Differential microbial composition in parasitic vs. questing ticks based on 16S next-generation sequencing.

Introduction: As tick-borne diseases rise to become the second most prevalent arthropod-transmitted disease globally, the increasing investigations focus on ticks correspondingly. Factors contributed to this increase include anthropogenic influences, changes in vertebrate faunal composition, social-recreational shifts, and climatic variation. Employing the 16S gene sequence method in next-generation sequencing (NGS) allows comprehensive pathogen identification in samples, facilitating the development of refined approaches to tick research omnidirectionally. Methods: In our survey, we compared the microbial richness and biological diversity of ticks in Wuwei City, Gansu province, differentiating between questing ticks found in grass and parasitic ticks collected from sheep based on 16S NGS method. Results: The results show Rickettsia, Coxiella, and Francisella were detected in all 50 Dermacentor nuttalli samples, suggesting that the co-infection may be linked to specific symbiotic bacteria in ticks. Our findings reveal significant differences in the composition and diversity of microorganisms, with the Friedmanniella and Bordetella genera existing more prevalent in parasitic ticks than in questing ticks (p < 0.05). Additionally, the network analysis demonstrates that the interactions among bacterial genera can be either promotive or inhibitive in ticks exhibiting different lifestyles with the correlation index |r| > 0.6. For instance, Francisella restrains the development of 10 other bacteria in parasitic ticks, whereas Phyllobacterium and Arthrobacter enhance colonization across all tick species. Discussion: By leveraging NGS techniques, our study reveals a high degree of species and phylogenetic diversity within the tick microbiome. It further highlights the potential to investigate the interplay between bacterial genera in both parasitic and questing ticks residing in identical habitat environments.

Alterations in the Composition of Intestinal DNA Virome in Patients With COVID-19.

Patients with Coronavirus Disease 2019 (COVID-19), due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection mainly present with respiratory issues and related symptoms, in addition to significantly affected digestive system, especially the intestinal tract. While several studies have shown changes in the intestinal flora of patients with COVID-19, not much information is available on the gut virome of such patients. In this study, we used the viromescan software on the latest gut virome database to analyze the intestinal DNA virome composition of 15 patients with COVID-19 and investigated the characteristic alternations, particularly of the intestinal DNA virome to further explore the influence of COVID-19 on the human gut. The DNA viruses in the gut of patients with COVID-19 were mainly crAss-like phages (35.48%), Myoviridae (20.91%), and Siphoviridae (20.43%) family of viruses. Compared with healthy controls, the gut virome composition of patients with COVID-19 changed significantly, especially the crAss-like phages family, from the first time of hospital admission. A potential correlation is also indicated between the change in virome and bacteriome (like Tectiviridae and Bacteroidaceae). The abundance of the viral and bacterial population was also analyzed through continuous sample collection from the gut of patients hospitalized due to COVID-19. The gut virome is indeed affected by the SARS-CoV-2 infection, and along with gut bacteriome, it may play an important role in the disease progression of COVID-19. These conclusions would be helpful in understanding the gut-related response and contribute to the treatment and prevention strategies of COVID-19.

Rational engineering of BaLal_16 from a novel Bacillus amyloliquefaciens strain to improve catalytic performance.

L-amino acid ligases (Lals) are promising biocatalysts for the synthesis of dipeptides with special biological properties. However, their poor (or broad) substrate specificity limits their industrial applications. To address this problem, a molecular engineering method for Lals was developed to enhance their catalytic performance. Based on substrate channeling, entrances to the active site for different substrates were identified, and the "gate" located around the active site pocket, which plays an essential role in substrate recognition, was then engineered to facilitate acceptance of L-Gln. Two mutants (L110Y and N108F/L110Y) were discovered to display significantly increased catalytic activity toward L-Ala and L-Gln in the biosynthesis of Ala-Gln. The catalytic efficiency (kcat/ Km) of the L110Y and N108F/L110Y mutants was improved by 2.64-fold and 4.06-fold, respectively, compared with that of the wild type. N108F/L110Y was then further applied for batch production of Ala-Gln, which showed that the released Pi yield was 694.47 µM, which was an increase of approximately 21.4 %, and the yield of Ala-Gln was approximately 2.59 mM-1 L-1 mg-1. Collectively, these findings suggest the potential practical application of this method in the rational design of Lals for increased catalytic performance.

