Proportion Of Isoforms Research Articles

Can non-invasive motor unit analysis reveal distinct neural strategies of force production in young with uncomplicated type 1 diabetes?

to investigate the early consequences of type 1 diabetes (T1D) on the neural strategies of muscle force production. motor unit (MU) activity was recorded from the vastus lateralis muscle with High-Density surface Electromyography during isometric knee extension at 20 and 40% of maximum voluntary contraction (MVC) in 8 T1D (4 males, 4 females, 30.5 ± 3.6years) and 8 matched control (4 males, 4 females, 27.3 ± 5.9years) participants. Muscle biopsies were also collected from vastus lateralis for fiber type analysis, including myosin heavy chain (MyHC) isoform content via protein and mRNA expression. MVC was comparable between groups as well as MU conduction velocity, action potentials' amplitude and proportions of MyHC protein isoforms. Nonetheless, MU discharge rate, relative derecruitment thresholds and mRNA expression of MyHC isoform I were lower in T1D. young people with uncomplicated T1D present a different neural control of muscle force production. Furthermore, differences are detectable non-invasively in absence of any functional manifestation (i.e., force production and fiber type distribution). These novel findings suggest that T1D has early consequences on the neuromuscular system and highlights the necessity of a better characterization of neural control in this population.

Accurate isoform quantification by joint short- and long-read RNA-sequencing.

Accurate quantification of transcript isoforms is crucial for understanding gene regulation, functional diversity, and cellular behavior. Existing RNA sequencing methods have significant limitations: short-read (SR) sequencing provides high depth but struggles with isoform deconvolution, whereas long-read (LR) sequencing offers isoform resolution at the cost of lower depth, higher noise, and technical biases. Addressing this gap, we introduce Multi-Platform Aggregation and Quantification of Transcripts (MPAQT), a generative model that combines the complementary strengths of different sequencing platforms to achieve state-of-the-art isoform-resolved transcript quantification, as demonstrated by extensive simulations and experimental benchmarks. By applying MPAQT to an in vitro model of human embryonic stem cell differentiation into cortical neurons, followed by machine learning-based modeling of transcript abundances, we show that untranslated regions (UTRs) are major determinants of isoform proportion and exon usage; this effect is mediated through isoform-specific sequence features embedded in UTRs, which likely interact with RNA-binding proteins that modulate mRNA stability. These findings highlight MPAQT's potential to enhance our understanding of transcriptomic complexity and underline the role of splicing-independent post-transcriptional mechanisms in shaping the isoform and exon usage landscape of the cell.

ACLY alternative splicing correlates with cancer phenotypes

ATP-citrate lyase (ACLY) links carbohydrate and lipid metabolism and provides nucleocytosolic acetyl-CoA necessary for protein acetylation. ACLY has two major splice isoforms: the full-length canonical “long” isoform and an uncharacterized “short” isoform in which exon 14 is spliced out. Exon 14 encodes 10 amino acids within a disordered region of the protein and includes at least 1 site that is dynamically phosphorylated. Both isoforms are expressed in healthy tissues to varying degrees. Analysis of human transcriptomic data revealed that the Percent Spliced In (PSI) of exon 14, i.e., the proportion of long isoform, is increased in several cancers and correlated with poorer overall survival in a pan-cancer analysis, though not in individual tumor types, which prompted us to explore potential biochemical and functional differences between ACLY isoforms.Here, we show that there are no discernible differences in enzymatic activity or stability between isoforms or phosphomutants of ACLY in vitro. Similarly, both isoforms and phosphomutants were able to rescue ACLY functions, including fatty acid synthesis and bulk histone acetylation, when re-expressed in Acly knockout cells. Deletion of Acly exon 14 in mice did not overtly impact development or metabolic physiology, nor did it attenuate tumor burden in a genetic model of intestinal cancer.Notably, expression of epithelial splicing regulatory protein 1 (ESRP1) is highly correlated with ACLY PSI. We report that ACLY splicing is regulated by ESRP1. In turn, both ESRP1 expression and ACLY PSI are correlated with specific immune signatures in tumors. Despite these intriguing patterns of ACLY splicing in healthy and cancer tissues, functional differences between the isoforms remain elusive.

