[Effects of Chinese Milk Vetch Returning Incorporated with Chemical Fertilizer Reduction on the Composition and Function of Soil Bacterial Communities in Paddy Fields].

Chinese milk vetch (Astragalus sinicus L.) is an important organic nutrient resource in the southern Henan rice-growing area. Thus, the effects of Chinese milk vetch (MV) returning incorporated with reduced chemical fertilizer on the physicochemical properties and bacterial community characteristics in paddy soil were studied. These results can provide a certain theoretical basis for the improvement of soil fertility and reduction of chemical fertilizer in this area. A field experiment was conducted for 12 consecutive years, involving six fertilization treatments (blank control, CK; 100% chemical fertilizer, F100; 80% chemical fertilizer+22.5 t·hm-2 MV, MV1F80; 80% chemical fertilizer+45 t·hm-2 MV, MV2F80; 60% chemical fertilizer+22.5 t·hm-2 MV, MV1F60; and 60% chemical fertilizer+45 t·hm-2 MV, MV2F60). The high-throughput sequencing method was used to compare the effects of different fertilization treatments on soil bacterial community diversity, composition, and structural characteristics. The FAPROTAX function prediction method was used to analyze the abundance differences of functional groups between different fertilization treatments. Additionally, combined with soil physicochemical properties and bacterial community characteristics, we explored the key soil environmental factors that changed the structure and functional characteristics of the soil bacterial community. Compared with that under CK, the soil bulk density (BD) under the MV returning incorporated with reduced chemical fertilizer treatment was decreased, whereas soil organic carbon (SOC), total nitrogen (TN), total phosphorus (TP), and total potassium (TK) were increased by 12.7%-35.5%, 38.2%-65.7%, 66.7%-95.2%, and 20.3%-31.6%, respectively. Compared with that under the F100 treatment, the Sobs index and Shannon diversity index of the bacterial community under the MV returning incorporated with reduced chemical fertilizer were decreased, and the Sobs index and Shannon diversity index were significantly positively correlated with BD (P<0.05) but significantly negatively correlated with SOC and TN (P<0.05). Compared with that under the F100 treatment, the relative abundances of Firmicutes under the MV1F80 and MV2F60 treatments were significantly increased by 82.2% and 67.4% (P<0.05), but the relative abundances of Acidobacteria were significantly reduced by 32.6% and 40.5% (P<0.05), respectively. The relative abundance of Actinobacteria under the MV2F60 treatment was significantly increased by 30.0% (P<0.05) compared with that under the F100 treatment. According to RDA analysis, soil SOC, TN, and TK were the main soil environmental factors that significantly affected bacterial community (P<0.05). Compared with that under CK and the F100 treatment, the abundance of functional groups of chemoheterotrophy, nitrogen fixation, fermentation, and ureolysis under the MV returning incorporated with reduced chemical fertilizer treatment were improved, whereas the abundance of functional groups of animal parasites or symbionts, all human pathogens, and human pathogen pneumonia were reduced, particularly under MV1F80 and MV2F60. To summarize, the long-term MV returning to the field incorporated with reduced chemical fertilizer improved the soil physical and chemical properties, thus changing the structure and functional characteristics of the soil bacterial communities, contributing to the improvement in the soil fertility, stability, and health of micro-ecosystems in paddy fields, thus ensuring the green and sustainable development of regional agriculture.

Regulation of the expression of proinflammatory cytokines induced by SARS-CoV-2.

An outbreak of a novel coronavirus was reported in Wuhan, China, in late 2019. It has spread rapidly through China and many other countries, causing a global pandemic. Since February 2020, over 28 countries/regions have reported confirmed cases. Individuals with the infection known as coronavirus disease-19 (COVID-19) have similar clinical features as severe acute respiratory syndrome first encountered 17 years ago, with fever, cough, and upper airway congestion, along with high production of proinflammatory cytokines (PICs), which form a cytokine storm. PICs induced by COVID-19 include interleukin (IL)-6, IL-17, and monocyte chemoattractant protein-1. The production of cytokines is regulated by activated nuclear factor-kB and involves downstream pathways such as Janus kinase/signal transducers and activators transcription. Protein expression is also regulated by post-translational modification of chromosomal markers. Lysine residues in the peptide tails stretching out from the core of histones bind the sequence upstream of the coding portion of genomic DNA. Covalent modification, particularly methylation, activates or represses gene transcription. PICs have been reported to be induced by histone modification and stimulate exudation of hyaluronic acid, which is implicated in the occurrence of COVID-19. These findings indicate the impact of the expression of PICs on the pathogenesis and therapeutic targeting of COVID-19.

