Long-term application of nitrogen and phosphorus fertilizers changes the process of community construction by affecting keystone species of crop rhizosphere microorganisms.

Keystone species of microbial communities play a very important role in community structure and ecosystem function; however, the effect of long-term nitrogen (N) and phosphorus (P) fertilizers on key taxa and the mechanisms of community construction of rhizosphere microbial communities remain unclear. In this study, the effect of nine fertilization treatments (N0P0, N0P1, N0P2, N1P0, N1P1, N1P2, N2P0, N2P1, and N2P2) on soil microbial community diversity, keystone species, and construction methods in the crop rhizosphere were studied in a loess hilly area after 26 years of fertilization. The results showed that fertilization significantly increased the nutrient contents of the rhizospheric soil and root system and significantly affected microbial community composition (based on the Bray-Curtis distance) and community construction process (ß-nearest taxon index: ßNTI). The decrease in the abundance of oligotrophic bacteria (from phyla Acidobacteriota and Chloroflexi) in the keystone species of bacterial communities shifted the community construction process from homogenizing dispersal to variable selection process and was significantly regulated by soil factors (total P and carbon-N ratio). However, the decrease in the abundance of keystone species (from phylum Basidiomycota) in the fungal communities did not have a significant effect on community construction, which was mainly affected by root characteristics (root N content and soluble sugar). This study found that long-term N and P fertilization changed the keystone species composition of bacterial communities by affecting the nutrient content of the rhizospheric soil, such as total P, so that the construction mode of communities changed from a stochastic to a deterministic process, and the N2 fertilization, especially the N1P2 treatment was better for increasing network stability (modularity and clustering coefficient).

A prediction model for COVID-19 liver dysfunction in patients with normal hepatic biochemical parameters.

Coronavirus disease 2019 (COVID-19) patients with liver dysfunction (LD) have a higher chance of developing severe and critical disease. The routine hepatic biochemical parameters ALT, AST, GGT, and TBIL have limitations in reflecting COVID-19-related LD. In this study, we performed proteomic analysis on 397 serum samples from 98 COVID-19 patients to identify new biomarkers for LD. We then established 19 simple machine learning models using proteomic measurements and clinical variables to predict LD in a development cohort of 74 COVID-19 patients with normal hepatic biochemical parameters. The model based on the biomarker ANGL3 and sex (AS) exhibited the best discrimination (time-dependent AUCs: 0.60-0.80), calibration, and net benefit in the development cohort, and the accuracy of this model was 69.0-73.8% in an independent cohort. The AS model exhibits great potential in supporting optimization of therapeutic strategies for COVID-19 patients with a high risk of LD. This model is publicly available at https://xixihospital-liufang.shinyapps.io/DynNomapp/.

LncRNA expression in idiopathic achalasia: New insight and preliminary exploration into pathogenesis.

Idiopathic achalasia is a primary esophageal motility disorder characterized by the absence of esophageal peristalsis and impaired relaxation of the lower esophageal sphincter (LES). However, the pathogenesis of idiopathic achalasia remains unclear. To further understand the pathogenesis, we conducted lncRNA and mRNA microarray analyses. LES specimens from 5 patients and 4 controls were used for microarray. Potential target genes with significantly changed lncRNA and mRNA were predicted using cis/trans-regulatory algorithms, followed by the Gene Ontology and KEGG pathway enrichment analysis to understand the biophysical effect. Finally, 7,133 significantly dysregulated mRNAs (3,136 increased and 3,997 decreased), along with 6,892 significantly dysregulated lncRNAs (4,900 increased and 1,992 decreased). Biophysical function analysis revealed that the cell adhesion molecule (CAM) pathway was a common pathway. The predicted lncRNA targets of NRXN1 (Down FC: 9.07), NTNG2 (UP FC: 2.75), CADM1 (Down FC: 2.26), NLGN1 (Down FC: 4.60), NEGR1 (Down FC: 2.335), CD22 (Down FC: 5.62), HLA-DQB1 (Down FC: 5.06), and HLA-DOA (Down FC: 2.31) were inputted in this pathway, which was mainly located in the synapse part of the neural system and immune system. Our study demonstrates the lncRNAs and corresponding mRNAs that may play important roles in idiopathic achalasia.

