Heavy Chain mRNA Research Articles

Expression of nonmuscle myosin IIC is regulated by non-canonical binding activity of miRNAs

The expression of mechanoresponsive nonmuscle myosin II (NMII)C is found to be inducible during tumor progression, but its mechanism is yet to be explored. Here, we report a group of microRNAs (mmu-miR-200a-5p, mmu-miR-532-3p, mmu-miR-680, and mmu-miR-1901) can significantly repress the expression of nonmuscle myosin IIC (NMIIC). Interestingly, these microRNAs have both canonical and non-canonical binding sites at 3/UTR and coding sequence (CDS) of NMIIC's heavy chain (HC) mRNA. Each of the miRNA downregulates NMHC-IIC to a different degree as assessed by dual-luciferase and immunoblot analyses. When we abolish the complementary base pairing at canonical binding site, mmu-miR-532-3p can still bind at non-canonical binding site and form Argonaute2 (AGO2)-miRNA complex to downregulate the expression of NMIIC. Modulating the expression of NMIIC by miR-532-3p in mouse mammary tumor cells, 4T1, increases its tumorigenic potential both invitro and invivo. Together, these studies provide the functional role of miRNA's non-canonical binding mediated NMIIC regulation in tumor cells.

Role of p53 in Cisplatin-Induced Myotube Atrophy.

Chemotherapy-induced sarcopenia is an unfavorable prognostic factor implicated in the development of postoperative complications and reduces the quality of life of patients with cancer. Skeletal muscle loss due to cisplatin use is caused by mitochondrial dysfunction and activation of muscle-specific ubiquitin ligases Atrogin-1 and muscle RING finger 1 (MuRF1). Although animal studies suggest the involvement of p53 in age-, immobility-, and denervation-related muscle atrophy, the association between cisplatin-induced atrophy and p53 remains unknown. Herein, we investigated the effect of a p53-specific inhibitor, pifithrin-alpha (PFT-α), on cisplatin-induced atrophy in C2C12 myotubes. Cisplatin increased the protein levels of p53, phosphorylated p53, and upregulated the mRNA expression of p53 target genes PUMA and p21 in C2C12 myotubes. PFT-α ameliorated the increase in intracellular reactive oxygen species production and mitochondrial dysfunction, and also reduced the cisplatin-induced increase in the Bax/Bcl-2 ratio. Although PFT-α also reduced the cisplatin-induced increase in MuRF1 and Atrogin-1 gene expression, it did not ameliorate the decrease in myosin heavy chain mRNA and protein levels and muscle-specific actin and myoglobin protein levels. We conclude that cisplatin increases muscle degradation in C2C12 myotubes in a p53-dependent manner, but p53 has minimal involvement in the reduction of muscle protein synthesis.

Immune response of large yellow croaker Larimichthys crocea towards a recombinant vaccine candidate targeting the parasitic ciliate Cryptocaryon irritans.

Cryptocaryon irritans, a parasitic ciliate, pose a major threat to marine teleost fish aquaculture. So far, no effective and safe control method is available. In this study, the protective efficacy of a recombinant truncated surface antigen of C. irritans (rCiSA32.6t) for large yellow croaker (Larimichthys crocea) against the parasite challenge with a sub-lethal dose of the infective theronts was evaluated by comparing the relative percent survivals (RPS), the specific antibody titers in sera, and the expression levels of the immune-related genes among the negative or adjuvant control fish, fish intraperitoneally immunized with rCiSA32.6t. The results showed that a RPS of 50.1% in rCiSA32.6t-immunized fish was achieved in comparison to negative control fish against C. irritans. A significant increase was noted in the antigen-specific immunoglobulin M (IgM) and immunoglobulin T (IgT) antibody levels in the sera of the rCiSA32.6t-vaccinated fish. Compared to the negative control fish, quantitative real-time PCR analysis indicated that the interleukin-1beta, IgT, and IgM heavy chain mRNA level in the fish head kidney, spleen, gill, and skin tissue were upregulated post-rCiSA32.6t immunization. This study indicates that the rCiSA32.6t can provide a high level of immune protection against C. irritans infection in grouper and is therefore pursued as a candidate C. irritans vaccine.

