Identification and Characterization of Legionella pneumophila Effector Proteins Important for in vivo Pathogenesis and Persistence

Legionella pneumophila is a natural pathogen of freshwater protozoa and an opportunistic pathogen of humans. Legionella is the causative agent of Legionnaires’ disease, a potentially fatal atypical pneumonia. This bacteria lives a unique intracellular lifestyle that involves internalization via phagocytosis, rapid avoidance of endocytic degradation, and subversion of host pathways to decorate its compartment into an ER-like, replication-permissive vacuole called the Legionella-Containing Vacuole (LCV). This process is largely mediated by a special secretion machinery called the Dot/Icm type IVb secretion system. This system is predicted to secrete over 300 effector proteins into the host cytosol during intracellular infection and is essential for virulence. Studies over the past two decades have focused on identifying effectors important for Legionella virulence and their specific interactions with the host. Although multiple effectors have been characterized and multiple pathways of Legionella subversion of host pathways have been described, the majority of effectors encoded by L. pneumophila remain uncharacterized. Here, I describe the establishment of insertion sequencing (INSeq) as a valuable tool to comprehensively study the virulence of effector proteins during infections of multiple hosts. Using INSeq in combination with a liquid handling robot, a clonally arrayed pool of Legionella pneumophila mutants was generated, and an effector mutant sublibrary was established and used to perform a high-throughput screen in multiple hosts. I verified effector mutants with virulence defects in this screen and further demonstrated the function of the effector RavY in promoting bacterial intracellular replication after the formation of a mature and protective vacuole. Additionally, I generated a Nlrc4-/- Myd88-/- mouse line that supports sustained replication of flagellated Legionella. Using the effector mutant pool, I performed another INSeq screen in this new mouse line and comprehensively examined the virulence of effector mutants in a 7-day infection. The enhanced resolution of this screen allowed the discovery of novel growth phenotypes, including a large number of effector mutants with fitness advantages during infection. This includes Lpg1354, an effector with predicted GDSL hydrolase activity that may be associated with the growth advantage of the mutant strain and its molecular activity. Together, the establishment of the L. pneumophila effector mutant pool, the Nlrc4-/- Myd88-/- mouse model for sustained in vivo infection, and the identification of novel effector mutants with virulence phenotypes in multiple hosts provide valuable insight on the importance of a large number of previously unstudied effector proteins during Legionella infection. These results may facilitate further characterization of Legionella effectors and shed light on their implications in Legionella pathogenesis and disease

Escherichia coli Uses Separate Enzymes to Produce H2S and Reactive Sulfane Sulfur From L-cysteine

Hydrogen sulfide (H2S) has been proposed to have various physiological functions, and it may function through reactive sulfane sulfur. Since the two sulfur forms often coexist, they are normally considered interchangeable. Here, we characterized the production of H2S and reactive sulfane sulfur in Escherichia coli MG1655 and found that they are not readily interchangeable. They are primarily produced from L-cysteine via different enzymes. L-Cysteine desulfhydrases consumed L-cysteine and directly generated H2S. The produced H2S was mainly lost through evaporation into the gas phase, as E. coli does not have enzymes that easily oxidize H2S to reactive sulfane sulfur. L-Cysteine desulfhydrases were also responsible for the degradation of exogenous L-cysteine, which is toxic at high levels. Conversely, L-cysteine aminotransferase and 3-mercaptopyruvate sulfurtransferase sequentially metabolized endogenous L-cysteine to produce cellular reactive sulfane sulfur; however, it was not a major route of H2S production during normal growth or during the metabolism of exogenous L-cysteine by the resting cells. Noticeably, the 3-mercaptopyruvate sulfurtransferase mutant contained less reactive sulfane sulfur and displayed a greater sensitivity to H2O2 than did the wild type. Thence, reactive sulfane sulfur is likely a common cellular component, involved in protein sulfhydration and protecting cells from oxidative stress

A Robust Hybrid Approach Based on Estimation of Distribution Algorithm and Support Vector Machine for Hunting Candidate Disease Genes

Microarray data are high dimension with high noise ratio and relatively small sample size, which makes it a challenge to use microarray data to identify candidate disease genes. Here, we have presented a hybrid method that combines estimation of distribution algorithm with support vector machine for selection of key feature genes. We have benchmarked the method using the microarray data of both diffuse B cell lymphoma and colon cancer to demonstrate its performance for identifying key features from the profile data of high-dimension gene expression. The method was compared with a probabilistic model based on genetic algorithm and another hybrid method based on both genetics algorithm and support vector machine. The results showed that the proposed method provides new computational strategy for hunting candidate disease genes from the profile data of disease gene expression. The selected candidate disease genes may help to improve the diagnosis and treatment for diseases

Photo-functionalized TiO2 nanotubes decorated with multifunctional Ag nanoparticles for enhanced vascular biocompatibility

