Study on the construction of nomogram prediction model for prognostic assessment of heart failure patients based on serological markers and echocardiography.

OBJECTIVE: We aimed to construct a nomogram prediction model for prognostic assessment of patients with heart failure (HF) based on serological markers and echocardiography. PATIENTS AND METHODS: A total of 200 HF patients admitted to the Second Affiliated Hospital of Nanchang University from January 2018 to January 2020 were selected as the research objects. According to the New York Heart Association (NYHA) cardiac function classification, they were divided into 3 groups, including 65 cases of grade II, 97 cases of grade III, and 38 cases of grade IV. Three groups of echocardiographic parameters were compared [including left ventricular ejection fraction (LVEF), left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular end-systolic volume (LVESV)], differences in serum markers brain natriuretic peptide (BNP), soluble growth-stimulating expression gene 2 (sST2) and the Modified Early Warning Score (MEWS). The patients were divided into two groups according to their clinical outcomes during the follow-up period, including 52 cases in the death group and 148 cases in the survival group. The clinical data of the two groups were compared, and multi-factor logistic regression analysis was performed to screen out the independent risk factors affecting the patient's death. A nomogram model of the patient's mortality risk was constructed based on the independent risk factors. Receiver operating characteristic (ROC) curves and calibration curves were used to evaluate the discrimination and accuracy of the nomogram model. RESULTS: As the cardiac function class of elderly chronic heart failure (CHF) patients increases, LVEDD, LVESD, sST2, and MEWS increase and LVEF decreases (p<0.05). Multifactor analysis results showed that LVEF, LVEDD, sST2, and MEWS were independent factors affecting the clinical outcome of patients. The AUCs predicted using LVEF, LVEDD, sST2, and MEWS alone were 0.738, 0.775, 0.717, 0.831, and 0.768, respectively. There is a certain degree of discrimination, and the model has extremely high accuracy. CONCLUSIONS: MEWS, LVEDD, and sST2 increase as the NYHA cardiac function grade of HF patients increases and LVEF decreases, which can reflect the severity of the disease to a certain extent. Additionally, the nomogram model established based on this has a high predictive value for the long-term prognosis of patients and can formulate effective intervention measures for quantitative values.

[Preparation and in vitro evaluation of FDM 3D printed theophylline tablets with personalized dosage].

OBJECTIVE: To explore the feasibility of preparing different doses of tablets for personalized treatment by fused deposition modeling (FDM) 3D printing technology, and to evaluate the in vitro quality of the FDM 3D printed tablets. METHODS: Three different sizes of hollow tablets were prepared by fused deposition modeling 3D printing technology with polyvinyl alcohol (PVA) filaments. Theophylline was chosen as the model drug. In the study, 20 mg, 50 mg and 100 mg of theophylline was filled into the cavity of the tablets, respectively. The microscopic morphology of the tablets was observed by scanning electron microscopy (SEM). The weight variation of the tablets was investigated by weighing method. The hardness of the tablets was measured by tablet hardness tester. The contents of the drugs in the tablets were determined by ultraviolet and visible spectrophotometry (UV-Vis), and the dissolution apparatus was used to assay the in vitro drug release of the tablets. RESULTS: The prepared FDM 3D printed tablets were all in good shape without printing defects. And there was no leakage phenomenon. The diameter and thickness of the tablets were consistent with the design. The layers were tightly connected, and the fine structure of the formulation could be clearly observed without printing defects by scanning electron microscopy. The average weight of the three sizes of tablets was (150.5±2.3) mg, (293.6±2.6) mg and (456.2±5.6) mg, respectively. The weight variation of the three sizes of tablets were boss less than 5%, which met the requirements; The hardness of the tablets all exceeded 200 N; The contents of theophylline in the three tablets were 98.0%, 97.2% and 97.9% of the dosage (20 mg, 50 mg and 100 mg), and the relative standard deviation (RSD) was 1.06%, 1.15% and 0.63% respectively; The time for 80% drug released from the three dosage of tablets was within 30 min. CONCLUSION: Three different dosages of theophylline tablets were successfully prepared by FDM 3D printing technology in this study. The exploration may bring beneficial for the preparation of personalized dose preparations. We expect that with the development of 3D printing technology, FDM 3D printed personalized tablets can be used in the clinic as soon as possible to provide personalized treatment for patients.

