The Conserved Cysteine-Rich Secretory Protein MaCFEM85 Interacts with MsWAK16 to Activate Plant Defenses.

Metarhizium anisopliae is an entomopathogenic fungus which may enhance plant growth and resistance when acting as an endophyte in host plants. However, little is known about the protein interactions nor their activating mechanisms. Common in fungal extracellular membrane (CFEM) proteins have been identified as plant immune regulators that suppress or activate plant resistance responses. Here, we identified a CFEM domain-containing protein, MaCFEM85, which was mainly localized in the plasma membrane. Yeast two-hybrid (Y2H), glutathione-S-transferase (GST) pull-down, and bimolecular fluorescence complementation assays demonstrated that MaCFEM85 interacted with the extracellular domain of a Medicago sativa (alfalfa) membrane protein, MsWAK16. Gene expression analyses showed that MaCFEM85 and MsWAK16 were significantly upregulated in M. anisopliae and M. sativa, respectively, from 12 to 60 h after co-inoculation. Additional yeast two-hybrid assays and amino acid site-specific mutation indicated that the CFEM domain and 52th cysteine specifically were required for the interaction of MaCFEM85 with MsWAK16. Defense function assays showed that JA was up-regulated, but Botrytis cinerea lesion size and Myzus persicae reproduction were suppressed by transient expression of MaCFEM85 and MsWAK16 in the model host plant Nicotiana benthamiana. Collectively, these results provide novel insights into the molecular mechanisms underlying interactions of M. anisopliae with host plants.

Kruppel Like Factor 5 Enhances High Glucose-Induced Renal Tubular Epithelial Cell Transdifferentiation in Diabetic Nephropathy.

Background - Diabetic nephropathy (DN) is a principal reason for kidney disease worldwide. High glucose (HG) is a major factor for DN. Kruppel like factor 5 (KLF5) participates in DN development. In the present study, we aim to elaborate the role of KLF5 in HG-induced renal tubular epithelial cell (RTEC) transdifferentiation in DN. Methods - RTECs (HK-2 cells) were treated with HG and were transfected with si-KLF5 or oe-HMGB1. Afterwards, expression of KLF5 and HMGB1 was detected, HK cell viability was determined, and levels of alpha-smooth muscle actin (α-SMA), E-cadherin, vimentin, and transforming growth factor beta 1 (TGF-ß1) were assessed. Additionally, the binding relation between KLF5 and HMGB1 was analyzed. Results - In HK-2 cells with HG treatment, expression of KLF5 and HMGB1 was upregulated; levels of α-SMA, vimentin, and TGF-ß1 were increased; and E-cadherin level was decreased. Moreover, KLF5 silencing resulted in down-regulated levels of α-SMA, vimentin, and TGF-ß1 but upregulated level of E-cadherin. On the other hand, KLF5 could bind to the HMGB1 promoter and activate HMGB1 transcription. HMGB1 overexpression partially counteracted the inhibitive effect of KLF5 silencing on HG-induced HK-2 transdifferentiation. Conclusion - HG induced overexpressed KLF5 in RTECs, and as a transcription factor, KLF5 could bind to the HMGB1 promoter, thereby promoting HMGB1 transcription and RTEC transdifferentiation.

Microbiological contamination in donor corneas preserved for medium-term.

