Molecular Characteristics of Antimicrobial Resistance and Virulence in Klebsiella pneumoniae Strains Isolated from Goose Farms in Hainan, China.

We retrospectively investigated 326 samples that were collected from goose farms in Hainan Province, China, in 2017. A total of 33 carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates were identified from 326 samples, and the 33 CRKP isolates were characterized based on whole-genome sequencing (WGS) data from the Illumina and Oxford Nanopore Technologies (ONT) platforms. All of these 33 CRKP isolates possessed blaNDM-5, and a single isolate coharbored mcr-1 and blaNDM-5, while 4 isolates carried multiple virulence and metal tolerance gene clusters. One CRKP strain (CMG-35-2) was selected for long sequence reading. A hybrid plasmid carrying the virulence, resistance, and metal resistance gene in the strain was found. It possessed 2 backbones [IncFIB(K)-IncFII(K)] within a single plasmid that were closely related to K. pneumoniae plasmids from a human-associated habitat in the United States and from a human isolate in Hong Kong. A mouse abdominal infection model indicated that that strain was of the moderate virulence phenotype. This study revealed that K. pneumoniae on goose farms is an important reservoir for blaNDM-5 and these bacteria are represented by a diversity of sequence types. The heterozygous multiple drug resistance genes carried on plasmids highlighted the genetic complexity of CRKP and the urgent need for continued active surveillance. IMPORTANCE CRKP is one of the most important pathogens, which can cause infection not only in humans but also in waterfowl. The discovery of blaNDM-5-producing K. pneumoniae in waterfowl farms in recent years suggests that waterfowl are an important reservoir for blaNDM-5-producing Enterobacteriaceae. However, there are few studies on the spread of blaNDM-5-producing bacteria in waterfowl farms. Our study showed that the IncX3 plasmid carrying blaNDM-5 in goose farms is widely present in K. pneumoniae isolates and a large number of resistance genes are accumulated in it. We found a transferable IncFIB-FII hybrid plasmid that combines virulence, resistance, and metal resistance genes, which allow transfer of these traits between bacteria in different regions. The results of this study contribute to a better understanding of the prevalence and transmission of carbapenem-resistant K. pneumoniae in goose farms.

Blood cell characterization and transcriptome analysis reveal distinct immune response and host resistance of different ploidy cyprinid fish following Aeromonas hydrophila infection.

Aeromonas hydrophila can pose a great threat to survival of freshwater fish. In this study, A. hydrophila infection could decrease blood cell numbers, promote blood cell damage as well as alter the levels of alkaline phosphatase (ALP), lysozyme (LZM), aspartate aminotransferase (AST), total antioxidant capacity (T-AOC), total superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) in immune-related tissues of red crucian carp (RCC, 2 N = 100) and triploid cyprinid fish (3 N fish, 3 N = 150). In addition, the significant alternation of antioxidant status was observed in PBMCs isolated from RCC and 3 N following LPS stimulation. The core differential expression genes (DEGs) involved in apoptosis, immunity, inflammation and cellular signals were co-expressed differentially in RCC and 3 N following A. hydrophila challenge. NOD-like receptor (NLR) signals appeared to play a critical role in A. hydrophila-infected fish. DEGs of NLR signals in RCCah vs RCCctl were enriched in caspase-1-dependent Interleukin-1ß (IL-1ß) secretion, interferon (IFN) signals as well as cytokine activation, while DEGs of NLR signals in 3Nah vs 3Nctl were enriched in caspase-1-dependent IL-1ß secretion and antibacterial autophagy. These results highlighted the differential signal regulation of different ploidy cyprinid fish to cope with bacterial infection.

Transcriptome analysis and co-expression network reveal the mechanism linking mitochondrial function to immune regulation in red crucian carp (Carassius auratus red var) after Aeromonas hydrophila challenge.