[Preliminary analysis on COVID-19 case spectrum and spread intensity in different provinces in China except Hubei province].

Objective: To analyze the characteristics of COVID-19 case spectrum and spread intensity in different provinces in China except Hubei province. Methods: The daily incidence data and case information of COVID-19 were collected from the official websites of provincial and municipal health commissions. The morbidity rate, severity rate, case-fatality rate, and spread ratio of COVID-19 were calculated. Results: As of 20 March, 2020, a total of 12 941 cases of COVID-19 had been conformed, including 116 deaths, and the average morbidity rate, severity rate and case-fatality rate were 0.97/100 000, 13.5% and 0.90%, respectively. The morbidity rates in Zhejiang (2.12/100 000), Jiangxi (2.01/100 000) and Beijing (1.93/100 000) ranked top three. The characteristics of COVID-19 case spectrum varied from province to province. The first three provinces (autonomous region, municipality) with high severity rates were Tianjin (45.6%), Xinjiang (35.5%) and Heilongjiang (29.5%). The case-fatality rate was highest in Xinjiang (3.95%), followed by Hainan (3.57%) and Heilongjiang (2.70%). The average spread ratio was 0.98 and the spread intensity varied from province to province. Tibet had the lowest spread ratio (0), followed by Qinghai (0.20) and Guangdong (0.23). Conclusion: The intervention measures were effective in preventing the spread of COVID-19 and improved treatment effect in China. However, there were significant differences among different regions in severity, case-fatality rate and spread ratio.

[The preliminary analysis on the characteristics of the cluster for the COVID-19].

Since December 2019, COVID-19, a new emerging infection disease, has spread in 27 countries and regions. The clusters of many cases were reported with the epidemic progresses. We collected currently available information for 377 COVID-19 clusters (1 719 cases), excluded the hospital clusters and Hubei cases, during the period from January 1 to February 20, 2020. There were 297 family clusters (79%), case median was 4; 39 clusters of dining (10%), case median was 5; 23 clusters of shopping malls or supermarkets (6%), case median was 13; 12 clusters of work units (3%), case median was 6, and 6 clusters of transportation. We selected 325 cases to estimate the incubation period and its range was 1 to 20 days, median was 7 days, and mode was 4 days. The analysis of the epidemic situation in a department store in China indicated that there was a possibility of patients as the source of infection during the incubation period of the epidemic. From February 5 to 21, 2020, 634 persons were infected on the Diamond Princess Liner. All persons are susceptible to the 2019 coronavirus. Age, patients during the incubation period and the worse environment might be the cause of the cases rising. The progress of the two typical outbreaks clearly demonstrated the spread of the early cases in Wuhan. In conclusion, screening and isolating close contacts remained essential other than clinical treatment during the epidemic. Especially for the healthy people in the epidemic area, isolation was the key.

[A surveillance study on CRISPR/Cas molecular biomarker in Escherichia coli].