HLA-G isoforms, HLA-C allotype and their expressions differ between early abortus and placenta in relation to spontaneous abortions

IntroductionSpontaneous abortion (SAB) affects approximately 10% of clinically recognized pregnancies. Fetal trophobalst invasion and remodeling of maternal spiral arteries is reported to be dependent on crosstalk between HLA-C/HLA-G expressed on extra villous trophoblast (EVTs)and Killer cell Immunoglobin like receptors (KIRs) of decidual NK (dNK). Immune dysfunction in decidua contributes to early miscarriage. MethodologyThe study used mother neonate paired cord blood and term placenta samples (n = 46), elective abortus (n = 17,gestational age = 10–12 weeks of pregnancy) and SAB abortus (n = 24, gestational age = 12–15 weeks of pregnancy) for HLA-G, KIR2D and HLA-C. In addition, term placenta was collected from women with history of spontaneous pregnancy loss (n = 24) and women with history of live birth (n = 32). SSP-PCR was used for genotyping, RT-PCR for gene expression, copy number variation (CNVs) and HLA-C allotyping and ELISA for protein expression studies. ResultsMembrane bound HLA-G4 isoform proportion was higher 39.28%, p = 0.02) in term placenta. SAB abortus had higher proportion of HLA-G3 (50%),while elective abortus exhibited higher proportion of soluble isoforms (HLA-G5, = 5.9, HLA-G6 = 5.9%, HLA-G7 = 11.8%).Higher inhibitory KIR2DL1 content and copy numbers with lower HLA-C2 in SAB contrasted with higher copy numbers of KIR2DS1(p = 0.001), KIR2DS1+/2DL1+- HLA-C2 combined genotype in healthy placenta. Elevated KIR2D protein levels (p = 0.001), and concurrently, HLA-C levels were upregulated in healthy placenta. ConclusionOur data supports lower cognate receptor ligand KIR2DS1+/2DL1+ HLA–C2 together with predominance of HLA-G3 isoform in SAB as confounding factors in spontaneous pregnancy loss. HLA-G isoforms and expression differed between first trimester abortus and term placenta suggesting temporal modulation and marks novelty of the study.

Detecting and Validating MAPT Mutations in Neurodegeneration Patients and Analysis of Exon Splicing Consequences.

Mutation of MAPT has been observed in patients with parkinsonism, progressive supranuclear palsy, and corticobasal degeneration and is a significant cause of frontotemporal dementia. In this chapter, we discuss considerations for next-generation sequencing analysis to identify MAPT mutations in patient genomic DNA and describe the validation of these mutations by Sanger sequencing. One of the most common effects of MAPT mutations is differential splicing of exon 10, which leads to an imbalance in the proportion of 3-repeat and 4-repeat tau isoforms. We describe how to investigate the effect of novel DNA variants on the splicing efficiency of this exon in vitro using the exon-trapping technique, also known as the splicing reporter minigene assay.

Gene expression and alternative splicing contribute to adaptive divergence of ecotypes.

Regulation of gene expression is a critical link between genotype and phenotype explaining substantial heritable variation within species. However, we are only beginning to understand the ways that specific gene regulatory mechanisms contribute to adaptive divergence of populations. In plants, the post-transcriptional regulatory mechanism of alternative splicing (AS) plays an important role in both development and abiotic stress response, making it a compelling potential target of natural selection. AS allows organisms to generate multiple different transcripts/proteins from a single gene and thus may provide a source of evolutionary novelty. Here, we examine whether variation in alternative splicing and gene expression levels might contribute to adaptation and incipient speciation of dune-adapted prairie sunflowers in Great Sand Dunes National Park, Colorado, USA. We conducted a common garden experiment to assess transcriptomic variation among ecotypes and analyzed differential expression, differential splicing, and gene coexpression. We show that individual genes are strongly differentiated for both transcript level and alternative isoform proportions, even when grown in a common environment, and that gene coexpression networks are disrupted between ecotypes. Furthermore, we examined how genome-wide patterns of sequence divergence correspond to divergence in transcript levels and isoform proportions and find evidence for both cis and trans-regulation. Together, our results emphasize that alternative splicing has been an underappreciated mechanism providing source material for natural selection at short evolutionary time scales.

CD19 Intron Retention Is Mechanism of CAR-T Treatment Resistance in Non-Hodgkin Lymphoma

