Conserved antigen structures and antibody-driven variations on foot-and-mouth disease virus serotype A revealed by bovine neutralizing monoclonal antibodies.

Foot-and-mouth disease virus (FMDV) serotype A is antigenically most variable within serotypes. The structures of conserved and variable antigenic sites were not well resolved. Here, a historical A/AF72 strain from A22 lineage and a latest A/GDMM/2013 strain from G2 genotype of Sea97 lineage were respectively used as bait antigen to screen single B cell antibodies from bovine sequentially vaccinated with A/WH/CHA/09 (G1 genotype of Sea97 lineage), A/GDMM/2013 and A/AF72 antigens. Total of 39 strain-specific and 5 broad neutralizing antibodies (bnAbs) were isolated and characterized. Two conserved antigenic sites were revealed by the Cryo-EM structures of FMDV serotype A with two bnAbs W2 and W125. The contact sites with both VH and VL of W125 were closely around icosahedral threefold axis and covered the B-C, E-F, and H-I loops on VP2 and the B-B knob and H-I loop on VP3; while contact sites with only VH of W2 concentrated on B-B knob, B-C and E-F loops on VP3 scattering around the three-fold axis of viral particle. Additional highly conserved epitopes also involved key residues of VP158, VP1147 and both VP272 / VP1147 as determined respectively by bnAb W153, W145 and W151-resistant mutants. Furthermore, the epitopes recognized by 20 strain-specific neutralization antibodies involved the key residues located on VP3 68 for A/AF72 (11/20) and VP3 175 position for A/GDMM/2013 (9/19), respectively, which revealed antigenic variation between different strains of serotype A. Analysis of antibody-driven variations on capsid of two virus strains showed a relatively stable VP2 and more variable VP3 and VP1. This study provided important information on conserve and variable antigen structures to design broad-spectrum molecular vaccine against FMDV serotype A.

Transcriptome and metabolome analyses reveal the regulatory role of MdPYL9 in drought resistance in apple.

BACKGROUND: The mechanisms by which the apple MdPYL9 gene mediates the response to drought stress remain unclear. Here, transcriptome and metabolome analyses of apple plants under drought were used to investigate the mechanisms by which MdPYL9 regulates the response to drought stress in apple. MdPYL9-overexpressed transgenic and non-transgenic apple histoculture seedlings were rooted, transplanted, and subjected to drought treatments to clarify the mechanisms underlying the responses of apples to drought stress through phenotypic observations, physiological and biochemical index measurements, and transcriptomic and metabolomic analyses. RESULTS: Under drought stress treatment, transgenic plants were less affected by drought stress than non-transgenic plants. Decreases in the net photosynthetic rate, stomatal conductance, and transpiration rate of transgenic apple plants were less pronounced in transgenic plants than in non-transgenic plants, and increases in the intercellular CO2 concentration were less pronounced in transgenic plants than in non-transgenic plants. The relative electrical conductivity and content of malondialdehyde, superoxide anion, and hydrogen peroxide were significantly lower in transgenic plants than in non-transgenic plants, and the chlorophyll content and activities of antioxidant enzymes (superoxide dismutase, peroxidase, and catalase) were significantly higher in transgenic plants than in non-transgenic plants. The number of differentially expressed genes (DEGs) involved in the response to drought stress was lower in transgenic plants than in non-transgenic plants, and the most significant and highly annotated DEGs in the transgenic plants were involved in the flavonoid biosynthesis pathway, and the most significant and highly annotated DEGs in control plants were involved in the phytohormone signal transduction pathway. The number of differentially accumulated metabolites involved in the response to drought stress was lower in transgenic plants than in non-transgenic plants, and up-regulated metabolites were significantly enriched in apigenin-7-O-glucoside in transgenic plants and in abscisic acid in non-transgenic plants. In the flavonoid biosynthetic pathway, the expression of genes encoding chalcone synthase (CHS) and chalcone isomerase (CHI) was more significantly down-regulated in non-transgenic plants than in transgenic plants, and the expression of the gene encoding 4-coumarate-CoA ligase (4CL) was more significantly up-regulated in transgenic plants than in non-transgenic plants, which resulted in the significant up-regulation of apigenin-7-O-glucoside in transgenic plants. CONCLUSIONS: The above results indicated that the over-expression of MdPYL9 increased the drought resistance of plants under drought stress by attenuating the down-regulation of the expression of genes encoding CHS and CHI and enhancing the up-regulated expression of the gene encoding 4CL, which enhanced the content of apigenin-7-O-glucoside.