Aeromonas hydrophila is a common pathogen of freshwater fish. In this study, A. hydrophila infection was shown to cause tissue damage, trigger physiological changes as well as alter the expression profiles of immune- and metabolic-related genes in immune tissues of red crucian carp (RCC). Transcriptome analysis revealed that acute A. hydrophila infection exerted a profound effect on mitochondrial oxidative phosphorylation linking metabolic regulation to immune response. In addition, we further identified cellular senescence, apoptosis, necrosis and mitogen-activated protein kinase signal pathways as crucial signal pathways in the kidney of RCC subjected to A. hydrophila infection. These findings may have important implications for understanding modulation of immunometabolic response to bacterial infection.

A Transposon-Associated CRISPR/Cas9 System Specifically Eliminates both Chromosomal and Plasmid-Borne mcr-1 in Escherichia coli.

The global spread of antimicrobial-resistant bacteria has been one of the most severe threats to public health. The emergence of the mcr-1 gene has posed a considerable threat to antimicrobial medication since it deactivates one last-resort antibiotic, colistin. There have been reports regarding the mobilization of the mcr-1 gene facilitated by ISApl1-formed transposon Tn6330 and mediated rapid dispersion among Enterobacteriaceae species. Here, we developed a CRISPR/Cas9 system flanked by ISApl1 in a suicide plasmid capable of exerting sequence-specific curing against the mcr-1-bearing plasmid and killing the strain with chromosome-borne mcr-1. The constructed ISApl1-carried CRISPR/Cas9 system either restored sensitivity to colistin in strains with plasmid-borne mcr-1 or directly eradicated the bacteria harboring chromosome-borne mcr-1 by introducing an exogenous CRISPR/Cas9 targeting the mcr-1 gene. This method is highly efficient in removing the mcr-1 gene from Escherichia coli, thereby resensitizing these strains to colistin. The further results demonstrated that it conferred the recipient bacteria with immunity against the acquisition of the exogenous mcr-1 containing the plasmid. The data from the current study highlighted the potential of the transposon-associated CRISPR/Cas9 system to serve as a therapeutic approach to control the dissemination of mcr-1 resistance among clinical pathogens.

Re-engineering a mobile-CRISPR/Cas9 system for antimicrobial resistance gene curing and immunization in Escherichia coli.

OBJECTIVES: In this study, we developed an IS26-based CRISPR/Cas9 system as a proof-of-concept study to explore the potential of a re-engineered bacterial translocatable unit (TU) for curing and immunizing against the replication genes and antimicrobial resistance genes. METHODS: A series of pIS26-CRISPR/Cas9 suicide plasmids were constructed, and specific guide RNAs were designed to target the replication gene of IncX4, IncI2 and IncHI2 plasmids, and the antibiotic resistance genes mcr-1, blaKPC-2 and blaNDM-5. Through conjugation and induction, the transposition efficiency and plasmid-curing efficiency in each recipient were tested. In addition, we examined the efficiency of the IS26-CRISPR/Cas9 system of cell immunity against the acquisition of the exogenous resistant plasmids by introducing this system into antimicrobial-susceptible hosts. RESULTS: This study aimed to eliminate the replication genes and antimicrobial resistance genes using pIS26-CRISPR/Cas9. Three plasmids with different replicon types, including IncX4, IncI2 and IncHI2 in three isolates, two pUC19-derived plasmids, pUC19-mcr-1 and pUC19-IS26mcr-1, in two lab strains, and two plasmids bearing blaKPC-2 and blaNDM-5 in two isolates were all successfully eliminated. Moreover, the IS26-based CRISPR/Cas9 system that remained in the plasmid-cured strains could efficiently serve as an immune system against the acquisition of the exogenous resistant plasmids. CONCLUSIONS: The IS26-based CRISPR/Cas9 system can be used to efficiently sensitize clinical Escherichia coli isolates to antibiotics in vitro. The single-guide RNAs targeted resistance genes or replication genes of specific incompatible plasmids that harboured resistance genes, providing a novel means to naturally select bacteria that cannot uptake and disseminate such genes.

Upregulation of oxidative stress by triphenyl phosphate (TPhP) exposure causes antioxidant insult and apoptotic process in Epithelioma papulosum cyprini (EPC) cells.