Alterations in the intestinal microbiome and mental health status of workers in an underground tunnel environment.

BACKGROUND: Working in an underground tunnel environment is unavoidable in professions such as miners and tunnel workers, and there is a concern about the health of these workers. Few studies have addressed alterations in the intestinal microbiome of workers within that environment. RESULTS: Fecal samples were collected from the workers before they entered the tunnel (baseline status, BS) and after they left the tunnel (exposed status, ES), respectively (a time period of 3 weeks between them). We analyzed 16S rRNA sequencing to show the changes in microbial composition and self-evaluation of mental health questionnaire was also performed. The results showed that Shannon and Simpson indices decreased significantly from BS to ES. A higher abundance was found in the phylum Actinobacteria, classes Actinobacteria and Deltaproteobacteria, orders Bifidobacteriales, Coriobacteriales, and Desulfovibrionales, families Bifidobacteriaceae, Peptostreptococcaceae, Coriobacteriaceae, Clostridiaceae_1, Desulfovibrionaceae, Pseudomonadaceae, and Microbacteriaceae, and genera Bifidobacterium, Romboutsia, Clostridium sensu stricto, and Leucobacter in ES, while BS showed greater levels of genera Faecalibacterium and Roseburia. The self-evaluation showed that at least one-half of the tunnel workers experienced one or more symptoms of mental distress (inattention, sleeplessness, loss of appetite, headache or dizziness, irritability) after working in the underground tunnel environment. CONCLUSIONS: Collectively, the underground tunnel environment led to alterations in the intestinal microbiome, which might be relevant to symptoms of mental distress in underground-tunnel workers.

Potential molecular targets of nonstructural proteins for the development of antiviral drugs against SARS-CoV-2 infection.

Outbreaks of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2 have produced high pathogenicity and mortality rates in human populations. However, to meet the increasing demand for treatment of these pathogenic coronaviruses, accelerating novel antiviral drug development as much as possible has become a public concern. Target-based drug development may be a promising approach to achieve this goal. In this review, the relevant features of potential molecular targets in human coronaviruses (HCoVs) are highlighted, including the viral protease, RNA-dependent RNA polymerase, and methyltransferases. Additionally, recent advances in the development of antivirals based on these targets are summarized. This review is expected to provide new insights and potential strategies for the development of novel antiviral drugs to treat SARS-CoV-2 infection.

Construction of an Efficient Nicotinate Dehydrogenase Expression System in Comamonas testosteroni CNB-2 with Multi-level N-Terminal Engineering.

Nicotinate dehydrogenase (NDHase) is a membrane protein with three subunits (ndhS, ndhL, and ndhM), which is difficult to express in a functional form using common hosts such as Escherichia coli, Bacillus subtilis, or Pichia pastoris. Comamonas testosteroni is a suitable microbial chassis for expressing multi-subunit membrane proteins. However, the expression of NDHase in C. testosteroni is extremely low. We have developed a systematic approach to create an efficient protein expression system in C. testosteroni CNB-2 using multi-level N-terminal engineering. We selected a strong promoter for the Mmp1 system that enables control of transcriptional strength in unconventional bacteria. This enhanced the expression of a green fluorescent reporter protein threefold. Following modification of the N-terminal Shine-Dalgarno sequence and rearrangement of amino acid sequence in the starting area of the gene encoding NDHase, enzyme activity increased from 90.6 to 165 U/L. These optimized N-terminal Shine-Dalgarno and amino acid sequences were used to enhance the expression of ndhL subunit and improve the balance expression of three subunits of NDHase, resulting in enzyme activity of 192 U/L that far surpasses the previously reported level. These results highlight a promising strategy for the development of other heterologous expression systems for challenging proteins using unconventional bacteria.

Efficient heterologous expression of nicotinate dehydrogenase in Comamonas testosteroni CNB-2 with transcriptional, folding enhancement strategy.