Neovagina Creation: A Novel Improved Laparoscopic Vecchietti Procedure in Patients with Mayer-Rokitansky-Küster-Hauster Syndrome.

STUDY OBJECTIVE: To report a new improved laparoscopic Vecchietti vaginoplasty in patients with congenital vaginal agenesis and to investigate its efficacy and safety. DESIGN: A retrospective descriptive and case-control study. SETTING: Single academic institution. PATIENTS: Women who were diagnosed with Mayer-Rokitansky-Küster-Hauster (MRKH) syndrome and underwent our new improved laparoscopic Vecchietti procedure from July 2010 to June 2019 were selected as the study group. The eligible participants had congenital vaginal agenesis with normal 46,XX karyotype and ovarian function. Age-matched, nulliparous, sexually active women were selected as the control group. INTERVENTIONS: Women with MRKH syndrome in the study group underwent the novel improved laparoscopic Vecchietti procedure. All participants in both groups were required to complete Female Sexual Function Index and Female Genital Self-Image Scale questionnaires. MEASUREMENTS AND MAIN RESULTS: The effects of our procedure, including the anatomic and functional efficacy of the neovagina, were the primary outcomes. The secondary outcomes consisted of the perioperative complications, surgical morbidities, and long-term postoperative discomfort. A total of 79 patients with MRKH syndrome underwent our new improved Vecchietti vaginoplasty, of whom 44 (55.7%) were diagnosed as Type I MRKH syndrome, whereas 35 (44.3%) were Type II MRKH syndrome. At a 30-month follow-up after surgery, an anatomic neovagina measuring 10.44 cm in length and 1.30 cm in width was achieved. All 79 patients obtained anatomic success with 92.41% of functional efficacy. Compared with 81 age-matched, nulliparous women in the control group, there was no statistical difference regardless of individual measure or total Female Sexual Function Index scores (p >.05). The Female Genital Self-Image Scale assessment showed a significantly lower score in patients undergoing the vaginoplasty (20.14 ± 3.05 vs 22.95 ± 2.12; p <.001). There were no severe perioperative complications except 1 mild bladder injury and 1 transient fever. CONCLUSION: Our novel improved laparoscopic Vecchietti vaginoplasty is a relatively safe and effective method for surgical treatment of congenital vaginal agenesis. It may be an alternative to neovagina creation for reaching satisfying anatomic and functional efficacy and improving patients' sexual function.

Elevation in the counts of IL-35-producing B cells infiltrating into lung tissue in mycobacterial infection is associated with the downregulation of Th1/Th17 and upregulation of Foxp3+Treg.

IL-35 is an anti-inflammatory cytokine and is thought to be produced by regulatory T (Treg) cells. A previous study found that IL-35 was upregulated in the serum of patients with active tuberculosis (ATB), and IL-35-producing B cells infiltrated to tuberculous granuloma of patients with ATB. Purified B cells from such patients generated more IL-35 after stimulation by antigens of Mycobacterium tuberculosis and secreted more IL-10. However, the function and the underlying mechanisms of IL-35-producing B cells in TB progression have not been investigated. The present study found that the expression of mRNA of IL-35 subsets Ebi3 and p35 was elevated in mononuclear cells from peripheral blood, spleen, bone marrow, and lung tissue in a mouse model infected with Mycobacterium bovis BCG, as tested by real-time polymerase chain reaction. Accordingly, the flow cytometry analysis showed that the counts of a subset of IL-35+ B cells were elevated in the circulating blood and in the spleen, bone marrow, and lung tissue in BCG-infected mice, whereas anti-TB therapy reduced IL-35-producing B cells. Interestingly, BCG infection could drive the infiltration of IL-35-producing B cells into the lung tissue, and the elevated counts of IL-35-producing B cells positively correlated with the bacterial load in the lungs. Importantly, the injection of exogenous IL-35 stimulated the elevation in the counts of IL-35-producing B cells and was associated with the downregulation of Th1/Th17 and upregulation of Foxp3+Treg.The study showed that a subset of IL-35-producing B cells might take part in the downregulation of immune response in mycobacterial infection.