OBJECTIVE: A new method related to molecular biomarker with CRISPR/Cas (clustered regularly interspaced short palindromic repeats-cas) in Escherichia (E.) coli was developed and used for surveillance programs. METHODS: CRISPR/Cas sequence that containing 135 strains with complete sequence and 203 strains with whole genome shotgun sequence of E. coli in GenBank by BLAST and 361 strains of E. coli (including 38 strains of E. coli O157∶H7) in laboratory were identified by PCR and analyzed with the CRISPR Finder. Spacers were compared with DANMAN and the phylogenetic trees of cas gene were constructed under Clustal Ⅹ and Mega 5.1. RESULTS: With new perspective, a descriptive method was developed targeting on the position of CRISPR/cas in E. coli. The CRISPR1 was detected in 77.04%, 100.00% and 75.62% and the CRISPR2 was detected in 74.81%, 100.00% and 92.24% and the CRISPR3 and CRISPR4 were detected in 11.85%, 0 and 1.39% for 135 strains with complete sequence, 203 strains with whole genome shotgun sequence and 361 strains in the laboratory, respectively. One strain downloaded in GenBank with whole genome sequencing and 2 strains in the our laboratory were identified that containing four CRISPR locus. The other E. coli strain was with insertion sequence in downstream of the non-cas CRISPR1. The unique CRISPR was found in 8 strains of O55∶H7, in 180 strains of O157∶H7, in 8 strains of O157∶HNM, in 40 strains of O104∶H4, in 4 strains of O145∶H28, in all the 699 E. coli strains. The phylogenetic tree could be divided into two groups-cas with type I-E or type I-F. CONCLUSIONS: CRISPR/Cas might be used as a valuable molecular biomarker in epidemiological surveillance studies to identify the high virulent strains or new strains of E. coli. Phage night be related to the missing or obtaining of spacers.

Comparative Efficacy of Four Treatments in Patients with Graves' Disease: a Network Meta-analysis.

BACKGROUND: The question of which treatment should be preferred for the treatment of Graves' disease is debatable, and pairwise meta-analyses could not obtain hierarchies of these treatments. Our intention was to integrate the evidence to provide hierarchies of the comparative efficacy of 4 treatments (radioiodine, radioiodine+prednisone, antithyroid drugs and surgery). METHODS: We conducted a Bayesian-framework network meta-analysis of randomized controlled trials (RCTs) to compare 4 treatments in patients with Graves' disease. The eligible RCTs were identified by searching Amed, the British Nursing Index, Embase, PubMed, the Cochrane Central Register of Controlled Trials (CENTRAL), Google scholar, SIGLE, the National Technical Information Service, the National Research Register (UK) and the Current Controlled Trials databases. The data for 2 outcomes (e.g., ophthalmopathy and recurrence) were independently extracted by 2 authors. RESULTS: A total of 4 RCTs were ultimately included. Radioiodine+prednisone therapy showed statistical significance in reducing the incidence of new or deteriorative ophthalmopathy comparing with the other 3 therapies. Compared with radioiodine, therapy with antithyroid drugs therapy as well as surgery significantly decreased the incidence of new or deteriorative ophthalmopathy. Radioiodine therapy significantly reduced the rate of recurrence when compared to therapy with antithyroid drugs or surgery. For decreasing the incidence of new or deteriorative ophthalmopathy, the 4 treatments were ranked as follows: radioiodine+prednisone therapy, therapy with antithyroid drugs, surgery and radioiodine therapy. For reducing the rate of recurrence, 3 treatments were ranked as follows: radioiodine therapy, therapy with antithyroid drugs and surgery. CONCLUSIONS: Radioiodine+prednisone therapy might have the least probability of leading to an exacerbation or new appearance of ophthalmopathy, and radioiodine therapy might have the least probability of causing a recurrence.

Linker length affects expression and bioactivity of the onconase fusion protein in Pichia pastoris.

The aim of this study was to analyze the effect of linker length on the expression and biological activity of recombinant protein onconase (ONC) in fusion with human serum albumin (HSA) in Pichia pastoris. Four flexible linkers with different lengths namely Linker L0, L1: (GGGGS)1, L2: (GGGGS)2, and L3:(GGGGS)3 were inserted into the fusion gene and referred to as HSA-n-ONC, where N = 0, 5, 10, or 15. The sequence of the fusion gene HSA-ONC was designed based on the GC content and codon bias in P. pastoris; the signal peptide of albumin was used as the secretion signal. Gene sequences coding for the fusion protein with different linkers were inserted into pPICZα-A to form recombinant plasmids pPICZα-A/HSA-n-ONC, which were then transformed into P. pastoris X-33 for protein expression. Ideal conditions for expression of the fusion proteins were optimized at a small scale, using shake flasks before proceeding to mass production in 10-L fermenters. The recombinant fusion proteins were purified by aqueous two-phase extraction coupled with DEAE anion exchange chromatography, and their cytotoxic effect on the tumor cell was evaluated by the sulforhodamine B assay. The results showed that the expressed amount of fusion proteins had no significant relationship with the length of different linkers and rHSA-0-ONC had no cytotoxic effect on the tumor cells. While rHSA-5-ONC and rHSA-10-ONC had a weak cytotoxic effect, rHSA-15-ONC could kill various tumor cells in vitro. In summary, the biological activity of the fusion protein gradually improved with increasing length of the linker.

