Arrhythmogenic mechanism of a novel ryanodine receptor mutation underlying sudden cardiac death.

AIMS: The ryanodine receptor 2 (RyR2) is essential for cardiac muscle excitation-contraction coupling; dysfunctional RyR2 participates in the development of inherited arrhythmogenic cardiac disease. In this study, a novel RyR2 mutation A690E is identified from a patient with family inheritance of sudden cardiac death, and we aimed to investigate the pathogenic basis of the mutation. METHODS AND RESULTS: We generated a mouse model that carried the A690E mutation. Mice were characterized by adrenergic-induced ventricular arrhythmias similar to clinical manifestation of the patient. Optical mapping studies revealed that isolated A690E hearts were prone to arrhythmogenesis and displayed frequency-dependence calcium transient alternans. Upon ß-adrenoceptor challenge, the concordant alternans was shifted towards discordant alternans that favour triggering ectopic beats and Ca2+ re-entry; similar phenomenon was also found in the A690E cardiomyocytes. In addition, we found that A690E cardiomyocytes manifested abnormal Ca2+ release and electrophysiological disorders, including an increased sensitivity to cytosolic Ca2+, an elevated diastolic RyR2-mediated Ca2+ leak, and an imbalance between Ca2+ leak and reuptake. Structural analyses reveal that the mutation directly impacts RyR2-FK506 binding protein interaction. CONCLUSION: In this study, we have identified a novel mutation in RyR2 that is associated with sudden cardiac death. By characterizing the function defects of mutant RyR2 in animal, whole heat, and cardiomyocytes, we demonstrated the pathogenic basis of the disease-causing mutation and provided a deeper mechanistic understanding of a life-threatening cardiac arrhythmia.

Pathogenic mechanism of a catecholaminergic polymorphic ventricular tachycardia causing-mutation in cardiac calcium release channel RyR2.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a condition that is characterized by an abnormal heart rhythm in response to physical or emotional stress. The majority CPVT patients carry mutations in the RYR2 gene that encodes the calcium release channel/ryanodine receptor (RyR2) in cardiomyocytes. The pathogenic mechanisms that account for the clinical phenotypes of CPVT are still elusive. We have identified a de novo mutation, A165D, from a CPVT patient. We found that CPVT phenotypes are recapitulated in A165D knock-in mice. The mutant RyR2 channels enhanced sarcoplasmic reticulum Ca2+ release, triggered delayed afterdepolarization in cardiomyocytes. Structural analysis revealed that the A165D mutation is located in a loop that is involved in inter-subunit interactions in the RyR2 tetrameric structure, it disrupted conformational stability of the RyR2, which favored a closed-to-open state transition, resulting in a leaky channel. The loop also harbors several other CPVT mutations, which suggests a common pathogenic molecular mechanism of CPVT-causing mutations. Our data illustrated disease-relevant functional defects and provide a deeper mechanistic understanding of a life-threatening cardiac arrhythmia.

SBP1 is an adhesion-associated factor without the involvement of virulence in Streptococcus suis serotype 2.

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that infects swine and humans with high mortality and morbidity. Although a number of virulence-associated factors have been reported, the understanding of the molecular mechanism underlying SS2 pathogenicity remains limited. Our previous studies revealed that srtBCD-associated protein 2' (SBP2') contributed to the pathogenesis of SS2, but the function of another member in the srtBCD cluster, srtBCD-associated protein 1 (SBP1) was still unknown. Here, we found that sbp1 was widely distributed among high virulent SS2 strains, suggesting that sbp1 may be involved in the pathogenesis of SS2. To investigate the function of SBP1, we firstly conducted Western blotting analyses to confirm that SBP1 was expressed in the high virulent SS2 strain ZY05719 both in vivo and in vitro, then constructed the deletion mutant of sbp1 by homologous recombination. Bacterial adhesion assay, indirect immunofluorescence assay and protein binding assay all demonstrated that SBP1 was associated with adhesion of SS2 to HEp-2 cells. However, SBP1 did not influence the invasion, phagocytosis or intracellular survival of SS2. Furthermore, infection assays in vivo showed that inactivation of sbp1 failed to impair the ability of SS2 to cause zebrafish and mouse mortality. Overall, these results indicate that SBP1 is an adhesion-associated factor without the involvement of virulence in Streptococcus suis serotype 2.