The complete mitochondrial genome of Xenopsylla cheopis (Siphonaptera: Pulicidae).

Xenopsylla cheopis, also called oriental rat flea, is an ectoparasite as well as disease vector for murine typhus and bubonic plague. In the study, the whole mitochondrial genome of X. cheopis was sequenced and assembled, which is the second report of mitochondrial genome in the family Pulicidae and the sixth mitochondrial genome in the order Siphonaptera (fleas). The mitochondrial genome is 18,902 bp in length, consisting of 40% A, 44% T, 6% G, and 10% C. Phylogenetic analysis of all available mitochondrial genomes from Siphonaptera indicated that X. cheopis clustered with Ctenocephalides felis since both species belonged to the family Pulicidae. The complete mitochondrial genome of X. cheopis could serve as useful genetic data for investigating the genetic relationship of fleas.

Label-Free Quantitative Proteomic Analysis of Three Strains of Viscerotropic Leishmania Isolated from Patients with Different Epidemiological Types of Visceral Leishmaniasis in China.

BACKGROUND: There are three epidemiological types of visceral leishmaniasis in China, which are caused by Leishmania strains belonging to the L. donovani complex. The mechanisms underlying their differences in the population affected, disease latency, and animal host, etc., remain unclear. We investigated the protein abundance differences among Leishmania strains isolated from three types of visceral leishmaniasis endemic areas in China. METHODS: Promastigotes of the three Leishmania strains were cultured to the log phase and harvested. The protein tryptic digests were analyzed with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), followed by label-free quantitative analysis. The MS experiment was performed on a Q Exactive mass spectrometer. Raw spectra were quantitatively analyzed with the MaxQuant software (ver 1.3.0.5) and matched with the reference database. Differentially expressed proteins were analyzed using the bioinformatics method. The MS analysis was repeated three times for each sample. RESULTS: A total of 5012 proteins were identified across the KS-2, JIASHI-5 and SC6 strains in at least 2 of the three samples replicate. Of them, 1758 were identified to be differentially expressed at least between 2 strains, including 349 with known names. These differentially expressed proteins with known names are involved in biological functions such as energy and lipid metabolic process, nucleotide acid metabolic process, amino acid metabolic process, response to stress, cell membrane/cytoskeleton, cell cycle and proliferation, biological adhesion and proteolysis, localization and transport, regulation of the biological process, and signal transduction. CONCLUSION: The differentially expressed proteins and their related biological functions may shed light on the pathogenicity of Leishmania and targets for the development of vaccines and medicines.

CircPVT1 Regulates Cell Proliferation, Apoptosis and Glycolysis in Hepatocellular Carcinoma via miR-377/TRIM23 Axis.

BACKGROUND: Recent studies reported that circular RNAs (circRNAs) exert essential functions in hepatocellular carcinoma (HCC) progression. However, the expression profile and function of circular RNA PVT1 (circPVT1) in HCC are not fully addressed. Thus, we aimed to probe into the function of circPVT1 in HCC development. METHODS: The levels of circPVT1, microRNA-377 (miR-377) and transcripts encoding tripartite motif containing 23 (TRIM23) were determined by qRT-PCR. The stability and localization of circPVT1 were examined by RNase R digestion assay and subcellular fraction assay, respectively. Cell proliferation and apoptosis were evaluated by MTT assay and flow cytometry analysis, respectively. The relationship between miR-377 and circPVT1 or TRIM23 was determined by dual-luciferase reporter assay and RNA immunoprecipitation (RIP). The protein expression of TRIM23 was measured by Western blot. The glycolysis level was assessed by specific kits and Seahorse Extracellular Flux Analyzer XF96. The function of circPVT1 in vivo was investigated in a murine xenograft model. RESULTS: CircPVT1 and TRIM23 levels were elevated, while miR-377 was decreased in HCC. CircPVT1 knockdown restrained proliferation and glycolysis, but enhanced apoptosis in HCC cells. CircPVT1 could bind to miR-377 and inhibition of miR-377 restored circPVT1 knockdown-mediated effect on HCC cells. TRIM23 was certified as a target of miR-377, and TRIM23 upregulation overturned the influence of miR-377 upregulation or circPVT1 silence on HCC progression. Moreover, circPVT1 knockdown restrained tumor growth in HCC in vivo. CONCLUSION: CircPVT1 aggravated the progression of HCC by upregulating TRIM23 via sponging miR-377.