To evaluate the characteristics of microbiological contamination in donor corneas preserved for medium-term. A total of 82 donated corneas from June 1, 2014 to November 30, 2014 were retrospectively analyzed. The corneas were preserved in cornea chambers medium-term solution at 4-8 °C for keratoplasty. After removal of the central corneas for transplantation, the corneoscleral rims were put back into the medium for 1 month at room temperature (20-25 °C). The suspicious contaminated storage solutions indicated with transparency or color change were examined with bacteria and fungi cultivation for strain identification. The data collected included gender, age, procurement site and causes of death of donors, and follow-up of recipients. Statistical analysis was performed using Microsoft Excel and SPSS 24.0. Significance level was set at a P value < 0.05. The overall pathogen positive rate was 9.8% (n = 8), including 7 (87.5%) fungi and 1 (12.5%) bacteria. They were 2 (2.44%) Fusarium, 2 (2.44%) Chromomycosis, 1 (1.22%) Candida albicans, 1 (1.22%) Aspergillus versicolor, 1 (1.22%) Acremonium species, and 1 (1.22%) Enterococcus. 5 contaminated corneas were used for penetrating keratoplasty; although four out of five (80%) had not been given antifungal drugs during more than 6 months following-up period, none of the recipients was infected with a graft. Donor age (P = 0.839), gender (P = 0.062), procurement sites (P = 0.713) and cause of death (P = 0.711) had no statistically significant influence on the contamination rate. All donor corneas have a possibility of microbiological contamination. Strict tissue preservation protocol but not antifungal drugs following keratoplasty seems necessary to prevent graft infection.

[Effect of Chlorambucil Combined with Ibrutinib on Mantle Cell Lymphoma Cell Line Jeko-1 and Its Related Mechanism].

OBJECTIVE: To investigate the toxic effect of chlorambucil combined with ibrutinib on mantle cell lymphoma (MCL) cell line Jeko-1 and its related mechanism. METHODS: The MCL cell line Jeko-1 was incubated with different concentrations of chlorambucil or ibrutinib or the combination of the two drugs, respectively. CCK-8 assay was used to detect the proliferation of the cells, and Western blot was used to measure the protein expression levels of BCL-2, caspase-3, PI3K, AKT and P-AKT. RESULTS: After Jeko-1 cells were treated with chlorambucil (3.125, 6.25, 12.5, 25, 50 µmol/L) and ibrutinib (3.125, 6.25, 12.5, 25, 50 µmol /L) alone for 24, 48, 72h respectively, the cell proliferation was inhibited in a time- and dose-dependent manner. Moreover, the two drugs were applied in combination at low doses (single drug inhibition rate<50%), and the results showed that the combination of two drugs had a more significant inhibitory effect (all P < 0.05). Compared with the control group, the apoptosis rate of the single drug group of chlorambucil (3.125, 6.25, 12.5, 25, 50 µmol/L) and ibutinib (3.125, 6.25, 12.5, 25, 50 µmol/L) was increased in a dose-dependent manner. The combination of the two drugs at low concentrations (3.125, 6.25, 12.5 µmol/L) could significantly increase the apoptosis rate compared with the corresponding concentration of single drug groups (all P < 0.05). Compared with control group, the protein expression levels of caspase-3 in Jeko-1 cells were upregulated, while the protein expression levels of BCL-2, PI3K, and p-AKT/AKT were downregulated after treatment with chlorambucil or ibrutinib alone. The combination of the two drugs could produce a synergistic effect on the expressions of the above-mentioned proteins, and the differences between the combination group and the single drug groups were statistically significant (all P < 0.05). CONCLUSION: Chlorambucil and ibrutinib can promote the apoptosis of MCL cell line Jeko-1, and combined application of the two drugs shows a synergistic effect, the mechanism may be associated with the AKT-related signaling pathways.

High efficiency removal of organic and inorganic iodine with ferrate[Fe(VI)] through oxidation and adsorption.

I- is a halogen species existing in natural waters, and the transformation of organic and inorganic iodine in natural and artificial processes would impact the quality of drinking water. Herein, it was found that Fe(VI) could oxidize organic and inorganic iodine to IO3-and simultaneously remove the resulted IO3- through Fe(III) particles. For the river water, wastewater treatment plant (WWTP) effluent, and shale gas wastewater treated by 5 mg/L of Fe(VI) (as Fe), around 63 %, 55 % and 71 % of total iodine (total-I) had been removed within 10 min, respectively. Fe(VI) was superior to coagulants in removing organic and inorganic iodine from the source water. Adsorption kinetic analysis suggested that the equilibrium adsorption amount of I- and IO3- were 11 and 10.1 µg/mg, respectively, and the maximum adsorption capacity of IO3- by Fe(VI) resulted Fe(III) particles was as high as 514.7 µg/mg. The heterogeneous transformation of Fe(VI) into Fe(III) effectively improved the interaction probability of IO3- with iron species. Density functional theory (DFT) calculation suggested that the IO3- was mainly adsorbed in the cavity (between the γ-FeOOH shell and γ-Fe2O3 core) of Fe(III) particles through electrostatic adsorption, van der Waals force and hydrogen bond. Fe(VI) treatment is effective for inhibiting the formation of iodinated disinfection by-products in chlor(am)inated source water.

