Are both distinct epithelial and stromal cells molecular analysis from phyllodes tumors versus fibroadenoma components affected in breast fibroepithelial progression?

Purpose: To determine molecular events involved in the tumorigenesis of phyllodes tumors (PT) and the role of each stromal (SC) and epithelial (EC) cell. Methods: Frozen breast samples enriched with epithelial and stromal cells from three fibroadenomas and 14 PT were retrieved and laser microdissected. Sanger and polymerase chain reaction-based sequencing of exon 2 MED12 and TERT promoter hotspot mutations were performed; 44K microarray platform was used to analyze gene expression. Results: All three fibroadenomas (FAs) presented mutations in MED12, but not in TERT, whose mutation was observed in five of the 14 PTs. EC and SC of each affected tumor displayed identical alterations. Of the total differentially expressed genes (DEG) (EC = 1,543 and SC = 850), 984 were EC-eDEGs and 291 were SC-eDEGs. We found a high similarity of diseases and functions enriched by both cell types, but dissimilarity in the number of enriched canonical pathways. Three signaling canonical pathways overlapping with EC and SC were predicted to be activated in one cell type and inactivated in the other, while no overlap in eDEGs was assigned to them. We also identified 13 EC-eDEGs and five SC-eDEGs enriched networks, in which the SC-eDEGs were able to segregate FA from PT samples. Conclusions: Identical TERT mutations from both SC and ES origins might affect the PTs tumorigenesis. Gene expression differences suggest coordinated molecular processes between these components with determinant differences acquired by SC, able to fully distinguish PTs from FAs lesions.

Biological function and clinical significance of long non-coding RNA LINC00342 in head and neck squamous carcinoma / 中华耳鼻咽喉头颈外科杂志

Objective: To investigate the relationship between the long-non-coding RNA LINC00342 expression and the clinicopathological parameters of head and neck squamous cell carcinoma (HNSCC) and the biological function of LINC00342 in HNSCC cells. Methods: The expression level of LINC00342 in the HNSCC was analyzed using transcriptome sequencing data from TCGA (The Cancer Genome Atlas) database, and the expressions of LINC00342 in laryngeal squamous cell carcinoma tissues (LSCC) of 27 patients in the First Hospital of Shanxi Medical University were detected by transcriptome sequencing. The expression levels of LINC00342 in human embryonic lung diploid cells 2BS, HNSCC cell lines FD-LSC-1, CAL-27 and Detroit562 were determined by real-time quantitative polymerase chain reaction (qPCR). RNAi (RNA interference) was used for LINC00342 knockdown in HNSCC cell lines, and the changes of malignant phenotype in the tumor cells after LINC00342 knockdown were examined by cell counting kit-8 (CCK-8), colony formation, flow cytometry, transwell invasion and migration assays. Bioinformatics analysis was performed to construct a LINC00342-centered competing endogenous RNA (ceRNA) regulatory network, and GO (Gene Ontology) enrichment analysis was performed. Statistical analysis and graphing were performed using SPSS 25.0 software and GraphPad Prism 6 software. Results: Mean LINC00342 levels in HNSCC tissues and TCGA database were higher than that in normal control tissues, but with no significantly statistical difference (P=0.522). LINC00342 expression levels were positively correlated with cervical lymph node metastasis and pathological grade in patients with HNSCC, with higher expression in male patients than in female patients (P<0.05). Transcriptome sequencing analysis showed that mean expression level of LINC00342 in LSCC tissues of 27 patients was significantly higher than that in the paired adjacent normal mucosa tissues (t=1.56, P=0.036). LINC00342 expression was significantly upregulated in HNSCC cell lines FD-LSC-1, CAL-27 and Detroit562 (t-values of -12.17, -23.26 and -388.57, respectively; all P<0.001). Knockdown of LINC00342 by transfecting si-LINC00342-1 and si-LINC00342-2 inhibited HNSCC cell proliferation (t-values of 8.95 and 4.84, 2.70 and 5.55, 2.02 and 3.70, respectively), colony formation (t-values of 6.66 and 6.17, 7.38 and 11.65, 4.90 and 5.79, respectively), migration (t-values of 8.21 and 7.19, 5.76 and 6.46, 6.28 and 9.92, respectively) and invasion abilities (t-values of 9.29 and 10.25, 11.30 and 11.36, 8.02 and 8.66, respectively), but promoting apoptosis in cell lines FD-LSC-1 and CAL-27 (t-values of -2.21 and -5.83, -3.05 and -5.25 respectively) (all P-values<0.05). The LINC00342-centered ceRNA network consists of 10 downregulated microRNA and 647 upregulated mRNA nodes. GO analysis results indicated that LINC00342-regulated mRNAs were enriched in 22 biological processes, 32 molecular functions, and 12 cellular components. Conclusion: High level of LINC00342 is associated with the malignant progression of HNSCC. LINC00342 promotes the proliferation, migration, invasion, and antagonizes apoptosis of HNSCC cells, which serves as a potential molecular marker in HNSCC.

