Characterization and Proteome Analysis of Inosine 5-Monophosphate Dehydrogenase in Epidemic Streptococcus suis Serotype 2

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that causes severe disease symptoms in pigs and humans. In the present study, we found one isogenic mutant lacking inosine 5-monophosphate dehydrogenase (IMPDH) ΔZY05719 was attenuated in pigs compared with the wild-type SS2 strain ZY05719. Comparative proteome analysis of the secreted proteins expression profiles between ZY05719 and ΔZY05719 allowed us to identify Triosephosphate isomerase (TPI) and glyceraldehyde phosphate dehydrogenase (GAPDH), which were down expressed in the absence of the IMPDH. Both of them are glycolytic enzymes participating in the glycolytic pathway. Compared with ZY05719, ΔZY05719 lost the ability of utilize mannose, which might relate to down expression of TPI and GAPDH. In addition, GAPDH is a well-known factor that involved in adhesion to host cells, and we demonstrated ability of adhesion to HEp-2 and PK15 by ΔZY05719 was significantly weakened, in contrast to ZY05719. The adhesion to host cells is the crucial step to cause infection for pathogen, and the reduction adhesion of ΔZY05719, to some extent illustrates the attenuated virulence of ΔZY05719.     

Edwardsiella piscicida type III protein EseJ suppresses apoptosis through down regulating type 1 fimbriae,which stimulate the cleavage of caspase‐8

The type III secretion system effector EseJ plays a regulatory role inside bacteria. It suppresses the adherence of Edwardsiella piscicida (E. piscicida) to host epithelial cells by down regulating type 1 fimbriae. In this study, we observed that more macrophages infected with ΔeseJ strain of E. piscicida detached as compared with those infected with the wild‐type (WT) strain. Terminal deoxynucleotidyl transferase dUTP nick‐end labelling (TUNEL) staining and cleaved caspase‐3 examination revealed that the detachment is due to increased apoptosis, suggesting that EseJ suppresses macrophage apoptosis. However, apoptosis inhibition by EseJ is not relative to a type III secretion system (T3SS) and is not related to EseJ's translocation. Since EseJ negatively regulates type 1 fimbriae, murine J774A.1 cells were infected with ΔeseJΔfimA or ΔeseJΔfimH strains. It was demonstrated that ΔeseJ stimulates macrophage apoptosis through type 1 fimbriae. Moreover, we found that infecting J774A.1 cells with the ΔeseJ strain increased levels of cleaved caspase‐8, caspase‐9, and caspase‐3, demonstrating that EseJ inhibits apoptosis through either an extrinsic or a combination of extrinsic and intrinsic pathways. Pre‐treatment of macrophages with caspase‐8 inhibitor prior to infection with the ΔeseJ strain decreased the levels of cleaved caspase‐8, caspase‐9, and caspase‐3, indicating that the ΔeseJ strain stimulates apoptosis, mainly through an extrinsic pathway by up regulating type 1 fimbriae. Zebrafish larvae or blue gourami fish infected with the ΔeseJ strain consistently exhibited higher apoptosis than those infected with the E. piscicida WT strain or ΔeseJΔfimA strain. Taken together, we revealed that the T3SS protein EseJ of E. piscicida inhibits host apoptosis, mainly through an extrinsic pathway by down regulating type 1 fimbriae.     

RNAi-mediated reversible opening of the blood-brain barrier

Background

The blood‐brain barrier (BBB) contains tight junctions (TJs) which reduce the space between adjacent endothelial cells lining the fine capillaries of the microvasculature of the brain to form a selective and regulatable barrier.

Methods

Using a hydrodynamic approach, we delivered siRNA targeting the TJ protein claudin‐5 to the endothelial cells of the BBB in mice.