Integrative physiological, metabolomic, and transcriptomic analysis reveals the drought responses of two apple rootstock cultivars.

BACKGROUND: Drought is considered the main environmental factor restricting apple production and thus the development of the apple industry. Rootstocks play an important role in enhancing the drought tolerance of apple plants. Studies of the physiology have demonstrated that 'ZC9-3' is a strong drought-resistant rootstock, whereas 'Jizhen-2' is a weak drought-resistant rootstock. However, the metabolites in these two apple rootstock varieties that respond to drought stress have not yet been characterized, and the molecular mechanisms underlying their responses to drought stress remain unclear. RESULTS: In this study, the physiological and molecular mechanisms underlying differences in the drought resistance of 'Jizhen-2' (drought-sensitive) and 'ZC9-3' (drought-resistant) apple rootstocks were explored. Under drought stress, the relative water content of the leaves was maintained at higher levels in 'ZC9-3' than in 'Jizhen-2', and the photosynthetic, antioxidant, and osmoregulatory capacities of 'ZC9-3' were stronger than those of 'Jizhen-2'. Metabolome analysis revealed a total of 95 and 156 differentially accumulated metabolites in 'Jizhen-2' and 'ZC9-3' under drought stress, respectively. The up-regulated metabolites in the two cultivars were mainly amino acids and derivatives. Transcriptome analysis revealed that there were more differentially expressed genes and transcription factors in 'ZC9-3' than in 'Jizhen-2' throughout the drought treatment. Metabolomic and transcriptomic analysis revealed that amino acid biosynthesis pathways play key roles in mediating drought resistance in apple rootstocks. A total of 13 metabolites, including L-α-aminoadipate, L-homoserine, L-threonine, L-isoleucine, L-valine, L-leucine, (2S)-2-isopropylmalate, anthranilate, L-tryptophan, L-phenylalanine, L-tyrosine, L-glutamate, and L-proline, play an important role in the difference in drought resistance between 'ZC9-3' and 'Jizhen-2'. In addition, 13 genes encoding O-acetylserine-(thiol)-lyase, S-adenosylmethionine synthetase, ketol-acid isomeroreductase, dihydroxyacid dehydratase, isopropylmalate isomerase, branched-chain aminotransferase, pyruvate kinase, 3-dehydroquinate dehydratase/shikimate 5-dehydrogenase, N-acetylglutamate-5-P-reductase, and pyrroline-5-carboxylate synthetase positively regulate the response of 'ZC9-3' to drought stress. CONCLUSIONS: This study enhances our understanding of the response of apple rootstocks to drought stress at the physiological, metabolic, and transcriptional levels and provides key insights that will aid the cultivation of drought-resistant apple rootstock cultivars. Especially, it identifies key metabolites and genes underlying the drought resistance of apple rootstocks.

Anti-inflammatory and anti-oxidative electrospun nanofiber membrane promotes diabetic wound healing via macrophage modulation.

