[SNP Panel Analysis of Ancestry Inference in East Asian Populations].

ABSTRACT: Objective To develop an SNP Panel for East Asian population, which has a high individual identification rate and the capability of ancestry analysis. Methods The 55 SNP Panel by Professor KIDD of Yale University and the 128 SNP Panel by Professor SELDIN of Davis School of California University, 170 SNP Panel in total was used as the basis and its test data in the East Asian population was collected. The genetic parameters of SNP loci were calculated and combined with the results of heatmap analysis to screen SNP loci suitable for East Asian population. Some Tibetan and Han samples were tested. The possibility of using the SNP loci in ancestry inference was analyzed by means of STRUCTURE analysis, principal component analysis and heatmap analysis. Results A Panel with 45 SNPs （45 SNP Panel） was screened out, and the average genetic parameters of each SNP were better than 170 SNP Panel, with the same ancestry analysis and inference ability. Conclusion In terms of ancestry inference information, the 45 SNP Panel can completely replace the 170 SNP Panel and achieve the same ancestry analysis and inference ability. In genetic parameters, 45 SNP Panel is better than 170 SNP Panel in the East Asian population, which shows its important potential forensic application value.

SSH3 promotes malignant progression of HCC by activating FGF1-mediated FGF/FGFR pathway.

OBJECTIVE: To investigate the impact of silencing SSH3 on the expression of FGF/FGFR pathway-related genes FGF1, FGFR1, and FGFR2 in hepatocellular carcinoma (HCC) cell line, so as to further understand the role of SSH3 in proliferation and apoptosis of HCC cells. PATIENTS AND METHODS: TWe first detected SSH3 expression in 51 pairs of tumor tissue specimens and adjacent tissues collected from HCC patients through quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and analyzed the interplay between SSH3 expression and clinical characteristics of HCC patients. In vitro, after SSH3-silenced human HCC cell line was constructed by lentiviral transfection, Cell Counting Kit-8 (CCK-8), cell cloning assay, and flow apoptosis methods were conducted to explore the HCC cell functions. Finally, whether SSH3 exerts its biological characteristics through the FGF/FGFR pathway and the mutual regulation mechanism between SSH3 and FGF1 were further uncovered. RESULTS: It was found that SSH3 expression was remarkably higher in tumor tissues of HCC patients than that in normal tissues. Meanwhile, in comparison to patients with low expression of SSH3, patients with high expression of SSH3 had higher pathological grade and larger tumor size. In addition, after silencing SSH3, HCC cell proliferation ability was attenuated while the apoptosis ability was enhanced in comparison to the control group. Moreover, the protein levels of FGF1/FGFR pathway-related genes FGF1, FGFR1, and FGFR2 were markedly inhibited by the downregulation of SSH3. Meanwhile, cell recovery experiment demonstrated that the overexpression of FGF1 reversed the impact of SSH3 silencing on the proliferation and apoptosis of HCC cells. CONCLUSIONS: In summary, SSH3 is capable of accelerating the malignant progression of HCC by activating FGF1-mediated FGF/FGFR pathway, thus becoming a new molecular target for HCC therapy.

Kyphoplasty for the treatment of very severe osteoporotic vertebral compression fracture.

OBJECTIVE: A retrospective evaluation of the clinical outcome and technical feasibility of kyphoplasty for the treatment of very severe osteoporotic vertebral compression fracture (vsOVCF). METHODS: Patients with vsOVCF were treated with kyphoplasty and followed-up for 1 year. Vertebral body height variation, kyphotic angle, back pain (visual analogue scale [VAS]) and Oswestry disability index (ODI) were evaluated preoperatively, postoperatively, 1 month, 3 months and 1 year after treatment. RESULTS: In total, 35 patients (49 vertebrae) were treated with kyphoplasty. There were no cases of spinal or extraspinal injury, infection, bleeding, pulmonary embolism, epidural cement leakage, stroke or cardiac arrest as a result of treatment. There were significant postoperative improvements in all outcome measures (vertebral body height variation, kyphotic angle, VAS and ODI); these improvements were maintained during the follow-up period. CONCLUSION: Kyphoplasty is an effective and minimally invasive procedure for the treatment of vsOVCF.