Results

We have shown a significant decrease in claudin‐5 mRNA levels 24 and 48 hours post‐delivery of siRNA, with levels of protein expression decreasing up to 48 hours post‐injection compared to uninjected, phosphate‐buffered saline (PBS)‐injected and non‐targeting siRNA‐injected mice. We observed increased permeability at the BBB to molecules up to 742 Da, but not 4400 Da, using tracer molecule perfusion and MRI analysis. To illustrate the functional efficacy of size‐selective and transient barrier opening, we have shown that enhanced delivery of the small neuropeptide thyrotropin‐releasing hormone (TRH) (MW 360 Da) to the brains of mice 48 hours post‐injection of siRNA targeting claudin‐5 significantly modifies behavioural output.

Conclusions

These data demonstrate that it is now possible to transiently and size‐selectively open the BBB in mice, allowing in principle the delivery of a wide range of agents for the establishment and treatment of experimental mouse models of neurodegenerative, neuropsychiatric and malignant diseases. Copyright © 2008 John Wiley & Sons, Ltd.     

Annexin A1 restores Aβ1‐42‐induced blood–brain barrier disruption through the inhibition of RhoA‐ROCK signaling pathway

The blood–brain barrier (BBB) is composed of brain capillary endothelial cells and has an important role in maintaining homeostasis of the brain separating the blood from the parenchyma of the central nervous system (CNS). It is widely known that disruption of the BBB occurs in various neurodegenerative diseases, including Alzheimer's disease (AD). Annexin A1 (ANXA1), an anti‐inflammatory messenger, is expressed in brain endothelial cells and regulates the BBB integrity. However, its role and mechanism for protecting BBB in AD have not been identified. We found that β‐Amyloid 1‐42 (Aβ42)‐induced BBB disruption was rescued by human recombinant ANXA1 (hrANXA1) in the murine brain endothelial cell line bEnd.3. Also, ANXA1 was decreased in the bEnd.3 cells, the capillaries of 5XFAD mice, and the human serum of patients with AD. To find out the mechanism by which ANXA1 recovers the BBB integrity in AD, the RhoA‐ROCK signaling pathway was examined in both Aβ42‐treated bEnd.3 cells and the capillaries of 5XFAD mice as RhoA was activated in both cases. RhoA inhibitors alleviated Aβ42‐induced BBB disruption and constitutively overexpressed RhoA‐GTP (active form of RhoA) attenuated the protective effect of ANXA1. When pericytes were cocultured with bEnd.3 cells, Aβ42‐induced RhoA activation of bEnd.3 cells was inhibited by the secretion of ANXA1 from pericytes. Taken together, our results suggest that ANXA1 restores Aβ42‐induced BBB disruption through inhibition of RhoA‐ROCK signaling pathway and we propose ANXA1 as a therapeutic reagent, protecting against the breakdown of the BBB in AD.     

The role of LINC00094/miR‐224‐5p (miR‐497‐5p)/Endophilin‐1 axis in Memantine mediated protective effects on blood‐brain barrier in AD microenvironment

The dysfunction of the blood‐brain barrier (BBB) is one of the main pathological features of Alzheimer's disease (AD). Memantine (MEM), an N‐methyl‐d ‐aspartate (NMDA) receptor antagonist, has been reported that been used widely for AD therapy. This study was performed to demonstrate the role of the MEM in regulating BBB permeability in AD microenvironment as well as its possible mechanisms. The present study showed that LINC00094 was dramatically increased in Abeta_1‐42‐incubated microvascular endothelial cells (ECs) of BBB model in vitro. Besides, it was decreased in MEM‐incubated ECs. Silencing LINC00094 significantly decreased BBB permeability, meanwhile up‐regulating the expression of ZO‐1, occludin and claudin‐5. Furthermore, silencing LINC00094 enhance the effect of MEM on decreasing BBB permeability in AD microenvironment. The analysis of the mechanism demonstrated that reduction of LINC00094 inhibited Endophilin‐1 expression by up‐regulating miR‐224‐4p/miR‐497‐5p, promoted the expression of ZO‐1, occludin and claudin‐5, and ultimately alleviated BBB permeability in AD microenvironment. Taken together, the present study suggests that the MEM/LINC00094/miR‐224‐5p (miR‐497‐5p)/Endophilin‐1 axis plays a crucial role in the regulation of BBB permeability in AD microenvironment. Silencing LINC00094 combined with MEM provides a novel target for the therapy of AD.     