RNA N6-methyladenosine modification is required for miR-98/MYCN axis-mediated inhibition of neuroblastoma progression.

Neuroblastoma (NB) is one of the most common malignant tumors of the sympathetic nervous system in childhood. NB severely threatens patient's health and life. However, more effective diagnosis and treatment methods are badly needed in clinics all over the world. MYCN is well recognized as a genetic biomarker of high risk and poor outcome in NB. miRNAs are small RNAs and miR-98 involved in the pathogenesis of various cancers. The role and mechanism of miR-98 in NB remains to be investigated. Here we found that miR-98 was decreased in human MYCN-high-expression NB tissues, and its down-regulation was associated with poor prognosis of NB. Over-expression of miR-98 inhibited cell proliferation, migration and invasion of NB cells. The analysis by employing the software of miRanda predicted the possible binding sites of miR-98 in the 3'-UTR of MYCN, and experimental data illustrated that miR-98 directly bound to MYCN 3'-UTR and decreased MYCN expression. Over-expression of MYCN rescued the decreased malignant phenotype caused by over-expression of miR-98 in NB. N6-methyladenosine modification in 3'-UTR of MYCN promoted its interaction with miR-98. The data collectively demonstrated that RNA m6A modification was required for miR-98/MYCN axis-mediated inhibition of neuroblastoma progression, and miR-98 might be novel targets for NB detection and treatment.

Control of eosinopilic meningitis caused by Angiostrongylus cantonensis in China.

Rat lungworm Angiostrongylus cantonensis is the major infective agent of human eosinophilic meningitis (EM) in the world. The parasite was first noted in China in 1933. However, the public health importance was not realized until several EM outbreaks occurred recent years. Such disease is considered as emerging infectious disease in the People's Republic of China (P.R. China) since the major source of infection is invasive snail species, particularly Pomacea spp. National Institute of Parasitic Diseases (NIPD) initiated a systematic implementation research on this disease since 2003. Our researchers in NIPD developed the lung-microscopy for detecting A. cantonensis larvae in Pomacea snails and further accomplished the atlas of larval morphology by this method. We studied the determinants in infection, which helped the field collection of snails and improved the infection procedure in laboratory. Our researches promoted the promulgation of diagnosis criteria of angiostrongyliasis cantonensis by the Ministry of Health. We explored the molecular diversity of rat lungworm and its major snail host for development of source-tracing technique. The transmission modelling could provide the vulnerable area for surveillance. All the studies supported the surveillance system of EM caused by A. cantonensis in P.R. China. Such implementation research will provide a case study for control of emerging infectious diseases.

Cancer-associated fibroblasts induce immunotherapy resistance in hepatocellular carcinoma animal model.

Hepatocellular carcinoma is known to be a common predominant cancer in adults, especially in eastern countries. Immune response and cancer-associated fibroblasts (CAFs) have significant influences on tumor development. However, the interaction between CAFs and immunotherapy is unclear in hepatocellular carcinoma. We measured the number of activated fibroblasts in hepatocellular carcinoma samples and samples taken from normal liver tissues. A total of 20 patients' fresh hepatocellular carcinoma and normal tissues which were surrounding the tumor were obtained from the surgery and used for evaluating alpha-SMA expression. We investigated the effects of CAFs in anti-tumor immunity in hepatocellular carcinoma animal model. The effects of CAFs in inducing anti-PD-1 treatment resistance were also measured in a preclinical animal model. Activated fibroblasts were highly accumulated in hepatocellular carcinoma tissues but not in surrounding normal tissues. CAFs showed a significant tumor-promoting effect in an immunocompetent model. The infiltration and function of some immune cells like myeloid-derived suppressive cells and T-cells were increased by CAFs. CAFs also reduced the number and activation of tumor-infiltrating cytotoxic T-cell in tumor tissue. In the treatment model, tumors with a higher amount of CAFs had been insensitive to therapy with anti-PD-1. CAFs are potent inducers of immunosuppression in hepatocellular carcinoma. Depleting CAFs rescued the antitumor immunity in the hepatocellular model and could be a novel treatment to combine with the existing immunotherapy.

