Distant relatives of a eukaryotic cell-specific toxin family evolved a complement-like mechanism to kill bacteria.

Cholesterol-dependent cytolysins (CDCs) comprise a large family of pore-forming toxins produced by Gram-positive bacteria, which are used to attack eukaryotic cells. Here, we functionally characterize a family of 2-component CDC-like (CDCL) toxins produced by the Gram-negative Bacteroidota that form pores by a mechanism only described for the mammalian complement membrane attack complex (MAC). We further show that the Bacteroides CDCLs are not eukaryotic cell toxins like the CDCs, but instead bind to and are proteolytically activated on the surface of closely related species, resulting in pore formation and cell death. The CDCL-producing Bacteroides is protected from the effects of its own CDCL by the presence of a surface lipoprotein that blocks CDCL pore formation. These studies suggest a prevalent mode of bacterial antagonism by a family of two-component CDCLs that function like mammalian MAC and that are wide-spread in the gut microbiota of diverse human populations.

Development of Consensus-Based Best Practice Guidelines for the Peri- and Post-operative Care of Pediatric Patients with Spinal Deformity and Programmable Implanted Devices.

STUDY DESIGN: Modified Delphi consensus study. OBJECTIVE: To develop consensus-based best practices for the care of pediatric patients who have implanted programmable devices (IPDs) and require spinal deformity surgery. SUMMARY OF BACKGROUND DATA: Implanted programmable devices (IPDs) are often present in patients with neuromuscular or syndromic scoliosis who require spine surgery. Guidelines for monitoring and interrogating these devices during the peri-operative period are not available. METHODS: A panel was assembled consisting of 25 experts (i.e., spinal deformity surgeons, neurosurgeons, neuro-electrophysiologists, cardiologists, and otolaryngologists). Initial postulates were based on literature review and results from a prior survey. Postulates addressed the following IPDs: vagal nerve stimulators (VNS), programmable ventriculo-peritoneal shunts (VPS), intrathecal baclofen pumps (ITBP), cardiac pacemakers and implantable cardioverter-defibrillators (ICD), deep brain stimulators (DBS), and cochlear implants. Cardiologist and otolaryngologists participants responded only to postulates on cardiac pacemakers or cochlear implants, respectively. Consensus was defined as ≥80% agreement, items that did not reach consensus were revised and included in subsequent rounds. A total of three survey rounds and one virtual meeting were conducted. RESULTS: Consensus was reached on 39 total postulates across six IPD types. Postulates addressed general spine surgery considerations, use of intraoperative monitoring and cautery, use of magnetically-controlled growing rods (MCGRs), and use of an external remote controller to lengthen MCGRs. Across IPD types, consensus for the final postulates ranged from 94.4-100%. Overall, experts agreed that MCGRs can be surgically inserted and lengthened in patients with a variety of IPDs and provided guidance for the use of intraoperative monitoring and cautery, which varied between IPD types. CONCLUSION: Spinal deformity correction surgery often benefits from the use of intraoperative monitoring, monopolar and bipolar cautery, and MCGRs. Final postulates from this study can inform the peri- and post-operative practices of spinal deformity surgeons who treat patients with both scoliosis and IPDs. LEVEL OF EVIDENCE: V- Expert opinion.

Pharmacologic hyperstabilisation of the HIV-1 capsid lattice induces capsid failure.

The HIV-1 capsid has emerged as a tractable target for antiretroviral therapy. Lenacapavir, developed by Gilead Sciences, is the first capsid-targeting drug approved for medical use. Here, we investigate the effect of lenacapavir on HIV capsid stability and uncoating. We employ a single particle approach that simultaneously measures capsid content release and lattice persistence. We demonstrate that lenacapavir's potent antiviral activity is predominantly due to lethal hyperstabilisation of the capsid lattice and resultant loss of compartmentalisation. This study highlights that disrupting capsid metastability is a powerful strategy for the development of novel antivirals.

A Retrospective, Cross-Sectional Analysis of Motor Development, Cognition, and Mood in 87 Patients With Childhood-Onset Hereditary Spastic Paraplegias.