Triphenyl phosphate (TPhP) is the predominant compound of organophosphate flame retardants (OPFRs), which can elicit a toxicological effect on physiological response and tissue development of fish. In this study, we investigated the effect of TPhP exposure on cell viability, antioxidant capacities, and apoptosis in EPC cells. Current study revealed that TPhP exposure could decrease cell viability and promote intracellular oxidative stress in EPC cells. In addition, high-dose TPhP exposure could facilitate antioxidant insults and cause mitochondrial collapse in a dose-dependent manner, along with increased gene expressions involved in apoptosis and unfolded protein response (UPR). These results indicated that reactive oxygen species (ROS)-induced cytotoxic stress and cell death were involved in antioxidant insults and apoptotic activation in TPhP-exposed fish cells.

Gut-liver immune and redox response in hybrid fish (Carassius cuvieri ♀ × Carassius auratus red var. ♂) after gut infection with Aeromonas hydrophila.

Aeromonas hydrophila can pose a great threat to fish survival. In this study, we investigated the differential immune and redox response in gut-liver axis of hybrid fish (WR) undergoing gut infection. WR anally intubated with A. hydrophila showed severe midgut injury with decreased length-to-width ratios of villi along with GC hyperplasia and enhanced antioxidant activities, but expression profiles of cytokines, chemokines, antibacterial molecules, redox sensors and tight junction proteins decreased dramatically. In contrast, immune-related gene expressions and antioxidant activities increased significantly in liver of WR following gut infection with A. hydrophila. These results highlighted the differential immune regulation and redox balance in gut-liver axis response to bacterial infection.

[Identification of sarcosaphagous Calliphorid flies by analyzing the sequence of 16S rDNA].

OBJECTIVE: To explore the application of a 289bp fragment of the 16S rDNA gene to identify various species of sarcosaphagous Calliphorid flies. METHODS: Twenty-six Calliphorid flies were collected from 14 Chinese provinces. All specimens were properly assigned into three genera and six species. The DNA of the pectoralis was extracted using CTAB method. Then PCR amplification was done for the 289 bp fragment of the 16S rDNA gene. The PCR products were then purified and sequenced, and the obtained sequences were uploaded to GenBank. The phylogenetic tree was built by the neighbor-joining method and intraspecific and interspecific divergences were calculated by sequence analysis. RESULTS: The above 26 sarcosaphagous flies could be well clustered according to different genera and species. The evolutional intraspecific values were all zero, the evolutional interspecific variations varied from 0.3% to 6.5%. CONCLUSION: The 289 bp fragment of the 16S rDNA of sarcosaphagous flies can be effectively used to identify most of the flies at species level. This method appears to be fast and low dissipative, which might be used to estimate postmortem interval by sarcosaphagous flies.

NDM-5-Producing Escherichia coli Co-Harboring mcr-1 Gene in Companion Animals in China.

Carbapenem and colistin are important antibiotics for the treatment of infections caused by multidrug-resistant Gram-negative pathogens. Here, we isolated the blaNDM-5-harboring Escherichia coli in companion animals in healthy or diseased companion animals from veterinary clinics in six cities in China from July to November 2016. A total of 129 rectal swabs of healthy or diseased dogs and cats were collected from veterinary clinics in six different cities in China, and the isolates were subjected to carbapenem and colistin susceptibility testing. Resistance genes were confirmed using PCR. Conjugation experiments were conducted to determine the transferability of antibiotic resistance genes (ARGs) in the strains. The isolated rate of blaNDM-5-harboring E. coli strains was 3.88% (five strains). These five strains were multidrug resistant to at least three antibiotics and corresponded to four sequence types including ST101. The blaNDM-5 gene was located on 46 kb IncX3 plasmids in these five strains, and the genetic contexts were shared and were nearly identical to the K. pneumoniae plasmid pNDM5-IncX3 from China. In addition, one strain (CQ6-1) co-harbored blaNDM-5-encoding-IncX3 plasmid along with a mcr-1-encoding-IncX4 plasmid, and their corresponding genetic environments were identical to the blaNDM-5-IncX3 and mcr-1-IncX4 hybrid plasmid reported previously from the same area and from the same clinic. The results indicated that the similar genetic contexts were shared between these isolates from companion animals, and the IncX3-type plasmids played a key role in the spread of blaNDM-5 among these bacteria.

