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Carbapenemase-producing Enterobacteriaceae (CPE) isolates are recognized as one of the most severe threats to public health. However, the population structure and genetic characteristics of CPE isolates among bloodstream infections (BSIs) are largely unknown. To address this knowledge gap, in this study, we included patients with clinically significant BSIs due to Enterobacterales isolates, recruited from 26 sentinel hospitals in China (2014–2015). CPE isolates were microbiologically and genomically characterized, including their susceptibility profiles, molecular typing, phylogenetic features, and genetic context analysis of carbapenemase-encoding genes. Of the 2569 BSI Enterobacterales isolates enrolled, 42 (1.6%) were carbapenemase-positive. Moreover, among the 2242 investigated isolates, 1111 (49.6%) extended-spectrum β-lactamase (ESBL)-producing isolates were identified in Escherichia (E.) coli, Klebsiella (K.) pneumoniae, Proteus (P.) mirabilis, and K. oxytoca. Whole genome sequencing analysis showed the clonal spread of K. pneumoniae carbapenemase (KPC)-2-producing K. pneumoniae sequence type 11 (ST11) and New Delhi metallo-β-lactamase (NDM)-5-producing E. coli ST167 in our collection. Plasmid analysis revealed that carbapenemase-encoding genes were located on multiple plasmids. A high prevalence of biofilm-encoding type 3 fimbriae clusters and yesiniabactin-associated genes was observed in K. pneumoniae isolates. This work demonstrates the high prevalence of ESBLs and the wide dissemination of CPE among BSI isolates in China, which represent real clinical threats. Moreover, our findings first illustrate a more comprehensive genome scenario of CPE isolates among BSIs. The clonal spread of KPC-2-producing K. pneumoniae ST11 and NDM-5-producing E. coli ST167 needs to be closely monitored.

Genome-editing techniques incorporating artificial nucleases develop rapidly and enable efficient and precise modification of genomic DNA of numerous organisms. The present research aimed to establish a rapid, sensitive and visual method for genotyping of germline genome-edited mutants with small genomic fragment deletion. The genome-edited pigs with 2-bp deletion and 11-bp deletion of Myostatin (MSTN) gene generated by TALENs system were used as test materials to check the proposed allele-specific PCR (AS-PCR) and lateral flow nucleic acid biosensor (LFNAB) cascade method. AS-PCR can produce products with different tags to distinguish genome-edited alleles and wild-type alleles. A LFNAB was applied to do visual detection of AS-PCR products without using additional instruments. Furthermore, we demonstrated that AS-PCR and LFNAB cascade could accurately and visually distinguish genome-edited pigs with small genomic fragment deletion of Myostatin (MSTN) gene and wild-type pigs with limit of detection (LOD) of 0.1 ng. The proposed AS-PCR and LFNAB cascade can do rapid and visual genotyping of genome-edited mutants with small genomic fragment deletion, serving as a platform for genome-edited animal genotyping.

The non-volant terrestrial fauna of the Ryukyu Archipelago is characterized by high endemism with island groups forming distinct biogeographical regions separated by straits. This endemism has resulted from complex histories of insularization caused by geological events and dispersal through land bridges or across the sea during a timeframe of several million years. However, little is known about the zoogeography of terrestrial invertebrates in this region. We presented the first study looking into the earthworm fauna between Taiwan and the Ryukyu Archipelago under a phylogeographical context, and investigated the systematics and phylogeography of the giant earthworms in the Metaphire formosae species group endemic to this region. We highlighted strong evidence supporting that giant earthworms collected from Yonagunijima, the westernmost island of the Ryukyus, represent a new member of the species group, and further described it as a new species, Metaphire dunan sp. nov. Phylogenetic analyses and divergence time estimation rejected the hypothesis that the separation between M. dunan and its Taiwanese sister species, M. formosae, took place 8 Ma in late Miocene, when Taiwan and the Ryukyu region were supposedly first separated from each other and from the Eurasian continent. Instead, our results suggested that the speciation within the M. formosae species group occurred during the arc-continent collision in Taiwan after 5 Ma and M. dunan diverged from its sister species soon after the diversification of the species group had started. In Taiwan, the divergence of mountain-dwelling species followed a north-to-south pattern, consistent with the southward-propagating arc-continent collision on the island.

Two previously undescribed 3(4), 8(9), 9(10)-trisecocycloartane triterpene dilactones with a featured eleven-membered ring, pseudoladilactones A and B (1-2), together with three known ones (4-5), were isolated from the 80 % ethanol extract of the cones of Pseudolarix amabilis. The structures of compounds 1 and 2 were determined on the basis of analysis of 1D- and 2D-NMR spectroscopic data. The absolute configuration of pseudoladilactones A (1) was elucidated by single crystal X-ray diffraction (CuKα). All isolates were tested for cytototoxicity against four human tumor cell lines A549, HCT116, SK-BR-3, and HepG2.

Early Carboniferous granitoids, distributed in the Yining and Central Tianshan blocks, Chinese western Tianshan, are important for deciphering their tectonic contexts and evolutionary history of the South Tianshan Ocean. Here we present LA-ICP-MS zircon U-Pb ages, in situ Hf isotopic data, and whole-rock geochemical data of the monzogranites and syenogranites from the Kalejun Mountains, southern part of the Yining Block, aiming to constrain their petrogenesis and geodynamic background. The results reveal that these monzogranites and syenogranites were emplaced at 356 Ma and 344 Ma, respectively. Geochemically, they are characterized by high SiO2 (68.74-75.76 wt.%) and K2O (4.58-6.18 wt%), low MgO (0.14-0.76 wt.%) and P2O5 (0.04-0.24 wt.%) contents. In addition, they are depleted in Ba, Sr, Eu, Ti, Nb, and Zr, and enriched in Rb, Cs, Th, and U. These features indicate that the Kalejun granites are high-silica I-type granites. The low positive epsilon(Hf)(t) values (+1.1 to +8.2) and old T-DM2(Hf) model ages (1294 to 827 Ma) of the studied zircons imply that the parental magma of these granites were derived from the reworking of Proterozoic basement with minor involvement of juvenile materials. Combined with the Late Devonian to Early Carboniferous magmatic activities in the Yining and Central Tianshan blocks (e.g. the discovery of A-type bubble rhyolites and high-Mg andesites), it is suggested that the Kalejun granites were likely related to the roll-back of the South Tianshan oceanic slab. Finally, we argue that the subduction of South Tianshan Ocean probably terminated at the end of the Early Carboniferous.

Blue flowers, which are rare, have long been a focus for researchers engaged in colored flower breeding. In this study, Plumbago auriculata, a plant with blue flowers, and its white flower forma Plumbago auriculata f. alba were used as research materials. Flower color quantification, environmental scanning electron microscopy (ESEM) observation of petal epidermal cells, and determination of the cell sap pH were carried out; moreover, metabolomic analysis was used to analyze the flavonoid metabolites of both types of petals. The results showed that the flower color of P. auriculata was bluish violet, and there was no significant difference in the epidermal cell distribution of the white and blue petals. The key substance that determined the bluish violet flower color was cyanidin-3-O-(6″-p-coumaroylglucoside) (Cy3-pC·G). Compared to the white petal cells, the blue petal cells exhibited up to 40 upregulated flavonoid compounds that were copigments. Together with the significantly increased pH (5.92 ± 0.12) of the petal cell sap, these copigments affected the color presentation of Cy3-pC·G, causing the final petal color of P. auriculata to be bluish violet. The results of this study indicate that cyanidin is an important anthocyanidin in blue flowers, which provides new insights into the breeding of blue flowers.

Plumbago auriculata Lam., a typical distylous plant, undergoes heteromorphic self-incompatibility (HetSI). The use of in vitro flowering allows the early identification of a plant's style morphology, and this process can be used to increase the efficiency of the breeding process and expedite research on the flowering mechanisms of P. auriculata. Here, we used seeds of P. auriculata as explants; obtained plantlets; and assessed the effects of temperature, thermoperiod, photoperiod, and light intensity on the in vitro flowering of the plants. The optimum temperature treatment was a diurnal alternation of 35 °C for 12 h and 25 °C for 12 h. Under these conditions, flower bud formation occurred within approximately 33 d, and the flowering percentage was as high as 91%. A photoperiod of 16 h and a light intensity of 200 µmol m−2 s−1 also promoted in vitro flowering. Under these conditions, flower buds formed within approximately 33 d, and the flowering percentage was approximately 90%. However, relatively long photoperiods and high light intensity suppressed flower bud development, reduced flowering, and shortened flower lifespan. The in vitro flowers had a lighter colour and smaller petals than did the in vivo flowers, although their floral structures (including their reproductive structures) were intact. This work demonstrates a stable system for the induction of in vitro flowering in P. auriculata and describes suitable temperature (35 °C/25 °C) and light (16 h photoperiod and 200 µmol m−2 s−1) conditions for rapid in vitro flowering. This system allows the efficient identification of long and short P. auriculata morphs and allows the formulation of a reasonable cultivation plan. The methods described here provide technical support for the development, utilization, and rapid propagation of excellent new P. auriculata varieties.

