Abstract： Objective　To elucidate the antigenic antibody reaction of recombinant expression of non-structural protein 1 (NS1) of Japanese encephalitis (JE) virus with various mosquito-borne flaviviruses, including JE virus, and the antigenic antibody reaction of serum samples of patients infected with JE virus in acute stage. Methods　In this study, Escherichia coli prokaryotic expression vector (pET) system was used to recombinant express Japanese encephalitis virus NS1 gene. Western Blot assay was performed to detect the antibody responses of the recombinantly expressed protein against a variety of mosquito-transmitted flaviviruses, including JE virus, as well as antigen-antibody reactions of serum from patients with acute JE virus infection. Results　The NS1 gene expression product of JE virus (P3 strain) was in the form of an inclusion body, and the denatured and renatured expression product was displayed as a single band in the denatured gel (polyacrylamide gel electrophoresis, PAGE), with a molecular weight of about 45 000. The results of further antigen-antibody analysis showed that the antigen/antibody hybridization reaction of the expression product with polyclonal or monoclonal antibody of JE virus (mosquito isolates, encephalitis isolates) and serum samples of patients with acute JE virus infection could be completely consistent. The recombinant product showed negative antigen/antibody hybridization reactions with mosquito-transmitted flaviviruses, such as dengue virus and yellow fever virus polyclonal antibodies, but positive reactions with polyclonal antibodies to West Nile virus and Murray Valley encephalitis virus. Conclusions　In this study, the recombinant expression of the NS1 protein of JE virus was successfully obtained, and the antigen/antibody reaction between the recombinant protein and samples of patients infected with mosquito-borne flavivirus and JE virus was analyzed. The study results provide important basic data for elucidating the antigen-antibody reaction between the NS1 protein of JE virus and mosquito-borne flavivirus. The recombinant expression protein obtained in this study provides an important material basis for further research on the function of JE virus NS1 protein.

Objective: To investigate the mechanism of vascular endothelial growth factor(VEGF) inducing tolerogenic dendritic cells(DCs) in oral squamous cell carcinoma (OSCC). Methods: The DCs were divided into four groups: Control group (DC), VEGF group (VEGF added into DC), Co-culture group (DC co-cultured with SCC7) and Anti-VEGF group (anti-VEGF antibody added into DC co-cultured with SCC7). Flow cytometry (FCM) was used to detect DC surface markers. To detect the effect of DC on proliferation activity of T lymphocyte, the experiment included five groups: Nc group (T lymphocyte), Control group (T lymphocyte added into DC), VEGF group (T lymphocyte + DC + VEGF), Co-culture group (T lymphocyte + DC + supernatant of SCC7) and Anti-VEGF group (T lymphocyte + DC + supernatant of SCC7 + anti-VEGF antibody). Subsequently, the mixed lymphocyte reaction(MLR) was conducted. The expression levels of indole-2, 3-doxygenase(IDO)and programmed cell death 1 ligand 1(PD-L1)in DC were detected by western blot, real time PCR and FCM respectively. For the cytotoxic lymphocyte (CTL) assay, SCC7 cells and CTLs were mixed and CTL-mediated SCC7 cells cytotoxicity was tested. The experiment included four groups: Control group (T lymphocyte + DC), IDO inhibition group (T lymphocyte + DC + IDO inhibitor), Anti-PD-L1 antibody group (T lymphocyte + DC + anti-PD-L1 antibody) and Combination group (T lymphocyte + DC + IDO inhibitor + anti-PD-L1 antibody). The SCC7 tumor-bearing mice treated with IDO inhibitor and the anti-PD-L1 antibody were sacrificed and the tumor inhibition rate and the spleen index were determined. Results: Compared with Control group, exogenous VEGF or SCC7 co-culture inhibited the relative number of DC expressing CD11C, CD80, CD86, CD40 and MHC Ⅱ. The positive DCs were increased in the Anti-VEGF group compared with VEGF or Co-culture group. In VEGF or Co-culture group, the number of T cells stimulated by SCC7-pulsed DCs was decreased compared with Control group. However, the ability of Anti-VEGF group to induce T cell proliferation was significantly increased compared with VEGF or Co-culture group. Significantly increased expression of IDO and PD-L1 were observed in VEGF and Co-culture group. However, this was partially reversed by addition of anti-VEGF antibody into the co-culture system. Compared with Control group, the expressions of CD11C and CD86 in DC in both the IDO inhibition group and Anti-PD-L1 antibody group were increased, and were significantly higher in the Combination group compared with the single drug groups. The similar results were exhibited in MLR and CTL assay. In vivo, the results revealed that the tumors obtained from the mice in three experimental groups were smaller than those in the control group. Furthermore, the tumor volume of the Combination group was the smallest. The spleen index of each group was calculated and the results showed the spleen index of the three experimental groups was significantly higher than that of Control group. Conclusion VEGF in OSCC micro-environment inhibits the maturation and function of DC that are transformed into tolerogenic DC by high expression of IDO and PD-L1.

