Objective:To investigate the expression of anaplastic lymphoma kinase (ALK), tropomyosin receptor kinase (TRK), and recombinant C-Ros oncogene 1, receptor tyrosine kinase (ROS1) in Spitz tumors, and to analyze their associations with clinical and histopathological features of Spitz tumors.Methods:Clinical and histopathological characteristics, as well as follow-up data, were collected from patients with Spitz tumors at Department of Pathology, Hospital of Dermatology, Chinese Academy of Medical Sciences from January 2017 to August 2023, and retrospectively analyzed. Immunohistochemical staining for ALK, pan-TRK, and ROS1 was performed on skin tissues, and associations between the expression of these kinase proteins and clinicopathological features were analyzed.Results:A total of 57 patients with Spitz tumors were collected, including 36 females and 21 males. Immunohistochemical staining showed that 30 (52.6%) patients were positive for ALK, 4 (7.0%) were positive for ROS1, only 2 (3.5%) were positive for TRK, and 21 (36.8%) were negative for the three kinase proteins. Among the 30 ALK-positive patients, the median age was 9.5 years, 21 (70.0%) were females, and 15 (50.0%) presented with lesions on the face, which mainly manifested as papules or nodules; histologically, 29 (96.7%) patients had hypopigmented tumors with an exophytic growth pattern, and the tumor cells were mainly large and long spindle cells arranged in long cord-like, plexiform or fascicular patterns. Among the 4 ROS1-positive patients, there were 3 females and 1 male, presenting with exophytic papules or polyps; histologically, tumor cells were mostly arranged in small nests, without obvious clefts around cell nests. Two TRK-positive patients were both males aged 20 and 50 years respectively, and presented with brown and skin-colored flat papules, respectively; histologically, the tumors were located superficially with a flat base, and tumor cells spread in a pagetoid pattern in the epidermis, with some epithelioid cells and small cell nests. Among the 21 patients negative for the 3 kinase proteins, 9 were males and 12 were females, and they clinically presented with macules, papules and polypoid lesions; histologically, most tumors were located superficially, consisting of a mixture of epithelioid cells and spindle cells, with rare cytological atypia and mitotic figures, and 2 cases showed mild tissue structural and cellular atypia. Fifty-seven patients were followed up for 2 - 83.3 months, with a median follow-up of 19.2 months. Only 1 ALK-positive child experienced a recurrence, and no recurrence or lymph node metastasis was observed in the other cases.Conclusions:Among the three kinase proteins, ALK showed the highest positive rate in Spitz tumors in this study, while TRK- and ROS1-positive cases were sporadic. Histopathologically, ALK-positive Spitz tumor cells were mainly long spindle cells arranged in long cord-like or plexiform patterns, while TRK- and ROS1-positive Spitz tumors tended to have small cell nests. Both the kinase protein-positive and -negative Spitz tumors mostly had a good prognosis.

To report the first case of cutaneous chronic active Epstein-Barr virus disease manifesting as persistent erythema multiforme in China. The 12-year-old female patient presented with recurrent erythema and blisters all over the body, accompanied by oral erosions for more than 5 months. Skin examination showed dark erythema scattered on the right upper eyelid and cheeks, as well as erosions and blisters arising in the dark erythema on the lower jaw; broad bean- to pigeon egg-sized blisters with clear fluids arising in erythema were scattered on the back, buttocks, and limbs, and some manifested as atypical targetoid lesions; there was a mung bean-sized erosion on the mucosa of the lower lip and the right buccal region each; the patient also presented with moon face and multiple striae atrophicae on the lower limbs. Histopathological examination of the skin lesions on the lower limb revealed basket-weave hyperkeratosis, epidermal necrosis, liquefaction degeneration of basal cells with subepidermal blister formation, and perivascular lymphocytic infiltration in the superficial dermis; direct immunofluorescence assay showed negative staining for IgG, IgM, IgA, and complement C3 among epidermal cells and at the basement membrane zone; enzyme-linked immunosorbent assay showed negative staining for serum antibodies against desmoglein 1/3 (Dsg1/3), BP180, and type Ⅶ collagen; immunohistochemical examination demonstrated partial positive staining for CD3, CD4, CD5, CD8, CD56, granzyme B, and Epstein-Barr virus-encoded RNA, positive staining for Ki67 (> 70%), but negative staining for CD20. The Epstein-Barr virus DNA level was measured to be 1.97 × 10 6 IU/ml in whole blood samples and 2.65 × 10 7 IU/ml in blister fluid samples. No mutation sites with functional significance were identified by whole-exome sequencing. Based on these findings, a diagnosis of cutaneous chronic active Epstein-Barr virus disease manifesting as persistent erythema multiforme was made. The patient was treated with methylprednisolone at a dose of 40 mg/d, intravenous drips of ganciclovir at 200 mg twice daily, etc., and discharged after improvement.

