Isoform Composition Research Articles

Identification of Giant Isoforms of Obscurin in Rat Striated Muscles Using Polyclonal Antibodies.

Using produced polyclonal antibodies specific to the N-terminal sequence (residues 61-298) of rat obscurin, we investigated the isoform composition of this protein in 4 striated muscles: myocardium of the left ventricle, diaphragm, skeletal m. gastrocnemius (containing mainly fast fibers), and m. soleus (containing mainly slow fibers). The m. gastrocnemius, m. soleus, and diaphragm were found to have 2 giant isoforms of obscurin: a smaller A-isoform and a larger B-isoform. Their molecular weights were ~870 and ~1150 kDa in the diaphragm and m. gastrocnemius and ~880 and ~1130 kDa in m. soleus, respectively. The B-isoform to A-isoform ratio was 1:3 in the diaphragm and m. soleus and 1:4 in the m. gastrocnemius. In the left-ventricular myocardium, A-isoform of obscurin with a molecular weight of ~880 kDa was found. No other obscurin isoforms or their fragments within the molecular weight range of 10 up to ~800 kDa were revealed in the investigated rat striated muscles. The antibodies produced are recommended for research into qualitative and quantitative changes of giant obscurin isoforms in rat striated muscles in the norm and during the development of pathological processes.

Differential expression of quick-to-court gene isoforms in Drosophila male and female

The quick-to-court (qtc) gene is expressed in both males and females but affects only the mating behavior of males, probably due to the different composition of isoforms in different sexes. We tested this hypothesis and examined the sex-specific expression of qtc transcripts in the tissues of male and female Oregon-R flies. It was found that some qtc transcripts, such as qtc-RM and qtc-RN, are testis-specific, while others like qtc-RH are found in ovaries but absent in testes. No sex-specific transcripts were identified in the brain, suggesting further investigation into specific brain structures may be needed. There is probably a complex regulation of qtc gene expression, which is potentially influenced by various factors in different tissues.

Single-Molecule Characterization of Heterogeneous Oligomer Formation during Co-Aggregation of 40- and 42-Residue Amyloid-β.

The two most abundant isoforms of amyloid-β (Aβ) are the 40- (Aβ40) and 42-residue (Aβ42) peptides. Since they coexist and there is a correlation between toxicity and the ratio of the two isoforms, quantitative characterization of their interactions is crucial for understanding the Aβ aggregation mechanism. In this work, we follow the aggregation of individual isoforms in a mixture using single-molecule FRET spectroscopy by labeling Aβ42 and Aβ40 with the donor and acceptor fluorophores, respectively. We found that there are two phases of aggregation. The first phase consists of coaggregation of Aβ42 with a small amount of Aβ40, while the second phase results mostly from aggregation of Aβ40. We also found that the aggregation of Aβ42 is slowed by Aβ40 while the aggregation of Aβ40 is accelerated by Aβ42 in a concentration-dependent manner. The formation of oligomers was monitored by incubating mixtures in a plate reader and performing a single-molecule free-diffusion experiment at several different stages of aggregation. The detailed properties of the oligomers were obtained by maximum likelihood analysis of fluorescence bursts. The FRET efficiency distribution is much broader than that of the Aβ42 oligomers, indicating the diversity in isoform composition of the oligomers. Pulsed interleaved excitation experiments estimate that the fraction of Aβ40 in the co-oligomers in a 1:1 mixture of Aβ42 and Aβ40 varies between 0 and 20%. The detected oligomers were mostly co-oligomers especially at the physiological ratio of Aβ42 and Aβ40 (1:10), suggesting the critical role of Aβ40 in oligomer formation and aggregation.

Alternative splicing of lncRNA LAIR fine-tunes the regulation of neighboring yield-related gene LRK1 expression in rice.