BACKGROUND: HSP is a heterogeneous group of rare genetic diseases. In childhood, little is known of the development and psychological manifestations. METHODS: Retrospective analysis of 87 patients with childhood-onset HSP. Patient consent was obtained and data regarding gross motor, fine motor, and language development; equipment usage; surgical procedures; cognition; and mood were collected at each clinic visit and by phone call and analyzed using mean, median, range, and interquartile ranges (IQRs). RESULTS: The cohort contained 18 genetic types of HSP. Participant data ranged from birth to 36 years. Follow-up was variable spanning from a single clinic visit to 24 years of longitudinal visits. The mean age in months of sitting = 7.37, median = 6, range = 5 to 48, IQR = 0; crawling mean = 9.6, median = 9, range 7 to 23, IQR = 0; pulling to stand mean = 10.7, median 9, range: 9 to 36, IQR = 0; and the age for walking was mean = 16.25, median = 15, range = 11 to 63 IQR = 6. Eighteen patients did not achieve independent ambulation. Twenty-five were noted to have initial gait abnormalities. Median age for first word spoken was 12 months. Fifty-five of 87 participants were enrolled in mainstream or honors classes. Twenty-two of 87 had attention deficit disorder. Patients reported experiencing sadness around their diagnoses, and 26 of 87 reported being diagnosed with anxiety or depression. CONCLUSIONS: In childhood-onset HSP, motor disorder is the predominant feature; however, screening for attention deficit, anxiety, and depression is indicated.

Diagnostic utility of different types of somatosensory evoked potential changes in pediatric idiopathic scoliosis correction surgery.

PURPOSE: To evaluate the diagnostic accuracy of intraoperative somatosensory evoked potential (SSEP) monitoring and types of SSEP changes in predicting the risk of postoperative neurological outcomes during correction surgery for idiopathic scoliosis (IS) in the pediatric age group (≤ 21 years). METHODS: Database review was performed to identify literature on pediatric patients with IS who underwent correction with intraoperative neuromonitoring. The sensitivity, specificity, and diagnostic odds ratio (DOR) of transient and persistent SSEP changes and complete SSEP loss in predicting postoperative neurological deficits were calculated. RESULTS: Final analysis included 3778 patients. SSEP changes had a sensitivity of 72.9%, specificity of 96.8%, and DOR of 102.3, while SSEP loss had a sensitivity of 41.8%, specificity of 99.3%, and DOR of 133.2 for predicting new neurologic deficits. Transient and persistent SSEP changes had specificities of 96.8% and 99.1%, and DORs of 16.6 and 59, respectively. CONCLUSION: Intraoperative SSEP monitoring can predict perioperative neurological injury and improve surgical outcomes in pediatric scoliosis fusion surgery. LEVEL OF EVIDENCE: Level 2. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Early-Onset and Severe Complex Hereditary Spastic Paraplegia Caused by De Novo Variants in SPAST.

BACKGROUND: Familial hereditary spastic paraplegia (HSP)-SPAST (SPG4) typically presents with a pure HSP phenotype. OBJECTIVE: The aim of this study was to delineate the genotypic and phenotypic spectrum of children with de novo HSP-SPAST. METHODS: This study used a systematic cross-sectional analysis of clinical and molecular features. RESULTS: We report the clinical and molecular spectrum of 40 patients with heterozygous pathogenic de novo variants in SPAST (age range: 2.2-27.7 years). We identified 19 unique variants (16/40 carried the same recurrent variant, p.Arg499His). Symptom onset was in early childhood (median: 11.0 months, interquartile range: 6.0 months) with significant motor and speech delay, followed by progressive ascending spasticity, dystonia, neurogenic bladder dysfunction, gastrointestinal dysmotility, and epilepsy. The mean Spastic Paraplegia Rating Scale score was 32.8 ± 9.7 (standard deviation). CONCLUSIONS: These results confirm that de novo variants in SPAST lead to a severe and complex form of HSP that differs from classic familial pure HSP-SPAST. Clinicians should be aware of this syndrome in the differential diagnosis for cerebral palsy. © 2022 International Parkinson and Movement Disorder Society.

Single-molecule analysis of the entire perfringolysin O pore formation pathway.

