Comparative Secretome Analysis of Magnaporthe oryzae Identified Proteins Involved in Virulence and Cell Wall Integrity.

Plant fungal pathogens secrete numerous proteins into the apoplast at the plant-fungus contact sites to facilitate colonization. However, only a few secretory proteins were functionally characterized in Magnaporthe oryzae, the fungal pathogen causing rice blast disease worldwide. Asparagine-linked glycosylation 3 (Alg3) is an α-1,3-mannosyltransferase functioning in the N-glycan synthesis of N-glycosylated secretory proteins. Fungal pathogenicity and cell wall integrity are impaired in Δalg3 mutants, but the secreted proteins affected in Δalg3 mutants are largely unknown. In this study, we compared the secretomes of the wild-type strain and the Δalg3 mutant and identified 51 proteins that require Alg3 for proper secretion. These proteins were predicted to be involved in metabolic processes, interspecies interactions, cell wall organization, and response to chemicals. Nine proteins were selected for further validation. We found that these proteins were localized at the apoplastic region surrounding the fungal infection hyphae. Moreover, the N-glycosylation of these proteins was significantly changed in the Δalg3 mutant, leading to the decreased protein secretion and abnormal protein localization. Furthermore, we tested the biological functions of two genes, INV1 (encoding invertase 1, a secreted invertase) and AMCase (encoding acid mammalian chinitase, a secreted chitinase). The fungal virulence was significantly reduced, and the cell wall integrity was altered in the Δinv1 and Δamcase mutant strains. Moreover, the N-glycosylation was essential for the function and secretion of AMCase. Taken together, our study provides new insight into the role of N-glycosylated secretory proteins in fungal virulence and cell wall integrity.

Phosphorylation-mediated Regulatory Networks in Mycelia of Pyricularia oryzae Revealed by Phosphoproteomic Analyses.

Protein phosphorylation is known to regulate pathogenesis, mycelial growth, conidiation and stress response in Pyricularia oryzae However, phosphorylation mediated regulatory networks in the fungal pathogen remain largely to be uncovered. In this study, we identified 1621 phosphorylation sites of 799 proteins in mycelia of P. oryzae, including 899 new p-sites of 536 proteins and 47 new p-sites of 31 pathogenicity-related proteins. From the sequences flanking the phosphorylation sites, 19 conserved phosphorylation motifs were identified. Notably, phosphorylation was detected in 7 proteins that function upstream of Pmk1, but not in Pmk1 and its downstream Mst12 and Sfl1 that have been known to regulate appressorium formation and infection hyphal growth of P. oryzae Interestingly, phosphorylation was detected at the site Ser240 of Pmp1, which is a putative protein phosphatase highly conserved in filamentous fungi but not characterized. We thus generated Δpmp1 deletion mutants and dominant allele PMP1S240D mutants. Phenotyping analyses indicated that Pmp1 is required for virulence, conidiation and mycelial growth. Further, we observed that phosphorylation level of Pmk1 in mycelia was significantly increased in the Δpmp1 mutant, but decreased in the PMP1S240D mutant in comparison with the wild type, demonstrating that Pmp1 phosphorylated at Ser240 is important for regulating phosphorylation of Pmk1. To our surprise, phosphorylation of Mps1, another MAP kinase required for cell wall integrity and appressorium formation of P. oryzae, was also significantly enhanced in the Δpmp1 mutant, but decreased in the PMP1S240D mutant. In addition, we found that Pmp1 directly interacts with Mps1 and the region AA180-230 of Pmp1 is required for the interaction. In summary, this study sheds new lights on the protein phosphorylation mediated regulatory networks in P. oryzae.

Anti-N-methyl-D-aspartate receptor encephalitis concomitant with multifocal subcortical white matter lesions on magnetic resonance imaging: a case report and review of the literature.