Spatial and temporal expression pattern of somatostatin receptor 2 in mouse / 生物工程学报

Somatostatin (SST) is an inhibitory polypeptide hormone that plays an important role in a variety of biological processes. Somatostatin receptor 2 (SSTR2) is the most widely expressed somatostatin receptor. However, the specific cell types expressing Sstr2 in the tissues have not been investigated. In this study, we detected the expression pattern of SSTR2 protein in mouse at different development stages, including the embryonic 15.5 days and the postnatal 1, 7, 15 days as well as 3 and 6 months, by multicolour immunofluorescence analyses. We found that Sstr2 was expressed in some specific cells types of several tissues, including the neuronal cells and astrocytes in the brain, the mesenchymal cells, the hematopoietic cells, the early hematopoietic stem cells, and the B cells in the bone marrow, the macrophages, the type Ⅱ alveolar epithelial cells, and the airway ciliated cells in the lung, the epithelial cells and the neuronal cells in the intestine, the hair follicle cells, the gastric epithelial cells, the hematopoietic stem cells and the nerve fibre in the spleen, and the tubular epithelial cells in the kidney. This study identified the specific cell types expressing Sstr2 in mouse at different developmental stages, providing new insights into the physiological function of SST and SSTR2 in several cell types.

Effects of neutrophil extracellular traps during human respiratory syncytial virus infection in vitro / Efeitos das redes extracelulares dos neutrófilos durante a infecção in vitro pelo vírus sincicial respiratório

Abstract The human respiratory syncytial virus (hRSV) is the most common cause of severe lower respiratory tract diseases in young children worldwide, leading to a high number of hospitalizations and significant expenditures for health systems. Neutrophils are massively recruited to the lung tissue of patients with acute respiratory diseases. At the infection site, they release neutrophil extracellular traps (NETs) that can capture and/or inactivate different types of microorganisms, including viruses. Evidence has shown that the accumulation of NETs results in direct cytotoxic effects on endothelial and epithelial cells. Neutrophils stimulated by the hRSV-F protein generate NETs that are able to capture hRSV particles, thus reducing their transmission. However, the massive production of NETs obstructs the airways and increases disease severity. Therefore, further knowledge about the effects of NETs during hRSV infections is essential for the development of new specific and effective treatments. This study evaluated the effects of NETs on the previous or posterior contact with hRSV-infected Hep-2 cells. Hep-2 cells were infected with different hRSV multiplicity of infection (MOI 0.5 or 1.0), either before or after incubation with NETs (0.5-16 μg/mL). Infected and untreated cells showed decreased cellular viability and intense staining with trypan blue, which was accompanied by the formation of many large syncytia. Previous contact between NETs and cells did not result in a protective effect. Cells in monolayers showed a reduced number and area of syncytia, but cell death was similar in infected and non-treated cells. The addition of NETs to infected tissues maintained a similar virus-induced cell death rate and an increased syncytial area, indicating cytotoxic and deleterious damages. Our results corroborate previously reported findings that NETs contribute to the immunopathology developed by patients infected with hRSV.

