[Preparation and Application of Monoclonal Antibody Against Human von Willebrand Factor Propeptide].

OBJECTIVE: To develop monoclonal antibodies that can specifically recognize human von Willebrand factor (VWF) propeptide (VWFpp) in plasma, and establish a rapid and reliable method for the detection of VWFpp antigen in plasma by using the double-antibody sandwich ELISA with the obtained anti-VWFpp monoclonal antibody. METHODS: The recombinant human VWFpp (D1 and D2 regions) protein expressed in eukaryotic cells was used as immunogen to immunize BALB/c mice with routine method, so as to obtain clones of fusion cells. After screening and identification, hybridoma cell lines secreting monoclonal antibodies against VWFpp were selected, and then double-antibody sandwich ELISA assay was used to construct VWFpp antigen detection kit for the determination of VWFpp in human plasma. The levels of VWFpp antigen in plasma of 12 leukemia patients who underwent bone marrow transplantation were dynamically detected. RESULTS: Two hybridoma cell lines that can be subcultured continuously and secrete monoclonal antibodies against VWFpp were obtained and named SZ175 and SZ176 respectively. Identified by ELISA and Western blot, the antibodies could both specifically recognize VWFpp but couldn't recognize mature VWF (without propeptide). Based on the principle of double-antibody sandwich ELISA, monoclonal antibodies SZ175 and SZ176 were successfully made into a kit for detecting VWFpp antigen. The plasma VWFpp levels of leukemia patients before and after bone marrow transplantation were dynamically detected. The results showed that the plasma VWFpp levels of the patients after transplantation were significantly higher than those before transplantation. CONCLUSION: Two monoclonal antibodies against VWFpp were successfully prepared, and a double-antibody sandwich ELISA detection kit for VWFpp antigen was constructed, which provides a powerful tool for further study on the biological function of VWFpp, the clinical diagnosis and classification of von Willebrand disease (VWD), and the prognostic monitoring of endothelial injury-related diseases.

Hematopoiesis reconstitution and anti-tumor effectiveness of Pai-Neng-Da capsule in acute leukemia patients with haploidentical hematopoietic stem cell transplantation.

BACKGROUND: With the rapid development of haploidentical hematopoietic stem cell transplantation (haplo-HSCT), primary poor graft function (PGF) has become a life-threatening complication. Effective therapies for PGF are inconclusive. New Chinese patent medicine Pai-Neng-Da (PND) Capsule exerts dual effect in promoting hematopoiesis recovery and regulating immunity. Still, the application of PND capsule in hematopoietic stem cell transplantation, especially in the haplo-HSCT setting, has not yet been reported. AIM: To evaluate the role of PND capsule in acute leukemia patients with haplo-HSCT. METHODS: We retrospectively collected data of acute leukemia patients who underwent haplo-HSCT at the Affiliated People's Hospital of Ningbo University between April 1, 2015 and June 30, 2020. Twenty-nine consecutive patients received oral PND capsule from the sixth day to the first month after haplo-HSCT were included in the PND group. In addition, 31 patients who did not receive PND capsule during haplo-HSCT were included in the non-PND group. Subsequently, we compared the therapeutic efficacy according to the western medical evaluation indexes and Chinese medical symptom scores, and the survival between the PND group and the non-PND group, using the chi-square test, Fisher's exact test, and the Kaplan-Meier method. RESULTS: The duration of platelet engraftment was shorter in the PND group than in the non-PND group (P = 0.039). The PND group received a lower frequency of red blood cells and platelet transfusions than the non-PND group (P = 0.033 and P = 0.035, respectively). In addition, PND capsule marginally reduced the rate of PGF (P = 0.027) and relapse (P = 0.043). After 33 (range, 4-106) months of follow-up, the 3-year relapse-free survival (P = 0.046) and progression-free survival (P = 0.049) were improved in the PND group than in the non-PND group. Also, the therapeutic efficacy of the PND group according to Chinese medical symptom scores was significantly better than that of the non-PND group (P = 0.022). Moreover, the adverse events caused by PND capsule were mild. Nevertheless, there were no significant differences in the duration of neutrophil engraftment, the risk of infection within 100 days after haplo-HSCT, the acute graft-versus-host disease, or the 3-year overall survival between the two groups. CONCLUSION: PND capsule could promote hematopoiesis reconstitution, improve the therapeutic efficacy of Chinese medical symptom scores, present anti-tumor effectiveness, and prolong the survival of acute leukemia patients with haplo-HSCT.

