Three-in-One: Molecular Engineering of D-A-π-A Featured Type I and Type II Near-Infrared AIE Photosensitizers for Efficient Photodynamic Cancer Therapy and Bacteria Killing.

Bacterial infections are closely correlated with the genesis and progression of cancer, and the elimination of cancer-related bacteria may improve the efficacy of cancer treatment. However, the combinatorial therapy that utilizes two or more chemodrugs will increase potential adverse effects. Image-guided photodynamic therapy is a highly precise and potential therapy to treat tumor and microbial infections. Herein, four donor-acceptor-π-bridge-acceptor (D-A-π-A) featured near-infrared (NIR) aggregation-induced emission luminogens (AIEgens) (TQTPy, TPQTPy, TQTC, and TPQTC) with type I and type II reaction oxygen species (ROS) generation capabilities are synthesized. Notably, TQTPy shows mitochondria targeted capacity, the best ROS production efficiency, long-term tumor retention capacity, and more importantly, the three-in-one fluorescence imaging guided therapy against both tumor and microbial infections. Both in vitro and in vivo results validate that TQTPy performs well in practical biomedical application in terms of NIR-fluorescence imaging-guided photodynamic cancer diagnosis and treatment. Moreover, the amphiphilic and positively charged TQTPy is able to specific and ultrafast discrimination and elimination of Gram-positive (G+) Staphylococcus aureus from Gram-negative (G-) Escherichia coli and normal cells. This investigation provides an instructive way for the construction of three-in-one treatment for image-guided photodynamic cancer therapy and bacteria elimination.

Cytosolic serpins act in a cytoprotective feedback loop that limits ESX-1-dependent death of Mycobacterium marinum-infected macrophages.

Serine protease inhibitors (serpins) constitute the largest family of protease inhibitors expressed in humans, but their role in infection remains largely unexplored. In infected macrophages, the mycobacterial ESX-1 type VII secretion system permeabilizes internal host membranes and causes leakage into the cytosol of host DNA, which induces type I interferon (IFN) production via the cyclic GMP-AMP synthase (cGAS) and stimulator of IFN genes (STING) surveillance pathway, and promotes infection in vivo. Using the Mycobacterium marinum infection model, we show that ESX-1-mediated type I IFN signaling in macrophages selectively induces the expression of serpina3f and serpina3g, two cytosolic serpins of the clade A3. The membranolytic activity of ESX-1 also caused leakage of cathepsin B into the cytosol where it promoted cell death, suggesting that the induction of type I IFN comes at the cost of lysosomal rupture and toxicity. However, the production of cytosolic serpins suppressed the protease activity of cathepsin B in this compartment and thus limited cell death, a function that was associated with increased bacterial growth in infected mice. These results suggest that cytosolic serpins act in a type I IFN-dependent cytoprotective feedback loop to counteract the inevitable toxic effect of ESX-1-mediated host membrane rupture. IMPORTANCE: The ESX-1 type VII secretion system is a key virulence determinant of pathogenic mycobacteria. The ability to permeabilize host cell membranes is critical for several ESX-1-dependent virulence traits, including phagosomal escape and induction of the type I interferon (IFN) response. We find that it comes at the cost of lysosomal leakage and subsequent host cell death. However, our results suggest that ESX-1-mediated type I IFN signaling selectively upregulates serpina3f and serpina3g and that these cytosolic serpins limit cell death caused by cathepsin B that has leaked into the cytosol, a function that is associated with increased bacterial growth in vivo. The ability to rupture host membranes is widespread among bacterial pathogens, and it will be of interest to evaluate the role of cytosolic serpins and this type I IFN-dependent cytoprotective feedback loop in the context of human infection.

Misinterpretation of sleep-induced second-degree atrioventricular block.