An experimental model for Staphylococcus aureus hepatic abscess in Bama minipig.

Pyogenic hepatic abscess (PHA) is a rare but potentially serious disease. Investigations of new therapeutic methods urgently need experimental support in corresponding animal models. However, to date, few studies have evaluated PHA in the minipig. The linear regression equation of the Staphylococcus aureus ATCC 25923 strain was established. PHA was successfully mocked, and S. aureus ATCC 29213 was the only pathogenic bacterium identified. The abscess formation stage was observed on the 21st day of the operation. This study will provide a baseline for further studies evaluating new treatment methods for PHA.

Polymorphism in heme oxygenase-1 (HO-1) promoter and alcohol are related to the risk of esophageal squamous cell carcinoma on Chinese males.

UNLABELLED: Chronic alcohol drinking is astrong risk factor for esophageal squamous cell carcinoma (ESCC). In this study, the correlation between the HO-1 gene promoter polymorphism and alcohol, along with the risk of ESCC on Chinese males, was analyzed.The case-control study was performed in 143 ESCC patients and 264 cancer-free controls. All subjects were males. Allelotypic frequencies of (GT)n repeat were examed by PCR-based genotyping and DNA sequencing. The frequencies of L-allele and L-allele carriers (S/L and L/L genotypes) was significantly higher in ESCC patients than in controls (p =0.001 and 0.004), The adjusted ORs for ESCC with S/L and L/L genotypes vs S/S genotype was 2.212 (95% CI 1.297-3.775, p= 0.004). The adjusted ORs for light, moderate and heavy drinking was 1.467, 5.215 and 9.525 respectively among L-allele carriers (S/L and L/L genotypes )and 1.389, 2.096 and 3.039 respectively for the S/S genotype. Length of a(GT)n repeat in the HO-1 gene promoter may be associated with the development of ESCC in Chinese male drinkers. Reducing alcohol intake might be most protective among L-allele carriers of this polymorphism. KEYWORDS: esophageal squamous cell carcinoma; heme oxygenase-1 promoter polymorphism; alcohol drinking.

Quetiapine ameliorates anxiety-like behavior and cognitive impairments in stressed rats: implications for the treatment of posttraumatic stress disorder.

The purpose of this study was to determine preventive and protective effects of chronic orally administration with quetiapine (QUE) against anxiety-like behavior and cognitive impairments in rats exposed to the enhanced single prolonged stress (ESPS), an animal model that is used to study post-traumatic stress disorder (PTSD), and to detect changes in the expression of cortical phosphorylated p44/42 extracellular-regulated protein kinase (pERK1/2). Before or after exposure to ESPS paradigm, consisting of 2-h constraint, 20-min forced swimming, ether-induced loss of consciousness, and an electric foot shock, rats were given orally QUE (10 mg/kg daily) for 14 days. Animals were then tested in the open field (OF), elevated plus-maze (EPM), and Morris water maze (MWM). Brains were removed for immunohistochemical staining of pERK1/2. ESPS exposure resulted in pronounced anxiety-like behavior compared to unexposed animals. ESPS-exposed animals also displayed marked learning and spatial memory impairments. However, QUE treatment (both before and after ESPS exposure) significantly ameliorated anxiety-like behavior, learning and spatial memory impairments. ESPS also markedly reduced the expression of pERK1/2 in the prefrontal cortex, medial amygdala nucleus, and cingulate gyrus. Both before and after ESPS exposure QUE treatments significantly elevated the reduced pERK1/2 expression in the three brain regions. QUE has preventive and protective effects against stress-associated symptoms and the changes in pERK1/2 functions may be associated with the pathophysiology of traumatic stress and the therapeutic efficacy of anti-PTSD therapy.