猪链球菌2型新的感染相关因子自溶素的鉴定与分析

根据Sanger研究所公布的猪链球菌2型(SS2)P1/7株的自溶素序列,设计检测引物,取SS2我国2次流行株、其它临床分离株和参考株,及猪链球菌1型、1/2型、7型和9型,共33株,分别以其DNA为模板,PCR扩增.结果表明,SS2除无毒株T15阴性外,其他临床分离株27株(含人源2株)均阳性;其它猪链球菌为,SS7阳性,SS1、SS1/2和SS9均阴性.同时设计引物向两侧扩增,以四川流行株ZY05719和江苏流行株HA9801的DNA为模板,扩增自溶素ORF完整的编码基因,软件分析结果显示,该基因含有6个重复的"GBS_Bsp-like"域和1个"N-乙酰胞壁酰-L-丙氨酸酰胺酶"域,与SS2欧洲株有较高同源性(99.8%),但与SS2加拿大株差异较大.在DNASTAR分析所编码蛋白的抗原性的基础上,另设计引物,以ZY05719株DNA为模板,PCR扩增具有良好免疫原性的片段基因,并定向克隆至表达载体pET30a( )中,进行重组表达,SDS-PAGE和Western blot表明,所获得重组自溶素具有良好反应原性.     

SopB‐ and SifA‐dependent shaping of the Salmonella‐containing vacuole proteome in the social amoeba Dictyostelium discoideum

The ability of Salmonella to survive and replicate within mammalian host cells involves the generation of a membranous compartment known as the Salmonella‐containing vacuole (SCV). Salmonella employs a number of effector proteins that are injected into host cells for SCV formation using its type‐3 secretion systems encoded in SPI‐1 and SPI‐2 (T3SS‐1 and T3SS‐2, respectively). Recently, we reported that S. Typhimurium requires T3SS‐1 and T3SS‐2 to survive in the model amoeba Dictyostelium discoideum. Despite these findings, the involved effector proteins have not been identified yet. Therefore, we evaluated the role of two major S. Typhimurium effectors SopB and SifA during D. discoideum intracellular niche formation. First, we established that S. Typhimurium resides in a vacuolar compartment within D. discoideum. Next, we isolated SCVs from amoebae infected with wild type or the ΔsopB and ΔsifA mutant strains of S. Typhimurium, and we characterised the composition of this compartment by quantitative proteomics. This comparative analysis suggests that S. Typhimurium requires SopB and SifA to modify the SCV proteome in order to generate a suitable intracellular niche in D. discoideum. Accordingly, we observed that SopB and SifA are needed for intracellular survival of S. Typhimurium in this organism. Thus, our results provide insight into the mechanisms employed by Salmonella to survive intracellularly in phagocytic amoebae.     

Ischemic neurons activate astrocytes to disrupt endothelial barrier via increasing VEGF expression

Blood–brain barrier (BBB) disruption occurring within the first few hours of ischemic stroke onset is closely associated with hemorrhagic transformation following thrombolytic therapy. However, the mechanism of this acute BBB disruption remains unclear. In the neurovascular unit, neurons do not have direct contact with the endothelial barrier; however, they are highly sensitive and vulnerable to ischemic injury, and may act as the initiator for disrupting BBB when cerebral ischemia occurs. Herein, we employed oxygen–glucose deprivation (OGD) and an in vitro BBB system consisting of brain microvascular cells and astrocytes to test this hypothesis. Neurons (CATH.a cells) were exposed to OGD for 3‐h before co‐culturing with endothelial monolayer (bEnd 3 cells), or endothelial cells plus astrocytes (C8‐D1A cells). Incubation of OGD‐treated neurons with endothelial monolayer alone did not increase endothelial permeability. However, when astrocytes were present, the endothelial permeability was significantly increased, which was accompanied by loss of occludin and claudin‐5 proteins as well as increased vascular endothelial growth factor (VEGF) secretion into the conditioned medium. Importantly, all these changes were abolished when VEGF was knocked down in astrocytes by siRNA. Our findings suggest that ischemic neurons activate astrocytes to increase VEGF production, which in turn induces endothelial barrier disruption.

