Nitrate assimilation compensates for cell wall biosynthesis in the absence of Aspergillus fumigatus phosphoglucose isomerase.

Phosphoglucose isomerase (PGI) links glycolysis, the pentose phosphate pathway (PPP), and the synthesis of cell wall precursors in fungi by facilitating the reversible conversion between glucose-6-phosphate (Glc6p) and fructose-6-phosphate (Fru6P). In a previous study, we established the essential role of PGI in cell wall biosynthesis in the opportunistic human fungal pathogen Aspergillus fumigatus, highlighting its potential as a therapeutic target. In this study, we conducted transcriptomic analysis and discovered that the Δpgi mutant exhibited enhanced glycolysis, reduced PPP, and an upregulation of cell wall precursor biosynthesis pathways. Phenotypic analysis revealed defective protein N-glycosylation in the mutant, notably the absence of glycosylated virulence factors DPP V and catalase 1. Interestingly, the cell wall defects in the mutant were not accompanied by activation of the MpkA-dependent cell wall integrity (CWI) signaling pathway. Instead, nitrate assimilation was activated in the Δpgi mutant, stimulating glutamine synthesis and providing amino donors for chitin precursor biosynthesis. Blocking the nitrate assimilation pathway severely impaired the growth of the Δpgi mutant, highlighting the crucial role of nitrate assimilation in rescuing cell wall defects. This study unveils the connection between nitrogen assimilation and cell wall compensation in A. fumigatus.IMPORTANCEAspergillus fumigatus is a common and serious human fungal pathogen that causes a variety of diseases. Given the limited availability of antifungal drugs and increasing drug resistance, it is imperative to understand the fungus' survival mechanisms for effective control of fungal infections. Our previous study highlighted the essential role of A. fumigatus PGI in maintaining cell wall integrity, phosphate sugar homeostasis, and virulence. The present study further illuminates the involvement of PGI in protein N-glycosylation. Furthermore, this research reveals that the nitrogen assimilation pathway, rather than the canonical MpkA-dependent CWI pathway, compensates for cell wall deficiencies in the mutant. These findings offer valuable insights into a novel adaptation mechanism of A. fumigatus to address cell wall defects, which could hold promise for the treatment of infections.

Microstructural Evolution and Mechanical Properties of a Ni-Based Alloy with High Boron Content for the Pre-Sintered Preform (PSP) Application.

The pre-sintered preform (PSP) is an advanced technology for repairing the Ni-based superalloy blade in a turbine. In general, boron is added to the Ni-based superalloys in small quantities (<0.1 wt.%) to increase boundary strength and cohesivity. Despite this, the effect of high B content (>1.0 wt.%) on the microstructure evolution and mechanical properties in Ni-based superalloys for the PSP application is rarely studied. The variety, composition and evolution of the precipitates during solution heat treatment in the alloy with high B content were determined by EBSD, EPMA and SEM. The results indicate that Cr, W and Mo-rich M5B3 type borides precipitate from the matrix and its area fraction reaches up to about 8%. The area fraction of boride decreases with the prolonging of solution time and the increase of temperature higher than 1120 °C. The borides nearly disappear after solution treatment at 1160 °C for 2 h. The redissolution of boride and eutectic results in the formation of B-rich area with low incipient melting (about 1189 °C). It can bond metallurgically with the blade under the melting point of the blade, which decreases the precipitation of harmful phases of the blade after PSP repairing. The microhardness within the grain in the PSP work-blank first decreases (lower than 1160 °C) and then increases (higher than 1185 °C) with the increase of solution heat treatment temperature due to the dissolving and precipitation of borides. The tensile strength of the combination of PSP work-blank and Mar-M247 matrix at room temperature after solution treatment is related to the area fraction of boride, incipient melting and the cohesion between PSP work-blank and Mar-M247 matrix.

Palladium-Phosphide-Modified Three-Dimensional Phospho-Doped Graphene Materials for Hydrogen Storage.