Objective To compare the differences in the composition and diversity of intestinal flora between knee osteoarthritis(KOA)patients with dampness-heat obstruction syndrome,cold-dampness blockage syndrome and healthy people;To explore the characteristics of intestinal flora of KOA patients with the above two TCM syndrome types.Methods According to the inclusion and exclusion criteria,dampness-heat obstruction syndrome group,cold-dampness blockage syndrome group,and healthy control group were screened,with 10 cases for each group,and then their fecal samples were collected.16S rDNA sequencing technology was used to compare the differences of intestinal flora among groups by Alpha and Beta diversity analysis.Results There was no significant difference in species richness of intestinal flora among the three groups,but there was statistical significance in species diversity between cold-dampness blockage syndrome group and dampness-heat obstruction syndrome group,and healthy control group(P<0.05).There were statistical significance among the three groups in intestinal flora composition(P= 0.001).At the phylum level,Bacteroidota and Firmicutes were significantly dominant,and at genus level,the abundance of Prevotella in dampness-heat obstruction syndrome group and cold-dampness blockage syndrome group increased.The abundance of Enterobacteriaceae,Lachnospira and Klebsiella was relatively large in dampness-heat obstruction syndrome group.The abundance of Prevotella and Pseudomonas in cold-dampness blockage syndrome group was large.Conclusion There are differences in the intestinal flora structure between KOA patients with dampness-heat obstruction syndrome and cold-dampness blockage syndrome.

Objective:To detect genetic mutations in a case of severe epidermolysis bullosa simplex.Methods:Clinical data and peripheral blood samples were collected from the patient and her parents, and genomic DNA was extracted. Whole exome sequencing was performed to identify causative gene mutations in the patient, and then Sanger sequencing to verify the mutations among the family members.Results:A heterozygous mutation c.1429G>A at position 1429 in exon 7 of the KRT5 gene was identified in the patient, which led to the substitution of glutamic acid by lysine at amino acid position 477 (p.Glu477Lys) of keratin 5 encoded by the KRT5 gene. The mutation was not detected in her unaffected parents.Conclusion:A causative mutation c.1429G>A (p.Glu477Lys) in the KRT5 gene was identified in the patient with severe epidermolysis bullosa simplex, which was a de novo mutation.

Objective:To identify the underlying gene mutation in a family with Legius syndrome mainly manifesting as multiple café-au-lait spots, and to confirm the diagnosis.Methods:Clinical data were collected from a proband with multiple café-au-lait spots as the main clinical manifestation, his parents and grandparents. Genomic DNA was extracted from peripheral blood samples of the above subjects. Whole-exome sequencing was performed to identify mutation sites in the proband, PCR and Sanger sequencing were performed to verify the candidate mutation among the family members, and to confirm the diagnosis.Results:The proband, a 12-year-old male patient, presented with more than 10 café-au-lait spots with a major diameter of > 5 mm on the trunk, and multiple freckles on the axilla and groin. Mutation analysis of the proband revealed a small heterozygous deletion mutation (c.1220_1238del) in exon 7 of the SPRED1 gene encoding Sprouty-related EVH1 domain-containing protein 1, causing a frameshift mutation in the amino acid sequence (p.L407fs*) . This mutation was detected in the affected mother of the proband, but not in his father or grandparents. The mutation in the proband was a novel mutation, and was inherited from his mother. The mutation was co-segregated with the disease in the family, and a diagnosis of Legius syndrome was made.Conclusion:Clinical symptoms of Legius syndrome are similar to those of early-stage neurofibromatosis type Ⅰ, and genetic testing is helpful for early diagnosis, prognosis prediction and follow-up scheduling.

