Interfacial Reduction Nucleation of Noble Metal Nanodots on Redox-Active Metal-Organic Frameworks for High-Efficiency Electrocatalytic Conversion of Nitrate to Ammonia.

Electrochemically converting nitrate to ammonia is a promising route to realize artificial nitrogen recycling. However, developing highly efficient electrocatalysts is an ongoing challenge. Herein, we report the construction of stable and redox-active zirconium metal-organic frameworks (Zr-MOFs) based on Zr6 nanoclusters and redox-reversible tetrathiafulvalene (TTF) derivatives as inorganic nodes and organic linkers, respectively. The redox-active Zr-MOF can facilitate the in situ reduction of noble metal precursors free of external reductants and realize the uniform nucleation of noble metal nanodots (NDs) on Zr-MOF, achieving the preparation of M-NDs/Zr-MOF (M = Pd, Ag, or Au). The highly porous Zr-MOF with good conductivity can facilitate the mass transfer process. Among the M-NDs/Zr-MOF catalysts, Pd-NDs/Zr-MOF exhibits the highest electrocatalytic activity, delivering a NH3 yield of 287.31 mmol·h-1·g-1cat. and a Faradaic efficiency of 58.1%. The proposed interfacial reduction nucleation strategy for anchoring M NDs on Zr-MOFs can be applied to other challenging energy conversion reactions.

Importin ß1 Mediates Nuclear Entry of EIN2C to Confer the Phloem-Based Defense against Aphids.

Ethylene Insensitive 2 (EIN2) is an integral membrane protein that regulates ethylene signaling towards plant development and immunity by release of its carboxy-terminal functional portion (EIN2C) into the nucleus. The present study elucidates that the nuclear trafficking of EIN2C is induced by importin ß1, which triggers the phloem-based defense (PBD) against aphid infestations in Arabidopsis. In plants, IMPß1 interacts with EIN2C to facilitate EIN2C trafficking into the nucleus, either by ethylene treatment or by green peach aphid infestation, to confer EIN2-dependent PBD responses, which, in turn, impede the phloem-feeding activity and massive infestation by the aphid. In Arabidopsis, moreover, constitutively expressed EIN2C can complement the impß1 mutant regarding EIN2C localization to the plant nucleus and the subsequent PBD development in the concomitant presence of IMPß1 and ethylene. As a result, the phloem-feeding activity and massive infestation by green peach aphid were highly inhibited, indicating the potential value of EIN2C in protecting plants from insect attacks.

Genome-wide DNA methylation profiles and small noncoding RNA signatures in sperm with a high DNA fragmentation index.

BACKGROUND: A growing number of studies have reported that sperm DNA fragmentation (SDF) is associated with male infertility. However, no studies have compared genome-wide DNA methylation profiles and sncRNA signatures between sperm with high and low sperm DNA fragmentation indices (DFIs). METHODS: Whole-genome bisulfite sequencing (WGBS) was performed on sperm samples from a weak group (DFI ≥ 30%, n = 6) and normal group (DFI ≤ 15%, n = 7). Small noncoding RNA (sncRNA) deep sequencing was conducted for sperm samples from the weak (DFI ≥ 30%, n = 13) and normal (DFI ≤ 15%, n = 17) groups. RESULTS: A total of 4939 differentially methylated regions (DMRs) were identified in the weak group sperm samples relative to normal group sperm samples, with 2072 (41.95%) of them located in promoter regions. The percentages of hypermethylated DMRs were higher than those of hypomethylated DMRs in all seven examined gene annotation groups. Hypermethylated DMRs were significantly enriched in terms associated with neurons and microtubules. Compared with the normal group, the global DNA methylation level of the weak group sperm showed a downward trend, with lower correlation for methylation in the weak group sperm; therefore, the chromosomes of high-DFI sperm may be loose. On average, 40.5% of sncRNAs were annotated as rsRNAs, 19.3% as tsRNAs, 10.4% as yRNAs, and 7.1% as miRNAs. A total of 27 miRNAs, 151 tsRNAs, and 70 rsRNAs were differentially expressed between the two groups of sperm samples. Finally, 7 sncRNAs were identified as candidate sperm quality biomarkers, and the target genes of the differentially expressed miRNAs are involved in nervous system development. CONCLUSION: Our findings suggest that genome-wide DNA methylation profiles and sncRNA signatures are significantly altered in high-DFI sperm. Our study provides potential biomarkers for sperm quality.

