Dopamine Receptor 1 Impedes ILC2-Mediated Antitumor Immunity.

Ever-growing evidence has revealed that group 2 innate lymphoid cells (ILC2s) exhibit pleiotropic effects in antihelminth immunity, allergy, tissue protection, and cancer. Currently, the role of ILC2s in cancer is highly controversial regarding the intricate tumor microenvironment (TME), and the tumor-promoting or antitumor immunological mechanisms of ILC2s remain largely unknown. In this study, we report that dopamine receptor 1 (DRD1) restrains ILC2 activity in the TME. DRD1 deficiency promotes ILC2 activation, which irritates eosinophil recruitment and cytotoxic CD8+ T cell expansion during ongoing malignancy. Consequently, DRD1-deficient mice exhibit delayed tumor growth and reduced tumor progression. Furthermore, fenoldopam, a selective DRD1 agonist, restrains the ILC2 response in the TME and aggravates tumor burden in mice. Taken together, our data elaborate that the DRD1 signal acts as an excitatory rheostat in regulating ILC2-dependent antitumor immunity.

M1 polarization of macrophages promotes stress-induced hair loss via interleukin-18 and interleukin-1ß.

Stress-induced hair loss is a prevalent health concern, with mechanisms that remain unclear, and effective treatment options are not yet available. In this study, we investigated whether stress-induced hair loss was related to an imbalanced immune microenvironment. Screening the skin-infiltrated immune cells in a stressed mouse model, we discovered a significant increase in macrophages upon stress induction. Clearance of macrophages rescues mice from stress-induced hair shedding and depletion of hair follicle stem cells (HFSCs) in the skin, demonstrating the role of macrophages in triggering hair loss in response to stress. Further flow cytometry analysis revealed a significant increase in M1 phenotype macrophages in mice under stressed conditions. In searching for humoral factors mediating stress-induced macrophage polarization, we found that the hormone Norepinephrine (NE) was elevated in the blood of stressed mice. In addition, in-vivo and in-vitro studies confirm that NE can induce macrophage polarization toward M1 through the ß-adrenergic receptor, Adrb2. Transcriptome, enzyme-linked immunosorbent assay (ELISA), and western blot analyses reveal that the NLRP3/caspase-1 inflammasome signaling and its downstream effector interleukin 18 (IL-18) and interleukin 1 beta (IL-1ß) were significantly upregulated in the NE-treated macrophages. However, inhibition of the NE receptor Adrb2 with ICI118551 reversed the upregulation of NLRP3/caspase-1, IL-18, and IL-1ß. Indeed, IL-18 and IL-1ß treatments lead to apoptosis of HFSCs. More importantly, blocking IL-18 and IL-1ß signals reversed HFSCs depletion in skin organoid models and attenuated stress-induced hair shedding in mice. Taken together, this study demonstrates the role of the neural (stress)-endocrine (NE)-immune (M1 macrophages) axis in stress-induced hair shedding and suggestes that IL-18 or IL-1ß may be promising therapeutic targets.

Exploring Shared Genetic Signatures of Alzheimer's Disease and Multiple Sclerosis: A Bioinformatic Analysis Study.

INTRODUCTION: Many clinical studies reported the coexistence of Alzheimer's disease (AD) and multiple sclerosis (MS), but the common molecular signature between AD and MS remains elusive. The purpose of our study was to explore the genetic linkage between AD and MS through bioinformatic analysis, providing new insights into the shared signatures and possible pathogenesis of two diseases. METHODS: The common differentially expressed genes (DEGs) were determined between AD and MS from datasets obtained from Gene Expression Omnibus (GEO) database. Further, functional and pathway enrichment analysis, protein-protein interaction network construction, and identification of hub genes were carried out. The expression level of hub genes was validated in two other external AD and MS datasets. Transcription factor (TF)-gene interactions and gene-miRNA interactions were performed in NetworkAnalyst. Finally, receiver operating characteristic (ROC) curve analysis was applied to evaluate the predictive value of hub genes. RESULTS: A total of 75 common DEGs were identified between AD and MS. Functional and pathway enrichment analysis emphasized the importance of exocytosis and synaptic vesicle cycle, respectively. Six significant hub genes, including CCL2, CD44, GFAP, NEFM, STXBP1, and TCEAL6, were identified and verified as common hub genes shared by AD and MS. FOXC1 and hsa-mir-16-5p are the most common TF and miRNA in regulating hub genes, respectively. In the ROC curve analysis, all hub genes showed good efficiency in helping distinguish patients from controls. CONCLUSION: Our study first identified a common genetic signature between AD and MS, paving the road for investigating shared mechanism of AD and MS.