     

Peripheral nerve pericytes modify the blood–nerve barrier function and tight junctional molecules through the secretion of various soluble factors

The objectives of this study were to establish pure blood–nerve barrier (BNB) and blood–brain barrier (BBB)‐derived pericyte cell lines of human origin and to investigate their unique properties as barrier‐forming cells. Brain and peripheral nerve pericyte cell lines were established via transfection with retrovirus vectors incorporating human temperature‐sensitive SV40 T antigen (tsA58) and telomerase. These cell lines expressed several pericyte markers such as α‐smooth muscle actin, NG2, platelet‐derived growth factor receptor β, whereas they did not express endothelial cell markers such as vWF and PECAM. In addition, the inulin clearance was significantly lowered in peripheral nerve microvascular endothelial cells (PnMECs) through the up‐regulation of claudin‐5 by soluble factors released from brain or peripheral nerve pericytes. In particular, bFGF secreted from peripheral nerve pericytes strengthened the barrier function of the BNB by increasing the expression of claudin‐5. Peripheral nerve pericytes may regulate the barrier function of the BNB, because the BNB does not contain cells equivalent to astrocytes which regulate the BBB function. Furthermore, these cell lines expressed several neurotrophic factors such as NGF, BDNF, and GDNF. The secretion of these growth factors from peripheral nerve pericytes might facilitate axonal regeneration in peripheral neuropathy. Investigation of the characteristics of peripheral nerve pericytes may provide novel strategies for modifying BNB functions and promoting peripheral nerve regeneration. J. Cell. Physiol. 226: 255–266, 2010. © 2010 Wiley‐Liss, Inc.     

Aβ1–42 oligomer‐induced leakage in an in vitro blood–brain barrier model is associated with up‐regulation of RAGE and metalloproteinases,and down‐regulation of tight junction scaffold proteins

Accumulating evidence indicates that abnormal deposition of amyloid‐β (Aβ) peptide in the brain is responsible for endothelial cell damage and consequently leads to blood–brain barrier (BBB) leakage. However, the mechanisms underlying BBB disruption are not well described. We employed an monolayer BBB model comprising bEnd.3 cell and found that BBB leakage was induced by treatment with Aβ_1–42, and the levels of tight junction (TJ) scaffold proteins (ZO‐1, Claudin‐5, and Occludin) were decreased. Through comparisons of the effects of the different components of Aβ_1–42, including monomer (Aβ_1–42‐Mono), oligomer (Aβ_1–42‐Oligo), and fibril (Aβ_1–42‐Fibril), our data confirmed that Aβ_1–42‐Oligo is likely to be the most important damage factor that results in TJ damage and BBB leakage in Alzheimer's disease. We found that the incubation of bEnd.3 cells with Aβ_1–42 significantly up‐regulated the level of receptor for advanced glycation end‐products (RAGE). Co‐incubation of a polyclonal antibody to RAGE and Aβ_1–42‐Oligo in bEnd.3 cells blocked RAGE suppression of Aβ_1–42‐Oligo‐induced alterations in TJ scaffold proteins and reversed Aβ_1–42‐Oligo‐induced up‐regulation of RAGE, matrix metalloproteinase (MMP)‐2, and MMP‐9. Furthermore, we found that these effects induced by Aβ_1–42‐Oligo treatment were effectively suppressed by knockdown of RAGE using small interfering RNA (siRNA) transfection. We also found that GM 6001, a broad‐spectrum MMP inhibitor, partially reversed the Aβ_1–42‐Oligo‐induced inhibitor effects in bEnd.3 cells. Thus, these results suggested that RAGE played an important role in Aβ‐induced BBB leakage and alterations of TJ scaffold proteins, through a mechanism that involved up‐regulation of MMP‐2 and MMP‐9.