The first complete mitogenome of skin beetles Omorgus chinensis (Coleoptera: Trogidae) with the phylogenetic implications.

The first complete mitogenome of Trogidae, Omorgus chinensis (Coleoptera: Trogidae) is sequenced using the next generation sequencing. The genomic structure is a circular molecule with 18682 bp in length, comprising 13 protein-coding genes, 22 transfer RNA genes (tRNAs), 2 ribosomal RNAs (rRNAs) and a control region. The nucleotide composition is A (39.44%), C (13.82%), T (36.78%), and G (9.96%) with an AT content of 76.22%. The phylogenetic analysis of 18 insects in Scarabaeoidae show that Omorgus chinensis shares a close ancestry with Lucanidae and Geotrupidae.

A multilocus assessment reveals two new synonymies for East Asian Cyclommatus stag beetles (Coleoptera, Lucanidae).

Cyclommatus scutellaris Möllenkamp, 1912, Cyclommatus elsae Kriesche, 1921 and Cyclommatus tamdaoensis Fujita, 2010 are East Asian stag beetle species with long-debated taxonomic relationships due to high intraspecific morphological variability. In this study, we applied multilocus phylogenetic analyses to reassess their relationships. Two mitochondrial genes (16S rDNA, COI) and two nuclear genes (28S rDNA, Wingless) were used to reconstruct the phylogeny through the Bayesian inference (BI) and Maximum Likelihood (ML) methods. Both topologies supported two clades: the clade C. scutellaris was sister to the clade (C. elsae + C. tamdaoensis) with the subclade C. tamdaoensis embedded in the subclade C. elsae. The Kimura 2-parameter (K2P) genetic distance analysis yielded a low mean value (≤0.035) among the three taxa, which was well below the minimum mean value between other Cyclommatus species (≥0.122). We also compared the accuracy and efficiency of two approaches, GMYC and ABGD, in delimitating the three lineages. The result shows that ABGD is a better approach than GMYC. Our molecular data recognizes the three species as different populations of a single species, ranging from Taiwan Island to the continent. Therefore, we propose two new junior synonyms for C. scutellaris: C. tamdaoensis, syn. nov. and C. elsae syn. nov.

Relationship between platelet/lymphocyte ratio and prognosis of patients with septic acute kidney injury: A pilot study.

BACKGROUND: To explore the potential role of the platelet/lymphocyte ratio (PLR) as a prognostic marker in septic patients with acute kidney injury (AKI) and to provide theoretical evidence for the epidemiological study of the prognosis of patients with septic AKI in its early stage. METHODS: A pilot study was conducted. A logistic regression analysis was conducted to screen the risk factors, and the selected factors were performed using multiple logistic regression analysis; a Receiver Operating Characteristic curve was used to determine the optimal cutoff value of the PLR and then to calculate the sensitivity and specificity of the PLR ratio. RESULTS: Mechanical ventilation, platelet count, PLR, and arterial blood lactate concentration have a correlation with sepsis (p < 0.05). An elevated PLR is significantly associated with a worse prognosis of sepsis-induced AKI (higher mortality). CONCLUSION: The PLR might be an effective factor in predicting a worse prognosis of septic AKI patients.

Clinical utility of characterizing intestinal flora in septic kidney injury.

The incidence of septic acute kidney injury (AKI) is increasing, it has become a major threat to human health because of its acute onset, poor prognosis, and high hospital costs. The most common cause of AKI in critical-care units is sepsis. Septic AKI is a complex and multi-factorial process; its pathogenesis is not fully understood. In sepsis, the destruction of mucosal barriers, intestinal flora disorders, intestinal ischemia/reperfusion injury, use of antibiotics, and lack of intestinal nutrients lead to an inflammatory reactions that in turn affects the metabolism and immunity of the host. Such changes further influence the occurrence and development of AKI. New technology is enabling various detection methods for intestinal flora. Clinical application of these methods in septic renal injury is expected to clarify the relationship among pathogenesis, disease progression mechanism, and intestinal flora.

Levosimendan does not reduce the mortality of critically ill adult patients with sepsis and septic shock: a meta-analysis.