A number of publications have claimed that Mobitz type II atrioventricular block (AVB) may occur during sleep. None of the reports defined type II AVB and representative electrocardiograms were either misinterpreted or missing. Relatively benign Wenckebach type I AVB is often misdiagnosed as Mobitz type II which is an indication for a pacemaker. Review of the published reports indicates that Mobitz type II AVB does not occur during sleep when it is absent in the awake state. Conclusion: There is no proof that sleep is associated with Mobitz type II AVB.

Loss of the long form of Plod2 phenocopies contractures of Bruck syndrome - osteogenesis imperfecta.

Bruck syndrome is an autosomal recessive form of osteogenesis imperfecta (OI) caused by biallelic variants in PLOD2 or FKBP10 and is characterized by joint contractures, bone fragility, short stature, and scoliosis. PLOD2 encodes LH2, which hydroxylates type I collagen telopeptide lysines, a critical step for collagen crosslinking. The Plod2 global knockout mouse model is limited by early embryonic lethality, thus the role of PLOD2 in skeletogenesis is not well understood. We generated a novel Plod2 mouse line modeling a variant identified in two unrelated individuals with Bruck syndrome: PLOD2 c.1559dupC, predicting a frameshift and loss of the long isoform LH2b. In the mouse, the duplication led to loss of LH2b mRNA as well as significantly reduced total LH2 protein. This model, Plod2fs/fs, survived up to E18.5 although in non-Mendelian genotype frequencies. The homozygous frameshift model recapitulated the joint contractures seen in Bruck syndrome and had indications of absent type I collagen telopeptide lysine hydroxylation in bone. Genetically labeling tendons with Scleraxis-GFP in Plod2fs/fs mice revealed the loss of extensor tendons in the forelimb by E18.5 and developmental studies showed extensor tendons developed through E14.5 but were absent starting at E16.5. Second harmonic generation showed abnormal tendon type I collagen fiber organization, suggesting structurally abnormal tendons. Characterization of the skeleton by µCT and Raman spectroscopy showed normal bone mineralization levels. This work highlights the importance of properly crosslinked type I collagen in tendon and bone, providing a promising new mouse model to further our understanding of Bruck syndrome.

Bruck syndrome is a rare disease where individuals have brittle bone as well as contracted or stiff joints. Mutations in two genes are associated with Bruck syndrome and, in this work, we focus on PLOD2. Mice without Plod2 die at an early embryonic stage, before they have a chance to fully develop. In this work, we created a mouse with a PLOD2 mutation seen in people with Bruck syndrome. Some of these new Bruck syndrome model mice survived to a later gestational age, but all died at birth. The Bruck syndrome mice were small and had contracted joints. We found they were missing tendons in their arms and had structurally abnormal tendons in their knees. Bone mineralization was normal, but there were indications that the modifications needed for normal type I collagen structure were absent. Overall, this is an advantageous new mouse model of Bruck syndrome that can be used to study this rare disease and highlights the importance of Plod2 in tendon.

The Guardian of Vision: Intelligent Bacteriophage-Based Eyedrops for Clinical Multidrug-Resistant Ocular Surface Infections.

Clinical multidrug-resistant Pseudomonas aeruginosa (MDR-PA) is the leading cause of refractory bacterial keratitis (BK). However, the reported BK treatment methods lack biosecurity and bioavailability, which usually causes irreversible visual impairment and even blindness. Herein, for BK caused by clinically isolated MDR-PA infection, armed phages are modularized with the type I photosensitizer (PS) ACR-DMT, and an intelligent phage eyedrop is developed for combined phagotherapy and photodynamic therapy (PDT). These eyedrops maximize the advantages of bacteriophages and ACR-DMT, enabling more robust and specific targeting killing of MDR-PA under low oxygen-dependence, penetrating and disrupting biofilms, and efficiently preventing biofilm reformation. Altering the biofilm and immune microenvironments alleviates inflammation noninvasively, promotes corneal healing without scar formation, protects ocular tissues, restores visual function, and prevents long-term discomfort and pain. This strategy exhibits strong scalability, enables at-home treatment of ocular surface infections with great patient compliance and a favorable prognosis, and has significant potential for clinical application.

