Development of a clinical chemiluminescent immunoassay for serum GPC3 and simultaneous measurements alone with AFP and CK19 in diagnosis of hepatocellular carcinoma.

BACKGROUND: Glypican-3 (GPC3) is an oncofetal antigen that shows great promise as a biomarker for diagnosis of hepatocellular carcinoma (HCC), but there is no reliable kit that can be used to detect it in clinics. The aim of this study is to develop a stable performance kit for GPC3 detection in clinics. DESIGN AND METHODS: The paired antibodies were identified through cycle-screening methods based on our previous research. Then, a double antibodies sandwich chemiluminescent immunoassay for detecting serum GPC3 was developed. The performance of the developed GPC3 diagnostic kit was evaluated by detecting the concentration of serum GPC3 and assessing its single or combined use with alpha fetoprotein (AFP) and cytokeratin 19 fragment (CK19) for HCC diagnosis. RESULTS: The assay demonstrated a linear range of 10-800 ng/ml, the cross-reactivity rate at 0.018% (AFP), 0.020% (carcino-embryonic antigen), and 0.021% (CK19), respectively. The minimum detectable concentration was 0.05 ng/ml; the intraassay coefficient of variation (CV) and interassay CV were both less than 10%, with good stability and reproducibility. GPC3 has a high sensitivity (54.2%) and specificity (99.4%) in diagnosing HCC. The level of GPC3 in HCC was robust higher than that in healthy or other liver diseases' sera (108.67 ± 230.04 ng/ml vs. 3.99 ± 7.68 ng/ml). The diagnostic sensitivity of GPC3 single or combined with CK19 and AFP for HCC was evaluated, and the rates were 54.2 and 90.6%, respectively. CONCLUSIONS: An applicable chemiluminescent immunoassay with stable performance against GPC3 in diagnosing HCC has been established and the combination of GPC3 with CK19 and AFP could improve the diagnostic sensitivity for HCC.

Peach genetic resources: diversity, population structure and linkage disequilibrium.

BACKGROUND: Peach (Prunus persica (L.) Batsch) is one of the most important model fruits in the Rosaceae family. Native to the west of China, where peach has been domesticated for more than 4,000 years, its cultivation spread from China to Persia, Mediterranean countries and to America. Chinese peach has had a major impact on international peach breeding programs due to its high genetic diversity. In this research, we used 48 highly polymorphic SSRs, distributed over the peach genome, to investigate the difference in genetic diversity, and linkage disequilibrium (LD) among Chinese cultivars, and North American and European cultivars, and the evolution of current peach cultivars. RESULTS: In total, 588 alleles were obtained with 48 SSRs on 653 peach accessions, giving an average of 12.25 alleles per locus. In general, the average value of observed heterozygosity (0.47) was lower than the expected heterozygosity (0.60). The separate analysis of groups of accessions according to their origin or reproductive strategies showed greater variability in Oriental cultivars, mainly due to the high level of heterozygosity in Chinese landraces. Genetic distance analysis clustered the cultivars into two main groups: one included four wild related Prunus, and the other included most of the Oriental and Occidental landraces and breeding cultivars. STRUCTURE analysis assigned 469 accessions to three subpopulations: Oriental (234), Occidental (174), and Landraces (61). Nested STRUCTURE analysis divided the Oriental subpopulation into two different subpopulations: 'Yu Lu' and 'Hakuho'. The Occidental breeding subpopulation was also subdivided into nectarine and peach subpopulations. Linkage disequilibrium (LD) analysis in each of these subpopulations showed that the percentage of linked (r2 > 0.1) intra-chromosome comparisons ranged between 14% and 47%. LD decayed faster in Oriental (1,196 Kbp) than in Occidental (2,687 Kbp) samples. In the 'Yu Lu' subpopulation there was considerable LD extension while no variation of LD with physical distance was observed in the landraces. From the first STRUCTURE result, LG1 had the greatest proportion of alleles in LD within all three subpopulations. CONCLUSIONS: Our study demonstrates a high level of genetic diversity and relatively fast decay of LD in the Oriental peach breeding program. Inclusion of Chinese landraces will have a greater effect on increasing genetic diversity in Occidental breeding programs. Fingerprinting with genotype data for all 658 cultivars will be used for accession management in different germplasms. A higher density of markers are needed for association mapping in Oriental germplasm due to the low extension of LD. Population structure and evaluation of LD provides valuable information for GWAS experiment design in peach.