The cholesterol-dependent cytolysin perfringolysin O (PFO) is secreted by Clostridium perfringens as a bacterial virulence factor able to form giant ring-shaped pores that perforate and ultimately lyse mammalian cell membranes. To resolve the kinetics of all steps in the assembly pathway, we have used single-molecule fluorescence imaging to follow the dynamics of PFO on dye-loaded liposomes that lead to opening of a pore and release of the encapsulated dye. Formation of a long-lived membrane-bound PFO dimer nucleates the growth of an irreversible oligomer. The growing oligomer can insert into the membrane and open a pore at stoichiometries ranging from tetramers to full rings (~35 mers), whereby the rate of insertion increases linearly with the number of subunits. Oligomers that insert before the ring is complete continue to grow by monomer addition post insertion. Overall, our observations suggest that PFO membrane insertion is kinetically controlled.

Cholesterol-dependent cytolysins: The outstanding questions.

The cholesterol-dependent cytolysins (CDCs) are a major family of bacterial pore-forming proteins secreted as virulence factors by Gram-positive bacterial species. CDCs are produced as soluble, monomeric proteins that bind specifically to cholesterol-rich membranes, where they oligomerize into ring-shaped pores of more than 30 monomers. Understanding the details of the steps the toxin undergoes in converting from monomer to a membrane-spanning pore is a continuing challenge. In this review we summarize what we know about CDCs and highlight the remaining outstanding questions that require answers to obtain a complete picture of how these toxins kill cells.

Ocular Manifestations in Patients with Fanconi Anemia: A Single-Center Experience Including 106 Patients.

OBJECTIVES: To describe the prevalence of acquired ocular manifestations in patients with Fanconi anemia (FA) and to describe and correlate the congenital ocular malformations with the genetic subtypes of the disease. STUDY DESIGN: This is a cross-sectional observational study of 106 consecutive patients with confirmed diagnosis of FA who were followed at the Hematopoietic Stem Cell Transplantation (HSCT) Service at the Federal University of Paraná, Curitiba, Parana, Brazil. Participants underwent a complete ophthalmologic evaluation and 84 patients underwent ocular ultrasound examination. This study was conducted between November 2014 and August 2017. RESULTS: The patients ranged in age from 6 months to 43 years of age. Microphthalmia was the most common congenital ocular abnormality (95.2%). A decrease in anthropometric measurements was observed, including palpebral fissure length (78/103 patients [76.5%]), microcornea (48/103 patients [46.6%]), and ptosis (31/103 patients [30.1%]). We identified a new ophthalmic condition in 15 patients with FA, that is, epiretinal tissue on the optic disc. The genetic subtype was identified in 78 patients (79.6%), the FA-A subtype was most prevalent (50%). The most common acquired ocular manifestation (non-graft-versus-host disease [GVHD] related) in patients who did not undergo HSCT (n = 44) was limbal neovascularization (13.6%), whereas in patients who underwent HSCT (n = 62), the GVHD-related manifestation was ocular GVHD (51.6%). The most frequent symptom of ocular GVHD was keratoconjunctivitis sicca (29%). CONCLUSIONS: Several ocular manifestations were identified in patients with FA.

In Situ Maturated Early-Stage Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes Improve Cardiac Function by Enhancing Segmental Contraction in Infarcted Rats.

The scant ability of cardiomyocytes to proliferate makes heart regeneration one of the biggest challenges of science. Current therapies do not contemplate heart re-muscularization. In this scenario, stem cell-based approaches have been proposed to overcome this lack of regeneration. We hypothesize that early-stage hiPSC-derived cardiomyocytes (hiPSC-CMs) could enhance the cardiac function of rats after myocardial infarction (MI). Animals were subjected to the permanent occlusion of the left ventricle (LV) anterior descending coronary artery (LAD). Seven days after MI, early-stage hiPSC-CMs were injected intramyocardially. Rats were subjected to echocardiography pre-and post-treatment. Thirty days after the injections were administered, treated rats displayed 6.2% human cardiac grafts, which were characterized molecularly. Left ventricle ejection fraction (LVEF) was improved by 7.8% in cell-injected rats, while placebo controls showed an 18.2% deterioration. Additionally, cell-treated rats displayed a 92% and 56% increase in radial and circumferential strains, respectively. Human cardiac grafts maturate in situ, preserving proliferation with 10% Ki67 and 3% PHH3 positive nuclei. Grafts were perfused by host vasculature with no evidence for immune rejection nor ectopic tissue formations. Our findings support the use of early-stage hiPSC-CMs as an alternative therapy to treat MI. The next steps of preclinical development include efficacy studies in large animals on the path to clinical-grade regenerative therapy targeting human patients.