Nicotinate dehydrogenase (NDHase) from Comamonas testosteroni JA1 catalyzes the C6 hydroxylation of 3-cyanopyridine with high regional selectivity, which is a very difficult and complex reaction for chemical synthesis. However, because NDHase is a membrane protein with three subunits (ndhS, ndhL and ndhM), it is difficult to express the enzyme in a functional form using common hosts such as Escherichia coli, Bacilus subtilis or Pichia pastoris. Furthermore, the enzyme requires special electron transfer chains in the membrane system for proper catalytic activity. Thus, we investigated the expression of NDHase in non-model bacterial strains, which are evolutionarily similar to C. testosteroni JA1, using several broad-host plasmids with different copy numbers as expression vectors. We successfully expressed NDHase in soluble from using the pVLT33 vector in C. testosteroni CNB-2, and found the activity of enzyme to be 40.6 U/L. To further improve the expression of NDHase in C. testosteroni CNB-2, we trialed a T7-like MmP1 system, composed of MmP1 RNA polymerase and an MmP1 promoter, which is used for transcriptional control in non-model bacteria. This increased protein expression and enzyme activity doubled to 90.5 U/L. A molecular chaperone was co-expressed using pBBR1 MCS-5 in the same host to improve the efficiency of folding and assembly of multi-subunit structures. The maximum activity was 115 U/L using the molecular chaperone GroES-EL, far surpassing the previously reported level, although expression was almost equivalent. These results indicate that a strategy involving the construction of a T7-like system and co-expression of a molecular chaperone offers an efficient approach for heterologous expression of enzymes that are difficult to express in functional forms using conventional hosts.

Transcriptome Based System Biology Exploration Reveals Homogeneous Tumorigenicity of Alimentary Tract Malignancy.

Malignancies of alimentary tract include esophageal carcinoma (ESCA), stomach adenocarcinoma (STAD), colon adenocarcinoma (COAD), and rectum adenocarcinoma (READ). Despite of their similarities in cancer development and progression, there are numerous researches concentrating on single tumor but relatively little on their common mechanisms. Our study explored the transcriptomic data of digestive tract cancers from The Cancer Genome Atlas database, yielding their common differentially expressed genes including 1,700 mRNAs, 29 miRNAs, and 362 long non-coding RNAs (lncRNAs). There were 12 mRNAs, 5 miRNAs, and 16 lncRNAs in the core competitive endogenous RNAs network by RNA-RNA interactions, highlighting the prognostic nodes of SERPINE1, hsa-mir-145, and SNHG1. In addition, the weighted gene co-expression network analysis (WGCNA) illustrated 20 gene modules associated with clinical traits. By taking intersections of modules related to the same trait, we got 67 common genes shared by ESCA and READ and screened 5 hub genes, including ADCY6, CXCL3, NPBWR1, TAS2R38, and PTGDR2. In conclusion, the present study found that SERPINE1/has-mir-145/SNHG1 axis acted as promising targets and the hub genes reasoned the similarity between ESCA and READ, which revealed the homogeneous tumorigenicity of digestive tract cancers at the transcriptome level and led to further comprehension and therapeutics for digestive tract cancers.

Effectiveness of advanced nursing care on depression in patients with ovarian cancer: A protocol of systematic review of randomized controlled trial.

BACKGROUND: This systematic review will assess the effectiveness of advanced nursing care (ANC) on depression in patients with ovarian cancer (OC). METHODS: We will identify any relevant randomized controlled trial from Cochrane Library, MEDLINE, Embase, Web of Science, Springer, Chinese Biomedical Literature Database, and China National Knowledge Infrastructure from their inceptions to March 5, 2019. The primary outcome includes depression. The secondary outcomes consist of anxiety, quality of life, and adverse events. Data that meets all the eligibility criteria will be extracted, pooled, and analyzed by using RevMan 5.3 software. Methodological quality for each eligible study will be assessed by using Cochrane risk of bias tool. RESULTS: This study will analyze depression, anxiety, quality of life, and adverse events of ANC on depression in patients with OC. CONCLUSION: The findings of this study will provide the latest evidence for the effectiveness and adverse events of ANC on depression in patients with OC. ETHICS AND DISSEMINATION: No ethic approval is required for this study, because all the data will be extracted from previous published studies. The results of this study will be presented at conference or will be published at a peer-reviewed journal. PROSPERO REGISTRATION NUMBER: PROSPERO CRD42019126374.

