Nomogram for evaluating coronary revascularisation necessity based on multi-parameter coronary CT angiography.

AIM: To build a nomogram model to improve the evaluation of revascularisation necessity using multi-parameter coronary computed tomography (CT) angiography (CCTA). MATERIALS AND METHODS: In this retrospective study, 335 patients who underwent CCTA and required revascularisation within 1 month were selected and allocated to the revascularisation group; 208 patients who did not undergo revascularisation were allocated to the non-revascularisation group. CCTA parameters, including CCTA stenosis, plaque qualitative-quantitative characteristics, and fractional flow reserve derived from CT angiography (CT-FFR), for both groups were analysed and compared. Independent risk factors for evaluating revascularisation were obtained using univariate and multivariable regression analysis, after which multi-parameter models were built. Finally, a nomogram was created with these independent risk factors using the R programming language. RESULTS: Plaque analysis was performed successfully for 543 patients with 1,072 target plaques. The performance of the multi-parameter model (AUC 0.894, p<0.001) was significantly higher than that of models based on stenosis (AUC 0.804, p<0.001), plaque qualitative/quantitative characteristics (AUC 0.754/0.789, p<0.001), or CT-FFR (AUC 0.848, p<0.001) alone, to evaluate the necessity of revascularisation. The independent risk factors were CCTA stenosis (OR 1.004, p=0.04), positive remodelling (OR 2.474, p<0.001), total plaque volume (OR 1.001, p<0.001), non-calcified plaque volume proportion (OR 1.019, p<0.001), and CT-FFR (OR 0.001, p<0.001). Subsequently, a nomogram based on these factors was created. CONCLUSION: The multi-parameter CCTA model improved performance in evaluating revascularisation necessity. The nomogram based on these factors is shows promise in clinical settings.

[Study on mosaicism of SCN1A gene mutation in parents of children with Dravet syndrome].

Objective: To investigate the clinical phenotypes and the mutant allele proportion of parents with SCN1A gene mutation mosaicism of Dravet syndrome (DS) children, thus to provide guidance for family reproduction and prenatal diagnosis. Method: The clinical data and peripheral blood DNA samples of DS patients with a SCN1A gene mutation proved by Sanger sequencing were collected prospectively from February 2005 to November 2016 in Department of Pediatrics, Peking University First Hospital. The same mutation was searched in parents and other available relatives. Parental somatic mosaicism was confirmed and quantified by Ion Torrent Personal Genome Machine (PGM) and Raindrop droplet digital PCR (ddPCR). The families were followed up and prenatal diagnosis was provided. Result: Mosaicisms of SCN1A gene mutation in parents were identified in 5.2% (30 out of 575) DS families. Seventeen were fathers and thirteen were mothers. The mutant allele proportion ranged from 1.7% to 32.9% by PGM and from 0.82% to 34.51% by ddPCR, respectively. In 30 parents with somatic mosaicism, thirteen were asymptomatic, ten had a history of febrile seizures (FS), five with epilepsy, one with febrile seizure plus and one had a history of afebrile seizure. Four families had two children with DS. Three siblings of the probands were confirmed genetically with the same pathogenic mutation. One deceased sister of the proband was assumed to have the same pathogenic mutation because she matched DS diagnosis after medical history review despite no blood sample. Two families received prenatal diagnosis. One second pregnancy was terminated because the fetus inherited the mutation as the mother's wish. Conclusion: Sanger sequencing detects parents of some children with DS are SCN1A mutation mosaics. PGM and ddPCR can be used for accurate quantification of mutant mosaics, which can provide accurate guidance for family genetic counseling.

Unraveling the Numerous Biosynthetic Products of the Marine Sediment-Derived Fungus, Aspergillus insulicola.

A new tripeptide, pre-sclerotiotide F (3), was isolated from a marine sediment-derived fungus, Aspergillus insulicola, along with five known compounds, one of which was new at the time of isolation, scerotiotide F (4). The absolute configuration elucidation of the new compound was determined using a combination of NMR, HR-ESI-MS, and optical rotation analyses. Cytotoxicities were measured in vitro against selected cancer cells. The effects of pre-sclerotiotide F (3) and sclerotiotide F (4) on LPS-induced NF-κB and iNOS expression were also measured.