A Novel Cerclage Insertion: Modified Laparoscopic Transabdominal Cervical Cerclage with Transvaginal Removing (MLTCC-TR).

STUDY OBJECTIVE: To report on our center's experience of a novel modified approach for laparoscopic cervical cerclage and to evaluate its safety and efficacy preliminarily. DESIGN: Retrospective descriptive study. SETTING: Single academic institution. PATIENTS: Pregnant and nonpregnant women who underwent the modified laparoscopic transabdominal cervical cerclage with transvaginal removing (MLTCC-TR) from June 2016 to April 2019. Eligible participants had multiple adverse obstetric histories or the short cervix and were not suitable for a second transvaginal cerclage. INTERVENTIONS: Preconceptional or postconceptional MLTCC-TR. MEASUREMENTS AND MAIN RESULTS: A total of 24 participants (including 3 first-trimester singleton pregnant women) underwent the MLTCC-TR, giving birth to 27 infants. Among 21 women who underwent preconceptional cerclage, 26 cases of postoperational pregnancies were noted, and the incidence of term labor was 73.07%, which was significantly higher than that in the precerclage group (p <.001). Their mean gestational age at delivery was 37.21 ± 5.05 weeks. Among 3 cases of postconceptional cerclage, the mean gestational age at cerclage was 10.90 ± 2.61 weeks, and all of them had term delivery. The overall neonatal survival rate was 100% (27/27), of which 81.48% (22/27) were term infants. There were no severe perioperative complications directly related to the insertion of cerclage. CONCLUSION: Our new approach of MLTCC-TR may be a relatively effective, feasible, and safe treatment for cervical insufficiency. It may be considered as an acceptable alternative to the traditional laparoscopic cervical cerclage with its superiority of transvaginal removing.

Construction of a recombinant eukaryotic expression vector containing DNM3 gene and its expression in colon cancer cells.

INTRODUCTION: Dynamin 3 (DNM3) is a large GTPase that possesses mechanochemical properties and has been shown to be involved in malignancies. However, most studies about DNM3 are observational, and knowledge of the precise molecular mechanism of DNM3 remains limited. MATERIALS AND METHODS: We constructed a PCDH-CMV-MCS-EF1a-GFP-Puro-DNM3 recombinant eukaryotic expression vector, which was then transfected into SW620 and LoVo cells. One cell line was divided into three groups. DNM3 mRNA and protein expression was analyzed by quantitative real-time PCR and Western blot assay. To investigate DNM3 biological activity in colon cancer SW620 and LoVo cell line, we performed cell proliferation, transwell migration, and invasion assay. Matrix metalloproteinase (MMP)-2 and MMP-9 protein expressions were detected by Western blot. RESULT: We successfully constructed a PCDH-CMV-MCS-EF1a-GFP-Puro-DNM3 recombinant eukaryotic expression vector, and stable DNM3 expression was observed in SW620 and LoVo cell lines. The vector overexpressing DNM3 inhibited the proliferation, weak invasion, and migration ability of colon cancer SW620 and LoVo cells relative to those in the control group (all P<0.001). DNM3 downregulated the protein expression of MMP-2 and MMP-9. CONCLUSION: DNM3 may weaken the malignant behavior of colon cancer and may have promoted the invasion and migration of colon cancer by regulating the expression of MMP-2 and MMP-9.

Surgical resection and post-operative radiotherapy in an adult renal neuroblastoma patient with multiple bone and joint metastases: A case report.