Structure of Bacillus subtilis YXKO--a member of the UPF0031 family and a putative kinase.

We determined the 1.6-A resolution crystal structure of a conserved hypothetical 29.9-kDa protein from the SIGY-CYDD intergenic region encoded by a Bacillus subtilis open reading frame in the YXKO locus. YXKO homologues are broadly distributed and are by and large described as proteins with unknown function. The YXKO protein has an alpha/beta fold and shows high structural homology to the members of a ribokinase-like superfamily. However, YXKO is the only member of this superfamily known to form tetramers. Putative binding sites for adenosine triphosphate (ATP), a substrate, and Mg(2+)-binding sites were revealed in the structure of the protein, based on high structural similarity to ATP-dependent members of the superfamily. Two adjacent monomers contribute residues to the active site. The crystal structure provides valuable information about the YXKO protein's tertiary and quaternary structure, the biochemical function of YXKO and its homologues, and the evolution of its ribokinase-like superfamily.

Structure of Thermotoga maritima stationary phase survival protein SurE: a novel acid phosphatase.

BACKGROUND: The rpoS, nlpD, pcm, and surE genes are among many whose expression is induced during the stationary phase of bacterial growth. rpoS codes for the stationary-phase RNA polymerase sigma subunit, and nlpD codes for a lipoprotein. The pcm gene product repairs damaged proteins by converting the atypical isoaspartyl residues back to L-aspartyls. The physiological and biochemical functions of surE are unknown, but its importance in stress is supported by the duplication of the surE gene in E. coli subjected to high-temperature growth. The pcm and surE genes are highly conserved in bacteria, archaea, and plants. RESULTS: The structure of SurE from Thermotoga maritima was determined at 2.0 A. The SurE monomer is composed of two domains; a conserved N-terminal domain, a Rossman fold, and a C-terminal oligomerization domain, a new fold. Monomers form a dimer that assembles into a tetramer. Biochemical analysis suggests that SurE is an acid phosphatase, with an optimum pH of 5.5-6.2. The active site was identified in the N-terminal domain through analysis of conserved residues. Structure-based site-directed point mutations abolished phosphatase activity. T. maritima SurE intra- and intersubunit salt bridges were identified that may explain the SurE thermostability. CONCLUSIONS: The structure of SurE provided information about the protein's fold, oligomeric state, and active site. The protein possessed magnesium-dependent acid phosphatase activity, but the physiologically relevant substrate(s) remains to be identified. The importance of three of the assigned active site residues in catalysis was confirmed by site-directed mutagenesis.

Crystal structure of the global regulator FlhD from Escherichia coli at 1.8 A resolution.

FlhD is a 13.3 kDa transcriptional activator protein of flagellar genes and a global regulator. FlhD activates the transcription of class II operons in the flagellar regulon when complexed with a second protein FlhC (21.5 kDa). FlhD also regulates other expression systems in Escherichia coli. We are seeking to understand this plasticity of FlhD's DNA-binding specificity and, to this end, we have determined the crystal structure of the isolated FlhD protein. The structure was solved by substituting seleno-methionine for natural sulphur-methionine in FlhD, crystallizing the protein and determining the structure factor phases by the method of multiple-energy anomalous dispersion (MAD). The FlhD protein is dimeric. The dimer is tightly coupled, with an intimate contact surface, implying that the dimer does not easily dissociate. The FlhD monomer is predominantly alpha-helical. The C-termini of both FlhD monomers (residues 83-116) are completely disrupted by crystal packing, implying that this region of FlhD is highly flexible. However, part of the C-terminus structure in chain A (residues 83-98) was modelled using a native FlhD crystal. What is seen in chain A suggests a classic DNA-binding, helix-turn-helix (HTH) motif. FlhD does not bind DNA by itself, so it may be that the DNA-binding HTH motif becomes rigidly defined only when FlhD forms a complex with some other protein, such as FlhC. If this were true, it might explain how FlhD exhibits plasticity in its DNA-binding specificity, as each partner protein with which it forms a complex could allosterically affect the binding specificity of its HTH motif. A disulphide bridge is seen between the unique cysteine residues (Cys-65) of FlhD native homodimers. Alanine substitution at Cys-65 does not affect FlhD transcription activator activity, suggesting that the disulphide bond is not necessary for either dimer stability or this function of FlhD. Electrostatic potential analysis indicates that dimeric FlhD has a negatively charged surface.