Toll-like receptor 10 expression in B cells is negatively correlated with the progression of primary Sjögren's disease.

Primary Sjögren's Disease (pSjD) is considered a B cell-mediated disease. Toll-like receptor 10 (TLR10) is highly expressed in human B cells, indicating that TLR10 probably plays a vital role in pSjD. We examined TLR10 expression in peripheral B subsets of pSjD patients and analyzed their association with disease activity. We observed that TLR10 expression in total, naïve, memory, and switched memory B cells was significantly increased in low-activity pSjD patients as compared with healthy controls and high-activity patients. TLR10 expression in the above mentioned B subsets (except naïve B) was negatively correlated with serum levels of anti-SSA antibody and BAFF, respectively. Moreover, a higher proportion of high-activity pSjD patients was observed in TLR10 low- than high-expressed patients. Our study concluded that TLR10 expression in CD19+ B and memory B was negatively correlated with pSjD disease activity, suggesting that TLR10 might take part in the progression of pSjD.

Nucleic acid therapy in pediatric cancer.

The overall survival, progress free survival, and life quality of cancer patients have improved due to the advance in minimally invasive surgery, precision radiotherapy, and various combined chemotherapy in the last decade. Furthermore, the discovery of new types of therapeutics, such as immune checkpoint inhibitors and immune cell therapies have facilitated both patients and doctors to fight with cancers. Moreover, in the context of the development in biocompatible and cell type targeting nano-carriers as well as nucleic acid-based drugs for initiating and enhancing the anti-tumor response have come to the age. The treatment paradigms utilization of nucleic acids, including short interfering RNA (siRNA), antisense oligonucleotides (ASO), and messenger RNA (mRNA), can target specific protein expression to achieve the therapeutic effects. Over ten nucleic acid therapeutics have been approved by the FDA and EMA in rare diseases and genetic diseases as well as dozens of registered clinical trails for varies cancers. Though generally less dangerous of pediatric cancers than adult cancers was observed during the past decades, yet pediatric cancers accounted for a significant proportion of child deaths which hurt those family very deeply. Therefore, it is necessary to pay more attention for improving the treatment of pediatric cancer and discovering new nucleic acid therapeutics which may help to improve the therapeutic effect and prognoses in turns to ameliorate the survival period and quality of life for children patient. In this review, we focus on the nucleic acid therapy in pediatric cancers.

Immunoglobulin D enhances immune surveillance by activating antimicrobial, proinflammatory and B cell-stimulating programs in basophils.

Immunoglobulin D (IgD) is an enigmatic antibody isotype that mature B cells express together with IgM through alternative RNA splicing. Here we report active T cell-dependent and T cell-independent IgM-to-IgD class switching in B cells of the human upper respiratory mucosa. This process required activation-induced cytidine deaminase (AID) and generated local and circulating IgD-producing plasmablasts reactive to respiratory bacteria. Circulating IgD bound to basophils through a calcium-mobilizing receptor that induced antimicrobial, opsonizing, inflammatory and B cell-stimulating factors, including cathelicidin, interleukin 1 (IL-1), IL-4 and B cell-activating factor (BAFF), after IgD crosslinking. By showing dysregulation of IgD class-switched B cells and 'IgD-armed' basophils in autoinflammatory syndromes with periodic fever, our data indicate that IgD orchestrates an ancestral surveillance system at the interface between immunity and inflammation.