[Analysis of anatomy features for patients with narrow anterior chamber angle and primary iridociliary body cysts].

OBJECTIVE: To investigate the prevalence and anatomy features of iridociliary body cysts in patients with narrow anterior chamber angle. METHODS: Retrospective case series study. The prevalence and anatomy features of iridociliary body cysts in 223 patients (402 eyes) were analyzed retrospectively with ultrasound biomicroscopy (UBM). All of the patients were examined for susceptive narrow anterior chamber angle without complaint. The age of the patients, the site, diameter and number of cysts, the anterior chamber angle and the central anterior chamber depth were measured. RESULTS: Iridociliary body cysts were found in 19 patients (23 eyes) out of 223 patients (402 eyes), the prevalence is 5.7%. Fifteen patients were unilateral and four patients bilateral. Two cases originated from the ciliary process, eighteen cases from the iris root, and three from both the root and posterior surface of the iris. Twenty one cases were single cysts while two cases were multiple cysts. The diameter of the cysts ranged from 0.5 to 3.1 mm, averaged (0.71 ± 0.53) mm. The average age and the central anterior chamber depth of the eyes with iridociliary body cysts were (55.32 ± 10.74) years and (2.25 ± 0.39) mm, with no significant difference (t = 0.534, 0.783; P > 0.05) as compared to that of patients without cysts, which were (57.46 ± 10.52) years and (2.14 ± 0.34) mm. The anterior chamber angle in iridociliary body cysts group was 8.2° (21.0°, 0.0°), with no significant difference (Z = -0.062, P > 0.05) as compared to that of patients without cysts, which was 8.9° (21.4°, 0.0°). CONCLUSIONS: The prevalence rate of iridociliary body cysts in this study is 5.7%, central anterior chamber depth and anterior chamber angle in patients with cysts do not differ form patients without cysts.

Bioinformatics Analysis and Functional Characterization of the CFEM Proteins of Metarhizium anisopliae.

The entomopathogen Metarhizium anisopliae is a facultative rhizosphere or endophytic fungus available for managing pests and improving plant growth. The CFEM (common in fungal extracellular membrane) proteins form a unique group in fungi but are rarely reported in entomopathogens. In this study, we cloned and identified 13 CFEM genes from M. anisopliae (MaCFEMs). Sequence alignment and WebLogo analysis showed that eight cysteines were the most conserved amino acids in their CFEM domain. Phylogenic analysis suggested that these 13 proteins could be divided into 4 clades based on the presence of the transmembrane region and the position of CFEM domain in the whole sequence. Six MaCFEM proteins with a signal peptide and without a transmembrane domain were considered candidate effector proteins. According to Phyre2 analysis, the MaCFEM88 and MaCFEM85 have the most homologous to Csa2 in Candida albicans. Subcellular localization analysis revealed that five effectors were located in the plasma membrane, while MaCFEM88 may locate in both plasma membrane and nucleus in the treated Nicotiana benthamiana. Expression pattern analysis showed that MaCFEM81, 85, 88, and 89 expression level was significantly higher in the sporulation stage compared to other growth stages. Furthermore, the yeast secretion assay showed that six candidate effectors were able to secrete out of the cell. All of the MaCFEMs couldn't affect INF1-induced programmed cell death (PCD), but MaCFEM85 and 88 could trigger a slight hypersensitive response both when applied separately or in combination with INF1 in N. benthamiana leaves. These findings showed that six MaCFEM potential effectors with various structures and subcellular localizations in host cells might be used to illustrate the roles of MaCFEM proteins during M. anisopliae-plant interactions.