[Telomerase activity inhibition and apoptosis induction of BEL-7404 human hepatoma cells by antisense oligonucleotides to telomerase RNA component].

Telomerase activity was detected in all of four human hepatoma cells but absent in normal liver tissue. Telomerase activity of BEL-7404 human hepatoma cells was inhibited effectively by antisense oligonucleotide to telomerase RNA component at final concentration of 1 mumol/L, whereas sense and missense oligonucleotides have no effects on its activity. The inhibition of telomerase activity was weakened, as the concentration of antisense oligonucleotide decreased. Treating BEL-7404 human hepatoma cells 96 hours continuously by the antisense oligomers at final concentration of 5 mumol/L, the morphology of treated cells changed and apoptosis percent of the cells increased markedly.

Antihepatoma effect of alpha-fetoprotein antisense phosphorothioate oligodeoxyribonucleotides in vitro and in mice.

AIM: To evaluate antihepatoma effect of antisense phosphorothioate oligodeoxyribonucleotides (S-ODNs) targeted to alpha-fetoprotein (AFP) genes in vitro and in nude mice. METHODS: AFP gene expression was examined by immunocytochemical method or enzyme-linked immunosorbent assay. Effect of S-ODNs on SMMC-7721 human hepatoma cell growth in vitro was determined using microculture tetrazolium assay. In vitro antitumor activities of S-ODNs were monitored by measuring tumor weight differences in treated and control mice bearing SMMC-7721 xenografts. Induction of cell apoptosis was evaluated by fluorescence-activated cell sorter (FACS) analysis. RESULTS: Antisense S-ODN treatment led to reduced AFP gene expression. Specific antisense S-ODNs, but not control S-ODNs, inhibited the growth of hepatoma cells in vitro. In vitro, only antisense S-ODNs exhibited obvious antitumor activities. FACS analysis revealed that the growth inhibition by antisense S-ODNs was associated with their cell apoptosis induction. CONCLUSION: Antisense S-ODNs targeted to AFP genes inhibit the growth of human hepatoma cells and solid hepatoma, which is related to their cell apoptosis induction.

Using a non-radioisotopic, quantitative TRAP-based method detecting telomerase activities in human hepatoma cells.

A non-radioisotopic, quantitative TRAP-based telomerase activity assay was established mainly by using SYBR Green-I staining instead of radioisotope. Comparing with conventional radioisotope based method, it was better in reproducibility and accuracy. Using this method, we found telomerase activities were absent in normal human liver cells, while detected in all of four human hepatoma cell lines (BEL-7404, SMMC-7721, QGY-7903 and HCCM) without significant differences.

Human hepatoma cell telomerase activity inhibition and cell cycle modulation by its RNA component antisense oligodeoxyribonucleotides.

AIM: To investigate the effects of human telomerase RNA component antisense oligodeoxyribonucleotides on telomerase activity of human hepatoma cells and their effects on cell cycle distribution. METHODS: Modified telomeric repeat amplification protocol was used to detect telomerase activity. Cell cycle was analyzed by flow cytometer. RESULTS: Telomerase activity was detected in all of four human hepatoma cell lines but absent in normal liver cells. Antisense oligomers to human telomerase RNA component (hTR) inhibited telomerase activity of BEL-7404 human hepatoma cells markedly in vitro. After in vitro treatment with antisense oligomers for 96 h, cell cycle of BEL-7404 human hepatoma cells was mainly arrested at G2/M phase. CONCLUSION: Antisense oligomers to hTR inhibited telomerase activity of BEL-7404 human hepatoma cells in vitro and resulted in cell cycle arrest at G2/M phase.