Molecular Epidemiology of New Delhi Metallo-ß-Lactamase-Producing Escherichia coli in Food-Producing Animals in China.

We conducted a molecular surveillance study for carbapenem-resistant Enterobacteriaceae (CRE) colonization in food-producing animals in China that included primarily swine and poultry for three consecutive years. A total of 2,771 samples from food-producing animals and their surrounding environments were collected from different regions in China from 2015 to 2017. Enrichment cultures supplemented with meropenem were used to isolate carbapenem non-susceptible isolates and these were subsequently identified by MALDI-TOF MS. Resistance phenotypes and genotypes were confirmed using antimicrobial susceptibility testing and molecular biological techniques. Genomic characteristics of the carbapenemase-producing isolates were investigated using whole genome sequencing (WGS) and bioinformatic analysis. In total, 88 NDM-positive Enterobacteriaceae were identified from 2,771 samples and 96.6% were Escherichia coli. The New Delhi metallo-ß-lactamase (NDM)-positive E. coli displayed a diversity of sequence types (ST), and ST48 and ST165 were the most prevalent. Three variants of bla NDM (bla NDM-1, bla NDM-4, and bla NDM-5) were detected and WGS indicated that bla NDM-5 predominated and was carried primarily on IncX3 plasmids. All these isolates were also multiply-drug resistant. These results revealed that food-producing animals in China are an important reservoir for NDM-positive E. coli and pose a potential threat to public health.

Acupuncture for post-stroke cognitive impairment: a systematic review and meta-analysis.

OBJECTIVES: The aim of this study was to evaluate the effectiveness and safety of acupuncture for the treatment of post-stroke cognitive impairment (PSCI). METHODS: The Cochrane Library, Embase, Medline, China National Knowledge Infrastructure (CNKI), Chinese Science and Technology Periodical (VIP), Wanfang, and Chinese Biological Medicine (CBM) databases were electronically searched from their inception to 10 April 2019. The Montreal Cognitive Assessment (MoCA) scale and Mini-Mental State Examination (MMSE) scale were used as outcomes to assess effectiveness with respect to cognitive function. Assessment of risk of bias (ROB) and Grading of Recommendations Assessment, Development, and Evaluation (GRADE) assessment were performed by two reviewers independently. Data were analyzed using Review Manager (RevMan) 5.3. RESULTS: A total of 28 trials with 2144 participants were included in the qualitative synthesis and meta-analysis. Four of the 28 trials (14%) were assessed as being at overall low ROB, 24 of the 28 trials (86%) were assessed as having overall high ROB. The quality of evidence for both MoCA and MMSE were deemed to be very low by the GRADE criteria. Results indicated that acupuncture groups may be benefiting more than non-acupuncture groups with respect to variation of MoCA scores (merged mean difference (MMD): 2.66, 95% confidence interval (CI): 2.18 to 3.13, p < 0.00001; heterogeneity: χ2 = 35.52, p = 0.0007, I2 = 63%), and the heterogeneity decreased in both subgroup analysis and sensitivity analysis. In addition, acupuncture groups might be benefiting more than non-acupuncture groups in terms of changes in MMSE score (MMD = 2.97, 95% CI = 2.13 to 3.80, p < 0.00001; heterogeneity: χ2 = 269.75; p < 0.00001; I2 = 92%), and the heterogeneity decreased in subgroup analysis. Only one RCT addressed adverse events, and the symptoms were mild and did not affect treatment and evaluation. CONCLUSION: Acupuncture could be effective and safe for PSCI. Nevertheless, the results should be interpreted cautiously due to the high ROB of included trials and very low quality of evidence for assessed outcomes.