BACKGROUND: Anti-N-methyl-D-aspartate receptor encephalitis is a severe autoimmune disorder characterized by severe psychiatric symptoms, seizures, decreased consciousness, autonomic dysregulation, and dyskinesias. Multifocal subcortical white matter lesions on fluid-attenuated inversion recovery and diffuse weighted images have rarely been reported in previous literature, and serial magnetic resonance imaging changes after plasma exchange have not been presented before. CASE PRESENTATION: A previously healthy 24-year-old Chinese woman presented with acute psychiatric symptoms characterized by fear and agitation followed by decreased consciousness, dyskinesias, and seizures. Magnetic resonance imaging revealed hyperintense lesions on fluid-attenuated inversion recovery and diffuse weighted images in bilateral subcortical white matter. Cerebrospinal fluid analysis revealed a mild pleocytosis with lymphocytic predominance. Protein and glucose levels were normal. Aquaporin-4 antibodies in serum and cerebrospinal fluid were negative. Identification of anti-N-methyl-D-aspartate receptor antibodies in serum and cerebrospinal fluid confirmed the diagnosis of anti-N-methyl-D-aspartate receptor encephalitis. She was initially treated with combined intravenous immunoglobulin and methylprednisolone without improvement. Plasma exchange was then initiated with good response; the patient made a full recovery after several cycles of plasma exchange. Repeat magnetic resonance imaging performed 1 month after plasma exchange showed partial resolution of the hyperintense lesions in bilateral subcortical white matter, and follow-up magnetic resonance imaging 2 months after plasma exchange showed complete resolution. CONCLUSION: Anti-N-methyl-D-aspartate receptor encephalitis may be concomitant with multifocal subcortical white matter lesions. Such lesions may resolve after appropriate immunotherapy.

Hyper-methylated miR-203 dysregulates ABL1 and contributes to the nickel-induced tumorigenesis.

Nickel compounds have been found to be carcinogenic based upon epidemiological, animal and cell culture studies. Previous studies suggest that epigenetic mechanisms play a role in Nickel-induced carcinogenesis such as DNA methylation and histone modification. In this study, we investigated the role of microRNAs (miRNAs) in nickel-induced carcinogenesis. The expression of several miRNAs which may function as tumor suppressor genes revealed a strong downregulation of miR-203 in Ni3S2-transformed 16HBE cells (NSTCs). Meanwhile, we observed hypermethylation of CpGs in miR-203 promoter and first exon area, and proved that the hyper-methylated miR-203 was involved in the Nickel-induced tumorigenesis. Moreover, we identified that miR-203 may suppress the tumorigenesis at least in part through negatively regulating its target gene ABL1. Our findings indicate that DNA methylation-associated silencing of tumor suppressor miRNAs contributes to the development of Nickel-induced cancer.

Anti-N-methyl-D-aspartate receptor encephalitis with serum anti-thyroid antibodies and IgM antibodies against Epstein-Barr virus viral capsid antigen: a case report and one year follow-up.

BACKGROUND: Anti-N-methyl-D-aspartate receptor encephalitis is an increasingly common autoimmune disorder mediated by antibodies to certain subunit of the N-methyl-D-aspartate receptor. Recent literatures have described anti-thyroid and infectious serology in this encephalitis but without follow-up. CASE PRESENTATION: A 17-year-old Chinese female patient presented with psychiatric symptoms, memory deficits, behavioral problems and seizures. She then progressed through unresponsiveness, dyskinesias, autonomic instability and central hypoventilation during treatment. Her conventional blood work on admission showed high titers of IgG antibodies to thyroglobulin, thyroid peroxidase and IgM antibodies to Epstein-Barr virus viral capsid antigen. An immature ovarian teratoma was found and removal of the tumor resulted in a full recovery. The final diagnosis of anti-N-methyl-D-aspartate receptor encephalitis was made by the identification of anti-N-methyl-D-aspartate receptor antibodies in her cerebral spinal fluid. Pathology studies of the teratoma revealed N-methyl-D-aspartate receptor subunit 1 positive ectopic immature nervous tissue and Epstein-Barr virus latent infection. She was discharged with symptoms free, but titers of anti-thyroid peroxidase and anti-thyroglobulin antibodies remained elevated. One year after discharge, her serum remained positive for anti-thyroid peroxidase and anti-N-methyl-D-aspartate receptor antibodies, but negative for anti-thyroglobulin antibodies and IgM against Epstein-Barr virus viral capsid antigen. CONCLUSIONS: Persistent high titers of anti-thyroid peroxidase antibodies from admission to discharge and until one year later in this patient may suggest a propensity to autoimmunity in anti- N-methyl-D-aspartate receptor encephalitis and support the idea that neuronal and thyroid autoimmunities represent a pathogenic spectrum. Enduring anti-N-methyl-D-aspartate receptor antibodies from admission to one year follow-up but seroreversion of Epstein-Barr virus viral capsid antigen IgM may raise the important issue of elucidating the triggers and boosters of anti- N-methyl-D-aspartate receptor encephalitis.