CLIC1 a novel biomarker of intraperitoneal metastasis in serous epithelial ovarian cancer.

Early diagnosis of intraperitoneal metastasis is a pivot for survival of patients with serous epithelial ovarian cancers (SEOC). However, to date, there is lack of efficient molecular biomarker for early metastasis of SEOC. Here, we found that the expression of chloride intracellular channel 1 (CLIC1) is highly correlative with intraperitoneal metastasis. There is very low expression of CLIC1 in normal ovaries (NO), benign ovarian tumor (BOT), and primary ovarian cancer without metastasis (POCNM); but its expression is remarkably high in primary ovarian cancer with metastasis (POCM) omentum and peritoneal metastasis. Furthermore, for clinic prediction of intraperitoneal metastasis of SEOC, the sensitivity and specificity of CLIC1 overexpression were 97.4 and 88.1 %, respectively. Collectively, CLIC1 may be a potential sensitive and specific molecular biomarker for early diagnose for SEOC metastasis.

Secretory Nogo-B regulates Th2 differentiation in the lung cancer microenvironment.

Nogo-B, a ubiquitously expressed member of the reticulon family, plays an important role in maintaining endoplasmic reticulum (ER) structure, regulating protein folding, and calcium homeostasis. In this study, we demonstrate that Nogo-B expression and secretion are upregulated in lung cancer and correlate to overall survival. Nogo-B is secreted by various cells, particularly lung cancer cells. ER stress and phosphorylation at serine 107 can induce Nogo-B secretion. Secretory Nogo-B suppresses the differentiation of Th2 cells and the release of type 2 cytokines, thus influencing the anti-tumor effects of Th2-related immune cells, including IgE+B cell class switching and eosinophil activation.

Characterization of SARS-CoV-2-specific humoral immunity and associated factors in the healthy population post-vaccination.

OBJECTIVES: To investigate factors that may influence humoral immunity post-vaccination with a COVID-19-inactivated vaccine (SC2IV). METHODS: A total of 1596 healthy individuals from the Seventh Affiliated Hospital, Sun Yat-sen University (1217) and Shenzhen Baotian Hospital (379) were enrolled in this study among which 694 and 218 participants were vaccinated with two-dose SC2IV, respectively. Physical examination indices were recorded. The levels of neutralizing antibody (NA), Spike IgG, receptor-binding domain (RBD) IgG, RBD IgG + IgM + IgA, and nucleocapsid IgG of SARS-CoV-2 were measured by a non-virus ELISA kit. Multiple statistical analyses were carried out to identify factors that influence humoral immunity post-vaccination. RESULTS: The two-dosage vaccination could induce NA in more than 90 % of recipients. The NA has the strongest correlation with anti-RBD IgG. Age is the most important independent index that affects the NA level, while basophil count, creatine kinase-MB, mean corpuscular hemoglobin, the ratio of albumin to urine creatinine, and thyroglobulin antibody have relatively minor contributions. Indices that affect the NA level were different between males and females. Antibodies targeting other epitopes of SARS-CoV-2 were detected in recipients without anti-RBD. CONCLUSIONS: The factors identified in association with the NA level post-vaccination may help to evaluate the protective effect, risk of re-infection, the severity of symptoms, and prognosis for vaccine recipients in clinical.

A Germinal Center Checkpoint of AIRE in B Cells Limits Antibody Diversification.

In response to antigens, B cells undergo affinity maturation and class switching mediated by activation-induced cytidine deaminase (AID) in germinal centers (GCs) of secondary lymphoid organs, but uncontrolled AID activity can precipitate autoimmunity and cancer. The regulation of GC antibody diversification is of fundamental importance but not well understood. We found that autoimmune regulator (AIRE), the molecule essential for T cell tolerance, is expressed in GC B cells in a CD40-dependent manner, interacts with AID and negatively regulates antibody affinity maturation and class switching by inhibiting AID function. AIRE deficiency in B cells caused altered antibody repertoire, increased somatic hypermutations, elevated autoantibodies to T helper 17 effector cytokines and defective control of skin Candida albicans. These results define a GC B cell checkpoint of humoral immunity and illuminate new approaches of generating high-affinity neutralizing antibodies for immunotherapy.