Role of the lipoxin A4 receptor in the development of neutrophil extracellular traps in Leishmania infantum infection.

BACKGROUND: Neutrophils play an immunomodulatory role through the release of neutrophil extracellular traps (NETs). NETs are released in response to Leishmania infection, but the mechanism of NET extrusion has not been elucidated. The lipoxin A4 receptor on neutrophils is crucial for the inflammatory response and immune regulation of many diseases, including Leishmania infection. Therefore, in the present study, we tried to explore whether Leishmania infantum promastigotes stimulate neutrophil activation and NET release via activating the lipoxin A4 receptor. RESULTS: Leishmania infantum promastigotes stimulated neutrophil activity, but blocking of the lipoxin A4 receptor with its antagonist Boc prior to L. infantum stimulation abrogated these effects. Neutrophils showed citrullinated histone H3 expression and simultaneous NET extrusion on L. infantum stimulation, but a decline in both was observed on blocking of the lipoxin A4 receptor. Moreover, differentiated HL-60 cells with lipoxin A4 receptor silencing showed a decrease in citrullinated histone H3 expression as compared to the unsilenced HL-60 samples on stimulation with promastigotes. CONCLUSIONS: Leishmania infantum promastigotes altered the characteristics of neutrophils and induced NET extrusion by activating the lipoxin A4 receptor. The lipoxin A4 receptor may have potential as a therapeutic target in relation to NET extrusion in the treatment of leishmaniasis, but its mechanisms of action need to be explored in more depth.

miR-134 suppresses the migration and invasion of non­small cell lung cancer by targeting ITGB1.

Most cancer-related deaths are caused by the development of metastatic disease. Thus, investigation of the underlying mechanisms of metastasis is urgent to design more effective targeted drugs and to treat metastatic disease more effectively. MicroRNAs (miRNAs) have emerged as potential targets for cancer treatment. In the present study, we aimed to identify the roles of miR-134 in non-small cell lung cancer (NSCLC) cell migration and invasion. We demonstrated that overexpression of miR-134 inhibited migration and invasion of A549 and H1299 cells. Further mechanistic investigations revealed that miR-134 inhibited epithelial-to-mesenchymal transition (EMT) evidenced by upregulation of E-cadherin expression and downregulation of vimentin expression. Using luciferase assays, we identified integrin ß1 (ITGB1) as a direct target of miR-134. Performing RNAi and rescue experiments, we confirmed that miR-134 exerted its migratory and invasive suppressive role partly by downregulating ITGB1. Finally, an in vivo experiment also, to some extent, suggested that miR-134 may function as a suppressor of metastasis. Taken together, our findings suggest that miR-134 suppresses migration and invasion of NSCLC by targeting ITGB1.

miR-134 inhibits non-small cell lung cancer growth by targeting the epidermal growth factor receptor.

The epidermal growth factor receptor (EGFR) is frequently activated in a wide range of solid tumours and represents an important therapeutic target. MicroRNAs (miRNAs) have recently been recognized as a rational and potential modality for anti-EGFR therapies. However, more EGFR-targeting miRNAs need to be explored. In this study, we identified a novel EGFR-targeting miRNA, miRNA-134 (miR-134), in non-small-cell lung cancer (NSCLC) cell lines. Luciferase assays confirmed that EGFR is a direct target of miR-134. In addition, the overexpression of miR-134 inhibited EGFR-related signaling and suppressed NSCLC cells proliferation by inducing cell cycle arrest and/or apoptosis, suggesting that miR-134 functions as a tumour suppressor in NSCLC. Further mechanistic investigation including RNAi and rescue experiments suggested that the down-regulation of EGFR by miR-134 partially contributes to the antiproliferative role of miR-134. Last, in vivo experiments demonstrated that miR-134 suppressed tumour growth of A549 xenograft in nude mice. Taken together, our findings suggest that miR-134 inhibits non-small cell lung cancer growth by targeting the EGFR.

miR-373 Inhibits Glioma Cell U251 Migration and Invasion by Down-Regulating CD44 and TGFBR2.