Cytochrome c (Cyt c), as an important biomarker, plays an important role in cell apoptosis and the mitochondrial electron transport chain. Here, we have developed a novel biosensor for a Cyt c assay based on polydopamine nanotubes (PDANTs) and an exonuclease I (Exo I) cyclic amplification strategy. The PDANTs can quickly adsorb FAM labeled Cyt c aptamer (P1) and quench its labeled fluorophore groups. When the Cyt c was introduced into the system, Cyt c can specifically bind to the FAM-labeled aptamer with high affinity and cause the recovery of the fluorescence signal due to the formation of a Cyt c-P1 complex. Exo I then could catalyze the cleavage of the aptamer sequence from the Cyt c-P1 complex and release the target Cyt c, and the released Cyt c could continue to bind to the Cyt c aptamer to achieve fluorescence signal amplification. The developed method demonstrates good sensitivity for Cyt c assays in the range from 0.01 to 100 μM with the detection limit of 0.003 μM. Moreover, the aptasensor has been successfully applied to detect Cyt c in a complex system.

ST59 is the predominant pathotype of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in China. As a variant of ST59, there is relatively little known about the detailed information of ST338. To address this issue, here, we described thirteen ST338 CA-MRSA strains isolated from severe bloodstream infection cases, and focused on their epidemiology, genetic features and virulence potential. Phylogenetic analysis showed the earliest isolated strain of this study is likely a predecessor of recent ST338 lineage (after year of 2014). Furthermore, the phylogenetic reconstruction and time estimation suggested that ST338 evolved from ST59 in 1991. Notably, the carrying patten of virulence factors of all ST338 strains were similar, and the genomic islands νSaα, νSaγ and SaPI and the core virulence factors like hla and psm were detected in ST338 isolates. However, all ST338 isolates lacked some adhesion factors such as clfA, clfB, eap, cna and icaD. Additionally, among these ST338 strains, one PVL-negative ST338 isolate was detected. Experiment on mice nose and human alveolar epithelial cell showed that the nasal colonization ability of ST338 was weaker than that of CA-MRSA MW2. In a mouse bloodstream infection model and skin infection model, PVL+ and PVL− strains had the similar virulence, which was dependent on upregulation of toxin genes rather than the presence of mobile genetic elements such as ΦSa2 carrying PVL. Our findings provide important insight into the epidemiology and pathogenicity of the novel and highly virulent ST338-SCCmec Vb clone.

In nature, many different factors cause plants to present variegated leaves. The purpose of this study was to reveal the changes in the green and albino leaves of Hydrangea macrophylla 'Maculata'. It was found that in the albino zone, the leaves became thinner, the chloroplast structure disappeared, and a large number of leucoplasts replaced chloroplasts. In addition, the albino zone of the leaves contained almost no chlorophyll and showed no function related to transforming and utilizing light energy, and more intense oxidative stress was observed in the albino zone of the leaves than in the green zone. RNA-seq analysis showed that the chlorophyll synthesis pathway of the albino zone of leaves was blocked. Upregulated expression of the hydrangea ringspot virus (HdRSV) coat protein (CP) gene was detected in albino tissue by RT-qPCR. Finally, combined UPLC-MS/MS and RNA-seq analyses revealed metabolic changes involving multiple pathways in albino leaf tissue, centered on the TCA cycle. We hypothesize that HdRSV may alter energy metabolism in the albino zone of leaves, including increased lipid metabolism, reduced sugar metabolism, and increased synthesis of amino acids and the viral capsid protein from ribosomes.

Background Candida tropicalis is the most common non-albicans Candida species identified in immunocompromised patients, which often appears with high mortality. However, data on the outcomes of treatment for Candida tropicalis fungemia in patients with neutropenia remain limited. Methods In the present study, 90 neutropenic adult patients with proven Candida tropicalis fungemia, who received initial antifungal therapy, were retrospectively analyzed. Results These results revealed that the overall 8-day and 30-day mortality among patients in the entire data set were 22.2% and 33.3%, respectively. However, there was no significant difference between the survival and death group, in terms of baseline characteristics. The univariate analysis of risk factors identified the treatment with azole as a predictor of mortality, while treatments that containing amphotericin B were associated with reduced mortality. In addition, the survival rate on day 30 was observed in 60.7% (17/28) of patients who were initially treated with echinocandins, while this was observed in 86.4% (19/22, P=0.039) and 100% (13/13, P=0.024) of patients treated with amphotericin B plus echinocandins and amphotericin B, respectively. Conclusion These data indicate for the first time that the initial therapy with amphotericin B-based agents was associated with a better survival rate and could be assessed as the optimal strategy for the treatment of Candida tropicalis fungemia in patients with neutropenia.

The effect of phlorotannin extracts (PTE) (from sporophyll of Undaria pinnatifida) added at different levels (0, 25, 125, 625 µmol/g protein) on the gel properties of mackerel (Scomberomorus niphonius) myofibrillar protein (MP) was studied with and without ultraviolet A (UVA) irradiation. The results showed that the gel strength and cooking yield increased in a PTE dose-dependent manner, and at the level of 625 µmol/g protein PTE, the highest gel strength of 308.43 ± 8.12 (mN·cm) and cooking yield of 76.16 ± 1.40% were obtained in the samples treated with UVA irradiation. The same samples also showed increased carbonyl content, decreased total sulfhydryl, unwinding of α-helix, and quenching of fluorescence intensity of endogenous tryptophan, all of which indicated that elevated protein oxidation in these samples led to enhanced protein cross-linking. Results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated aggregation of myosin heavy chains (MHCs) in the UVA-treated gels with PTE, also evidenced by the dense three-dimensional network structure in these samples visualized by scanning electron microscopy (SEM). Electron spin resonance (ESR) and spin trapping results indicated that free radicals were produced during the gelation process, possibly originated from UVA-treated PTE, which played a critical role of oxidizing fish MPs, and eventually led to the improvement of the textural properties of the mackerel MP gel. PRACTICAL APPLICATION: Brown algae are a family of high-yield marine algae. Phlorotannin extracts are highly active natural substances extracted from brown algae that can have many applications. Ultraviolet A (UVA) as a green and environmentally friendly physical processing method has been widely used in food processing in recent years. The method proposed in this study could be utilized to improve properties of fish protein gel made from poorly performing low-priced fishes, and provide workable guidance for industry to expand the application of brown algae in food processing to better meet consumer's demand for high-quality marine foods.

To explore the clinical effect and adverse reactions of Strychnos nux-vomica in bortezomib-induced peripheral neuropathy (BIPN) of patients with multiple myeloma (MM).A total of 19 MM patients with BIPN were enrolled and Nux Vomica Capsule (NVC, 0.4 g, thrice daily) were orally administrated for 30 days. Comparative analysis on parameters between pre- and post-therapy, including peripheral neuropathy (PN) grade, neurotoxicity score, Chinese medicine (CM) syndrome score, total neuropathy score (TNS), coagulation function, and serum nerve growth factor (NGF) levels were conducted. The adverse events were monitored.In BIPN of MM patients who received NVC, PN grade was lowered, neurotoxicity score was obviously decreased (P⩽0.01), and both CM syndrome score and TNS were remarkably decreased (P0.01). After the therapy, activated partial thromboplastin time was prolonged (P0.01) and fibrinogen was declined (P0.05), showing improvement in the hypercoagulable state of patients. No significant difference of NGF recovery degrees was detected between pre- and post-therapy (P0.05). No evident adverse reactions were observed during the course of treatment.Strychnos nux-vomica L. has significantly effect with a good safety in treatment of BIPN in MM patients.