Ten-eleven translocation (Tet) and thymine DNA glycosylase (TDG), components of the demethylation pathway, are direct targets of miRNA-29a.

The ten-eleven translocation family of proteins (Tet1/2/3, Tets) converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which can be further oxidized and repaired by thymine DNA glycosylase (TDG), to influence gene transcription in embryonic and adult tissues. However the mechanisms of how Tets and TDG levels are regulated are unknown. We show that miR-29 can directly regulate Tet1-3 and TDG mRNA levels through binding to their 3'UTRs. miR-29 mimic decreases global 5hmC levels, a hallmark of Tet activity. Moreover, the mRNA levels for Tet3 and TDG are inversely correlated with the levels of miR-29 in aged mouse aorta implying that aging may affect methylation patterns via miRNA. In summary, our data show that Tets and TDG are direct targets of miR-29 and unravel a novel regulatory role for this miRNA in epigenetic DNA demethylation pathways.

Hypoxia-inducible factor-1 drives annexin A2 system-mediated perivascular fibrin clearance in oxygen-induced retinopathy in mice.

Oxygen-induced retinopathy (OIR) is a well-characterized model for retinopathy of prematurity, a disorder that results from rapid microvascular proliferation after exposure of the retina to high oxygen levels. Here, we report that the proliferative phase of OIR requires transcriptional induction of the annexin A2 (A2) gene through the direct action of the hypoxia-inducible factor-1 complex. We show, in addition, that A2 stabilizes its binding partner, p11, and promotes OIR-related angiogenesis by enabling clearance of perivascular fibrin. Adenoviral-mediated restoration of A2 expression restores neovascularization in the oxygen-primed Anxa2(-/-) retina and reinstates plasmin generation and directed migration in cultured Anxa2(-/-) endothelial cells. Systemic depletion of fibrin repairs the neovascular response to high oxygen treatment in the Anxa2(-/-) retina, whereas inhibition of plasminogen activation dampens angiogenesis under the same conditions. These findings show that the A2 system enables retinal neoangiogenesis in OIR by enhancing perivascular activation of plasmin and remodeling of fibrin. These data suggest new potential approaches to retinal angiogenic disorders on the basis of modulation of perivascular fibrinolysis.

The prognostic and immunological role of FKBP1A in an integrated muti-omics cancers analysis, especially lung cancer.

BACKGROUND AND AIM: FKBP1A, a gene encoding the FK506-binding protein 1A, has emerged as a significant player in cancer progression and prognosis. This study aimed to comprehensively investigate the multifaceted role of FKBP1A in cancer, focusing on its differential expression patterns, prognostic implications, genetic alterations, and associations with the tumor microenvironment. METHODS AND RESULTS: Using large-scale datasets, including GTEx, TCGA, HPA, and cBioPortal, we analyzed FKBP1A expression across normal tissues and various cancer types. Our findings revealed that FKBP1A exhibited aberrant upregulation in most human cancers, making it a potential biomarker for malignancy. Moreover, FKBP1A expression correlated with poor overall survival, disease-specific survival, disease-free interval, and progression-free interval in several cancers, indicating its prognostic significance. Genetic alteration analysis showed that FKBP1A gene amplification was prevalent, particularly in ovarian cancer. Furthermore, FKBP1A expression was associated with tumor mutational burden and microsatellite instability, highlighting its potential involvement in tumor-immune response. Notably, FKBP1A expression positively correlated with stromal and immune cell scores, suggesting its role in shaping the tumor microenvironment. Additionally, according to the functional enrichment analysis, experimental validation in lung adenocarcinoma confirmed the role of FKBP1A through the regulation of EGFR signaling by apoptosis, which is consistent with drug sensitivity analysis to some extent. CONCLUSION: In conclusion, FKBP1A exhibits differential expression in cancer, serves as a prognostic indicator, undergoes genetic alterations, and influences the tumor-immune microenvironment. These findings shed light on the multifaceted role of FKBP1A in cancer development and progression, suggesting its potential as a therapeutic target and guidance of clinical drugs selection, and provide valuable insights into patient prognosis for interventions based on pharmaceuticals.