Identification of the Plasmid-Mediated Colistin Resistance Gene mcr-1 in Escherichia coli Isolates From Migratory Birds in Guangdong, China.

We determined the prevalence and transmission characteristics of mcr-1-positive Escherichia coli (MCRPEC) isolates from migratory birds Anser indicus in Guangdong, China. We identified 22 MCRPEC from 303 A. indicus fecal samples (7.3%) in Guangzhou, Zhaoqing, and Futian. The mcr-1 gene coexisted with 24 other types of antibiotic resistance genes (ARG), and 11 ARGs were highly prevalent at levels >50%. The MCRPEC displayed a diversity of sequence types (ST), and 19 distinct STs were identified with ST10, ST1146, and ST1147 as the most prevalent. In addition, these MCRPEC from birds were closely related phylogenetically to those from other sources in China. Whole-genome sequencing analysis demonstrated that mcr-1 was located on IncX4 (n=9, 40.9%), IncI2 (n=5, 22.7%) and IncP (n=1, 4.5%) plasmids and the latter shared an identical plasmid backbone with other sources. These results highlight the significance of migratory birds in the transmission of antibiotic resistance and provide powerful evidence that migratory birds are potential transmitters of antibiotic resistance.

Acupuncture for amnestic mild cognitive impairment: A pilot multicenter, randomized, parallel controlled trial.

INTRODUCTION: Patients with amnesic mild cognitive impairment (aMCI) are more likely to develop Alzheimer disease than corresponding age normal population. Because Alzheimer disease is irreversible, early intervention for aMCI patients seems important and urgent. We have designed a pilot multicenter, randomized, parallel controlled trial to assess the efficacy and safety of acupuncture on aMCI, explore the feasibility of acupuncture in the treatment of aMCI, so as to provide a reference for large-sample clinical trials in the next stage. METHOD: We designed a pilot multicenter, randomized, parallel controlled trial. This trial aims to test the feasibility of carrying out a large-sample clinical trial. In this trial, 50 eligible patients with aMCI will be included and allocated to acupuncture group (n = 25) or sham acupuncture group (n = 25) at random. Subjects will accept treatment 2 times a week for 12 weeks continuously, with a total of 24 treatment sessions. We will select 6 acupoints (GV20, GV14, bilateral BL18, bilateral BL23). For the clinical outcomes, the primary outcome is Montreal cognitive assessment, which will be assessed from baseline to the end of this trial. And the secondary outcomes are Mini-mental State Examination, Delayed Story Recall, Clinical Dementia Rating scale, Global Deterioration Scale, Activity of Daily Life, Alzheimer Disease Assessment Scale-Cognitive Section, brain magnetic resonance imaging, brain functional magnetic resonance imaging, and event-related potential P300, which will be assessed before and after treatment. In addition, we will assess the safety outcomes from baseline to the end of this trial and feasibility outcome after treatment. We will evaluate neuropsychological assessment scale (Montreal cognitive assessment, Mini-mental State Examination, Alzheimer Disease Assessment Scale-Cognitive Section) at 3 months and 6 months after treatment. DISCUSSION: This pilot trial aims to explore the feasibility of the trial, verify essential information of its efficacy and safety. This pilot study will provide a preliminary basis for carrying out a larger clinical trial of acupuncture on aMCI in near future.

Prevalence of tet(X4) in Escherichia coli From Duck Farms in Southeast China.

