Isoform Shift Research Articles

Progerin mRNA expression in non-HGPS patients is correlated with widespread shifts in transcript isoforms.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a premature aging disease caused primarily by a C1824T mutation in LMNA. This mutation activates a cryptic splice donor site, producing a lamin variant called progerin. Interestingly, progerin has also been detected in cells and tissues of non-HGPS patients. Here, we investigated progerin expression using publicly available RNA-seq data from non-HGPS patients in the GTEx project. We found that progerin expression is present across all tissue types in non-HGPS patients and correlated with telomere shortening in the skin. Transcriptome-wide correlation analyses suggest that the level of progerin expression is correlated with switches in gene isoform expression patterns. Differential expression analyses show that progerin expression is correlated with significant changes in genes involved in splicing regulation and mitochondrial function. Interestingly, 5' splice sites whose use is correlated with progerin expression have significantly altered frequencies of consensus trinucleotides within the core 5' splice site. Furthermore, introns whose alternative splicing correlates with progerin have reduced GC content. Our study suggests that progerin expression in non-HGPS patients is part of a global shift in splicing patterns.

Molecular mechanisms of PI3K isoform dependence in embryonic growth.

The phosphoinositide 3-kinase (PI3K) pathway is an important signaling mechanism for cell proliferation and metabolism. Mutations that activate PIK3CA may make cells p110α dependent, but when phosphatase tensin homolog (PTEN) is lost, the p110β isoform of PI3Ks becomes more important. However, the exact mechanism underlying the prevalence of p110s remains unclear. In this study, our aim was to elucidate the processes behind PI3K isoform dependency in a cellular model of embryonic development. In order to understand PI3K isoform prevalence, mouse embryonic fibroblasts (MEFs) were used and p110β, PTEN and Rac1 activity was modulated using retroviral plasmids. Expression levels and cellular growth were assessed by performing immunoblots and crystal violet assays. The levels of PTEN had only a partial effect on the prevalence of PI3K isoforms in MEFs. The dependency on p110α diminished when PTEN was depleted. Of note, when PTEN expression was repressed, there was no full transition in dependency from one PI3K isoform to the other. Interestingly, the viability of PTEN-depleted MEFs became less dependent on p110α and more dependent on p110β when p110β was overexpressed. Nevertheless, the overexpression of p110β in conjunction with PTEN knock-downs did not result in a complete shift of isoforms in PI3Ks. Finally, we investigated Rac1 activation with a mutant allele and determined a more potent increase in p110β prominence in MEFs. These findings suggest that multiple cellular parameters, including PTEN status, PI3K isoform levels, and Rac1 activity, combine to influence PI3K isoform prevalence, rather than a single determinant.

Changes in the Bile Acid Pool and Timing of Female Puberty: Potential Novel Role of Hypothalamic TGR5.

The regulation of pubertal timing and reproductive axis maturation is influenced by a myriad of physiologic and environmental inputs yet remains incompletely understood. To contrast differences in bile acid isoform profiles across defined stages of reproductive maturity in humans and a rat model of puberty and to characterize the role of bile acid signaling via hypothalamic expression of bile acid receptor populations in the rodent model. Secondary analysis and pilot studies of clinical cohorts, rodent models, ex vivo analyses of rodent hypothalamic tissues. Bile acid concentrations is the main outcome measure. Lower circulatory conjugated:deconjugated bile acid concentrations and higher total secondary bile acids were observed in postmenarcheal vs pre-/early pubertal adolescents, with similar shifts observed in infantile (postnatal day [PN]14) vs early juvenile (PN21) rats alongside increased tgr5 receptor mRNA expression within the mediobasal hypothalamus of female rats. 16S rRNA gene sequencing of the rodent gut microbiome across postnatal life revealed changes in the gut microbial composition predicted to have bile salt hydrolase activity, which was observed in parallel with the increased deconjugated and increased concentrations of secondary bile acids. We show that TGR5-stimulated GnRH release from hypothalamic explants is mediated through kisspeptin receptors and that early overexpression of human-TGR5 within the arcuate nucleus accelerates pubertal onset in female rats. Bile acid isoform shifts along stages of reproductive maturation are conserved across rodents and humans, with preclinical models providing mechanistic insight for the neuroendocrine-hepatic-gut microbiome axis as a potential moderator of pubertal timing in females.