A fungal transcription factor essential for starch degradation affects integration of carbon and nitrogen metabolism.

In Neurospora crassa, the transcription factor COL-26 functions as a regulator of glucose signaling and metabolism. Its loss leads to resistance to carbon catabolite repression. Here, we report that COL-26 is necessary for the expression of amylolytic genes in N. crassa and is required for the utilization of maltose and starch. Additionally, the Δcol-26 mutant shows growth defects on preferred carbon sources, such as glucose, an effect that was alleviated if glutamine replaced ammonium as the primary nitrogen source. This rescue did not occur when maltose was used as a sole carbon source. Transcriptome and metabolic analyses of the Δcol-26 mutant relative to its wild type parental strain revealed that amino acid and nitrogen metabolism, the TCA cycle and GABA shunt were adversely affected. Phylogenetic analysis showed a single col-26 homolog in Sordariales, Ophilostomatales, and the Magnaporthales, but an expanded number of col-26 homologs in other filamentous fungal species. Deletion of the closest homolog of col-26 in Trichoderma reesei, bglR, resulted in a mutant with similar preferred carbon source growth deficiency, and which was alleviated if glutamine was the sole nitrogen source, suggesting conservation of COL-26 and BglR function. Our finding provides novel insight into the role of COL-26 for utilization of starch and in integrating carbon and nitrogen metabolism for balanced metabolic activities for optimal carbon and nitrogen distribution.

Neuroprotective effect of salidroside against central nervous system inflammation-induced cognitive deficits: A pivotal role of sirtuin 1-dependent Nrf-2/HO-1/NF-κB pathway.

Central nervous system (CNS) inflammation occurs in cognitive dysfunctions, but the underlying mechanisms remain unclear. Here, we investigated the role of sirtuin 1 (SIRT1) and salidroside in CNS inflammation-induced cognitive deficits model. In vivo, CNS inflammation was initiated by a single intracerebroventricular injection of lipopolysaccharide (LPS). The levels of inflammatory cytokines and the capability of free radial scavenging were determined after the LPS challenge. In vivo, salidroside and nicotinamide, a SIRT1 inhibitor, were used in PC12 cell. Of note, with the treatment of salidroside, LPS-induced learning and memory impairments were effectively improved. Salidroside also remarkably inhibited the inflammatory cytokines, up-regulated the concentration of superoxide dismutase and inhibited the vitalities of malondialdehyde in serum, hippocampus, and cell supernatant. Besides, the expression of Sirt1, Nrf-2, HO-1, Bax, Bcl-2, caspase-9, and caspase-3 and the phosphorylation of AMPK, NF-κBp65, and IκBα were increased accompanying with the LPS-induced cognitive impairments, which were significantly suppressed by salidroside treatment. In PC12 cell model, nicotinamide significantly abrogated the beneficial effects of salidroside, as indicated by the antioxidant, anti-inflammatory, and antiapoptosis signaling. Together, our results showed that salidroside may be a novel therapy drug in neurodegenerative diseases, and the protective effect was involved in SIRT1-dependent Nrf-2/HO-1/NF-κB pathway.

Study on the relation between tissues pathologies and traditional chinese medicine syndromes in knee osteoarthritis: Medical image diagnostics by preoperative X-ray and surgical arthroscopy.

OBJECTIVE: This study aims to investigate whether integration of traditional Chinese medicine and modern medicine has advantage in achieving the improved diagnosis and treatment of knee osteoarthritis. METHODS: 90 patients with knee osteoarthritis were selected from The Department of Minimal Invasive Joint of The Third Affiliated Hospital of Beijing University of Chinese Medicine from June 2013 to June 2015. They were divided into 3 groups with 30 cases per group in accordance to the syndrome differentiation of traditional Chinese medicine. The patients underwent arthroscopic surgery, and we categorized the patients having the same characterization in each group, and those having distinct difference into the three groups. Based on the arthroscopic analysis, we performed analysis of statistical data in order to analyze the relation between knee osteoarthritis under arthroscope and traditional Chinese medicine syndromes. RESULTS: There are three syndromes according to traditional Chinese medicine that can be categorized into various different groups. The synovial proliferation can be seen mostly in the syndrome of stagnation of blood stasis. The slight damage of knee joint cartilage can be seen in the syndrome of yang deficiency and cold stagnation, the severe one in the syndrome of kidney-marrow deficiency. We found that there are different pathological expressions with the various degree of the tissues damage at the knee and we categorized the knee according to their syndrome. CONCLUSION: For knee osteoarthritis, different syndromes of traditional Chinese medicine presents different tissues pathological changes at the knee joint under arthroscopy, which will provide objective basis for the diagnosis of this medical condition.