UNDETECTABLE IGE AS A SENTINEL BIOMARKER FOR HUMORAL IMMUNODEFICIENCY

<h3>Introduction</h3> The clinical significance of undetectable IgE (<2 IU/mL) in patients with otherwise normal immunoglobulins is unclear. We hypothesize that undetectable IgE reflects an impairment in isotype switching that could portend future development of a broader defect in humoral immunity. <h3>Methods</h3> We recruited 17 healthy adults with IgE <2 IU/mL. We compared their response to immunization with a Salmonella typhi polysaccharide vaccine relative to 16 randomly selected adults with IgE >2 IU/mL. We assessed pre-and post-vaccine IgG titers 4-6 weeks after vaccination, with a normal Salmonella typhi IgG index (STIGG index) defined as a >2-fold increase in titers. We isolated peripheral blood lymphocytes from 14 patients with undetectable IgE and induced IgE production <i>in vitro</i> via stimulation with CD40 ligand (CD40L) and IL-4. IgE production was assessed through semi-quantitative PCR of ε-germline and IgE heavy chain transcripts. <h3>Results</h3> 4/17 patients with undetectable IgE had a STIGG index ≤2, compared with 0/16 patients in the control group (p-value = 0.0582, Fisher's exact test). ε-germline and IgE heavy chain mRNA were detected in peripheral lymphocytes from all of the assessed patients with undetectable IgE. <h3>Conclusion</h3> An impaired polysaccharide vaccine response occurred more frequently in patients with undetectable IgE than in patients with an IgE >2 IU/mL, but the difference in this small pilot study was nonsignificant. A larger sample size is needed to clarify these findings. Peripheral blood lymphocytes isolated from patients with undetectable IgE are capable of producing IgE transcripts in response to CD40L/IL-4 stimulation.

Detection of intracellular sodium ions based on phenotype-specific activation of NaA43 DNAzyme

The intracellular sodium ion is one of the crucial elements for regulating physiological functions such as action potential and muscle contractions. However, detecting sodium ions in live cells is challenging because false signals may arise from the abundant sodium ions in the extracellular environment when introducing the detection agents. To minimize it, we report a DNAzyme-based detection of sodium ions in live cells via activation by endogenous mRNA. The substrate strand of DNAzyme first hybridizes to a blocking strand that prevents undesired cleavage of DNAzyme when delivered. Once entering cells, an endogenous mRNA biomarker binds to the toehold region of the blocking strand and displaces it, allowing the proper formation of the DNAzyme, which specifically catalyzes the cleavage of the substrate strand in the presence of intracellular Na+ and produces fluorescence signals. Using differentiating skeletal muscle cells as the model system, we demonstrated the successful delivery and phenotype-specific detection of intracellular sodium ions only in differentiated myotubes with highly-expressed myosin heavy chain mRNA. Moreover, using a drug cocktail to increase the permeability of the cell membrane, elevated levels of intracellular sodium ion was observed. This platform offers a broad and promising strategy for detecting intracellular metal ions, suggesting a great potential for understanding its role in cell/tissue physiology.

Puerarin attenuates isoproterenol‑induced myocardial hypertrophy via inhibition of the Wnt/β‑catenin signaling pathway.

Myocardial hypertrophy (MH) is an independent risk factor for cardiovascular disease, which in turn lead to arrhythmia or heart failure. Therefore, attention must be paid to formulation of therapeutic strategies for MH. Puerarin is a key bioactive ingredient isolated from Pueraria genera of plants that is beneficial for the treatment of MH. However, its molecular mechanism of action has not been fully determined. In the present study, 40 µM puerarin was demonstrated to be a safe dose for human AC16 cells using Cell Counting Kit-8 assay. The protective effects of puerarin against MH were demonstrated in AC16 cells stimulated with isoproterenol (ISO). These effects were characterized by a significant decrease in surface area of cells (assessed using fluorescence staining) and mRNA and protein expression levels of MH-associated biomarkers, including atrial and brain natriuretic peptide, assessed using reverse transcription-quantitative PCR and western blotting, as well as β-myosin heavy chain mRNA expression levels. Mechanistically, western blotting demonstrated that puerarin inhibited activation of the Wnt signaling pathway. Puerarin also significantly decreased phosphorylation of p65; this was mediated via crosstalk between the Wnt and NF-κB signaling pathways. An inhibitor (Dickkopf-1) and activator (IM-12) of the Wnt signaling pathway were used to demonstrate that puerarin-mediated effects alleviated ISO-induced MH via the Wnt signaling pathway. The results of the present study demonstrated that puerarin pre-treatment may be a potential therapeutic strategy for preventing ISO-induced MH and managing MH in the future.