Antioxidant phenolic glucosides from Gentiana piasezkii.

An extract of Gentiana piasezkii afforded a new arbutin derivative 6'-O-vanilloylarbutin (1) and a new flavone-C-glucoside 7-O-feruloylorientin (2), together with four known flavonoids lutonarin (3), saponarin (4), isoorient (5) and luteolin (6). Their structures were established based on spectroscopic methods including 2D NMR (COSY and gHMBC) techniques. Compounds 1, 2, 5 and 6 were evaluated for the antioxidant activity in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay system.

Nontuberculous mycobacterial cutaneous infection confirmed by biochemical tests, polymerase chain reaction-restriction fragment length polymorphism analysis and sequencing of hsp65 gene.

We report a woman in whom a slow-growing scotochromogenic strain of Mycobacterium was cultured from skin lesions. According to its phenotypic and biochemical characteristics we could predict only that it might be M. szulgai, M. scrofulaceum or M. gordonae. Polymerase chain reaction amplification of the hsp65 gene and subsequent restriction fragment length polymorphism analysis on the isolated strain showed that its restriction pattern differed from both M. scrofulaceum and other scotochromogenic species. Ninety-nine per cent similarity was detected between the isolated strain and M. gordonae by sequencing of the hsp65 gene. This result suggests that the isolated strain may be either a slow-growing scotochromogenic Mycobacterium most resembling M. gordonae or a novel mycobacterial species.

Fatal anaphylaxis following jack jumper ant sting in southern Tasmania.

The "jack jumper" ant (Myrmecia pilosula) is a major cause of anaphylaxis in Tasmania. We describe four deaths attributed to stings by this ant between 1980 and 1999. All victims were men aged 40 years or over with significant comorbidities; two were taking angiotensin-converting enzyme inhibitors, which may increase risk of severe anaphylaxis. Three victims had known ant-sting allergy, but only one carried adrenaline, which he did not use. Another believed he was protected by previous attempts at hyposensitisation with whole ant-body extract. There is potential to prevent deaths by careful education of people with known allergy, prescribing of adrenaline for auto-injection and development of an effective hyposensitisation therapy.

Phylogenetic and biogeographic relationships of eastern Asian and eastern North American disjunct Suillus species (fungi) as inferred from nuclear ribosomal RNA ITS sequences.

Species of Suillus produce fleshy, pored mushrooms. They are important symbiotic (ectomycorrhizal) partners of many coniferous trees. The genus includes several putative eastern Asian and eastern North American disjunct species, i.e., the S. americanus-S. sibiricus and S. decipiens-S. spraguei complexes. Phylogenetic relationships among the groups were determined to further understand the biogeographic pattern. Analyses were based on 40 sequences of the ITS region of the nuclear ribosomal RNA tandem repeats, representing 18 distinct species/populations. Our phylogenetic analyses suggested that: (1) Chinese and United States' (U.S.) S. spraguei plus S. decipiens form a strongly supported monophyletic group, with North American S. decipiens and Chinese S. spraguei being sister taxa; (2) S. americanus, Asian and U.S. S. sibiricus, plus S. umbonatus form a clade supported by a high bootstrap value; and (3) little ITS sequence divergence exists within the latter group compared to the S. decipiens-S. spraguei clade. Phylogenetic patterns revealed by this study imply a close phylogenetic relationship between eastern Asian and eastern North American disjunct population/species of Suillus. These fungi display relatively high host fidelity (at least to the host subgenus level), suggesting potential coevolutionary/comigratory trends.

Cytotoxicity of pilosulin 1, a peptide from the venom of the jumper ant Myrmecia pilosula.

The synthetic peptide pilosulin 1, corresponding to the largest defined allergenic polypeptide found in the venom of the jumper ant Myrmecia pilosula, inhibited the incorporation of [methyl-3H]thymidine into proliferating Epstein-Barr transformed (EBV) B-cells. The LD50 was four-fold lower in concentration than melittin, a cytotoxic peptide found in honey bee venom. Loss of cell viability was assessed by flow cytometry by measuring the proportion of cells that fluoresced in the presence of the fluorescent dye 7-aminoactinomycin D. Examination of proliferating EBV B-cells indicated that the cells lost viability within a few minutes exposure to pilosulin 1. Partial peptides of pilosulin 1 were less efficient in causing loss of cell viability and the results suggest that the 22 N-terminal residues are critical to the cytotoxic activity of pilosulin 1. Normal blood white cells were also labile to pilosulin 1. T- and B-lymphocytes, monocytes and natural killer cells, however, were more labile than granulocytes. Analysis of pilosulin 1 using circular dichroism indicated that, in common with melittin and other Hymenoptera venom toxins, it had the potential to adopt an alpha-helical secondary structure.