BACKGROUND: Previous studies on whether or not levosimendan improved the prognosis of patients with sepsis and septic shock have been inconsistent. We aimed to provide an updated analysis of the therapeutic value of levosimendan in adult patients with sepsis and septic shock, in order to provide evidence-based medical evidence for its use. METHODS: PubMed, Embase, Cochrane Library, Wanfang Data, and CNKI were searched until August 2018 without language restriction. Randomized controlled studies of levosimendan with either inotropic drugs or placebo for the treatment of sepsis or septic shock were enrolled. The primary outcome was mortality, and cardiac index and serum lactate levels were the secondary outcomes. RESULTS: A total of 20 randomized controlled studies were included in this meta-analysis, including 1467 patients, with 738 patients in the experimental group (levosimendan group) and 729 patients in the control group (other inotropic drugs or placebo). There were no significant differences in mortality between the levosimendan and control groups (fixed-effect relative risk [RR] = 0.90, 95% confidence interval [CI] [0.79, 1.03], P = 0.13). Levosimendan increased the cardiac index (VMD [weighted mean difference] = 0.51, 95% CI [0.06, 0.95], P = 0.02); and serum lactate levels were lower (VMD = -1.04, 95% CI [-1.47, -0.60], P < 0.00001). CONCLUSIONS: Based on current clinical evidence, levosimendan does not reduce mortality in adult critically ill patients with sepsis and septic shock. Physicians should use levosimendan with caution in patients with sepsis and septic shock.

Thermal Decomposition Behavior of Hydroxytyrosol (HT) in Nitrogen Atmosphere Based on TG-FTIR Methods.

The thermal decomposition behavior of olive hydroxytyrosol (HT) was first studied using thermogravimetry (TG). Cracked chemical bond and evolved gas analysis during the thermal decomposition process of HT were also investigated using thermogravimetry coupled with infrared spectroscopy (TG-FTIR). Thermogravimetry-Differential thermogravimetry (TG-DTG) curves revealed that the thermal decomposition of HT began at 262.8 °C and ended at 409.7 °C with a main mass loss. It was demonstrated that a high heating rate (over 20 K·min-1) restrained the thermal decomposition of HT, resulting in an obvious thermal hysteresis. Furthermore, a thermal decomposition kinetics investigation of HT indicated that the non-isothermal decomposition mechanism was one-dimensional diffusion (D1), integral form g(x) = x², and differential form f(x) = 1/(2x). The four combined approaches were employed to calculate the activation energy (E = 128.50 kJ·mol-1) and Arrhenius preexponential factor (ln A = 24.39 min-1). In addition, a tentative mechanism of HT thermal decomposition was further developed. The results provide a theoretical reference for the potential thermal stability of HT.

The application of MutMap in forward genetic studies based on whole-genome sequencing.

Classical forward genetic analysis relies on construction of complicated progeny populations and development of many molecular markers for linkage analysis in genetic mapping, which is both time- and cost-consuming. The recently developed MutMap is a new forward genetic approach based on high-throughput next-generation sequencing technologies. It is more efficient and affordable than traditional methods. Moreover, new extended methods based on MutMap have been developed: MutMap+, which is based on self-crossing; MutMap-Gap, which is used to recognize the causative variations occurring in genome gap regions; QTL-seq, a method similar to MutMap for mapping quantitative trait loci. These methods are free from constructing complicated mapping population, genetic hybridization and linkage information. They have greatly accelerated the identification of genetic elements associated with interested phenotypic variation. Here, we review the basic principles of MutMap, and discuss their future applications in next generation sequencing-based forward genetic mapping and crop improvement.

Preparation, Characterization, and Antioxidant Activity Evaluation of Liposomes Containing Water-Soluble Hydroxytyrosol from Olive.

Due to the multiple hydroxyl groups in its structure, hydroxytyrosol (HT) is very sensitive to air and light and has very strong instability and hydrophilicity that affect its biological activity. This study attempted to prepare liposomes containing water-soluble HT to improve the bioavailability and biocompatibility of the target drug. The preparation process factors (temperature, mass ratio of phospholipid (PL) and cholesterol (CH), Tween-80 volume, HT mass) were studied and response surface methodology (RSM) was applied to optimize the conditions. The results demonstrated that by using a temperature of 63 °C, mass ratio of PL and CH 4.5:1, HT mass 5 mg and Tween-80 volume of 6 mL, HT liposomes with an encapsulation efficiency (EE) of 45.08% were prepared. It was found that the particle sizes of the HT liposomes were well distributed in the range of 100-400 nm. Compared to free HT, prepared HT liposomes had better stability and a distinct slow release effect in vitro. Besides, HT liposomes presented better DPPH radical scavenging activity than free HT, which could be due to the fact that HT was encapsulated fully inside the liposomes. In addition, the encapsulation mechanism of HT was evaluated. In summary, the results indicated that HT liposome could enhance the antioxidant activity and was a promising formulation for prolonging the biological activity time of the target drug.