Antigen-derived peptides engage the ER stress sensor IRE1α to curb dendritic cell cross-presentation.

Dendritic cells (DCs) promote adaptive immunity by cross-presenting antigen-based epitopes to CD8+ T cells. DCs process internalized protein antigens into peptides that enter the endoplasmic reticulum (ER), bind to major histocompatibility type I (MHC-I) protein complexes, and are transported to the cell surface for cross-presentation. DCs can exhibit activation of the ER stress sensor IRE1α without ER stress, but the underlying mechanism remains obscure. Here, we show that antigen-derived hydrophobic peptides can directly engage ER-resident IRE1α, masquerading as unfolded proteins. IRE1α activation depletes MHC-I heavy-chain mRNAs through regulated IRE1α-dependent decay (RIDD), curtailing antigen cross-presentation. In tumor-bearing mice, IRE1α disruption increased MHC-I expression on tumor-infiltrating DCs and enhanced recruitment and activation of CD8+ T cells. Moreover, IRE1α inhibition synergized with anti-PD-L1 antibody treatment to cause tumor regression. Our findings identify an unexpected cell-biological mechanism of antigen-driven IRE1α activation in DCs, revealing translational potential for cancer immunotherapy.

Correlation between Meat Quality and Muscle Fiber Types of Tibetan and Large White Pigs

This research was designed to explore the differences of meat quality and their causes of pigs in different regions, In this study, natural raised Tibetan pigs in different regions (Linzhi of Tibet, Gannan of Gansu, and Aba of Sichuan) and large white pigs were selected, the meat quality of their longissimus muscle (pH, color, cooking loss, shear force), myoglobin concentration, hydroxyproline content, relative expression of different Myosin Heavy Chain (MYHC) mRNA were determined, and correlation analysis were adopted. The results showed that the pH3h of Tibetan pigs was significantly lower than that of large white pigs 6.0 (P<0.05), and there was no significant difference among different regions (5.7~5.8),The redness value (a*) and myoglobin content of Tibetan pigs were significantly higher than those of large white pigs (2.12±0.87, 0.004 μmol/g). Among them, Aba Tibetan pigs had the highest a* (8.10±0.99). The shear force and hydroxyproline content of Tibetan pigs were significantly higher than those of large white pigs (41.81±0.83、5.92±0.52%) (P<0.05), the proportion of type IIa muscle fibers in Tibetan pigs was significantly higher and the proportion of type IIb muscle fibers is significantly lower than their of large white pigs (P<0.05); correlation analysis shown that a* was significantly negatively correlated with L* and type IIb muscle fibers (P<0.01), and significantly positively correlated with myoglobin content, type I and type IIa muscle fibers (P<0.01). Shear force and hydroxyproline content were extremely significantly correlated (P<0.01). In conclusion, the color of Tibetan pig is redder and the tenderness was worse than that of large white pig, the great differences between Tibetan pigs and large white pigs and the differences among Tibetan pigs from different regions in meat quality are highly related to their muscle fiber type.

Cytoplasmic factories for axonemal dynein assembly.