Human cytomegalovirus-IgM seropositivity is not associated with atherogenic alterations of lipid profiles and inflammatory status in ischemic stroke patients: a preliminary study.

BACKGROUND: Past exposure to human cytomegalovirus has been suggested to participate in the pathogenetic events associated with atherosclerotic lesion establishment and progression. However, whether ongoing human cytomegalovirus infection is related to plaque instability, and subsequent acute cerebral ischemia, is relatively unknown. The purpose of this study was to evaluate the potential relationships between active human cytomegalovirus infection and ischemic stroke, especially in regard to metabolism and inflammation. METHODS: Ninety-nine acute ischemic stroke patients, associated with large artery atherosclerosis, were divided into two groups based on the presence or absence of human cytomegalovirus immunoglobulin M (IgM) (human cytomegalovirus-IgM-positive/human cytomegalovirus-IgM-negative = 33:66). Baseline clinical characteristics, inflammatory factors, and biochemical assessments were compared in both groups. Then, all patients and human cytomegalovirus-IgM-positive patients were divided into quartiles according to their high-sensitivity C-reactive protein levels, respectively, and risk factors were compared. Finally, correlations between inflammatory factors (high-sensitivity C-reactive protein and white blood cell count) and other atherosclerosis risk factors in both human cytomegalovirus-IgM-positive and -negative subjects were evaluated. RESULTS: An association between human cytomegalovirus-IgM seropositivity and atherogenic modification of metabolism and inflammatory status were not found in this study. Both age and white blood cell count increased across quartiles of high-sensitivity C-reactive protein in all subjects (P = 0.001), while age and low-density lipoprotein cholesterol increased across quartiles of high-sensitivity C-reactive protein in the human cytomegalovirus-IgM-positive group (P = 0.02 and 0.007, respectively). Multivariate linear regression analysis showed that high-sensitivity C-reactive protein was associated with age in human cytomegalovirus-IgM-positive group (P = 0.002), while no other factor was associated with white blood cell count in these subjects. CONCLUSION: Our study provided no evidence for the direct implication of active systemic human cytomegalovirus infection, represented by human cytomegalovirus-IgM positivity, in the pathogenesis of acute ischemic strokes, particularly those involving plaque instability and metabolic disorders.

[Analysis of 62 adult patients with viral meningitis].

OBJECTIVE: To investigate the clinical manifestation, cerebrospinal fluid (CSF) and auxiliary examination findings of adult viral meningitis. METHODS: 62 adult patients with viral meningitis were retrospectively analyzed. RESULTS: Headache occurred in all the 62 (100%) patients, fever occurred in 61 (98%) patients, meningeal irritation sign occurred in 48 (77%) patients. The abduction of left eye was limited in one patient. Seizure occurred in 2 patients. The mean duration time was 17 days, 93% patients less than 30 days. The pressure of CSF increased in 80% patients, leukocyte counts increased in 91% patients, protein level increased in 81% patients, chloride level was normal in 35% patients and slightly lower in 65% patients, glucose level was normal in 94% patients. 7 patients had positive IgM antibody of Coxackievirus B group both in serum and CSF, one patient had positive IgM antibody of EB virus in CSF. Cranial CT scan had no special findings in all patients. 23 patients performed MRI examination, meningeal enhancement occurred in 9 patients. 52% patients had abnormal EEG, mainly increased local or diffuse slow waves. CONCLUSION: Adult viral meningitis was a kind of self-limited disease, chloride level was slightly lower in more than half patients, meningeal enhancement was detected in MRI in part patients.