Resumo O vírus sincicial respiratório humano (hRSV) é a causa mais comum de doenças graves do trato respiratório inferior em crianças pequenas em todo o mundo, resultando em grande número de hospitalizações e gastos significativos para os sistemas de saúde. Neutrófilos são recrutados em massa para o tecido pulmonar de pacientes com doenças respiratórias agudas. No local da infecção, eles liberam armadilhas extracelulares de neutrófilos (NETs) que podem capturar e/ou inativar diferentes tipos de microrganismos, incluindo vírus. Evidências demonstraram que o acúmulo de NETs resulta em efeitos citotóxicos diretos nas células endoteliais e epiteliais. Os neutrófilos estimulados pela proteína F do vírus sincicial respiratório (hRSV-F) geram NETs que são capazes de capturar partículas virais, reduzindo assim sua transmissão. No entanto, a produção maciça de NETs obstrui as vias aéreas e aumenta a gravidade da doença. Assim, um maior conhecimento sobre os efeitos das NETs durante as infecções por hRSV é essencial para o desenvolvimento de novos tratamentos específicos e eficazes. Este estudo avaliou os efeitos das NETs no contato prévio ou posterior à infecção de células Hep-2 com hRSV. As células Hep-2 foram infectadas com diferentes quantidades de hRSV (multiplicidade de infecção ou MOI 0,5 ou 1,0), antes ou após a incubação com NETs (0,5-16 μg/mL). Células infectadas e não tratadas mostraram redução da viabilidade celular e intensa coloração com azul de tripano, que foi acompanhada pela formação de sincícios numerosos e grandes. O contato prévio entre as NETs e as células não resultou em efeito protetor. As células em monocamadas mostraram um número e área de sincícios reduzidos, mas a morte celular foi semelhante àquela apresentada por células infectadas e não tratadas. A adição de NETs aos tecidos infectados manteve taxa de morte celular e formação de sincícios semelhantes àqueles induzidos pelo vírus em células não tratadas, indicando danos citotóxicos e deletérios. Nossos resultados corroboram achados relatados anteriormente de que as NETs contribuem para a imunopatologia desenvolvida por pacientes infectados com hRSV.

Clinicodemographic and computed tomography scan findings associated with thyroid gland invasion among patients with laryngeal squamous cell carcinoma

Objective@#We aim to determine the association between preoperative CT scan findings of thyroid cartilage invasion, cricoid cartilage invasion, and paraglottic space involvement with tumor extension to the thyroid gland on final histopathology among patients with laryngeal squamous cell carcinoma (SCC) in a tertiary hospital in the Philippines.@*Methods@#Patients with histopathologically-confirmed laryngeal SCC who underwent total laryngectomy with thyroidectomy while admitted at the public ward of the Department of Otorhinolaryngology, Philippine General Hospital, from January 2013 to December 2019 were included. CT scans were reviewed by 2 blinded independent radiologists. CT scan data including subsite/s involved, thyroid cartilage/cricoid cartilage erosion, paraglottic space involvement, thyroid gland involvement, and thyroid gland involvement on final histopathology were gathered. Clinicopathologic data such as age, T Stage, early tracheostomy, and time between CT scan and surgery were also collected and analyzed. Fisher’s exact test was computed for both clinicopathologic and CT scan data with a significant value having p<0.05. Cramer’s V and phi coefficient were computed for nondichotomous and dichotomous variables, respectively. Odds ratio was also computed for dichotomous variables with p<0.05 on Fisher’s exact test.@*Results@#Fifty-nine (59) men and 4 women were included, aged 43 to 81 years old. Most patients were classified as T3 (n=18) and T4 (n=41) (total=93.7%), with most having transglottic primary tumors (n=29, 46%), and only 8 (12.7%) patients having subglottic tumor involvement on final histopathology. Thyroid cartilage erosion or invasion were noted in 63.4% (n=40) of patients’ CT scans and 58.7% (n=37) of patients’ final histopathology reports. Cricoid cartilage and paraglottic space involvement were often not reported on final histopathology results. Thyroid gland involvement was only noted in the final histopathology reports of 5 patients (7.9%). Significant association between cricoid cartilage findings (p=0.032, Cramer’s V=0.318) or thyroid gland involvement on CT scan (p=0.018, Phi=0.384; OR 13 95% CI 1.797, 94.035) with thyroid gland involvement on final histopathology was noted. The rest of the variables had no significant association with thyroid gland involvement on final histopathology.@*Conclusion@#Cricoid cartilage involvement (erosion or invasion) and thyroid gland involvement on CT scan are associated with thyroid gland involvement on final histopathology in patients with laryngeal SCC. Patients with these CT scan findings may stand to benefit more from thyroidectomy to ensure good margins of resection.