X-ray crystallography shines a light on pore-forming toxins.

A common form of cellular attack by pathogenic bacteria is to secrete pore-forming toxins (PFTs). Capable of forming transmembrane pores in various biological membranes, PFTs have also been identified in a diverse range of other organisms such as sea anemones, earthworms and even mushrooms and trees. The mechanism of pore formation by PFTs is associated with substantial conformational changes in going from the water-soluble to transmembrane states of the protein. The determination of the crystal structures for numerous PFTs has shed much light on our understanding of these proteins. Other than elucidating the atomic structural details of PFTs and the conformational changes that must occur for pore formation, crystal structures have revealed structural homology that has led to the discovery of new PFTs and new PFT families. Here we review some key crystallographic results together with complimentary approaches for studying PFTs. We discuss how these studies have impacted our understanding of PFT function and guided research into biotechnical applications.

New Cardiomyokine Reduces Myocardial Ischemia/Reperfusion Injury by PI3K-AKT Pathway Via a Putative KDEL-Receptor Binding.

Background CDNF (cerebral dopamine neurotrophic factor) belongs to a new family of neurotrophic factors that exert systemic beneficial effects beyond the brain. Little is known about the role of CDNF in the cardiac context. Herein we investigated the effects of CDNF under endoplasmic reticulum-stress conditions using cardiomyocytes (humans and mice) and isolated rat hearts, as well as in rats subjected to ischemia/reperfusion (I/R). Methods and Results We showed that CDNF is secreted by cardiomyocytes stressed by thapsigargin and by isolated hearts subjected to I/R. Recombinant CDNF (exoCDNF) protected human and mouse cardiomyocytes against endoplasmic reticulum stress and restored the calcium transient. In isolated hearts subjected to I/R, exoCDNF avoided mitochondrial impairment and reduced the infarct area to 19% when administered before ischemia and to 25% when administered at the beginning of reperfusion, compared with an infarct area of 42% in the untreated I/R group. This protection was completely abrogated by AKT (protein kinase B) inhibitor. Heptapeptides containing the KDEL sequence, which binds to the KDEL-R (KDEL receptor), abolished exoCDNF beneficial effects, suggesting the participation of KDEL-R in this cardioprotection. CDNF administered intraperitoneally to rats decreased the infarct area in an in vivo model of I/R (from an infarct area of ≈44% in the I/R group to an infarct area of ≈27%). Moreover, a shorter version of CDNF, which lacks the last 4 residues (CDNF-ΔKTEL) and thus allows CDNF binding to KDEL-R, presented no cardioprotective activity in isolated hearts. Conclusions This is the first study to propose CDNF as a new cardiomyokine that induces cardioprotection via KDEL receptor binding and PI3K/AKT activation.

A Key Motif in the Cholesterol-Dependent Cytolysins Reveals a Large Family of Related Proteins.