[Establishment of a model for predicting the postoperative morbidity in patients with pelvic and acetabulum fractures].

OBJECTIVE: To establish a model for predicting the postoperative morbidity in patients with pelvic and acetabulum fractures. METHODS: The clinical of 94 patients with pelvic and acetabulum fractures were retrospectively reviewed for analysis of the postoperative complications and duration of hospital stay. The preoperative physiological scores (PS) and operative severity scores (OS) of the patients were selected as the variables, and their regression coefficients were calculated by logistic regression analysis to establish the model for predicting the postoperative morbidities. The predictive value of the model was evaluated according to the ROC curve. RESULTS: The model for predicting postoperative morbidities of pelvic and acetabulum fractures, ln P/(1-P)=-13.83+0.47x PS+0.33xOS, has an area under ROC curve of 0.888-/+0.037 with the cut-off point of 37%, sensitivity of 84.21%, specificity of 82.14%, concordance rate of 82.98%, misclassification rate of 15.79% and omission classification rate of 17.86%. CONCLUSION: This model possesses a high predictive capability for postoperative morbidities of pelvic and acetabulum fractures, and can be helpful for clinical therapeutic decision-making and lower the risks in operation.

[Effect of cytomegalovirus infection on expression of matrix metalloproteinase-2 in human endothelial cells].

OBJECTIVE: To investigate the effect of cytomegalovirus infection on expression of matrix metalloproteinase-2 in human endothelial cells. METHODS: Human umbilical vein endothelial cells were cultured in vitro. Cells between 3-6 passages were infected with cytomegalovirus for different time. Expression of matrix metalloproteinase-2 mRNA was analyzed by reverse transcription-polymerase chain reaction, matrix metalloproteinase-2 activity was detected by gel zymography. RESULTS: Expression of matrix metalloproteinase-2 mRNA and its activity 6 hours after infection was almost equal to control, and was greatly enhanced 12 and 24 hours after infection. CONCLUSION: Cytomegalovirus infection up-regulates expression of matrix metalloproteinase-2 in human endothelial cells. It might be one of the mechanisms that cytomegalovirus is involved in atherosclerosis.

[Application of skin flaps with supra-carpal cutaneous branch of the ulnar artery and dorsal carpal branch of the anterior interosseous artery in reconstruction of complicated amputaled palm].

OBJECTIVE: To investigate the clinical efficacy of carpal branch of ulnar artery skin flap and dorsal carpal branch of anterior interosseous artery skin flap in replantation of complicated severed hand. METHODS: From August 1999 to October 2005, 18 patients with complicated severed hands were treated in our department. According to the skin defect of severed hand, the palm of the hand was restored with upper carpal branch of ulnar artery skin flap (2-4 cm x 3-6 cm), followed by restoration of the back of the hand with dorsal carpal branch of anterior interosseous artery skin flap (4-6 cm x 5-8 cm) and replantation of the hand was performed simultaneously. RESULTS: All the replanted hands and the skip flaps survived after the surgery with satisfactory shape and function. The surgery achieved an over 70% recovery of the hand function in all cases. CONCLUSIONS: For complicated severed hands, upper carpal branch of the ulnar artery skin flap and dorsal carpal branch of the anterior interosseous artery skin flap are effective to repair the skin defect, and this procedure provides a good option for reconstructing shape and function of the severed hands.