Yifei Jianpi recipe improves cigarette smoke-induced inflammatory injury and mucus hypersecretion in human bronchial epithelial cells by inhibiting the TLR4/NF-κB signaling pathway / 南方医科大学学报

OBJECTIVE@#To explore the mechanism of Yifei Jianpi recipe for improving cigarette smoke- induced inflammatory injury and mucus hypersecretion in cultured human bronchial epithelial cells.@*METHODS@#Serum samples were collected from 40 SD rats treated with Yifei Jianpi recipe (n=20) or normal saline (n=20) by gavage. Cultured human bronchial epithelial 16HBE cells were stimulated with an aqueous cigarette smoke extract (CSE), followed by treatment with the collected serum at different dilutions. The optimal concentration and treatment time of CSE and the medicated serum for cell treatment were determined with CCK-8 assay. The expressions of TLR4, NF-κB, MUC5AC, MUC7, and muc8 at both the mRNA and protein levels in the treated cells were examined with RT- qPCR and Western blotting, and the effects of TLR4 gene silencing and overexpression on their expressions were assessed. The expressions of TNF-α, IL-1 β, IL-6 and IL-8 in the cells were detected using ELISA.@*RESULTS@#At the optimal concentration of 20%, treatment with the medicated serum for 24 h significantly lowered the mRNA and protein expressions of TLR4, NF- κB, MUC5AC, MUC7, and MUC8 in CSE- exposed 16HBE cells, and these effects were further enhanced by TLR4 silencing in the cells. In 16HBE cells with TLR4 overexpression, the expressions of TLR4, NF-κB, MUC5AC, MUC7, and MUC8 were significantly increased after CSE exposure and were lowered following treatment with the medicated serum (P < 0.05). The medicated serum also significantly lowered the levels of TNF-α, IL-1β, IL-6 and IL-8 in CSE-exposed 16HBE cells (P < 0.05).@*CONCLUSIONS@#In the 16HBE cell model of chronic obstructive pulmonary disease (COPD), treatment with Yifei Jianpi recipe-medicated serum improves inflammation and mucus hypersecretion possibly by reducing MUC secretion and inhibiting the TLR4/NF-κB signaling pathway.

Dermatophagoides farinae induces conjunctival epithelial cell damage to promote neutrophil migration and neutrophil extracellular traps formation / 中国血吸虫病防治杂志

OBJECTIVE@#To investigate the mechanisms underlying allergic conjunctivitis caused by conjunctival epithelial cell damage, neutrophil migration and neutrophil extracellular traps (NETs) formation induced by crude extracts of Dermatophagoides farinae mite (CDM).@*METHODS@#Human conjunctival epithelial cells were stimulated with 500, 1 000, 2 000, 4 000 ng/mL, and the expression levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and IL-8 were detected using quantitative real-time PCR (qPCR) assay and enzyme-linked immunosorbent assay (ELISA). The culture supernatant of human conjunctival epithelial cells was collected and co-cultured with neutrophils. Neutrophil migration was measured using Transwell migration assay, and the expression of NETs markers myeloperoxidase (MPO) and citrullinated histone H3 (CitH3) was quantified using immunofluorescence staining. Neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA), and then NETs were collected for treatment of human conjunctival epithelial cells. Cell apoptosis was detected using flow cytometry, and the levels of IL-6, TNF-α, IFN-γ and IL-8 were measured in the cell culture supernatant using ELISA.@*RESULTS@#Treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL up-regulated IL-6, TNF-α, IFN-γ and IL-8 expression in human conjunctival epithelial cells. Following treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL, the culture supernatant of human conjunctival epithelial cells promoted neutrophil migration and induced increases in the staining intensity of MPO and CitH3. In addition, increased NETs triggered the apoptosis of human conjunctival epithelial cells and IL-6, TNF-α, IFN-γ and IL-8 secretion in the culture supernatant of human conjunctival epithelial cells.@*CONCLUSIONS@#CDM induces human conjunctival epithelial cell damages, thereby promoting neutrophil migration and NETs formation, while the release of NETs further aggravates human conjunctival epithelial cell damages.

The role of Nrf2 in the alteration of tight junction protein expression in choroid plexus epithelial cells created by lanthanum-activated MMP9 / 中华劳动卫生职业病杂志