SOX7 interferes with ß-catenin activity to promote neuronal apoptosis.

SOX7 mediates various developmental processes. However, its role in neuronal apoptosis remains unclear. In the present study, we investigated the expression pattern and role of SOX7 in potassium deprivation-induced rat cerebellar granule neuron apoptosis. Our results showed that both mRNA and protein levels of SOX7 were upregulated when potassium was deprived. SOX7 overexpression promoted neuronal apoptosis, whereas knockdown of SOX7 protected neurons against apoptosis. Moreover, we found that ß-catenin activity was suppressed during apoptosis and that ß-catenin inhibition was crucial for potassium deprivation-induced neuronal apoptosis. This suppression was mediated by an interaction between SOX7 and ß-catenin but not by protein degradation. Lastly, we showed that ß-catenin inhibition mediated the pro-apoptotic effect of SOX7. Together, our findings demonstrated that SOX7 interfered with ß-catenin activity to promote neuronal apoptosis, which acted as a novel signaling mechanism in neuronal cell death.

Characterization and homologous overexpression of an N-acetylglucosaminidase Nag1 from Trichoderma reesei.

Trichoderma reesei is thought to be a promising recombinant host for the production and secretion of complex proteins due to its ability to secrete large amounts of proteins. In this study we identified a functional N-acetyl-ß-glucosaminidase (NAGase) gene Nag1 in T. reesei. Nag1, a putative gene encoding a GH 20 family NAGase in T. reesei, was cloned and homologous overexpressed in the T. reesei RutC30ΔU3 with a strong cellobiohydrolase1 gene (cbh1) promoter. Nag1 was secreted in its active form and the highest expression level was around 499.85IU/ml. Nag1 has a molecular mass of 80kDa. The optimum pH and temperature were 4.0 and 60°C, respectively.

A novel major facilitator transporter TrSTR1 is essential for pentose utilization and involved in xylanase induction in Trichoderma reesei.

Trichoderma reesei (teleomorph Hypocrea jecorina) is an industrially important filamentous fungus for glycoside hydrolases production, with its xylanolytic enzymes widely applied in many areas. However, the molecular mechanisms underlying xylanase expression are still insufficiently understood. In particular, the effect of sugar transporter on the induction of xylanase expression is unclear. In this work, we identified a novel major facilitator transporter TrSTR1 that is capable of transporting xylose by using a xylose utilization system in Saccharomyces cerevisiae. In T. reesei, TrSTR1 is essential for the utilization of d-xylose, l-arabinose, and even their downstream metabolites D-xylitol and L-arabitol. TrSTR1 is also involved in the induction of xylanase expression since both the xylanase activity and extracellular protein concentration in the Tu6△str1 strain were decreased, which further confirmed by a qRT-PCR analysis of the transcript levels of the key transcriptional regulators. Our observations provide new insights into connections between pentose utilization and xylanase production in T. reesei.

An Ime2-like mitogen-activated protein kinase is involved in cellulase expression in the filamentous fungus Trichoderma reesei.

OBJECTIVES: Eukaryotic mitogen-activated protein kinases (MAPKs) play crucial roles in transducing environmental and developmental signals inside the cell and regulating gene expression, however, the roles of MAPKs remain largely unknown in Trichoderma reesei. RESULTS: T. reesei ime2 (TrIme2) encodes an Ime2-like MAPK in T. reesei. The deletion of the TrIme2 gene led to 90% increase in cellulase activity against filter paper during earlier period time of cellulase induction as well as the extracellular protein production. Compared to the parent strain, the transcriptional levels of the three major cellulase genes cbh1,cbh2, egl1 were increased by about 9 times, 4 times, 2 times, respectively, at 8 h after cellulase induction in the ΔTrIme2 mutant. In addition, the disruption of TrIme2 caused over 50% reduction of the transcript levels of cellulase transcriptional regulators cre1 and xyr1. CONCLUSION: TrIme2 functions in regulation of the expression of cellulase gene in T.reesei, and is a good candidate for genetically engineering of T. reesei for higher cellulase production.