This study sought to discuss the correlation between the third-generation cephalosporins (3GC)-resistant Escherichia coli and Klebsiella pneumoniae and antibiotic consumption intensity from 143 Chinese tertiary hospitals in 2014. With a retrospective design, the correlation between antibiotic consumption and 3GC-resistant E. coli and K. pneumoniae were performed. 3GC-resistant E. coli was significantly correlated with the consumption of all antibiotics (r = 0.252, p

Background Carbapenem-resistant Klebsiella pneumoniae (CRKP) has become a major problem among nosocomial infections, and it is a serious threat to patients. The clinical characteristics and outcome of CRKP bloodstream infection (BSI) in nontransplant patients remains unelucidated. The aim of this study was as follows: identify the risk factors of CRKP infection; generate new ideas for prevention; and generate new ideas for the most effective therapeutic management in nontransplant patients. Methods The study retrospectively analyzed the clinical and microbiological data of nontransplant patients with K pneumoniae (KP) bacteremia from January 2013 to December 2015 to identify risk factors, clinical features, and outcomes using multivariate logistic regression analysis. Results Of the 371 patients with KP-BSI in nontransplant patients included in this study, 28.0% (N = 104) had CRKP. The 28-day mortality was higher in patients infected with CRKP (55.8%) than in those with carbapenem-susceptible KP (13.9%) (P < .001). Multivariate analysis showed previous gastric catheterization, previous use of carbapenems, hypoproteinemia, and high Acute Physiologic Assessment and Chronic Health Evaluation II scores as independent risk factors for CRKP-BSIs. Carbapenem-resistant KP infection, severe illness, and tigecycline therapy were independent risk factors for death from KP-BSIs. Taken together, inappropriate antibiotic treatment both in empirical and definitive therapy and imipenem minimum inhibitory concentrations (MICs) of >8 mg/L were associated with poor clinical outcome. Conclusions Nontransplant patients with CRKP-BSI had higher mortality. Carbapenems exposure was an independent risk factor for CRKP infection. Imipenem MICs of >8 mg/L, tigecycline therapy, and inappropriate treatments increased the 28-day mortality of KP-BSI patients.

The present study aimed to determine the taxonomic positions of strains designated R-5-52-3T, R-5-33-5-1-2, R-5-48-2 and R-5-51-4 isolated from hot spring water samples. Cells of these strains were Gram-stain-negative, non-motile and rod-shaped. The strains shared highest 16S rRNA gene sequence similarity with Vulcaniibacterium thermophilum KCTC 32020T (95.1%). Growth occurred at 28–55 °C, at pH 6–8 and with up to 3 % (w/v) NaCl. DNA fingerprinting, biochemical, phylogenetic and 16S rRNA gene sequence analyses suggested that R-5-52-3T, R-5-33-5-1-2, R-5-48-2 and R-5-51-4 were different strains but belonged to the same species. Hence, R-5-52-3T was chosen for further analysis and R-5-33-5-1-2, R-5-48-2 and R-5-51-4 were considered as additional strains of this species. R-5-52-3T possessed Q-8 as the only quinone and iso-C15:0, iso-C11:0, C16 : 0 and iso-C17 : 0 as major fatty acids. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, unidentified polar lipids and two unidentified phospholipids. The genomic G+C content was 71.6 mol%. Heat shock proteins (e.g. Hsp20, GroEL, DnaK and Clp ATPases) were noted in the R-5-52-3T genome, which could suggest its protection in the hot spring environment. Pan-genome analysis showed the number of singleton gene clusters among Vulcaniibacterium members varied. Average nucleotide identity (ANI) values between R-5-52-3T, Vulcaniibacterium tengchongense YIM 77520T and V. thermophilum KCTC 32020T were 80.1–85.8 %, which were below the cut-off level (95–96 %) recommended as the ANI criterion for interspecies identity. Thus, based on the above results, strain R-5-52-3T represents a novel species of the genus Vulcaniibacterium , for which the name Vulcaniibacterium gelatinicum sp. nov. is proposed. The type strain is R-5-52-3T (=KCTC 72061T=CGMCC 1.16678T).

We aimed to clarify the epidemiologic and clinical importance of evolutionary events that occurred in carbapenem-resistant Klebsiella pneumoniae (CRKP). We collected 203 CRKP causing bloodstream infections in a tertiary hospital in China during 2013-2017. We detected a subclonal shift in the dominant clone sequence type (ST) 11 CRKP in which the previously prevalent capsular loci (KL) 47 had been replaced by KL64 since 2016. Patients infected with ST11-KL64 CRKP had a significantly higher 30-day mortality rate than other CRKP-infected patients. Enhanced virulence was further evidenced by phenotypic tests. Phylogenetic reconstruction demonstrated that ST11-KL64 is derived from an ST11-KL47-like ancestor through recombination. We identified a pLVPK-like virulence plasmid carrying rmpA and peg-344 in ST11-KL64 exclusively from 2016 onward. The pLVPK-like-positive ST11-KL64 isolates exhibited enhanced environmental survival. Retrospective screening of a national collection identified ST11-KL64 in multiple regions. Targeted surveillance of this high-risk CRKP clone is urgently needed.

Since 2010, methicillin-resistant Staphylococcus aureus (MRSA) ST59 began to increase in prevalence in China, gradually replacing ST239 and has become the dominant clone in most hospitals in China. Here, we investigated the changing epidemiology, phylogenetic reconstruction, and genomic characterization of MRSA clones in China to identify the genomic driving factors in the prevalence of ST59. Most MRSA isolates were identified as ST59 (36.98%; 277/749), which increased from 25.09% in 2014 to 35.53% in 2019. The phylogenetic analysis of the 749 MRSA isolates showed a high level of diversity and the copresence of hospital-associated, community-associated, livestock-associated, and hypervirulent clones. Furthermore, minimum spanning trees revealed that ST59 MRSA clones from different hospitals and regions were integrated, suggesting that frequent exchanges had occurred between regions and hospitals. ST59 clones displayed higher susceptibility to antimicrobials than did ST239 and ST5 MRSA clones, indicating that resistance to non-β-lactam and fluoroquinolone antibiotics may be not critical for the epidemic success of ST59 clones. Virulence factors detection showed that sak and chp genes enriched in MRSA ST59 may be associated with the enhanced spreading success of ST59, whereas qacA may have contributed to the predominance of ST5 in East China. Our refined analysis of different clones among ST239, ST5, ST59, and ST398 demonstrated the existence of potential driving factors for the evolution of nosocomial MRSA populations and diversity of the evolutionary events surrounding clonal replacement. IMPORTANCE As a developing country, China has an unbalanced health care system due to regional differences in economic development. However, China is also a country worthy of study with regard to the population dynamics of MRSA within the more resource-rich health care systems. In this study, we carried out genomic analysis to investigate the genomic epidemiology and characterization of MRSA isolated from bloodstream infections over a timespan of 6 years. Our refined analysis of different MRSA clones among ST59, ST5, ST239, and ST398 demonstrated the existence of driving factors for the evolution of nosocomial MRSA populations and diversity of the evolutionary events surrounding clonal replacement.

Background Various stimuli, including Clonorchis sinensis infection, can cause liver fibrosis. Liver fibrosis is characterized by the activation of hepatic stellate cells (HSCs) with massive production of extracellular matrix (ECM). Our previous study showed that the TGF-β1-induced Smad signaling pathway played a critical role in the activation of HSCs during liver fibrosis induced by worm infection; however, the mechanisms that modulate the TGF-β/Smad signaling pathway are still poorly understood. Accumulating evidence demonstrates that miRNAs act as an important regulator of activation of HSCs during liver fibrosis. Methods The target of miR-497 was determined by bioinformatics analysis combined with a dual-luciferase activity assay. LX-2 cells were transfected with miR-497 inhibitor and then stimulated with TGF-β1 or excretory/secretory products of C. sinensis (CsESPs), and activation of LX-2 was assessed using qPCR or western blot. In vivo, the mice treated with CCl4 were intravenously injected with a single dose of adeno-associated virus serotype 8 (AAV8) that overexpressed anti-miR-497 sequences or their scramble control for 6 weeks. Liver fibrosis and damage were assessed by hematoxylin and eosin (H&E) staining, Masson staining, and qPCR; the activation of the TGF-β/Smad signaling pathway was detected by qPCR or western blot. Results In the present study, the expression of miR-497 was increased in HSCs activated by TGF-β1 or ESPs of C. sinensis. We identified that Smad7 was the target of miR-497 using combined bioinformatics analysis with luciferase activity assays. Transfection of anti-miR-497 into HSCs upregulated the expression of Smad7, leading to a decrease in the level of p-Smad2/3 and subsequent suppression of the activation of HSCs induced by TGF-β1 or CsESPs. Furthermore, miR-497 inhibitor delivered by highly-hepatotropic (rAAV8) inhibited TGF-β/smads signaling pathway by targeting at Smad7 to ameliorate CCL4-induced liver fibrosis. Conclusions The present study demonstrates that miR-497 promotes liver fibrogenesis by targeting Smad7 to promote TGF-β/Smad signaling pathway transduction both in vivo and in vitro, which provides a promising therapeutic strategy using anti-miR-497 against liver fibrosis. Graphical Abstract

Heteromorphic self-incompatibility (HetSI), which is regulated by sporophytes, occurs in some species as a strategy to promote cross-pollination. This research aimed to reveal metabolic changes occurring in HetSI. We used fluorescence microscopy as a tool to compare growth behavior in self-incompatible (SI) and self-compatible (SC) pollination in both pin and thrum flowers of Plumbago auriculata and to identify the ideal timepoint for sample collection for subsequent experiments. We also employed scanning electron microscopy (SEM) to evaluate intermorph structural differences in the pollen grains and stigmas in relation to HetSI. Importantly, UPLC-MS/MS was applied in this study to identify metabolites, compare metabolic differences between pin and thrum styles and monitor metabolic changes in SC and SI pollinations in the two types of flowers. The metabolites mainly included amino acids/peptides, flavonoids, glycosides/sugars, phenols, other organic acids, fatty acids (derivatives)/lipids, amines, aldehydes, alkaloids, alcohols and other compounds. Surprisingly, energy-related nutrients such as amino acids/peptides and tricarboxylic acid cycle-related metabolites were found at higher levels in SI pollinations than in SC pollinations. This result indicates that physiological changes in pollen-stigma interactions differ in pin and thrum styles and SC and SI pollinations and that energy deficiency is not one of the reasons for HetSI.