Objective: To investigate the effect of nuclear factor erythroid 2-related factor 2 (Nrf2) in the alteration of tight junction protein expression in choroid plexus epithelial cells created by lanthanum-activated matrix metalloproteinase 9 (MMP9) . Methods: In October 2020, immortalized rat choroid plexus epithelial cell line (Z310) cells were used as the blood-cerebrospinal fluid barrier in vitro, and were divided into control group and 0.125, 0.25, 0.5 mmol/L lanthanum chloride (LaCl(3)) treatment group. After treating Z310 cells with different concentrations of LaCl(3) for 24 hours, the morphological changes of Z310 cells were observed under inverted microscope, the protein expression levels of MMP9, occludin and zonula occludens-1 (ZO-1) were observed by cellular immunofluorescence method, and the protein expression levels of MMP9, tissue inhibitors of metalloproteinase1 (TIMP1) , occludin, ZO-1 and Nrf2 were detected by Western blotting. The level of reactive oxygen species (ROS) in cells was detected by flow cytometry. Results: Compared with the control group, Z310 cells in the LaCl(3) treatment group were smaller in size, with fewer intercellular junctions, and more dead cells and cell fragments. The expression level of MMP9 protein in cells treated with 0.25 and 0.5 mmol/L LaCl(3) was significantly higher than that in the control group (P<0.05) , and the expression level of TIMP1 and tight junction proteins occudin and ZO-1 was significantly lower than that in the control group (P<0.05) . Compared with the control group, the ROS production level in the 0.25, 0.5 mmol/L LaCl(3) treatment group was significantly increased (P<0.05) , and the Nrf2 protein expression level in the 0.125, 0.25, 0.5 mmol/L LaCl(3) treatment group was significantly decreased (P<0.05) . Conclusion: Lanthanum may increase the level of ROS in cells by down regulating the expression of Nrf2, thus activating MMP9 to reduce the expression level of intercellular tight junction proteins occludin and ZO-1.

Effect of sulfur fumigation on quality and safety of Lilii Bulbus / 中国中药杂志

Lilii Bulbus is a commonly used Chinese herbal medicine with both medicinal and edible values, while the market products usually has the problem of sulfur fumigation. Therefore, the quality and safety of Lilii Bulbus products deserve attention. In this study, ultra-high performance liquid chromatography-time of flight-tandem mass spectrometry(UPLC-Q-TOF-MS/MS) was combined with principal component analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA) to analyze the differential components of Lilii Bulbus before and after sulfur fumigation. We identified ten markers generated after sulfur fumigation, summarized their mass fragmentation and transformation patterns, and verified the structures of phenylacrylic acid markers of sulfur fumigation. At the same time, the cytotoxicity of the aqueous extracts of Lilii Bulbus before and after sulfur fumigation was evaluated. The results showed that in the concentration range of 0-800 mg·L~(-1), the aqueous extract of Lilii Bulbus after sulfur fumigation had no significant effect on the viability of human liver LO2 cells, human renal proximal tubular HK-2 cells, and rat adrenal pheochromocytoma PC-12 cells. Moreover, the viability of the cells exposed to the aqueous extract of Lilii Bulbus before and after sulfur fumigation showed no significant difference. This study identified phenylacrylic acid and furostanol saponins as markers of sulfur-fumigated Lilii Bulbus for the first time, and made clear that proper sulfur fumigation of Lilii Bulbus would not produce cytotoxicity, providing a theoretical basis for the rapid identification and quality and safety control of sulfur-fumigated Lilii Bulbus.

Development of an olfactory epithelial organoid culture system based on small molecule screening / 生物工程学报

Olfactory epithelium, which detects and transmits odor signals, is critical for the function of olfactory system. Olfactory epithelium is able to recover spontaneously after injury under normal circumstances, but this ability is dampened in certain diseases or senility, which causes olfactory dysfunction. The olfactory epithelium consists of basal cells, sustentacular cells and olfactory sensory neurons. In order to develop an olfactory epithelial organoid containing multiple olfactory cell types in vitro, we used three-dimensional culture model and small molecules screening. This organoid system consists of horizontal basal-like cells, globose basal-like cells, sustentacular-like cells and olfactory sensory neurons-like cells. Through statistical analysis of clone diameter, immunofluorescence staining and qPCR detection of the expression level of related marker genes. We identified a series of growth factors and small molecule compounds that affected the proliferation, composition and gene expression of the organoids. CHIR-99021, an activator of Wnt signaling pathway, increased the colony formation and proliferation rate of olfactory epithelial organoids and the expression level of marker genes of olfactory sensory neurons-like cells. In addition, each factor in the culture system increased the proportion of c-Kit-positive globose basal-like cell colonies in organoids. Moreover, EGF and vitamin C were both beneficial to the expression of horizontal basal-like cell marker genes in organoids. The established olfactory epithelial organoid system mimicked the process of olfactory epithelial stem cells differentiating into various olfactory epithelial cell types, thus providing a research model for studying olfactory epithelial tissue regeneration, the pathological mechanism of olfactory dysfunction and drug screening for olfactory dysfunction treatment.

