The global regulator of pathogenesis PnCon7 positively regulates Tox3 effector gene expression through direct interaction in the wheat pathogen Parastagonospora nodorum.

To investigate effector gene regulation in the wheat pathogenic fungus Parastagonospora nodorum, the promoter and expression of Tox3 was characterised through a series of complementary approaches. Promoter deletion and DNase I footprinting experiments identified a 25 bp region in the Tox3 promoter as being required for transcription. Subsequent yeast one-hybrid analysis using the DNA sequence as bait identified that interacting partner as the C2H2 zinc finger transcription factor PnCon7, a putative master regulator of pathogenesis. Silencing of PnCon7 resulted in the down-regulation of Tox3 demonstrating that the transcription factor has a positive regulatory role on gene expression. Analysis of Tox3 expression in the PnCon7 silenced strains revealed a strong correlation with PnCon7 transcript levels, supportive of a direct regulatory role. Subsequent pathogenicity assays using PnCon7-silenced isolates revealed that the transcription factor was required for Tox3-mediated disease. The expression of two other necrotrophic effectors (ToxA and Tox1) was also affected but in a non-dose dependent manner suggesting that the regulatory role of PnCon7 on these genes was indirect. Collectively, these data have advanced our fundamental understanding of the Con7 master regulator of pathogenesis by demonstrating its positive regulatory role on the Tox3 effector in P. nodorum through direct interaction.

Structural insight into Golgi membrane stacking by GRASP65 and GRASP55 proteins.

The stacking of Golgi cisternae involves GRASP65 and GRASP55. The oligomerization of the N-terminal GRASP domain of these proteins, which consists of two tandem PDZ domains, is required to tether the Golgi membranes. However, the molecular basis for GRASP assembly is unclear. Here, we determined the crystal structures of the GRASP domain of GRASP65 and GRASP55. The structures reveal similar homotypic interactions: the GRASP domain forms a dimer in which the peptide-binding pockets of the two neighboring PDZ2 domains face each other, and the dimers are further connected by the C-terminal tail of one GRASP domain inserting into the binding pocket of the PDZ1 domain in another dimer. Biochemical analysis suggests that both types of contacts are relatively weak but are needed in combination for GRASP-mediated Golgi stacking. Our results unveil a novel mode of membrane tethering by GRASP proteins and provide insight into the mechanism of Golgi stacking.

Rationally constructed ZnCdS-HDCs@In2S3-HNRs double-hollow heterojunction with promoted light capture capability for photoelectrochemical biosensing.

The construction of novel heterojunction is regarded as an operative scheme to promote the transport of photogenerated carriers and reduce electron-hole pair recombination to enhance the photoelectrochemical (PEC) performances. Herein, ZnCdS hollow dodecahedral nanocages (ZnCdS-HDCs) and In2S3 hollow nanorods (In2S3-HNRs), which were derived from two different of metal-organic frameworks (MOFs) by solvothermal sulfidation method and were constructed an original double-hollow heterostructure ZnCdS-HDCs@In2S3-HNRs. The intrinsic mechanism of In2S3-HNRs benefiting from unique morphology to boost the photochemical properties under visible light irradiation was illustrated. Meanwhile, the mechanism of the novel type II heterojunction with staggered matching levels was revealed, which could effectively restrict electron-hole pair reassociation separation, and accelerated charge separation and transfer. Therefore, based on the excellent PEC performance of ZnCdS- HDCs@In2S3-HNRs double-hollow heterostructure, a signal-off PEC biosensor platform without labeled was constructed for the detection of CA15-3, which manifested acceptable specificity, reproducibility and stability. Additionally, the expected PEC biosensors showed a linear response range from 1.0 × 10-5 to 10 U·mL-1 in addition to an ultralow detection limit of 3.78 × 10-6 U·mL-1. This study innovatively constructed and prepared a new double-hollow heterojunction material with superior PEC nature for the application of PEC biosensing, which exhibits a broad application prospect.

The causal-effect of bed rest and post-dural puncture headache in patients receiving diagnostic lumbar puncture: A prospective cohort study.