Type II mode of JAK2 inhibition and destabilization are potential therapeutic approaches against the ruxolitinib resistance driven myeloproliferative neoplasms.

Background: Ruxolitinib has been approved by the US FDA for the treatment of myeloproliferative neoplasms such as polycythemia vera and primary myelofibrosis. Ruxolitinib will remain a main stay in the treatment of MPN patients due to its effective therapeutic benefits. However, there have been instances of ruxolitinib resistance in MPN patients. As JAK2 is a direct target of ruxolitinib, we generated ruxolitinib-resistant clones to find out the mechanism of resistance. Methods: Cell-based screening strategy was used to detect the ruxolitinib-resistant mutations in JAK2. The Sanger sequencing method was used to detect the point mutations in JAK2. Mutations were re-introduced using the site-directed mutagenesis method and stably expressed in Ba/F3 cells. Drug sensitivities against the JAK2 inhibitors were measured using an MTS-based assay. JAK2 and STAT5 activation levels and total proteins were measured using immunoblotting. Computational docking studies were performed using the Glide module of Schrodinger Maestro software. Results: In this study, we have recovered seven residues in the kinase domain of JAK2 that affect ruxolitinib sensitivity. All these mutations confer cross-resistance across the panel of JAK2 kinase inhibitors except JAK2-L983F. JAK2-L983F reduces the sensitivity towards ruxolitinib. However, it is sensitive towards fedratinib indicating that our screen identifies the drug-specific resistance profiles. All the ruxolitinib-resistant JAK2 variants displayed sensitivity towards type II JAK2 inhibitor CHZ-868. In this study, we also found that JAK1-L1010F (homologous JAK2-L983F) is highly resistant towards ruxolitinib suggesting the possibility of JAK1 escape mutations in JAK2-driven MPNs and JAK1 mutated ALL. Finally, our study also shows that HSP90 inhibitors are potent against ruxolitinib-resistant variants through the JAK2 degradation and provides the rationale for clinical evaluation of potent HSP90 inhibitors in genetic resistance driven by JAK2 inhibitors. Conclusion: Our study identifies JAK1 and JAK2 resistance variants against the type I JAK2 inhibitors ruxolitinib, fedratinib, and lestaurtinib. The sensitivity of these resistant variants towards the type II JAK2 inhibitor CHZ-868 indicates that this mode of type II JAK2 inhibition is a potential therapeutic approach against ruxolitinib refractory leukemia. This also proposes the development of potent and specific type II JAK2 inhibitors using ruxolitinib-resistance variants as a prototype.

SPATS2L is a positive feedback regulator of the type I interferon signaling pathway and plays a vital role in lupus.

Through genome-wide association studies (GWAS) and integrated expression quantitative trait locus (eQTL) analyses, numerous susceptibility genes ("eGenes", whose expressions are significantly associated with common variants) associated with systemic lupus erythematosus (SLE) have been identified. Notably, a subset of these eGenes is correlated with disease activity. However, the precise mechanisms through which these genes contribute to the initiation and progression of the disease remain to be fully elucidated. In this investigation, we initially identify SPATS2L as an SLE eGene correlated with disease activity. eSignaling and transcriptomic analyses suggest its involvement in the type I interferon (IFN) pathway. We observe a significant increase in SPATS2L expression following type I IFN stimulation, and the expression levels are dependent on both the concentration and duration of stimulation. Furthermore, through dual-luciferase reporter assays, western blot analysis, and imaging flow cytometry, we confirm that SPATS2L positively modulates the type I IFN pathway, acting as a positive feedback regulator. Notably, siRNA-mediated intervention targeting SPATS2L, an interferon-inducible gene, in peripheral blood mononuclear cells (PBMCs) from patients with SLE reverses the activation of the interferon pathway. In conclusion, our research highlights the pivotal role of SPATS2L as a positive-feedback regulatory molecule within the type I IFN pathway. Our findings suggest that SPATS2L plays a critical role in the onset and progression of SLE and may serve as a promising target for disease activity assessment and intervention strategies.

