhibit TMPRSS2 (B3-TMP) was most potent. It was more effective than A1AT for TMPRSS2 enzyme inhibition (with an eighteen-fold improvement in the second order inhibition rate constant) and for blocking SARS-CoV-2 viral replication. These findings advance the potential for serpin RCL mutagenesis to generate new inhibitors, and may lead to novel anti-viral biological molecules.

Abstract LB094: First of its kind T cell receptors and functional proteomics detected from the same single cells to advance cancer immunology discovery

Abstract T cell receptor repertoire diversity and T cell cytokine production are the important attributes for the assessment of effective T cell immunity against cancer and pathogens, yet existing single-cell genomics platforms cannot interrogate TCR sequences and proteomic cytokines simultaneously. IsoPlexis’ functional proteomics has demonstrated the values in cell therapy and cancer immunology against blood cancers and solid tumors. The ability to connect the T cell receptor diversity to the most potent single cells enables a wide variety of applications for tumor infiltrating lymphocytes, personalized neoantigen TCR’s, cancer immunology in terms of understanding antigen specificity, as well as self-potency. IsoPlexis single-cell TCR Duomic platform provide a unique measurement for connecting T cell receptor sequences to those T cell cytokine profiles, for the first time. Human PBMC-derived T cell line established from healthy donor was stimulated with PMA/ionomycin (2ul/ml, Biolegend) for 6hrs, stained and resuspended in Reverse transcription premix containing reverse transcriptase with buffer and protein phosphatase inhibitor at 1 x 106/ml and then loaded on Duomic chips. Ran on IsoLight overnight, cDNA was collected from the chip followed by cleanup and amplification, proceeded with TCR amplification and sequencing on NextSeq 1000/2000. Proteomic data from single cells was automatically generated and analyzed by IsoSpeak software. The single-cell TCR sequencing data on the IsoPlexis TCR Duomics platform shows that 78.9% cells have TCR alpha chain recombination and 94.4% cells have TCR beta chain recombination. Totally 77.7% cells have combination on both alpha and beta chain. VDJ Usage Plot further displays the top10 most common V (D) J combination flow between T cell receptor alpha chain and beta chain among the single cells, revealing the diversity of TCR repertoire at the single-cell level. In addition, the proteomics data identifies the dominant proteins among the single cells are Granzyme B, GM-CSF, IFN-g and TNF-a. Furthermore, we define 3 distinct cell clusters based on functional proteins and an association of VDJ distribution with these cell clusters, suggesting the occurrence of V(D)J sequences in the most common reconstructed alpha and beta sequence. IsoPlexis single-cell TCR Duomics platform, for the first time, enables simultaneous profiling of functional proteomics and TCR repertoires/TCR clonotypes as well as identification of specific TCR V(D)J combination with functional cell clusters across the same individual T cells. The combination of these two analytes at the single cells provides a key new modality of tracking the most potent and also the antigen specific immune cells together to fight cancer and infectious disease, for the first time. Citation Format: Jennifer Yang, Jing Zhou, Sean Mackay. First of its kind T cell receptors and functional proteomics detected from the same single cells to advance cancer immunology discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB094.

Abstract 2810: Modeling pHLA:TCR interactions for effective TCR therapies: Leveraging AI and molecular dynamics

Abstract Background: The purpose of our study is to aid the development of adoptive T-cell receptor (TCR) -based cancer therapies with an AI platform that models binding between peptide-human leukocyte antigen (pHLA) complexes and TCRs. One of the main challenges in the field is finding the right pHLA target and the TCR that binds it. The development of in silico methods that can reduce the cost and time of laboratory research is the ‘holy grail’ of novel TCR therapies design. Molecular Dynamics (MD) simulation is a computational method allowing to gain a direct insight into the movements of protein complexes and giving the possibility to study the dynamics of the pHLA:TCR interaction. The combination of the MD and AI models helps us to increase the accuracy of the pHLA:TCR binding prediction. Methods: We conducted large-scale MD simulations of the crystal structures of 130 pHLA:TCR complexes available in the Protein Data Bank (PDB). Next, we analyzed the obtained trajectories in terms of formed bonds and other physico-chemical properties. This led us to postulate an MD model of interaction further used as an inductive bias for the AI binding prediction model, in which we applied deep learning on public single-cell pHLA:TCR datasets to estimate the probability of binding.In order to validate and improve our platform, we are building a pHLA:TCR interaction database with paired alpha and beta chain TCR sequences from a cohort of 100 colorectal cancer patients (NCT04994093). Results: We established the agretopicity and epitopicity of peptide residues within the pHLA:TCR systems based on the characteristics of the bonds formed between peptides and HLAs or TCRs in MD simulations. Additionally, the incorporation of other molecular properties, e.g. solvent accessible surface area, improved our AI pHLA:TCR model. In order to refine our results, we also account for the HLA type and physico-chemical properties of peptides. The introduction of the detailed TCR-peptide interaction matrix allowed the model to generalize to previously unseen pHLA targets. Conclusions: Rational design of effective TCR therapies can benefit from novel pHLA:TCR models for which the predictive accuracy is driven by the synergistic AI-MD approach. Citation Format: Alexander Myronov, Sławomir Stachura, Anna Sanecka-Duin, Oskar Gniewek, Łukasz Grochowalski, Maciej Jasiński, Paulina Król, Joanna Marczyńska-Grzelak, Piotr Skoczylas, Daniel Wojciechowski, Giovanni Mazzocco, Mikołaj Mizera, Jan Kaczmarczyk, Agnieszka Blum. Modeling pHLA:TCR interactions for effective TCR therapies: Leveraging AI and molecular dynamics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2810.