BACKGROUND: In the inflammatory milieu of diabetic chronic wounds, macrophages undergo substantial metabolic reprogramming and play a pivotal role in orchestrating immune responses. Itaconic acid, primarily synthesized by inflammatory macrophages as a byproduct in the tricarboxylic acid cycle, has recently gained increasing attention as an immunomodulator. This study aims to assess the immunomodulatory capacity of an itaconic acid derivative, 4-Octyl itaconate (OI), which was covalently conjugated to electrospun nanofibers and investigated through in vitro studies and a full-thickness wound model of diabetic mice. RESULTS: OI was feasibly conjugated onto chitosan (CS), which was then grafted to electrospun polycaprolactone/gelatin (PG) nanofibers to obtain P/G-CS-OI membranes. The P/G-CS-OI membrane exhibited good mechanical strength, compliance, and biocompatibility. In addition, the sustained OI release endowed the nanofiber membrane with great antioxidative and anti-inflammatory activities as revealed in in vitro and in vivo studies. Specifically, the P/G-CS-OI membrane activated nuclear factor-erythroid-2-related factor 2 (NRF2) by alkylating Kelch-like ECH-associated protein 1 (KEAP1). This antioxidative response modulates macrophage polarization, leading to mitigated inflammatory responses, enhanced angiogenesis, and recovered re-epithelization, finally contributing to improved healing of mouse diabetic wounds. CONCLUSIONS: The P/G-CS-OI nanofiber membrane shows good capacity in macrophage modulation and might be promising for diabetic chronic wound treatment.

Discovery of potent heat shock protein 90 (Hsp90) inhibitors: structure-based virtual screening, molecular dynamics simulation, and biological evaluation.

Heat shock protein 90 (Hsp90) is considered an attractive therapeutic target for cancer treatment due to its high expression in many cancers. In this study, four potent Hsp90 inhibitors (HPs 1-4) were identified using structure-based virtual screening. Among them, HP-4 exhibited the most potent inhibitory effects (IC50 = 17.64 ± 1.45 nM) against the Hsp90 protein, which was about 7.7 times stronger than that of MPC-3100 (a positive inhibitor targeting Hsp90). In vitro cytotoxicity assay suggested that HP-4 could effectively inhibit the proliferation of a series of tumour cells, including HCT-116, HeLa, A549, A2780, DU145, HepG2 and A498. Furthermore, in vivo assay displayed that HP-4 had significant anti-tumour effects on HCT-116 cell-derived xenograft models. These data demonstrate that HP-4 could be a potential lead compound for the further investigation of anti-tumour drugs.

Structures of Foot-and-Mouth Disease Virus with Bovine Neutralizing Antibodies Reveal the Determinant of Intraserotype Cross-Neutralization.

Foot-and-mouth disease virus (FMDV) exhibits broad antigenic diversity with poor intraserotype cross-neutralizing activity. Studies of the determinant involved in this diversity are essential for the development of broadly protective vaccines. In this work, we isolated a bovine antibody, designated R55, that displays cross-reaction with both FMDV A/AF/72 (hereafter named FMDV-AAF) and FMDV A/WH/09 (hereafter named FMDV-AWH) but only has a neutralizing effect on FMDV-AWH. Near-atomic resolution structures of FMDV-AAF-R55 and FMDV-AWH-R55 show that R55 engages the capsids of both FMDV-AAF and FMDV-AWH near the icosahedral 3-fold axis and binds to the ßB and BC/HI-loops of VP2 and to the B-B knob of VP3. The common interaction residues are highly conserved, which is the major determinant for cross-reaction with both FMDV-AAF and FMDV-AWH. In addition, the cryo-EM structure of the FMDV-AWH-R55 complex also shows that R55 binds to VP3E70 located at the VP3 BC-loop in an adjacent pentamer, which enhances the acid and thermal stabilities of the viral capsid. This may prevent capsid dissociation and genome release into host cells, eventually leading to neutralization of the viral infection. In contrast, R55 binds only to the FMDV-AAF capsid within one pentamer due to the VP3E70G variation, which neither enhances capsid stability nor neutralizes FMDV-AAF infection. The VP3E70G mutation is the major determinant involved in the neutralizing differences between FMDV-AWH and FMDV-AAF. The crucial amino acid VP3E70 is a key component of the neutralizing epitopes, which may aid in the development of broadly protective vaccines. IMPORTANCE Foot-and-mouth disease virus (FMDV) causes a highly contagious and economically devastating disease in cloven-hoofed animals, and neutralizing antibodies play critical roles in the defense against viral infections. Here, we isolated a bovine antibody (R55) using the single B cell antibody isolation technique. Enzyme-linked immunosorbent assays (ELISA) and virus neutralization tests (VNT) showed that R55 displays cross-reactions with both FMDV-AWH and FMDV-AAF but only has a neutralizing effect on FMDV-AWH. Cryo-EM structures, fluorescence-based thermal stability assays and acid stability assays showed that R55 engages the capsid of FMDV-AWH near the icosahedral 3-fold axis and informs an interpentamer epitope, which overstabilizes virions to hinder capsid dissociation to release the genome, eventually leading to neutralization of viral infection. The crucial amino acid VP3E70 forms a key component of neutralizing epitopes, and the determination of the VP3E70G mutation involved in the neutralizing differences between FMDV-AWH and FMDV-AAF could aid in the development of broadly protective vaccines.