Dickkopf-1 inhibits the secretion of MUC5AC induced by Mycoplasma pneumoniae P1-C in mouse lung epithelial cells / 生物工程学报

Mycoplasma pneumoniae is the most common pathogen of respiratory tract infection in children and adults. Clinical observation shows that M. pneumoniae infection can cause massive mucus secretion in the respiratory tract, which makes the breathing of patients difficult. Studies have shown that M. pneumoniae infection can cause massive secretion of mucin 5AC (MUC5AC). Adhesin P1 plays an important role in the pathogenesis of M. pneumoniae infection by mediating the adhesion of pathogens to host cells, and the C-terminal residues of P1 (P1-C) are immunogenic. This study investigated the molecular mechanism of Wnt/β-catenin signaling pathway inhibitor Dickkopf-1 (DKK1) in the secretion of MUC5AC in mouse airway epithelial cells (MAECs) induced by P1-C. Scanning electron microscope and hematoxylin-eosin staining were used to observe the effect of P1-C on mucus secretion of MAECs. Protein chip was used to detect the secretion of cytokines and analyse the enrichment of related signaling pathways induced by P1-C in MAECs. Periodic acid schiff stain (PAS) staining, Tunel staining and Masson staining were used to detect the damage of the lungs of mouse exposed to P1-C. Immunohistochemistry was used to detect the secretion of MUC5AC expression, and Western blotting was used to reveal the molecular mechanism of DKK1-regulated secretion of MUC5AC induced by P1-C protein in MACES. The results showed that P1-C induced the massive secretion of mucus and inflammatory factors in MAECs. During P1-C infection, DKK1 down-regulated janus kinase 2 (JAK2), phosphorylation signaling and transcription activator 1 (p-STAT1) and phosphorylation signaling and activator of transcription 3 (p-STAT3) expression. Overexpression of DKK1 significantly up-regulated the expression of MUC5AC repressor transcription factor fork-head box protein A2 (FOXA2). At the same time, the expression of MUC5AC induced by P1-C was inhibited significantly. It is speculated that DKK1 can effectively reduce the secretion of MUC5AC in MAECs induced by P1-C by inhibiting the JAK/STAT1-STAT3 signaling pathway and up-regulating the expression of FOXA2.

Giant myoepithelial carcinoma of the nuchal region: a case report and literature review / 临床耳鼻咽喉头颈外科杂志

Myoepithelioma, also known as malignant myoepithelioma, is a rare malignant tumor originating from myoepithelial cell. This article reports a patient with a huge tumor in the neck and left elbow who underwent fine needle aspiration under local anesthesia. The pathological diagnosis was a myoepithelioma. Under general anesthesia, giant tumors in the lower neck, posterior cranial fossa, neck, and left elbow were removed, and postoperative pathology showed that they were all myoepithelial tumors. Immunohistochemistry showed AE1/AE3 (+), P63 (+), CK7 (+), CK5 (+), and CD138 (+). The clinical characteristics and diagnosis and treatment process of this case are reported and relevant literature is reviewed.

Progress in the relationship between head and neck squamous cell carcinom and the microbial community / 临床耳鼻咽喉头颈外科杂志

Microorganisms are one of the important factors which maintain the homeostasis of human health. Despite recent advances, the relationship between microorganisms and head and neck squamous cell carcinoma （HNSCC） is still unclear, and the impact of microorganisms on the incidence and prognosis of HNSCC cannot be neglected. Therefore, this article provides a systematic and comprehensive review summarizing the epidemiological evidence of microbial dysbiosis related to HNSCC and discusses the associations between them.

Central Granular Cell Odontogenic Tumor: A Literature Review of Cases Reported in the Last 71 Years with a New Case Report / 中国医学科学杂志(英文版)

Central granular cell odontogenic tumors (CGCOTs) are rare, benign, slowly growing odontogenic neoplasms. Due to their uncertain histogenesis, CGCOTs are still not included as a distinct entity in the WHO classification (2017) of odontogenic tumors. We report a case of CGCOT involving the right side of maxillary anterior region of a 39-year-old white female. Immunohistochemical staining showed that granular cells positively expressed CD68 and vimentin, and negatively expressed S-100 protein. Meanwhile, we searched PubMed, Google Scholar, and Scopus databases to summary the clinico-pathological features of 51 reported cases of CGCOT. The results showed that the granular cells of 28.6% cases were immunopositive for vimentin and CD68, and odontogenic epithelial cells were positive immunoreactivity for cytokeratin. These findings reinforced the mesenchymal origin of granular cells and the odontogenic nature of epithelium islands.

