[Pathological features and immune microenvironment in HER-2 intratumoral heterogeneous breast cancers].

Objective: To observe the clinical pathology features, and immune microenvironment of HER-2 intratumoral heterogeneity breast cancer. Methods: Thirty cases of HER-2 intratumoral heterogeneous breast cancer were retrospectively analyzed in Tianjin Medical University Cancer Institute and Hospital from November 2017 to June 2020. HER-2 expression was detected by immunohistochemistry and verified by dual color silver-enhanced in-situ hybridization (D-SISH). HER-2 intratumoral positive and negative regions were divided. The pathological characteristics, subtype, and the level of tumor infiltrating lymphocytes (TILs) and the expression of programmed cell death-ligand 1 (PD-L1) were evaluated respectively. Results: The proportion of HER-2 positive cells of the breast cancer ranged from 10% to 90%. The pathological type was mainly invasive non-special typecarcinoma. Six cases presented different pathological types between HER-2 positive and negative regions. The HER-2-positive areas included 2 cases of carcinoma with apocrine differentiation, and the negative areas included 2 cases of invasive micropapillary carcinoma, 1 case of invasive papillary carcinoma, and 1 case of carcinoma with apocrine differentiation. In HER-2 positive regions, 17 cases were Luminal B and 13 cases were HER-2 overexpressed types. There were 22 cases of Luminal B and 8 cases of triple negative tumors in the HER-2 negative areas. The levels of TILs in HER-2 positive and negative areas accounted for 53.3% (16/30) and 26.7% (8/30), respectively, with a statistically significant difference (P=0.035). The positive expression of PD-L1 in HER-2 positive area and HER-2 negative area were 6 cases and 9 cases, respectively. Among 8 cases with HER-2 negative regions containing triple negative components, 4 cases were positive for PD-L1 expression. Conclusions: In the case of HER-2 intratumoral heterogeneity, it is necessary to pay attention to both HER-2 positive and negative regions, and evaluate subtype separately as far as possible. For HER-2 intratumoral heterogeneous breast cancer containing triple negative components, the treatment mode can be optimized by refining the intratumoral expression of PD-L1.

Construction and identification of a model for HJURP gene defect expression in human embryo villus cells.

OBJECTIVES: To construct a lentiviral vector for RNA interference (RNAi) of the HJURP gene and to identify the silencing efficiency in the human embryo villus cells and to provide a human embryo villus cells multiplication and chromosome segregation. MATERIALS AND METHODS: In accordance with the study, three specific sequences of siRNA targeting HJURP gene were designed, synthesized, then the complementary DNA containing both sense and antisense oligonucleotides of the targeting sequences were annealed and inserted into the lentiviral vector.The correct clonings were confirmed by PCR and sequencing. The most effective recombinant lentivirus vector was screened, and the recombinant plasmids with the lentivirus packaging mixes were co-transfected into 293T cells to obtain packaged lentivirus particles. Then viral titer was determined. The silencing efficiency of target gene in human embryo villus cells was detected by Real-Time PCR. RESULTS: DNA sequencing showed that the shRNA sequence was successfully inserted into the lentivirus vector. The recombinant lentiviral vector was successfully transfected into 293T cells. The recombinant lentivirus had a titer of 108 PFU/ml. After silencing HJURP gene in human embryo villus cells, the expression level of HJURP mRNA decreased significantly and the RNAi efficiency was greater than 70%. CONCLUSION: A lentiviral shRNA expression vector targeting the HJURP gene was successfully constructed and may effectively silence the target gene at a cellular level, which provides a experimental model for the influence of HJURP gene expressing inhibition on human embryo villus cells multiplication and chromosome segregation.

Case Report Identification of a novel SLC45A2 mutation in albinism by targeted next-generation sequencing.

Albinism is a diverse group of hypopigmentary disorders caused by multiple-genetic defects. The genetic diagnosis of patients affected with albinism by Sanger sequencing is often complex, expensive, and time-consuming. In this study, we performed targeted next-generation sequencing to screen for 16 genes in a patient with albinism, and identified 21 genetic variants, including 19 known single nucleotide polymorphisms, one novel missense mutation (c.1456 G>A), and one disease-causing mutation (c.478 G>C). The novel mutation was not observed in 100 controls, and was predicted to be a damaging mutation by SIFT and Polyphen. Thus, we identified a novel mutation in SLC45A2 in a Chinese family, expanding the mutational spectrum of albinism. Our results also demonstrate that targeted next-generation sequencing is an effective genetic test for albinism.

Relationships Between Oleic and Linoleic Acid Content and Seed Colonization by Cercospora kikuchii and Diaporthe phaseolorum.