Complete Response to Combined Chemotherapy and Anti-PD-1 Therapy for Recurrent Gallbladder Carcinosarcoma: A Case Report and Literature Review.

Background: Gallbladder carcinosarcoma (GBCS) is a rare and aggressive malignancy with extremely poor prognosis. Although surgery is regarded as the primary therapy for GBCS, the effective therapeutic strategies for unresected lesions have been poorly defined. Case Presentation: We presented a case of a 74-year-old male who underwent radical resection of gallbladder carcinoma at a local hospital. Seven months later, he was admitted to our hospital due to right upper abdominal discomfort. Postoperative radiological examinations showed multiple hepatic lesions, hilar lymph node metastasis, and main portal vein tumor thrombus. The pathological consultation results confirmed GBCS and immunohistochemical examinations revealed PD-L1 expression in 20% of tumor cells. Then, the patient received chemotherapy (Gemcitabine plus Oxaliplatin, GEMOX) in combination with anti-PD-1 therapy. After nine courses of the combination therapy, complete regression of the tumors was achieved with no evidence of relapse till now. Conclusions: We, for the first time, reported a patient with recurrent GBCS who benefited from the combined chemotherapy and immunotherapy, providing a potential effective management strategy for the refractory malignant tumor.

[Analysis of multifocal electroretinogram first-order kernel P(1) wave in anisometropic amblyopia].

OBJECTIVE: To investigate the features of the multifocal electroretinogram (mfERG) first-order kernel P(1) wave in anisometropic amblyopic eyes. METHODS: mfERG of fifteen anisometropic amblyopic eyes and their normal control eyes were recorded with ROLAND RETIscan visual evoked response system, the mean amplitude density and eclipse period of the P(1) wave were analyzed and compared between the amblyopic and normal eyes with t-test. RESULTS: The P(1) wave amplitude density of mfERG first-order kernel in amblyopic eyes (164.7 ± 73.1) nV×deg(-2) was significantly attenuated in the central region of the visual field as compared with that of the control eyes (227.0 ± 61.3) nV×deg(-2) (t = 2.554, P = 0.016). The eclipse period of the amblyopic eyes (30.3 ± 4.3) ms was shorter than that of the control eyes (34.4 ± 3.2) ms (t = 2.92, P = 0.007). CONCLUSIONS: The significant change of the multifocal electroretinogram (mfERG) first-order kernel P(1) wave may reflect the abnormality of the retinal on-bipolar cells function and the visual information transmission, whether the P(1) wave could be used as an objective index of amblyopic eyes is a promising research topic.

Identification of the key genes involved in the regulation of symbiotic pathways induced by Metarhizium anisopliae in peanut (Arachis hypogaea) roots.

We detected and compared the mRNA and protein expression levels of immunity-associated and symbiosis-associated genes in peanut (Arachis hypogaea) roots inoculated with entomopathogenic fungus M. anisopliae or the phytopathogenic fungus Fusarium oxysporum, by RT-qPCR and parallel reaction monitoring (PRM). The selected genes were mainly associated with plant-fungus interactions, signal transduction, regulation of cell death, nitrogen or iron metabolism, nutrient acquisition or transport, and compound synthesis based on previous transcriptome analysis. The results showed that the host basal defense responses were significantly inhibited by both M. anisopliae and F. oxysporum, which suggests that both fungi actively suppress the host immunity for successful colonization and infection. However, only F. oxysporum induced a strong host hypersensitivity, which indicates that the host is strongly resisting F. oxysporum but potentially allowing M. anisopliae. Additionally, the genes (SYMRK, CaM, CCaMK, FRI2, ABCC2, F6H1, SCT, NRT24 and LTP1) related to symbiosis and growth were distinctively observed with an up-regulated expression following M. anisopliae treatment, which implies that the host was actively initiating the establishment of symbiosis with the fungus. This study revealed a synergistic relationship between host immunosuppression and the promotion of symbiosis during interactions with M. anisopliae. It suggested that M. anisopliae benefited plant for symbiotic relationship, in addition to controlling herbivorous insects as an entomopathogen.