The development of efficient hydrogen storage materials is crucial for advancing hydrogen-based energy systems. In this study, we prepared a highly innovative palladium-phosphide-modified P-doped graphene hydrogen storage material with a three-dimensional configuration (3D Pd3P0.95/P-rGO) using a hydrothermal method followed by calcination. This 3D network hindering the stacking of graphene sheets provided channels for hydrogen diffusion to improve the hydrogen adsorption kinetics. Importantly, the construction of the three-dimensional palladium-phosphide-modified P-doped graphene hydrogen storage material improved the hydrogen absorption kinetics and mass transfer process. Furthermore, while acknowledging the limitations of primitive graphene as a medium in hydrogen storage, this study addressed the need for improved graphene-based materials and highlighted the significance of our research in exploring three-dimensional configurations. The hydrogen absorption rate of the material increased obviously in the first 2 h compared with two-dimensional sheets of Pd3P/P-rGO. Meanwhile, the corresponding 3D Pd3P0.95/P-rGO-500 sample, which was calcinated at 500 °C, achieved the optimal hydrogen storage capacity of 3.79 wt% at 298 K/4 MPa. According to molecular dynamics, the structure was thermodynamically stable, and the calculated adsorption energy of a single H2 molecule was -0.59 eV/H2, which was in the ideal range of hydrogen ad/desorption. These findings pave the way for the development of efficient hydrogen storage systems and advance the progress of hydrogen-based energy technologies.

Development and Evaluation of Two Double-Antibody Sandwich ELISAs for Detecting Severe Fever with Thrombocytopenia Syndrome Virus Infection.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly emerging tick-borne virus with a fatality rate between 12% and 50%. Currently, effective vaccines or antiviral drugs are not available to treat SFTSV infection, and a diagnostic method for its detecting is urgently needed. Monoclonal (MAb) and polyclonal antibodies (PAbs) against SFTSV were prepared by immunizing animals with the SFTSV nucleocapsid protein (NP). We developed 2 double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) to detect the SFTSV NP, which was captured using the MAbs and PAbs generated. Both methods were applicable for the diagnosis of SFTSV-infected patients, as confirmed by a quantitative polymerase chain reaction. Furthermore, the sensitivity and specificity of the 2 assays for diagnosing severe fever with thrombocytopenia syndrome were 100% and the antibodies did not react with recombinant Dabieshan NP or recombinant dengue virus NS1 subtype 1 and 2 proteins. In addition, 2 standard curves were established for quantitative detection of the NP: the monoclonal antibody-based ELISA (MAb-based ELISA) had a lower limit of detection than the polyclonal-based ELISA. Therefore, the MAb-based ELISA can be employed for convenient and effective detection of SFTSV.

The Effect of Stress Ratios on the Very High Cycle Fatigue Behavior of 9%Cr Turbine Steel at 630 °C.

Effects of the stress ratio on the very high cycle fatigue behaviors of 9%Cr turbine steel have been investigated at 630 °C. The experimental results show that the S-N curve has a continuous downward trend and has no fatigue limit with the increasing in the cycles at 630 °C. Meanwhile, according to the analysis of microstructure, there are two failure modes that were observed at different stress ratios (R = -1 and 0.1), including surface crack failure and internal crack failure, respectively. Besides, the theoretical threshold value of the crack growth is compared with the calculated value of the fracture surface. To decrease the difference between the threshold value of internal crack initiation and the corresponding theoretical value, a new model for the crack growth threshold of the interior-induced fracture at different stress ratios is proposed and discussed.

A novel small RNA contributes to restrain cellular chain length and anti-phagocytic ability in Streptococcus suis 2.

Streptococcus suis serotype 2 (SS2) is an important porcine and human pathogen. Regulatory small non-coding RNAs (sRNAs) play an essential role in diverse physiological processes, although they remain poorly understood in SS2. In this study, we identified eight novel sRNAs through a combination of computational strategies and experimental identification. To explore roles of these novel sRNAs, sRNA34 was preferentially selected to assess phenotypes of the deletion strain in vitro and in vivo. The inactivation of sRNA34 significantly elongated the cellular chain, remarkably increased sensitivity to phagocytosis by RAW264.7, and attenuated virulence in a mouse infection model. Transcriptomic analysis revealed that inactivation of sRNA34 altered expression of multiple genes contributing to cellular chain formation and elongation, indicating a potential mechanism of sRNA34 in maintaining proper bacterial chain length to resist phagocytosis by the host cell. In summary, sRNA34 is a novel sRNA that contributes to cellular chain regulation and the anti-phagocytosis ability of SS2.