Safety profile evaluation of different herbicides used on peach rootstock seedlings.

To screen out suitable herbicides for peach nurseries, we treated the potted seedlings of the peach rootstock 'Nemaguard' with eleven herbicides under recommended doses to investigate the changes of physiological indices and comprehensively evaluate the safety of different herbicides using principal component analysis (PCA). The results showed that soil application of quizalofop-p exhibited no detectable phytotoxicity on rootstock seedlings, while the remaining herbicides generated multiple symptoms, including green loss, wilting, spot, and withering. Starane caused rapid wilting and death, with a 100.0% phytotoxicity index (PI). Soil application of n-(phosphonomethyl)glycine, glufosinate-ammonium, acetochlor, and MCPA-Na showed a PI＞65.0%. As compared with the control, all herbicides inhibited leaf area growth to varying degrees, with a 10.0%-56.2% and 5.8%-44.4% reduction in young leaf area and mature leaf area, respectively. All herbicides, except quizalofop-p, increased the electrolyte permeability of leaf and root tip cells by 21.2%-145.0% and 36.9%-291.4%, respectively, and significantly inhibited root growth. The total root length, root surface area, root volume, and the number of root tips significantly decreased by 37.3%-75.3%, 35.7%-83.0%, 44.3%-89.9%, and 42.6%-73.7%, respectively. Although net photosynthetic rate (Pn) and transpiration rate (Tr) of leaves were not significantly affected by quizalofop-p, mesotrione-atrazine, MCPA-Na·bentazone, bensulfuron-methyl·quinclorac, and bensulfuron-methyl·acetochlor, there was significant reduction of 29.6%, 28.9%, 28.4% and 27.9% in Pn and 21.9%, 29.2%, 26.4%, and 19.7% in Tr post soil application of n-(phosphonomethyl)glycine, glufosinate-ammonium, acetochlor, and MCPA-Na. The overall safety ranking of the 11 examined herbicides is as follows: quizalofop-p>bensulfuron-methyl·acetochlor>bensulfuron-methyl·quinclorac>esotrione·atrazine> auizalofop-p·fluoroglycofen>acetochlor>MCPA-Na·bentazone>MCPA-Na>n-(phosphonomethyl)glycine>glufosinate-ammonium>sterane.

[Significance of Lymphocyte-to-Monocyte Ratio and Corrected Levels of Serum Calcium as Prognostic Markers in Patients with Newly Diagnosed Multiple Myeloma].