BACKGROUND: Post-dural puncture headache (PDPH) is the most common complication of lumbar puncture. Patients who received lumbar puncture were previously suggested to remain in bed for a certain time to prevent PDPH; however, this concept was challenged by recent studies. We aimed to investigate whether ambulation instead of resting supine following lumbar puncture could increase the risk of PDPH. METHODS: The study used a prospective cohort design, applying convenience sampling among patients who received diagnostic lumbar puncture between January and September 2018 in the neurology ward of a tertiary medical center. The patients who fulfilled the inclusion criteria were informed that the current practice suggests lying supine for 6 to 8 hours after lumbar puncture, but they were allowed to either follow the suggestion or ambulate by their wills. The timing of bed rest was recorded, in addition to other possible risk factors of PDPH. The study endpoint is the presence or absence of PDPH within 48 hours of lumbar puncture. RESULTS: A total of 137 patients who received lumbar puncture were enrolled, including 103 with bed rest following lumbar puncture and 34 without. There was no difference in demographics between the two groups. PDPH was found in 21 patients, with a total follow-up period of 5959 person-hours and an incidence density of 0.35%. There was no significant difference between the incidence of PDPH between the two groups (non-bed rest group 5.9% vs bed rest group 18.4%; p = 0.078), nor was incidence density (non-bed rest group 0.13% vs bed rest group 0.43%, p = 0.113). The results remained the same after adjusting for age. CONCLUSION: Bed rest following lumbar puncture does not prevent PDPH, and even leads to a marginally increased risk of PDPH. Amendment to the current practice guideline post-lumbar puncture care might be needed to improve patient care.

Engineering skeletal muscle tissues with advanced maturity improves synapse formation with human induced pluripotent stem cell-derived motor neurons.

To develop effective cures for neuromuscular diseases, human-relevant in vitro models of neuromuscular tissues are critically needed to probe disease mechanisms on a cellular and molecular level. However, previous attempts to co-culture motor neurons and skeletal muscle have resulted in relatively immature neuromuscular junctions (NMJs). In this study, NMJs formed by human induced pluripotent stem cell (hiPSC)-derived motor neurons were improved by optimizing the maturity of the co-cultured muscle tissue. First, muscle tissues engineered from the C2C12 mouse myoblast cell line, cryopreserved primary human myoblasts, and freshly isolated primary chick myoblasts on micromolded gelatin hydrogels were compared. After three weeks, only chick muscle tissues remained stably adhered to hydrogels and exhibited progressive increases in myogenic index and stress generation, approaching values generated by native muscle tissue. After three weeks of co-culture with hiPSC-derived motor neurons, engineered chick muscle tissues formed NMJs with increasing co-localization of pre- and postsynaptic markers as well as increased frequency and magnitude of synaptic activity, surpassing structural and functional maturity of previous in vitro models. Engineered chick muscle tissues also demonstrated increased expression of genes related to sarcomere maturation and innervation over time, revealing new insights into the molecular pathways that likely contribute to enhanced NMJ formation. These approaches for engineering advanced neuromuscular tissues with relatively mature NMJs and interrogating their structure and function have many applications in neuromuscular disease modeling and drug development.

A specific fungal transcription factor controls effector gene expression and orchestrates the establishment of the necrotrophic pathogen lifestyle on wheat.

The fungus Parastagonospora nodorum infects wheat through the use of necrotrophic effector (NE) proteins that cause host-specific tissue necrosis. The Zn2Cys6 transcription factor PnPf2 positively regulates NE gene expression and is required for virulence on wheat. Little is known about other downstream targets of PnPf2. We compared the transcriptomes of the P. nodorum wildtype and a strain deleted in PnPf2 (pf2-69) during in vitro growth and host infection to further elucidate targets of PnPf2 signalling. Gene ontology enrichment analysis of the differentially expressed (DE) genes revealed that genes associated with plant cell wall degradation and proteolysis were enriched in down-regulated DE gene sets in pf2-69 compared to SN15. In contrast, genes associated with redox control, nutrient and ion transport were up-regulated in the mutant. Further analysis of the DE gene set revealed that PnPf2 positively regulates twelve genes that encode effector-like proteins. Two of these genes encode proteins with homology to previously characterised effectors in other fungal phytopathogens. In addition to modulating effector gene expression, PnPf2 may play a broader role in the establishment of a necrotrophic lifestyle by orchestrating the expression of genes associated with plant cell wall degradation and nutrient assimilation.