Axonemal dyneins power the beating of motile cilia and flagella. These massive multimeric motor complexes are assembled in the cytoplasm, and subsequently trafficked to cilia and incorporated into the axonemal superstructure. Numerous cytoplasmic factors are required for the dynein assembly process, and, in mammals, defects lead to primary ciliary dyskinesia, which results in infertility, bronchial problems and failure to set up the left-right body axis correctly. Liquid-liquid phase separation (LLPS) has been proposed to underlie the formation of numerous membrane-less intracellular assemblies or condensates. In multiciliated cells, cytoplasmic assembly of axonemal dyneins also occurs in condensates that exhibit liquid-like properties, including fusion, fission and rapid exchange of components both within condensates and with bulk cytoplasm. However, a recent extensive meta-analysis suggests that the general methods used to define LLPS systems in vivo may not readily distinguish LLPS from other mechanisms. Here, I consider the time and length scales of axonemal dynein heavy chain synthesis, and the possibility that during translation of dynein heavy chain mRNAs, polysomes are crosslinked via partially assembled proteins. I propose that axonemal dynein factory formation in the cytoplasm may be a direct consequence of the sheer scale and complexity of the assembly process itself.

YTHDF2 alleviates cardiac hypertrophy via regulating Myh7 mRNA decoy

BackgroundPathological cardiac hypertrophy is a major contributor of heart failure (HF), which seriously threatens human’s health world widely. Deregulation of m6A RNA methylation, and m6A methyltransferases and de-methyltransferases have been demonstrated to act essential roles in cardiac hypertrophy and HF. Here, we studied the potential roles and its underlying mechanisms of m6A Reader YTHDF proteins in HF. In this study, we constructed HF mouse model by transverse aortic constriction surgery. Primary cardiomyocytes were isolated and stimulated with isoproterenol (ISO) or phenylephrine (PHE) to induce myocardial hypertrophy.ResultsThrough single-cell RNA-seq analysis, immunofluorescent staining, HE staining, Western blotting, and real time-PCR detections, we found that YTHDF2 mRNA and protein level, but not YTHDF1 or YTHDF3, was significantly increased during HF development. YTHDF2 overexpression could efficiently alleviate cardiac hypertrophy. Furthermore, through immunoprecipitation accompanied with mass spectrometry analysis, Gene Ontology (GO) analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, we found that ISO stimulation did not evidently affect YTHDF2-interacting proteins. However, ISO or PHE stimulation significantly increased YTHDF2 protein interacting with Myh7 (beta-myosin heavy chain) mRNA, an important cardiac hypertrophy marker, in an m6A-dependent manner. Knockdown of Myh7 or deletion of the YTH domain of YTHDF2 reversed the protective effects of YTHDF2 on cardiac hypertrophy. Finally, we found that ISO or PHE stimulation promoted YTHDF2 protein expression through enhancing Ythdf2 mRNA stability in an m6A-dependent manner in cardiomyocytes.ConclusionsOverall, our results indicate that the m6A Reader YTHDF2 suppresses cardiac hypertrophy via Myh7 mRNA decoy in an m6A-dependent manner. This study highlights the functional importance of YTHDF2-dependent cardiac m6A mRNA regulation during cardiac hypertrophy, and provides a novel mechanistic insight into the therapeutic mechanisms of YTHDF2.

Preliminary Investigation of In vitro, Bidirectional Vocal Fold Muscle-Mucosa Interactions.

Oversimplified clinical dogma suggests that laryngeal diseases fall into two broad, mutually exclusive diagnostic categories-mucosal injury or neuromuscular/functional disorders. Extensive investigation in the lower airway as well as other organ systems suggest complex interactions between tissue types underlying both tissue health and pathological states. To date, no such relationship has been described in the vocal folds, likely the most bioactive organ in the body. We hypothesize interactions between the vocal fold muscle and mucosa likely contribute to aberrant phonatory physiology and warrant further investigation to ultimately develop novel therapeutic strategies. Primary culture of myoblasts from rat thyroarytenoid muscle and fibroblasts from the vocal fold mucosa were established. Co-culture and conditioned media experiments were performed to established bidirectional interactions between cell types. Transforming Growth Factor (TGF)-β was employed to stimulate a fibrotic phenotype in culture. In addition to quantitative PCR, standard migration and proliferation assays were performed as well as immunocytochemistry. Bidirectional cell-cell interactions were observed. Without TGF-β stimulation, myoblast conditioned media inhibited fibroblast migration, but enhanced proliferation. Conversely, fibroblast conditioned media increased both myoblast proliferation and migration. Myoblast conditioned media decreased TGF-β-mediated gene expression and of particular interest, ACTA2 mRNA expression. In both co-culture and in response to fibroblast conditioned media, myosin heavy chain (Myh2) mRNA expression decreased in myoblasts. These data are the first to describe interactions between cell types within the vocal fold. The implications for these interactions in vivo warrant further investigation to develop and refine optimal treatment strategies.