Objectives Most vancomycin-intermediate Staphylococcus aureus (VISA) and heterogeneous VISA (hVISA) are derived from hospital-associated MRSA due to treatment failure; however, the prevalence of hVISA/VISA in community settings remains unclear. Methods Four hundred and seventy-six community-associated isolates were collected between 2010 and 2011 during national surveillance for antimicrobial resistance in 31 county hospitals across China. Drug susceptibility evaluation and mecA detection were performed by using broth microdilution and PCR analysis, respectively. hVISA/VISA were identified by using macro-Etest and a modified population analysis profile (PAP)-AUC method. The genetic features of all hVISA/VISA isolates were genotyped. Results Among 476 isolates, MRSA and MSSA accounted for 19.7% (n = 94) and 80.3% (n = 382), respectively. Two VISA and 36 hVISA isolates were identified by PAP-AUC testing. The VISA isolates and 29 of the hVISA isolates were MRSA. The proportion of hVISA/VISA was significantly higher in MRSA (30.9%) than in MSSA (1.8%). The hVISA/VISA isolates were assigned to 18 STs classified into seven clonal complexes (CCs). CC121 (n = 12) followed by ST239 (n = 11) was the most prevalent hVISA/VISA clone. All ST239-hVISA/VISA were MRSA, while 12 CC121-hVISA isolates included 6 MSSA and 6 MRSA isolates. SCCmec III was predominant among MRSA-hVISA/VISA isolates. agr I and agr IV were detected in ST239 and CC121, respectively. All except two strains were positive for Panton–Valentine leucocidin genes. Conclusions To the best of our knowledge, this is the first report of CC121 as a prevalent hVISA clone in community settings, highlighting the necessity of surveillance and stricter infection control measures for this globally disseminated lineage.

This is a study of microstructure changes and Soybean milk colloidal particles degradation during in vitro digestion. Soybean milk passed through the digestion stages of the Infogest protocol, while its colloidal particles were examined in terms of size decrease/free surface evolution kinetics and of surface charge. Colloidal protein break-down was determined in relation to the digestion stage, and the shape and structure of the soybean milk particles were monitored with confocal microscopy. Gastric digestion significantly influenced the soybean milk particles, increasing the particle size and decreasing absolute surface charge, hydrolyzing protein and accumulating aminoacids. Similar breakdown was not observed in the absence of pepsin, so the protein break-down can be primarily attributed to pepsin-induced hydrolysis. Correlation of the aminoacids release kinetics with the changes in surface area of the soybean milk proteins showed a direct relation between aminoacids release and solid-water interface formation. A sequence of events can be proposed where proteins are exposed and hydrolyzed throughout the simulated gastrointestinal tract as new interface (substrate) is created in the soybean milk colloidal systems.

Background: The epidemic new strain NAP1/BI/027/ST-1 of Clostridioides difficile (C. difficile) causes more severe coliti and a higher mortality rate than historical strains. However, C. difficile NAP1/BI/027/ST-1 (C. difficile RT027) infections have been rarely reported in Asia, particularly in China. Purpose: The objective of this study was to strengthen the understanding of the molecular characterizations of C. difficile RT027 in China. Patients and methods: Two C. difficile NAP1/BI/027/ST-1 were detected from two patients, and no additional isolates were found. Whole genome sequencing (WGS) was used to characterize two C. difficile RT027 isolates and control strain CD6 (from Hong Kong), and comparative genomic analysis was performed to compare genomic differences between seven isolates from Mainland China, CD6, and 10 isolates from North America and Europe. Results: The comparative genomic analysis revealed that isolates obtained from Mainlan China were outside of the two epidemic lineages, FQR1 and FQR2, and might have decreased virulence and transmissibility for outbreak. Furthermore, unique SNP mutations were detected in isolates obtained from Mainland China, which may affect the biological function of C. difficile. Conclusion: We speculate that C. difficile RT027 isolates in Mainland China may have different features, compared to those in North America and Europe.

The aim of this study is to discuss the non-catechin flavonoids (NCF) from Camellia sinensis (L.) O. Kuntze seed improving TNF-α impaired insulin stimulated glucose uptake and insulin signaling. Flavonoids had anti-metabolic syndrome and anti-inflammatory properties. It had widely been known for biological activity of catechins in tea, but very few research reports discussed the biological activity of non-catechin flavonoids in tea seed. We used HepG2 cell to treat with 5 μM insulin or with 5 μM insulin + 30 ng/ml TNF-α. Detecting the glucose concentration of medium, insulin decreased the glucose levels of medium meant that insulin promoted glucose uptake into cells, but TNF-α inhibited the glucose uptake effect of insulin. Furthermore, insulin increased the protein expressions of IR, IRS-1, IRS-2, PI3K-α, Akt/PKB, GLUT-2, AMPK, GCK, pyruvate kinase, and PPAR-γ. TNF-α activated p65 and MAPKs (p38, JNK1/2 and ERK1/2), iNOS and COX-2 which worsened the insulin signaling expressions of IR, IRS-1, IRS-2, PI3K-α, Akt/PKB, GLUT-2, AMPK, GCK, pyruvate kinase, and PPAR-γ. We added NCF (500, 1000, 2000 ppm) to cell with insulin and TNF-α. Not only glucose levels of medium were lowered, and the protein expressions of insulin signaling were increased, but p38, JNK1/2, iNOS and COX-2 were also reduced. NCF could ameliorate TNF-α induced insulin resistance through inhibiting p38, JNK1/2, iNOS and COX-2, and suggested that it might be used in the future to help control insulin resistance. This finding is the first report to present the discovery. Keywords: Flavonoids, Camellia sinensis (L.) O. Kuntze seed, TNF-α, Insulin resistance

Adult Chrysoperla sinica Tjeder is a common pollen feeder in maize fields. They are thus directly exposed to insecticidal proteins by consumption of genetically engineered maize pollen containing Bacillus thuringiensis (Bt) proteins. Here we assessed the potential effects of Cry1Ab/2Aj- or Cry1Ac-containing Bt maize pollen on the fitness of adult C. sinica via a dietary-exposure assay under laboratory conditions. Survival, pre-oviposition, fecundity and adult dry weight did not differ between adult C. sinica consuming Bt or the corresponding non-Bt maize pollen. The stability of the Cry protein in the food sources and uptake of the Cry protein by adult C. sinica during the feeding experiment were confirmed by ELISA. These results demonstrate that adult C. sinica are not affected by the consumption of Cry1Ab/2Aj- or Cry1Ac-containing maize pollen, suggesting that production of Bt maize expressing cry1Ab/2Aj or cry1Ac genes will pose a negligible risk to adult C. sinica.

Hypervirulent Klebsiella pneumoniae strains are typically associated with severe infections and susceptible to most antimicrobial agents. In 2017, a carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP) strain was isolated from the sputum of a chronic obstructive pulmonary disease (COPD) patient in Zhejiang, China. The goal of the present study was to characterize the molecular features of the CR-hvKP isolate ZJ27003 and its blaKPC-2-harboring plasmid p27003_KPC. Antimicrobial susceptibility was evaluated using the broth microdilution and agar dilution methods. String tests, serum-killing and mouse survival assays were performed to assess virulence, and plasmid conjugation was performed by filter mating. The complete genome sequence of ZJ27003 was acquired using a hybrid assembly of Illumina and Nanopore platform data. The sequence type (ST) of this CR-hvKP isolate was identified as ST23, which exhibits hypervirulence with high serum resistance and murine infection model. The strain is also resistant to carbapenems (imipenem, meropenem and ertapenem), aztreonam and cephalosporins. Additionally, the CR-hvKP isolate carries a 36,708-bp blaKPC-2-harboring plasmid, named p27003_KPC, belonging to the P1 incompatibility (Inc) group. The backbone of p27003_KPC is similar to that of a blaGES-5-harboring Pseudomonas aeruginosa plasmid, in which the blaGES-5 and its surrounding regions were replaced by a blaKPC-2-containing translocatable unit derived from Enterobacteriaceae. The results of a conjugation assay revealed that p27003_KPC can be transferred from K. pneumoniae to P. aeruginosa PAO1 and make the recipient resistant against carbapenem. The identification of a carbapenem-resistant hypervirulent K. pneumoniae isolate carrying and disseminating the blaKPC-2 gene highlights a severe threat to public health.