Background: Chimeric antigen receptor T-cell (CAR-T) therapy has become a viable treatment option for patients (pts) with relapsed or refractory diffuse large B-cell lymphoma (R/R DLBCL). Transcriptomic markers for CD19 directed CAR-T failure have been characterized in pre-treatment B-cell acute lymphoblastic leukemia (B-ALL) but not in DLBCL. Here, we report the identification of RNA alternative splicing (AS) events associated with CD19 directed CAR-T response in pre-treatment DLBCL pts and of RNA binding proteins (RNABP) regulating these AS events. Methods: To identify putative AS markers for CAR-T treatment outcome, we performed differential AS on bulk RNA-seq samples of pre-treatment tumor biopsies in pts that had a durable response (DR), defined via remission up to 9 months post-treatment, and pts that had a non-durable response (NDR) with rMATS-turbo (Shen et al. PNAS. 2014). Intron retention (IR) events within CD19 were quantified with a secondary, more robust algorithm to ensure reproducibility and accuracy. Survival analyses were done using the survival and survminer libraries in R. Validation assays for the downstream effects CD19 IR were conducted in EJ1, Jeko-1, OCI-Ly3, SU-DHL-6, and Toledo cell lines. The surface protein expression of CD19 was assayed via flow cytometry (FC) followed by Quantibrite PE (BD Biosciences, San Jose, CA) quantification. Total CD19 protein expression was measured via western blot (WB) in the aforementioned cell lines. Analyses of CD19 IR was done via RT-PCR followed by electrophoresis, and bands were quantified with iBright Analysis Software (Thermo Fisher Scientific, Carlsbad, CA). The effect of RNABP was assessed via the creation of CRISPR/Cas9 knockout (KO) cell lines followed by WB of the target gene and quantification of CD19 surface protein expression by FC. Results: Introns 2 and 6 of CD19 are more highly expressed in NDR pts. A combined metric estimating the proportion of normal CD19 isoforms that do not express introns 2 or 6 (normal CD19) was created and DR pts expressed normal CD19 isoforms significantly higher than NDR pts (Figure 1). Normal CD19 isoforms classification performance was assessed via leave one out cross validation and achieved an overall accuracy of 67.6%. Sensitivity (classifying NDR) was 61.1% and specificity (classifying DR) was 81.3%. The expression of normal CD19 relative to the median level within the cohort was significantly associated with progress free survival (Figure 2). The expression of CD19 intron 6 was negatively correlated with CD19 surface protein expression in DLBCL cell lines. Cell lines were sorted by cells that exhibited high CD19 surface expression (bright) and low expression (dim). Cell lines sorted into the dim category expressed higher levels of intron 6 than bright cells. The correlation of RNABP expression with CD19 intron 6 expression was done in this patient cohort and the NCICCR-DLBCL cohort. Six RNABP were significantly correlated in both cohorts (r 2 > 0.2 and p-value <0.01). NOP2, one of the RNABP that was positively correlated with intron 6 expression, was knocked out in Jeko-1 cell lines to validate the putative correlation with CD19 protein expression. The resulting knock out cells exhibited lower levels of intron 6 retention and higher CD19 protein expression by FC and WB than non-transformed cells. Conclusion: Pre-treatment tumors of R/R DLBCL patients who relapse before 9 months following CD19 directed CAR-T therapy have higher expression of CD19 isoforms that retain introns 2 and 6. The upregulated expression of the CD19 isoforms retaining these introns is correlated with CD19 surface protein expression and is a marker for sustained remission following CAR-T treatment. NOP2 is correlated with CD19 intron retention and surface protein expression and could be a target for modulating CD19 expression in conjunction with CAR-T treatment. Figure 1: The expression of normal CD19 isoforms in pre-treatment tumor samples of DR (red) and NDR patients (blue). ΔPSI denotes the difference in medians between the two groups. P-value yielded by a Wilcoxon test between the two groups is reported. Figure 2: Progress free survival (PFS) stratified by expression of normal CD19 isoforms. Red - above cohort median expression of normal CD19 isoforms. Blue - below cohort median expression of normal CD19 isoforms. P-value yielded via log-rank test.

The influence of skeletal muscle mitochondria and sex on critical torque and performance fatiguability in humans.