Fibrillarin RNA methylase is an interacting protein of Cryptosporidium parvum calmodulin-like protein (CpCML).

Cryptosporidium parvum is an obligate protozoan parasite invading epithelial cells of small intestine of human and animals, and causing diarrheal disease. In apicomplexan parasites, calcium signaling can regulate many essential biological processes such as invasion and migration. As the main intracellular receptor for calcium ions, calmodulins control the activities of hundreds of enzymes and proteins. Calmodulin-like protein (CML) is an important member of the calmodulin family and may play a key role in C. parvum, however, the actual situation is still not clear. The present study aimed to identify the parasite interaction partner proteins of C. parvum calmodulin-like protein (CpCML). By constructing the cpcml bait plasmid, 5 potential CpCML - interacting proteins in C. parvum oocyst were screened by yeast-two-hybrid system (Y2H). Bimolecular fluorescence complementation (BiFC) and Co-immunoprecipitation (Co-IP) were performed as subsequent validations. Fibrillarin RNA methylase (FBL) was identified via this screening method as CpCML interacting protein in C. parvum. The identification of this interaction made it possible to get a further understanding of the function of CpCML and its contribution to the pathogenicity of C. parvum.

Curcumin mitigates Cryptosporidium parvum infection through modulation of gut microbiota and innate immune-related genes in immunosuppressed neonatal mice.

Cryptosporidium parvum is a major cause of diarrheal disease in immature or weakened immune systems, mainly in infants and young children in resource-poor settings. Despite its high prevalence, fully effective and safe drugs for the treatment of C. parvum infections remain scarce, and there is no vaccine. Meanwhile, curcumin has shown protective effects against C. parvum infections. However, the mechanisms of action and relationship to the gut microbiota and innate immune responses are unclear. Immunosuppressed neonatal mice were infected with oocysts of C. parvum and either untreated or treated with a normal diet, curcumin or paromomycin. We found that curcumin stopped C. parvum oocysts shedding in the feces of infected immunosuppressed neonatal mice, prevented epithelial damage, and villi degeneration, as well as prevented recurrence of infection. Curcumin supplementation increased the relative abundance of Bacteroidetes and decreased the relative abundance of Firmicutes and Proteobacteria in mice infected with C. parvum as shown by 16S rRNA gene sequencing analysis. The relative abundance of Lactobacillus, Bacteroides, Akkermansia, Desulfovibrio, Prevotella, and Helicobacter was significantly associated with C. parvum infection inhibited by curcumin. Curcumin significantly (P < 0.01) suppressed IFN-γ and IL -18 gene expression levels in immunosuppressed neonatal C. parvum-infected mice. We demonstrate that the therapeutic effects curcumin are associated with alterations in the gut microbiota and innate immune-related genes, which may be linked to the anti-Cryptosporidium mechanisms of curcumin.

Hierarchical Self-Assembly of Nanowires on the Surface by Metallo-Supramolecular Truncated Cuboctahedra.