[Treatment of children with EB virus infection by Chinese medicine: a clinical study].

OBJECTIVE: To provide evidence for Chinese medical treatment of children with EB virus infection by exploring its clinical efficacy from multiple angles. METHODS: Totally 81 children patients were randomly assigned to the treatment group (46 cases) and the control group (35 cases). Patients in the treatment group took Chinese medical decoction, while those in the control received intravenous dripping of Ganciclovir and oral administration of pidotimod. The treatment period for the two groups was 2 weeks. Patients were followed-up till the 12th week. Clinical symptoms such as fever, lymphadenopathy and hepatosplenomegaly, as well as lab indices such as abnormal lymphocyte percentage, EB virus antibody, virus DNA load, T cell subsets, immunoglobulin, and so on were observed before and after treatment, at week 4 and 12 of follow-ups. RESULTS: (1) The total effective rate at week 2 was 95.6% in the treatment group, higher than that of the control group (94.3%), but there was no statistical difference between the two groups. (2) The time for defervescence, duration of pharyngeal hyperemia, duration of swollen tonsils was shorter in the treatment group than in the control group (P<0.05). The subsidence of lymphadenopathy, hepatomegaly, and abnormal lymphocytes was better in the treatment group than in the control group (P < 0.05). (3) The positive cases of peripheral blood hetero-lymphocyte was significantly reduced after treatment, at week 4 and 12 of follow-ups both in the treatment group and the control group (P < 0.01). The expression of IgA and IgM decreased after treatment in the two groups when compared with before treatment in the same group (P < 0.05, P < 0.01). IgG in the treatment group also obviously decreased after treatment, at week 4 and 12 of follow-ups (P < 0.05, P < 0.01), while it decreased only after treatment in the control group (P < 0.05). Activities of AST and ALT in the treatment group and the AST activity in the control group were markedly improved when compared with those before treatment (P < 0.05). Compared with the control group, the abnormal lymphocyte positive case number obviously decreased in the treatment group after treatment, at week 4 and 12 of follow-ups (P < 0.05). (4) After treatment, at week 4 and 12 of follow-ups, CD3+ and CD8+ significantly decreased; CD4+, CD4/CD8, and B cells significantly increased in the two groups, when compared with before treatment (P < 0.05). NK cells significantly increased more in the treatment group after treatment, at week 4 and 12 of follow-ups, higher than before treatment as well as the control group (P < 0.05). (5) EB viral DNA and EB viral CA-IgM negative conversion case numbers significantly increased in the two groups after treatment, at week 4 and 12 of follow-ups (P < 0.05). Compared with the control group, EB viral DNA and EB viral CA-IgM negative conversion case numbers significantly increased in the treatment group after treatment and at week 4 of follow-ups (P < 0.05). CONCLUSIONS: Treatment of EB virus infection by Chinese medical treatment was effective. It could promote the recovery of EB viral infection, and reduce the risk of vicious disease after EB viral infection.

[Filamentous morphology: a new frontier for genetic modification of filamentous fungal cell factories].

Filamentous fungi are a group of eukaryotic microorganisms widely found in nature. Some filamentous fungi have been developed as "cell factories" and extensively used for the production of recombinant proteins, organic acids, and secondary metabolites due to their strong protein secretion capabilities or effective synthesis of many natural products. The growth morphology of filamentous fungi significantly influences the quality and quantity of fermented products. Previous research conducted by the authors' group revealed that an increase in hyphal branches leads to enhanced protein secretion during liquid fermentation. With the development of morphological engineering of filamentous fungi, an increasing number of studies have focused on modifying fungal mycelium morphology to improve the yield of target metabolites during fermentation. While there have been a few reviews on the relationship between fungal fermentation morphology and productivity, research in this area is rapidly developing and requires updates. The paper presents a comprehensive review of domestic and international research reports, along with the authors' own research findings, to systematically review the morphological patterns of filamentous fungi, the impact of fungal morphology on industrial fermentation, as well as methods and strategies for regulating mycelial morphology. The aim of this review is to enhance the understanding of relevant domestic scholars regarding the morphological development of filamentous fungi and provide ideas for the rational engineering of fungal strains suitable for industrial fermentation.