OBJECTIVE: To investigate the significance of peripheral blood lymphocyte to monocyte ratio (LMR) and corrected levels of serum calcium (cCa) as prognostic markers for the newly diagnosed multiple myeloma (MM) patients. METHODS: The clinical data of 114 newly diagnosed MM patients in the Second Affiliated Hospital of Kunming Medical University from January 2013 to March 2020 were retrospectively analyzed. Receiver operating characteristic (ROC) curve analysis was used to identify the optimal cutoff value, and the patients were divided into high LMR group and low LMR group (LMR≥3.35 and LMR < 3.35). Moreover, the patients were divided into four groups according to initial diagnosis LMR and LMR after four courses of treatment (LMR4): Group A (LMR≥3.35, LMR4≥3.35), Group B (LMR≥3.35, LMR4 < 3.35), Group C (LMR < 3.35, LMR4≥3.35), and group D (LMR < 3.35, LMR4 < 3.35). The simple prognosis model was established by combined with LMR and cCa, the patients were divided into Group a (no risk factor), group b (1 risk factor) and Group c (2 risk factors). Independent sample T-test, Pearson Chi-square test or Mann-Whitney U test were used to evaluate the differences between various parameters, and Kaplan-Meier method and Cox regression were used for survival analysis. RESULTS: The median follow-up time was 13.05（0.1-72.5）months. Survival analysis showed that the patients with low LMR predicted poor prognosis, the overall survival (OS) time of the patients with low LMR was significantly shorter (17 vs 50.5 months, P=0.006) than the patients with high LMR, the difference was also significant between group A and Group D (56.5 vs 30.5 months, P=0.043). The OS of the patients was also significantly shorter in the high cCa group (≥2.75 mmol/L) compared with normal group (8.5 vs 34 months, P=0.006). Multivariate survival analysis showed that LMR < 3.35 (P=0.028) and cCa≥2.75 mmol/L (P=0.036) were the independent risk factors affecting prognosis of MM patients. The comparison of risk factors showed that the median OS of Group a, b and c was 50, 20, and 8.5 months, respectively. The prognosis of the patients without risk factors was better than that of patients with 1-2 risk factors (Group a vs Group b, P < 0.0001; Group a vs Group c, P=0.002). CONCLUSION: LMR and cCa are the independent risk factors affecting the prognosis of newly diagnosed MM patients, and the development of a simple prognosis system combining them can quickly identify the prognosis of newly diagnosed MM patients.

PTEN status is related to cell proliferation and self-renewal independent of CD133 phenotype in the glioma-initiating cells.

CD133 is extensively used as a surface marker to identify and isolate glioma-initiating cells (GICs) from malignant brain tumors; however, instances of CD133(-) cells exhibiting similar properties have also been reported. To clarify the availability of CD133 as the GIC marker, we first evaluated the ratio of CD133(+) cells and malignancy of glioma spheroids GIC1 and GIC2, respectively. GIC1, which showed a lower percentage of CD133(+) cells, exhibited a highly aggressive behavior in comparison with GIC2. The following experiments demonstrated that tumor suppressor PTEN was lost in GIC1, resulting in the activation of AKT pathway. Overexpression of recombinant PTEN in GIC1 suppressed its proliferation and self-renew without significant effect on CD133 expression level, indicating that the inconsistence between the ratio of CD133(+) cells and proliferation and self-renewal capacity of GIC1 and GIC2 was caused by PTEN deficiency. To further validate our conclusion, a series of GICs were analyzed and the percentages of CD133(+) cells could not reflect the degrees of cell proliferation and self-renewal characteristics in the PTEN deficient GICs, suggesting that the application of CD133 as the GIC maker was restricted by PTEN loss. Furthermore, down-regulation of PTEN in the PTEN-expressing GICs could break the positive correlation between the ratio of CD133(+) cells and proliferation and self-renewal capacity. Our results demonstrated that PTEN status is related to cell proliferation and self-renewal independent of CD133 phenotype in the glioma-initiating cells, resulting in the limitations of CD133 as a biomarker for PTEN deficient GICs.

Effects of different components of Mao Dongqing's total flavonoids and total saponins on transient ischemic attack (TIA) model of rats.

Objective: To study the effects of the different components of the total flavonoids and total saponins from Mao Dongqing's active site on the rats of TIA model, determine the optimal reactive components ratio of Mao Dongqing on the rats of TIA. Methods: TIA rat model was induced by tail vein injection of tert butyl alcohol, the blank group was injected with the same amount of physiological saline, then behavioral score wasevaluated. Determination the level of glutamic acid in serum, the activity of Na+-K+-ATP enzyme, CA++-ATP enzyme and Mg++-ATP enzyme in Brain tissue, observe the changes of hippocampus in brain tissue, the comprehensive weight method was used to evaluate the efficacy of each component finally. Results: The contents of total flavonoids and total saponins in the active part of Mao Dongqing can significantly improve the pathological changes of brain tissue in rats, improve the activity of Na+-K+-ATP enzyme, Ca++-ATP enzyme and Mg++-ATP enzyme in the brain of rats, and reduce the level of glutamic acid in serum. The most significant of the contents was the ratio of 10:6. CONCLUSION: The different proportions of total flavonoids and total saponins in the active part of Mao Dongqing all has a better effect on the rats with TIA, and the ratio of 10:6 is the best active component for preventing and controlling TIA.