UCP1-dependent brown adipose activation accelerates cardiac metabolic remodeling and reduces initial hypertrophic and fibrotic responses to pathological stress.

Brown adipose tissue (BAT) is correlated to cardiovascular health in rodents and humans, but the physiological role of BAT in the initial cardiac remodeling at the onset of stress is unknown. Activation of BAT via 48 h cold (16°C) in mice following transverse aortic constriction (TAC) reduced cardiac gene expression for LCFA uptake and oxidation in male mice and accelerated the onset of cardiac metabolic remodeling, with an early isoform shift of carnitine palmitoyltransferase 1 (CPT1) toward increased CPT1a, reduced entry of long chain fatty acid (LCFA) into oxidative metabolism (0.59 ± 0.02 vs. 0.72 ± 0.02 in RT TAC hearts, p < .05) and increased carbohydrate oxidation with altered glucose transporter content. BAT activation with TAC reduced early hypertrophic expression of β-MHC by 61% versus RT-TAC and reduced pro-fibrotic TGF-β1 and COL3α1 expression. While cardiac natriuretic peptide expression was yet to increase at only 3 days TAC, Nppa and Nppb expression were elevated in Cold TAC versus RT TAC hearts 2.7- and 2.4-fold, respectively. Eliminating BAT thermogenic activation with UCP1 KO mice eliminated differences between Cold TAC and RT TAC hearts, confirming effects of BAT activation rather than autonomous cardiac responses to cold. Female responses to BAT activation were blunted, with limited UCP1 changes with cold, partly due to already activated BAT in females at RT compared to thermoneutrality. These data reveal a previously unknown physiological mechanism of UCP1-dependent BAT activation in attenuating early cardiac hypertrophic and profibrotic signaling and accelerating remodeled metabolic activity in the heart at the onset of cardiac stress.

Equisetum arvense standardized dried extract hinders age-related osteosarcopenia

Age-associated osteosarcopenia is an unresolved syndrome characterized by the concomitant loss of bone (osteopenia) and skeletal muscle (sarcopenia) tissues increasing falls, immobility, morbidity, and mortality. Unbalanced resorption of bone in the remodeling process and excessive protein breakdown, especially fast type II myosin heavy chain (MyHC-II) isoform and myofiber metabolic shift, are the leading causes of bone and muscle deterioration in the elderly, respectively. Equisetum arvense (EQ) is a plant traditionally recommended for many pathological conditions due to its anti-inflammatory properties. Thus, considering that a chronic low-grade inflammatory state predisposes to both osteoporosis and sarcopenia, we tested a standardized hydroalcoholic extract of EQ in in vitro models of muscle atrophy [C2C12 myotubes treated with proinflammatory cytokines (TNFα/IFNγ), excess glucocorticoids (dexamethasone), or the osteokine, receptor activator of nuclear factor kappa-B ligand (RANKL)] and osteoclastogenesis (RAW 264.7 cells treated with RANKL). We found that EQ counteracted myotube atrophy, blunting the activity of several pathways depending on the applied stimulus, and reduced osteoclast formation and activity. By in silico target fishing, IKKB-dependent nuclear factor kappa-B (NF-κB) inhibition emerges as a potential common mechanism underlying EQ′s anti-atrophic effects. Consumption of EQ (500 mg/kg/day) by pre-geriatric C57BL/6 mice for 3 months translated into: i) maintenance of muscle mass and performance; ii) restrained myofiber oxidative shift; iii) slowed down age-related modifications in osteoporotic bone, significantly preserving trabecular connectivity density; iv) reduced muscle- and spleen-related inflammation. EQ can preserve muscle functionality and bone remodeling during aging, potentially valuable as a natural treatment for osteosarcopenia.

RNA-binding proteins regulating the CD44 alternative splicing.