Critical torque (CT) represents the highest oxidative steady state for intermittent knee extensor exercise, but the extent to which it is influenced by skeletal muscle mitochondria and sex is unclear. Vastus lateralis muscle biopsy samples were collected from 12 females and 12males -matched for relative maximal oxygen uptake normalized to fat-free mass (FFM) (F: 57.3 (7.5) ml(kg FFM)-1 min-1 ; M: 56.8 (7.6) ml(kg FFM)-1 min-1 ; P=0.856) - prior to CT determination and performance fatiguability trials. Males had a lower proportion of myosin heavy chain (MHC) I isoform (40.6 (18.4)%) compared to females (59.5 (18.9)%; P=0.021), but MHC IIa and IIx isoform distributions and protein markers of mitochondrial content were not different between sexes (P > 0.05). When normalized to maximum voluntary contraction (MVC), the relative CT (F: 42.9 (8.3)%; M: 37.9 (9.0)%; P=0.172) and curvature constant, W' (F: 26.6 (11.0) Nms(Nm)-1 ; M: 26.4 (6.5) Nms(Nm)-1 ; P=0.962) were not significantly different between sexes. All protein biomarkers of skeletal muscle mitochondrial content, as well as the proportion of MHC I isoform, positively correlated with relative CT (0.48<r<0.70; P<0.05), and the proportion of MHC IIx isoform correlated positively with relative W' (r=0.57; P=0.007). Indices of performance fatiguability were not different between males and females for MVC- and CT-controlled trials (P > 0.05). Greater mitochondrial protein abundance was associated with attenuated declines in potentiated twitch torque for exercise at 60% MVC (P<0.05); however, the influence of mitochondrial protein abundance on performance fatiguability was reduced when exercise was prescribed relative to CT. Whether these findings translate to whole-body exercise requires additional research. KEY POINTS: The quadriceps critical torque represents the highest intensity of intermittent knee extensor exercise for which an oxidative steady state is attainable, but its relationship with skeletal muscle mitochondrial protein abundance is unknown. Matching males and females for maximal oxygen uptake relative to fat-free mass facilitates investigations of sex differences in exercise physiology, but studies that have compared critical torque and performance fatiguability during intermittent knee extensor exercise have not ensured equal aerobic fitness between sexes. Skeletal muscle mitochondrial protein abundance was correlated with critical torque and fatigue resistance for exercise prescribed relative to maximum voluntary contraction but not for exercise performed relative to the critical torque. Differences between sexes in critical torque, skeletal muscle mitochondrial protein abundance and performance fatiguability were not statistically significant. Our results suggest that skeletal muscle mitochondrial protein abundance may contribute to fatigue resistance by influencing the critical intensity of exercise.

A combined top-down and bottom-up LC-HRMS/MS method for the quantification of human growth hormone in plasma and serum

ObjectiveThe precise and accurate quantification of human growth hormone (GH) in plasma/ serum is crucial for the diagnosis and treatment of diseases like GH deficiency or acromegaly. However, the ligand-binding assays (LBAs) currently used for routine testing show considerable methodological variability. Here, we present a complementary, combined top-down and bottom-up LC-MS-based method to quantify (intact) GH in plasma and serum, which concurrently provides a basis for a MS-based analysis of GH in doping controls. DesignExtraction of GH from plasma/ serum was accomplished by protein precipitation, followed by an immunocapture step using protein A-coupled magnetic beads and a polyclonal anti-GH antibody. The intact protein was subsequently analyzed top-down on a 2D-LC-HRMS/MS system. In addition, sample extracts were digested with trypsin and analyzed for signal peptides corresponding to ‘total’, 22 kDa and 20 kDa GH (bottom-up). Both assays were validated according to current guidelines and compared to the GH isoform differential immunoassay used in routine doping control analysis. GH concentrations in serum samples of healthy adults, patients with acromegaly, and in samples obtained after administration of recombinant GH were analyzed as proof-of-principle. ResultsThe intact monomeric 22 kDa isoform of GH was selectively quantified in a representative working range of 0.5 to 10 ng/ml by top-down LC-HRMS/MS. Subsequent bottom-up analysis provided additional data on ‘total’ and 20 kDa GH. Top-down and bottom-up assay results for the 22 kDa isoform correlated well with the corresponding immunoassay results (R2 > 0.95). For a possible application of the method in an anti-doping context, the ratio between 22 kDa and ‘total’ GH was evaluated, indicating differences between the various donor groups, but only with limited significance. ConclusionThe top-down and bottom-up LC-HRMS/MS method developed here presents a valuable tool for the quantification of GH in plasma/ serum complementary to established LBAs used at present in clinical measurements. Albeit the examination of the GH isoform proportions by the LC-MS method does not yet allow for the assessment of GH abuse, the obtained findings provide an important basis to enable LC-MS-based GH analysis of doping control samples in the future.

Human lncRNA SUGCT-AS1 Regulates the Proinflammatory Response of Macrophage.

Macrophages are the major primary immune cells that mediate the inflammatory response. In this process, long non-coding RNAs (lncRNAs) play an important, yet largely unknown role. Therefore, utilizing several publicly available RNA sequencing datasets, we predicted and selected lncRNAs that are differentially expressed in M1 or M2 macrophages and involved in the inflammatory response. We identified SUGCT-AS1, which is a human macrophage-specific lncRNA whose expression is increased upon M1 macrophage stimulation. Conditioned media of SUGCT-AS1-depleted M1 macrophages induced an inflammatory phenotype of vascular smooth muscle cells, which included increased expression of inflammatory genes (IL1B and IL6), decreased contractile marker proteins (ACTA2 and SM22α), and increased cell migration. Depletion of SUGCT-AS1 promoted the expression and secretion of proinflammatory cytokines, such as TNF, IL1B, and IL6, in M1 macrophages, and transcriptomic analysis showed that SUGCT-AS1 has functions related to inflammatory responses and cytokines. Furthermore, we found that SUGCT-AS1 directly binds to hnRNPU and regulates its nuclear-cytoplasmic translocation. This translocation of hnRNPU altered the proportion of the MALT1 isoforms by regulating the alternative splicing of MALT1, a mediator of NF-κB signaling. Overall, our findings suggest that lncRNAs can be used for future studies on macrophage regulation. Moreover, they establish the SUGCT-AS1/hnRNPU/MALT1 axis, which is a novel inflammatory regulatory mechanism in macrophages.