Non-conventional yeasts such as Yarrowia lipolytica, Pichia pastoris, Kluyveromyces marxianus, Rhodosporidium toruloides and Hansenula polymorpha have proven to be efficient cell factories in producing a variety of natural products due to their wide substrate utilization spectrum, strong tolerance to environmental stresses and other merits. With the development of synthetic biology and gene editing technology, metabolic engineering tools and strategies for non-conventional yeasts are expanding. This review introduces the physiological characteristics, tool development and current application of several representative non-conventional yeasts, and summarizes the metabolic engineering strategies commonly used in the improvement of natural products biosynthesis. We also discuss the strengths and weaknesses of non-conventional yeasts as natural products cell factories at current stage, and prospects future research and development trends.
Yeasts/genetics* ; Yarrowia/metabolism* ; Gene Editing ; Metabolic Engineering

Objective To investigate the relationship between subgroups of central lymph node metastasis (sCLNM) and lateral lymph node metastasis (LNM) of unilatal papillary thyroid carcinoma (uPTC) with cervical lymph node negative(cN0).Methods The clinical and pathological data of 161 patients with cN0-uPTC who underwent total thyroidectomy+central lymph node dissection+lateral lymph node dissection from Jan.2016 to Dec.2016 were retrospectively analyzed.The relationship between the lymph node metastasis of each subarea in the central area of the affected side and the lymph node metastasis of the affected side was investigated.Results Binary logistic regression analysis of cN0-uPTC subregions in the affected central region showed:pre-laryngeal lymph node metastasis,pre-tracheal lymph node metastasis and paratracheal lymph node metastasis were independent risk factors for lymph node metastasis in the affected lateral region(P=0.008,0.016,0.035,respectively).Prelaryngeal lymph node metastasis was an independent risk factor for lymph node metastasis in the affected area Ⅱ (P=0.015).Pre-tracheal lymph node metastasis was an independent risk factor for lymph node metastasis in affected area Ⅲ (P=0.004).Pre-tracheal and para-tracheal lymph node metastasis were independent risk factors for lymph node metastasis in the affected Ⅳ area (P=0.035,0.011,respectively).Conclusions The lymph node metastasis pathway of thyroid cancer had certain regularity.The pre-laryngeal lymph node metastasis has the prediction value for the lymph node metastasis of the affected area Ⅱ.The pre-tracheal lymph node metastasis has the prediction value for the lymph node metastasis of the affected area Ⅲ.The pre-tracheal and paratracheal lymph node metastasis have the prediction value for lymph node metastasis of the affected area Ⅳ.Lymph node dissection in affected areas Ⅲ and Ⅳ needs to be considered in patients with pre-tracheal or paratracheal lymph node metastases.On this basis,lymph node dissection on the affected areas Ⅱ,Ⅲ,and Ⅳ might be considered if there is pre-laryngeal lymph node metastasis at the same time.

Objective: To investigate the clinicopathologic characteristics, immunophenotypes, and differential diagnostic features of extra-pleural solitary fibrous tumor (SFT) with uncommon histology. Methods: Seven cases of extra-pleural SFT with uncommon histology were collected during January 2015 and December 2016 in Zhejiang Provincal People′s Hospital; the clinical and radiologic features, histomorphology, immunophenotype and prognosis were analyzed. EnVision method was used for immunohistochemical staining of STAT6, CD34 and other differential diagnosis associated markers. Results: There were five male and two female patients, age from 23 to 54 years (mean=39 years). Three tumors were located in the soft tissue of head and neck, two in trunk subcutaneous soft tissue, one in sella region, and one in the kidney. Grossly the tumors ranged from 0.4 to 8.0 cm (mean=3.1 cm). Microscopically, all three head and neck cases resembled giant cell angiofibroma/giant cell subtype SFT, and one case showed sheet-like pattern of the multinucleated syncytial cells, creating a biphasic arrangement similar to myofibroma. Both truncal tumor resembled lipomatous type SFT, with one similar to dermatofibrosarcoma protuberans and the other to atypical spindle cell lipomatous tumor. The sella tumor showed morphology of a conventional SFT with high grade sarcomatous transformation. The renal tumor demonstrated a malignant SFT with entrapped benign renal tubules, mimicking a biphase synovial sarcoma or a malignant mixed epithelial and stromal tumor. By immunohistochemistry, all seven SFTs showed diffuse and strong nuclear reactivity to antibody against STAT6. Conclusions Extra-pleural SFTs show a significant heterogeneity of morphology and biological behavior which could cause differential confusion.Careful attention to its characteristic histomorphology with the use of STAT6 immunohistochemistry can help distinguish this tumor from its many mimickers.