Influence of Metarhizium anisopliae (IMI330189) and Mad1 protein on enzymatic activities and Toll-related genes of migratory locust.

Efficacy of Metarhizium anisopliae strain (IMI330189) and Mad1 protein alone or in combination by feeding method to overcome immune-related enzymes and Toll-like pathway genes was investigated in migratory locust. M. anisopliae (IMI330189) is a potent and entomopathogenic fungal strain could be effectively used against insect pests. Similarly, Mad1 protein adheres to insect cuticle, causing virulence to insects. We confirmed maximum 55% of mortality when M. anisopliae (IMI330189) and Mad1 was applied in combination. Similarly, increased PO activity was observed in locust with combined dose of Mad1 + IMI330189 whereas PO, POD, and SOD activities reduced using Mad1 independently. Four Toll-like signaling pathway genes (MyD88, Cactus, Pelle, and CaN) were investigated from midgut and body of the migratory locust after 72 h of treatments. Subsequently, the expression of MyD88 in the midgut and body significantly decreased with the application of Mad1 and Mad1 + IMI330189. Performance of these treatments was absolutely non-consistent in both parts of insects. Meanwhile, IMI330189 significantly raised the expression of Cactus in both midgut and body. However, the combined treatment (Mad1 + IMI330189) significantly reduced the Cactus expression in both body parts. Pelle expression was significantly increased in the midgut with the application of independent treatment of Mad1 and IMI330189 whereas the combined treatment (Mad1 + IMI330189) suppressed the Pelle expression in midgut. Its expression level was absolutely higher in body with the application of IMI330189 and Mad1 + IMI330189 only. On the other hand, Mad1 significantly increased the expression of CaN in midgut. However, all three treatments significantly affected and suppressed the expression of CaN gene in body of locust. This shows that the applications of M. anisopliae and Mad1 protein significantly affected Toll signaling pathway genes, which ultimately increased level of susceptibility of locust. However, their effect was significantly different in both parts of locust which recommends that the Toll-related genes are conserved in midgut instead of locust body.

[Studies on extraction of active fraction from Rhizoma et Radix Ligustici by supercritical CO2 extraction].

OBJECTIVE: To study the conditions of extraction of active fraction in Rhizoma et Radix Ligustici by supercritical CO2 extraction (SFE). METHODS: Using the content of ferulic acid as the index, conditions for the extraction including extracting pressure, extracting temperature, temperature of resolution and extracting time were optimized by uniform design. RESULTS: The best SFE conclusions were as follows: cosolvent was 50% ethanol (ml/g), extracting pressure was 40 MPa, extracting temperature was 50 degrees C, pressure of resolution was 6 MPa, temperature of resolution was 50 degrees C, extracting time was 3.5 hours. CONCLUSIONS: The optimum extraction has high extraction ratio of active fraction in Rhizoma et Radix Ligustici, so it is reasonable and practicable.

[The investigation of tranilast on the proliferation and migration of human Tenon's capsule fibroblasts].