Hepatitis B virus serum RNA transcript isoform composition and proportion in chronic hepatitis B patients by nanopore long-read sequencing.

Serum hepatitis B virus (HBV) RNA is a promising new biomarker to manage and predict clinical outcomes of chronic hepatitis B (CHB) infection. However, the HBV serum transcriptome within encapsidated particles, which is the biomarker analyte measured in serum, remains poorly characterized. This study aimed to evaluate serum HBV RNA transcript composition and proportionality by PCR-cDNA nanopore sequencing of samples from CHB patients having varied HBV genotype (gt, A to F) and HBeAg status. Longitudinal specimens from 3 individuals during and following pregnancy (approximately 7 months between time points) were also investigated. HBV RNA extracted from 16 serum samples obtained from 13 patients (73.3% female, 84.6% Asian) was sequenced and serum HBV RNA isoform detection and quantification were performed using three bioinformatic workflows; FLAIR, RATTLE, and a GraphMap-based workflow within the Galaxy application. A spike-in RNA variant (SIRV) control mix was used to assess run quality and coverage. The proportionality of transcript isoforms was based on total HBV reads determined by each workflow. All chosen isoform detection workflows showed high agreement in transcript proportionality and composition for most samples. HBV pregenomic RNA (pgRNA) was the most frequently observed transcript isoform (93.8% of patient samples), while other detected transcripts included pgRNA spliced variants, 3' truncated variants and HBx mRNA, depending on the isoform detection method. Spliced variants of pgRNA were primarily observed in HBV gtB, C, E, or F-infected patients, with the Sp1 spliced variant detected most frequently. Twelve other pgRNA spliced variant transcripts were identified, including 3 previously unidentified transcripts, although spliced isoform identification was very dependent on the workflow used to analyze sequence data. Longitudinal sampling among pregnant and post-partum antiviral-treated individuals showed increasing proportions of 3' truncated pgRNA variants over time. This study demonstrated long-read sequencing as a promising tool for the characterization of the serum HBV transcriptome. However, further studies are needed to better understand how serum HBV RNA isoform type and proportion are linked to CHB disease progression and antiviral treatment response.

New Perspectives in Dried Blood Spot Biomarkers for Lysosomal Storage Diseases.

Dried blood spots (DBSs) biomarkers are convenient for monitoring for specific lysosomal storage diseases (LSDs), but they could have relevance for other LSDs. To determine the specificity and utility of glycosphingolipidoses biomarkers against other LSDs, we applied a multiplexed lipid liquid chromatography tandem mass spectrometry assay to a DBS cohort of healthy controls (n = 10) and Gaucher (n = 4), Fabry (n = 10), Pompe (n = 2), mucopolysaccharidosis types I-VI (n = 52), and Niemann-Pick disease type C (NPC) (n = 5) patients. We observed no complete disease specificity for any of the markers tested. However, comparison among the different LSDs highlighted new applications and perspectives of the existing biomarkers. We observed elevations in glucosylceramide isoforms in the NPC and Gaucher patients relative to the controls. In NPC, there was a greater proportion of C24 isoforms, giving a specificity of 96-97% for NPC, higher than 92% for the NPC biomarker N-palmitoyl-O-phosphocholineserine ratio to lyso-sphingomyelin. We also observed significantly elevated levels of lyso-dihexosylceramide in Gaucher and Fabry disease as well as elevated lyso-globotriaosylceramide (Lyso-Gb3) in Gaucher disease and the neuronopathic forms of Mucopolysaccharidoses. In conclusion, DBS glucosylceramide isoform profiling has increased the specificity for the detection of NPC, thereby improving diagnostic accuracy. Low levels of lyso-lipids can be observed in other LSDs, which may have implications in their disease pathogenesis.

New insights into the role of microheterogeneity of ZP3 during structural maturation of the avian equivalent of mammalian zona pellucida.