Objective: To investigate the clinicopathologic characteristics, immunophenotype, differential and diagnostic features of atypical spindle cell lipomatous tumor (ASLT). Methods: Three cases of ASLT were collected from January 2010 to March 2017 at Zhejiang Provincial People′s Hospital. The clinical and imaging features, histomorphology, immunophenotype and prognosis were analyzed. Fluorescence in situ hybridization (FISH) was used to detect MDM2 gene amplification, and relevant literature was reviewed. Results: All three patients were adult males, aged 38, 43 and 54 years, respectively. One tumor originated in the subcutaneous soft tissue in the head and neck, one was located in the left primary bronchus and one in the latissimus dorsi muscle. Grossly, all three tumors were circumscribed and ranged from 4.0 to 5.8 cm in size. Microscopically, all showed a focally infiltrative front. These tumors were composed of variable proportions of spindle-shaped and adipocytic cells in a background of variable fibrous and edematous matrix. Scattered lipoblasts were easily seen. One tumor was composed predominately of spindle tumor cells, one of adipocytic cells, and one of equally mixed cell populations. The spindle tumor cells were generally bland-appearing with focal nuclear enlargement and hyperchromasia noted in one case. Mitosis was not seen in neither the spindle cells nor the adipocytic cells. By immunohistochemistry, diffuse and strong reactivity to CD34 of the spindle cells was noted in all cases, definite loss of Rb expression was noted in one of three cases, and S-100 protein was expressed only in the adipocytic cells. INI-1 was intact and Ki-67 index was 1% to 3%. All other markers including CDK4, MDM2, STAT6, SOX10, CD99, bcl-2, β-catenin, CD117, GFAP, CK, EMA, SMA and desmin were negative. FISH of MDM2 was done in two cases, and both showed no amplification. The ASLT in the head and neck had two recurrences during 17 months of follow-up, whereas the tumor in the latissimus dorsi was free of disease during 33 months of follow-up. Conclusions ASLT is a rare subtype of low-grade adipocytic neoplasm and is distinctive from atypical lipomatous tumor/well-differentiated liposarcoma. The histomorpholgy of ASLT has significant heterogeneity and forms a continuous spectrum. ASLT needs to be distinguished from a series of benign and malignant soft tissue tumors.

Objective: To investigate the clinicopathologic and molecular genetic features of secretory carcinoma of salivary gland (SCSG). Methods: Six cases of SCSG were collected from Zhejiang Provincial People's Hospital from January 2011 to March 2018. The clinical, histopathological and immunohistochemical features were analyzed and fluorescence in situ hybridization (FISH) was used to detect ETV6 gene rearrangement. Results: Four out of 6 tumors originated in the parotid gland and one of each in the minor salivary glands of soft palate and the buccal mucosa. Grossly, 4 cases were solid and 2 were partially cystic with maximum diameter ranging from 1.0 to 4.0 cm. Microscopically, 5 tumors showed typical features of low grade SCSG with tumor divided by thin fibrous septa into lobules composed of solid acinar, microcystic, follicular and papillary structures with abundant extracellular mucinous secretions. The tumor cells had cuolated or hobnail cytoplasm with low-grade nuclei and scarce mitoses. Perineural invasion was present in 1 case. The remaining tumor showed about 30% of the tumor areas with high-grade transformation characterized by proliferation of a distinct population of anaplastic cells arranged in irregular glandular, small nested and single cell patterns that were surrounded by desmoplastic stroma and invaded into surface mucosa with ulceration. Immunohistochemistry showed that all 6 tumors had diffuse and strong reactivities to S100 protein and cytokeratin 7, and 4 cases showed focal reactivity to gross cystic disease fluid protein 15 (GCDFP15), all were negative for discovered on gist 1 (DOG1), cytokeratin 20, p63 and calponin. High grade transformation cases were analysed, the high grade SCSG components showed a significantly increased Ki-67 index and cyclin D1 positive tumor cells compared to the conventional SCSG components. FISH analyses showed that 4 cases had ETV6 gene rearrangement. Eleven to seventy one months' follow-up showed no evidence of tumor recurrence nor metastasis. Conclusions SCSG harbors characteristic genetic abnormalities with ETV6 gene rearrangement and typically shows a low grade morphology with occasionally, high grade transformation can be present.