Associations of dietary antioxidant micronutrients with the prevalence of obesity in adults.

Background: Antioxidant micronutrients have a therapeutic potential for clinical treatment of obesity. NO research, however, has examined the connection between the complex level of dietary antioxidants and obesity. Materials and methods: We mainly aimed to investigate the relationship between a combination of antioxidants and obesity using the database of the national health and nutrition examination survey (NHANES). This cross-sectional study contains a survey of 41,021 people (≥18 years) in total ranging from 2005 to 2018. Multivariate logistic and weighted quantile sum (WQS) regression were performed to investigate the associations between these antioxidants, both individually and collectively, and the prevalence of obesity. The restricted cubic spline (RCS) regression was also utilized to analyze the linearity of these associations. Results: According to multivariate logistic models, we found that the levels of most antioxidants in the highest quartile were independently related to a lower prevalence of obesity, while a reverse result was observed in selenium (p for trend <0.05). The WQS index revealed that a total of the 11 antioxidants is negatively related to the prevalence of obesity and abdominal obesity (all p<0.001), and iron/vitamin C have the greatest weight in the negative associations between antioxidant complex and obesity, as well as abdominal obesity. In addition, the RCS regression showed that retinol, vitamin A, α-carotene, ß-carotene, ß-cryptoxanthin, vitamin C, iron, and copper all had a non-linear association with obesity. Threshold effect analysis demonstrated that the inflection points of retinol, vitamin A, α-carotene, ß-carotene, ß-cryptoxanthin, vitamin C, iron, and cooper were 235.57, 374.81, 58.89, 891.44, 30.70, 43,410.00, 11,240.00, and 990.00 µg/day, respectively. Conclusion: Our study found that a high level of a complex of 11 dietary antioxidants is related to a lower prevalence of obesity and abdominal obesity, among this inverse associations iron and vitamin C have the greatest weight.

Feedback regulation of endothelial cell surface plasmin generation by PKC-dependent phosphorylation of annexin A2.

In response to blood vessel injury, hemostasis is initiated by platelet activation, advanced by thrombin generation, and tempered by fibrinolysis. The primary fibrinolytic protease, plasmin, can be activated either on a fibrin-containing thrombus or on cells. Annexin A2 (A2) heterotetramer (A2·p11)(2) is a key profibrinolytic complex that assembles plasminogen and tissue plasminogen activator and promotes plasmin generation. We now report that, in endothelial cells, plasmin specifically induces activation of conventional PKC, which phosphorylates serine 11 and serine 25 of A2, triggering dissociation of the (A2·p11)(2) tetramer. The resulting free p11 undergoes ubiquitin-mediated proteasomal degradation, thus preventing further translocation of A2 to the cell surface. In vivo, pretreatment of A2(+/+) but not A2(-/-) mice with a conventional PKC inhibitor significantly reduced thrombosis in a carotid artery injury model. These results indicate that augmentation of fibrinolytic vascular surveillance by blockade of serine phosphorylation is A2-dependent. We also demonstrate that plasmin-induced phosphorylation of A2 requires both cleavage of A2 and activation of Toll-like receptor 4 on the cell surface. We propose that plasmin can limit its own generation by triggering a finely tuned "feedback" mechanism whereby A2 becomes serine-phosphorylated, dissociates from p11, and fails to translocate to the cell surface.

The annexin A2/S100A10 system in health and disease: emerging paradigms.