Flow cytometric analysis of cell killing by the jumper ant venom peptide pilosulin 1.

Pilosulin 1 is a synthetic 56-amino acid residue polypeptide that corresponds to the largest allergenic polypeptide found in the venom of the jumper ant Myrmecia pilosula. Initial experiments showed that pilosulin 1 lysed erythrocytes and killed proliferating B cells. Herein, we describe how flow cytometry was used to investigate the cytotoxicity of the peptide for human white blood cells. Cells were labeled with fluorochrome-conjugated antibodies, incubated with the peptide and 7-aminoactinomycin D (7-AAD), and then analyzed. The effects of varying the peptide concentration, serum concentration, incubation time, and incubation temperature were measured, and the cytotoxicity of pilosulin 1 was compared with that of the bee venom peptide melittin. The antibodies and the 7-AAD enabled the identification of cell subpopulations and dead cells, respectively. It was possible, using the appropriate mix of antibodies and four-color analysis, to monitor the killing of three or more cell subpopulations simultaneously. We found that 1) pilosulin 1 killed cells within minutes, with kinetics similar to those of melittin; 2) pilosulin 1 was a slightly more potent cytotoxic agent than melittin; 3) both pilosulin 1 and melittin were more potent against mononuclear leukocytes than against granulocytes; and 4) serum inhibited killing by either peptide.

Introduction of apoptosis by high proinsulin and glucose in cultured human umbilical vein endothelial cells is mediated by reactive oxygen species.

There is much evidence that diabetes and hyperglycaemia contribute to the impairment of endothelial function and induce severe changes in the proliferation, the adhesive and synthetic properties of endothelial cells. Induction of apoptosis could represent one mechanism to prevent the new accumulation of those vascular defects and to allow generation of vascular endothelium. In this study, we demonstrate that high concentrations of glucose or proinsulin induce apoptosis in human umbilical endothelial cells by three independent methods (DNA fragmentation, fluorescence activated cell sorting analysis, and morphology). The number of apoptotic cells was increased by glucose (30 mmol/l or proinsulin (100 nmol/l) from less than 10% to about 30%. Activation of protein kinase C (PKC) largely prevented the induction of apoptosis, whereas inhibition of PKC further increased the number of apoptotic cells. Similar changes as induced by glucose were also observed after incubation of the cells with the non-metabolisable 3-O-methylglucose. These findings indicate that hyperglycaemic conditions stimulate the induction of apoptosis in endothelial cells by a mechanism which is independent from the formation of diacylglycerol and the activation of PKC. The induction of apoptosis by the non-metabolisable glucose suggests that formation of oxygen derived radicals by autoxidative processes is involved and may lead to an activation of transcription factors such as nuclear transcription factor-kappaB (NF-kappaB) transferring the activation signal into the nucleus and leading to changes in gene expression necessary for induction of apoptosis.

[Detection of Mycobacterium tuberculosis using a nested-primer gene amplification assay].

A nested-primer gene amplification assay (NPGAA) was established by using two pairs of primers, an outside pair of primers and an inside pair of primers, sequences of which were from M. tuberculosis(Mtb) gro EL genes. The outside pair of primers should amplify a 576-bp piece of the Mtb gro EL gene that contains sites for the inside pair of primers, which should amplify a 344-bp piece. The results shows NPGAA'S detectable limitation was 1 organism/ml and no amplification products were produced from DNAs of other mycobacterium species tested in this study. For detecting Mtb, the entire NPGAA, from sample preparation to data analysis, can be completed within 6-8 hours. When identifying AFB cultures isolated from lesions of skin diseases, the same positive size patterns were obtained.

[Detection of antibodies to sulpholipid IV from mycobacterium tuberculosis in pulmonary tuberculosis patients].