Disruption of glucose homeostasis by bacterial infection orchestrates host innate immunity through NAD+/NADH balance.

Metabolic reprogramming is crucial for activating innate immunity in macrophages, and the accumulation of immunometabolites is essential for effective defense against infection. The NAD+/NADH (ratio of nicotinamide adenine dinucleotide and its reduced counterpart) redox couple serves as a critical node that integrates metabolic pathways and signaling events, but how this metabolite couple engages macrophage activation remains unclear. Here, we show that the NAD+/NADH ratio serves as a molecular signal that regulates proinflammatory responses and type I interferon (IFN) responses divergently. Salmonella Typhimurium infection leads to a decreased NAD+/NADH ratio by inducing the accumulation of NADH. Further investigation shows that an increased NAD+/NADH ratio correlates with attenuated proinflammatory responses and enhanced type I IFN responses. Conversely, a decreased NAD+/NADH ratio is linked to intensified proinflammatory responses and restrained type I IFN responses. These results show that the NAD+/NADH ratio is an essential cell-intrinsic factor that orchestrates innate immunity, which enhances our understanding of how metabolites fine-tune innate immunity.

Use of Marsupialization as a Definitive Treatment for Large-sized Dentigerous Cysts in a Patient with Mucopolysaccharidosis Type I.

The correct diagnosis is fundamental for the appropriate treatment to be employed in a particular pathology. The best treatment is not the one that solves only local problems, fragmenting the patient, and therefore, it is necessary to integrate the entire systemic condition of the individual before initiating any local treatment. This context inevitably requires dentistry to participate in a multidisciplinary approach, where the role of the dentist is expanded in concepts that encompass ethics, human dignity, and professional valorization. This article describes a clinical case of a patient with mucopolysaccharidosis type I, whose treatment of cystic lesions present in the mandible was exclusively performed through marsupialisation. The objective of this study is to demonstrate, within the complexity of this rare syndrome, the difficulties of diagnosis and the need for evaluation of the patient beyond the limits of the oral cavity, as well as to report two cases of large dentigerous cysts, surgically treated conservatively through marsupialisation, without the need for re-approach for enucleation and without recurrences over a 20-year period.

Iterative Data-adaptive Autoregressive (IDAR) whitening procedure for long and short TR fMRI.

Introduction: Functional magnetic resonance imaging (fMRI) has become a fundamental tool for studying brain function. However, the presence of serial correlations in fMRI data complicates data analysis, violates the statistical assumptions of analyses methods, and can lead to incorrect conclusions in fMRI studies. Methods: In this paper, we show that conventional whitening procedures designed for data with longer repetition times (TRs) (>2 s) are inadequate for the increasing use of short-TR fMRI data. Furthermore, we comprehensively investigate the shortcomings of existing whitening methods and introduce an iterative whitening approach named "IDAR" (Iterative Data-adaptive Autoregressive model) to address these shortcomings. IDAR employs high-order autoregressive (AR) models with flexible and data-driven orders, offering the capability to model complex serial correlation structures in both short-TR and long-TR fMRI datasets. Results: Conventional whitening methods, such as AR(1), ARMA(1,1), and higher-order AR, were effective in reducing serial correlation in long-TR data but were largely ineffective in even reducing serial correlation in short-TR data. In contrast, IDAR significantly outperformed conventional methods in addressing serial correlation, power, and Type-I error for both long-TR and especially short-TR data. However, IDAR could not simultaneously address residual correlations and inflated Type-I error effectively. Discussion: This study highlights the urgent need to address the problem of serial correlation in short-TR (< 1 s) fMRI data, which are increasingly used in the field. Although IDAR can address this issue for a wide range of applications and datasets, the complexity of short-TR data necessitates continued exploration and innovative approaches. These efforts are essential to simultaneously reduce serial correlations and control Type-I error rates without compromising analytical power.