Compared with standard cultivars, seed of mid-oleic soybean genotypes sometimes have shown increased colonization by Cercospora kikuchii in the field as judged by increased levels of purple-stained seed. To examine relationships between oleic and linoleic acid levels in soybean seed and postharvest seed colonization by two fungal seed pathogens, we inoculated seed with differing oleic:linoleic acid (O/L) ratios. Seed with defined O/L ratios were produced by allowing seed development of two isogenic soybean lines to occur in three different air temperature environments. Seed produced in these environments were harvested, individually analyzed for fatty acid composition, and inoculated with mycelium preparations of the fungal seed pathogens C. kikuchii or Diaporthe phaseolorum var. sojae. Fungal biomass of infected seed was quantified by measuring in vitro ergosterol content. For both soybean lines, colonization by C. kikuchii was positively correlated with the O/L ratio (r = 0.55, P < 0.03) and oleic acid content (r = 0.61, P < 0.02), and negatively correlated with linoleic (r = -0.60, P < 0.02) and linolenic (r = -0.58, P < 0.03) acid content. No association was found between the extent of seed colonization by D. phaseolorum and the seed O/L ratio. Our data suggest that the O/L ratio may be related to soybean seed colonization by C. kikuchii, but there is no evidence of a relationship with D. phaseolorum var. sojae colonization.

Ergosterol as a Quantifiable Biomass Marker for Diaporthe haseolorum and Cercospora kikuchi.

The relationship between ergosterol content and biomass was determined for the soybean fungal pathogens Diaporthe phaseolorum (Cooke & Ellis) Sacc. var. sojae, causal agent of Phomopsis seed decay, and Cercospora kikuchii (Matsumoto & Tomoy.), causal agent of leaf blight and purple seed stain. Biomass was manipulated by varying incubation period, and ergosterol was quantified by high-pressure liquid chromatography. Fungal dry mass was linearly correlated with ergosterol content (r2 = 0.90, P < 0.05 for D. phaseolorum, and r2 = 0.95, P < 0.01 for C. kikuchii). In vitro ergosterol content of fungi was 3.16 µg/mg for D. phaseolorum and 2.85 µg/mg for C. kikuchii. Ergosterol content of inoculated seed was qualitatively correlated with observed seed colonization by both pathogens. Soybean variety had a significant effect on fungal colonization by D. phaseolorum and ergosterol content. Results show that ergosterol content can be used to quantify colonization of soybean seed by both pathogens.

Aflatoxin production in peanut lines selected to represent a range of linoleic acid concentrations.

To determine whether concentrations of linoleate in peanut (Arachis hypogaea L.) seed oil could be used to predict an ability to support aflatoxin production, seeds of genotypes representing a range of linoleate content were inoculated with Aspergillus flavus Link ex Fries and assayed for aflatoxin content. Seeds were blanched and quartered, inoculated with conidia of A. flavus, placed on moistened filter paper in petri dishes, and incubated for 8 days at 28 degrees C. Multiple regression analysis was used to account for the variation among lines with the use of fatty acid concentrations as independent variables. In test 1, linoleate accounted for 39 to 44% of the variation among lines for aflatoxin B1 and B2 and total aflatoxin (26 to 27% after log transformation). Oleate accounted for substantial additional variation (27 to 29%) among lines (20 to 23% after log transformation). Other fatty acids accounted for small fractions of among-line variation. In test 2, linoleate accounted for about 35 to 44% of the variation among entries across traits (29 to 37% for log-transformed data); arachidate accounted for 19 to 29% (27 to 33% after log transformation). Eicosenoate accounted for a small part of the total entry variation. In both experiments, residual variation among entries was significant. Low-linoleate lines consistently contained more aflatoxin, whereas normal- to high-linoleate lines contained variable amounts. Although fatty acid concentrations accounted for significant portions of genetic variation, it is not practical to use them as predictors for susceptibility to aflatoxin contamination, especially for lines in the normal range for oleate and linoleate.

Comparison of Aflatoxin Production in Normal- and High-Oleic Backcross-Derived Peanut Lines.

The effect of the high-oleate trait of peanut on aflatoxin production was tested by comparing normal oleic lines with high-oleic backcross-derived lines. Seeds were blanched, quartered, and inoculated with Aspergillus flavus conidia, placed on moistened filter paper in petri dishes, and incubated for 8 days. In one experiment, dishes were stacked in plastic bags in a Latin square design with bags and positions in stacks as blocking variables. High-oleic lines averaged nearly twice as much aflatoxin as normal lines. Background genotype had no significant effect on aflatoxin content, and interaction between background genotype and oleate level was not detected. In a second experiment, dishes were arranged on plastic trays enclosed in plastic bags and stacked with PVC spacers between trays. Fungal growth and aflatoxin production were greater than in the first experiment. Background genotype, oleate level, and their interaction were significant. The mean of high-oleic lines was almost twice that of normal lines, but the magnitude of the difference varied with background genotype. Special care should be taken with high-oleic lines to prevent growth of Aspergillus spp. and concomitant development of aflatoxin contamination.