Whole-Genome Analysis of Diversity and SNP-Major Gene Association in Peach Germplasm.

Peach was domesticated in China more than four millennia ago and from there it spread world-wide. Since the middle of the last century, peach breeding programs have been very dynamic generating hundreds of new commercial varieties, however, in most cases such varieties derive from a limited collection of parental lines (founders). This is one reason for the observed low levels of variability of the commercial gene pool, implying that knowledge of the extent and distribution of genetic variability in peach is critical to allow the choice of adequate parents to confer enhanced productivity, adaptation and quality to improved varieties. With this aim we genotyped 1,580 peach accessions (including a few closely related Prunus species) maintained and phenotyped in five germplasm collections (four European and one Chinese) with the International Peach SNP Consortium 9K SNP peach array. The study of population structure revealed the subdivision of the panel in three main populations, one mainly made up of Occidental varieties from breeding programs (POP1OCB), one of Occidental landraces (POP2OCT) and the third of Oriental accessions (POP3OR). Analysis of linkage disequilibrium (LD) identified differential patterns of genome-wide LD blocks in each of the populations. Phenotypic data for seven monogenic traits were integrated in a genome-wide association study (GWAS). The significantly associated SNPs were always in the regions predicted by linkage analysis, forming haplotypes of markers. These diagnostic haplotypes could be used for marker-assisted selection (MAS) in modern breeding programs.

Gene regulation of anthocyanin biosynthesis in two blood-flesh peach (Prunus persica (L.) Batsch) cultivars during fruit development.

The blood-flesh peach has become popular in China due to its attractive anthocyanin-induced pigmentation and antioxidant properties. In this study, we investigated the molecular mechanisms underlying anthocyanin accumulation by examining the expression of nine genes of the anthocyanin biosynthesis pathway found in the peach mesocarp. Expression was measured at six developmental stages in fruit of two blood-flesh and one white-flesh peach cultivars, using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results show that the expression of the chalcone synthase (CHS) gene was closely related to anthocyanin accumulation in both of the blood-flesh peaches. In the white-flesh peach, we found that the transcription level of phenylalanine ammonia-lyase (PAL) during fruit development was much lower than that in the blood-flesh peach, even though all other genes of the anthocyanin biosynthesis pathway were highly expressed, suggesting that the PAL gene may be limiting in anthocyanin production in the white-flesh peach. Moreover, the transcription levels of the CHS and UDP-glucose-flavonoid 3-O-glucosyltransferase (UFGT) genes were markedly up-regulated at three days after bag removal (DABR) in the blood-flesh peach, suggesting that CHS and UFGT are the key genes in the process of anthocyanin biosynthesis for both of the blood-flesh peaches. The present study will be of great help in improving understanding of the molecular mechanisms involved in anthocyanin accumulation in blood-flesh peaches.

Preparation and characterization of monoclonal antibody against glypican-3.

Glypican-3 (GPC3) has been reported as a novel serum and histochemical marker for hepatocellular carcinoma (HCC) by several groups. As an oncofetal protein, it is expressed abundantly in the fetal liver, inactive in the normal adult liver, and frequently reactivated in HCC. Immunology reagents are urgently needed to proceed with mechanism-related research, clinical validation, and application. In this report, monoclonal antibodies (MAbs) against GPC3 were made from hyperimmune BALB/c mice by injecting 100 µg of purified antigen intraperitoneally. Hybridomas were screened by indirect enzyme-linked immunosorbent assay (ELISA) using purified protein. Finally 13 mouse hybridomas producing MAbs to GPC3 were established. The MAbs obtained were fully characterized using Western blot analysis, immunofluorescence, and immunohistochemistry. The results showed that these antibodies could be used for preliminary application of the next step mechanism-related research and GPC3 expression level analysis.