A-to-I RNA Editing and Hematopoiesis.

Adenosine to inosine (A-to-I) RNA editing plays essential roles in modulating normal development and homeostasis. This process is catalyzed by Adenosine Deaminase Acting on RNA (ADAR) family proteins. The most well-understood biological processes modulated by A-to-I editing are innate immunity and neurological development, attributed to ADAR1 and ADAR2 respectively. A-to-I editing by ADAR1 is also critical in regulating hematopoiesis. This review will focus on the role of A-to-I RNA editing and ADAR enzymes, particularly ADAR1, during normal hematopoiesis in humans and mice. Furthermore, we will discuss Adar1 mouse models that have been developed to understand the contribution of ADAR1 to hematopoiesis and its role in innate immune pathways.

Case Series of 6 Fetuses With Osteogenesis Imperfecta Type II: A Retrospective Study of Heart Pathology.

INTRODUCTION: Osteogenesis imperfecta (OI) is a rare genetic disorder characterized by bone fragility. While skeletal manifestations are well documented, few studies have explored the effect of OI on the fetal heart. This retrospective case series investigates cardiac pathology in OI type II fetuses, aiming to address this gap. METHODS: Medical records and autopsy reports of 6 genetically confirmed OI type II cases were examined. Fetuses had pathogenic variants in COL1A1 or PPIB, inducing structural defects in collagen type I. In addition to hematoxylin and eosin and Elastic van Gieson staining, the expression of collagen type I, COL1A1 and COL1A2 chains was examined by immunohistochemistry. RESULTS: Immunohistochemistry confirmed robust expression of collagen type I throughout the heart. Five fetuses had normal heart weight, while 1 had a low heart weight in the context of generalized growth retardation. None displayed structural heart anomalies. CONCLUSION: This study reveals robust collagen type I expression in the hearts of OI type II fetuses without structural anomalies. We hypothesize that collagen type I abnormalities may not be causative factors for heart anomalies during early embryonic development. Instead, their impact may be conceivably related to an increased susceptibility to degenerative changes later in life.

STRAP upregulates antiviral innate immunity against PRV by targeting TBK1.

Serine/threonine kinase receptor-associated protein (STRAP) serves as a scaffold protein and is engaged in a variety of cellular activities, although its importance in antiviral innate immunity is unknown. We discovered that STRAP works as an interferon (IFN)-inducible positive regulator, facilitating type I IFN signaling during pseudorabies virus infection. Mechanistically, STRAP interacts with TBK1 to activate type I IFN signaling. Both the CT and WD40 7 - 6 domains contribute to the function of STRAP. Furthermore, TBK1 competes with PRV-UL50 for binding to STRAP, and STRAP impedes the degradation of TBK1 mediated by PRV-UL50, thereby increasing the interaction between STRAP and TBK1. Overall, these findings reveal a previously unrecognized role for STRAP in innate antiviral immune responses during PRV infection. STRAP could be a potential therapeutic target for viral infectious diseases.

Whole paternal uniparental disomy of chromosome 4 with a novel homozygous IDUA splicing variant, c.159-9T>A, in a Chinese patient with mucopolysaccharidosis type I.

BACKGROUND: Mucopolysaccharidosis type I (MPS-I) is a rare autosomal recessive genetic lysosomal storage disorder that is caused by pathogenic variants of the α-L-iduronidase (IDUA) gene. This study aimed to identify the genetic causes of MPS-I in a Chinese patient and construct a minigene of IDUA to analyze its variants upon splicing. METHODS: Whole-exome sequencing (WES) and Sanger sequencing were used to confirm the potential causative variants. Single-nucleotide polymorphism (SNP) array was subsequently performed to confirm uniparental disomy (UPD). Minigene assay was performed to analyze the effect on splicing of mRNA. We meanwhile explored the conservative analysis and protein homology simulation. RESULTS: A novel homozygous splicing mutation of IDUA, c.159-9T>A, was identified in an individual presenting with overlapping features of MPS-I. Interestingly, only the father and sisters, but not the mother, carried the variant in a heterozygous state. WES and SNP array analyses validated paternal UPD on chromosome 4. Minigene splicing revealed two aberrant splicing events: exon 2 skipping and intron 1 retention. Moreover, the specific structure of the mutant protein obviously changed according to the results of the homologous model. CONCLUSIONS: This study describes a rare autosomal recessive disorder with paternal UPD of chromosome 4 leading to the homozygosity of the IDUA splicing variant in patients with MPS-I for the first time. This study expands the variant spectrum of IDUA and provides insights into the splicing system, facilitating its enhanced diagnosis and treatment.