Experimental and Numerical Studies on Recrystallization Behavior of Single-Crystal Ni-Base Superalloy.

The recrystallization (RX) behavior of superalloy during standard solution heat treatment (SSHT) varies significantly with deformation temperature. Single-crystal (SX) samples of Ni-base superalloy were compressed to 5% plastic deformation at room temperature (RT) and 980 °C, and the deformed samples were then subjected to SSHT process which consists of 1290 °C/1 h, 1300 °C/2 h, and 1315 °C/4 h, air cooling. RT-deformed samples showed almost no RX grains until the annealing temperature was elevated to 1315 °C, while 980 °C-deformed samples showed a large number of RX grains in the initial stage of SSHT. It is inferred that the strengthening effect of γ' phases and the stacking faults in them increase the driving force of RX for 980 °C-deformed samples. The RX grains nucleate and grow in dendritic arms preferentially when the microstructural inhomogeneity is not completely eliminated by SSHT. A model coupling crystal plasticity finite element method (CPFEM) and cellular automaton (CA) method was proposed to simulate the RX evolution during SSHT. One ({111} <110>) and three ({111} <110>, {100} <110>, {111} <112>) slip modes were assumed to be activated at RT and 980 °C in CPFEM calculations, respectively. The simulation takes the inhomogeneous as-cast dendritic microstructure into consideration. The simulated RX morphology and density conform well to experimental results.

Two novel regulators of N-acetyl-galactosamine utilization pathway and distinct roles in bacterial infections.

Bacterial pathogens can exploit metabolic pathways to facilitate their successful infection cycles, but little is known about roles of d-galactosamine (GalN)/N-acetyl-d-galactosamine (GalNAc) catabolism pathway in bacterial pathogenesis. Here, we report the genomic reconstruction of GalN/GalNAc utilization pathway in Streptococci and the diversified aga regulons. We delineated two new paralogous AgaR regulators for the GalN/GalNAc catabolism pathway. The electrophoretic mobility shift assays experiment demonstrated that AgaR2 (AgaR1) binds the predicted palindromes, and the combined in vivo data from reverse transcription quantitative polymerase chain reaction and RNA-seq suggested that AgaR2 (not AgaR1) can effectively repress the transcription of the target genes. Removal of agaR2 (not agaR1) from Streptococcus suis 05ZYH33 augments significantly the abilities of both adherence to Hep-2 cells and anti-phagocytosis against RAW264.7 macrophage. As anticipated, the dysfunction in AgaR2-mediated regulation of S. suis impairs its pathogenicity in experimental models of both mice and piglets. Our finding discovered two novel regulators specific for GalN/GalNAc catabolism and assigned them distinct roles into bacterial infections. To the best of our knowledge, it might represent a first paradigm that links the GalN/GalNAc catabolism pathway to bacterial pathogenesis.

Development of reverse transcription loop-mediated isothermal amplification assays to detect Hantaan virus and Seoul virus.

We developed two assays based on one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) to identify Hantaan virus (HTNV) and Seoul virus (SEOV), members of the Hantavirus genus that cause hemorrhagic fever with renal syndrome (HFRS). Our results showed that these assays can be conducted within 30min under isothermal conditions. The detection limit for HTNV was around 10 copies per reaction, similar to detection levels for quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays. The detection limit for SEOV was 100 copies per reaction, a sensitivity that was 10-fold lower than that for qRT-PCR assays but 10-fold higher than that for RT-PCR assays. The method we developed was specific for both HTNV and SEOV without any cross-reaction with other pathogens. We conclude that RT-LAMP assays could be useful for the rapid and direct detection of HTNV and SEOV clinically, and for the epidemiological investigation of HFRS.

The ß-galactosidase (BgaC) of the zoonotic pathogen Streptococcus suis is a surface protein without the involvement of bacterial virulence.