Alternative splicing is often deregulated in cancer, and cancer-specific isoform switches are part of the oncogenic transformation of cells. Accumulating evidence indicates that isoforms of the multifunctional cell-surface glycoprotein CD44 play different roles in cancer cells as compared to normal cells. In particular, the shift of CD44 isoforms is required for epithelial to mesenchymal transition (EMT) and is crucial for the maintenance of pluripotency in normal human cells and the acquisition of cancer stem cells phenotype for malignant cells. The growing and seemingly promising use of splicing inhibitors for treating cancer and other pathologies gives hope for the prospect of using such an approach to regulate CD44 alternative splicing. This review integrates current knowledge about regulating CD44 alternative splicing by RNA-binding proteins.

A 3D biomimetic model of lymphatics reveals cell–cell junction tightening and lymphedema via a cytokine-induced ROCK2/JAM-A complex

Impaired lymphatic drainage and lymphedema are major morbidities whose mechanisms have remained obscure. To study lymphatic drainage and its impairment, we engineered a microfluidic culture model of lymphatic vessels draining interstitial fluid. This lymphatic drainage-on-chip revealed that inflammatory cytokines that are known to disrupt blood vessel junctions instead tightened lymphatic cell-cell junctions and impeded lymphatic drainage. This opposing response was further demonstrated when inhibition of rho-associated protein kinase (ROCK) was found to normalize fluid drainage under cytokine challenge by simultaneously loosening lymphatic junctions and tightening blood vessel junctions. Studies also revealed a previously undescribed shift in ROCK isoforms in lymphatic endothelial cells, wherein a ROCK2/junctional adhesion molecule-A (JAM-A) complex emerges that is responsible for the cytokine-induced lymphatic junction zippering. To validate these in vitro findings, we further demonstrated in a genetic mouse model that lymphatic-specific knockout of ROCK2 reversed lymphedema in vivo. These studies provide a unique platform to generate interstitial fluid pressure and measure the drainage of interstitial fluid into lymphatics and reveal a previously unappreciated ROCK2-mediated mechanism in regulating lymphatic drainage.

Hierarchically goal-oriented prediction of skeletal muscle tissue constitutive behavior considering histological characteristics

Skeletal muscles provide essential mechanical support and motion generation for living beings. Developing prediction models heavily rely on extracting quantitative information from complex and extensive experimental data. However, intrinsically large statistical variations due to physiological differences and experimental uncertainties pose significant challenges reliable and robust prediction of tissue mechanical behavior.To address these issues, we develop a hierarchically goal-oriented framework for high-confidence prediction of the target quantity of interest (QoI), i.e., the constitutive behavior of skeletal muscle tissue in the present study. The histological characterization of skeletal muscle is quantified using immunofluorescence staining and image processing in order to take account of physiological differences. Mechanical experiments are used to correlate the myosin heavy chain (MHC) isoform shift with tissue mechanical properties. With the gathered experimental data, a novel histological parameter is introduced to enhance a classical constitutive model with discriminability in muscle histological characteristics. To tackle high-confidence prediction, the proposed predictive framework is holistically devised with Bayesian inference, parameter compression, correlation analysis, and uncertainty quantification of QoI. We benchmark the model predictions against experiments subjected to disparate histological differences originated from sites, individuals, or species.

Linking myosin heavy chain isoform shift to mechanical properties and fracture modes in skeletal muscle tissue.

Muscle fibers play a crucial role in the mechanical action of skeletal muscle tissue. However, it is unclear how the histological variations affect the mechanical properties of tissues. In this study, the shift of myosin heavy chain (MHC) isoforms is used for the first time to establish a linkage between tissue histological variation and passive mechanical properties. The shift of MHC isoform is found not only to induce significant differences in skeletal muscle passive mechanical properties, but also to lead to differences in strain rate responses. Non-negligible rate dependence is observed even in the conventionally defined quasi-static regime. Fidelity in the estimated constitutive parameters, which can be impacted due to variation in MHC isoforms and hence in rate sensitivity, is enhanced using a Bayesian inference framework. Subsequently, scanning electron microscopy and fluorescence microscopy are used to characterize the fracture morphology of muscle tissues and fibers. The fracture mode of both MHC I and II muscle fibers exhibited shearing of endomysium. Results show that the increase in strain rate only leads to stronger rebounding of the muscle fibers during tissue rupture without changing fracture modes.