Mini-subvastus versus a standard approach in total knee arthroplasty: a prospective, randomized, controlled study.

This prospective randomized study compared the clinical and radiological results of primary total knee arthropasty (TKA) using a mini-subvastus approach (group I; n = 35) or a standard approach (group II; n = 33). A posterior-stabilized prosthesis was used in both groups by the same surgeon. Mean follow-up was 18 months (range 14 - 26 months). Patients in group I lost less blood and experienced less pain 1 day post-operatively. They achieved an active straight leg raise earlier and underwent less lateral retinacular releases. Functional outcome and the range of knee movements were significantly better in group I up to 9 months post-operatively, but there was no significant difference between the groups at 1 year post-operatively or at final follow-up. Reduced access and visibility in group I prolonged the operation time and resulted in five technical errors on radiographic evaluation. Based on these results, the authors currently only use the mini-subvastus approach for minimally invasive TKA in selected cases.

Quantitative diagnosis of Schistosoma infections by measurement of circulating antigens in serum and urine.

Recent research on the detection of the schistosome circulating antigens, circulating anodic antigen (CAA) and circulating cathodic antigen (CCA), has greatly expanded the scope of applications. In the present paper aspects of assay development, in particular of highly sensitive assays and of field-applicable assays, are discussed. The progress in the use of CAA- and CCA-detection for diagnosis, follow-up of chemotherapy and sero-epidemiological studies is evaluated.

Dot immunoassay with biotinylated antigen for determination of antibodies against the circulating cathodic antigen (CCA) in schistosomiasis japonica.

Based on the fact that schistosomiasis patients in both the acute and chronic phase of the infection show a strong humoral immune response against the gut-associated circulating cathodic antigen, a simple and sensitive dot immunobinding assay for schistosomiasis japonica was developed. Circulating cathodic antigen that had been purified by immunoadsorption using monoclonal antibody was biotinylated with biotin aminocaproylhydrazide via the carbohydrate moiety of the antigen. Serum samples dotted onto nitrocellulose strips were then tested in an assay involving a combined incubation step of biotinylated antigen and streptavidin peroxidase, and a subsequent staining; the total assay time was 1.5 hr. Assaying the sera of 105 uninfected controls and 104 Schistosoma japonicum-infected individuals showed a specificity of 99.0%, and sensitivities of 96.2% (acute infections) and 94.1% (chronic infections). The described assay is economic, rapid, and reproducible and lends itself to use under field conditions.

Diagnostic evaluation of dot-binding assays for circulating cathodic antigen (CCA) and anti-CCA determinations in schistosomiasis japonica using defined biotinylated conjugates.

Using an affinity purified circulating cathodic antigen (CCA) preparation and an anti-CCA monoclonal IgM antibody, we modified dot-binding assays for anti-CCA and CCA detections with biotinylated conjugates. A study on 3 groups of 138 schistosomiasis japonica patients and 105 healthy individuals demonstrated a high predictive rate of over 95% in both assays, highly comparable to those of circumoral precipitin test (COPT). A blind test in 342 samples of various groups revealed higher detection rates in Ab-binding assay with both acute and chronic case groups (over 90%), but comparatively lower rates in Ag-binding assay with chronic groups (50%-76%). Distinct reductions of either GMRT and dot-indexes were found in 48 praziquantel treated patients whose sera were collected 9 months after chemotherapy. The major target molecules detected by the two binding assays were proved to be the protein incorporated moieties readily precipitated by trichloroacetic acid (TCA), ammonium sulphate and higher concentration of polyethylene glycol (PEG) suggestive of pathological, specific immunoglobulins in the free or complex forms. Non-specific dot reactions were found in some acute and chronic patients with non-relevant bovine serum albumin (BSA) conjugated peroxidase and streptavidin-PO controls, and also in normal sera with the McAb peroxidase conjugate or when working concentration of the biotinylated McAb was not properly titrated. The reliability and proper management of the dot binding assay as an immunodiagnostic tool were discussed.