The diversity in alternative splicing of long noncoding RNAs (lncRNAs) poses a challenge for functional annotation of lncRNAs. Moreover, little is known on the effects of alternatively spliced lncRNAs on crop yield. In this study, we cloned nine isoforms resulting from the alternative splicing of the lncRNA LAIR in rice. The LAIR isoforms are generated via alternative 5'/3' splice sites and different combinations of specific introns. All LAIR isoforms activate the expression of the neighboring LRK1 gene and enhance yield-related rice traits. In addition, there are slight differences in the binding ability of LAIR isoforms to the epigenetic modification-related proteins OsMOF and OsWDR5, which affect the enrichment of H4K16ac and H3K4me3 at the LRK1 locus, and consequently fine-tune the regulation of LRK1 expression and yield-related traits. These differences in binding may be caused by polymorphic changes to the RNA secondary structure resulting from alternative splicing. It was also observed that the composition of LAIR isoforms was sensitive to abiotic stress. These findings suggest that the alternative splicing of LAIR leads to the formation of a functional transcript population that precisely regulates yield-related gene expression, which may be relevant for phenotypic polymorphism-based crop breeding under changing environmental conditions.

Characterization of neurofibrillary tangle immunophenotype signatures to classify tangle maturity in Alzheimer's disease.

Tau aggregation into neurofibrillary tangles in Alzheimer's disease (AD) is a dynamic process involving changes in tau phosphorylation, isoform composition, and morphology. To facilitate studies of tangle maturity, we developed an image analysis pipeline to study antibody labeling signatures that can distinguish tangle maturity levels in AD brain tissue. Using fluorescent immunohistochemistry, we co-labeled AD brain tissue with four antibodies that bind different tau epitopes. Mean fluorescence intensity of each antibody was measured, and spectral clustering was used to identify tangle immunophenotypes. Five distinct tangle populations were identified, and different tangle maturity immunophenotypes were identified with increasing Braak stage. Early tangle immunophenotypes were more prevalent in later affected regions and advanced immunophenotypes were associated with ghost morphology. Our findings indicate that tangle populations characterized by advanced tau immunophenotypes are associated with higher Braak stage and more mature morphology, providing a new framework for defining tangle maturity levels using tau antibody signatures. Populations of neurofibrillary tangles exist in Alzheimer's disease. The immunophenotype of neurofibrillary tangle populations relates to their maturity. The most advanced immunophenotypes are associated with higher Braak stage. The most advanced immunophenotypes are associated with ghost morphology. The most immature immunophenotypes are associated with later affected regions.

Kbtbd13R408C-knockin mouse model reveals impaired relaxation kinetics as novel pathomechanism for NEM6 cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): VENI-Nederlandse organisatie voor Wetenschappelijk Onderzoek (NWO) We identified a novel cardiomyopathy gene: KBTBD13 encoding kelch repeat and BTB (POZ) domain containing 13. The Dutch founder variant KBTBD13R408C not only affects skeletal muscle function resulting in nemaline myopathy type 6 (NEM6), but also affects the heart. In the Dutch NEM6 cohort, various cardiac abnormalities were observed, including systolic dysfunction, diastolic dysfunction, atrial fibrillation, ventricular tachycardia and three patients died of sudden cardiac death. The majority of NEM6 patients harbors the Dutch founder variant, c.1222C>T, p.Arg408Cys (KBTBD13R408C). To provide insight in the mechanism underlying cardiac dysfunction in NEM6, here we assessed cardiac structure and function in Kbtbd13R408C-knockin mice, which closely recapitulate the well characterized skeletal muscle phenotype, i.e. impaired relaxation kinetics and mitochondrial dysfunction. Pressure-volume loop analyses showed that the end-diastolic pressure-volume relation was steeper in Kbtbd13R408C-knockin mice, indicating diastolic dysfunction. Histological evaluation of cardiac structure revealed no increase in fibrosis in Kbtbd13R408C-knockin mice. Also, no alterations in titin isoform composition or myosin heavy chain composition were observed that could account for the increased diastolic stiffness. Next, we studied the contraction and relaxation kinetics in wild type (WT) and Kbtbd13R408C-knockin mice at the intact single cardiomyocyte level by a high-throughput contractility set-up. Our data showed a prolonged relaxation time in Kbtbd13R408C-knockin mice compared to WT mice, indicating impaired relaxation kinetics. No differences in the magnitude of contraction (percentage of shortening) was found between cardiomyocytes of Kbtbd13R408C-knockin and WT mice. In parallel, calcium-handling was assessed by calcium indicator Fura-2AM. These studies reveal that calcium-release is not affected, but calcium-reuptake is slower in Kbtbd13R408C-knockin mice, which might contribute to the impaired relaxation kinetics. Current studies focus on how KBTBD13R408C affects calcium-reuptake kinetics and sarcomere kinetics in NEM6 cardiomyocytes. Hence, our studies provide the first insights in the pathomechanism underlying cardiac dysfunction in NEM6.