Molecular and cellular evidence for the impact of a hypertrophic cardiomyopathy-associated RAF1 variant on the structure and function of contractile machinery in bioartificial cardiac tissues

Noonan syndrome (NS), the most common among RASopathies, is caused by germline variants in genes encoding components of the RAS-MAPK pathway. Distinct variants, including the recurrent Ser257Leu substitution in RAF1, are associated with severe hypertrophic cardiomyopathy (HCM). Here, we investigated the elusive mechanistic link between NS-associated RAF1S257L and HCM using three-dimensional cardiac bodies and bioartificial cardiac tissues generated from patient-derived induced pluripotent stem cells (iPSCs) harboring the pathogenic RAF1 c.770 C > T missense change. We characterize the molecular, structural, and functional consequences of aberrant RAF1–associated signaling on the cardiac models. Ultrastructural assessment of the sarcomere revealed a shortening of the I-bands along the Z disc area in both iPSC-derived RAF1S257L cardiomyocytes and myocardial tissue biopsies. The aforementioned changes correlated with the isoform shift of titin from a longer (N2BA) to a shorter isoform (N2B) that also affected the active force generation and contractile tensions. The genotype-phenotype correlation was confirmed using cardiomyocyte progeny of an isogenic gene-corrected RAF1S257L-iPSC line and was mainly reversed by MEK inhibition. Collectively, our findings uncovered a direct link between a RASopathy gene variant and the abnormal sarcomere structure resulting in a cardiac dysfunction that remarkably recapitulates the human disease.

Cardiac myosin motor deficits are associated with left ventricular dysfunction in human ischemic heart failure.

During ischemic heart failure (IHF), cardiac muscle contraction is typically impaired, though the molecular changes within the myocardium are not fully understood. Thus, we aimed to characterize the biophysical properties of cardiac myosin in IHF. Cardiac tissue was harvested from 10 age-matched males, either with a history of IHF or nonfailing (NF) controls that had no history of structural or functional cardiac abnormalities. Clinical measures before cardiac biopsy demonstrated significant differences in measures of ejection fraction and left ventricular dimensions. Myofibrils and myosin were extracted from left ventricular free wall cardiac samples. There were no changes in myofibrillar ATPase activity or calcium sensitivity between groups. Using isolated myosin, we found a 15% reduction in the IHF group in actin sliding velocity in the in vitro motility assay, which was observed in the absence of a myosin isoform shift. Oxidative damage (carbonylation) of isolated myosin was compared, in which there were no significant differences between groups. Synthetic thick filaments were formed from purified myosin and the ATPase activity was similar in both basal and actin-activated conditions (20 µM actin). Correlation analysis and Deming linear regression were performed between all studied parameters, in which we found statistically significant correlations between clinical measures of contractility with molecular measures of sliding velocity and ELC carbonylation. Our data indicate that subtle deficits in myosin mechanochemical properties are associated with reduced contractile function and pathological remodeling of the heart, suggesting that the myosin motor may be an effective pharmacological intervention in ischemia.NEW & NOTEWORTHY Ischemic heart failure is associated with impairments in contractile performance of the heart. This study revealed that cardiac myosin isolated from patients with ischemic heart failure had reduced mechanical activity, which correlated with the impaired clinical phenotype of the patients. The results suggest that restoring myosin function with pharmacological intervention may be a viable method for therapeutic intervention.