Interferonopathies: From concept to clinical practice.

The horror autoinflammaticus derived from aberrant type I interferon secretion determines a special group of autoinflammatory diseases named interferonopathies. Diverse mechanisms involved in nucleic acids sensing, metabolizing or the lack of interferon signaling retro-control are responsible for the phenotypes associated to Aicardi-Goutières Syndrome (AGS), Proteasome-Associated Autoinflammatory Diseases (PRAAS), STING-Associated Vasculopathy with Infancy Onset (SAVI) and certain forms of monogenic Systemic lupus erythematosus (SLE). This review approaches interferonopathies from the basic immunogenetic concept to diagnosis and treatment.

Versatile and efficient mammalian genome editing with Type I-C CRISPR System of Desulfovibrio vulgaris.

CRISPR-Cas tools for mammalian genome editing typically rely on single Cas9 or Cas12a proteins. While type I CRISPR systems in Class I may offer greater specificity and versatility, they are not well-developed for genome editing. Here, we present an alternative type I-C CRISPR system from Desulfovibrio vulgaris (Dvu) for efficient and precise genome editing in mammalian cells and animals. We optimized the Dvu type I-C editing complex to generate precise deletions at multiple loci in various cell lines and pig primary fibroblast cells using a paired PAM-in crRNA strategy. These edited pig cells can serve as donors for generating transgenic cloned piglets. The Dvu type I-C editor also enabled precise large fragment replacements with homology-directed repair. Additionally, we adapted the Dvu-Cascade effector for cytosine and adenine base editing, developing Dvu-CBE and Dvu-ABE systems. These systems efficiently induced C-to-T and A-to-G substitutions in human genes without double-strand breaks. Off-target analysis confirmed the high specificity of the Dvu type I-C editor. Our findings demonstrate the Dvu type I-C editor's potential for diverse mammalian genome editing applications, including deletions, fragment replacement, and base editing, with high efficiency and specificity for biomedicine and agriculture.

Epstein-Barr virus nuclear antigen EBNA3A modulates IRF3-dependent IFNß expression.

Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, persistently infects over 90% of the human adult population and is associated with several human cancers. To establish life-long infection, EBV tampers with the induction of type I interferon (IFN I)-dependent antiviral immunity in the host. How various EBV genes help orchestrate this crucial strategy is incompletely defined. Here, we reveal a mechanism by which the EBV nuclear antigen 3A (EBNA3A) may inhibit IFNß induction. Using proximity biotinylation we identify the histone acetyltransferase P300, a member of the IFNß transcriptional complex, as a binding partner of EBNA3A. We further show that EBNA3A also interacts with the activated IFN-inducing transcription factor IRF3 that collaborates with P300 in the nucleus. Both events are mediated by the N-terminal domain of EBNA3A. We propose that EBNA3A limits binding of IRF3 to the IFNß promoter, thereby hampering downstream IFN I signaling. Collectively, our findings suggest a new mechanism of immune evasion by EBV, affected by its latency gene EBNA3A.

Anaesthetic management of obstetric patients with Chiari type I malformation: a retrospective case series and literature review.