[Detection of circulating antigen in urine from mice infected with Toxoplasma tachyzoites].

Urine samples collected from three groups of mice infected experimentally with different numbers doses of Toxoplasma trophozoites were detected for the presence of Toxoplasma circulating antigen by using fast ELISA. The results showed that presence of circulating antigen in all three infected groups of mice in comparison with the normal control group. Toxoplasma circulating antigen was detected on days 5, 4 and 3 after infection in light-, moderate- and heavy-infection groups, respectively. The concentration of circulating antigen was on a parallel with the duration of infection. Western blot analysis of the Toxoplasma circulating antigen in urine revealed the existence of seven specific bands with molecular weights of 75, 67, 55, 43, 30, 28 and 22 kDa.

Detection of IgM antibodies directed against the gut-associated circulating cathodic antigen in sera from Schistosoma mansoni infected patients. Development and comparison of three enzyme-linked immunoassays.

The majority of the human IgM antibodies detected with an immunofluorescence assay (IFA) on adult worms are directed against the gut-associated circulating cathodic antigen (CCA). In order to study this phenomenon further we developed and evaluated three related ELISA methods to specifically detect IgM antibodies against purified CCA. The assays employed: 1) direct coating of CCA, 2) indirect coating of CCA via a monoclonal antibody, and 3) IgM antibody-capture by rabbit anti-mu chain antibodies. Using a group of 46 positive sera, it was found that the three ELISA's and the IFA were significantly correlated. To discriminate between positive and negative sera we used a cut-off level of average reactivity + 3 standard deviations of 50 negative sera. False negative reactions were not found in any of the ELISA's, while both in the direct and indirect ELISA one false positive reaction occurred. For further studies or diagnostic use the antibody-capture ELISA is recommended.

[Diagnosis of toxoplasmosis by polymerase chain reaction].

Based on the partial sequences of the specific DNA cloned fragment from T. gondii (ZS2 strain) a specific primer of the oligonucleotide for the Toxoplasma gondii DNA sequence has been designed and synthesized in our laboratory. The method of the DNA diagnosis for toxoplasmosis by polymerase chain reaction (PCR) has been established. A specific amplified band was shown in the PCR products from DNAs of T. gondii and seven manifold terata. The DNAs from the peripheral blood leukocytes of fifty normal individuals and seventy-five patients as infants with hepatitis syndrome and pregnant women with previous abnormal birth histories were diagnosed by PCR. Among the seventy-five diagnosed cases, ten were positive. The normal individuals all were negative. Using 32P-cloned T. gondii specific DNA fragment as probe and Southern blot assay, the results showed that the probe only hybridized to the specific amplified DNA bands, but did not hybridize to the amplified DNA products of negative cases. Our PCR method is a rapid, highly specific and sensitive one for detecting toxoplasmosis as compared with DNA probing, immunoassay and animal inoculation.

Construction of a genomic DNA library of the Toxoplasma gondii ZS2 strain, screening of specific clones, and DNA diagnosis of toxoplasmosis.

We have constructed a genomic DNA library of the Toxoplasma gondii ZS2 strain and isolated a specific cloned DNA sequence from this organism. The restriction map of this cloned 1.1-kb DNA fragment was analyzed. Southern and dot-blot analyses showed that the 32P-labeled DNA fragment hybridized to parasite DNA, to DNAs from peripheral blood leukocytes and the thymus of baby pigs that were artificially infected with T. gondii, and to DNAs of T. gondii-positive anencephalic and hydrocephalic fetuses. It did not hybridize with DNA from controls, (i.e., normal human and baby pig peripheral blood leukocytes, spleen of normal mice, Plasmodium falciparum, Pneumocystis carinii, and pBR322). As few as 100 T. gondii parasites or 500 pg of purified DNA from T. gondii can be detected by dot-blot hybridization. This probe method was specific and sensitive, and has been used successfully in detecting various clinical cases of toxoplasmosis with T. gondii.