The egg coat including mammalian zona pellucida (ZP) and the avian equivalent, i.e., inner-perivitelline layer (IPVL), is a specialized extracellular matrix being composed of the ZP glycoproteins and surrounds both pre-ovulatory oocytes and ovulated egg cells in vertebrates. The egg coat is well known for its potential importance in both the reproduction and early development, although the underlying molecular mechanisms remain to be fully elucidated. Interestingly, ZP3, one of the ZP-glycoprotein family members forming scaffolds of the egg-coat matrices with other ZP glycoproteins, exhibits extreme but distinctive microheterogeneity to form a large number of isoelectric-point isoforms at least in the chicken IPVL. In the present study, we performed three-dimensional confocal imaging and two-dimensional polyacrylamide-gel electrophoresis (2D-PAGE) of chicken IPVLs that were isolated from the ovarian follicles at different growth stages before ovulation. The results suggest that the relative proportions of the ZP3 isoforms are differentially altered during the structural maturation of the egg-coat matrices. Furthermore, tandem mass spectrometry (MS/MS) analyses and ZP1 binding assays against separated ZP3 isoforms demonstrated that each ZP3 isoform contains characteristic modifications, and there are large differences among ZP3 isoforms in the ZP1 binding affinities. These results suggest that the microheterogeneity of chicken ZP3 might be regulated to be associated with the formation of egg-coat matrices during the structural maturation of chicken IPVL. Our findings may provide new insights into molecular mechanisms of egg-coat assembly processes.

Regulation of Arp5 expression by alternative splicing coupled to nonsense-mediated RNA decay

Actin-related protein 5 (ARP5) inhibits the differentiation of skeletal, smooth, and cardiac muscle tissues, and ARP5 expression increases or decreases according to physiological and pathological changes in the muscle differentiation status. However, the regulatory mechanisms of ARP5 expression are largely unknown. Here, we identified a novel Arp5 mRNA isoform that contains premature termination codons in alternative exon 7b and is thus targeted by nonsense-mediated mRNA decay (NMD). In mouse skeletal muscle cells, switching from the canonical Arp5 isoform, i.e., Arp5(7a), to the NMD-targeted isoform Arp5(7b) occurred during differentiation, suggesting that Arp5 expression is regulated by alternative splicing coupled to NMD (AS-NMD). We developed an original method to accurately quantify the proportion of both Arp5 isoforms and measured higher levels of Arp5(7b) in muscle and brain tissues, where ARP5 is less expressed. The 3′ splice site in Arp5 exon 7 has an unusual acceptor sequence that often leads to the skip of the authentic splice site and the use of the cryptic splice site localized 16 bases downstream. When the unusual acceptor sequence was mutated to the usual one, the Arp5(7b) isoform was barely detectable. The expression of several splicing factors involved in 3′ splice site recognition was reduced after muscle differentiation. Additionally, knockdown of splicing factors increased the levels of Arp5(7b) and decreased the expression of Arp5(7a). Furthermore, strong positive correlations were found between Arp5 expression and the levels of these splicing factors in human skeletal and cardiac muscle tissues. Thus, Arp5 expression in muscle tissues is most likely regulated by the AS-NMD pathway.

2140. INO-4800, a DNA Plasmid Vaccine Encoding the Ancestral SARS-CoV-2 Spike Protein, Is Stable Over a Range of Temperatures Not Requiring Ultra-Cold Chain Storage

Abstract Background Effective vaccines deployable globally are needed to control the COVID-19 pandemic. INO-4800, a plasmid DNA vaccine encoding the ancestral SARS-CoV-2 spike (S) protein, has demonstrated safety and immunogenicity. We report the stability of multiple INO-4800 lots used in Phase 3 clinical trials. Methods Lots of INO-4800 stored at the test temperatures (storage temperature [2–8°C] and accelerated temperature [25 ± 2°C]) were sampled every 3 months. Sample stability at each timepoint was assessed in terms of 2 parameters: i) purity as measured by DNA isoform homogeneity by capillary gel electrophoresis (CGE) to quantify relative proportions of different plasmid topologies (supercoiled, open circular, and linear), and ii) potency as measured by a cell-based assay for flow cytometric detection of fluorescently labeled S protein. Results At 2–8°C, all tested lots of INO-4800 retained structural homogeneity for ≥12 months (Fig. 1A). Proportions of circular and supercoiled plasmid isoforms were ≥96% and ≥89%, respectively, remaining above minimum regulatory authority-approved specifications (≥85% for circular and ≥80% for supercoiled isoforms). At 25 ± 2°C, all tested lots retained structural homogeneity within specifications for ≥6 months (Fig. 1B), with proportions of circular and supercoiled plasmid isoforms being ≥96% and ≥81%, respectively. Moreover, structural homogeneity of 1/2 (50%) lots was within specification at 9 months at 25 ± 2°C. Potency of all tested lots was ≥84% for ≥6 months at both 2–8°C and 25 ± 2°C, remaining above the regulatory authority-approved minimum of 77% (Figs. 1C and 1D). Moreover, 3/3 (100%) of tested lots were within potency specification at 12 months at 2–8°C and 2 lots and 1 lot, respectively, were within specification at 9 and 12 months at 25 ± 2°C. Data from additional timepoints are accruing and will be presented. Figure 1:Stability of INO-4800 lots used in Phase 3 trials as measured by isoform homogeneity (A and B) and potency (C and D). Shaded areas represent regulatory authority-approved specifications. Conclusion INO-4800 is stable across temperatures ranging from 2°C to 25 ± 2°C and does not require ultra-cold storage. This demonstrated stability confers upon INO-4800 the potential to be deployed globally even in resource-constrained settings lacking cold chain infrastructure, thereby contributing to pandemic preparedness and control. Disclosures Robert J. Juba, Jr., MS Chemical Engineering Practice, BS Chemical Engineering, Inovio Pharmaceuticals: Employee|Inovio Pharmaceuticals: Stocks/Bonds Katherine Seals, n/a, Inovio Pharmaceuticals: Employee|Inovio Pharmaceuticals: Stocks/Bonds Peggy Kinney, MS Quality Management, Inovio Pharmaceuticals: Employee|Inovio Pharmaceuticals: Stocks/Bonds.