Parastichy, the spiral arrangement of plant organs, is an example of the long-range apparent order seen in biological systems. These ordered arrangements provide scientists with both an aesthetic challenge and a mathematical inspiration. Synthetic efforts to replicate the regularity of parastichy may allow for molecular-scale control over particle arrangement processes. Here we report the packing of a supramolecular truncated cuboctahedron (TCO) into double-helical (DH) nanowires on a graphite surface with a non-natural parastichy pattern ascribed to the symmetry of the TCOs and interactions between TCOs. Such a study is expected to advance our understanding of the design inputs needed to create complex, but precisely controlled, hierarchical materials. It is also one of the few reported helical packing structures based on Platonic or Archimedean solids since the discovery of the Boerdijk-Coxeter helix. As such, it may provide experimental support for studies of packing theory at the molecular level.

Isolation and characterization of porcine monoclonal antibodies revealed two distinct serotype-independent epitopes on VP2 of foot-and-mouth disease virus.

Pigs are susceptible to foot-and-mouth disease virus (FMDV), and the humoral immune response plays an essential role in protection against FMDV infection. However, little information is available about FMDV-specific mAbs derived from single B cells of pigs. This study aimed to determine the antigenic features of FMDV that are recognized by antibodies from pigs. Therefore, a panel of pig-derived mAbs against FMDV were developed using fluorescence-based single B cell antibody technology. Western blotting revealed that three of the antibodies (1C6, P2-7E and P2-8G) recognized conserved antigen epitopes on capsid protein VP2, and exhibited broad reactivity against both FMDV serotypes A and O. An alanine-substitution scanning assay and sequence conservation analysis elucidated that these porcine mAbs recognized two conserved epitopes on VP2: a linear epitope (2KKTEETTLL10) in the N terminus and a conformational epitope involving residues K63, H65, L66, F67, D68 and L81 on two ß-sheets (B-sheet and C-sheet) that depended on the integrity of VP2. Random parings of heavy and light chains of the IgGs confirmed that the heavy chain is predominantly involved in binding to antigen. The light chain of porcine IgG contributes to the binding affinity toward an antigen and may function as a support platform for antibody stability. In summary, this study is the first to reveal the conserved antigenic profile of FMDV recognized by porcine B cells and provides a novel method for analysing the antibody response against FMDV in its natural hosts (i.e. pigs) at the clonal level.

The novel function of miR-3195 for mutant PROK2 (c.223-4C>A) degradation.

Kallmann syndrome (KS) is a rare human genetic disorder characterized by hypogonadotropic hypogonadism with the reduction or absence of olfactory sense. Mutations in multiple genes, including chemokine prokineticin-2 (PROK2), are considered to contribute to the abnormal migration of gonadotropin-releasing hormone neurons in the embryonic stage. However, the mechanisms of the different inheritance modes of KS have not been comprehensively determined. In this article, we present the case of one KS patient with the same mutation in PROK2 (c.223-4C>A) as his mother. RNA sequencing analysis of his leukocytes showed a new transcript of PROK2, which contained a partial intron (192 bp) compared to those of his parents. Furthermore, we observed that hsa-miR-3195 was expressed at low levels in his and his father's sera compared to his mother's. Unexpectedly, hsa-miR-3195 was also identified to specifically target the 192 bp intron of the aberrant PROK2 transcript of this patient. We determined that high expression of hsa-miR-3195 could efficiently target aberrant PROK2 and stabilize the normal function of PROK2 in vitro, which provided a probable explanation for the different phenotypes of the patient and his mother with the same genotype.

Mechanistic Investigation of a Self-Assembling Peptide against Escherichia coli.