Serum IgG and IgM antibodies against a novel 2,3-diacyl-trehalose-2'-sulphate (SLIV) antigen using ELISA were determined in control (n = 112) and in pulmonary tuberculosis (n = 120) patients. The specificity and sensitivity of SLIV-IgG-ELISA were 96% and 52%, respectively. Those of SLIV-IgM-ELISA were 95% and 33%, respectively. Compared with PPD-IgG-ELISA, SLIV-IgG-ELISA showed that the specificity was about the same but the sensitivity was higher.

[Production of monoclonal antibodies against Mycobacterium leprae].

A series of hybridoma cell lines, which secrete monoclonal antibodies (McAbs), were produced by means of fusion between mouse myeloma cells SP2/O and spleen cells from BALB/C mice immunized with whole M. leprae plus unique phenolic glycolipid I(PGL-I) of M. leprae and M. leprae sonicates supernatant fluid (MLSS) as immunogen. Primary identification indicated that H2 cell line can secrete McAb against the epitope of PGL-I; IIIE10 cell line can secrete McAb against PGL-I and MLSS and (5) 24 D6C8 cell line only against whole M. leprae. The uses of these McAbs in serodiagnosis of leprosy, identification of M. leprae, analysis and purification of M. leprae antigens, and key problems in technology for producing McAbs against M. leprae were also discussed.

Rapid serodiagnosis for leprosy--a preliminary study on latex agglutination test.

In this study, we have developed two latex agglutination tests (LATs) with phenolic glycolipid-I (PGL-I) and natural disaccharide-octyl-bovine serum albumin (ND-O-BSA) as antigens in 110 leprosy patients (LL = 30, BL = 30, BT = 30, and TT = 20), 50 tuberculosis cases, and 30 normal controls. These two LATs were compared with corresponding ELISAs (ND-O-BSA ELISA and PGL-I ELISA) and analyzed by the chi-squared test. There were no significant differences between the two LATs (PGL-I LAT and ND-O-BSA LAT) and their corresponding ELISAs. There was an increase in the proportion of positive cases detectable which coincided with the clinical classification of leprosy, i.e., lepromatous cases were more likely to be positive than tuberculoid cases. LATs are more simple and rapid than ELISAs and have high sensitivity (77% in ND-O-BSA LAT, 80.5% in PGL-I LAT) and specificity (99% in both LATs). LATs may become useful tools for the immunodiagnosis of leprosy in the field. The stability and repeatability of LATs are discussed in detail.

A preliminary study of the inhibiting mechanism of anisodamine on rabbit platelets activated by E. coli endotoxin.

In this study, we observed the endotoxin induced platelet aggregation, 5-HT and beta-g releases, cellular cAMP decrease, and also the changes of morphology and membrane fluidity of platelet. These changes play an important role in septic shock, especially in disseminated intravascular coagulation (DIC) and respiratory distress syndrome (RDS). After anisodamine (654) intervention all the parameters mentioned above were improved to a certain extent. This preliminary study of the inhibiting mechanism of 654 on rabbit platelets activated by ET supports the clinical possibility of using 654 for relieving DIC and RDS.

[Study on rapid serodiagnosis for leprosy].

In this article, we have developed two latex agglutination reaction tests (LAR) with PGL-1 and ND-O-BSA as antigens in 80 cases of leprosy patients (LL = 20, BL = 20, BT = 20, TT = 20), 20 cases of tuberculosis and 25 cases of normal control. These two latex agglutination tests were compared with relevant ELISAs (ND-O-BSA- and PGL-1-ELISA), and analysed by Chi-square (chi 2) test. The results indicated that the sensitivity and specificity of the two LARs (PGL-1- and ND-O-BSA-LAR) are similar to their relevant ELISAs respectively (PGL-1-LAR Vs ND-O-BSA-LAR, chi 2 = 1.785, P greater than 0.10; PGL-1-LAR Vs PGL-1-ELISA, chi 2 = 2.0, P greater than 0.10; ND-O-BSA-LAR Vs ND-O-BSA-ELISA, chi 2 = 0.972, P greater than 0.25), and the increase of positive rate is coincident with clinical classification of leprosy. Since LARs are more simple and rapid than ELISAs, LARs may become more useful tools for immunodiagnosis of leprosy in the field. Also conditions to ensure the stability and reproducibility of LARs are discussed in detail.