Concerns regarding carbapenem-resistant Klebsiella pneumoniae (CR-Kp), especially in bloodstream infections (BSIs), are continuing to increase worldwide. Several novel agents with activity against BSI CR-Kp have been approved or are in late-stage clinical development. In this study, the antibacterial effects of ceftazidime/avibactam (CZA), aztreonam/avibactam (AZA), meropenem/vaborbactam (MEV), imipenem-cilastatin/relebactam (ICR) and eravacycline (ERV) against three colistin-resistant CR-Kp (COLR-Kp) and four CZA-resistant CR-Kp (CZAR-Kp) were tested by time-kill assay. Klebsiella pneumoniae ATCC® BAA-1705TM was used as a control strain. Two COLR-Kp isolates carried the blaKPC-2 gene and four CAZR-Kp isolates carried metallo-β-lactamase genes. The results revealed that ERV resulted in re-growth of seven tested isolates. CZA and MEV showed a bactericidal effect against isolates harbouring blaKPC-2. ICR reduced the population of six isolates to >5 log10 CFU/mL compared with the initial count. AZA showed a bactericidal effect (>5 log10 CFU/mL) against seven isolates and a bacteriostatic effect (

Klebsiella pneumoniae strains carrying OXA-48-like carbapenemases are increasingly prevalent across the globe. There is thus an urgent need to better understand the mechanisms that underpin the dissemination of bla OXA-48-like carbapenemases. To this end, four ertapenem-resistant K. pneumoniae isolates producing OXA-48-like carbapenemases were isolated from two patients. Genome sequencing revealed that one sequence type (ST) 17 isolate carried bla OXA-181, whilst three isolates from a single patient, two ST76 and one ST15, carried bla OXA-232. The 50514 bp bla OXA-181-harbouring plasmid, pOXA-181_YML0508, was X3-type with a conjugation frequency to Escherichia coli of 1.94×10−4 transconjugants per donor. The bla OXA-232 gene was located on a 6141 bp ColKP3-type plasmid, pOXA-232_WSD, that was identical in the ST76 and ST15 K. pneumoniae isolates. This plasmid could be transferred from K. pneumoniae to E. coli at low frequency, 8.13×10−6 transconjugants per donor. Comparative analysis revealed that the X3 plasmid acquired the bla OXA-48-like gene via IS3000-mediated co-integration of the ColKP3-type plasmid. Our study highlights how plasmid integration and rearrangements can contribute to the spread of bla OXA-48-like genes, which provides important clues for clinical prevention of the dissemination of K. pneumoniae strains carrying bla OXA-48-like carbapenemases.

Plumbago auriculata Lam. is an ornamental plant native to South Africa and widely cultivated in China, but the cultivated plants are dominated by a single variety. The development of new varieties is of great commercial interest, and genetic diversity is the foundation of breeding programs. In this study, 85 progenies were obtained by crosses between Plumbago auriculata and Plumbago auriculata f. alba. The genetic diversity of these hybrids was evaluated using horticultural traits and ISSR and SRAP markers. Of the 25 horticultural traits evaluated, the largest variation was found in the beginning of the blooming period, and sepal length was the least variable trait. Correlation analysis showed that the wider the plant, the greater the number of inflorescences and the earlier the flowering. Seven factors explained 65.171% of the total variance; the first factor was leaf morphology, and the second factor was flower morphology. The genetic diversity of the 85 progenies was analyzed using seven pairs of SRAP primers and eight ISSR primers. The average number of effective alleles for 85 hybrids was 1.638, and the average Shannon index value was 0.507. The Nei genetic similarity coefficient indicated that the similarity between WLBS and WSBL was the highest, while that between BLWS and BSWL was the lowest. Analysis of molecular variance (AMOVA) indicated that the main variation was within populations. Cluster analysis based on horticultural traits and molecular markers divided all progenies into seven and five groups, respectively, and there were obvious differences between the two clusters. In this study, we created intermediate materials for future breeding, taking the first step in the cross-breeding of P. auriculata.

Clostridioides difficile is the most common pathogen causing antibiotic-associated diarrhea. Previous studies showed that diverse sources, aside from C. difficile infection (CDI) patients, played a major role in C. difficile hospital transmission. This study aimed to investigate relationships and transmission potential of C. difficile strains from different sources. A prospective study was conducted both in the intensive care unit (ICU) and six livestock farms in China in 2018–2019. Ninety-eight strains from CDI patients (10 isolates), asymptomatic hospitalized carriers (55), the ICU environment (12), animals (14), soil (4), and farmers (3) were collected. Sequence type (ST) 3/ribotype (RT) 001, ST35/RT046, and ST48/RT596 were dominant types, distributed widely in multiple sources. Core-genome single-nucleotide polymorphism (cgSNP) analysis showed that hospital and farm strains shared several common clonal groups (CGs, strains separated by ≤ 2 cgSNPs) (CG4/ST3/RT001, CG7/ST35/RT046, CG11/ST48/RT596). CDI patients, asymptomatic carriers, and the ICU environment strains also shared several common CGs. The number of virulence genes was not statistically different between strains from different sources. Multi-source strains in the same CG carried identical virulence gene sequences, including pathogenicity genes at the pathogenicity locus and adhesion-related genes at S-layer cassette. Resistance genes (ermB, tetM, etc.) were widespread in multiple sources, and multi-source strains in the same CG had similar resistance phenotypes and carried consistent transposons and plasmid types. The study indicated that interspecies and cross-regional transmission of C. difficile occurs between animals, the environment, and humans. Community-associated strains from both farms and asymptomatic hospitalized carriers were important reservoirs of CDI in hospitals.

Profiling of polyphenolics in four types of brown macroalgae, namely Laminaria japonica (L. japonica), Undaria pinnatifida (U. pinnatifida), Sargassum fusiforme (S. fusiforme), and Ascophyllum nodosum (A. nodosum), and their effect on oxidation resistance were investigated for the first time. Polyphenolic extracts from marine brown macroalgae were shown to effectively remove oxidants from cells and cellular systems. A. nodosum showed the highest antioxidant activity among evaluated brown macroalgae, showing a better scavenging effect on 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical and alleviating oxidative damage caused by hydrogen peroxide to human keratinocytes (HaCaT) cells. Through Q-Exactive HF-X mass spectrometry analysis, 12 polyphenolic compounds were preliminarily identified, including phlorotannins, phenolic acids, and flavonoids. Significant differences in content and variety of polyphenolics were found in evaluated brown macroalgae, which could be related to differences in antioxidant activity in vivo and in vitro. Moreover, the antioxidant activity might be related to the total phenolic content and the types of polyphenolics, especially phlorotannins. The findings presented in this study indicate that A. nodosum could be used as an important substitute for functional ingredients in foods and pharmaceutical preparations, as well as a raw material for phlorotannins research.

Background 4-Hydroxyphenylacetic acid (4HPAA) is an important raw material for the synthesis of drugs, pesticides and biochemicals. Microbial biotechnology would be an attractive approach for 4HPAA production, and cofactors play an important role in biosynthesis. Results We developed a novel strategy called cofactor engineering based on clustered regularly interspaced short palindromic repeat interference (CRISPRi) screening (CECRiS) for improving NADPH and/or ATP availability, enhancing the production of 4HPAA. All NADPH-consuming and ATP-consuming enzyme-encoding genes of E. coli were repressed through CRISPRi. After CRISPRi screening, 6 NADPH-consuming and 19 ATP-consuming enzyme-encoding genes were identified. The deletion of the NADPH-consuming enzyme-encoding gene yahK and the ATP-consuming enzyme-encoding gene fecE increased the production of 4HPAA from 6.32 to 7.76 g/L. Automatically downregulating the expression of the pabA gene using the Esa-PesaS quorum-sensing-repressing system further improved the production of 4HPAA. The final strain E. coli 4HPAA-∆yfp produced 28.57 g/L of 4HPAA with a yield of 27.64% (mol/mol) in 2-L bioreactor fed-batch fermentations. The titer and yield are the highest values to date. Conclusion This CECRiS strategy will be useful in engineering microorganisms for the high-level production of bioproducts.