Identification and therapeutic rescue of autophagosome and glutamate receptor defects in C9ORF72 and sporadic ALS neurons.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease with diverse etiologies. Therefore, the identification of common disease mechanisms and therapeutics targeting these mechanisms could dramatically improve clinical outcomes. To this end, we developed induced motor neuron (iMN) models from C9ORF72 and sporadic ALS (sALS) patients to identify targets that are effective against these types of cases, which together comprise ~90% of patients. We find that iMNs from C9ORF72 and several sporadic ALS patients share two common defects - impaired autophagosome formation and the aberrant accumulation of glutamate receptors. Moreover, we show that an anticoagulation-deficient form of activated protein C, 3K3A-APC, rescues these defects in both C9ORF72 and sporadic ALS iMNs. As a result, 3K3A-APC treatment lowers C9ORF72 dipeptide repeat protein (DPR) levels, restores nuclear TDP-43 localization, and rescues the survival of both C9ORF72 and sporadic ALS iMNs. Importantly, 3K3A-APC also lowers glutamate receptor levels and rescues proteostasis in vivo in C9ORF72 gain- and loss-of-function mouse models. Thus, motor neurons from C9ORF72 and at least a subset of sporadic ALS patients share common, early defects in autophagosome formation and glutamate receptor homeostasis and a single therapeutic approach may be efficacious against these disease processes.

Sec61ß facilitates the maintenance of endoplasmic reticulum homeostasis by associating microtubules.

Sec61ß, a subunit of the Sec61 translocon complex, is not essential in yeast and commonly used as a marker of endoplasmic reticulum (ER). In higher eukaryotes, such as Drosophila, deletion of Sec61ß causes lethality, but its physiological role is unclear. Here, we show that Sec61ß interacts directly with microtubules. Overexpression of Sec61ß containing small epitope tags, but not a RFP tag, induces dramatic bundling of the ER and microtubule. A basic region in the cytosolic domain of Sec61ß is critical for microtubule association. Depletion of Sec61ß induces ER stress in both mammalian cells and Caenorhabditis elegans, and subsequent restoration of ER homeostasis correlates with the microtubule binding ability of Sec61ß. Loss of Sec61ß causes increased mobility of translocon complexes and reduced level of membrane-bound ribosomes. These results suggest that Sec61ß may stabilize protein translocation by linking translocon complex to microtubule and provide insight into the physiological function of ER-microtubule interaction.

ACR11 modulates levels of reactive oxygen species and salicylic acid-associated defense response in Arabidopsis.

The ACT domain (aspartate kinase, chorismate mutase and TyrA), an allosteric effector binding domain, is commonly found in amino acid metabolic enzymes. In addition to ACT domain-containing enzymes, plants have a novel family of ACT domain repeat (ACR) proteins, which do not contain any recognizable catalytic domain. Arabidopsis has 12 ACR proteins, whose functions are largely unknown. To study the functions of Arabidopsis ACR11, we have characterized two independent T-DNA insertion mutants, acr11-2 and acr11-3. RNA gel-blot analysis revealed that the expression of wild-type ACR11 transcripts was not detectable in the acr11 mutants. Interestingly, a lesion-mimic phenotype occurs in some rosette leaves of the acr11 mutants. In addition, high levels of reactive oxygen species (ROS), salicylic acid (SA), and callose accumulate in the mutant leaves when grown under normal conditions. The expression of several SA marker genes and the key SA biosynthetic gene ISOCHORISMATE SYNTHASE1 is up-regulated in the acr11 mutants. Furthermore, the acr11 mutants are more resistant to the infection of bacterial pathogen Pseudomonas syringae pathovar tomato DC3000. These results suggest that ACR11 may be directly or indirectly involved in the regulation of ROS and SA accumulation, which in turn modulates SA-associated defense responses and disease resistance in Arabidopsis.

Haploinsufficiency leads to neurodegeneration in C9ORF72 ALS/FTD human induced motor neurons.

An intronic GGGGCC repeat expansion in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the pathogenic mechanism of this repeat remains unclear. Using human induced motor neurons (iMNs), we found that repeat-expanded C9ORF72 was haploinsufficient in ALS. We found that C9ORF72 interacted with endosomes and was required for normal vesicle trafficking and lysosomal biogenesis in motor neurons. Repeat expansion reduced C9ORF72 expression, triggering neurodegeneration through two mechanisms: accumulation of glutamate receptors, leading to excitotoxicity, and impaired clearance of neurotoxic dipeptide repeat proteins derived from the repeat expansion. Thus, cooperativity between gain- and loss-of-function mechanisms led to neurodegeneration. Restoring C9ORF72 levels or augmenting its function with constitutively active RAB5 or chemical modulators of RAB5 effectors rescued patient neuron survival and ameliorated neurodegenerative processes in both gain- and loss-of-function C9ORF72 mouse models. Thus, modulating vesicle trafficking was able to rescue neurodegeneration caused by the C9ORF72 repeat expansion. Coupled with rare mutations in ALS2, FIG4, CHMP2B, OPTN and SQSTM1, our results reveal mechanistic convergence on vesicle trafficking in ALS and FTD.