DIP-MS: ultra-deep interaction proteomics for the deconvolution of protein complexes

Most proteins are organized in macromolecular assemblies, which represent key functional units regulating and catalyzing most cellular processes. Affinity purification of the protein of interest combined with liquid chromatography coupled to tandem mass spectrometry (AP–MS) represents the method of choice to identify interacting proteins. The composition of complex isoforms concurrently present in the AP sample can, however, not be resolved from a single AP–MS experiment but requires computational inference from multiple time- and resource-intensive reciprocal AP–MS experiments. Here we introduce deep interactome profiling by mass spectrometry (DIP-MS), which combines AP with blue-native-PAGE separation, data-independent acquisition with mass spectrometry and deep-learning-based signal processing to resolve complex isoforms sharing the same bait protein in a single experiment. We applied DIP-MS to probe the organization of the human prefoldin family of complexes, resolving distinct prefoldin holo- and subcomplex variants, complex–complex interactions and complex isoforms with new subunits that were experimentally validated. Our results demonstrate that DIP-MS can reveal proteome modularity at unprecedented depth and resolution.

B-esterases activities in several tissues of the marine fish: sea bass and hake, as potential biomarkers of bisphenol A derivatives

In the marine environment, a new threat linked to plastic pollution relates to plastic additives. This threat encompasses multiple chemical compound groups with a high bioaccumulation potential for these chemical mixtures. Hence, informative biomarkers are needed to indicate the effects of environmentally realistic mixtures of these additives. This study proposes an in vitro approach using tissue homogenates of two marine fish, the European sea bass and hake, which are both of interest in aquaculture and fisheries. The selected biomarkers are B-esterase activities comprising acetylcholinesterase (AChE) and carboxylesterases (CEs). The physiological role of AChE in brain and muscle is mainly neural transmission, while CEs participate in liver detoxification processes. However, B-esterases are also widely distributed in other tissues/organs, where their role is yet to be determined, but their inhibition may have undesired biological consequences. Here, we compared the interaction of the plastic additives, bisphenol A and some of its derivatives, like tetrabromobisphenol A (TBBPA), with B-esterase activities. We particularly focused not only on the robust and broadly distributed CE enzymes in brain, gonad, liver, kidney, and plasma tissues of two marine fish, but also on the use of two commercial substrates, p-nitrophenyl butyrate and α-naphthyl butyrate, tentatively representing two CE isoforms. The results evidenced specific species and tissue responses that could be due to a diverse isoform composition. They identified sea bass as better protected against neurotoxic exposures, at least in terms of B-esterase composition. The flame retardant TBBPA was the most reactive to B-esterases inhibition, although Bisphenol A bis (2,3-dihydroxy propyl) ether and Bisphenol F bis (3-chloro-2-hydroxypropyl) ether warrant further toxicology assessments.

Unilateral nasal obstruction mediates reversible morphological and phenotypic changes in masticatory muscles of growing rats.