     

猪链球菌2型Dbp缺失株的构建及特性分析

【目的】通过构建DNA结合膜蛋白(DNA-binding membrance protein,Dbp)基因的缺失株,探究Dbp基因对猪链球菌2型强毒株毒力的影响。【方法】通过PCR检测Dbp基因的分布。利用同源重组原理构建Dbp基因上下游片段的重组质粒,将构建好的质粒电转入ZY05719感受态细胞中,筛选Dbp缺失突变株,通过PCR及测序分析对其进行验证。生物学特性分析比较缺失株?Dbp和野毒株ZY05719在生长速率、形态特征、毒力等方面的差异。【结果】Dbp基因为猪链球菌2型强毒株中相对保守基因,构建了Dbp基因缺失株?Dbp。体外实验结果显示Dbp基因缺失株的生长速率在对数期减慢,并且缺失株?Dbp的荚膜同野毒株存在显著差异,斑马鱼实验结果表明缺失株?Dbp毒力下降。【结论】Dbp与猪链球菌2型的毒力相关,在猪链球菌2型的致病过程中起一定的作用,这丰富了对该菌致病机理的认识。     

Notoginsenoside R1 intervenes degradation and redistribution of tight junctions to ameliorate blood-brain barrier permeability by Caveolin-1/MMP2/9 pathway after acute ischemic stroke

BackgroundThe leakage of blood-brain barrier (BBB) is main pathophysiological change in acute stage of ischemic stroke, which not only deteriorates neurological function, but also increases the risk of hemorrhagic transformation after thrombolysis.Purpose/Study DesignThis article investigates the efficacy of Notoginsenoside R1, an active ingredient of Panax notoginseng, on BBB permeability and explores related mechanisms after acute ischemic stroke.MethodsIn vivo, male Sprague-Dawley rats (260–280 g) were selected and randomly divided into 6 groups: sham group, model group, low, middle and high doses of Notoginsenoside R1 groups and positive drug Dl-3-n-Butylphthalide group. Except for sham group, rats were performed with permanent middle cerebral artery occlusion model in each group. Twelve hours later, rats were evaluated for Bederson neurological function, and BBB integrity by Evans blue leak imaging; Triphenyltetrazolium chloride staining was used to detect the volume of cerebral infarction. Frozen sections of rats’ brain tissue were prepared for detection of MMPs activity in situ zymography. Peripheral tissue of cerebral infarction was collected and tested the expression of MMP2, 9 and tight junction proteins (zo1, claudin5, occludin) by western blot. In vitro, transwell endothelial barrier model was established by bEnd.3 cells. Oxygen glucose deprivation (OGD) was chosen to simulate the hypoxic environment. Suitable OGD stimulation time as well as Notoginsenoside R1 and Dl-3-n-Butylphthalide optimal dose concentrations were determined through transwell leakage and CCK8 assay. Furthermore, endothelial subcellular component proteins were extracted. The change of zo1, claudin5, occludin and caveolin1 was detected by western blot.ResultsNotoginsenoside R1 treatment significantly reduced BBB leakage and cerebral infarction volume, weakened neurological deficits in post-stroke rats. Moreover, it inhibited the activity of MMPs in infarcted cortex and striatum, down-regulated MMP2, 9 and up-regulated zo1 and claudin5 expressions in penumbra. In vitro, Notoginsenoside R1 treatment decreased OGD-induced endothelial barrier permeability, restored expressions of zo1, claudin5 on cellular membrane and cytoplasm, as well as mediated membrane redistribution of occludin and caveolin1 from actin cytoskeletal fraction.ConclusionsNotoginsenoside R1 treatment attenuates BBB permeability, cerebral infarction volume and neurological impairments in rats with acute cerebral ischemia. The mechanisms might be related to intervening degradation and redistribution of zo1, caludin5 and occludin by caveolin1/ MMP2/9 pathway. More effects and mechanisms of Notoginsenoside R1 on rehabilitation of stroke are worthy to be explored in the future.     