Transcriptomic profiles of muscular dystrophy with myositis (mdm) in extensor digitorum longus, psoas, and soleus muscles from mice

BackgroundTitinopathies are inherited muscular diseases triggered by genetic mutations in the titin gene. Muscular dystrophy with myositis (mdm) is one such disease caused by a LINE repeat insertion, leading to exon skipping and an 83-amino acid residue deletion in the N2A-PEVK region of mouse titin. This region has been implicated in a number of titin—titin ligand interactions, hence are important for myocyte signaling and health. Mice with this mdm mutation develop a severe and progressive muscle degeneration. The range of phenotypic differences observed in mdm mice shows that the deletion of this region induces a cascade of transcriptional changes extending to numerous signaling pathways affected by the titin filament. Previous research has focused on correlating phenotypic differences with muscle function in mdm mice. These studies have provided understanding of the downstream physiological effects resulting from the mdm mutation but only provide insights on processes that can be physiologically observed and measured. We used differential gene expression (DGE) to compare the transcriptomes of extensor digitorum longus (EDL), psoas and soleus muscles from wild-type and mdm mice to develop a deeper understand of these tissue-specific responses.ResultsThe overall expression pattern observed shows a well-differentiated transcriptional signature in mdm muscles compared to wild type. Muscle-specific clusters observed within the mdm transcriptome highlight the level of variability of each muscle to the deletion. Differential gene expression and weighted gene co-expression network analysis showed a strong directional response in oxidative respiration-associated mitochondrial genes, which aligns with the poor shivering and non-shivering thermogenesis previously observed. Sln, which is a marker associated with shivering and non-shivering thermogenesis, showed the strongest expression change in fast-fibered muscles. No drastic changes in MYH expression levels were reported, which indicated an absence of major fiber-type switching events. Overall expression shifts in MYH isoforms, MARPs, and extracellular matrix associated genes demonstrated the transcriptional complexity associated with mdm mutation. The expression alterations in mitochondrial respiration and metabolism related genes in the mdm muscle dominated over other transcriptomic changes, and likely account for the late stage cellular responses in the mdm muscles.ConclusionsWe were able to demonstrate that the complex nature of mdm mutation extends beyond a simple rearrangement in titin gene. EDL, psoas and soleus exemplify unique response modes observed in skeletal muscles with mdm mutation. Our data also raises the possibility that failure to maintain proper energy homeostasis in mdm muscles may contribute to the pathogenesis of the degenerative phenotype in mdm mice. Understanding the full disease-causing molecular cascade is difficult using bulk RNA sequencing techniques due to intricate nature of the disease. The development of the mdm phenotype is temporally and spatially regulated, hence future studies should focus on single fiber level investigations.

Age and Blood Pressure Contribute to Aortic Cell and Tissue Stiffness Through Distinct Mechanisms.

Aortic stiffening is strongly associated with both aging and hypertension, but the underlying mechanisms remain unclear. We hypothesized that aging-induced aortic stiffness is mediated by a mechanism differing from hypertension. We conducted comprehensive in vivo and in vitro experiments using multiple rat models to dissect the different mechanisms of aortic stiffening mediated by aging and hypertension. A time-course study in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) normotensive rats showed more pronounced aging-associated aortic stiffening in SHR versus WKY. Angiotensin II-induced hypertension was associated with more significant aortic stiffening in older versus young WKY rats. Hypertension aggravated aging effects on aortic wall thickness and extracellular matrix content, indicating combinational effects of aging and hypertension on aortic stiffening. Intrinsic stiffness of isolated aortic vascular smooth muscle cells (VSMCs) increased with age in WKY rats, although no significant difference between older SHR and older WKY VSMCs was observed in 2-dimensional culture, reconstituted 3-dimensional tissues were stiffer for older SHR versus older WKY. A selective inhibitor that reduced hypertension-mediated aortic stiffening did not decrease age-related stiffening in aortic VSMCs and aortic wall. Integrin β1 and SM22 (smooth muscle-specific SM22 protein) expression were negligibly changed in WKY VSMCs during aging but were markedly increased by hypertension in older versus young WKY VSMCs. A notable shift of filamin isoforms from B to A was detected in older WKY VSMCs. Our results indicate distinct mechanisms mediating aging-associated aortic VSMC and vessel stiffness, providing new insights into aortic stiffening and the pathogenesis of hypertension in the elderly.

Gaining insight into the role of FoxO1 in the progression of disuse-induced skeletal muscle atrophy.