Mouth breathing as a result of nasal obstruction affects craniofacial growth and development. This study aimed to investigate the effects of unilateral nasal obstruction and its recovery, along with the role of nitric oxide (NO) in masticatory muscle physiology. Forty-eight 4-week-old male rats were divided into control and experimental groups. The five experimental groups were subjected to left-sided nasal obstruction by suturing the external nostril, and the sutures were removed after 1, 3, 5, 7, or 9 weeks to allow for varying recovery periods. We assessed morphological changes in masseter, temporalis, and digastric muscle, by examining cross-sectional area (CSA) and myosin heavy chain (MHC) isoform composition of muscle fibers. Reverse transcription-quantitative real-time polymerase chain reaction to measure messenger RNA (mRNA) levels for tumor necrosis factor-α (TNF-α), glucose transporter 4 (GLUT4), and neuronal nitric oxide synthase (nNOS) were conducted. The SpO2, CSA, and fibers showing MHC-2b isoforms were significantly lower, while RT-PCR showed higher mRNA levels in TNF-α and nNOS, and a decrease in GLUT4 mRNA in the jaw-closing muscles in the long-term nasal obstruction groups than that in the control group. The study findings should be interpreted cautiously because of the functional differences between rodents and humans in terms of respiratory mechanisms. Unilateral nasal obstruction affects the morphology and contractile characteristics of the rat masticatory muscles during development, with possible involvement of NO in muscle hypofunction. These changes may revert to baseline levels if the nasal obstruction is eliminated before puberty in rats.

Mechanical interaction of myosin and native thin filament in the disused rat soleus muscle

The disuse of skeletal limb muscles occurs in a variety of conditions, yet our comprehension of the molecular mechanisms involved in adaptation to disuse remains incomplete. We studied the mechanical characteristics of actin-myosin interaction using an in vitro motility assay and isoform composition of myosin heavy and light chains by dint of SDS-PAGE in soleus muscle of both control and hindlimb-unloaded rats. 14 days of hindlimb unloading led to the increased maximum sliding velocity of actin, reconstituted, and native thin filaments over rat soleus muscle myosin by 24 %, 19 %, and 20 %, respectively. The calcium sensitivity of the “pCa-velocity” relationship decreased. There was a 26 % increase in fast myosin heavy chain IIa (MHC IIa), a 22 % increase in fast myosin light chain 2 (MLC 2f), and a 13 % increase in fast MLC 1f content. The content of MLC 1s/v, typical for slow skeletal muscles and cardiac ventricles did not change. At the same time, MLC 1s, typical only for slow skeletal muscles, disappeared. The maximum velocity of soleus muscle native thin filaments was 24 % higher compared to control ones sliding over the same rabbit myosin. Therefore, both myosin and native thin filament kinetics could influence the mechanical characteristics of the soleus muscle. Additionally, the MLC 1s and MLC 1s/v ratio may contribute to the mechanical characteristics of slow skeletal muscle, along with MHC, MLC 2, and MLC 1 slow/fast isoforms ratio.

Acute Exercise Promptly Normalizes Myocardial Myosin Heavy-Chain Isoform mRNA Composition in Diabetic Rats: Implications for Diabetic Cardiomyopathy.

Background and Objectives: The acute effects of exercise on the myosin heavy-chain (MHC) isoform mRNA expression and the upstream transcription factors in diabetic and non-diabetic hearts remain unexplored. We aimed to determine the acute effect of a single exercise session on the expression of left ventricular MHC, MHC-α and MHC-β, and thyroid receptor (TR), TR-α1 and TR-β, isoform mRNA in diabetic and non-diabetic rats. Materials and Methods: Sprague-Dawley rats were assigned to four groups: non-diabetic control (CS), diabetic exercise (DIEX), sedentary diabetic (DIS), and non-diabetic exercise (CEX). Diabetes was induced via streptozotocin injection (55 mg/kg). DIEX and CEX rats performed an exercise session (60 min at 50 m/min and 0% grade) 6-7 weeks after diabetes induction. Results: MHC-α mRNA was lower in DIS (p = 0.03) and not different in DIEX (p = 0.1) relative to CS. DIS showed higher MHC-β mRNA than the non-diabetic rats, CS and CEX (p = 0.02 and p = 0.009, respectively). MHC-β mRNA in DIEX was normalized to non-diabetic levels in CS (p = 0.3). TR-α1 was higher in DIS and not different in DIEX relative to CS and CEX (p = 0.03 and p = 1.0, respectively). In CEX, exercise did not change MHC-α, MHC-β, and TR-α1 relative to CS (p = 1.0). TR-β was not different between groups. Conclusion: In conclusion, exercise appears to acutely normalize the myocardial MHC and TR isoform mRNA expression only in the diabetic heart. These responses may induce therapeutic mechanisms other than changing the MHC isoform composition.