Expression of CEACAM1 or CEACAM5 in AZ‐521 cells restores the type IV secretion deficiency for translocation of CagA by Helicobacter pylori

Helicobacter pylori represents an important pathogen involved in diseases ranging from gastritis, peptic ulceration, to gastric malignancies. Prominent virulence factors comprise the vacuolating cytotoxin VacA and the cytotoxin‐associated genes pathogenicity island (cagPAI)‐encoded type IV secretion system (T4SS). The T4SS effector protein CagA can be translocated into AGS and other gastric epithelial cells followed by phosphorylation through c‐Src and c‐Abl tyrosin kinases to hijack signalling networks. The duodenal cell line AZ‐521 has been recently introduced as novel model system to investigate CagA delivery and phosphorylation in a VacA‐dependent fashion. In contrast, we discovered that AZ‐521 cells display a T4SS incompetence phenotype for CagA injection, which represents the first reported gastrointestinal cell line with a remarkable T4SS defect. We proposed that this deficiency may be due to an imbalanced coexpression of T4SS receptor integrin‐β₁ or carcinoembryonic antigen‐related cell adhesion molecules (CEACAMs), which were described recently as novel H. pylori receptors. We demonstrate that AZ‐521 cells readily express integrin‐β₁, but overexpression of integrin‐β₁ constructs did not restore the T4SS defect. We further show that AZ‐521 cells lack the expression of CEACAMs. We demonstrate that genetic introduction of either CEACAM1 or CEACAM5, but not CEACAM6, in AZ‐521 cells is sufficient to permit injection and phosphorylation of CagA by H. pylori to degrees observed in the AGS cell model. Expression of CEACAM1 or CEACAM5 in infected AZ‐521 cells was also accompanied by tyrosine dephosphorylation of the cytoskeletal proteins vinculin and cortactin, a hallmark of H. pylori‐infected AGS cells. Our results suggest the existence of an integrin‐β₁‐ and CEACAM1‐ or CEACAM5‐dependent T4SS delivery pathway for CagA, which is clearly independent of VacA. The presence of two essential host protein receptors during infection with H. pylori represents a unique feature in the bacterial T4SS world. Further detailed investigation of these T4SS functions will help to better understand infection strategies by bacterial pathogens.     

Interaction between cFLIPL and Itch,a ubiquitin ligase,is obstructed in Trypanosoma cruzi‐infected human cells

Death receptor‐mediated host cell apoptosis, a defense strategy for elimination by the immune system of parasite‐infected cells, is inhibited by Trypanosoma cruzi, the causative agent of Chagas' disease. It has previously been reported by us that, in infected cells, T. cruzi upregulates and exploits cFLIP_L, a mammalian inhibitor of death receptor signaling. Here it is shown that ubiquitination of cFLIP_L, leading to proteasomal degradation, is inhibited in parasite‐infected cells. The extent of expression of Itch, a protein thought to be an ubiquitin ligase for cFLIP_L, was found to be equivalent in T. cruzi‐infected and in uninfected cells. However, co‐immunoprecipitation analysis showed that the interaction between cFLIP_L and Itch is strongly inhibited in T. cruzi‐infected cells. This unique parasite strategy, which has not been reported in any other pathogen‐infected cells, may allow the host cell to accumulate cFLIP_L, eventually resulting in the inhibition of apoptosis of T. cruzi‐infected cells.     