OBJECTIVES: Carbapenems, colistin, and tigecycline are critically important antibiotics in clinics. After the global appearance of bla NDM and mcr mediating the resistance to carbapenems and colistin, respectively, tigecycline becomes the last-resort drug against severe human infections caused by multidrug-resistant bacteria. Recently, a mobile tigecycline resistance gene tet(X4) has been identified in Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii that causes high resistance to tigecycline and other tetracyclines. In this study, the prevalence of tet(X4) in E. coli isolates from duck and goose farms in Southeast China was identified and characterized. METHODS: Feces, soil, sewage, and dust samples were collected from duck and goose farms along with the southeast coast provinces of China. Antimicrobial susceptibility testing and polymerase chain reaction screening were performed to investigate the phenotype and genotype of tigecycline resistance. Conjugation, S1 pulsed-field gel electrophoresis (PFGE), and whole-genome sequencing were used to determine the transferability, genetic location, and the genomic characteristics of tet(X4). RESULTS: In total, 1,716 samples were collected, and 16 isolates (0.9%) recovered from Guangdong, Shandong, and Jiangsu were positive for tet(X4) gene with tigecycline minimum inhibitory concentrations ≥16 mg/L. Notably, among these tet(X4)-positive E. coil isolates, seven of them were from the environment samples (soil and sewage). PFGE and multilocus sequence typing demonstrated that ST3997 was the most prevalent sequence type (eight isolates, 50%) in Jiangsu province. By conjugation assays, 11 isolates were able to transfer tet(X4) plasmid to E. coli C600 recipient, and these plasmids belonged to IncHI1 and IncX1 detected by sequence analysis. tet(X4) was found adjacent to an insertion sequence ISCR2 downstream and a catD gene upstream for all isolates. In addition, multiple-drug resistance to tigecycline, chlortetracycline, ampicillin, florfenicol, ciprofloxacin, gentamicin, trimethoprim/sulfamethoxazole, and fosfomycin was profiled in most of the tet(X4)-positive isolates. CONCLUSION: The identification of tet(X4) harboring E. coli strains in duck farms and their surrounding environment enlarges our knowledge of the variety and prevalence of tigecycline resistance. The prevalence of tet(X4) raises concern for the use of tetracyclines in animal farming, and the tet(X4) gene should be listed as primary gene for resistance surveillance.

Acupuncture therapy for poststroke spastic hemiplegia: A systematic review and meta-analysis of randomized controlled trials.

BACKGROUND: Hemiplegia after stroke is one of the main dysfunctions in stroke patients. Acupuncture had been widely used for poststroke spastic hemiplegia (PSSH), but the efficiency was unclear. This study aimed to examine the efficiency and safety of acupuncture for individuals with PSSH. METHODS: We searched nine databases from their inception to 27th July 2019. Randomized controlled trials (RCTs) of acupuncture for the treatment of PSSH met the screening criteria. The quality of methodology was evaluated by Cochrane's risk of bias tool. RevMan 5.3 was used to perform the meta-analysis. The primary outcome was the Fugl-Myer Assessment (FMA) score, and the secondary outcomes were the Ashworth Scale for Spasticity (ASS) and Barthel Index (BI) scores. To evaluated the safety of acupuncture therapy, researchers scanned the full text to collect adverse events. RESULTS: Researchers retrieved 2452 articles in total, after screening, thirty-eight studies with 2628 participants of were included. In this meta-analysis, twenty-seven trials revealed that acupuncture therapy was associated with an increase in FMA scores compared with rehabilitation training (RT) (MD: 8.43, 95% CI, 6.57 to 10.28, p < 0.00001, I2 = 75%). According to the analysis of subgroup of interventions, ten trials showed that manual acupuncture (MA) plus RT was associated with an increase in FMA compared with RT (MD: 10.84, 95% CI, 9.29 to 13.29, p < 0.00001, I2 = 24%), three trials showed that electroacupuncture (EA) plus RT was associated with an improvement in FMA compared with RT (MD: 9.44, 95% CI, 1.00 to 17.88, p = 0.03, I2 = 81%), twelve trials showed that MA was associated with an increase in FMA compared with RT (MD: 5.48, 95% CI, 2.07 to 8.89, p = 0.002, I2 = 74%), and one trials showed that EA was associated with an improvement in FMA compared with RT (MD: 11.35, 95% CI, 5.03 to 17.67, p = 0.0004). According to the analysis of subgroup of treatment duration, four trials used acupuncture therapy for more than 1 month, revealed that acupuncture therapy was associated with an increase in FMA scores compared with RT (MD: 9.24, 95% CI, 0.42 to 18.06, p = 0.04, I2 = 93%). Thirty-two trials used acupuncture therapy for less than or equal to 1 month, revealed that acupuncture therapy was associated with an increase in FMA scores compared with RT (MD: 8.32, 95% CI, 6.56 to 10.09, p < 0.00001, I2 = 61%). Six trials indicated that acupuncture therapy was better than RT in terms of the ASS (MD: 0.46, 95% CI, -0.65 to -0.27, p < 0.00001, I2 = 67%), twenty-four trials indicated that acupuncture therapy was better than RT in terms of the BI scores (MD: 8.32, 95% CI, 5.30 to 11.35, p < 0.00001, I2 = 88%). In general, the methodologies of the RCTs were of poor quality. Two RCTs reported no adverse events, one trial reported five adverse events without severe influence, others did not mention. CONCLUSIONS: This review discovered that acupuncture might be a safe and effective adjuvant therapy for individuals with PSSH. Nevertheless, there were methodological limitations in the included RCTs, and well-designed and large-scale studies should be carried out to confirm our results.