Adaptive restructuring of grape metabolism in winter period

In the unstable conditions of the Anapo-Taman zone of the Krasnodar Krai, the urgency of the problem of winter hardiness of grapes increases due to an increase in the average annual air temperature against the background of an increase in the frequency of low critical air temperatures in the winter. The adaptive rearrangements of grape metabolism associated with resistance to winter stresses have been studied. Objects of research: grape varieties of different ecological and geographical origin: Crystal, Dostoyny, Krasnostop AZOS, Vostorg, Aligote, Zarif. The electrophoretic separation of peroxidases in polyacrylamide gel in the studied grape varieties revealed that the quantitative and qualitative composition of isoforms changed during the winter period and depended on the variety and the influence of the stress factor. During the autumn-winter period, the varieties Crystal, Krasnostop AZOS, Vostorg revealed an increased total content of anthocyanins in (13.2-14.4 conventional units), ascorbic acid in shoots (13.7-18.4 µg/g of raw weight) in contrast to the varieties Aligote, Zarif. According to the research, it was found that the Crystal grape variety has increased frost resistance, followed in descending order by Krasnostop AZOS, Vostorg, Dostoyny. These varieties have great adaptive capabilities in unstable conditions of a changing climate and are recommended for cultivation in the Anapo-Taman zone, as well as for use in breeding as sources of frost resistance. Varieties Aligote, Zarif are singled out as less frost-resistant.

Actin Isoform Composition and Binding Factors Fine-Tune Regulatory Impact of Mical Enzymes

Actin Isoform Composition and Binding Factors Fine-Tune Regulatory Impact of Mical Enzymes

Development of a scalable single process for producing SARS-CoV-2 RBD monomer and dimer vaccine antigens.

We have developed a single process for producing two key COVID-19 vaccine antigens: SARS-CoV-2 receptor binding domain (RBD) monomer and dimer. These antigens are featured in various COVID-19 vaccine formats, including SOBERANA 01 and the licensed SOBERANA 02, and SOBERANA Plus. Our approach involves expressing RBD (319-541)-His6 in Chinese hamster ovary (CHO)-K1 cells, generating and characterizing oligoclones, and selecting the best RBD-producing clones. Critical parameters such as copper supplementation in the culture medium and cell viability influenced the yield of RBD dimer. The purification of RBD involved standard immobilized metal ion affinity chromatography (IMAC), ion exchange chromatography, and size exclusion chromatography. Our findings suggest that copper can improve IMAC performance. Efficient RBD production was achieved using small-scale bioreactor cell culture (2L). The two RBD forms - monomeric and dimeric RBD - were also produced on a large scale (500L). This study represents the first large-scale application of perfusion culture for the production of RBD antigens. We conducted a thorough analysis of the purified RBD antigens, which encompassed primary structure, protein integrity, N-glycosylation, size, purity, secondary and tertiary structures, isoform composition, hydrophobicity, and long-term stability. Additionally, we investigated RBD-ACE2 interactions, in vitro ACE2 recognition of RBD, and the immunogenicity of RBD antigens in mice. We have determined that both the monomeric and dimeric RBD antigens possess the necessary quality attributes for vaccine production. By enabling the customizable production of both RBD forms, this unified manufacturing process provides the required flexibility to adapt rapidly to the ever-changing demands of emerging SARS-CoV-2 variants and different COVID-19 vaccine platforms.