Titanium dioxide (TiO2) has a long history of application in blood contact materials, but it often suffers from insufficient anticoagulant properties. Recently, we have revealed the photocatalytic effect of TiO2 also induces anticoagulant properties. However, for long-term vascular implant devices such as vascular stents, besides anticoagulation, also anti-inflammatory, anti-hyperplastic properties, and the ability to support endothelial repair, are desired. To meet these requirements, here, we immobilized silver nanoparticles (AgNPs) on the surface of TiO2 nanotubes (TiO2-NTs) to obtain a composite material with enhanced photo-induced anticoagulant property and improvement of the other requested properties. The photo-functionalized TiO2-NTs showed protein-fouling resistance, causing the anticoagulant property and the ability to suppress cell adhesion. The immobilized AgNPs increased the photocatalytic activity of TiO2-NTs to enhances its photo-induced anticoagulant property. The AgNP density was optimized to endow the TiO2-NTs with anti-inflammatory property, a strong inhibitory effect on smooth muscle cells (SMCs), and low toxicity to endothelial cells (ECs). The in vivo test indicated that the photofunctionalized composite material achieved outstanding biocompatibility in vasculature via the synergy of photo-functionalized TiO2-NTs and the multifunctional AgNPs, and therefore has enormous potential in the field of cardiovascular implant devices. Our research could be a useful reference for further designing of multifunctional TiO2 materials with high vascular biocompatibility

Clinical features of neonatal hyperthyroidism: a retrospective analysis in southwestern China

PurposeThis study aimed to explore the clinical characteristics and evaluate the different types of thyroid dysfunction in babies with neonatal hyperthyroidism.MethodsThe clinical data of 19 neonates with hyperthyroidism admitted to the Children's Hospital of Chongqing Medical University between January 2012 and April 2021 were retrospectively analyzed.ResultsFifteen (78.9%) infants were born to mothers with Graves’ disease. Eleven (57.9%) infants were premature; two babies were born at small for gestational age. The age at diagnosis ranged from 3 to 34 days, with a mean of 18.53 ± 6.85 days. The majority of the babies presented with goiter (84.2%) and tachycardia (94.7%) after birth. Nine (47.4%) of them presented with abnormal weight gain, seven (36.8%) presented with stare or ocular protrusion, six (31.6%) presented with hyperexcitability, four (21.1%) presented with jaundice and liver dysfunction, two (10.5%) presented with sweating, one (5.3%) presented with fever, and one case presented without any symptoms. Transient hyperthyroidism was the main thyroid dysfunction in our study. Overt hyperthyroidism was diagnosed in 13 (68.4%) neonates. Another three babies (15.8%) presented with hyperthyroidism with slightly elevated free triiodothyronine levels, normal thyroxine (T4) levels, and low thyroid-stimulating hormone (TSH) levels. Normal thyroid hormone levels with low TSH levels were observed in three (15.8%) neonates. Ten children were treated with antithyroid drugs. Eighteen children recovered normal thyroid function at 1–3 months of age; one baby in the study group required further levothyroxine supplementation due to primary hypothyroidism (HT). One child was found to have developmental delay at 2 years of age during follow-up.ConclusionsOur study highlights the need for prolonged monitoring of thyroid function in suspected patients. A single normal screening for hyperthyroidism or the absence of a maternal history of hyperthyroidism cannot exclude this disease

Natural compounds protect against the pathogenesis of osteoarthritis by mediating the NRF2/ARE signaling

Osteoarthritis (OA), a chronic joint cartilage disease, is characterized by the imbalanced homeostasis between anabolism and catabolism. Oxidative stress contributes to inflammatory responses, extracellular matrix (ECM) degradation, and chondrocyte apoptosis and promotes the pathogenesis of OA. Nuclear factor erythroid 2-related factor 2 (NRF2) is a central regulator of intracellular redox homeostasis. Activation of the NRF2/ARE signaling may effectively suppress oxidative stress, attenuate ECM degradation, and inhibit chondrocyte apoptosis. Increasing evidence suggests that the NRF2/ARE signaling has become a potential target for the therapeutic management of OA. Natural compounds, such as polyphenols and terpenoids, have been explored to protect against OA cartilage degeneration by activating the NRF2/ARE pathway. Specifically, flavonoids may function as NRF2 activators and exhibit chondroprotective activity. In conclusion, natural compounds provide rich resources to explore the therapeutic management of OA by activating NRF2/ARE signaling

The pattern of late gadolinium enhancement by cardiac MRI in fulminant myocarditis and its prognostic implication: a two-year follow-up study

BackgroundMyocardial fibrosis, as quantified by late gadolinium enhancement (LGE) in cardiac magnetic resonance (CMR), provides valuable prognostic information for patients with myocarditis. However, due to the low incidence rate of fulminant myocarditis (FM) and accordingly small sample size, the knowledge about the role of LGE to patients with FM is limited.Methods and resultsA total of 44 adults with viral-FM receiving the Chinese treating regimen were included in this retrospective study. They were divided into the low LGE group and the high LGE group according to the ratio of LGE to left ventricular mass (LGE mass%). CMR exams and LGE were performed after hemodynamic assistance at discharge in all patients with FM. Routine echocardiography parameters and global longitudinal strain (GLS) at discharge and at 2-year follow-up were obtained and then compared. Both left ventricular ejection fraction (LVEF) and GLS showed no significant difference in both groups at discharge, whereas significant differences were observed at 2-year follow-up between two groups. Moreover, there were significant improvements of LVEF and GLS in the low LGE group, but not in the high LGE group during the 2-year period. Furthermore, LGE mass% was negatively correlated with GLS and LVEF.ConclusionsThere were two distinct forms of LGE presentation in patients with FM. Moreover, the cardiac function of patients with low LGE was significantly better than those with high LGE at 2-year follow-up. LGE mass% at discharge provided significant prognosis information about cardiac function of patients with FM