Expression of FoxO transcription factors increases during certain forms of atrophy. In a dephosphorylated state, FoxOs participate in ubiquitin-mediated proteasomal degradation through the transcriptional activation of E3-ubiquitin ligases such as MAFbx/atrogin-1 and MuRF1. There is exhaustive research demonstrating that FoxO3a is sufficient to induce MAFbx/atrogin-1 and MuRF-1 expressions. In contrast, the data are conflicting on the requirement of FoxO1 signaling in the activation of the E3-ubiquitin ligases. Moreover, no reports currently exist on the particular role of FoxO1 in the molecular mechanisms involved in the progression of physiological muscle wasting. Here, we have applied the most extensively used rodent model of microgravity/functional unloading to stimulate disuse-induced skeletal muscle atrophy such as rat hindlimb suspension (HS). We showed that inhibition of FoxO1 activity by a selective inhibitor AS1842856 completely reversed an increase in expression of MuRF-1, but not MAFbx/atrogin-1, observed upon HS. Furthermore, we demonstrated that FoxO1 induced upregulation of another E3-ubiquitin-ligase of a MuRF protein family MuRF-2 in skeletal muscle subjected to disuse. Prevention of the MuRF increase upon HS impeded upregulation of transcript expression of a negative regulator of NFATc1 pathway calsarcin-2, which was associated with a partial reversion of MyHC-IId/x and MyHC-IIb mRNA expressions. Importantly, FoxO1 inhibition induced a marked increase in p70S6k phosphorylation, an important stage in the initiation of protein translation, concomitant with the restoration of global protein synthesis in the skeletal muscle of the HS rats. Examination of eIF3f expression and the eEF2k/eEF2 pathway, other factors controlling translation initiation and elongation respectively, did not reveal any impact of FoxO1 on their activity. Lastly, we observed a decrease in transcript levels of Sesn3, but not Sesn1 and Sesn2, upon disuse, which was completely reversed by FoxO1 inhibition. These data demonstrate that FoxO1 signaling contributes to the development of disuse-induced skeletal muscle atrophy, including slow to fast MyHC isoform shift, mostly through upregulation of MuRF-1 and MuRF-2 expression. Furthermore, FoxO1 inhibition is required to recover Sesn3 mRNA expression in atrophic conditions, which likely contributes to the enhanced p70S6k activity and restoration of the protein synthesis rate.

HnRNPC induces isoform shifts in miR-21-5p leading to cancer development

MicroRNA (miRNA) processing is a critical step in mature miRNA production. Its dysregulation leads to an increase in miRNA isoforms with heterogenous 5′-ends (isomiRs), which can recognize distinct target sites because of their shifted seed sequence. Although some miRNA genes display productive expression of their 5′-isomiRs in cancers, how their production is controlled and how 5′-isomiRs affect tumor progression have yet to be explored. In this study, based on integrative analyses of high-throughput sequencing data produced by our group and publicly available data, we demonstrate that primary miR-21 (pri-miR-21) is processed into the cancer-specific isomiR isomiR-21-5p | ±1, which suppresses growth hormone receptor (GHR) in liver cancer. Treatment with antagomirs against isomiR-21-5p | ±1 inhibited the in vitro tumorigenesis of liver cancer cells and allowed the recovery of GHR, whereas the introduction of isomiR-21-5p | ±1 mimics attenuated these effects. These effects were validated in a mouse model of spontaneous liver cancer. Heterogeneous nuclear ribonucleoprotein C and U2 small nuclear RNA auxiliary factor 2 were predicted to bind upstream of pre-miR-21 via a poly-(U) motif and influence Drosha processing to induce the production of isomiR-21-5p | ±1. Our findings suggest an oncogenic function for the non-canonical isomiR-21-5p | ±1 in liver cancer, and its production was shown to be regulated by hnRNPC.

Molecular mechanisms by which splice modulator GEX1A inhibits leukaemia development and progression.