Glioblastoma multiforme (GBM) is the most malignant glioma, unveiling the underlying mechanisms of its aggressiveness could promote the discovery of potential targets for effective treatment. MicroRNAs (miRNAs) are important participants in both development and disease, its involvement in cancers has long been recognized. In this study, we investigated the role of miRNA-373 (miR-373) in GBM cell line U251, demonstrated that although miR-373 does not affect cell growth of U251, it inhibits migration and invasion of U251. Forced expression of miR-373 down-regulates the expressions CD44 and TGFBR2, while knockdown of CD44 and TGFBR2 presents the similar phenotype as miR-373 overexpression, suggesting that CD44 and TGFBR2 are functional targets of miR-373, down-regulation of CD44 and TGFBR2 by miR-373 are partly responsible for the migration, and invasion suppressive role of miR-373 in U251.

Diverse functions of miR-373 in cancer.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression post-transcriptionally. They are involved in almost all cellular processes, and many have been described as potential oncogenes or tumor suppressors. MicroRNA-373 (miR-373), which was first identified as a human embryonic stem cell (ESC)-specific miRNA, is suggested to be implicated in the regulation of cell proliferation, apoptosis, senescence, migration and invasion, as well as DNA damage repair following hypoxia stress. Deregulation of miR-373 has been demonstrated in a number of cancers, whether it acts as an oncogene or a tumor suppressor, however, seems to be context dependent. In this review, we focus on the diverse functions of miR-373 and its implication in cancers.

The complete mitochondrial genome of the rodent intra-arterial nematodes Angiostrongylus cantonensis and Angiostrongylus costaricensis.

The two rodent intra-arterial nematodes, Angiostrongylus cantonensis and Angiostrongylus costaricensis, can cause human ill-health. The present study aimed to characterize and compare the mitochondrial (mt) genomes of these two species, and clarify their phylogenetic relationship and the position in the phylum Nematoda. The complete mt genomes of A. cantonensis and A. costaricensis are 13,497 and 13,585 bp in length, respectively. Hence, they are the smallest in the class of Chromadorea characterized thus far. Like many nematode species in the class of Chromadorea, they encode 12 proteins, 22 transfer RNAs, and two ribosomal RNAs. All genes are located on the same strand. Nucleotide identity of the two mt genomes is 81.6%, ranging from 77.7% to 87.1% in individual gene pairs. Our mt genome-wide analysis identified three major gene arrangement patterns (II-1, II-2, and II-3) from 48 nematode mt genomes. Both patterns II-1 and II-2 are distinct from pattern II-3, which covers the Spirurida, supporting a closer relationship between Ascaridida and Strongylida rather than Spirurida. Thymine (T) was highly concentrated on coding strands in Chromadorea, but balanced between the two strands in Enoplea, probably due to the gene arrangement pattern. Interestingly, the gene arrangement pattern of mt genomes and phylogenetic analysis based on concatenated amino acids indicated a closer relationship between the order Ascaridida and Rhabditida rather than Spirurida as indicated in previous studies. These discrepancies call for new research, reassessing the position of the order of Ascaridida in the phylogenetic tree. Once consolidated, the findings are important for population genetic studies and target identification.

[Investigation on Babesia in ticks infested on police dogs in selected areas of China].

399 tick specimens were collected from the body surface of police dogs in Chongqing municipality, provinces of Fujian, Guangdong, Hainan, Guangxi, Hebei, Henan, Shanxi, Jiangsu and Zhejiang. Nested-PCR and sequence testing were taken to investigate the prevalence of Babesia sp. in ticks. The results showed that Babesia vogeli was found in ticks infested on the body surface of police dogs, with a positive rate of 5.3%. The prevalence in Chongqing, Guangdong, Guangxi, Hainan and Zhejiang was 4/16, 3.6% (1/28), 12.5%(11/88), 3.3% (4/121) and 1/15, respectively. It suggested that there was a certain rate of infected ticks infested on the body suriface of police dogs, which contributed to the potential threat to staff. The prevention and control measures should be strengthened.