Differential expression and cellular distribution of γ‐tubulin and βIII‐tubulin in medulloblastomas and human medulloblastoma cell lines

The breakdown of the blood–brain barrier (BBB) has been considered to be a key step in the disease process of a number of neurological disorders such as cerebral ischemia and Alzheimer's disease. Many in vitro BBB models derived from animal tissues have been established to elucidate the mechanism of BBB insufficiency. However, only a few human immortalized in vitro BBB models have been reported. In the present study, a temperature‐sensitive SV40‐T antigen was introduced to immortalize cells using a retrovirus to obtain a better human in vitro BBB model which sustains physiological properties. This endothelial cell (EC) line, termed TY08, showed a spindle‐shaped morphology. The cells expressed all key tight junctional proteins, such as occludin, claudin‐5, zonula occludens (ZO)‐1 and ZO‐2 at their cell‐to‐cell boundaries, and had low permeability to inulin across its monolayer. The cells also expressed various influx and efflux transporters and exhibited the functional expression of p‐glycoprotein. Furthermore, the TY08 cells grew and proliferated well under the permissive temperature and stopped growing under the non‐permissive temperature to serve as physiological ECs forming the BBB. Thus, conditionally immortalized TY08 cells retaining the in vivo BBB functions should facilitate analyses for determining the pathophysiology of various neurological diseases. J. Cell. Physiol. 225: 519–528, 2010. © 2010 Wiley‐Liss, Inc.     

Proteomic identification of potential target proteins regulated by the SCFFbp1‐mediated proteolysis pathway in Fusarium oxysporum

F‐box proteins function in the recruitment of proteins for SCF ubiquitination and proteasome degradation. Here, we studied the role of Fbp1, a nonessential F‐box protein of the tomato pathogen Fusarium oxysporum f. sp. lycopersici. The Δfbp1 mutant showed a significant delay in the production of wilt symptoms on tomato plants and was impaired in invasive growth on cellophane membranes and on living plant tissue. To search for target proteins recruited by Fbp1, a combination of sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and matrix‐assisted laser desorption/ionization time‐of‐flight/time‐of‐flight (MALDI‐TOF/TOF) was used to compare proteins in mycelia of the wild‐type and Δfbp1 mutant. The proteomic approach identified 41 proteins differing significantly in abundance between the two strains, 17 of which were more abundant in the Δfbp1 mutant, suggesting a possible regulation by proteasome degradation. Interestingly, several of the identified proteins were related to vesicle trafficking. Microscopic analysis revealed an impairment of the Δfbp1 strain in directional growth and in the structure of the Spitzenkörper, suggesting a role of Fbp1 in hyphal orientation. Our results indicate that Fbp1 regulates protein turnover and pathogenicity in F. oxysporum.     

The E3 ubiquitin ligase HECTD3 regulates ubiquitination and degradation of Tara

Tara was identified as an interacting partner of guanine nucleotide exchange factor Trio and TRF1. Tara is proposed to be involved in many important fundamental cellular processes, ranging from actin remodeling, directed cell movement, to cell cycle regulation. Yet, its exact roles required further elucidation. Here, we identify a novel Tara-binding protein HECTD3, a putative member of HECT E3 ubiquitin ligases. HECTD3 directly binds Tara in vitro and forms a complex with Tara in vivo. Overexpression of HECTD3 enhances the ubiquitination of Tara in vivo and promotes the turnover of Tara, whereas depletion of HECTD3 by small interfering RNA decreases Tara degradation. Furthermore, depletion of HECTD3 leads to multipolar spindle formation. All these findings suggest that HECTD3 may facilitate cell cycle progression via regulating ubiquitination and degradation of Tara.     