Because of their distinctive mode of action in targeting bacterial cell membranes, antimicrobial peptides (AMPs) are increasingly regarded as a potential candidate for the development of novel antibiotics to combat the wide spread of bacterial resistance. To date, understanding of the exact molecular process by which AMPs act on the real bacterial envelope remains challenging. Simultaneously, the aggregated state of AMPs upon interaction with bacterial envelopes is still elusive. Previously, we have demonstrated that the potent antibacterial activity of a designed surfactant-like peptide Ac-A9K-NH2 benefited greatly from its high self-assembling ability and appropriate self-assembled morphologies and sizes. By using high-resolution atomic force microscopy, we here not only follow the variations of the Escherichia coli cell envelope in the presence of Ac-A9K-NH2 but also characterize the peptide aggregates on the bacterial surface as well as on the substrate surface. The results, together with those from fluorescence, zeta potential, circular dichroism, and scanning electron microscopy measurements, indicate that both the positively charged peptide monomers and self-assembled nanostructures can directly act on the negatively charged bacterial surface, followed by their insertion into the bacterial membrane, the formation of surface nanopores, and membrane lysis. The mechanism of Ac-A9K-NH2 against E. coli is thus consistent with the detergent-like mode of action. This work enhances our mechanistic understanding of the antibacterial behaviors of self-assembling peptides that will be valuable in exploring their biomedical applications.

Implication of Broadly Neutralizing Bovine Monoclonal Antibodies in the Development of an Enzyme-Linked Immunosorbent Assay for Detecting Neutralizing Antibodies against Foot-and-Mouth Disease Virus Serotype O.

Vaccination with inactivated vaccines is still the main measure to control foot-and-mouth disease (FMD) in areas where the disease is endemic, and the level of neutralizing antibody in vaccinated animals is directly related to their protection against virus challenge. Currently, neutralizing antibody is mainly detected using the virus neutralization test (VNT) based on cell culture, which is laborious and time-consuming and requires restrictive biocontainment facilities. In this study, two broadly neutralizing antibodies (bnAbs), E46 and F128, were successfully produced using techniques for the isolation of single B cells from peripheral blood mononuclear cells (PBMCs) from bovines sequentially immunized with three topotypes of foot-and-mouth disease virus (FMDV) serotype O. Based on these bnAbs, a blocking enzyme-linked immunosorbent assay (ELISA) for detecting neutralizing antibodies (NA-ELISA) against FMDV serotype O was developed. The specificity and sensitivity of the test were estimated to be 99.21% and 100%, respectively. A significant correlation (P < 0.01) was observed between the NA-ELISA titers and the VNT titers for all sera from vaccinated animals and for all tested strains, suggesting that the NA-ELISA could detect neutralizing antibodies against FMDV serotype O strains of wide antigenic and molecular diversity and could be used for the evaluation of protective immunity.

Functional analysis of the UL24 protein of suid herpesvirus 1.

The UL24 homologous genes are conserved in alphaherpesviruses. However, the proximity of the UL24 gene and the UL23 gene encoding for thymidine kinase (TK) in the genome of suid herpesvirus 1 (SuHV-1) makes it difficult to mutate UL24 without affecting the expression of the TK gene, and thus functional studies of the UL24 gene have lagged behind. In this study, CRISPR/Cas9 and homologous recombination were adopted to generate UL24 and TK mutant viruses. Deletion of either the UL24 or the TK gene resulted in significantly reduced SuHV-1 replication and spread capacity in Vero cells. However, UL24-deleted virus still maintained a certain degree of lethality in mice, while TK-deleted viruses completely lost their lethality in mice. Similarly, neurovirulence of UL24-deleted virus in mice was not significantly affected compared to parental virus. In comparison, infection with the TK-deleted viruses resulted in significantly reduced neurovirulence and complete loss of lethality. In addition, and for the first time, viral UL24 protein was found to be expressed late during SuHV-1 infection; enhanced green fluorescence protein (eGFP) labeled UL24 protein was shown to be localized in the nucleus via heterologous expression. In conclusion, the UL24 gene of SuHV-1 encodes a nuclear-localized viral protein and acts as a minor virulence-associated factor compared to the TK gene.

Proteome and Transcriptome Reveal Involvement of Heat Shock Proteins and Indoleacetic Acid Metabolism Process in Lentinula Edodes Thermotolerance.