[Determination of trace chromium(VI) by catalytic spectrofluorimetry using pyronine Y-H2O2-Cr(VI) system].

A simple and highly sensitive new kinetic catalytic fluorimetric method was proposed for the determination of trace chromium (VI), based on the catalytic effect of trace amounts of chromium (VI) on the oxidation of Pyronine Y by hydrogen peroxide in acetic acid-sodium acetate buffer medium leading to a decrease in the fluorescence intensity. The optimum conditions and kinetic properties of the catalytic reaction were also studied. The apparent activation energy and the apparent rate constant are 159. 92 k * x mol(-1) and 5. 7X10-2 s-1 respectively. The linear range of the calibration curve is 0. 02-0. 24 microg x mL(-1) and the detection limit is 0. 012 microg x mL(-1). The present method was applied to the determination of chromium(VI) in river water, and industrial and electroplating waste water with good results.

Cathodic-controlled and near-infrared organic upconverter for local blood vessels mapping.

Organic materials are used in novel optoelectronic devices because of the ease and high compatibility of their fabrication processes. Here, we demonstrate a low-driving-voltage cathodic-controlled organic upconverter with a mapping application that converts near-infrared images to produce images of visible blood vessels. The proposed upconverter has a multilayer structure consisting of a photosensitive charge-generation layer (CGL) and a phosphorescent organic light-emitting diode (OLED) for producing clear images with a high resolution of 600 dots per inch. In this study, temperature-dependent electrical characterization was performed to analyze the interfacial modification of the cathodic-controlled upconverter. The result shows that the upconverter demonstrated a high conversion efficiency of 3.46% because of reduction in the injection barrier height at the interface between the CGL and the OLED.

Transparent organic upconversion devices for near-infrared sensing.

Transparent organic upconversion devices are shown in a night-vision demonstration of a real object under near-infrared (NIR) illumination in the dark. An extraordinarily high current gain - reflecting the on-off switching effect - greater than 15 000 at a driving voltage of 3 V is demonstrated, indicating the high sensitivity to NIR light and potential of using the proposed upconverter in practical applications. A maximum luminance exceeding 1500 cd m(-2) at 7 V is achieved. Unlike previous studies, where 2D aperture projection is reported, the current study shows 3D images of real objects under NIR illumination in the dark.

Antisense proline-arginine RAN dipeptides linked to C9ORF72-ALS/FTD form toxic nuclear aggregates that initiate in vitro and in vivo neuronal death.

Expanded GGGGCC (G4C2) nucleotide repeats within the C9ORF72 gene are the most common genetic mutation associated with both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Sense and antisense transcripts of these expansions are translated to form five dipeptide repeat proteins (DRPs). We employed primary cortical and motor neuron cultures, live-cell imaging, and transgenic fly models and found that the arginine-rich dipeptides, in particular Proline-Arginine (PR), are potently neurotoxic. Factors that anticipated their neurotoxicity included aggregation in nucleoli, decreased number of processing bodies, and stress granule formation, implying global translational dysregulation as path accountable for toxicity. Nuclear PR aggregates were also found in human induced motor neurons and postmortem spinal cord tissues from C9ORF72 ALS and ALS/FTD patients. Intronic G4C2 transcripts, but not loss of C9ORF72 protein, are also toxic to motor and cortical neurons. Interestingly, G4C2 transcript-mediated neurotoxicity synergizes with that of PR aggregates, suggesting convergence of mechanisms.

A novel electrochemiluminescence ethanol biosensor based on tris(2,2'-bipyridine) ruthenium (II) and alcohol dehydrogenase immobilized in graphene/bovine serum albumin composite film.

We developed a novel electrochemiluminescence (ECL) ethanol biosensor based on Ru(bpy)(3)(2+) and alcohol dehydrogenase (ADH) immobilized by graphene/bovine serum albumin composite film. The graphene film was directly formed on a glassy carbon electrode surface via an in situ reduction of graphene oxide (GO) and Ru(bpy)(3)(2+) was immobilized during its formation. The graphene film acted as both a decorating agent for immobilization of Ru(bpy)(3)(2+) and a matrix to immobilize bovine serum albumin (BSA), meanwhile BSA not only acted as a reductant to reduce GO, but also provided a friendly environment for ADH immobilization. Furthermore, ADH was separated from Ru(bpy)(3)(2+) by the electron-conductive graphene/BSA composite film to retain its enzymatic activity. The experimental results indicated that the biosensor had excellent electrochemical activity, ECL response to ethanol and stability. Such a design of Ru(bpy)(3)(2+)-graphene/BSA film to modify electrode holds a great promise as a new biocompatible platform for the development of enzyme-based ECL biosensors.