A TLR4-Targeting Bioactive Peptide Hydrogel to Regulate Immune-Microenvironment for Diabetic Wound Repair.

Persistent inflammation and disrupted immunoregulation are critical factors in impeding diabetic wound healing. While immunoregulatory hydrogel dressings hold significant promise for clinical applications in diabetic wound healing, the current application often demands intricate interventions and high-cost treatments involving cytokines and cell therapies. The development of single component immunoregulatory hydrogels remains a complex challenge. To address this issue, an active peptide hydrogel with immunoregulatory properties targeting the TLR4/NF-kB pathway, aiming to promote rapid diabetic wound healing, is engineered. The hydrogel sequence comprises naphthalene derivative, phenylalanine, and glycine to modulate hydrophilic/hydrophobic characteristics. The amino group on arginine contributes to tissue adhesion and regulation of intermolecular forces, ultimately yielding stable gels. The results underscore the formation of the peptide hydrogel (NFA) via the physical crosslinking of self-assembled nanofibers in water, thereby affording both excellent injectability and tissue adhesion. Notably, NFA demonstrates significant potential in promoting wound healing in a mouse model with full-thickness wounds by regulating macrophage responses in the inflammatory microenvironment through the TLR4/NF-kB pathway.

Pan-genome analysis of Streptococcus suis serotype 2 revealed genomic diversity among strains of different virulence.

Streptococcus suis (SS) is an emerging zoonotic pathogen that causes severe infections in swine and humans. Among the 33 known serotypes, serotype 2 is most frequently associated with infections in pigs and humans. To better understand the virulence characterization of S. suis serotype 2 (SS2) and discriminate the difference between virulent and avirulent strains in SS2, characterization of the genomic features of strains with different virulence is required. The result showed that Streptococcus suis have an open pan-genome. The pan-genome shared by the 19 S. suis serotype 2 strains was composed of 1,239 core genes and 2,436 accessory genes. COG analysis indicated that core genes are involved in the basic physiological function, but accessory genes related to tachytely evolution. Comparative analysis between core genomes of virulent strains and 9 avirulent strains suggested that srtBCD pilus cluster was a significant discrepancy between virulent and avirulent strains. Analysis between high virulent and group B low virulent strains showed 53 and 58 genes specific to each other. Moreover, genomes of avirulent strains tend to be larger than virulent strains; avirulent strains tend to possess more prophages sequences than virulent strains. Our findings could be contributed to a better understanding of the genomics of S. suis serotype 2.

Somatotype and Its Impact on Asymptomatic Target Organ Damage in the Elderly Chinese: The Northern Shanghai Study.

OBJECTIVE: To investigate the relationship between asymptomatic target organ damage (TOD) and different somatotypes in a population of elderly from Chinese community-dwelling. METHODS: A total of 2098 Chinese senior residents from northern Shanghai older than 65 years were recruited in the research. The following somatotype parameters were recorded and analyzed: body mass index, waist circumference, hip circumference, and waist-hip ratio were recorded and calculated. Asymptomatic TOD, including urine albumin/creatinine ratio, estimated glomerular filtration rate (eGFR), intima-media thickness (IMT), left ventricular mass index (LVMI), left ventricular diastolic function, and carotid-femoral pulse wave velocity (PWV) was recorded using the MyLab30 Gold CV system and SphygmoCor. RESULTS: Of all 2098 residents, 817 (38.9%) were overweight and 289 (13.8%) were obese. All somatotype measures were significantly correlated with TOD parameters (p<0.05). After adjustment for age and male gender, in total population, LVMI (p<0.001), cardiac diastolic function (E/Ea, p<0.001), PWV (p<0.001), eGFR (p=0.03), and urine albumin/creatinine ratio (p<0.001) changed gradually and significantly correlated with increasing BMI values. Obesity and overweight were independently related to the incidence of LVH, LVDD, artery stiffness, carotid arterial plaque, and microalbuminuria. CONCLUSION: The incidence of asymptomatic TOD was significantly correlated with overweight and obesity, especially in women, whereas the underweight may favor in the prevention of TOD.