Since its discovery as a src kinase substrate more than three decades ago, appreciation for the physiologic functions of annexin A2 and its associated proteins has increased dramatically. With its binding partner S100A10 (p11), A2 forms a cell surface complex that regulates generation of the primary fibrinolytic protease, plasmin, and is dynamically regulated in settings of hemostasis and thrombosis. In addition, the complex is transcriptionally upregulated in hypoxia and promotes pathologic neoangiogenesis in the tissues such as the retina. Dysregulation of both A2 and p11 has been reported in examples of rodent and human cancer. Intracellularly, A2 plays a critical role in endosomal repair in postarthroplastic osteolysis, and intracellular p11 regulates serotonin receptor activity in psychiatric mood disorders. In human studies, the A2 system contributes to the coagulopathy of acute promyelocytic leukemia, and is a target of high-titer autoantibodies in patients with antiphospholipid syndrome, cerebral thrombosis, and possibly preeclampsia. Polymorphisms in the human ANXA2 gene have been associated with stroke and avascular osteonecrosis of bone, two severe complications of sickle cell disease. Together, these new findings suggest that manipulation of the annexin A2/S100A10 system may offer promising new avenues for treatment of a spectrum of human disorders.

Presence of Viable, Clinically Relevant Legionella Bacteria in Environmental Water and Soil Sources of China.

The distribution of pathogenic Legionella in the environmental soil and water of China has not been documented yet. In this study, Legionella was detected in 129 of 575 water (22.43%) and 41 of 442 soil samples (9.28%) by culture. Twelve Legionella species were identified, of which 11 were disease-associated. Of the Legionella-positive samples, 109 of 129 (84.50%) water and 29 of 41 (70.73%) soil were positive for L. pneumophila, which accounted for about 75% of Legionella isolates in both water and soil, suggesting L. pneumophila was the most frequent species. Soil showed a higher diversity of Legionella spp. as compared with water (0.6279 versus 0.4493). In contrast, serogroup (sg) 1 was more prevalent among L. pneumophila isolates from water than from soil (26.66% versus 12.21%). Moreover, many disease-associated sequence types (STs) of L. pneumophila were found in China. Intragenic recombination was acting on L. pneumophila from both water and soil. Phylogeny, population structure, and molecular evolution analyses revealed a probable existence of L. pneumophila isolates with a special genetic background that is more adaptable to soil or water sources and a small proportion of genetic difference between water and soil isolates. The detection of viable, clinically relevant Legionella demonstrates soil as another source for harboring and dissemination of pathogenic Legionella bacteria in China. Future research should assess the implication in public health with the presence of Legionella in the soil and illustrate the genetic and pathogenicity difference of Legionella between water and soil, particularly the most prevalent L. pneumophila. IMPORTANCE Pathogenic Legionella spp. is the causative agent of Legionnaires' disease (LD), and L. pneumophila is the most common one. Most studies have focused on L. pneumophila from water and clinical samples. However, the soil is another important reservoir for this bacterium, and the distribution of Legionella spp. in water and soil sources has not been compared and documented in China yet. Discovering the distribution of Legionella spp. and L. pneumophila in the two environments may help a deep understanding of the pathogenesis and molecular evolution of the bacterium. Our research systematically uncovered the distributions of Legionella spp. in different regions and sources (e.g., water and soil) of China. Moreover, phylogeny, population structure, and molecular evolution study revealed the possible existence of L. pneumophila with a special genetic background that is more adaptable to soil or water sources, and genetic difference may exist.

Elderly Male With Cardiovascular-Related Comorbidities Has a Higher Rate of Fatal Outcomes: A Retrospective Study in 602 Patients With Coronavirus Disease 2019.