OBJECTIVE: To establish a non-invasive method for beta-thalassemia by detecting parental CD41-42 mutation in cell-free DNA derived from maternal plasma with droplet digital PCR (ddPCR). METHODS: Beta-actin gene and beta-thalassemia gene CD41-42 mutation were respectively set as the reference and target sequences. A novel method was established based on Bio-Rad ddPCR technique with specific primers and TaqMan probes for the two genes. The accuracy, sensitivity and detective linearity range of the developed method were evaluated by detection of the target gene gradient concentration samples. The applicability was also evaluated by testing 20 maternal plasma samples. RESULTS: The ddPCR method could accurately detect the beta-thalassemia CD41-42 mutation in cell-free DNA derived from maternal plasma. Within the target sequence concentration ratio of 5.00%-0.50%, the relative errors were all < 0.05, the linear regression equation was Y=1.0101-X-0.0071 and R=0.9994. The results of 20 maternal plasma cell-free DNA samples were all consistent with those of the follow-up testing. CONCLUSION A ddPCR method for detecting parental CD41-42 mutation in cell-free DNA from maternal plasma was developed. The method is simple, rapid, accurate, and can be applied for non-invasive prenatal diagnosis for couples simultaneously carrying the CD41-42 mutation.
Cell-Free Nucleic Acids ; DNA ; blood ; Female ; Humans ; Mutation ; Polymerase Chain Reaction ; Pregnancy ; Prenatal Diagnosis ; methods ; beta-Thalassemia ; diagnosis ; genetics

Objective:To analyze the factors related to metastasis of contralateral central lymph node (CLN) in cN0 papillary thyroid car-cinoma (PTC) and discuss the indications for CLN dissection. Methods:We enrolled 149 unilateral PTC patients who underwent total thyroidectomy and prophylactic bilateral (CLN) dissection. This work analyzed the relationship of gender, age, extrathyroidal extension, multifocality, thyroiditis, ipsilateral central lymph nodes, and prelaryngeal lymph node with CLNs. Results:The rates of metastasis to ip-silateral and contralateral central compartments were 73.2%and 23.5%, respectively. In univariate analysis, gender, age, tumor size, multifocality, and thyroiditis were not important in predicting contralateral central compartment lymph node metastasis (P=0.792, 0.097, 0.531, 0.269, and 1.000, respectively);by contrast, extrathyroidal extension (P=0.017), prelaryngeal lymph nodes (P=0.006), and ipsilateral CLNs (P<0.001) are related to CLN metastasis. However, multivariate analysis showed that ipsilateral central metastasis was an independent risk factor for lymph node metastasis in the contralateral central region when the number of ipsilateral central metas-tases is≥3 (P=0.010). Conclusion:Extracapsular invasion, prelaryngeal lymph nodes, and ipsilateral CLN influence the metastases of CLN. Bilateral CLN dissection should be performed when the number of ipsilateral central metastases is≥3 and there is merger of ex-tra-laryngeal lymph nodes or capsule invasion.

Objective:To retrospectively analyze the regularity and risk factors of skip metastasis (central lymph node negative and lat-eral lymph node positive) in papillary thyroid carcinoma (PTC). Methods:A total of 521 PTC patients underwent total thyroidectomy and central plus lateral lymph node dissection at The First Affiliated Hospital of Chongqing Medical University from January 2013 to De-cember 2016. Clinicopathological characteristics of the patients were collected and analyzed. Results:Skip metastasis rate of PTC was 8.3％(43/521). Tumors in the upper lobe (OR=3.401, 95％CI:1.770-6.536;P=0.001) and in the lateral part (OR=3.424, 95％CI:1.182-9.920;P=0.023) of the thyroid, as well as age above 45 (OR=2.856, 95％CI:1.488-5.482;P=0.002), were independent risk factors for skip metastases for this disease. Clinically node-negative (cN0) PTC patients with tumors in the upper lobe had higher possibility of skip metastases than those with clinically involved lateral neck nodes(cN1b) (P=0.022). Conclusion:Skip metastasis of PTC is not un-common. Thus, preoperative clinical assessment and imaging examination for lateral lymph node is necessary, especially for PTC pa-tients who are above 45 years old and with tumors in the upper lobe and/or unilateral area of thyroid. The lateral lymph node dissec-tion should be performed when necessary.