Renal neuroblastoma is uncommon, particularly in adults, with only a few cases having been reported in studies published in the English language. The incidence is only 0.12 cases/1 million individuals in those aged >20 years. Studies of the pathogenesis, biological characteristics, treatment and prognosis of renal neuroblastoma are limited due to this low incidence. The present study reports the case of a 22-year-old adult female who was diagnosed with a left renal neuroblastoma by computed tomography (CT), bone scan and pathological examination. The patient underwent a left nephroureterectomy, ipsilateral lymph node dissection and post-operative radiotherapy, prior to discharge 60 days after admittance. At the nine-month follow-up examination, the patient showed no evidence of recurrence, progression or metastatic disease on the CT scans of the chest, abdomen and pelvis. Renal neuroblastoma is extremely uncommon in adults. The diagnosis and treatment of renal neuroblastoma is complicated by the overall low incidence, lack of specific treatment guidelines, advanced disease state due to late presentation, and its associated co-morbidities. Further study of the pathogenesis, biological and clinical characteristics, and treatment of renal neuroblastoma is required to provide an optimal treatment for patients and to improve the patient's quality of life.

Effects of α-enolase (ENO1) over-expression on malignant biological behaviors of AGS cells.

OBJECTIVE: To investigate the effects of α-Enolase (ENO1) over-expression on the proliferative and migratory abilities of AGS cells. METHODS: The target gene was cloned and mounted to the eukaryotic expression vector pcDNA3.1(+), then was transfected into gastric cancer cell lines AGS. mRNA and protein level of ENO1 in AGS cells were verified by real-time quantitative RT-PCR and Western Blot, respectively. The effects of over-expression of ENO1 on proliferative and migratory abilities of AGS cells were detected by the experiments of CCK-8, colony formation and wound healing assays. RESULTS: The eukaryotic expression vector pcDNA3.1(+)/eno1 was successfully constructed, and verified by sequencing. It was shown from the cell proliferation curves that the proliferative ability of AGS-ENO1 transfected group was higher than that of the control group after 72 hours (t = 3.44, P = 0.04), meanwhile, the number of the cell-colonies of the AGS-ENO1 group were significantly greater than that of the control group (t = 5.26, P = 0.01). For the ability of migration, it was significantly enhanced in the over-expression ENO1 cells than in the negative cells (t = 7.35, P < 0.001). CONCLUSION: The over-expression of ENO1 protein can enhance the abilities of proliferation and migration in gastric cancer cells of AGS, which indicates that ENO1 may be an important potential tumor-marker associated with the development of gastric cancer.

Effect of stable transfection with PHD3 on growth and proliferation of HepG2 cells in vitro and in vivo.

OBJECTIVE: To establish a human hepatoma HepG2 cell line with stable expression of Prolyl hydroxylase domain 3 (PHD3) gene and study its effect of growth and proliferation in nude mice xenograft tumor. METHODS: Eukaryotic expression vectors of pcDNA 3.1-PHD3 was constructed. HepG2 cells were transfected with recombinant plasmid pcDNA 3.1-PHD3 and empty vector plasmid pcDNA 3.1 by lipofectamine 2000 as transfected group, control group respectively, while the HepG2 cell without any operation was considered as parental group. Steady expression cells were gotten by G418 selecting. RT-PCR and agarose gel electrophoresis were used to confirm the expression of PHD3 in HepG2 cells and transfection successfully. The growth of these cells in vivo were also observed by injecting three groups of cell into nude mice, and volume were measured and compared. RESULTS: The recombinant plasmid pcDNA 3.1-PHD3 and empty vector plasmid pcDNA 3.1 were successfully transfected into human hepatoma HepG2 cell line and showed stable expression in this cell line. Tumors were observed in nude mice when the transfectant cells were xenografted successfully, The average tumor size of PCDNA (3.1)-PHD3 groups are significant different compared with other two groups (P < 0.001). CONCLUSION: The PHD3 gene may have negative influence of growth and proliferation on HepG2 cells in vitro. The PHD3 may be a potentially tumor suppressor.