Molecular basis for sculpting the endoplasmic reticulum membrane.

The endoplasmic reticulum (ER) is involved in many critical processes, including protein and lipid synthesis and calcium storage. Morphologically, the ER can be divided into two subdomains: a network of interconnected tubules and interspersed sheets. Until recently, how these different compartments form in a continuous membrane system was unclear. Several classes of integral membrane proteins have been identified in the ER; the reticulons and DP1/Yop1p play roles in the generation of ER tubules, and possibly in stabilizing ER sheets, atlastins and Sey1p are dynamin-like GTPases that facilitate tubular network formation by mediating ER membrane fusion, and Climp63, p180, and kinectin are enriched in ER sheets and influence their formation. In this review, we summarize recent advances in our understanding of how these proteins participate in ER shaping. We also discuss possible mechanisms for regulating ER morphology via the cytoskeleton. Lessons learned about sculpting the ER membrane may be applicable to other organelles.

Clinical utility of 18F-FDG PET parameters in patients with advanced nasopharyngeal carcinoma: predictive role for different survival endpoints and impact on prognostic stratification.

OBJECTIVE: To investigate the prognostic impact of different 2-[fluorine-18]fluoro-2-deoxy-D-glucose positron emission tomography (¹8F-FDG PET) parameters in patients with advanced nasopharyngeal carcinoma (NPC). PATIENTS AND METHODS: A total of 196 patients with primary stage III-IVb NPC were included in the study. The following parameters derived from pretreatment ¹8F-FDG PET were determined: metabolic tumor volume and total lesion glycolysis (TLG) of the primary tumor, maximal standardized uptake value of the primary tumor and the neck lymph nodes. Multivariable Cox proportional hazards models were used to identify independent predictors of survival. RESULTS: Multivariable analysis demonstrated that TLG values greater than 330 independently predicted overall survival (P=0.0014) and disease-free survival (P=0.0005). We identified IVa-b stage and TLG values greater than 330 as independent predictors of local failure-free survival. In addition, a high maximal standardized uptake value of the neck lymph nodes (P=0.005), male sex (P=0.041), and stage IVa-b (P=0.009) independently predicted distant failure-free survival. A TLG cutoff value of 330 allowed a better stratification of overall survival and disease-free survival rates. A scoring system combining significant PET parameters and traditional prognostic factors was formulated to define distinct prognostic groups for local failure-free survival and distant failure-free survival. There was a stepwise decrease in the 5-year local (97.7, 90.4, and 47.3%, P<0.0001) and distant control rates (96.8, 88.5, 73.9, and 36.4%, P<0.0001) according to the distinct prognostic scores. CONCLUSION: In patients with advanced NPC, the prognostic significance of ¹8F-FDG PET parameters seems to depend on the specific endpoint. The combination of PET metabolic parameters with traditional risk factors may significantly improve prognostic stratification for this group of patients.

Nonaqueous capillary electrophoresis for rapid and sensitive determination of fangchinoline and tetrandrine in radix Stephaniae tetrandrae and its medicinal preparations.

A simple, rapid, and sensitive non-aqueous capillary electrophoresis procedure with head-column field-amplified sample stacking concentration for the analysis of fangchinoline and tetrandrine is established. Optimum separation and stacking conditions were obtained when the sample was injected at 8 kV for 50 s after preliminary pressure injection of ethanol (16.9 kPa) for 0.6 s and separated with the buffer containing 50 mM ammonium acetate, 0.5% (v/v) acetic acid, and 50% (v/v) acetonitrile in methanol medium at 24 kV applied voltage. The analytes were detected by UV at 214 nm. The two bisbenzylisoquinoline alkaloids can be separated within 6 min and quantified with high sensitivity. The detection limits were 0.30 ng mL(-1) for fangchinoline and 0.34 ng mL(-1) for tetrandrine, which indicated that the sensitivities were at least 1000-fold enhanced over those reported in the literature as obtained by UV detection. The method was applied to the analysis of fangchinoline and tetrandrine in Radix Stephaniae tetrandrae and its medicinal preparations with good results.