Decreased vascular smooth muscle contractility in Hutchinson\u2013Gilford Progeria Syndrome linked to defective smooth muscle myosin heavy chain expression

Children with Hutchinson–Gilford Progeria Syndrome (HGPS) suffer from multiple cardiovascular pathologies due to the expression of progerin, a mutant form of the nuclear envelope protein Lamin A. Progerin expression has a dramatic effect on arterial smooth muscle cells (SMCs) and results in decreased viability and increased arterial stiffness. However, very little is known about how progerin affects SMC contractility. Here, we studied the LaminAG609G/G609G mouse model of HGPS and found reduced arterial contractility at an early age that correlates with a decrease in smooth muscle myosin heavy chain (SM-MHC) mRNA and protein expression. Traction force microscopy on isolated SMCs from these mice revealed reduced force generation compared to wild-type controls; this effect was phenocopied by depletion of SM-MHC in WT SMCs and overcome by ectopic expression of SM-MHC in HGPS SMCs. Arterial SM-MHC levels are also reduced with age in wild-type mice and humans, suggesting a common defect in arterial contractility in HGPS and normal aging.

Utilization of the human gamma-satellite insulator for the enhancement of anti-PCSK9 monoclonal antibody expression in Chinese hamster ovary cells.

Monoclonal antibodies (mAbs) are widely employed as invaluable therapeutics for a vast number of human disorders. Several approaches have been introduced for the improvement of mAb production in Chinese hamster ovary (CHO) cells due to the increasing demand for these products. In this regard, various chromatin-modifying elements such as insulators have been incorporated in the expression vectors to augment mAb expression. In this study, human gamma-satellite insulator containing vectors were utilized for the expression of an anti-proprotein convertase subtilisin/kexin type 9 (PCSK9) mAb in CHO-K1 cells. To this aim, dual expression vectors encoding the antibody light chain (LC) and heavy chain (HC) with or without the insulator element were constructed, and mAb expression was evaluated in transient and stable expression. Based on the results, mAb expression significantly increased in the stable cell pool, and clonal cells developed using the human gamma-satellite insulator. In contrast, transient antibody expression was not affected by the insulator element. Finally, the enhancement of LC and HC mRNA levels was found in the insulator containing stable cell pools using the quantitative real-time-polymerase chain reaction (qRT-PCR). Our findings showed the positive effect of the human gamma-satellite insulator on the stable expression of an anti-PCSK9 immunoglobulin G1 (IgG1) mAb in CHO-K1 cells using dual expression vectors.

Smyd1 is essential for myosin expression and sarcomere organization in craniofacial, extraocular, and cardiac muscles

Skeletal and cardiac muscles are striated myofibers that contain highly organized sarcomeres for muscle contraction. Recent studies revealed that Smyd1, a lysine methyltransferase, plays a key role in sarcomere assembly in heart and trunk skeletal muscles. However, Smyd1 expression and function in craniofacial muscles are not known. Here, we analyze the developmental expression and function of two smyd1 paralogous genes, smyd1a and smyd1b, in craniofacial and cardiac muscles of zebrafish embryos. Our data show that loss of smyd1a (smyd1amb5) or smyd1b (smyd1bsa15678) has no visible effects on myogenic commitment and expression of myod and myosin heavy-chain mRNA transcripts in craniofacial muscles. However, myosin heavy-chain protein accumulation and sarcomere organization are dramatically reduced in smyd1bsa15678 single mutant, and almost completely diminish in smyd1amb5; smyd1bsa15678 double mutant, but not in smyd1amb5 mutant. Similar defects are also observed in cardiac muscles of smyd1bsa15678 mutant. Defective craniofacial and cardiac muscle formation is associated with an upregulation of hsp90α1 and unc45b mRNA expression in smyd1bsa15678 and smyd1amb5; smyd1bsa15678 mutants. Together, our studies indicate that Smyd1b, but not Smyd1a, plays a key role in myosin heavy-chain protein expression and sarcomere organization in craniofacial and cardiac muscles. Loss of smyd1b results in muscle-specific stress response.