Comparison of Vitamin E Isoforms Among Plasma, Breast Milk, and Dietary Intake Measure of Mother-Infant Dyads

ObjectivesVitamin E is a fat-soluble nutrient with four isoforms: α-, β-, δ-, and γ-tocopherol. These isoforms differentially modulate inflammation and are related to important perinatal outcomes such as preterm delivery and Apgar scores. Understanding the dietary consumption of these isoforms and their respective prevalence in biological samples is an important component to optimize nutritional recommendations. The purpose of this study is to compare tocopherol isoform proportions among maternal, cord, and neonatal plasma; maternal breast milk; and maternal dietary intake.MethodsSamples of maternal breast milk and maternal, cord, and neonatal plasma were obtained within 1 month following delivery for maternal-infant dyads (N = 17) from the neonatal intensive care unit. Maternal dietary intake was assessed using the Harvard Willett Food Frequency Questionnaire. Relative proportions of α-, δ-, and γ-tocopherol in each sample type were measured and median tocopherol concentrations were compared using Kruskal-Wallis tests. A p-value < 0.05 was statistically significant.ResultsTotal tocopherol concentrations were significantly different across sample groups (P < 0.001). Concentrations were highest in maternal serum, followed by neonatal serum, maternal breast milk, and umbilical cord serum. In all samples, α-tocopherol had the highest relative proportion, followed by γ- and δ-tocopherol, respectively. Compared to all biological samples, the dietary intake proportion of γ-tocopherol was significantly higher (P < 0.001), while the proportion of α-tocopherol was significantly lower (p = 0.04).ConclusionsWe observed significant differences in tocopherol concentrations across related biological samples, with maternal plasma containing the highest concentration and umbilical cord plasma the lowest. Mothers also consumed significantly higher percentages of γ-tocopherol than those found in both their plasma and breast milk. This suggests that proportions of individual tocopherol isoforms are influenced by factors other than dietary intake. Additional research should explore these associations in a larger cohort and analyze the effects of supplementation on tocopherol concentrations.Funding SourcesUNMC Pediatrics Department; Child Health Research Institute.

Genetic engineering of the branched-chain fatty acid biosynthesis pathway to enhance surfactin production from Bacillus subtilis.

Surfactin, which is composed of a β-hydroxy fatty acid chain and a peptide ring, has drawn considerable attention due to its potential applications in the biomedicine, bioremediation, and petroleum industries. However, the low yield of surfactin from wild strains still restricts its industrial applications. In this study, eight genes relevant to the fatty acid biosynthesis pathway were targeted to enhance surfactin production, and high surfactin-yielding strains with potential industrial applications were obtained. When ldeHA and acc were co-overexpressed, the surfactin yield of recombinant strains TDS8 and TPS8 increased to 1.55- and 1.19-fold of their parental strains, respectively, again proving that the conversion of acetyl-coenzyme A (CoA) to malonyl-CoA is the rate-limiting step in fatty acid biosynthesis. Furthermore, changes in surfactin isoforms of recombinant strain TPS8 suggest that the fatty acid precursor synthesis pathway can be modified to improve the proportion of different isoforms. In addition, the deletion of lpdV, which is responsible for the conversion of α-ketoacyl-CoA precursors, resulted in a sharp decrease in surfactin production, further demonstrating the importance of branched-chain fatty acid biosynthesis in surfactin production. This work will facilitate the design and construction of more efficiently engineered strains for surfactin production and further extend industrial applications.

Genotype Score for Iron Status Is Associated with Muscle Fiber Composition in Women.