Quantification of circulating schistosome antigen by monoclonal antibody-based enzyme-linked immunosorbent assay.

A two-site enzyme-linked immunosorbent assay (2S-ELISA) was developed for the quantification of gut-associated schistosome circulating anodic proteoglycan (GASCAP) of Schistosoma japonicum using a monoclonal antibody (McAb M120), purified by one-step hydroxylapatite (HAP) chromatography, as both solid phase and peroxidase-conjugated reagent. In this system, a high detectable level of serum GASCAP concentration (0.25 microgram/L) could be achieved with the immunoaffinity purified GASCAP being used as the standard. The specificity of the assay was 97.2%, with minimized interference of rheumatoid factor. The intra-assay (4.01%) and interassay (6.09%) coefficients of variation and the mean analytical recovery (101.4%) for GASCAP demonstrated the precision and accuracy of the assay.

[Preparation and diagnostic application of monoclonal antibodies against Schistosoma japonicum].

The present paper reported on an anti-CCA monoclonal antibody, McAb-IIID 10, which could be used in determinations of both parasite-oriented circulating antigens and specific anti-CCA antibodies. The established competitive ELISA (C-ELISA) using McAb-IIID 10 to detect schistosome-antibodies showed high sensitivity and specificity in the diagnosis of schistosomiasis with few cross-reactions. In field trials, coincident rates in 3 separate batches of serum samples when subjected to double-blind detections were obtained. A total of 1,915 serum samples had been determined by C-ELISA, among them 113 acute cases achieved a 100% positive rate, 765 chronic and 25 late cases showed 96.3% and 72% positive respectively. 70% of the 66 cured schistosomiasis cases turned to be negative. None of the 750 normal individuals showed positive reactions. No cross reaction was found in 27 sera from hydatidosis, whereas 1 and 2 positive reactions were found in 43 paragonimiasis sera and 126 clonorchiasis sera respectively. The established McAb-IIID 10 involved Dot-ELISA was found of value in the assessment of effective chemotherapy and showed a high negative conversion rate of 97.9% in 48 cured schistosomiasis patients. In 16 experimentally infected rabbits, 12 became negative in Dot-ELISA determinations at the 8th week post treatment, and the remaining 4 treated ones, the titer as well as the reaction intensity were also found reduced. A good coincidence rate was also found between C-ELISA and Dot-ELISA, their detection results may be complementary each other.

[Construction of a genomic DNA library of Toxoplasma gondii (ZS2 strain), screening of specific clone and DNA diagnosis of toxoplasmosis].

We have constructed a genomic DNA library of Toxoplasma gondii (ZS2 strain) and screened out a specific DNA sequence for T. gondii. The restriction map of the cloned DNA fragment (1.1kb) was analysed. The Southern and dot-blot analyses showed that the 32P-labeled cloned DNA fragment hybridized to the parasite DNA, DNAs from peripheral white blood cells and thymus of baby pigs artificially infected with T. gondii and DNAs of T. gondii- positive anencephalus and hydrocephalus, but did not hybridize to DNAs from controls, i. e., normal human and baby pig peripheral white blood cells, spleen of normal mouse, Plasmodium falciparum, Pneumocystis carinii and pBR322. As few as 100 T. gondii parasites or 500pg purified DNA from T. gondii can be detected by dot blot hybridization. This established DNA probe method was specific and sensitive and has been successfully used in detecting various cases infected with T. gondii.

Quantitative determination of circulating antigens in human schistosomiasis mansoni using an indirect hemagglutination assay.

In serum and urine specimens collected from a group of Schistosoma mansoni infected individuals from Makundju, Zaire, the schistosome circulating anodic antigen (CAA) and the circulating cathodic antigen (CCA) were quantitatively determined using an indirect hemagglutination reaction with sheep erythrocytes sensitized with mouse IgM monoclonal antibodies directed against these circulating antigens. Levels of CAA in serum (up to 5 ng/ml) and CCA in serum and urine (up to 50 ng/ml) were strongly correlated with egg excretion and with each other. No correlation was found between egg excretion and antibody levels against the circulating antigens. Antigen was detectable only in patients excreting greater than 500 eggs per gram of feces.