Aim: Angiogenesis plays an important role in the initiation, development, and metastasis of malignant tumors. Antiangiogenic drugs combined with immune therapy are considered to have a synergistic effect on anti-tumor strategy. Weichang’an formula (WCAF) is a prescription of traditional Chinese medicine (TCM) based on pharmaceutical screening and clinical experience. The aim of this study is to examine the effect of WCAF and its combined action with Bevacizumab (BEV) in colorectal cancer, and to identify the possible mechanism of action.Methods: A human colon cancer cell (HCT 116) subcutaneous xenograft model was established in BALB/c-nu/nu mice. Tumor-bearing mice were randomized into each of four groups: control, WCAF treated, BEV treated, and WCAF plus BEV treated. Apoptosis was detected by TUNEL assay. Western blot was used to assess the protein levels of Leptin-R, STAT3, p-STAT3, BCL-2, and VEGFR-1. Immunohistochemistry was used to detect the micro-vessel density (MVD) and AKT1. Leptin and Vascular endothelial growth factor A (VEGF-A) mRNA expression were detected by Real-time PCR (RT-PCR). A network pharmacology study and validation assay were carried out to find the underlying molecular targets of WCAF related to immune regulation.Results: Compared with the control group, WCAF reduced tumor weight and volume, as well as promoted tumor cell apoptosis. WCAF treatment decreased the mRNA expression of Leptin and VEGF-A, while the protein levels of CD31, LEP-R, VEGFR-1, STAT3, and p-STAT3 were decreased in tumor tissues. In addition, VEGFR-1 protein expression was decreased in the WCAF group and the WCAF plus BEV group but not in the BEV group. The combination of WCAF and BEV demonstrated a partial additive anti-tumor effect in vivo. The pharmacological network also found there are 26 WCAF target proteins related to cancer immune and 12 cancer immune related pathways. The AKT1 protein expression in the WCAF and WCAF + BEV groups were significantly lower than the that in the control group (p < 0.01).Conclusion: WCAF can inhibit tumor growth and promote apoptosis and inhibit tumor angiogenesis in subcutaneous xenografts of human colon cancer HCT-116 in nude mice. WCAF also makes up for the deficiency of BEV by inhibiting VEGFR-1. The VEGFR-1 expression between the combination group and BEV alone achieved statistically significant difference (p < 0.01). Combined with BEV, WCAF showed a partial additive anti-tumor effect. The mechanism may be related to Leptin/STAT3 signal transduction, VEGF-A, VEGFR-1 and WCAF target proteins related to cancer immune such as leptin and AKT1.

Ceratostigma willmottianum (Plumbaginaceae) is a perennial herb native to China. Many species of Plumbaginaceae have been reported to exhibit heterostyly. Determining the functional breeding system of C. willmottianum can improve our understanding of the reproductive ecology of heterostylous plants. We investigated the floral traits and pollen and stigma characteristics in a natural population, and artificial pollination was carried out in an artificial population. It was found that C. willmottianum was distylous with short (S)- and long (L)-styled morphs, did not exhibit precise reciprocal herkogamy and was partially self-compatible but primarily outcrossing. In the artificial pollination experiments, the pollen tubes reached the base of the style under intermorph pollination, whereas they rarely penetrated the style under intramorph pollination and self-pollination. Both the L and S morphs exhibited a high seed set after intermorph pollination, whereas the seed set under intramorph pollination was lower. Therefore, C. willmottianum may spread the chance of receiving pollen between the two morphs by nonreciprocal heterostyly, which may be a unique mode of ecological adaptation in Plumbaginaceae. We believe that our discovery could provide new ideas regarding the origin and evolution of heterostyly.

To assess the genomic profiles of tigecycline (Tgc)-resistant Acinetobacter baumannii, including antibiotic resistance (AR) genes and virulence factors (VF), whole-genome shotgun sequencing was performed on 19 Tgc-resistant (TgcR) A. baumannii strains collected in a tertiary hospital during the early phase of the clinical introduction of Tgc in China from late 2012 to mid-2014. The major sample types containing TgcR strains were sputum and drain fluid. Data from an average of 624 Mbp of sequence was generated on each bacterial genome, with Q30 quality of 90%, and an average coverage of 96.6%. TCDC-AB0715 was used as a reference genome. The genome sequences were annotated for functional elements including AR genes, VFs, genome islands, and inserted sequences before they were comparatively analyzed. The antibiotic susceptibility phenotypes of the strains were examined by a broth microdilution method to determine the minimal inhibitory concentration (MIC) of strains against major clinical antibiotics. The AR genes (ARGs) were annotated using the Comprehensive Antibiotic Resistance Database (CARD). Thirty-three ARGs were shared by all 19 TgcR strains, and 24 ARGs were distributed differently among strains. A total of 391 VFs were found to be diversely distributed in all TgcR strains. Based on ARG number distribution, the 19 TgcR strains were divided into several groups. Highly differentiated genes included gpi, mphG, armA, msrE, adec, catB8, aadA, sul1, bla OXA- 435, aph3i, and bla TEM- 1, which may represent gene markers for TgcR A. baumannii sub-types. In addition, when compared with Tgc-sensitive (TgcS) strains collected during the same period, TgcR strains featured enrichment of ARGs including aph6id, aph3ib, and teta. Compared with 26 other whole-genome sequences of A. baumannii deposited in GeneBank, TgcR strains in this study commonly lacked the EF-Tu mutation for elfamycin resistance. Previous investigation of three A. baumannii strains isolated from one patient indicated genomic exchange and a homologous recombination event associated with generation of tigecycline resistance. This study further analyzed additional TgcR strains. Phylogenetic analysis revealed a close evolutionary relationship between 19 TgcR strains and to isolates in East and Northeast China. In short, the comprehensive functional and comparative genomic analysis of 19 clinical TgcR A. baumannii strains isolated in the early stage of Tgc usage in China revealed their close phylogenetic relationship yet variable genetic background involving multiple resistance mechanisms. Using a simple ARG or VF gene number diversity method and marker genes, TgcR strain sub-types can be identified. The distinct characteristics of TgcR A. baumannii strains with versatile genomic resistance and regulation patterns raise concern regarding prediction and control of Tgc resistance in the clinic.

China has been experiencing a rapid increase in the number of nosocomial infections caused by carbapenem-resistant Klebsiella pneumoniae ST11 (ST11-CRKP) for decades. The emergence of hypervirulent ST11-CRKP (ST11-CR-HvKP) strains is expected to become a serious public health issue in China, considering that carbapenem resistance and virulence have converged in an epidemic clone. K. pneumoniae strains that colonize the human intestinal tract may become a reservoir of virulence and carbapenemase-encoding genes. Here, we first characterized the genotypes and antimicrobial phenotypes of ST11-CR-HvKP strains isolated from diarrheal stool samples of inpatients in Zhejiang Province, China. Active surveillance approaches based on the findings of the present study should be implemented, particularly in intensive care units, to combat the spread of ST11-CR-HvKP and to improve treatment., The emergence and spread of carbapenem-resistant hypervirulent Klebsiella pneumoniae sequence type 11 (ST11-CR-HvKP) in China are a great concern in the public health community. However, the underlying mechanism that enables its wide dissemination in China remains unclear. Here, we investigated the prevalence of carriage of carbapenemase-producing Enterobacteriaceae (CPE) among inpatients with diarrhea in a teaching hospital over 1 year to identify ST11-CR-HvKP reservoirs and to understand the genetic background and plasmid profiles of these pathogens. As assessed by stool analysis, the CPE colonization rate (12.4%) among the inpatients with diarrhea was high (12.4%). Antibiotic exposure, surgical history, and CPE positivity were correlated. Genomic investigation of 65 carbapenem-resistant K. pneumoniae isolates indicated a shared bacterial population in various wards. According to maximum likelihood phylogenetic tree analysis, these isolates were partitioned into three major clades. Analysis of the wzi locus revealed three different K types (KL105, KL47, and K64) among the ST11 isolates, indicating the genetic diversity of these isolates. Genetic and sequence mapping revealed the complexity of virulence and resistance plasmid sets harbored by the isolates. These observations indicate that the dissemination of resistant bacteria is more complex than initially anticipated and possibly involves multiple K. pneumoniae ST11 lineages and a variety of virulence plasmids. Collectively, we show for the first time that stool may be a source of ST11-CR-HvKP isolates. Furthermore, the findings reveal the silent dissemination of ST11-CR-HvKP bacteria in Zhejiang Province, China. Future investigations are warranted to determine the association between rectal colonization by ST11-CR-HvKP and clinical infections. IMPORTANCE China has been experiencing a rapid increase in the number of nosocomial infections caused by carbapenem-resistant Klebsiella pneumoniae ST11 (ST11-CRKP) for decades. The emergence of hypervirulent ST11-CRKP (ST11-CR-HvKP) strains is expected to become a serious public health issue in China, considering that carbapenem resistance and virulence have converged in an epidemic clone. K. pneumoniae strains that colonize the human intestinal tract may become a reservoir of virulence and carbapenemase-encoding genes. Here, we first characterized the genotypes and antimicrobial phenotypes of ST11-CR-HvKP strains isolated from diarrheal stool samples of inpatients in Zhejiang Province, China. Active surveillance approaches based on the findings of the present study should be implemented, particularly in intensive care units, to combat the spread of ST11-CR-HvKP and to improve treatment.