Human muscle fiber composition is heterogeneous and mainly determined by genetic factors. A previous study reported that experimentally induced iron deficiency in rats increases the proportion of fast-twitch muscle fibers. Iron status has been reported to be affected by genetic factors. As the TMPRSS6 rs855791 T/C and HFE rs1799945 C/G polymorphisms are strongly associated with iron status in humans, we hypothesized that the genotype score (GS) based on these polymorphisms could be associated with the muscle fiber composition in humans. Herein, we examined 214 Japanese individuals, comprising of 107 men and 107 women, for possible associations of the GS for iron status with the proportion of myosin heavy chain (MHC) isoforms (I, IIa, and IIx) as markers of muscle fiber composition. No statistically significant correlations were found between the GS for iron status and the proportion of MHC isoforms in all participants. When the participants were stratified based on sex, women showed positive and negative correlations of the GS with MHC-IIa (age-adjusted p = 0.020) and MHC-IIx (age-adjusted p = 0.011), respectively. In contrast, no correlation was found in men. In women, a 1-point increase in the GS was associated with 2.42% higher MHC-IIa level and 2.72% lower MHC-IIx level. Our results suggest that the GS based on the TMPRSS6 rs855791 T/C and HFE rs1799945 C/G polymorphisms for iron status is associated with muscle fiber composition in women.

Comprehensive discovery of salt-responsive alternative splicing events based on Iso-Seq and RNA-seq in grapevine roots

Alternative splicing (AS) can enhance the adaptive capacity of organisms to respond to abiotic stresses via transcriptome plasticity; however, the mechanism underlying the responses of AS events to salt remains unclear in grapevine roots. In this study, an optimized genome annotation file was generated using Iso-Seq and RNA-seq data of grapevine roots. Based on the updated file, a total of 8,453 AS events across five types were identified in A17 grapevine roots, of which retained intron (RI) was the most dominant type. A total of 1,466 AS events affiliated with 1,113 genes responded to salt stress in grapevine roots. A global decrease in the proportion of inclusion isoforms was demonstrated, indicating that salt stress promoted the production of shorter isoforms. Only 206 salt-responsive AS events were affiliated with differentially expressed genes, indicating little overlap between transcriptional regulation and post-transcriptional regulation. A total of 672 and 410 AS events affected the length of protein products and miRNA binding, respectively. Differentially spliced genes were primarily related to RNA processing, nucleic acid binding, and ion channel activity. In addition, the salt-induced spliceosomal protein genes LSM2 and SRSF1 were identified, and their possible roles in regulating AS events were inferred. Moreover, we drafted an approach to explore possible interactions between AS and alternative polyadenylation (APA) using Iso-Seq data. Overall, these findings provide a comprehensive understanding of salt-responsive AS events in grapevine roots, providing insight into the molecular mechanism of salt tolerance in grapevine.

Developmental dynamics of voltage-gated sodium channel isoform expression in the human and mouse brain

BackgroundGenetic variants in the voltage-gated sodium channels SCN1A, SCN2A, SCN3A, and SCN8A are leading causes of epilepsy, developmental delay, and autism spectrum disorder. The mRNA splicing patterns of all four genes vary across development in the rodent brain, including mutually exclusive copies of the fifth protein-coding exon detected in the neonate (5N) and adult (5A). A second pair of mutually exclusive exons is reported in SCN8A only (18N and 18A). We aimed to quantify the expression of individual exons in the developing human brain.MethodsRNA-seq data from 783 human brain samples across development were analyzed to estimate exon-level expression. Developmental changes in exon utilization were validated by assessing intron splicing. Exon expression was also estimated in RNA-seq data from 58 developing mouse neocortical samples.ResultsIn the mature human neocortex, exon 5A is consistently expressed at least 4-fold higher than exon 5N in all four genes. For SCN2A, SCN3A, and SCN8A, a brain-wide synchronized 5N to 5A transition occurs between 24 post-conceptual weeks (2nd trimester) and 6 years of age. In mice, the equivalent 5N to 5A transition begins at or before embryonic day 15.5. In SCN8A, over 90% of transcripts in the mature human cortex include exon 18A. Early in fetal development, most transcripts include 18N or skip both 18N and 18A, with a transition to 18A inclusion occurring from 13 post-conceptual weeks to 6 months of age. No other protein-coding exons showed comparably dynamic developmental trajectories.ConclusionsExon usage in SCN1A, SCN2A, SCN3A, and SCN8A changes dramatically during human brain development. These splice isoforms, which alter the biophysical properties of the encoded channels, may account for some of the observed phenotypic differences across development and between specific variants. Manipulation of the proportion of splicing isoforms at appropriate stages of development may act as a therapeutic strategy for specific mutations or even epilepsy in general.