High Recovery Chromatographic Purification of mRNA at Room Temperature and Neutral pH.

Messenger RNA (mRNA) is becoming an increasingly important therapeutic modality due to its potential for fast development and platform production. New emerging RNA modalities, such as circular RNA, drive the need for the development of non-affinity purification approaches. Recently, the highly efficient chromatographic purification of mRNA was demonstrated with multimodal monolithic chromatography media (CIM® PrimaS), where efficient mRNA elution was achieved with an ascending pH gradient approach at pH 10.5. Here, we report that a newly developed chromatographic material enables the elution of mRNA at neutral pH and room temperature. This material demonstrates weak anion-exchanging properties and an isoelectric point of 5.3. It enables the baseline separation of mRNA (at least up to 10,000 nucleotides (nt) in size) from parental plasmid DNA (regardless of isoform composition) with both a NaCl gradient and ascending pH gradient approach, while mRNA elution is achieved in a pH range of 5-7. In addition, the basic structure of the novel material is a chromatographic monolith, enabling convection-assisted mass transfer of large RNA molecules to and from the active surface. This facilitates the elution of mRNA in 3-7 column volumes with more than 80% elution recovery and uncompromised integrity. This is demonstrated by the purification of a model mRNA (size 995 nt) from an in vitro transcription reaction mixture. The purified mRNA is stable for at least 34 days, stored in purified H2O at room temperature.

Worldwide Population Genomics Reveal Long-Term Stability of the Mitochondrial Genome Architecture in a Keystone Marine Plant.

Mitochondrial genomes (mitogenomes) of flowering plants are composed of multiple chromosomes. Recombination within and between the mitochondrial chromosomes may generate diverse DNA molecules termed isoforms. The isoform copy number and composition can be dynamic within and among individual plants due to uneven replication and homologous recombination. Nonetheless, despite their functional importance, the level of mitogenome conservation within species remains understudied. Whether the ontogenetic variation translates to evolution of mitogenome composition over generations is currently unknown. Here we show that the mitogenome composition of the seagrass Zostera marina is conserved among worldwide populations that diverged ca. 350,000 years ago. Using long-read sequencing, we characterized the Z. marina mitochondrial genome and inferred the repertoire of recombination-induced configurations. To characterize the mitochondrial genome architecture worldwide and study its evolution, we examined the mitogenome in Z. marina meristematic region sampled in 16 populations from the Pacific and Atlantic oceans. Our results reveal a striking similarity in the isoform relative copy number, indicating a high conservation of the mitogenome composition among distantly related populations and within the plant germline, despite a notable variability during individual ontogenesis. Our study supplies a link between observations of dynamic mitogenomes at the level of plant individuals and long-term mitochondrial evolution.

Myosin expression and contractile function are altered by replating stem cell-derived cardiomyocytes.

Myosin heavy chain (MyHC) is the main determinant of contractile function. Human ventricular cardiomyocytes (CMs) predominantly express the β-isoform. We previously demonstrated that ∼80% of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) express exclusively β-MyHC after long-term culture on laminin-coated glass coverslips. Here, we investigated the impact of enzymatically detaching hESC-CMs after long-term culture and subsequently replating them for characterization of cellular function. We observed that force-related kinetic parameters, as measured in a micromechanical setup, resembled α- rather than β-MyHC-expressing myofibrils, as well as changes in calcium transients. Single-cell immunofluorescence analysis revealed that replating hESC-CMs led to rapid upregulation of α-MyHC, as indicated by increases in exclusively α-MyHC- and in mixed α/β-MyHC-expressing hESC-CMs. A comparable increase in heterogeneity of MyHC isoform expression was also found among individual human induced pluripotent stem cell (hiPSC)-derived CMs after replating. Changes in MyHC isoform expression and cardiomyocyte function induced by replating were reversible in the course of the second week after replating. Gene enrichment analysis based on RNA-sequencing data revealed changes in the expression profile of mechanosensation/-transduction-related genes and pathways, especially integrin-associated signaling. Accordingly, the integrin downstream mediator focal adhesion kinase (FAK) promoted β-MyHC expression on a stiff matrix, further validating gene enrichment analysis. To conclude, detachment and replating induced substantial changes in gene expression, MyHC isoform composition, and function of long-term cultivated human stem cell-derived CMs, thus inducing alterations in mechanosensation/-transduction, that need to be considered, particularly for downstream in vitro assays.