Vitamin D and selenium for type 2 diabetes mellitus with Hashimoto's thyroiditis: Dosage and duration insights.

This letter discusses the publication by Feng et al. Iodine, selenium, and vitamin D are closely associated with thyroid hormone production in humans; however, the efficacy of selenium and vitamin D supplementation for type 2 diabetes mellitus (T2DM) patients with Hashimoto's thyroiditis (HT) remains controversial. In the retrospective study we discuss herein, the authors highlighted significant improvements in thyroid function, thyroid antibodies, blood glucose, and blood lipid in T2DM patients with HT following addition of vitamin D and selenium to their antidiabetic regimens, underscoring the value of these supplements. Our team is currently engaged in research exploring the relationship between micronutrients and HT, and we have obtained invaluable insights from the aforementioned study. Based on this research and current literature, we recommend a regimen of 4000 IU/day of vitamin D and 100-200 µg/day of selenium for over three months to six months for patients with HT, particularly for those with concurrent T2DM.

Total ankle replacement versus ankle fusion for end-stage ankle arthritis: A meta-analysis.

PURPOSE: This study aims to systematically review the efficacy and safety of total ankle replacement (TAR) and ankle fusion (AF) as treatment options for end-stage ankle arthritis. METHODS: A comprehensive literature search was conducted on data from multiple databases, including PubMed, The Cochrane Library, Construction and Building Materials, Embase, Web of Science, and Scopus for RCTs and prospective cohort studies comparing TAR and AF in patients with end-stage ankle arthritis from inception up to June, 2023. Our primary outcomes of interest included patients' clinical function scores and complications. We employed Review Manager 5.4 and Stata/MP 14.0 software for the meta-analysis. RESULTS: Our analysis incorporated 13 comparative studies, including 11 prospective studies, one pilot RCT, and one RCT. The pooled results revealed no significant difference in postoperative Short Form-36 scores between the TAR and AF groups (MD = -1.19, 95% CI: -3.89 to 1.50, p = .39). However, the postoperative Foot and Ankle Ability Measure scores in the AF group were significantly higher than in the TAR group (MD = 8.30, 95% CI: 1.01-15.60, p = .03). There was no significant difference in postoperative complication rates between the TAR and AF groups (RR = 0.95, 95% CI: 0.59 to 1.54, p = .85). CONCLUSION: Currently available evidence suggests no significant disparity in postoperative outcomes between TAR and AF. In the short term, TAR demonstrates better clinical scores than AF and lower complication rates. Conversely, in the long term, AF exhibits superior clinical scores and lower complication rates, although this difference is not statistically significant.

Albiflorin alleviates neuroinflammation of rats after MCAO via PGK1/Nrf2/HO-1 signaling pathway.

Ischemic stroke is acknowledged as one of the most frequent causes of death and disability, in which neuroinflammation plays a critical role. Emerging evidence supports that the PGK1/Nrf2/HO-1 signaling can modulate inflammation and oxidative injury. Albiflorin (ALB), a main component of Radix paeoniae Alba, possesses anti-inflammatory and antioxidative properties. However, how it exerts a protective role still needs further exploration. In our study, the middle cerebral artery occlusion (MCAO) model was established, and the Longa score was applied to investigate the degree of neurological impairment. Dihydroethidium (DHE) staining and Malondialdehyde (MDA) assay were used to detect the level of lipid peroxidation. 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining was used to measure the infarct area. Evans blue staining was employed to observe the integrality of the blood-brain barrier (BBB). The injury of brain tissue in each group was observed via HE staining. Immunofluorescence staining, enzyme-linked immunosorbent assay (ELISA) and western blot assay were used for the measurement of inflammatory factors and protein levels. We finally observed that ALB relieved cerebral infarction symptoms, attenuated oxidative damage in brain tissues, and reduced neuroinflammation and cell injury in MCAO rats. The overexpression of PGK1 abrogated the protective effect of ALB after experimental cerebral infarction. ALB promoted PGK1 degradation and induced Nrf2 signaling cascade activation for subsequent anti-inflammatory and antioxidant damage. Generally speaking, ALB exerted a protective role in treating cerebral ischemia, and it might target at PGK1/Nrf2/HO-1 signaling. Thus, ALB might be a potential therapeutic agent to alleviate neuroinflammation and protect brain cells after cerebral infarction.