The cholesterol-dependent cytolysins (CDCs) are bacterial, ß-barrel, pore-forming toxins. A central enigma of the pore-forming mechanism is how completion of the prepore is sensed to initiate its conversion to the pore. We identified a motif that is conserved between the CDCs and a diverse family of nearly 300 uncharacterized proteins present in over 220 species that span at least 10 bacterial and 2 eukaryotic phyla. Except for this motif, these proteins exhibit little similarity to the CDCs at the primary structure level. Studies herein show this motif is a critical component of the sensor that initiates the prepore-to-pore transition in the CDCs. We further show by crystallography, single particle analysis, and biochemical studies of one of these CDC-like (CDCL) proteins from Elizabethkingia anophelis, a commensal of the malarial mosquito midgut, that a high degree of structural similarity exists between the CDC and CDCL monomer structures and both form large oligomeric pore complexes. Furthermore, the conserved motif in the E. anophelis CDCL crystal structure occupies a nearly identical position and makes similar contacts to those observed in the structure of the archetype CDC, perfringolysin O (PFO). This suggests a common function in the CDCs and CDCLs and may explain why only this motif is conserved in the CDCLs. Hence, these studies identify a critical component of the sensor involved in initiating the prepore-to-pore transition in the CDCs, which is conserved in a large and diverse group of distant relatives of the CDCs.IMPORTANCE The cholesterol-dependent cytolysins' pore-forming mechanism relies on the ability to sense the completion of the oligomeric prepore structure and initiate the insertion of the ß-barrel pore from the assembled prepore structure. These studies show that a conserved motif is an important component of the sensor that triggers the prepore-to-pore transition and that it is conserved in a large family of previously unidentified CDC-like proteins, the genes for which are present in a vast array of microbial species that span most terrestrial environments, as well as most animal and human microbiomes. These studies establish the foundation for future investigations that will probe the contribution of this large family of CDC-like proteins to microbial survival and human disease.

Acute Myocardial Infarction Reduces Respiration in Rat Cardiac Fibers, despite Adipose Tissue Mesenchymal Stromal Cell Transplant.

Adipose-derived mesenchymal stromal cell (AD-MSC) administration improves cardiac function after acute myocardial infarction (AMI). Although the mechanisms underlying this effect remain to be elucidated, the reversal of the mitochondrial dysfunction may be associated with AMI recovery. Here, we analyzed the alterations in the respiratory capacity of cardiomyocytes in the infarcted zone (IZ) and the border zone (BZ) and evaluated if mitochondrial function improved in cardiomyocytes after AD-MSC transplantation. Female rats were subjected to AMI by permanent left anterior descending coronary (LAD) ligation and were then treated with AD-MSCs or PBS in the border zone (BZ). Cardiac fibers were analyzed 24 hours (necrotic phase) and 8 days (fibrotic phase) after AMI for mitochondrial respiration, citrate synthase (CS) activity, F0F1-ATPase activity, and transmission electron microscopy (TEM). High-resolution respirometry of permeabilized cardiac fibers showed that AMI reduced numerous mitochondrial respiration parameters in cardiac tissue, including phosphorylating and nonphosphorylating conditions, respiration coupled to ATP synthesis, and maximal respiratory capacity. CS decreased in IZ and BZ at the necrotic phase, whereas it recovered in BZ and continued to drop in IZ over time when compared to Sham. Exogenous cytochrome c doubled respiration at the necrotic phase in IZ. F0F1-ATPase activity decreased in the BZ and, to more extent, in IZ in both phases. Transmission electron microscopy showed disorganized mitochondrial cristae structure, which was more accentuated in IZ but also important in BZ. All these alterations in mitochondrial respiration were still present in the group treated with AD-MSC. In conclusion, AMI led to mitochondrial dysfunction with oxidative phosphorylation disorders, and AD-MSC improved CS temporarily but was not able to avoid alterations in mitochondria function over time.

Loss-of-Function Variants in PPP1R12A: From Isolated Sex Reversal to Holoprosencephaly Spectrum and Urogenital Malformations.

In two independent ongoing next-generation sequencing projects for individuals with holoprosencephaly and individuals with disorders of sex development, and through international research collaboration, we identified twelve individuals with de novo loss-of-function (LoF) variants in protein phosphatase 1, regulatory subunit 12a (PPP1R12A), an important developmental gene involved in cell migration, adhesion, and morphogenesis. This gene has not been previously reported in association with human disease, and it has intolerance to LoF as illustrated by a very low observed-to-expected ratio of LoF variants in gnomAD. Of the twelve individuals, midline brain malformations were found in five, urogenital anomalies in nine, and a combination of both phenotypes in two. Other congenital anomalies identified included omphalocele, jejunal, and ileal atresia with aberrant mesenteric blood supply, and syndactyly. Six individuals had stop gain variants, five had a deletion or duplication resulting in a frameshift, and one had a canonical splice acceptor site loss. Murine and human in situ hybridization and immunostaining revealed PPP1R12A expression in the prosencephalic neural folds and protein localization in the lower urinary tract at critical periods for forebrain division and urogenital development. Based on these clinical and molecular findings, we propose the association of PPP1R12A pathogenic variants with a congenital malformations syndrome affecting the embryogenesis of the brain and genitourinary systems and including disorders of sex development.