Hantavirus Gc induces long-term immune protection via LAMP-targeting DNA vaccine strategy.

Hemorrhagic fever with renal syndrome (HFRS) occurs widely throughout Eurasia. Unfortunately, there is no effective treatment, and prophylaxis remains the best option against the major pathogenic agent, hantaan virus (HTNV), which is an Old World hantavirus. However, the absence of cellular immune responses and immunological memory hampers acceptance of the current inactivated HFRS vaccine. Previous studies revealed that a lysosome-associated membrane protein 1 (LAMP1)-targeting strategy involving a DNA vaccine based on the HTNV glycoprotein Gn successfully conferred long-term immunity, and indicated that further research on Gc, another HTNV antigen, was warranted. Plasmids encoding Gc and lysosome-targeted Gc, designated pVAX-Gc and pVAX-LAMP/Gc, respectively, were constructed. Proteins of interest were identified by fluorescence microscopy following cell line transfection. Five groups of 20 female BALB/c mice were subjected to the following inoculations: inactivated HTNV vaccine, pVAX-LAMP/Gc, pVAX-Gc, and, as the negative controls, pVAX-LAMP or the blank vector pVAX1. Humoral and cellular immunity were assessed by enzyme-linked immunosorbent assays (ELISAs) and 15-mer peptide enzyme-linked immunospot (ELISpot) epitope mapping assays. Repeated immunization with pVAX-LAMP/Gc enhanced adaptive immune responses, as demonstrated by the specific and neutralizing antibody titers and increased IFN-γ production. The inactivated vaccine induced a comparable humoral reaction, but the negative controls only elicited insignificant responses. Using a mouse model of HTNV challenge, the in vivo protection conferred by the inactivated vaccine and Gc-based constructs (with/without LAMP recombination) was confirmed. Evidence of pan-epitope reactions highlighted the long-term cellular response to the LAMP-targeting strategy, and histological observations indicated the safety of the LAMP-targeting vaccines. The long-term protective immune responses induced by pVAX-LAMP/Gc may be due to the advantage afforded by lysosomal targeting after exogenous antigen processing initiation and major histocompatibility complex (MHC) class II antigen presentation trafficking. MHC II-restricted antigen recognition effectively primes HTNV-specific CD4+ T-cells, leading to the promotion of significant immune responses and immunological memory. An epitope-spreading phenomenon was observed, which mirrors the previous result from the Gn study, in which the dominant IFN-γ-responsive hot-spot epitopes were shared between HLA-II and H2d. Importantly, the pan-epitope reaction to Gc indicated that Gc should be with potential for use in further hantavirus DNA vaccine investigations.

Predictive value of non-fasting remnant cholesterol for short-term outcome of diabetics with new-onset stable coronary artery disease.

BACKGROUND: The relationship between non-fasting remnant cholesterol and cardiovascular outcome in the era of potent statin therapy remained to be elucidated. METHODS: A cohort study of three hundred and twenty eight diabetics diagnosed with new-onset stable coronary artery disease (CAD) by coronary angiography were enrolled. All cases were followed up for an average duration of twelve months. The association between baseline remnant cholesterol levels and major cardiovascular outcomes were evaluated using the receivers operating characteristic (ROC) curves and Cox proportional hazards regression analysis. RESULTS: During a period of 12-month's follow-up, 14.3% patients (47/328) underwent pre-specified adverse outcomes. The remnant cholesterol associated with high sensitivity C-reactive protein, neutrophil count and fibrinogen (R 2 = 0.20, 0.12 and 0.14; P = 0.000, 0.036 and 0.010 respectively). Area under the ROC curves (AUC) indicated discriminatory power of the remnant cholesterol to predict the adverse outcomes for this population (AUC = 0.64, P < 0.005). Kaplan-Meier curve suggested that the lower levels of remnant cholesterol showed relatively lower cardiac events for diabetic patients with stable CAD (Log rank X 2 = 8.94, P = 0.04). However, according to multivariate Cox proportional hazards regression, apart from hemoglobin A1C (Hazard ratio [H.R.] =1.38, 95% CI: 1.14-1.66, P = 0.001) and Gensini scores (H.R. = 1.00, 95% CI: 1.00-1.02; P = 0.035), remnant cholesterol did not qualify as an independent predictor of adverse prognosis in these settings (H.R. = 1.05, 95% CI: 0.46-2.37, P = 0.909). CONCLUSIONS: Non-fasting remnant cholesterol was associated with inflammatory biomarkers and high incidence of revascularization, but not qualified as an independent predictor for short-term prognosis of diabetics with new-onset stable coronary artery disease.