Tumor suppressor TSLC1 inhibits growth, proliferation, invasiveness and angiogenesis in nude mice xenografted tumor of Eca109 cells.

Tumor suppressor in lung cancer 1 (TSLC1) is a novel tumor suppressor gene whose inactivation is implicated in the occurrence, invasion, metastasis and prognosis of esophageal cancer. TSLC1 was studied by comparing the tumor formation of TSLC1 transfectant and control cells in nude mice. Compared with blank group and mock group, tumor size and infiltrating range of transfected group was less, differentiation of tumor tissue was slightly better, and differences of tumor angiogenesis was worse. There was no obvious difference between blank group and mock group. We have shown TSLC1 gene inhibited the growth proliferation, infiltration and angiogenesis of Eca109 cells.

Increased glutathione and mitogen-activated protein kinase phosphorylation are involved in the induction of doxorubicin resistance in hepatocellular carcinoma cells.

AIM: The human hepatocellular carcinoma (HCC) cell line HepG2 can easily acquire resistance to doxorubicin. However, the mechanism of action is unclear. METHODS: In the present study, we used confocal microscopy, flow cytometry and other methods to reveal the mechanisms by which HepG2 cells acquire doxorubicin resistance. RESULTS: Our results showed that R-HepG2 cells, a doxorubicin-resistant sub-line of HepG2, exhibited decreased intracellular accumulation of doxorubicin and increased expression of P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 when compared with HepG2 cells. R-HepG2 cells also harbored higher levels of glutathione and increased expression of glutathione peroxidase. Furthermore, we demonstrated that the phosphorylation of mitogen-activated protein kinases (p38 and c-jun-N-terminal kinases), IkBα and CREB were increased in R-HepG2 cells. Specific p38 inhibitor SB203580 decreased P-gp expression. The multi-kinase inhibitor sorafenib tosylate also significantly suppressed the phosphorylation of these proteins and inhibited the expression of P-gp. CONCLUSION: These findings reveal that the drug resistance could be acquired through mitogen-activated protein kinase-dependent upregulation of P-gp. This mechanism protects R-HepG2 cells from the anticancer action of doxorubicin.

Perfluorocarbon attenuates lipopolysaccharide-mediated inflammatory responses of alveolar epithelial cells in vitro.

BACKGROUND: Toll-like receptor-4 (TLR-4) is integrally involved in lipopolysaccharide (LPS) signaling and has a requisite role in the activation of nuclear factor-κB (NF-κB). The exact mechanisms that lend perfluorocarbon (PFC) liquids a cytoprotective effect have yet to be elucidated. Therefore we examined in an in vitro model the cytoprotective effect of PFC on LPS-stimulated alveolar epithelial cellls (AECs). METHODS: AECs (A549 cells, human lung adenocarcinoma cell line) were divided into four groups: control, PFC, LPS and LPS + PFC (coculture group) groups. Intercellular adhesion molecule-1 (ICAM-1) was detected by ELISA, tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) were detected by radioimmunological methods. The expression of TLR-4 mRNA and protein was detected by real time PCR and Western blotting, respectively. The activation of NF-κB was detected by Western blotting (proteins of I-κBa and NF-κB p65). RESULTS: ICAM-1, TNF-α and IL-8 were significantly increased in LPS-stimulated AECs groups. The expression of TLR-4 mRNA and protein in LPS-stimulated groups was markedly increased. Meanwhile, NF-κB was activated as indicated by the significant degradation of IκB-α and the significant release of NF-κB P65 and its subsequent translocation into the nucleus. There were no significant effects of PFC alone on any of the factors studied while the coculture group showed significant downregulation of the secretion of ICAM-1, TNF-α and IL-8, the expression of TLR-4 mRNA and the activity of NF-κB. CONCLUSIONS: Taken together, our results demonstrate that LPS can induce AEC-related inflammatory injury via the activation of TLR-4 and subsequent activation of NF-κB. PFC is able to protect AECs from LPS-induced inflammatory injury by blocking the initiation of the LPS signaling pathway, which is indicated by the significant decrease of TLR-4 expression and NF-κB activation.