Functional Profile of Human Cytomegalovirus Genes and Their Associated Diseases: A Review.

The human cytomegalovirus (HCMV), whose genome is 235 ± 1.9 kbp long, is a common herpesvirus. However, the functions of many of its genes are still unknown. HCMV is closely associated with various human diseases and infects 60-90% of the global population. It can infect various human cells, including fibroblasts, epithelial cells, endothelial cells, smooth muscle cells, and monocytes. Although HCMV infection is generally asymptomatic and causes subtle clinical symptoms, it can generate a robust immune response and establish a latent infection in immunocompromised individuals, including those with AIDS, transplant recipients, and developing fetuses. Currently available antivirals approved for the treatment of HCMV-associated diseases are limited by dose-limiting toxicity and the emergence of resistance; however, vaccines and immunoglobulins are unavailable. In this review, we have summarized the recent literature on 43 newly identified HCMV genes. We have described their novel functions on the viral replication cycle, latency, and host immune evasion. Further, we have discussed HCMV-associated diseases and current therapeutic targets. Our review may provide a foundational basis for studies aiming to prevent and develop targeted therapies for HCMV-associated diseases.

Infection and adaption-based proteomic changes of Streptococcus suis serotype 2 in a pig model.

Streptococcus suis (S. suis) is an emerging zoonotic agent that is responsible for significant economic losses to the porcine industry worldwide. However, most research regarding the pathogenic mechanisms has used in vitro cultures of S. suis, which may not provide an accurate representation of the in vivo biological activities. In this study, 188 differential abundance S. suis proteins were identified in in vivo samples obtained from the blood of the infected pigs. These were compared with in vitro samples by a Tandem Mass Tags (TMT) experiment. Thus, a virulence associated network was established using the enriched differential abundance proteins (obtained via bioinformatics analysis in this study) and the previously reported putative virulence factors associated with in vivo infection. One of the most important up-regulated hubs in this network, adhE (an acetaldehyde-CoA/alcohol dehydrogenase) was found. Furthermore, knocking out adhE in S. suis serotype 2 strain ZY05719 decreased virulence. Cell culture experiments and far-western blot analysis showed that adhE is involved in adhesion to Caco-2 cells; Hsp60 could be one of the receptors for this protein. SIGNIFICANCE: This study is a systematical research to identify in vivo regulated virulence associated proteins of S. suis in pigs. It constructs a network consisting of in vivo infection related factors for the first time to get to know the coordinated actions of a multitude of factors that lead to host pathogenicity and filter the most important hubs. The individual factors that contribute to infection is also identified. A novel differential protein adhE which is one of the most important hubs of this network and is up-regulated in abundance in vivo is found to moonlight as an important adhesion by binding Hsp60 and finally contributes to virulence.

Fibronectin-/fibrinogen-binding protein (FBPS) is not a critical virulence factor for the Streptococcus suis serotype 2 strain ZY05719.

Fibronectin-/fibrinogen-binding protein (FBPS) of Streptococcus suis serotype 2 (SS2) is an atypical anchorless microbial surface components recognizing adhesive matrix molecules (MSCRAMM) for which the role in bacterial infection are not clearly established. To investigate the biological functions of FBPS, an fbps knockout mutant was constructed in SS2 strain ZY05719 to explore the phenotypic changes between the wild-type and mutant strains. Cell morphology analyses combined with the basic growth curves showed that deletion of fbps does not significantly influence neither the thickness of the capsule of SS2 nor the cell growth characteristic. In addition to three previously identified host components fibronectin (FN), factor H (FH) and fibrinogen (FG), we also found that both laminin (LN) and immunoglobulin G (IgG) could bind specifically to FBPS. Furthermore, we confirmed that FBPS play an important role in adherence of SS2 to host cells. The in vitro assays demonstrated that an inactivation of fbps does not inhibit the intracellular survival of SS2 in RAW246.7 macrophages, attenuate the ability of invasion of host cells or the growth ability in pig blood. Additionally, the fbps mutation failed to decrease the virulence of SS2 in both BALB/c mice and zebrafish. Finally, immunization with recombinant FBPS showed no significant difference from the control groups in terms of murine viability after SS2 challenge. Taken together, we concluded that FBPS is not a critical virulence factor for the SS2 strain ZY05719.