Plasma homocysteine levels predict early recurrence after catheter ablation of persistent atrial fibrillation.

AIMS: To assess the association and the predictive value of plasma homocysteine (Hcy) with early recurrence in persistent atrial fibrillation patients after a single ablation procedure. METHODS AND RESULTS: Two hundred and fifty-seven consecutive patients with persistent atrial fibrillation who underwent successful catheter ablation were enrolled. Early recurrence of atrial tachyarrhythmia was documented within 3 months after ablation. The logistic regression analysis and Kaplan-Meier curve analysis were used to evaluate the association of Hcy with early recurrence. During the 3-month follow-up, 75 (29.2%) patients experienced recurrence. Patients with early recurrence were older, more likely to have larger left atrial diameter and higher CHA2DS2-VASc score (all P< 0.001). Plasma Hcy levels were significantly elevated in patients with early recurrence compared with those without early recurrence (15.1 ± 4.1 vs. 12.4 ± 3.7 µmol/L, P< 0.001). In multivariate analysis, Hcy was significantly associated with early recurrence (OR 1.188, 95% CI 1.097-1.286, P< 0.001). Hcy demonstrated a predictive value with AUC of 0.688 (95% CI 0.623-0.753, P< 0.001). The optimal cut-off value was 14 µmol/L for Hcy (sensitivity 69%, specificity 59%). Patients with Hcy ≥14 µmol/L had higher early recurrence rate compared with those with Hcy <14 µmol/L (41 vs. 22%, P= 0.006). CONCLUSION: Plasma Hcy levels are associated with early recurrence of atrial tachyarrhythmia after catheter ablation in persistent atrial fibrillation patients, thus it should be taken into account in prediction of early recurrence.

Culprit vessel only versus "one-week" staged percutaneous coronary intervention for multivessel disease in patients presenting with ST-segment elevation myocardial infarction.

OBJECTIVE: To explore the impact of a "one-week" staged multivessel percutaneous coronary intervention (PCI) versus culprit-only PCI on deaths and major adverse cardiac events (MACE). METHODS: We retrospectively analyzed 447 patients with multivessel disease who experienced a ST-segment elevation myocardial infarction (STEMI) within 12 h before undergoing PCI between July 26, 2008 and September 25, 2011. After completion of PCI in the infarct artery, 201 patients still in the hospital agreed to undergo PCI in non-infarct arteries with more than 70% stenosis for a "one-week" staged multivessel PCI. A total of 246 patients only received intervention for the culprit vessel. Follow-up ended on September 9, 2014. This study examined the differences in deaths from any cause (i.e., cardiac and noncardiac) and MACE between the two treatment groups. RESULTS: Compared to a culprit-only PCI treatment approach, the "one-week" staged multivessel PCI was strongly associated with greater benefits for 55-month all cause death [41 (16.7%) vs.13 (6.5%), P = 0.004] and MACE [82 (33.3%) vs. 40 (19.9%), P = 0.002] rates. In addition, there were significant differences in the number of myocardial infarctions [43 (17.5%) vs. 20 (10.0%), P = 0.023], coronary-artery bypass grafting [CABG; 20 (8.1%) vs. 6 (3.0%), P = 0.021], and PCI [31 (12.6%) vs. 12 (6.0%), P = 0.018]. Patients undergoing culprit-only PCI compared to "one-week" PCI had the same number of stent thrombosis events [7 (2.8%) vs. 3 (1.5%), P = 0.522]. CONCLUSIONS: Compared to a culprit-only PCI treatment approach, "one-week" staged multi-vessel PCI was a safe and effective selection for STEMI and multi-vessel PCI.