Splice modulators have been assessed clinically in treating haematologic malignancies exhibiting splice factor mutations and acute myeloid leukaemia. However, the mechanisms by which such modulators repress leukaemia remain to be elucidated. The primary goal of this assessment was to assess the molecular mechanism by which the natural splice modulator GEX1A kills leukaemic cells in vitro and within in vivo mouse models. Using human leukaemic cell lines, we assessed the overall sensitivity these cells have to GEX1A via EC50 analysis. We subsequently analysed its effects using in vivo xenograft mouse models and examined whether cell sensitivities were correlated to genetic characteristics or protein expression levels. We also utilised RT-PCR and RNAseq analyses to determine splice change and RNA expression level differences between sensitive and resistant leukaemic cell lines. We found that, in vitro, GEX1A induced an MCL-1 isoform shift to pro-apoptotic MCL-1S in all leukaemic cell types, though sensitivity to GEX1A-induced apoptosis was negatively associated with BCL-xL expression. In BCL-2-expressing leukaemic cells, GEX1A induced BCL-2-dependent apoptosis by converting pro-survival BCL-2 into a cell killer. Thus, GEX1A + selective BCL-xL inhibition induced synergism in killing leukaemic cells, while GEX1A + BCL-2 inhibition showed antagonism in BCL-2-expressing leukaemic cells. In addition, GEX1A sensitised FLT3-ITD+ leukaemic cells to apoptosis by inducing aberrant splicing and repressing the expression of FLT3-ITD. Consistently, in in vivo xenografts, GEX1A killed the bulk of leukaemic cells via apoptosis when combined with BCL-xL inhibition. Furthermore, GEX1A repressed leukaemia development by targeting leukaemia stem cells through inhibiting FASTK mitochondrial isoform expression across sensitive and non-sensitive leukaemia types. Our study suggests that GEX1A is a potent anti-leukaemic agent in combination with BCL-xL inhibitors, which targets leukaemic blasts and leukaemia stem cells through distinct mechanisms.

FOXP3 Isoforms and Human Regulatory T Cells

Background: &#x0D; Regulatory T-cells (Tregs) are critical to maintaining immune tolerance, thus prevent autoimmunity and allergic diseases. The master transcription factor FOXP3, expressed in humans as two isoforms through mRNA alternative splicing, controls the development and function of Tregs. However, the functions of the two isoforms remain unclear. We hypothesized that the oligonucleotides, developed by the lab, would efficiently shift FOXP3 to its shorter isoform, lacking exon 2 (FOXP3 ΔE2), which enables us to study how the FOXP3 ΔE2 isoform affects Treg function. &#x0D; &#x0D; Methods: &#x0D; Human peripheral blood mononuclear cells (PBMC) were obtained from healthy donors and cultured in the presence of 1 µM isoform-shifting oligonucleotides for three weeks. Expression of the FOXP3 isoforms, CD25, CTLA-4, CD40L, was examined through flow cytometry. Another goal was to study how the FOXP3 ΔE2 isoform shift affects the Treg response to inflammatory cytokines. &#x0D; &#x0D; Results: &#x0D; The oligonucleotides are highly effective in shifting the FOXP3 to the FOXP3 ΔE2 isoform. The majority of Tregs express only the FOXP3 ΔE2 isoform after two weeks of culturing in 1 µM oligo and had reduced expression of CD25, which plays a role in Treg differentiation, function, and homeostasis. Tregs display reduced expression of CTLA-4, a negative regulator of T-cell activation, but elevated CD40L, which costimulates and regulates the immune response. The FOXP3 ΔE2 isoform shift also sensitizes the Tregs to proinflammatory cytokine stimulation, allowing for increased IL-4, IL-17A, and IFN-γ production. &#x0D; &#x0D; Conclusion: &#x0D; Our studies demonstrate that FOXP3 ΔE2 Tregs are less stable because of the lower CD25 expression and less suppressive as determined by lower CTLA-4 but higher CD40L expression. In the inflammatory environment, the FOXP3 ΔE2 Tregs also produce more inflammatory cytokines, which further reduces their regulatory function. This study establishes an understanding of FOXP3ΔE2 Tregs’ role in autoimmunity and provides a potential novel target to treating autoimmune diseases. &#x0D; &#x0D; Acknowledgements: &#x0D; This study was funded, in part, with support from the Immunology and Infectious Diseases Training Program Grant funded, in part by T32 AI 060519 from the National Institutes of Health to RS and NIH AI159804 to BZ.