Streptococcal pathogens have evolved to express exoglycosidases, one of which is BgaC ß-galactosidase, to deglycosidate host surface glycolconjucates with exposure of the polysaccharide receptor for bacterial adherence. The paradigm BgaC protein is the bgaC product of Streptococcus, a bacterial surface-exposed ß-galactosidase. Here we report the functional definition of the BgaC homologue from an epidemic Chinese strain 05ZYH33 of the zoonotic pathogen Streptococcus suis. Bioinformatics analyses revealed that S. suis BgaC shared the conserved active sites (W240, W243 and Y454). The recombinant BgaC protein of S. suis was purified to homogeneity. Enzymatic assays confirmed its activity of ß-galactosidase. Also, the hydrolysis activity was found to be region-specific and sugar-specific for the Gal ß-1,3-GlcNAc moiety of oligosaccharides. Flow cytometry analyses combined with immune electron microscopy demonstrated that S. suis BgaC is an atypical surface-anchored protein in that it lacks the "LPXTG" motif for typical surface proteins. Integrative evidence from cell lines and mice-based experiments showed that an inactivation of bgaC does not significantly impair the ability of neither adherence nor anti-phagocytosis, and consequently failed to attenuate bacterial virulence, which is somewhat similar to the scenario seen with S. pneumoniae. Therefore we concluded that S. suis BgaC is an atypical surface-exposed protein without the involvement of bacterial virulence.

Rapid and sensitive detection of H7N9 avian influenza virus by use of reverse transcription-loop-mediated isothermal amplification.

An epidemic of human H7N9 influenza virus infection recently emerged in China whose clinical features include high mortality and which has also resulted in serious economic loss. The novel reassortant avian-origin influenza A (H7N9) virus which was the causative agent of this epidemic raised the possibility of triggering a large-scale influenza pandemic worldwide. It seemed likely that fast molecular detection assays specific for this virus would be in great demand. Here, we report a one-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) method for rapid detection of the hemagglutinin (HA) and neuraminidase (NA) genes of H7N9 virus, the minimum detection limit of which was evaluated using in vitro RNA transcription templates. In total, 135 samples from clinical specimens (from either patients or poultry) were tested using this method in comparison with the real-time PCR recommended by the World Health Organization (WHO). Our results showed that (i) RT-LAMP-based trials can be completed in approximately 12 to 23 min and (ii) the detection limit for the H7 gene is around 10 copies per reaction, similar to that of the real-time PCR, whereas the detection limit for its counterpart the N9 gene is 5 copies per reaction, a 100-fold-higher sensitivity than the WHO-recommended method. Indeed, this excellent performance of our method was also validated by the results for a series of clinical specimens. Therefore, we believe that the simple, fast, and sensitive method of RT-LAMP might be widely applied for detection of H7N9 infections and may play a role in prevention of an influenza pandemic.

Rapid visual detection of highly pathogenic Streptococcus suis serotype 2 isolates by use of loop-mediated isothermal amplification.

Streptococcus suis serotype 2 (S. suis 2) is an important zoonotic pathogen that causes considerable economic losses to the pig industry and significantly threatens public health worldwide. The highly pathogenic S. suis 2, which contains the 89K pathogenicity island (PAI), has caused large-scale outbreaks of infections in humans, resulting in high mortality rates. In this study, we established two loop-mediated isothermal amplification (LAMP)-based assays that can rapidly detect S. suis 2 and the 89K PAI and can be performed simultaneously under the same conditions. Further, based on the findings of these two LAMP assays and using the same set of serially diluted DNA samples, we compared the sensitivities of different LAMP product detection methods, including SYBR green detection, gel electrophoresis, turbidimetry, calcein assays, and hydroxynaphthol blue detection. The results suggest that target genes can be amplified and detected within 48 min under 63°C isothermal conditions. The sensitivity of tests for S. suis 2 detection varies between detection methods and reaction systems, indicating that for each LAMP reaction system, multiple detection methods should be performed to select the optimal one. The sensitivities of the optimized methods (7.16 copies/reaction) in the present study were identical to those of the real-time PCR assay, and the test results for reference strains and clinical samples showed that these LAMP systems have high specificities. Thus, since the LAMP systems established in this study are simple, fast, and sensitive, they may have good clinical potential for detecting the highly pathogenic S. suis 2.