The peripartum management of obstetric patients with Chiari type I malformation remains a challenge due to the degree of cerebellar tonsillar herniation and a paucity of published evidence. There is concern about neuraxial anaesthetic blocks and uncertainty regarding the optimum mode of delivery. We systematically searched the literature for the obstetric management of patients with Chiari type I malformation, independent of publication date and language. We also searched our local hospital database from December 2009 to December 2022 for all deliveries to patients with this condition. We identified 137 cases comprising 103 deliveries described in 40 publications that met our inclusion criteria; 34 deliveries were identified in our local database. There were 84 spontaneous vaginal deliveries, 52 caesarean deliveries, and one delivery by unknown modality. Sixty neuraxial blocks were performed; approximately half of these were epidural procedures for labour analgesia. Six patients had new or worsened symptoms following delivery, but it is unclear whether these were related to their Chiari malformation. We identified no cases with brainstem herniation or severe symptoms. We discuss our findings in relation to other published literature and address the concerns described. Our review reveals the use of a variety of modes of delivery and anaesthetic techniques and that most patients suffered no neurological complication. We conclude there is no of evidence to avoid any one approach to labour analgesia, delivery and anaesthesia. We propose a holistic, individualised and patient-centred approach with an appraisal of the risks and benefits to support shared-decision making.

Autoinflammatory Contributors to Cytokine Storm.

Cytokine Storm is a complex and heterogeneous state of life-threatening systemic inflammation and immunopathology. Autoinflammation is a mechanistic category of immune dysregulation wherein immunopathology originates due to poor regulation of innate immunity. The growing family of monogenic Systemic Autoinflammatory Diseases (SAIDs) has been a wellspring for pathogenic insights and proof-of-principle targeted therapeutic interventions. There is surprisingly little overlap between SAID and Cytokine Storm Syndromes, and there is a great deal to be inferred from those SAID that do, and do not, consistently lead to Cytokine Storm. This chapter will summarize how illustrations of the autoinflammatory paradigm have advanced the understanding of human inflammation, including the role of autoinflammation in familial HLH. Next, it will draw from monogenic SAID, both those with strong associations with cytokine storm and those without, to illustrate how the cytokine IL-18 links innate immune dysregulation and cytokine storm.

Reduced myotube diameter induced by combined inhibition of transforming growth factor-ß type I receptors Acvr1b and Tgfbr1 is associated with enhanced ß1-syntrophin expression.

Simultaneous inhibition of transforming growth factor-ß (TGF-ß) type I receptors Acvr1b and Tgfbr1 signalling has been associated with excessive skeletal muscle hypertrophy in vivo. However, it remains unclear whether the increased muscle mass in vivo is a direct result of inhibition of intracellular TGF-ß signalling or whether this is an indirect effect of an altered extracellular anabolic environment. Here, we tested whether individual or simultaneous knockdown of TGF-ß type I receptors in C2C12 myotubes was sufficient to induce muscle hypertrophy. The expression levels of TGF-ß type I receptors Acvr1b and Tgfbr1 in myotubes were knocked down individually or in combination in the absence or presence of TGF-ß1 and myostatin. Knocking down either Acvr1b or Tgfbr1 did not significantly change cell phenotype. Unexpectedly, simultaneous knockdown of both receptors reduced C2C12 myotube diameter, mRNA expression levels of Hgf, Ccn2 and Mymx with or without TGF-ß1 and myostatin administration. In spite of decreased phosphorylation of Smad2/3, phosphorylation of P70S6K was reduced. In addition, the gene expression level of ß1-syntrophin (Sntb1), which encodes a protein associated with the dystrophin-glycoprotein complex, was increased. Parallel experiments where Sntb1 gene expression was reduced showed an increase in myotube diameter and fusion of C2C12 myoblasts. Together, these results indicate that the knockdown of both TGF-ß type I receptors reduced myotube diameter. This atrophic effect was attributed to reduced protein synthesis signalling and an increased expression of ß1-syntrophin. These results have implications for our fundamental understanding of how TGF-ß signalling regulates skeletal muscle size.