[Spatio-temporal dynamics of woody plants seed rains in broad-leaved Korean pine mixed forest in Changbai Mountains form 2006 to 2017, China.] / 2006­2017年长白山阔叶红松林木本植物种子雨的时空动态.

Seeds are the basis for forest regeneration. To examine the composition and spatio-temporal dynamics of seed rains, a total of 150 seed traps of 0.5 m2 were installed in a 25 hm2 broad-leaved Korean pine (Pinus koraiensis) mixed forest plot in Changbai Mountains. With a total of 252 collections from May 2006 to September 2017, we collected 764299 mature and immature seeds which were belonged to 27 species, 17 genera, and 12 families. More than 90% of all collected seeds (704231 seeds) were from 13 canopy species. Seeds of four tree species, including Tilia amurensis, Fraxinus mandschurica, Acer mono, and Acer pseudo-sieboldianum could be collected every year from each trap. Mast-seeding was found in every canopy layer, but it happened one to two years earlier in the overstorey layer than midstorey and understorey layer. Almost all species produced seeds in autumn, with considerable spatiotemporal variation. Generally, the spatial variation of seeds was larger than temporal variation. Compared with annual variation coefficient of seeds in tropical forest of the Barro Colorado Island (BCI) and subtropical evergreen forest in the Gutianshan, annual variation coefficient of seeds in Changbai Mountains was higher, which supported the hypothesis that annual variation in seed rains would be lower in the tropics than that in higher latitudes.

The role of breeding system in community dynamics: Growth and mortality in forests of different successional stages.

Plant sexual systems appear to play an important role in community assembly: Dioecious species are found to tend to have a higher propensity to colonize communities in early successional stages. Here, we test two demographic hypotheses to explain this pattern in temperate forests. First, we test demographic differences between hermaphrodite and dioecious species in stressful younger successional stages: Previous theory predicts that hermaphrodite seed production is more harmed in stressful environments than that of dioecious populations leading to an advantage for females of dioecious species. Second, in primary forest, we hypothesized that dioecious species would show demographic advantage over monomorphic ones. We used data from two temperate forest plots in Northeast China surveyed over 10 years to compare the rates of growth and mortality of tree species with contrasting breeding systems in both secondary and primary forests. We assessed the effect of breeding system on the growth-mortality trade-off, while controlling for other traits usually considered as correlates of growth and mortality rates. We show that in the secondary forest, dioecious species showed weak advantage in demographic rates compared with monomorphic species; dioecious species showed considerably both lower relative growth and mortality rates compared to the hermaphrodites in the primary forest over 10 years, consistent with a priori predictions. Hermaphrodites showed strong growth-mortality trade-offs across forest stages, even when possibly confounding factors had been accounted for. These results suggest that sexual system influences community succession and assembly by acting on the rates of growth and mortality, and the trade-off between them. As vegetation develops, the demographic differences between breeding systems are much larger. Our results demonstrate the association between breeding system, succession, and community assembly and that this relationship is succession-stage dependent. Our findings support the suggestion that the demographic advantage of dioecious species facilitates the coexistence of sexual systems in primary forest.