[Detection of specific circulating immune complexes in leprosy patients by mouse monoclonal antibody against phenolic glycolipid-I].

In this article, we first report the development of a new test for detecting specific circulating immune complexes (SCIC) in the sera of leprosy patients: This test was named monoclonal antibody specific binding assay (McAb/SBA). We screened for SCIC (PGLI-IgG, PGLI-IgM) in 200 serum batches from 140 leprosy patients, 20 tuberculosis patients and 40 normal controls, and compared the McAb/SBA with PGL1/ELISA. The results indicated that: 1) McAb/SBA was highly sensitive (90%) and specific (95%). Its Youden's Index was 90. Except for its specificity of 95%, the sensitivity (85%) and Youden's Index (85) of the PGL1/ELISA test were lower than those of McAB/SBA; 2) The positive rate (34/40) in paucibacillary patients using McAb/SBA was higher than that (28/40) in PGL1/ELISA; 3) The increase and decline of MOD values in McAb/SBA were associated with BI. McAb/SBA is a new method for detecting SCIC (PGL1-IgG, PGL1-IgM) in the sera of leprosy patients, and it is more sensitive than PGL1-ELISA.

[Studies on the serological activity of ND-O-BSA and its application in serodiagnosis of leprosy].

In this article, optimum conditions of ND-O-BSA-ELISA were determined and comparisons made of the serologic activity of 3 different types of antigens (WML, PGI, ND-O-BSA) in leprosy (151) and tuberculosis (20) patients and normal controls (42). The results indicated that: 1) optimum conditions for ND-O-BSA-ELISA were ND-O-BSA and OPD concentration of 0.1 and 400 micrograms/ml respectively with HRP-IgM and test sera dilutions of 1:1000 and 1:200; and 2) serological activity showed highly significant positive correlations between 3 types of antigens (WML vs PGI: r = 0.88, P less than 0.0005; PGI vs ND-O-BSA: r = 0.86, P less than 0.0005), all of which were highly sensitive and specific for leprosy serodiagnosis with positive rates of 100% (PGI) and 97.4% (WML, ND-O-BSA) in leprosy patients. None were positive in normal controls, but crossreactivity of different degree were present in tuberculosis patients. The authors suggest that PGI-ELISA and ND-O-BSA-ELISA be used in combination in the evaluation of clinical therapeutic efficacy and determination of disease stages, as well as in epidemiologic survey of subclinical leprosy infection in "high-risk" areas.

Evaluation of fluorescent leprosy antibody absorption test versus enzyme-linked immunosorbent assay with phenolic glycolipid 1 and their use in immuno-epidemiological studies on leprosy.

We systematically conducted comparative studies on the validity, reliability and practicality of FLA-ABS.T/PGI-ELISA in large samples. Namely, 284 leprosy patients, 20 tuberculosis patients, 172 normal controls (from nonendemic area of leprosy), 425 leprosy household contacts (HC) and 2573 random samples from the general population (RS) were involved. The results indicated that FLA-ABS.T/PGI-ELISA are highly sensitive and specific for detecting antibodies against M. leprae. Their Youden's indexes (YI) are greater than 90%, and the positive predicative and negative values are 90%. The test results agreed with immuno-epidemiological studies: 1. The positive rates using FLA-ABS.T/PGI-ELISA increased gradually from TT to LL leprosy patients (in HC, the positive rates of PGI-ELISA were much higher in contacts of multibacillary patients than in contacts of paucibacillary patients); 2. The positive rates detected by FLA-ABS.T were identical to those of PGI-ELISA both in HC and in RS; 3. Among RS, the positive rates detected by FLA-ABS. T/PGI-ELISA were similar in each district and were in concordance with the general prevalence rates. Thus, both FLA-ABS.T and PGI-ELISA are useful tests in diagnosing leprosy and detecting subclinical infection with M. leprae. However, because the PGI-ELISA is simple, it will be more practical than FLA-ABS.T in the future. The authors emphasize that the methodology of obtaining dried blood from ear lobes is important for the immuno-epidemiological study of leprosy on a large scale.