BACKGROUND/AIMS: Heat stress could cause huge losses for Lentinula edodes in China and other Asian cultivation areas. Yet our understanding of mechanism how to defend to heat stress is incomplete. METHODS: Using heat-tolerant and heat-sensitive strains of L. edodes, we reported a combined proteome and transcriptome analysis of L. edodes response to 40 °C heat stress for 24 h. Meanwhile, the effect of LeDnaJ on the thermotolerance and IAA (indoleacetic acid) biosynthesis in L. edodes was analyzed via the over-expression method. RESULTS: The proteome results revealed that HSPs (heat shock proteins) such as Hsp40 (DnaJ), Hsp70, Hsp90 and key enzymes involved in tryptophan and IAA metabolism process LeTrpE, LeTrpD, LeTam-1, LeYUCCA were more highly expressed in S606 than in YS3357, demonstrating that HSPs and tryptophan as well as IAA metabolism pathway should play an important role in thermotolerance. Over-expression of LeDnaJ gene in S606 strains showed better tolerance to heat stress. It was also documented that intracellular IAA accumulation of S606 (8-fold up) was more than YS3357 (2-fold up), and exogenous IAA enhanced L. edodes tolerance to heat stress. CONCLUSION: Our data support the interest of LeTrpE, LeDnaJ, tryptophan and IAA could play a pivotal role in enhancing organism thermotolerance.

Temperature Difference Triggering Controlled Growth of All-Inorganic Perovskite Nanowire Arrays in Air.

All-inorganic perovskites have attracted increasing worldwide interest due to its significantly improved stability in atmospheric environment compared to organic-inorganic hybrid perovskites, which renders it infinitely applicable in many fields such as electronics, optoelectronics, and energy storage. However, all-inorganic perovskites have to confront the challenges from fabrication before their wide utilization in the aforementioned applications. Liquid-phase synthesis holds the advantage of mass production and easy modulation of composition but with the deficiencies of relatively low crystallinity and disordered products. Interestingly, gas-phase growth has complementary characteristics compared to the liquid-phase method. In this work, it is proposed that a novel temperature difference triggers growth strategy to integrate the merits of the liquid- and gas-phase methods, and the feasibility of this strategy via a simple lab-use hot plate is demonstrated. High quality all-inorganic perovskites, cesium lead halide (CsPbX3 ) nanowire arrays, can be epitaxially grown as in a gas-phase method, but at the same time, the composition of products can be easily modulated by predesigning the recipe of precursors as in the liquid-phase method on a large scale. Notably, the as-fabricated CsPbX3 perovskite nanowire arrays demonstrate excellent stability and good optoelectronic properties in air. It is believed that this novel strategy can strikingly prompt the development of perovskites fabrication and applications in future.

The heat shock protein 40 LeDnaJ regulates stress resistance and indole-3-acetic acid biosynthesis in Lentinula edodes.

DnaJ proteins, termed heat shock proteins based on their molecular weight, function as molecular chaperones that play critical roles in regulating organism growth and development as well as adaptation to the environment. However, little has been reported on their gene function in higher basidiomycetes. Here, the heat shock protein 40 (LeDnaJ) gene was cloned and characterized from Lentinula edodes. RNA interference was used to explore the function of LeDnaJ in response to heat stress and Trichoderma atroviride. Integration of the target gene into the L. edodes genome was confirmed by Southern blot analysis, and the silence efficiency of LeDnaJ was analyzed by qRT-PCR. The results revealed that LeDnaJ silence caused defects in mycelial growth and resistance to heat stress and T. atroviride, but increased the mycelial density compared with the wild type (WT) strain S606. Additionally, the IAA content showed a more than 10-fold increase in the WT after heat stress, but an about two-fold increase in the two LeDnaJ RNAi transfortants (LeDnaJ-i-6 and LeDnaJ-i-8). Previous study has shown that enhanced IAA (indole-3-acetic acid) content enhanced the thermotolerance of the heat-sensitive strain YS3357. In this study, it was documented that IAA amendments could partly restore the resistance to T. atroviride and thermotolerance of the two LeDnaJ RNAi transformants. Overall, LeDnaJ is nvolved in fungal growth, T. atroviride resistance, and thermotolerance by regulating the IAA biosynthesis in L. edodes.