A 15-year-old female patient presented with multiple café-au-lait spots all over the body for 15 years and scoliosis for more than 1 year. One year prior to the presentation, the patient underwent tumor resection due to occipital arteriovenous malformation. Skin examination showed multiple scattered café-au-lait spots of various sizes all over the body with the largest café-au-lait spot measuring about 3 cm × 4 cm, and freckles on the axillae and groins. Whole exome sequencing revealed a frameshift mutation due to a heterozygous one-base deletion (c.3328delT) in exon 26 of the NF1 gene in the patient, which was not identified in the patient′s parents and younger brother. The patient was diagnosed with neurofibromatosis type 1 complicated by occipital arteriovenous malformation and scoliosis.

Objective To construct a recombinant lentiviral vector carrying the casitas B-lineage lymphoma (Cbl)-b short hairpin RNA (shRNA),and to evaluate its effect on the biological behavior of A375 melanoma cells in vitro.Methods Three specific shRNAs targeting Cbl-b gene and a negative control shRNA were designed and synthesized,and recombinant lentiviral vectors were constructed.A375 cells were divided into 5 groups to be transfected with 3 kinds of lentiviral vector expressing Cbl-b genespecific shRNAs (CBLB-shRNA-1 group,CBLB-shRNA-2 group and CBLB-shRNA-3 group),a lentiviral vector containing negative control shRNA (negative control group),and an empty vector (blank control group).Real-time fluorescence-based quantitative PCR and Western blot analysis were performed to determine the silencing efficiency at 72 hours after transfection.Cell counting kit-8 (CCK-8)assay was conducted to evaluate cellular proliferative activity at 24,48,72 and 96 hours after transfection,flow cytometry to detect cell apoptosis and cell cycle at 72 hours after transfection,and Transwell invasion assay to assess cellular invasive activity at 72 hours after transfection.Results Three recombinant lentiviral vectors containing Cbl-b shRNA were constructed successfully.As Western blot analysis revealed,the CBLB-shRNA-3 showed the highest silencing efficiency.CCK-8 assay indicated that the proliferative activity of A375 cells was significantly lower in the CBLB-shRNA-3 group than in the negative control group and blank control group at 72 and 96 hours after transfection(all P ＜ 0.01).Flow cytometry showed that the apoptosis rate of A375 cells was significantly higher in the CBLB-shRNA-3 group (22.73％ ± 6.58％) than in the negative control group (6.08％ ± 1.35％,P ＜ 0.01) and blank control group (6.34％ ± 1.07％,P ＜ 0.01).The CBLB-shRNA-3 group showed a significantly higher proportion of A375 cells at G1 phase,but a significantly lower proportion of A375 cells at S phase compared with the negative control group and blank control group(all P ＜ 0.01).Transwell assay showed that there were significant differences in the number of A375 cells crossing the artificial basement membrane (matrigel) at 72 hours after transfection among the negative control group,blank control group and CBLB-shRNA-3 group (76.60 ± 1.82,73.20 ± 3.83,19.60 ± 1.14,respectively;F =794.50,P ＜ 0.01).Conclusions A recombinant CBLB-shRNA-3-expressing lentiviral vector which can efficiently silence Cbl-b gene has been successfully constructed.It can inhibit the proliferation,cell cycle progression and invasive activity of A375 cells,but promote the apoptosis of A375 cells.