Immunofluorescent localization of Schistosoma mansoni circulating cathodic antigen in tissues of infected mice using monoclonal antibody.

In the present study the kinetics of the uptake and deposition of the major circulating cathodic antigen (CCA) of Schistosoma mansoni in liver, spleen, and kidney of S. mansoni infected Swiss mice was investigated in relation to the duration of infection and infection dose (50, 100, 200 cercariae). The presence of antigen was studied with a direct immunofluorescence reaction on frozen sections of the mouse organs, using a fluorescein isothiocyanate (FITC)-labelled mouse IgM monoclonal antibody recognizing a repeating epitope of CCA. CCA was demonstrable from 2 weeks post infection (p.i.) onwards in Kupffer cells in the liver, from 3-4 weeks p.i. onwards in macrophages in the marginal zones in the spleen and from 8 weeks p.i. onwards in kidney glomeruli. The immunofluorescence reactions on CCA in kidney glomeruli, however, remained relatively weak.

Schistosoma japonicum: immunological response to circulating polysaccharide antigens in rabbits with a light infection.

To study the detectability of circulating polysaccharide antigens and the immunological response to such antigens in rabbits with a light Schistosoma japonicum infection, sera of five rabbits infected with 50 cercariae were studied up to 29 weeks post infection (p.i.). While one rabbit developed no worm burden, the other rabbits developed low worm burdens (4 to 16 worms). In the sera of these rabbits, the only polysaccharide antigen demonstrable with immunoelectrophoresis (IEF), was the circulating anodic antigen (CAA). With the enzyme-linked immunosorbent assay (ELISA), CAA was detectable from 5 to 6 weeks p.i. in the sera of the two rabbits with the highest number of worm couples. The lowest CAA level which was detectable in unconcentrated sera from which serum proteins had been removed was 125 ng CAA/ml, corresponding with a worm burden of 4.5 worm/kg body wt. During the entire infection, CAA-specific immune complexes were only demonstrable in very low concentrations. Antibodies against polysaccharide antigens were assessed with immunofluorescent antibody (IFA) on Rossman's fixed sections of adult worms, with the ELISA, and with IEF. Specific IgA, IgG, and IgM antibodies were detectable from 2 to 3 weeks p.i. with IFA and ELISA. These early antibodies were shown to be directed against gut-associated antigens, while antibodies against parenchyma-associated antigens were found later in the infection. With IEF, antibodies against two trichloroacetic acid (TCA)-soluble antigens were detectable, including the major, S. japonicum-specific antigen 2.

Schistosoma japonicum: immunological characterization and detection of circulating polysaccharide antigens from adult worms.

The antigenic constituents of a trichloroacetic acid (TCA)-soluble fraction of adult Schistosoma japonicum were studied with immunoelectrophoresis, and compared with those of Schistosoma mansoni. Eight TCA-soluble antigens of S. japonicum were demonstrated, five of which showed immunological identity with S. mansoni antigens. Of the eight antigens, five antigens with anodic motility were found as circulating antigens in S. japonicum-infected hamster and rabbit sera; the major circulating antigen was the circulating anodic antigen (CAA). Two other antigens, with cathodic motility, including the circulating cathodic antigen (CCA), were demonstrable as circulating antigens in S. mansoni infections, but not in S. japonicum infections. Most of the circulating antigens were shown to be gut-associated. Only one antigen, line 2, which was not demonstrable as circulating antigen and which was present in the parenchyma of the worms, was found to be specific for S. japonicum. Using an ELISA for the detection of CAA in the sera of S. japonicum-infected rabbits, a lower detection level of 100 ng CAA/ml serum was achieved. Moreover, at 7-8 weeks after infection, a direct relationship between worm burden and CAA level was demonstrated.