Dynamic response and optimal design of curved metallic sandwich panels under blast loading.

It is important to understand the effect of curvature on the blast response of curved structures so as to seek the optimal configurations of such structures with improved blast resistance. In this study, the dynamic response and protective performance of a type of curved metallic sandwich panel subjected to air blast loading were examined using LS-DYNA. The numerical methods were validated using experimental data in the literature. The curved panel consisted of an aluminum alloy outer face and a rolled homogeneous armour (RHA) steel inner face in addition to a closed-cell aluminum foam core. The results showed that the configuration of a "soft" outer face and a "hard" inner face worked well for the curved sandwich panel against air blast loading in terms of maximum deflection (MaxD) and energy absorption. The panel curvature was found to have a monotonic effect on the specific energy absorption (SEA) and a nonmonotonic effect on the MaxD of the panel. Based on artificial neural network (ANN) metamodels, multiobjective optimization designs of the panel were carried out. The optimization results revealed the trade-off relationships between the blast-resistant and the lightweight objectives and showed the great use of Pareto front in such design circumstances.

[Erythropoietin inhibits angiotensin II induced cardiomyocyte hypertrophy in vitro via activating PI3K/Akt-eNOS pathway].

OBJECTIVE: To explore the effect of erythropoietin (EPO) on angiotensin II (AngII) induced neonatal rat cardiomyocyte hypertrophy and the association with PI3K/Akt-eNOS signaling pathway. METHODS: Cardiomyocytes were isolated from new-born Sprague-Dawley rats and stimulated by AngII in vitro. The cell surface area and mRNA expression of atrial natriuretic factor (ANF) of cardiomyocytes were determined in the presence and absence of various concentrations of EPO, phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002 and nitric oxide synthase (NOS) inhibitor L-NAME. Intracellular signal molecules, such as Akt, phosphorylated Akt, eNOS and phosphorylated eNOS protein expressions were detected by western blot. Nitric oxide (NO) level in the supernatant of cultured cardiomyocytes was assayed by NO assay kit. RESULTS: EPO (20 U/ml) significantly inhibited AngII induced cardiomyocyte hypertrophy as shown by decreased cell surface area and ANF mRNA expression (all P < 0.05). EPO also activated Akt and enhanced the expression of eNOS and its phosphorylation (all P < 0.05), increased the NO production (P < 0.01). These effects could be partially abolished by cotreatment with LY294002 or L-NAME (all P < 0.05). CONCLUSION: EPO attenuates AngII induced cardiomyocytes hypertrophy via activating PI3K-Akt-eNOS pathway and promoting NO production.

Erythropoietin attenuates hypertrophy of neonatal rat cardiac myocytes induced by angiotensin-II in vitro.

OBJECTIVE: Erythropoietin (EPO) is a haematopoietic hormone that has been confirmed as a novel cardioprotective agent. In this study, we test the hypothesis that EPO inhibits angiotensin-II (Ang-II)-induced hypertrophy in cultured neonatal rat cardiomyocytes. MATERIAL AND METHODS: Cultured neonatal rat cardiomyocytes were used to evaluate the effects of EPO on Ang-II-induced hypertrophy in vitro. The surface area and mRNA expression of atrial natriuretic (ANF) myocytes were employed to detect cardiac hypertrophy. A phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002 and an endothelial nitric oxide synthase (eNOS) inhibitor L-NAME were also employed to detect the underlying mechanism of EPO. Intracellular signal molecules, such as Akt (PKB), phosphorylated Akt, eNOS and transforming growth factor-beta1 (TGF-beta1) protein expression were determined by Western blot. Nitric oxide (NO) levels in the supernatant of cultured cardiomyocytes were assayed using an NO assay kit. RESULTS: The results indicate that EPO significantly attenuates Ang-II-induced hypertrophy shown as inhibition of increases in cell surface area and ANF mRNA levels. NO production was also increased proportionally in the EPO-treated group. EPO enhanced Akt activation and eNOS protein expression, whereas LY294002 or L-NAME partially abolished the anti-hypertrophic effect of EPO, accompanied by a decrease in Akt activation, eNOS protein expression and/or a reduction of NO production. EPO also down-regulated the protein expression of TGF-beta1. CONCLUSION: We conclude that EPO attenuates cardiac hypertrophy via activation of the PI3K-Akt-eNOS-NO pathway and the down-regulation of TGF-beta1.