PUFA Treatment Affects C2C12 Myocyte Differentiation, Myogenesis Related Genes and Energy Metabolism.

Polyunsaturated fatty acids (PUFAs) are the main components of cell membrane affecting its fluidity, signaling processes and play a vital role in muscle cell development. The effects of docosahexaenoic acid (DHA) on myogenesis are well known, while the effects of arachidonic acid (AA) are largely unclear. The purpose of this study is to evaluate the effect of two PUFAs (DHA and AA) on cell fate during myogenic processes, Wnt signaling and energy metabolism by using the C2C12 cells. The cells were treated with different concentrations of AA or DHA for 48 h during the differentiation period. PUFA treatment increased mRNA level of myogenic factor 5 (Myf5), which is involved in early stage of myoblast proliferation. Additionally, PUFA treatment prevented myoblast differentiation, indicated by decreased myotube fusion index and differentiation index in parallel with reduced mRNA levels of myogenin (MyoG). After PUFA withdrawal, some changes in cell morphology and myosin heavy chain mRNA levels were still observed. Expression of genes associated with Wnt signaling pathway, and energy metabolism changed in PUFA treatment in a dose and time dependent manner. Our data suggests that PUFAs affect the transition of C2C12 cells from proliferation to differentiation phase by prolonging proliferation and preventing differentiation.

Beneficial Effects of a Mixture of Algae and Extra Virgin Olive Oils on the Age-Induced Alterations of Rodent Skeletal Muscle: Role of HDAC-4.

Aging is associated with a progressive decline in skeletal muscle mass, strength and function (sarcopenia). We have investigated whether a mixture of algae oil (25%) and extra virgin olive oil (75%) could exert beneficial effects on sarcopenia. Young (3 months) and old (24 months) male Wistar rats were treated with vehicle or with the oil mixture (OM) (2.5 mL/kg) for 21 days. Aging decreased gastrocnemius weight, total protein, and myosin heavy chain mRNA. Treatment with the OM prevented these effects. Concomitantly, OM administration decreased the inflammatory state in muscle; it prevented the increase of pro-inflammatory interleukin-6 (IL-6) and the decrease in anti-inflammatory interleukin-10 (IL-10) in aged rats. The OM was not able to prevent aging-induced alterations in either the insulin-like growth factor I/protein kinase B (IGF-I/Akt) pathway or in the increased expression of atrogenes in the gastrocnemius. However, the OM prevented decreased autophagy activity (ratio protein 1A/1B-light chain 3 (LC3b) II/I) induced by aging and increased expression of factors related with muscle senescence such as histone deacetylase 4 (HDAC-4), myogenin, and IGF-I binding protein 5 (IGFBP-5). These data suggest that the beneficial effects of the OM on muscle can be secondary to its anti-inflammatory effect and to the normalization of HDAC-4 and myogenin levels, making this treatment an alternative therapeutic tool for sarcopenia.