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.

Phytosterol-mediated glycerosomes combined with peppermint oil enhance transdermal delivery of lappaconitine by modulating the lipid composition of the stratum corneum.

Although the introduction of glycerosomes has enriched strategies for efficient transdermal drug delivery, the inclusion of cholesterol as a membrane stabilizer has limited their clinical application. The current study describes the development and optimization of a new type of glycerosome (S-glycerosome) that is formed in glycerol solution with β-sitosterol as the stabilizer. Moreover, the transdermal permeation properties of lappaconitine (LA)-loaded S-glycerosomes and peppermint oil (PO)-mediated S-glycerosomes (PO-S-glycerosomes) are evaluated, and the lipid alterations in the stratum corneum are analyzed via lipidomics. The LA-loaded S-glycerosomes prepared by the preferred formulation from the uniform design have a mean size of 145.3 ± 7.81nm and an encapsulation efficiency of 73.14 ± 0.35%. Moreover, the addition of PO positively impacts transdermal flux, peaking at 0.4% (w/v) PO. Tracing of the fluorescent probe P4 further revealed that PO-S-glycerosomes penetrate deeper into the skin than S-glycerosomes and conventional liposomes. Additionally, treatment with PO-S-glycerosomes alters the isoform type, number, and composition of sphingolipids, glycerophospholipids, glycerolipids, and fatty acids in the stratum corneum, with the most notable effect observed for ceramides, the main component of sphingolipids. Furthermore, the transdermal administration of LA-loaded PO-S-glycerosomes improved the treatment efficacy of xylene-induced inflammation in mice without skin irritation. Collectively, these findings demonstrate the feasibility of β-sitosterol as a stabilizer in glycerosomes. Additionally, the inclusion of PO improves the transdermal permeation of S-glycerosomes, potentially by altering the stratum corneum lipids.

Ca2+ Dynamics of Gap Junction Coupled and Uncoupled Deiters' Cells in the Organ of Corti in Hearing BALB/c Mice.

ATP, as a paracrine signalling molecule, induces intracellular Ca2+ elevation via the activation of purinergic receptors on the surface of glia-like cochlear supporting cells. These cells, including the Deiters' cells (DCs), are also coupled by gap junctions that allow the propagation of intercellular Ca2+ waves via diffusion of Ca2+ mobilising second messenger IP3 between neighbouring cells. We have compared the ATP-evoked Ca2+ transients and the effect of two different gap junction (GJ) blockers (octanol and carbenoxolone, CBX) on the Ca2+ transients in DCs located in the apical and middle turns of the hemicochlea preparation of BALB/c mice (P14-19). Octanol had no effect on Ca2+ signalling, while CBX inhibited the ATP response, more prominently in the middle turn. Based on astrocyte models and using our experimental results, we successfully simulated the Ca2+ dynamics in DCs in different cochlear regions. The mathematical model reliably described the Ca2+ transients in the DCs and suggested that the tonotopical differences could originate from differences in purinoceptor and Ca2+ pump expressions and in IP3-Ca2+ release mechanisms. The cochlear turn-dependent effect of CBX might be the result of the differing connexin isoform composition of GJs along the tonotopic axis. The contribution of IP3-mediated Ca2+ signalling inhibition by CBX cannot be excluded.