Isoform cell-type specificity in the mouse primary motor cortex

Full-length SMART-seq1 single-cell RNA sequencing can be used to measure gene expression at isoform resolution, making possible the identification of specific isoform markers for different cell types. Used in conjunction with spatial RNA capture and gene-tagging methods, this enables the inference of spatially resolved isoform expression for different cell types. Here, in a comprehensive analysis of 6,160 mouse primary motor cortex cells assayed with SMART-seq, 280,327 cells assayed with MERFISH2 and 94,162 cells assayed with 10x Genomics sequencing3, we find examples of isoform specificity in cell types—including isoform shifts between cell types that are masked in gene-level analysis—as well as examples of transcriptional regulation. Additionally, we show that isoform specificity helps to refine cell types, and that a multi-platform analysis of single-cell transcriptomic data leveraging multiple measurements provides a comprehensive atlas of transcription in the mouse primary motor cortex that improves on the possibilities offered by any single technology.

Advances in cirrhotic cardiomyopathy.

Cirrhotic cardiomyopathy (CCM) is a well-recognized entity. When patients with CCM encounter challenges such as liver transplantation, overt cardiac dysfunction manifests, leading to morbidity and mortality. Although revised diagnostic criteria for CCM have recently been proposed, these still need to be validated. Previous reviews have summarized the mechanisms of CCM, such as abnormalities of the β-adrenergic pathway, cardiac plasma membrane biophysical and biochemical properties, and electrophysiological changes. Cardiomyocyte apoptosis, inflammation, and oxidative stress also play important roles. The present review details further mechanisms of CCM, which include myosin heavy chain isoform shifts and abnormalities in cellular calcium transients. Additionally, we review recent studies on therapeutic strategies. Recent work underscores the importance of CCM in the natural history of the immediate and medium-term postoperative period after liver transplantation. Appropriate management strategies for CCM remain the area of greatest unmet need, requiring much further research. CCM is a clinically relevant syndrome affecting patients with cirrhosis, leading to increased morbidity and mortality. New diagnostic criteria have been recently proposed by an expert working group. The pathogenic mechanisms remain incompletely clarified and optimal management strategies need much further study.

Suppression of myofilament cross-bridge kinetic in the heart of orchidectomized rats

AimsThe increased incidence of heart failure with reduced ejection fraction in men compared with women suggests that male sex hormones significantly impact myocardial contractile activation. This study aims to examine associations among molecular alterations, cellular modulations and in vivo cardiac contractile function upon deprivation of testicular hormones. Main methodsMyocardial structure and functions were compared among sham-operated control and twelve-week orchidectomized (ORX) male rats with and without testosterone supplementation. Key findingsEchocardiography and pressure-volume relationships demonstrated a decreased left ventricular ejection fraction compared with sham-operated controls. The percentage of contractility reduction was generally similar to the decrease in tension development detected in both right ventricular trabeculae and skinned isolated left ventricular cardiomyocytes of ORX rats. Reductions in tension cost and the rate constant of tension redevelopment (ktr) in ORX samples suggested a decrease in the rate of cross-bridge formation, reflecting a reduced number of cross-bridges. Slow cross-bridge detachment in ORX rat hearts could result from a shift of myosin heavy chain isoforms towards a slower ATPase activity β-isoform and reductions in the phosphorylation levels of cardiac troponin I and myosin binding protein-C. All the changes in the ORX rat heart, including ejection fractions and myofilament protein expression and phosphorylation, were completed attenuated by a physiological dose of testosterone. SignificanceTestosterone plays a critical role in regulating the mechanical and contractile dynamics of the heart. Deprivation of male sex hormones cause the loss of normal preserved cardiac contractile function leading to a high risk of severe cardiomyopathy progression.