The Structural Basis for a Transition State That Regulates Pore Formation in a Bacterial Toxin.

The cholesterol-dependent cytolysin (CDC) genes are present in bacterial species that span terrestrial, vertebrate, and invertebrate niches, which suggests that they have evolved to function under widely different environmental conditions. Using a combination of biophysical and crystallographic approaches, we reveal that the relative stability of an intramolecular interface in the archetype CDC perfringolysin O (PFO) plays a central role in regulating its pore-forming properties. The disruption of this interface allows the formation of the membrane spanning ß-barrel pore in all CDCs. We show here that the relative strength of the stabilizing forces at this interface directly impacts the energy barrier posed by the transition state for pore formation, as reflected in the Arrhenius activation energy (Ea) for pore formation. This change directly impacts the kinetics and temperature dependence of pore formation. We further show that the interface structure in a CDC from a terrestrial species enables it to function efficiently across a wide range of temperatures by minimizing changes in the strength of the transition state barrier to pore formation. These studies establish a paradigm that CDCs, and possibly other ß-barrel pore-forming proteins/toxins, can evolve significantly different pore-forming properties by altering the stability of this transitional interface, which impacts the kinetic parameters and temperature dependence of pore formation.IMPORTANCE The cholesterol-dependent cytolysins (CDCs) are the archetype for the superfamily of oligomeric pore-forming proteins that includes the membrane attack complex/perforin (MACPF) family of immune defense proteins and the stonefish venom toxins (SNTX). The CDC/MACPF/SNTX family exhibits a common protein fold, which forms a membrane-spanning ß-barrel pore. We show that changing the relative stability of an extensive intramolecular interface within this fold, which is necessarily disrupted to form the large ß-barrel pore, dramatically alters the kinetic and temperature-dependent properties of CDC pore formation. These studies show that the CDCs and other members of the CDC/MACPF/SNTX superfamily have the capacity to significantly alter their pore-forming properties to function under widely different environmental conditions encountered by these species.

Studying Munc18:Syntaxin Interactions Using Small-Angle Scattering.

The interaction between the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein syntaxin (Sx) and regulatory partner Sec/Munc18 (SM) protein is a critical step in vesicle fusion. The exact role played by SM proteins, whether positive or negative, has been the topic of much debate. High-resolution structures of the SM:Sx complex have shown that SM proteins can bind syntaxin in a closed fusion incompetent state. However, in vitro and in vivo experiments also point to a positive regulatory role for SM proteins that is inconsistent with binding syntaxin in a closed conformation. Here we present protocols we used for the expression and purification of the SM proteins Munc18a and Munc18c and syntaxins 1 and 4 along with procedures used for small-angle X-ray and neutron scattering that showed that syntaxins can bind in an open conformation to SM proteins. We also describe methods for chemical cross-linking experiments and detail how this information can be combined with scattering data to obtain low-resolution structural models for SM:Sx protein complexes.

Cholesterol-dependent cytolysins: from water-soluble state to membrane pore.

The cholesterol-dependent cytolysins (CDCs) are a family of bacterial toxins that are important virulence factors for a number of pathogenic Gram-positive bacterial species. CDCs are secreted as soluble, stable monomeric proteins that bind specifically to cholesterol-rich cell membranes, where they assemble into well-defined ring-shaped complexes of around 40 monomers. The complex then undergoes a concerted structural change, driving a large pore through the membrane, potentially lysing the target cell. Understanding the details of this process as the protein transitions from a discrete monomer to a complex, membrane-spanning protein machine is an ongoing challenge. While many of the details have been revealed, there are still questions that remain unanswered. In this review, we present an overview of some of the key features of the structure and function of the CDCs, including the structure of the secreted monomers, the process of interaction with target membranes, and the transition from bound monomers to complete pores. Future directions in CDC research and the potential of CDCs as research tools will also be discussed.