Elderly with comorbidities have shown a higher rate of fatal outcomes when suffering coronavirus disease 2019 (COVID-19). However, a delineation of clinical significances of hematologic indices and underlying comorbidities in the progression and outcome of COVID-19 remains undefined. Six hundred two COVID-19 patients with established clinical outcomes (discharged or deceased) from Hankou Hospital of Wuhan, China between January 14, 2020 and February 29, 2020 were retrospectively analyzed. Of the 602 patients with COVID-19, 539 were discharged and 63 died in the hospital. The deceased group showed higher leukocyte and neutrophil counts but lower lymphocyte and platelet counts. Longer activated partial thromboplastin time (APTT) and prothrombin time (PT), as well as higher D-dimer and C-reactive protein levels, were found in non-survivors. Our observations suggest that these parameters could serve as potential predictors for the fatal outcome and in the discharged group. A higher neutrophil count and D-dimer level but lower lymphocyte were associated with a longer duration of hospitalization. A multivariable Cox regression analysis showed that higher neutrophil count, prolonged PT, and low lymphocyte count were risk factors for patients with COVID-19. Also, we found an association of lower lymphocyte count and higher C-reactive protein levels with the elderly group and those with cardiovascular-related comorbidities. The significantly different hematologic profiles between survivors and non-survivors support that distinct hematologic signatures in COVID-19 patients will dictate different outcomes as a prognostic marker for recovery or fatality. Lymphopenia and aggressive inflammatory response might be major causes for fatal outcomes in the elderly male and especially those with cardiovascular-related comorbidities.

Development and Characterization of High Efficacy Cell-Penetrating Peptide via Modulation of the Histidine and Arginine Ratio for Gene Therapy.

Cell-penetrating peptides (CPPs), as non-viral gene delivery vectors, are considered with lower immunogenic response, and safer and higher gene capacity than viral systems. In our previous study, a CPP peptide called RALA (arginine rich) presented desirable transfection efficacy and owns a potential clinic use. It is believed that histidine could enhance the endosome escaping ability of CPPs, yet RALA peptide contains only one histidine in each chain. In order to develop novel superior CPPs, by using RALA as a model, we designed a series of peptides named HALA (increased histidine ratio). Both plasmid DNA (pDNA) and siRNA transfection results on three cell lines revealed that the transfection efficacy is better when histidine replacements were on the C-terminal instead of on the N-terminal, and two histidine replacements are superior to three. By investigating the mechanism of endocytosis of the pDNA nanocomplexes, we discovered that there were multiple pathways that led to the process and caveolae played the main role. During the screening, we discovered a novel peptide-HALA2 of high cellular transfection efficacy, which may act as an exciting gene delivery vector for gene therapy. Our findings also bring new insights on the development of novel robust CPPs.

Virulence effector SidJ evolution in Legionella pneumophila is driven by positive selection and intragenic recombination.

Effector proteins translocated by the Dot/Icm type IV secretion system determine the virulence of Legionella pneumophila (L. pneumophila). Among these effectors, members of the SidE family (SidEs) regulate several cellular processes through a unique phosphoribosyl ubiquitination mechanism mediated by another effector, SidJ. Host-cell calmodulin (CaM) activates SidJ to glutamylate the SidEs of ubiquitin (Ub) ligases and to make a balanced Ub ligase activity. Given the central role of SidJ in this regulatory process, studying the nature of evolution of sidJ is important to understand the virulence of L. pneumophila and the interaction between the bacteria and its hosts. By studying sidJ from a large number of L. pneumophila strains and using various molecular evolution algorithms, we demonstrated that intragenic recombination drove the evolution of sidJ and contributed to sidJ diversification. Additionally, we showed that four codons of sidJ which are located in the N-terminal (NTD) (codons 58 and 200) and C-terminal (CTD) (codons 868 and 869) domains, but not in the kinase domain (KD) had been subjected to strong positive selection pressure, and variable mutation profiles of these codons were identified. Protein structural modeling of SidJ provided possible explanations for these mutations. Codons 868 and 869 mutations might engage in regulating the interactions of SidJ with CaM through hydrogen bonds and affect the CaM docking to SidJ. Mutation in codon 58 of SidJ might affect the distribution of main-chain atoms that are associated with the interaction with CaM. In contrast, mutations in codon 200 might influence the α-helix stability in the NTD. These mutations might be important to balance Ub ligase activity for different L. pneumophila hosts. This study first reported that intragenic recombination and positive Darwinian selection both shaped the genetic plasticity of sidJ, contributing to a deeper understanding of the adaptive mechanisms of this intracellular bacterium to different hosts.