[Stratified application of gene expression in diagnosis of thyroid nodules].

Objective: To investigate the value of BRAF V600E and multigene detection and stratified application for the diagnosis of thyroid nodules. Methods: A total of 1 117 patients with thyroid nodules resection at Nanjing Gulou Hospital from December 2020 to July 2022 were enrolled in the study. Fine needle aspiration (FNA) and core biopsy samplings were performed for cytopathologic examination and genetic testings; the findings were combined with BSRTC classification. The diagnostic performance of BRAF V600E and multigene detection were compared. Results: Among the 1, 117 patients who underwent thyroid nodules resection, 285 were male and 832 were female, with a median age of 46 years (range: 24-76 years). Postoperative histopathologic examination confirmed 1 040 cases of thyroid cancer and 77 cases of benign nodules. The sensitivity (87.0% vs. 80.8%, P<0.01) and diagnostic accuracy (87.9% vs. 82.1%, P<0.01) of multigene detection were significantly higher than those of BRAF V600E detection. The result of multigene detection showed that BRAF V600E mutation was the most common finding, followed by CCDC6-RET (E1-E12) fusion, ETV6-NTRK3 fusion, and KRAS mutation. Multigene detection had a higher sensitivity (81.9% vs. 72.8%, P<0.01) and lower cancer risk in wild-type (47.6% vs. 57.7%, P=0.069) than BRAF V600E detection in BSRTCⅠ-Ⅴ lesions. Compared with BRAF V600E detection, multigene had no significant difference of sensitivity in BSRTC Ⅰ lesions, but significantly higher sensitivity (86.3% vs 74.0%, P<0.01) in BSRTC Ⅲ lesions. Conclusions: Genetic detection can be used as an effective tool for the diagnosis of thyroid nodules. A stratified application of molecular markers in the diagnosis of thyroid nodules is proposed. Combined with FNA, single gene or multigene detection both can effectively assist in the diagnosis of thyroid nodules. Moreover, multigene detection is superior to single gene detection. For BSRTC Ⅲ lesion with wild-type BRAF, multigene detection can be considered with a repeated FNA.

[Antimicrobial susceptibility and drug-resistance genes of Yersinia spp. of retailed poultry in 4 provinces of China].

Objective: To monitor the antimicrobial resistance and drug-resistance genes of Yersinia enterocolitis, Y. intermedia and Y. frederiksenii recovered from retailed fresh poultry of 4 provinces of China. Methods: The susceptibility of 25 isolated Yersinia spp. to 14 classes and 25 kinds of antibiotics was determined by broth microdilution method according to CLSI (Clinical and Laboratory Standards Institute). The antibiotic resistance genes were predicted with antibiotic resistance genes database (ARDB) using whole genome sequences of Yersinia spp. Results: In all 22 Y. enterocolitis tested, 63.7% (14 isolates), 22.8% (5 isolates), 4.6% and 4.6% of 1 isolates exhibited the resistance to cefoxitin, ampicillin-sulbactam, nitrofurantoin and trimethoprim-sulfamethoxazole, respectively. All the 25 isolates were multi-drug resistant to more than 3 antibiotics, while 64.0% of isolates were resistant to more than 4 antibiotics. A few Y. enterocolitis isolates of this study were intermediate to ceftriaxone and ciprofloxacin. Most Yersinia spp. isolates contained antibiotic resistance genes mdtG, ksgA, bacA, blaA, rosAB and acrB, and 5 isolates recovered from fresh chicken also contained dfrA1, catB2 and ant3ia. Conclusion: The multi-drug resistant Yersinia spp. isolated from retailed fresh poultry is very serious in the 4 provinces of China, and their contained many kinds of drug-resistance genes.

[Myofibroma/myofibromatosis: a clinicopathologic analysis of 9 cases].

Objective: To investigate the clinical and histological features, diagnosis and differential diagnosis of myofibroma/myofibromatosis. Methods: The clinical data and pathology features of nine cases of myofibroma/myofibromatosis were collected from August 2011 to November 2016 in Affiliated Drum Tower Hospital, Nanjing University Medical School and Children's Hospital of Nanjing Medical University. Immunohistochemistry(IHC), PDGFRB molecular analysis and ETV6-NTRK3 gene fusion were performed and relevant literature reviewed. Results: There were 7 males and 2 females, with age ranging from 3 days to 18 years (mean 5 years). The tumors were located in head and neck (eight cases) and trunk (one case). Clinically, the tumors presented as freely movable nodules. Microscopically, they appeared biphasic with alternating light- and dark-staining areas. The light-staining area consisted mainly of plump myoid spindle cells with eosinophilic cytoplasm arranged in nodules, short fascicles, or whorls.The dark-staining area was composed of round or polygonal cells with slightly hyperchromatic nuclei or small spindle cells arranged around a distinct hemangiopericytoma-like vascular pattern. IHC showed the tumor cells in the light-staining area were strongly positive for vimentin and SMA, while cells in dark-staining area were strongly positive for vimentin, and weakly for SMA. Tumor cells were negative for desmin, S-100 protein, h-Caldesmon, CD34 and STAT6. Analysis of PDGFRB mutations was performed in seven cases. Two cases showed 12 exon point mutation c. 1681 c>T(p.R561C), one case showed 14 exon point mutation c. 1998C>G (p.N666K). ETV6-NTRK3 gene fusion was not detected by fluorescence in situ hybridization in four patients under three years old. All cases were followed for 6 to 68 months, with two recurrences. Conclusions: Myofibroma/myofibromatosis is an uncommon benign myofibroblastic tumor of infancy and childhood. The tumor can appear biphasic, and may show PDGFRB point mutation which is of potential diagnostic value.