Objective To analyze differentially expressed proteins in A375 melanoma cells before and after short hairpin RNA (shRNA)-mediated Cbl-b gene silencing.Methods The label-free quantitative proteomics approach was performed to identify differentially expressed proteins between A375 cells transfected with lentiviral vectors containing Cbl-b shRNA (Cbl-b shRNA group) and those with control lentiviral vectors (control group).Then,the properties of differentially expressed proteins were analyzed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) enrichment analysis.Western blot analysis was conducted to determine the expression of differential proteins (EphA2 and GSK3β) and phosphorylated protein kinase (p-AKT) after shRNA-mediated Cbl-b gene silencing.Statistical analysis was carried out by t test of two independent-samples for comparison of protein expression abundance between the two groups with SPSS 23.0 software.Additionally,the results of GO and KEGG enrichment analysis were analyzed by Fisher's exact test.Results A total of 3 449 proteins were identified and quantified,and 74 of them were differentially expressed between the Cbl-b shRNA group and control group.Compared with the control group,52 proteins were up-regulated and 22 were down-regulated in the Cbl-b shRNA group.GO enrichment analysis of differential proteins revealed that the top five significantly enriched biological processes were integrin-mediated cell adhesion,single-organism metabolic process,regulation of integrin-mediated cell adhesion,regulation of protein-targeting mitochondria and nucleic acid metabolic process.The top five significantly enriched molecular functions included DNA binding,2-iron,2-sulfur cluster binding,signaling receptor activity,cadherin binding and cell adhesion molecule binding.The top five significantly enriched cell components included nucleosome,DNA packaging complex,photoreceptor connecting cilium,DNA-protein complex and extracellular region part.KEGG enrichment analysis demonstrated that the top five significantly enriched melanoma-related signaling pathways were folate biosynthesis,axon guidance,extracellular matrix-receptor interaction,adherens junction and Wnt signaling pathways.As Western blot analysis revealed,the Cbl-b shRNA group showed lower protein expression of EphA2 (0.369),but higher protein expression of GSK3β (3.524) compared with the control group (1),which were consistent with the results of proteomics analysis.Additionally,the protein expression of p-AKT was down-regulated in Cbl-b shRNA group (0.453) compared with the control group (1).Conclusion Cbl-b may be involved in the occurrence of melanoma through a variety of biological pathways,and the EphA2/PI3K/AKT signaling pathway may be one important pathway.

Objective: To investigate the different pattern of neck lymph node metastasis, the choice of surgical methods and prognosis in early tongue squamous cell carcinoma. Methods: A total of 157 patients with early oral tongue squamous cell carcinoma were included in this study. Statistical analysis was performed to identify the pattern of lymph node metastasis, to determine the best surgical procedure and to analyze the prognosis. Results: The occurrence of cervical lymph node metastasis rate was 31%(48/157). Neck lymphatic metastasis was significantly related to tumor size (P=0.026) and histology differentiation type (P=0.022). The rate of metastasis was highest in level Ⅱ [33% (16/48)]. In level Ⅳ, the incidence of lymph node metastasis was 5%(7/157), and there was no skip metastases. The possibility of level Ⅳ metastasis was higher, when level Ⅱ (P=0.000) or Ⅲ (P=0.000) involved. The differentiation tumor recurrence, neck lymphatic metastasis and adjuvant radiotherapy were prognostic factors (P<0.05). Multivariate analyses revealed histology differentiation type, neck lymphatic metastases and adjuvant radiotherapy were the independent prognostic factors. Conclusions Neck lymphatic metastasis rate is high in early tongue squamous cell carcinoma, simultaneous glossectomy and neck dissection should be performed. Level Ⅳ metastasis rate is extremely low, so supraomohyoid neck dissection is sufficient for most of the time. The histology differentiation type, neck lymphatic metastasis and adjuvant radiotherapy are independent prognostic factors.

Syringaresinol-4---d-glucoside (SSG), a furofuran-type lignan, was found to modulate lipid and glucose metabolism through an activity screen of lipid accumulation and glucose consumption, and was therefore considered as a promising candidate for the prevention and treatment of metabolic disorder, especially in lipid and glucose metabolic homeostasis. In this study, the effects of SSG on lipogenesis and glucose consumption in HepG2 cells and C2C12 myotubes were further investigated. Treatment with SSG significantly inhibited lipid accumulation by oil red O staining and reduced the intracellular contents of total lipid, cholesterol and triglyceride in HepG2 cells. No effect was observed on cell viability in the MTT assay at concentrations of 0.1-10 μmol/L. SSG also increased glucose consumption by HepG2 cells and glucose uptake by C2C12 myotubes. Furthermore, real-time quantitative PCR revealed that the beneficial effects were associated with the down-regulation of sterol regulatory element-binding proteins-1c, -2 (), fatty acid synthase (), acetyl CoA carboxylase () and hydroxyl methylglutaryl CoA reductase (), and up-regulation of peroxisome proliferator-activated receptors alpha and gamma (and). SSG also significantly elevated transcription activity oftested by luciferase assay. These results suggest that SSG is an effective regulator of lipogenesis and glucose consumption and might be a candidate for further research in the prevention and treatment of lipid and glucose metabolic diseases.