Cell survival and death program modulated by LMP1: implication in antitumor immunity.

The genome of Epstein-Barr virus (EBV) encodes proteins essential for malignant transformation, for example, latent membrane protein 1(LMP1). Whereas, LMP1 up-regulates anti-apoptotic proteins to support viral replication, it also potentiates apoptosis, suggesting that a viral protein contributes to the survival of the virus, and it also elicit host defense leading to the destruction of the infected cells. The antitumor immunity is exerted by infiltrated CD8+ T cells elaborating cytotoxic effectors, like Fas ligand (FasL, CD95L or CD178). As a nuclear factor-kappaB (NF-kappaB)-dependent molecule, Fas is induced by LMP1, and LMP1 enhances Fas-mediated apoptosis, according to our finding of stimulus-dependent apoptosis regulation by LMP1. Data has shown that FasL-mediated cytotoxicity has significant therapeutic effect on EBV-associated nasopharyngeal carcinoma (NPC). Recent reports suggest that mutations affecting the Fas-mediated apoptotic pathway reduce individuals' susceptibility to cancers, but cytokine-targeting therapy which precisely regulates the Fas level on tumor cells could still contribute to enhancement of antitumor immunity in cancer patients.

Ethyl 2-(3-chloro-2-pyridyl)-5-oxopyrazolidine-3-carboxylate.

In the mol-ecule of the title compound, C(11)H(12)ClN(3)O(3), the five membered ring adopts an envelope conformation. In the crystal structure, inter-molecular N-H⋯O hydrogen bonds link the mol-ecules into centrosymmetric dimers.

[Hypoxia increases the expression of heparitinase and the invasiveness through the hypoxia inducible factor-1alpha dependent pathway in human ovarian cancer cell line SKOV3].

OBJECTIVE: To investigate the heparitinase (HPA) expression and cell invasiveness in human ovarian cancer cell line SKOV3 during hypoxia, and explore their relationship with hypoxia inducible factor-1alpha (HIF-1alpha). METHODS: SKOV3 cells were incubated with normoxia, hypoxia, and hypoxia plus rapamycin, respectively. SKOV3 cells of hypoxia group were incubated in 5% CO2 + 1% O2. Cells in hypoxia plus rapamycin group were incubated with 10 ng/ml of rapamycin before cultured under hypoxic condition. Cells in each group were collected for analysis after incubated with hypoxia for 12, 24, and 36 hours, respectively. Western blotting and RT-PCR were performed to detect the expressions of HIF-1alpha and HPA. Cell invasiveness was measured by matrigel invasion assay. RESULTS: Western blotting showed that the expression of HIF-1alpha significantly increased compared with normoxic group (P < 0.05). However, hypoxia had no obvious impact on HIF-1alpha mRNA expression. The expressions of HPA protein and mRNA of SKOV3 cells of hypoxia group were significantly higher than normoxic group (P < 0.05). The up-regulation of HPA expression in hypoxic group decreased after the utilization of rapamycin. The cell invasion of hypoxic group was significantly higher than that of normoxic group (P < 0.05); meanwhile, the expression of HPA was positively correlated with the invasiveness of SKOV3 cells (r = 0.9863, P < 0.05). CONCLUSION: Hypoxia may promote the expression of HPA and the invasiveness of SKOV3 cells through the HIF-1alpha pathway, which plays an important role in the pathogenesis of ovarian cancer.

N-Hydr-oxy-N-o-tolyl-acetamide.

In the mol-ecule of the title compound, C(9)H(11)NO(2), the methyl C atom bonded to the ring and the N atom lie in the benzene ring plane. An intra-molecular O-H⋯O hydrogen bond results in the formation of a five-membered planar ring, which is oriented at a dihedral angle of 81.37 (3)° with respect to the benzene ring. In the crystal structure, inter-molecular O-H⋯O hydrogen bonds link the mol-ecules stacked along the b axis. There are also π-π inter-actions between benzene rings with a face-to-face stacking distance of 3.434 Å.