SBP2 plays an important role in the virulence changes of different artificial mutants of Streptococcus suis.

Streptococcus suis (SS) is an important bacterial zoonotic pathogen, which can cause infections in pigs and humans. However, the pathogenesis of this bacterium remains unclear, even though some putative virulence factors (VFs) have been reported. Comparative proteomics could be used to identify markers that can distinguish bacterial strains with different virulence; however, the application of this method is restricted by the genome diversities existing in different strains. In this study, two mutants, WT ΔpepT and WT ΔrfeA, which were generated from the same wild-type (WT) strain, ZY05719, and showed opposite virulence tendencies, were constructed. Combining two proteomics assays, two-dimensional difference gel electrophoresis (2D-DIGE) and label-free proteomics, we identified 38 differentially abundant proteins in the mutants compared with their parent, including five known VFs of S. suis and 33 novel elements. One of the novel proteins, a putative pilus protein, named SBP2, was considered as the most promising VF, because SBP2 was not only linked with the known VFs in the virulence interaction network and was proposed to be located on the cell surface, but also showed enriched distribution among highly virulent strains of SS. SBP2 could also bind fibronectin and laminin, two important extracellular matrix proteins of the host, to facilitate the process of adhesion. Thus, spb2 was identified as encoding a promising virulence-associated candidate associated with the pathogenesis of SS, and a comprehensive virulence interaction network of SS was established for the first time.

Improved antifungal activity of amphotericin B-loaded TPGS-b-(PCL-ran-PGA) nanoparticles.

To develop amphotericin B-loaded biodegradable TPGS-b-(PCL-ran-PGA) nanoparticles (PLGA-TPGS-AMB NPs) for fungal infection treatment, PLGA-TPGS NPs and PLGA NPs were synthesized by a modified double emulsion method and characterized in terms of size and size distribution, morphology and zeta potential. Drug encapsulation efficiency, in vitro drug release, and in vitro/vivo tests against Candida glabrata were completed. The data showed that both of the two AMB-loaded NPs (PLGA-AMB NPs, PLGA-TPGS-AMB NPs) achieved significantly higher level of antifungal effects than water suspended AMB. In comparison with PLGA-AMB NPs, PLGA-TPGS-AMB NPs had a stronger protective effect against candidiasis and gained an advantage of prolonged antifungal efficacy. In conclusion, PLGA-TPGS-AMB NPs system significantly improves AMB bioavailability by increasing the aqueous dispersibility and improving the antifungal activity. And this would be an excellent choice for the antifungal treatment of the entrapped drug because of its low toxicity and higher effectiveness.

Anti-transferrin receptor-modified amphotericin B-loaded PLA-PEG nanoparticles cure Candidal meningitis and reduce drug toxicity.

Fatal fungal infections in central nervous system (CNS) can occur through hematogenous spread or direct extension. At present, hydrophobic amphotericin B (AMB) is the most effective antifungal drug in clinical trials. However, AMB is hydrophobic and therefore penetrates poorly into the CNS, and therapeutic levels of AMB are hard to achieve. The transferrin receptor (TfR/CD71) located at the blood-brain barrier mediates transferrin transcytosis. In order to enhance the receptor-mediated delivery of AMB into CNS with therapeutic level, an anti-TfR antibody (OX26)-modified AMB-loaded PLA (poly[lactic acid])-PEG (polyethylene glycol)-based micellar drug delivery system was constructed. The prepared OX26-modified AMB-loaded nanoparticles (OX26-AMB-NPs) showed significant reduction of CNS fungal burden and an increase of mouse survival time. In conclusion, OX26-AMB-NPs represent a promising novel drug delivery system for intracerebral fungal infection.