Embryonic stem cell-derived cardiomyocytes for the treatment of doxorubicin-induced cardiomyopathy.

BACKGROUND: Doxorubicin (Dox) is a chemotherapy drug with limited application due to cardiotoxicity that may progress to heart failure. This study aims to evaluate the role of cardiomyocytes derived from mouse embryonic stem cells (CM-mESCs) in the treatment of Dox-induced cardiomyopathy (DIC) in mice. METHODS: The mouse embryonic stem cell (mESC) line E14TG2A was characterized by karyotype analysis, gene expression using RT-PCR and immunofluorescence. Cells were transduced with luciferase 2 and submitted to cardiac differentiation. Total conditioned medium (TCM) from the CM-mESCs was collected for proteomic analysis. To establish DIC in CD1 mice, Dox (7.5 mg/kg) was administered once a week for 3 weeks, resulting in a cumulative Dox dose of 22.5 mg/kg. At the fourth week, a group of animals was injected intramyocardially with CM-mESCs (8 × 105 cells). Cells were tracked by a bioluminescence assay, and the body weight, echocardiogram, electrocardiogram and number of apoptotic cardiomyocytes were evaluated. RESULTS: mESCs exhibited a normal karyotype and expressed pluripotent markers. Proteomic analysis of TCM showed proteins related to the negative regulation of cell death. CM-mESCs presented ventricular action potential characteristics. Mice that received Dox developed heart failure and showed significant differences in body weight, ejection fraction (EF), end-systolic volume (ESV), stroke volume (SV), heart rate and QT and corrected QT (QTc) intervals when compared to the control group. After cell or placebo injection, the Dox + CM-mESC group showed significant increases in EF and SV when compared to the Dox + placebo group. Reduction in ESV and QT and QTc intervals in Dox + CM-mESC-treated mice was observed at 5 or 30 days after cell treatment. Cells were detected up to 11 days after injection. The Dox + CM-mESC group showed a significant reduction in the percentage of apoptotic cardiomyocytes in the hearts of mice when compared to the Dox + placebo group. CONCLUSIONS: CM-mESC transplantation improves cardiac function in mice with DIC.

Revisiting interaction specificity reveals neuronal and adipocyte Munc18 membrane fusion regulatory proteins differ in their binding interactions with partner SNARE Syntaxins.

The efficient delivery of cellular cargo relies on the fusion of cargo-carrying vesicles with the correct membrane at the correct time. These spatiotemporal fusion events occur when SNARE proteins on the vesicle interact with cognate SNARE proteins on the target membrane. Regulatory Munc18 proteins are thought to contribute to SNARE interaction specificity through interaction with the SNARE protein Syntaxin. Neuronal Munc18a interacts with Syntaxin1 but not Syntaxin4, and adipocyte Munc18c interacts with Syntaxin4 but not Syntaxin1. Here we show that this accepted view of specificity needs revision. We find that Munc18c interacts with both Syntaxin4 and Syntaxin1, and appears to bind "non-cognate" Syntaxin1 a little more tightly than Syntaxin4. Munc18a binds Syntaxin1 and Syntaxin4, though it interacts with its cognate Syntaxin1 much more tightly. We also observed that when bound to non-cognate Munc18c, Syntaxin1 captures its neuronal SNARE partners SNAP25 and VAMP2, and Munc18c can bind to pre-formed neuronal SNARE ternary complex. These findings reveal that Munc18a and Munc18c bind Syntaxins differently. Munc18c relies principally on the Syntaxin N-peptide interaction for binding Syntaxin4 or Syntaxin1, whereas Munc18a can bind Syntaxin1 tightly whether or not the Syntaxin1 N-peptide is present. We conclude that Munc18a and Munc18c differ in their binding interactions with Syntaxins: Munc18a has two tight binding modes/sites for Syntaxins as defined previously but Munc18c has just one that requires the N-peptide. These results indicate that the interactions between Munc18 and Syntaxin proteins, and the consequences for in vivo function, are more complex than can be accounted for by binding specificity alone.