Huangqi Guizhi Wuwu decoction promotes M2 microglia polarization and synaptic plasticity via Sirt1/NF-κB/NLRP3 pathway in MCAO rats.

Huangqi Guizhi Wuwu decoction (HGWD) has been demonstrated to ameliorate cerebral ischemia-reperfusion injury in clinical application. Nevertheless, the exact mechanisms of HGWD have not been conclusively elucidated. This study aimed to investigate the potential role and mechanism of HGWD on neurological deficits in a rat model of middle cerebral artery occlusion (MCAO). Our results showed that HGWD significantly alleviated neurological deficits in MCAO rats, evidenced by high mNSS score, reduced cerebral infarction area, and improved brain pathological injury. Besides, HGWD reduced the levels of TNF-α, IL-1ß, IL-6, SOD, MDA and GSH in the brain tissue. Further study suggested that HGWD promoted microglia polarization towards M2 by inhibiting M1 activation (Iba1+/CD16+, iNOS) and enhancing M2 activation (Iba1+/CD206+, Arg-1). Additionally, HGWD increased dendritic spine density and enhanced levels of synapse marker proteins (PSD95, Synapsin I). HGWD also up-regulated Sirt1 expression while inhibited p-NF-κB, NLRP3, ASC, and cleaved caspase-1 level in the hippocampus of MCAO rats. Sirt1 specific inhibitor EX527 notably weakened the neuroprotective efficacy of HGWD against cerebral ischemia, and significantly abolished its modulation on microglia polarization and synaptic plasticity in vivo. Collectively, our findings suggested that HGWD ameliorated neuronal injury in ischemic stroke by modulating M2 microglia polarization and synaptic plasticity, at least partially, via regulating Sirt1/NF-κB/NLRP3 pathway, further supporting HGWD as a potential therapy for neuroprotection after ischemic stroke.

Novel Human Meningioma Organoids Recapitulate the Aggressiveness of the Initiating Cell Subpopulations Identified by ScRNA-Seq.

High-grade meningioma has an unsatisfactory outcome despite surgery and postoperative radiotherapy; however, the factors driving its malignancy and recurrence remain largely unknown, which limits the development of systemic treatments. Single-cell RNA sequencing (scRNA-Seq) technology is a powerful tool for studying intratumoral cellular heterogeneity and revealing the roles of various cell types in oncogenesis. In this study, scRNA-Seq is used to identify a unique initiating cell subpopulation (SULT1E1+ ) in high-grade meningiomas. This subpopulation modulates the polarization of M2-type macrophages and promotes meningioma progression and recurrence. A novel patient-derived meningioma organoid (MO) model is established to characterize this unique subpopulation. The resulting MOs fully retain the aggressiveness of SULT1E1+ and exhibit invasiveness in the brain after orthotopic transplantation. By targeting SULT1E1+ in MOs, the synthetic compound SRT1720 is identified as a potential agent for systemic treatment and radiation sensitization. These findings shed light on the mechanism underlying the malignancy of high-grade meningiomas and provide a novel therapeutic target for refractory high-grade meningioma.

MYB44 regulates PTI by promoting the expression of EIN2 and MPK3/6 in Arabidopsis.

The plant signaling pathway that regulates pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) involves mitogen-activated protein kinase (MAPK) cascades that comprise sequential activation of several protein kinases and the ensuing phosphorylation of MAPKs, which activate transcription factors (TFs) to promote downstream defense responses. To identify plant TFs that regulate MAPKs, we investigated TF-defective mutants of Arabidopsis thaliana and identified MYB44 as an essential constituent of the PTI pathway. MYB44 confers resistance against the bacterial pathogen Pseudomonas syringae by cooperating with MPK3 and MPK6. Under PAMP treatment, MYB44 binds to the promoters of MPK3 and MPK6 to activate their expression, leading to phosphorylation of MPK3 and MPK6 proteins. In turn, phosphorylated MPK3 and MPK6 phosphorylate MYB44 in a functionally redundant manner, thus enabling MYB44 to activate MPK3 and MPK6 expression and further activate downstream defense responses. Activation of defense responses has also been attributed to activation of EIN2 transcription by MYB44, which has previously been shown to affect PAMP recognition and PTI development. AtMYB44 thus functions as an integral component of the PTI pathway by connecting transcriptional and posttranscriptional regulation of the MPK3/6 cascade.