OBJECTIVE: To investigate the effect of tranilast, N-(3,4-dimethoxycinnamoyl) anthramilic acid, on the proliferation and migration of human Tenon's capsule fibroblasts. METHODS: fibroblasts were cultured from Tenon's tissue of a glaucoma patient after trabeculectomy. The subcultured cells were incubated with DMEM medium containing different concentrations of tranilast for 72 h. The growth of fibroblasts was measured by methyl thiazlyl tetrazolium (MTT) assay and the cell count, and migration was evaluated by crutch method. The expression level of Protein kinase C (PKC) in fibroblasts was tested by immunohistochemical associated with image biological analysis (IBAS) methods. RESULTS: Treated with tranilast varying from 12.5 mg/L to 100.0 mg/L concentration, the proliferation of fibroblasts declined in a dose dependent manner. The migration of fibroblasts decreased from 40.20 +/- 5.83 to 22.50 +/- 4.21 and 9.80 +/- 2.14 cells/per field (P < 0.05) at 50.0 and 100.0 mg/L, respectively, and PKC expression was suppressed by tranilast from 0.2591 +/- 0.0038 to 0.2375 +/- 0.0106 and 0.1273 +/- 0.0573 Absorption value (P < 0.05) at 50.0 and 100.0 mg/L, respectively. CONCLUSION: Tranilast inhibited the proliferation as well as migration of fibroblasts in vitro, at least in part, by downregulation of PKC expression.

Promotion of minTBP-1-PRGDN on the attachment, proliferation and collagen I synthesis of human keratocyte on titanium.

AIM: To investigate the influence of minTBP-1-PRGDN on the attachment, proliferation and collagen I synthesis of human keratocyte on titanium (Ti) surface. METHODS: The chimeric peptide RKLPDAPRGDN (minTBP-1-PRGDN) was synthesized by connecting RKLPDA (minTBP-1) to the N-terminal of PRGDN, the influence of minTBP-1-PRGDN on the attachment, proliferation and collagen I synthesis of human keratocyte on Ti surface were tested using PRGDN and minTBP-1as controls. The keratocytes attached to the surface of Ti were either stained with FITC-labeled phalloidin and viewed with fluorescence microscope or quantified with alamar Blue method. The proliferation of keratocytes on Ti were quantified with 3-(4,5-dim- ethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide up-taking methods. The secretion of type I collagen were determined using an ELISA kit. RESULTS: The results showed that minTBP-1-PRGDN at a concentration of 100ng/mL was the most potent peptide to enhance the attachment of human keratocytes to the surface of Ti (1.40±0.03 folds, P=0.003), to promote the proliferation (1.26±0.05 folds, P=0.014) and the synthesis of type I collagen (1.530±0.128, P=0.008). MinTBP-1 at the same concentration could only promote the attachment (1.13±0.04 folds, P=0.020) and proliferation(1.15±0.06 folds, P=0.021), while PRGDN had no significant influence (P>0.05). CONCLUSION: Our data shows that the novel chimeric peptide minTBP-1-PRGDN could promote the attachment, proliferation and type I collagen synthesis of human keratocytes on the surface of Ti.

Gene polymorphisms and risk of adult early-onset ischemic stroke: A meta-analysis.

INTRODUCTION: Genetic studies restricted to young adult ischemic stroke patients may help in excluding the potentially confounding variables encountered with advanced age; thus, allowing a more precise risk evaluation derived from the inherited mutations alone. Through meta-analysis, this study was conducted to determine the genetic risk contributed by each susceptibility gene polymorphism, particularly in adult early-onset ischemic stroke patients. MATERIALS AND METHODS: Electronic databases were searched for all the case-control studies relating to any candidate genes for ischemic stroke. The range of age was 18-50 years for cases. Fixed or random effects model was used depending on the heterogeneity between studies. RESULTS: Twenty-six studies were finally included in this meta-analysis; these studies focused on 7 candidate genes. A significant but modest association was identified for 2 polymorphisms, namely, methylenetetrahydrofolate reductase (MTHFR) C677T (OR = 1.44, 95% CI = 1.14-1.80) and apolipoprotein E (ApoE) epsilon2-4 (OR = 2.53, 95% CI = 1.71-3.73). Although the pooled analysis for platelet glycoprotein Ia (GPIa) C807T showed a positive association (OR = 1.50, 95% CI=1.10-2.05), the Egger's test indicated the existence of publication bias (t=5.27, P>|t|=0.034). CONCLUSIONS: Genetic abnormalities specific to homocysteine and lipid metabolism increase the risk for ischemic stroke at an early age. These data may offer important implications for future genetic association studies for stroke.