[Proliferation and regeneration effects of constructed human beta-nerve growth factor gene on transfected cat corneal endothelial cells in vitro].

OBJECTIVE: To explore the mechanisms of proliferation and regeneration effects of a human nerve growth factor (beta-NGF) expression vector (pcDNA4-beta-NGF) on the transfected cat corneal endothelial cells in vitro. To provide a new method for long term cultivation of human corneal endothelial cells in vitro and to establish theoretical basis of gene therapy for corneal endothelial defects. METHODS: It was a experimental study. The human pcDNA4-beta-NGF expression vector was constructed and transfected into cultured cat corneal endothelial cells by Effectene lipofectine transfection technique. The expression of the reporter gene pcDNA4-beta-LacZ expression was used to determine the transfection efficiency 48 hours after the transfection. RT-PCR and immunohistochemistry techniques were used to check the transient expression status at mRNA and protein levels in cat corneal endothelial cells. Mitotic index and methyl thiazolyl tetrazolium (MTT) value were measured and cell numbers at different stages of cell cycles were determined by flow cytometer 96 hours after transfection. An in vitro quantitative cat corneal endothelial cell traumatic model was established which was used for observing the effect of human beta-NGF expression product on the DNA synthesis of cat endothelial cells and healing process of traumatized endothelial cells. RESULTS: A human nerve growth factor (beta-NGF) expression vector (pcDNA4-beta-NGF)was successfully constructed and confirmed by sequence analysis. Single layered pure cat corneal endothelial cells were obtained by a modified sliced tissue culture technique and confirmed by morphological analysis, neurone specific enolase immunohistochemistry study and transmission electronic microscope. Effectene lipofectine mediated transfection efficiency of pcDNA4-beta-NGF into cat corneal endothelial cells in vitro was 11.3%. The human beta-NGF could be highly expressed in the transfected corneal endothelial cells at mRNA and protein levels. Mitotic index, MTT value and G1 stage cell numbers, as well as traumatically defected endothelial cells numbers during the healing process of human beta-NGF transfected corneal endothelial cells were statistically differed from the pre-transfected cells and control groups. CONCLUSIONS: Effectene lipofectine transfection technique could be effectively used for transfecting pcDNA4-beta-NGF into cat corneal endothelial cells in vitro with good efficacy and the gene could stably express to improve the proliferation and regeneration of the cat corneal endothelial cells. This method could be managed as an experimental basis to be applied in the experimental study for transfecting the human beta-NGF gene into human corneal endothelial cells. Therefore a new method for resolving the problem of impossible regeneration of corneal endothelial cells could become possible.

[Infection status and clinical significance of Epstein-Barr virus in pediatric leukemia---a report of 35 cases].

BACKGROUND & OBJECTIVE: Epstein-Barr virus (EBV) is associated with genesis of many human tumors. This study was to detect EBV infection in pediatric leukemia, and to explore its clinical significance. METHODS: EBV DNA in peripheral blood mononuclear cells in 35 pediatric leukemia patients, including 26 cases of acute lymphoblastic leukemia (ALL) (24 received initial treatment and 2 received retreatment), 8 cases of acute non-lymphocytic leukemia (ANLL) and 1 case of chronic lymphocytic leukemia (CLL), and in 14 healthy children was detected by fluorescent quantitative polymerase chain reaction (FQ-PCR). Its clinical significance was analyzed according to the clinical manifestations, prednisone sensitivity test, and complete remission (CR) rate after induction chemotherapy. RESULTS: EBV DNA was detected in 8 (22.86%) of the 35 pediatric leukemia patients. The positive rate of EBV DNA was 26.92% (7/26) in ALL with quantity of (5.144+/-6.91)x10(5) copies/ml, and 12.5% (1/8) in ANLL patients with quantity of 4.031x10(3) copies/ml. No EBV DNA was detected in CLL patients and healthy controls. The occurrence rates of peripheral leukocytosis and hepatosplenomegaly were significantly higher in the patients with EBV infection than in the patients without EBV infection (P <0.001). In ALL, the rate of no response to prednisone was significantly higher in the patients with EBV infection than in the patients without EBV infection (100% vs. 26.32%, P =0.001); CR rate after induction chemotherapy was significantly lower in the patients with EBV infection than in the patients without EBV infection (28.57% vs. 84.21%, P=0.003). In ANLL, the differences of CR rate and relapse rate were not significant between the patients with and without EBV infection (P=0.5). CONCLUSIONS: Pediatric leukemia patients with EBV infection have higher incidence of peripheral leukocytosis and hepatosplenomegaly. ALL patients with EBV infection have poor prednisone response and low CR rate.