With its rare and naturally blue corolla, Plumbago auriculata Lam. has received attention and been introduced and planted worldwide. However, it is sensitive to low temperatures. Continuous low temperatures cause the stagnation of plant growth and the disappearance of ornamental traits. Currently, there is no report on the response of P. auriculata to low temperature. In this study, the chlorophyll fluorescence parameters of P. auriculata leaves were analysed after 10 days of low-temperature stress, and 943 differentially expressed genes were identified via high-throughput RNA sequencing. P. auriculata copes with low-temperature stress by changing the pathways of sugar metabolism, glutathione metabolism and lipid metabolism; however, the decrease in the assimilation level caused by damage to the photoreaction system is an important reason for plant growth stagnation. In addition, we identified 47 differentially expressed transcription factors, including the upregulated MYBS3 transcription factor, which is involved in the MYBS3-dependent pathway that is distinct from the DREB/CBF pathway. Our data reveal the cause of P. auriculata stagnation at low temperatures and provide valuable information for further studies on cold-response mechanisms and the genetic improvement of cold tolerance in P. auriculata.

Herein, a novel fluorescence method is reported for the detection of T4 polynucleotide kinase (T4 PNK) based on a FRET between dye-labeled DNA and polydopamine nanotubes (PDANTs). PDANTs can bind and quench the fluorescent dye-labeled single-stranded DNA (ssDNA) probes, whereas they have low affinity towards double-stranded DNA (dsDNA). In the presence of T4 PNK and ATP, λ exonuclease initiated the hydrolysis of the 5-phosphoryl termini of dsDNA and released ssDNA, which could strongly adsorb on the PDANTs and decrease the fluorescence intensity; on the basis of this concept, we successfully established a method for the detection of T4 PNK. The method displayed the detection limit of 0.01 U mL−1 with a linear range from 0.01 to 50 U mL−1. In addition, we investigated the effect of several inhibitors including Na2HPO4, (NH4)2SO4 and NaCl. The method provides a platform for the detection of T4 PNK and evaluation of inhibitors.

Background The purpose of the study is to discuss the correlation between the resistance rate of gram negative bacteria to fluoroquinolones (FQ) and antibiotic consumption intensity of 145 China tertiary hospitals in 2014. Methods This retrospective study adopted national monitoring data from 2014. Each participating hospital required to report annual consumption of each antibiotic, and the resistance rate of gram negative bacteria to FQ. Then the correlation between antibiotic usage and fluoroquinolones –resistant (FQR) rate was consequently investigated. Results One hundred forty-five hospitals were included in the study, and the median antibiotic consumption intensity was 46.30 (23.93–115.39) defined daily dosages (DDDs) per 100 patient-days. Cephalosporins ranks first in the antibiotics consumption, followed by fluoroquinolones, penicillins, and carbapenems. Fluoroquinolones resistance rate varied from hospital to hospital. The correlation analysis showed significant relationship between the percentage of FQR Escherichia coli and the consumption of FQs (r = 0.308, ppKlebsiella pneumoniae, not only FQs (r = 0.291, pppPseudomonas aeruginosa was observed to be correlated with the consumption of all antibiotics (r = 0.260, ppAcinetobacter baumannii was significantly correlated with the consumption of all antibiotics (r = 0.282, pppA. baumannii and the antibiotics consumption was not found. Conclusions A strong correlation was demonstrated between the antibiotic consumption and the rates of FQR gram-negative bacteria. As unreasonable antibiotics usage remains crucial in the proceeding of resistant bacteria selection, our study could greatly promote the avoidance of unnecessary antibiotic usage.

The objective of this study was to analyze erythromycin and clindamycin resistance patterns among different MRSA lineages in China. Antimicrobial susceptibility testing, resistance determinant screening, plasmid electroporation and sequence comparisons were performed. High rates of clindamycin (92.5%, 270/292) and erythromycin (92.8%, 271/292) resistance were observed. Additionally, 88.2% (60/68) of the ST59 MRSA isolates and 78.9% (15/19) of the ST239 MRSA isolates had constitutive resistance to clindamycin, while 82.0% (123/150) of the ST5 MRSA isolates showed inducible clindamycin resistance. The ermB gene was identified in 80.9% (55/68) of the ST59 isolates but was not detected in ST5 and ST239 MRSA isolates. Detection rates of ermA were high in the ST5 (99.3%, 149/150) and ST239 (89.5%, 17/19) MRSA isolates, but no ermA-positive ST59 MRSA isolates were identified. The ermC gene, observed to be harbored on similar, transmissible plasmids ranging in size from 2402 to 2473 bp, were found in different MRSA lineages. Summarily, high erythromycin and clindamycin resistance rates were observed in MRSA isolates. ST59 and ST239 MRSA isolates primarily exhibited constitutive resistance, while ST5 MRSA isolates showed inducible resistance phenotypes. ermA and ermB genes were frequently carried by specific MRSA clones, while ermC gene was present within small transmissible plasmids in all lineages. Erythromycin and clindamycin resistance genes transfer between MRSA isolates in healthcare settings remains a problem, and infection control procedures should be applied.

Catalpol, a major bioactive component from Rehmannia glutinosa, which has been used to treat diabetes. The present study was designed to elucidate the anti-diabetic effect and mechanism of action for catalpol in db/db mice. The db/db mice were randomly divided into six groups (10/group) according to their blood glucose levels: db/db control, metformin (positive control), and four dose levels of catalpol treatment (25, 50, 100, and 200 mg·kg-1), and 10 db/m mice were used as the normal control. All the groups were administered orally for 8 weeks. The levels of fasting blood glucose (FBG), random blood glucose (RBG), glucose tolerance, insulin tolerance, and glycated serum protein (GSP) and the globe gene expression in liver tissues were analyzed. Our results showed that catalpol treatment obviously reduced water intake and food intake in a dose-dependent manner. Catalpol treatment also remarkably reduce fasting blood glucose (FBG) and random blood glucose (RBG) in a dose-dependent manner. The RBG-lowering effect of catalpol was better than that of metformin. Furthermore, catalpol significantly improved glucose tolerance and insulin tolerance via increasing insulin sensitivity. Catalpol treatment significantly decreased GSP level. The comparisons of gene expression in liver tissues among normal control mice, db/db mice and catalpol treated mice (200 and 100 mg·kg-1) indicated that there were significant increases in the expressions of 287 genes, whichwere mainly involved in lipid metabolism, response to stress, energy metabolism, and cellular processes, and significant decreases in the expressions of 520 genes, which were mainly involved in cell growth, death, immune system, and response to stress. Four genes expressed differentially were linked to glucose metabolism or insulin signaling pathways, including Irs1 (insulin receptor substrate 1), Idh2 (isocitrate dehydrogenase 2 (NADP+), mitochondrial), G6pd2 (glucose-6-phosphate dehydrogenase 2), and SOCS3 (suppressor of cytokine signaling 3). In conclusion, catalpol ecerted significant hypoglycemic effect and remarkable therapeutic effect in db/db mice via modulating various gene expressions.

The Eastern Tianshan orogen contains many late Paleozoic porphyry Cu and magmatic Cu-Ni deposits. Recent studies demonstrate that several early Paleozoic volcanogenic massive sulfide (VMS) Cu-polymetallic and porphyry Cu deposits were discovered in the northern part of Eastern Tianshan. This study presents zircon U-Pb, whole-rock geochemical, and Sr-Nd isotopic data for granites and diorites from the Huangtupo VMS Cu-Zn deposit, northern part of the Eastern Tianshan. Our results can provide constraints on the genesis of intermediate and felsic intrusions as well as early Paleozoic geodynamic setting of the northern part of Eastern Tianshan. LA-ICP-MS zircon U-Pb analyses suggest that the granites and diorites were formed at 435 ± 2 Ma and 440 ± 2 Ma, respectively. Geochemical characteristics suggest that the Huangtupo granites and diorites are metaluminous rocks, exhibiting typical subduction-related features such as enrichment in LILE and LREE and depletion in HFSE. The diorites have moderate Mg#, positive eNd(t) values (+6.4 to +7.3), and young Nd model ages, indicative of a depleted mantle origin. The granites exhibit mineral assemblages and geochemical characteristics of I-type granites, and they have positive eNd(t) values (+6.7 to +10.2) and young Nd model ages, suggesting a juvenile crust origin. The early Paleozoic VMS Cu-polymetallic and porphyry Cu deposits in the northern part of Eastern Tianshan were genetically related. The formation of the early Paleozoic magmatic rocks as well as VMS and porphyry Cu deposits in the northern part of Eastern Tianshan was due to a southward subduction of the Junggar oceanic plate.