Combined treatment of sodium ferulate,n‐butylidenephthalide,and ADSCs rehabilitates neurovascular unit in rats after photothrombotic stroke

The remodelling of structural and functional neurovascular unit (NVU) becomes a central therapeutic strategy after cerebral ischaemic stroke. In the present study, we investigated the effect of combined therapy of sodium ferulate (SF), n‐butylidenephthalide (BP) and adipose‐derived stromal cells (ADSCs) to ameliorate the injured NVU in the photochemically induced thrombotic stroke in rats. After solely or combined treatment, the neovascularization, activation of astrocytes, neurogenesis, expressions of vascular endothelial growth factor (VEGF) and claudin‐5 were assessed by immunohistochemical or immunofluorescence staining. In order to uncover the underlying mechanism of therapeutic effect, signalling of protein kinase B/mammalian target of rapamycin (AKT/mTOR), extracellular signal‐regulated kinase 1/2 (ERK1/2), and Notch1 in infarct zone were analysed by western blot. ¹⁸F‐2‐deoxy‐glucose/positron emission tomography, magnetic resonance imaging, Evans blue staining were employed to evaluate the glucose metabolism, cerebral blood flow (CBF), and brain‐blood barrier (BBB) permeability, respectively. The results showed that combined treatment increased the neovascularization, neurogenesis, and VEGF secretion, modulated the astrocyte activation, enhanced the regional CBF, and glucose metabolism, as well as reduced BBB permeability and promoted claudin‐5 expression, indicating the restoration of structure and function of NVU. The activation of ERK1/2 and Notch1 pathways and inhibition of AKT/mTOR pathway might be involved in the therapeutic mechanism. In summary, we have demonstrated that combined ADSCs with SF and BP, targeting the NVU remodelling, is a potential treatment for ischaemic stroke. These results may provide valuable information for developing future combined cellular and pharmacological therapeutic strategy for ischaemic stroke.     

Consequences of flagellin export through the type III secretion system of Pseudomonas syringae reveal a major difference in the innate immune systems of mammals and the model plant Nicotiana benthamiana

Bacterial flagellin is perceived as a microbe (or pathogen)‐associated molecular pattern (MAMP or PAMP) by the extracellular pattern recognition receptors, FLS2 and TLR5, of plants and mammals respectively. Flagellin accidently translocated into mammalian cells by pathogen type III secretion systems (T3SSs) is recognized by nucleotide‐binding leucine‐rich repeat receptor NLRC4 as a pattern of pathogenesis and induces a death‐associated immune response. The non‐pathogen Pseudomonas fluorescens Pf0‐1, expressing a Pseudomonas syringae T3SS, and the plant pathogen P. syringae pv. tomato DC3000 were used to seek evidence of an analogous cytoplasmic recognition system for flagellin in the model plant Nicotiana benthamiana. Flagellin (FliC) was secreted in culture and translocated into plant cells by the T3SS expressed in Pf0‐1 and DC3000 and in their ΔflgGHI flagellar pathway mutants. ΔfliC and ΔflgGHI mutants of Pf0‐1 and DC3000 were strongly reduced in elicitation of reactive oxygen species production and in immunity induction as indicated by the ability of challenge bacteria inoculated 6 h later to translocate a type III effector–reporter and to elicit effector‐triggered cell death. Agrobacterium‐mediated transient expression in N. benthamiana of FliC with or without a eukaryotic export signal peptide, coupled with virus‐induced gene silencing of FLS2, revealed no immune response that was not FLS2 dependent. Transiently expressed FliC from DC3000 and Pectobacterium carotovorum did notinduce cell death in N. benthamiana, tobacco or tomato leaves. Flagellin is the major Pseudomonas MAMP perceived by N. benthamiana, and although flagellin secretion through the plant cell wall by the T3SS may partially contribute to FLS2‐dependent immunity, flagellin in the cytosol does not elicit immune‐associated cell death. We postulate that a death response to translocated MAMPs would produce vulnerability to the many necrotrophic pathogens of plants, such as P. carotovorum, which differ from P. syringae and other (hemi)biotrophic pathogens in benefitting from death‐associated immune responses.