[Expression of the human papillomavirus type 16L/E7 fusion protein in E. coli and observation of its immunogenicity in mice].

BACKGROUND: Many epidemiological and experimental evidences prove that cervical cancers are strongly associated with genital high-risk types of human papillomavirus (HPV). HPV16 is present in 50% of the tumor specimens. Thus, it is important to develop vaccines against HPV16 and cervical cancer. The authors studied the expression of the HPV16 L1DeltaCE7N fusion protein in E. coli and observed its immunogenicity. METHODS: The fragment of HPV16 L1DeltaC gene and the E7N gene were amplified by PCR separately; the fusion gene named L1DeltaCE7N was generated by fusing E7N to the C terminal of L1DeltaC then the chimeric gene was cloned into prokaryotic expression vector pGEX-2T and expressed in E. coli strain JM109. The L1DeltaCE7N protein expressed were detected by Western blot. Finally its immunogenicity was characterized in immunized mice. RESULTS: It was proved that the sequence and open reading frame of fusion gene L1DeltaE7N was correct by sequencing; SDA-PAGE gel analysis showed that HPV16 L1/E7 fusion protein was highly expressed in E. coli; the protein was expressed as soluble form and the molecular weight was about 85 x 10(3). The fusion protein could be purified by affinity chromatography and gel filtration. The ELISA result indicated that L1/E7 could elicit specific antibodies against L1 and E7 in immunized mice. In vivo tumor protection test indicated that tumor formation was retarded or prevented in the mice after vaccination with L1/E7, when C57 BL/6 mice were challenged by syngeneic HVP16E6 and E7 transformed tumor cells. CONCLUSION: HPV16L1/E7 fusion protein was expressed in E. coli, it can be a candidate for prophylactic and therapeutic vaccine for HPV16-associated infection and tumors.

[Analysis of the factors which influence allograft calcification after aorta transplantation of allogenetic rat].

AIM: To explore the factors which influence the calcification of homograft after aorta transplantation of allogenetic rat. METHODS: The research was devided into 2 groups: allogene group and isogenenic group. Allogene group: SD -->Wistar. Isogenenic group: Wistar to Wistar. Aortic valve homograft was heterotopically allografted onto abdominal aorta. The rats were sacrificed in batches at 2, 4, 8, 12 and 16 weeks postoperatively. Blood samples were obtained for accessing the expression of CD25, CD71, and AVH was obtained for accessing the calcium level and the expression of CD40. At the same time, the change of endotheliocyte and smooth muscle cells were observed with transmission electron microscope. RESULTS: (1)Compared with the isogene group, the expression of CD40, CD25 and CD71 in allogene group was much higher at each time point and reached peak at 2-4 weeks after operation. (2)The calcium level in allogene group increased at 4 weeks after operation and reached the peak at 12 weeks after operation. No significant difference in calcium level was found in isogenenic group over 5 different periods. (3)Exfoliation of endotheliacytes as well as necrosis of smooth muscle cells were observed in the graft in allogene group. CONCLUSION: The calcium level of homograft had a relation with immunological rejection. The calcification began at 4 weeks postoperation; the calcium level of homogaft increased gradually and reached the peak at 12 weeks postoperation and then maintained on a stable level.