This study examined the effects of eugenosedin-A (Eu-A) in a streptozotocin (STZ)/nicotinamide-induced rat model of type II diabetes mellitus (T2DM). Six-week-old Sprague–Dawley rats were randomly divided into three groups: (1) RD group, normal rats fed a regular diet (RD), (2) DM group, T2DM rats fed a high-fat diet, and (3) Eu-A group, T2DM rats fed a high fat diet plus oral Eu-A (5 mg/kg/day). After 30 days, the DM group had higher body weight, higher blood glucose and lower insulin levels than the RD group. The DM group also had increased protein expression of glycogen synthase kinase (GSK) in liver and skeletal muscle and decreased protein expression of insulin receptor (IR), insulin receptor substrate-1 (IRS-1), IRS-2, AMP-activated protein kinase (AMPK), glucose transporter-4 (GLUT-4), glucokinase (GCK), and peroxisome proliferator-activated receptor γ (PPAR-γ). STZ/nicotinamide-induced T2DM increased the expression of mitogen-activated protein kinases (MAPKs: p38, ERK, JNK) and inflammatory p65 protein. In the Eu-A treated T2DM rats, however, blood glucose was attenuated and the insulin concentration stimulated. Changes in IR, IRS-1 and IRS-2 proteins as well as AMPK, GLUT-4, GCK, GSK, PPAR-γ, MAPKs, and inflammatory p65 proteins were ameliorated. These results suggested that Eu-A alleviates STZ/nicotinamide-induced hyperglycemia by improving insulin levels and glucose metabolism, and inhibiting the MAPKs- and p65-mediated inflammatory pathway.

Objective. The aim of this study was to perform molecular characterization for and determine the antimicrobial susceptibility profiles of Clostridium difficile collected from hospitals during a 4-year period (2009–2013) in China. Methods. Strains of toxigenic C. difficile were isolated from patients with diarrhoea, and this was followed by typing using multilocus sequence typing (MLST) and testing for susceptibility to 10 antimicrobials by using the E-test. The mechanisms of resistance to moxifloxacin, erythromycin, clindamycin and tetracycline were investigated by PCR. Results. A total of 405 non-duplicate toxigenic C. difficile isolates were identified, while 31 sequence types (STs) were identified. A predominant type, ST-54, accounted for 20.2 % of the STs, followed by ST-35 (16.3 %) and ST-37 (13.6 %). We found that 6.2 % of the isolates were binary toxin genes-positive, and 83.7 % of these belonged to ST-5. All of the isolates demonstrated 100 % susceptibility to first-line Clostridium difficile infection (CDI) therapies (i.e. metronidazole and vancomycin), while the resistance rates varied for the other antibiotics tested. Two hundred and ninety three (72.3 %) isolates were susceptible to moxifloxacin. All 112 moxifloxacin-resistant isolates had mutations resulting in an amino acid substitution in gryA and/or gyrB. The ermB gene was detected in 86.7 % (241/278) of the erythromycin- and clindamycin-resistant isolates, while the tetM gene was present in 97.1 % (85/87) of the tetracycline-resistant isolates. Conclusion. MLST typing revealed a wide variety of STs causing CDI, while ST-54 was the most common ST. All of the isolates were susceptible to metronidazole and vancomycin, while the resistance rates varied for the other antibiotics tested. There were no changes in the trends for the STs and antibiotic susceptibility profiles over 4 years.

BACKGROUND Hepatocellular carcinoma (HCC) is the most important cause of cancer-related deaths worldwide. Pirfenidone is an orally available small molecule with therapeutic potential for fibrotic diseases. MATERIAL AND METHODS In this study, we analyzed the effects of different pirfenidone concentrations on the proliferation of HepG2 HCC cells using Cell Counting Kit-8 (CCK-8) and colony formation assays. Flow cytometry was performed to measure the apoptotic effects of pirfenidone on HepG2 cells. Western blot analysis was performed to detect the expression of β-catenin and p-β-catenin. RESULTS Pirfenidone inhibited proliferation and promoted HepG2 cell apoptosis. In addition, Western blot results indicated that pirfenidone suppressed b-catenin expression in HepG2 cells. To assess the mechanism, we treated HepG2 cells with pirfenidone, and pirfenidone plus the β-catenin activator, SB-216763. The results revealed that SB-216763 accelerated proliferation and inhibited apoptosis in HepG2 cells treated with pirfenidone. Western blot results showed that SB-216763 upregulated β-catenin expression in HepG2 cells treated with pirfenidone. CONCLUSIONS In conclusions, pirfenidone may be a potential drug for HCC treatment.

Physiological responses of rice (Oryza sativa L. cv. XZX 45) seedlings to N,N-dimethyl formamide (DMF) were evaluated using chemometric modeling. Thirteen selected physiological parameters of the plants were monitored to evaluate the phytotoxicity from DMF exposure. The data matrix was further treated with cluster analysis and principal component analysis (PCA). Cluster analysis (CA) rendered two distinct clusters, suggesting different responses to DMF exposure between the root and shoot. PCA results suggested that relative growth rate (RGR), water use efficiency (WUE), malondialdehyde (MDA), soluble sugar (SS), proline, protein, Evan blue uptake (EBU), catalase (CAT), and peroxidase (POD) were dominant in root tissues, whereas carotenoids and total chlorophyll were most common in the shoot of DMF-treated rice seedlings. Additionally, analysis of red list index (RLI) was conducted to calculate the sensitivity index among all selected parameters in the shoots and roots at DMF EC10 and EC50 concentrations. The most sensitive parameter to DMF exposure in the shoots was proline and RGR at EC50 concentration, whereas it was EBU in root tissues. Superoxide dismutase (SOD) and CAT activities were the most sensitive parameters to DMF stress in the roots and shoots at EC10 concentration, respectively. Chemometric analysis suggested that DMF exposure caused phytotoxic stress to young rice seedlings, but plant tissues responded differently.

Objectives: To investigate the population structure, drug resistance mechanisms and plasmids of community-associated Enterobacter cloacae complex (CA-ECC) isolates in China.Methods: Sixty-two CA-ECC isolates collected from 31 hospitals across China were typed by hsp60 typing and MLST. ESBL and AmpC-overexpression phenotype was determined by double-disc synergy test. Replicon typing and conjugation were performed for plasmid analysis. All ESBL-positive isolates and representative conjugants were subjected to detailed characterization by WGS.Results: Enterobacter hormaechei and Enterobacter kobei were predominant in our collections. MLST distinguished 46 STs with a polyclonal structure. ST591 was the most prevalent clone detected in northern China. Twenty-two isolates (35.5%) were ESBL positive and half of them were E. kobei. ESBL positivity was related to ESBL production (15/22) and to AmpC overexpression (18/22). Core-genome phylogenetic analysis identified intra- and inter-regional dissemination of ESBL-producing E. kobei clones. ESBL producers were exclusively classified as E. hormaechei and E. kobei, and blaCTX-M-3 was the most prevalent ESBL genotype (10/15) detected in four different environments. In the ESBL-positive population, the ESBL producers encoded more drug resistance genes (8-24 genes) by carrying more plasmids (1-3 plasmids) than the non-ESBL-producing isolates, resulting in an inter-group difference in drug susceptibilities. IncHI-type plasmids were prevalent in the ESBL producers (12/15). All IncHI2-type plasmids (n = 11) carried ESBL genes and shared a similar backbone to p09-036813-1A_261 recovered from Salmonella enterica in Canada.Conclusions: The species-specific distribution, species-dependent ESBL mechanism and endemic plasmids identified in our study highlight the necessity for tailored surveillance of CA-ECC in the future.

The increasing prevalence of KPC-producing Klebsiella pneumoniae (KPC-Kp) strains poses a serious threat to patients. Therapeutic options are limited to colistin, fosfomycin, tigecycline and selected aminoglycosides. Although the combination of fosfomycin with other antimicrobials is recommended, data regarding possible synergistic activity in vitro and in vivo appear inconsistent. Here we report that five drug combinations (fosfomycin combined with imipenem, ertapenem, tigecycline, colistin or amikacin) had a significant additive effect against 136 KPC-Kp strains in an in vitro chequerboard assay. In addition, time-kill assays revealed that fosfomycin enhanced the bactericidal activity of the five other antimicrobial agents. Moreover, owing to its persistent bactericidal effect, the combination of fosfomycin plus amikacin is an effective therapeutic candidate for infections by KPC-producing organisms.