Exercise Training and Vitamin C Supplementation Affects Ferritin mRNA in Leukocytes without Affecting Prooxidative/Antioxidative Balance in Elderly Women

Physical training and antioxidant supplementation may influence iron metabolism through reduced oxidative stress and subsequent lowering of mRNA levels of genes that are easily induced by this stress, including those responsible for iron homeostasis. Fifteen elderly women participated in our 12-week experiment, involving six weeks of training without supplementation and six weeks of training supported by oral supplementation of 1000 mg of vitamin C daily. The participants were divided into two groups (n = 7 in group 1 and n = 8 in group 2). In group 1, we applied vitamin C supplementation in the first six weeks of training, while in group 2 during the remaining six weeks of training. In both phases, the health-related training occurred three times per week. Training accompanied by vitamin C supplementation did not affect prooxidative/antioxidative balance but significantly decreased ferritin heavy chain (FTH) and ferritin light chain (FTL) mRNA in leukocytes (for FTH mRNA from 2^64.24 to 2^11.06, p = 0.03 in group 1 and from 2^60.54 to 2^16.03, p = 0.01 in group 2, for FTL mRNA from 2^20.22 to 2^4.53, p = 0.01 in group 2). We concluded that vitamin C supplementation might have caused a decrease in gene expression of two important antioxidative genes (FTH, FTL) and had no effect on plasma prooxidative/antioxidative balance.

High Levels of Class I Major Histocompatibility Complex mRNA Are Present in Epstein-Barr Virus-Associated Gastric Adenocarcinomas.

Epstein–Barr virus (EBV) is responsible for approximately 9% of stomach adenocarcinomas. EBV-encoded microRNAs have been reported as reducing the function of the class I major histocompatibility complex (MHC-I) antigen presentation apparatus, which could allow infected cells to evade adaptive immune responses. Using data from nearly 400 human gastric carcinomas (GCs), we assessed the impact of EBV on MHC-I heavy and light chain mRNA levels, as well as multiple other components essential for antigen processing and presentation. Unexpectedly, mRNA levels of these genes were as high, or higher, in EBV-associated gastric carcinomas (EBVaGCs) compared to normal control tissues or other GC subtypes. This coordinated upregulation could have been a consequence of the higher intratumoral levels of interferon γ in EBVaGCs, which correlated with signatures of increased infiltration by T and natural killer (NK) cells. These results indicate that EBV-encoded products do not effectively reduce mRNA levels of the MHC-I antigen presentation apparatus in human GCs.

Allele-Selective Knockdown of MYH7 Using Antisense Oligonucleotides

Hundreds of dominant-negative myosin mutations have been identified that lead to hypertrophic cardiomyopathy, and the biomechanical link between mutation and disease is heterogeneous across this patient population. To increase the therapeutic feasibility of treating this diverse genetic population, we investigated the ability of locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) to selectively knock down mutant myosin transcripts by targeting single-nucleotide polymorphisms (SNPs) that were found to be common in the myosin heavy chain 7 (MYH7) gene. We identified three SNPs in MYH7 and designed ASO libraries to selectively target either the reference or alternate MYH7 sequence. We identified ASOs that selectively knocked down either the reference or alternate allele at all three SNP regions. We also show allele-selective knockdown in a mouse model that was humanized on one allele. These results suggest that SNP-targeting ASOs are a promising therapeutic modality for treating cardiac pathology.

Longitudinal analysis of the antibody repertoire of a Zika virus-infected patient revealed dynamic changes in antibody response

ABSTRACT The Zika virus (ZIKV) is a mosquito-borne flavivirus that causes neonatal abnormalities and other disorders. Antibodies to the ZIKV envelope (E) protein can block infection. In this study, next-generation sequencing (NGS) of immunoglobulin heavy chain (IgH) mRNA transcripts was combined with single-cell PCR cloning of E-binding monoclonal antibodies for analysing antibody response in a patient from the early stages of infection to more than one year after the clearance of the virus. The patient's IgH repertoire 14 and 64 days after symptom onset showed dramatic dominant clonal expansion but low clonal diversity. IgH repertoire 6 months after disease-free status had few dominant clones but increased diversity. E-binding antibodies appeared abundantly in the repertoire during the early stages of infection but quickly declined after clearance of the virus. Certain VH genes such as VH5-10-1 and VH4-39 appeared to be preferentially enlisted for a rapid antibody response to ZIKV infection. Most of these antibodies require relatively few somatic hypermutations to acquire the ability to bind to the E protein, pointing to a possible mechanism for rapid defence against ZIKV infection. This study provides a unique and holistic view of the dynamic changes and characteristics of the antibody response to ZIKV infection.