Single-base methylome analysis reveals dynamic epigenomic differences associated with water deficit in apple.

Cytosine methylation is an essential feature of epigenetic regulation and is involved in various biological processes. Although cytosine methylation has been analysed at the genomic scale for several plant species, there is a general lack of understanding of the dynamics of global and genic DNA methylation in plants growing in environments challenged with biotic and abiotic stresses. In this study, we mapped cytosine methylation at single-base resolution in the genome of commercial apple (Malus x domestica), and analysed changes in methylation patterns associated with water deficit in representative drought-sensitive and drought-tolerant cultivars. We found that the apple genome exhibits ~54%, ~38% and ~8.5% methylation at CG, CHG and CHH sequence contexts, respectively. We additionally documented changes in gene expression associated with water deficit in an attempt to link methylation and gene expression changes. Global methylation and transcription analysis revealed that promoter-unmethylated genes showed higher expression levels than promoter-methylated genes. Gene body methylation appears to be positively correlated with gene expression. Water deficit stress was associated with changes in methylation at a multitude of genes, including those encoding transcription factors (TFs) and transposable elements (TEs). These results present a methylome map of the apple genome and reveal widespread DNA methylation alterations in response to water deficit stress. These data will be helpful for understanding potential linkages between DNA methylation and gene expression in plants growing in natural environments and challenged with abiotic and biotic stresses.

Generation and characterization of UL41 null pseudorabies virus variant in vitro and in vivo.

BACKGROUND: The alphaherpesvirus virion host shutoff (vhs) gene, UL41, can induce degradation of host mRNAs and shut off host protein synthesis. The roles of vhs in HSV-1 and HSV-2 have been studied extensively in previous studies, however, relatively little is known about the vhs protein of PRV. METHODS: A novel method combining CRISPR/Cas9 and Gibson assembly was developed to generate UL41 null PRV variant. The properties of UL41 null PRV in vitro and in vivo were further characterized. And the vhs activity of UL41 protein of PRV variant was evaluated by luciferase assay, Western-blot and RT-qPCR. RESULTS: Gibson assembly based on homologous recombination can accomplish one-step insertion of viral DNA fragments into donor plasmids efficiently (> 80%). Cas9/gRNA further largely enhanced the efficiency of homologous recombination. Using this method we were able to rapidly generate the UL41 null and revertant viruses of PRV variant. Compared to wild type (JS-2012), the UL41 null virus showed significantly smaller plaques and lower titers in Vero cells and impaired lethality and neuroinvasion in mice. Further the UL41 protein from different PRV strains exhibited unequal vhs activity in vitro, which of JS-2012 showed significantly weaker vhs activity than that of European-American strains. In addition UL41 null virus can also significantly decrease the expression of host genes during the early period of infection, which suggests other viral factors may be also involved in host shutoff. CONCLUSIONS: CRISPR/Cas9 combined with Gibson assembly efficiently generated UL41 null PRV. Compared to wild type, UL41 null PRV showed impaired both replication capability in vitro and neuroinvasion in vivo. Further UL41 protein of PRV variant showed significantly weaker vhs activity than that of PRV SC (European-American-like strain), suggesting the deficiency of vhs activity by the PRV variant UL41 protein.

Temperature dependence of Raman responses of few-layer PtS2.

Platinum disulfide (PtS2) is a newly emerging 2D material, which possesses relatively high carrier mobility, a widely tunable band gap from 0.25 to 1.6 eV, and ultra-high air stability, showing a potential in electronics and optoelectronics. Here, for the first time, we study the temperature-dependent Raman spectra on PtS2 with different thicknesses. It was found that with the temperature increase from 80 to 298 K, the [Formula: see text] and [Formula: see text] modes of all samples show linear softening. Moreover, the linear softening with temperature of PtS2 is much smaller than other 2D transition metal dichalcogenides, which could be attributed to the stronger interlayer coupling in PtS2. Our work gives fundamental temperature-dependent vibrational information of PtS2, which will be useful in future PtS2-based electronic devices.