In Vivo and In Vitro Toxicity Evaluation of Polyprenols Extracted from Ginkgo biloba L. Leaves.

Polyprenols of Ginkgo biloba L. leaves (GBP) are a new type of lipid with 14-24 isoprenyl units, which in humans have strong bioactivity like the dolichols. A large amount of work showed that GBP had good antibacterial activity and powerful protective effects against acute hepatic injury induced by carbon tetrachloride and alcohol, as well as antitumor activity, but the safety of GBP was not considered. The current study was designed to evaluate the toxicity of these polyprenols. Acute toxicity in mice was observed for 14 days after GBP oral dosing with 5, 7.5, 10, 15 and 21.5 g/kg body weight (b. wt.) Further, an Ames toxicity assessment was carried out by plate incorporation assay on spontaneous revertant colonies of TA97, TA98, TA100 and TA102, with GBP doses designed as 8, 40, 200, 1000 and 5000 µg/dish, and subchronic toxicity was evaluated in rats for 91 days at GBP doses of 500, 1000 and 2000 mg/kg b. wt./day. The weight, food intake, hematological and biochemical indexes, the ratio of viscera/body weight, and histopathological examinations of tissue slices of organs were all investigated. The results showed that no animal behavior and appearance changes and mortality were seen during the observation period with 21.5 g/kg GBP dose in the acute toxicity test. Also, no mutagenicity effects were produced by GBP (mutation rate < 2) on the four standard Salmonella strains (p > 0.05) in the Ames toxicity test. Furthermore, the no observed adverse effect level (NOAEL) of GBP was 2000 mg/kg for 91 days feeding of rats in the subchronic toxicity tests. Results also showed the hematological and biochemical indexes as well as histopathological examination changed within a small range, and all clinical observation indexes were normal. No other distinct impacts on cumulative growth of body weight, food intake and food utilization rate were discovered with GBP. No significant difference was discovered for the rats' organ weight and the ratio of viscera to body weight (p > 0.05). Reversible pathological changes in the histopathological examinations of tissue slices of organs were not observed. GBP could therefore be considered as a safe material with minor side effects.

Enzymatic hydrolysis of oleuropein from Olea europea (olive) leaf extract and antioxidant activities.

Oleuropein (OE), the main polyphenol in olive leaf extract, is likely to decompose into hydroxytyrosol (HT) and elenolic acid under the action of light, acid, base, high temperature. In the enzymatic process, the content of OE in olive leaf extract and enzyme are key factors that affect the yield of HT. A selective enzyme was screened from among 10 enzymes with a high OE degradation rate. A single factor (pH, temperature, time, enzyme quantity) optimization process and a Box-Behnken design were studied for the enzymatic hydrolysis of 81.04% OE olive leaf extract. Additionally, enzymatic hydrolysis results with different substrates (38.6% and 81.04% OE) were compared and the DPPH antioxidant properties were also evaluated. The result showed that the performance of hydrolysis treatments was best using hemicellulase as a bio-catalyst, and the high purity of OE in olive extract was beneficial to biotransform OE into HT. The optimal enzymatic conditions for achieving a maximal yield of HT content obtained by the regression were as follows: pH 5, temperature 55 °C and enzyme quantity 55 mg. The experimental result was 11.31% ± 0.15%, and the degradation rate of OE was 98.54%. From the present investigation of the antioxidant activity determined by the DPPH method, the phenol content and radical scavenging effect were both decreased after enzymatic hydrolysis by hemicellulase. However, a high antioxidant activity of the ethyl acetate extract enzymatic hydrolysate (IC50 = 41.82 µg/mL) was demonstated. The results presented in this work suggested that hemicellulase has promising and attractive properties for industrial production of HT, and indicated that HT might be a valuable biological component for use in pharmaceutical products and functional foods.