[Investigation of ticks parasitizing on bodies of dogs in Shanghai].

OBJECTIVE: To investigate the ticks which parasitize on the bodies of dogs in Shanghai. METHODS: The tick samples were collected from the dogs in 18 districts and counties of Shanghai from March to December in 2011, and the ticks were identified for the species through the morphological analysis by the dissecting microscope in laboratory. RESULTS: Totally 1 950 dogs were investigated for ticks parasitizing on the bodies of districts including Jiading, Minhang, Pudong, Songjiang, Huangpu and Jinshan. These ticks were belong to 2 species of 2 genera by morphological analysis in laboratory. Rhipicephalus sanguineus and Haemaphysalis longicornis were the ticks parasitizing on the bodies of pet dogs. R. sanguineus was the main ticks parasitizing on the bodies of police dogs and laboratory dogs. CONCLUSION: R. sanguineus is the domain tick parasitizing on dogs in Shanghai, and H. longicornis is a newly found hard tick species parasitizing on dogs in Shanghai. We should strengthen the prevention and control of ticks.

[Investigation on transmitting vectors of visceral leishmaniasis in Jiashi County, Xinjiang].

OBJECTIVE: To investigate sandfly vectors transmitting visceral leishmaniasis, including species and seasonal distribution in Jiashi county of Xinjiang Uygur Autonomous Region. METHODS: Sandflies were collected in the field, counted and identified. The specimens were dissected to analyze the gonotrophic cycle and to find infection of promastigotes. The resting places were observed by using oil-paper and sandfly-capturing trap. RESULTS: 4540 sandflies were collected with 99.9% of Phlebotomus wui and only 0.1% Sergentomyia minutus sinkiangensis. On the seasonal distribution, the first peak appeared by the end of May and the first ten-day of June, and the second peak was in the middle of August. Observation showed that the activity of sandflies occurred mainly from 22:00 to 4:00, reaching to the maximum in the midnight. Analysis on the gonotrophic cycle revealed that Ph. wui was an exophilic species and appeared nocturnally for feeding with preference to human blood. Natural infection with promastigotes was found in 4 sandflies, more in the field than the residential area. Resting places included the aperture on the wall of livestock sheds and in the caves. CONCLUSION: Ph. wui is the predominant species in Jiashi, with higher infection rate of natural promastigotes in the field and with two life generations annually.

[Multiplex PCR assay for the detection of Angiostrongylus cantonensis larvae in Pomacea canaliculata].

OBJECTIVE: To establish a multiplex PCR assay for detecting Angiostrongylus cantonensis larvae in Pomacea canaliculata. METHODS: A pair of specific primers was designed based on the sequences of the small subunit rDNA of A. cantonensis (GenBank jAY295804), in combination with 16s rDNA specific primers of P. canaliculata, a multiplex PCR was developed. The PCR was performed on positive and negative snails, and the amplified products were analyzed by agarose gel electrophoresis and DNA sequencing. DNA template of 200 III stage larvae of A. cantonensis was diluted by negative snail DNA (1200 ng/microl, 120 ng/microl, 12 ng/microl, 1200 pg/microl, 120 pg/microl and 12 pg/microl), to find the minimum detectable level. Single blind method was used to evaluate the accuracy. After being detected by lung microscopy, 172 snails from field were tested by the multiplex PCR to assess the sensitivity and specificity. RESULTS: Agarose gel electrophoresis and DNA sequencing analysis indicated that the target sequences were efficiently amplified by the PCR assay (550 bp for P. canaliculata, 405 bp for A. cantonensis). The minimum detectable level was 120 pg/microl. The coincidence between the two methods stood for 84.3% (145/172), including 45 positives and 100 negatives. 24 snails were PCR positive and microscopy negative, 3 snails were PCR negative and microscopy positive. The sensitivity and specificity of multiplex PCR was 93.8% and 80.6%, respectively. Its positive rate (40.1%, 69/172) was significant higher than that of lung-microscopy (27.9%, 48/172)(chi2 = 14.8, P < 0.01). CONCLUSION: A multiplex PCR method has been developed for the detection of A. cantonensis larvae in P. canaliculata.