Conspecific density dependence and community structure: Insights from 11 years of monitoring in an old-growth temperate forest in Northeast China.

Forest community structure may be influenced by seedling density dependence, however, the effect is loosely coupled with population dynamics and diversity in the short term. In the long term the strength of conspecific density dependence may fluctuate over time because of seedling abundance, yet few long-term studies exist. Based on 11 years of seedling census data and tree census data from a 25-ha temperate forest plot in Northeast China, we used generalized linear mixed models to test the relative effects of local neighborhood density and abiotic factors on seedling density and seedling survival. Spatial point pattern analysis was used to determine if spatial patterns of saplings and juveniles, in relation to conspecific adults, were in accordance with patterns uncovered by conspecific negative density dependence at the seedling stage. Our long-term results showed that seedling density was mainly positively affected by conspecific density, suggesting dispersal limitation of seedling development. The probability of seedling survival significantly decreased over 1 year with increasing conspecific density, indicating conspecific negative density dependence in seedling establishment. Although there was variation in conspecific negative density dependence at the seedling stage among species and across years, a dispersed pattern of conspecific saplings relative to conspecific adults at the local scale (<10 m) was observed in four of the 11 species examined. Overall, sapling spatial patterns were consistent with the impacts of conspecific density on seedling dynamics, which suggests that conspecific negative density dependence is persistent over the long term. From the long-term perspective, conspecific density dependence is an important driver of species coexistence in temperate forests.

[Factors influencing tree radial growth of three common species in broad-leaved Korean pine mixed forests in Changbai Mountains, China]. / 长白山阔叶红松林3ä¸ªå¸¸è§æ ‘ç§å¾„å‘ç”Ÿé•¿çš„å½±å“å› ç´ .

To understand the factors influencing tree radial growth, we analyzed the seasonal dynamics of tree growth of 3 common species (Pinus koraiensis, Tilia amurensis, Quercus mongolica), compared interspecific growth rates and explored the effects of size, neighborhood competition, soil and topography based on five years dendrometer bands monitoring data of the 3 common species in a broad-leaved Korean pine (P. koraiensis) mixed forest plot in Changbai Mountain, Northeast China. The results showed that the growth dynamics of 3 species were consistent. Trees began to grow in late May, thrived in July, grew at declining rates from late August and stopped growing in late October. Annual relative growth rates were significantly different among the species. Q. mongolica tended to grow faster than the other two species, and the differences of growth rates among the 3 species were especially large for small and medium trees. Tree growth rates of P. koraiensis and Q. mongolica were strongly decreased by neighborhood competition, while tree growth rate of T. amurensis was significantly related to tree size, soil and topography.

Integrated analysis of microRNA and mRNA expression profiles in HBx-expressing hepatic cells.

AIM: To identify the miRNA-mRNA regulatory network in hepatitis B virus X (HBx)-expressing hepatic cells. METHODS: A stable HBx-expressing human liver cell line L02 was established. The mRNA and miRNA expression profiles of L02/HBx and L02/pcDNA liver cells were identified by RNA-sequencing analysis. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis was performed to investigate the function of candidate biomarkers, and the relationship between miRNA and mRNA was studied by network analysis. RESULTS: Compared with L02/pcDNA cells, 742 unregulated genes and 501 downregulated genes were determined as differentially expressed in L02/HBx cells. Gene ontology analysis suggested that the differentially expressed genes were relevant to different biological processes. Concurrently, 22 differential miRNAs were also determined in L02/HBx cells. Furthermore, integrated analysis of miRNA and mRNA expression profiles identified a core miRNA-mRNA regulatory network that is correlated with the carcinogenic role of HBx. CONCLUSION: Collectively, the miRNA-mRNA network-based analysis could be useful to elucidate the potential role of HBx in liver cell malignant transformation and shed light on the underlying molecular mechanism and novel therapy targets for hepatocellular carcinoma.