Characterization of two multidrug-resistant Klebsiella pneumoniae harboring tigecycline-resistant gene tet(X4) in China.

Objectives: Tigecycline is recognized as one of the last-line antibiotics to treat serious bacterial infection caused by carbapenem-resistant Klebsiella pneumoniae (CRKP). The plasmid-borne gene tet(X4) mediates high resistance to tigecycline. However, the prevalence and genetic context of tet(X4) in K. pneumoniae from various sources are not fully understood. Here, we investigated the prevalence of tet(X4)-positive K. pneumoniae and characterized the genetic context of tet(X4)-bearing plasmids in K. pneumoniae isolates. Methods: Polymerase chain reaction (PCR) was used to detect the tet(X4) gene. The transferability of the tet(X4)-carrying plasmids was tested by conjugation assays. The Galleria mellonella infection model was used to test virulence of tet(X4)-positive strains. Whole-genome sequencing and genome-wide analysis were performed to identify the antimicrobial resistance and the virulence genes, and to clarify the genetic characteristics of the tet(X4)-positive isolates. Results: Among 921 samples, we identified two tet(X4)-positive K. pneumoniae strains collected from nasal swabs of two pigs (0.22%, 2/921). The two tet(X4)-positive isolates exhibited high minimum inhibitory concentrations to tigecycline (32-256 mg/L) and tetracycline (256 mg/L). The plasmids carrying the tet(X4) gene can transfer from the donor strain K. pneumoniae to the recipient strain Escherichia coli J53. Genetic analysis of the complete sequence of two tet(X4)-carrying plasmids pTKPN_3-186k-tetX4 and pTKPN_8-216k-tetX4 disclosed that the tet(X4) gene was flanked by delta ISCR2 and IS1R, which may mediate the transmission of the tet(X4) gene. Conclusion: The prevalence of tet(X4)-positive K. pneumoniae among different sources was low. ISCR2 and IS1R may contribute to the horizontal transfer of tet(X4) gene. Effective measures should be taken to prevent the transmission of tet(X4)-producing K. pneumoniae in humans or animals.

[Overexpression and characterization of a laccase gene from Pleurotus ostreatus in Trichoderma reesei].

OBJECTIVE: Expression, purification and characterization of a laccase gene from Pleurotus ostreatus in Trichoderma reesei. METHODS: The strong promoter and terminator of cellobiohydrolase I (cbh1) gene from T. reesei were amplified by PCR and inserted into pBluescriptIISK(+) to form vector pSKCST. The laccase gene from Pleurotus ostreatus was de novo synthesized according to T. reesei condon bias and cloned into the vector pLacdt resulting in the expression vector pSKLDT. The linearized pSKLDT was introduced into T. reesei strain Tu6 by protoplast-mediated transformation. The screened laccase expression transformants were grown in shake flasks on minimal medium and the recombinant laccase was purified and characterized. RESULTS: Transformants were isolated in selective screening medium plate and identified by PCR. The enzyme activity of laccase in transformant LC-7 was 237.134 U/mL which was 28.6 -fold higher than that in P. ostreatus. The specific activity of the purified enzyme was 9852 IU/mg. Enzymatic assay revealed that the optimum temperature for its activity was 50 degrees C and pH was 3.0. The optimum substrate was ABTS and the K(m) and V(max) for ABTS were 7.58 x 10(-2) mmol/L and 9.752 x 10(-3) mmol/L/min. Metal ions like Cu2+, Zn2+, Fe3+, Mn2+, Ba2+, Mg2+ and Fe2+ had different inhibitory effect on purified laccase. CONCLUSIONS: Under the regulation of cbh1 promoter and cbh1 signal peptide, heterologous laccase was successfully overexpressed in T. reesei.