[Expression of erythropoietin in neovascular glaucoma and its role in the angiogenesis in vitro].

Objective: To explore the expression of erythropoietin (EPO) in neovascular glaucoma (NVG) and its role in the angiogenesis in vitro. Methods: The levels of EPO in aqueous humor and vitreous body of NVG patients (10 cases) and non-NVG patients (10 cases) were detected by enzyme-linked immunosorbent assay (ELISA). Human umbilical vein endothelial cells (HUVECs) were used as the research object, and were divided into EPO 0.1, 1, 10 U/ml group according to EPO concentration, on the basis of treatment of HUVECs cells with 10 ng/ml vascular endothelial growth factor (VEGF). Effect of different concentrations of recombinant human EPO and VEGF on the proliferation, angiogenesis and the formation of capillary like structures of HUVECs in vitro was explored, meanwhile, protein and mRNA expression of hypoxia inducible factor-1α (HIF-1α) was detected by Western blot and RT-PCR. Results: In the study, no patients discontinued the trial. EPO level of aqueous humor in non-NVG patients was (29.4±0.6) ng/L, while EPO level in aqueous humor of NVG patients reached (307.5±7.6) ng/L, and the difference was statistically significant (t=2.186, P=0.039); vitreous EPO levels in non-NVG patients was (16.7±1.2) ng/L, while vitreous EPO levels in NVG patients reached (267.5±11.4) ng/L, with a statistically significant difference (t=2.517, P=0.019). Compared with control group, 0.1, 1, 10 U/ml EPO and 10 ng/ml VEGF promoted the proliferation of HUVECs, and formation of vascular lumen-like and capillary-like structure in vitro, and up-regulated the protein and mRNA expression of HIF-1α (all P<0.05). Conclusion: EPO was highly expressed in the NVG patients, and exogenous EPO could significantly promote the proliferation of HUVEC and formation of vascular lumen-like and capillary-like structure, which may be related to up-regulation of HIF-1 expression.

Microarray-based gene expression profiles in rabbit retina due to negative pressure suction.

We investigated a possible molecular pathogenesis involving retinal ganglion cell apoptosis following transient high intraocular pressure. Changes in the gene expression profiles of the retina were detected via gene chip methodology. Twelve New Zealand white rabbits were randomly assigned to control and 3-min negative pressure suction groups. The control group was treated only with a laser, and the experimental group was also treated with suction for 3 min, using a negative pressure generator. Total RNA was then extracted from the retinal tissue at different recovery stages to analyze gene expression profiles using the Agilent rabbit one-way gene chip. The groups were then compared. Immediately after negative pressure suction induction, 704 genes were differentially expressed. Among these, 485 genes were upregulated, and 219 were downregulated. Expression of the genes encoding CRYAA, CRYAB, and TLR3 genes, which are involved in apoptosis, was elevated. The KRT18 gene, which is involved in apoptosis, had reduced expression. Seven days after negative pressure suction, 482 genes were differentially expressed. Among these, 178 genes were upregulated, and 304 were downregulated. Expression of the genes encoding CRYAB, IL1-BETA and IL1R1, which are involved in apoptosis, was upregulated. Ten days after negative pressure suction, 402 genes were differentially expressed. Of these, 213 genes were upregulated, and 189 were downregulated. Apoptosis genes CRYAB, CRYBA3, CRYBB2, IL1- BETA, and IL1R1 showed higher expression levels. We concluded that negative pressure suction for long periods of time (for example, 3 min) results in changes in gene expression. Genes with higher fold changes help protect retinal ganglion cells from apoptosis. We suggest that promoting the expression of these genes should be considered as a new means for treating ischemic-hypoxic retinopathy.

Experimental research on the use of an antineoplastic drug with a bone implant.

In this experiment, we impregnated antineoplastic drugs into bone cement and polyporous ceramics in an attempt to develop a new material which could release the drug, as well as filling a postoperative bony defect. It was shown with cell culture that the drug can be delivered from these materials in vitro. Adriamycin-bone cement can release the drug over a period of 14 days in vitro and for 35 days in vivo in a goat. The times for the adriamycin-ceramic implant were respectively 16 days and 35 days. Microscopy showed only a mild foreign body macrocytic infiltration with the adriamycin-bone cement and a mild nonspecific inflammatory reaction with adriamycin-ceramic implant 9 weeks after operation. The results of treating rat sarcoma 180 with this drug-implant showed a tumour suppression rate of about 54% which is similar to that obtained with systemic chemotherapy. The implant not only fills the postoperative defect, but a high drug concentration locally is maintained, together a low systemic concentration.