Mechanism of gigantol in transmembrane transport in human lens epithelial cells / 中国中药杂志

Gigantol is a phenolic component of precious Chinese medicine Dendrobii Caulis, which has many pharmacological activities such as prevent tumor and diabetic cataract. This paper aimed to investigate the molecular mechanism of gigantol in transmembrane transport in human lens epithelial cells(HLECs). Immortalized HLECs were cultured in vitro and inoculated in the laser scanning confocal microscopy(LSCM) medium at 5 000 cells/mL. The fluorescence distribution and intensity of gigantol marked by fluorescence in HLECs were observed by LSCM, and the absorption and distribution of gigantol were expressed as fluorescence intensity. The transmembrane transport process of gigantol in HLECs were monitored. The effects of time, temperature, concentration, transport inhibitors, and different cell lines on the transmembrane absorption and transport of gigantol were compared. HLECs were inoculated on climbing plates of 6-well culture plates, and the ultrastructure of HLECs was detected by atomic force microscopy(AFM) during the transmembrane absorption of non-fluorescent labeled gigantol. The results showed that the transmembrane absorption of gigantol was in time and concentration-dependent manners, which was also able to specifically target HLECs. Energy and carrier transport inhibitors reduced gigantol absorption by HLECs. During transmembrane process of gigantol, the membrane surface of HLECs became rougher and presented different degrees of pits, indicating that the transmembrane transport of gigantol was achieved by active absorption of energy and carrier-mediated endocytosis.

Leonurine inhibits ferroptosis in renal tubular epithelial cells by activating p62/Nrf2/HO-1 signaling pathway / 中国中药杂志

To investigate the protective effect and the potential mechanism of leonurine(Leo) against erastin-induced ferroptosis in human renal tubular epithelial cells(HK-2 cells), an in vitro erastin-induced ferroptosis model was constructed to detect the cell viability as well as the expressions of ferroptosis-related indexes and signaling pathway-related proteins. HK-2 cells were cultured in vitro, and the effects of Leo on the viability of HK-2 cells at 10, 20, 40, 60, 80 and 100 μmol·L~(-1) were examined by CCK-8 assay to determine the safe dose range of Leo administration. A ferroptosis cell model was induced by erastin, a common ferroptosis inducer, and the appropriate concentrations were screened. CCK-8 assay was used to detect the effects of Leo(20, 40, 80 μmol·L~(-1)) and positive drug ferrostatin-1(Fer-1, 1, 2 μmol·L~(-1)) on the viability of ferroptosis model cells, and the changes of cell morphology were observed by phase contrast microscopy. Then, the optimal concentration of Leo was obtained by Western blot for nuclear factor erythroid 2-related factor 2(Nrf2) activation, and transmission electron microscope was further used to detect the characteristic microscopic morphological changes during ferroptosis. Flow cytometry was performed to detect reactive oxygen species(ROS), and the level of glutathione(GSH) was measured using a GSH assay kit. The expressions of glutathione peroxidase 4(GPX4), p62, and heme oxygenase 1(HO-1) in each group were quantified by Western blot. RESULTS:: showed that Leo had no side effects on the viability of normal HK-2 cells in the concentration range of 10-100 μmol·L~(-1). The viability of HK-2 cells decreased as the concentration of erastin increased, and 5 μmol·L~(-1) erastin significantly induced ferroptosis in the cells. Compared with the model group, Leo dose-dependently increased cell via-bility and improved cell morphology, and 80 μmol·L~(-1) Leo promoted the translocation of Nrf2 from the cytoplasm to the nucleus. Further studies revealed that Leo remarkably alleviated the characteristic microstructural damage of ferroptosis cells caused by erastin, inhibited the release of intracellular ROS, elevated GSH and GPX4, promoted the nuclear translocation of Nrf2, and significantly upregulated the expression of p62 and HO-1 proteins. In conclusion, Leo exerted a protective effect on erastin-induced ferroptosis in HK-2 cells, which might be associated with its anti-oxidative stress by activating p62/Nrf2/HO-1 signaling pathway.

Selective photodynamic effects on cervical adenocarcinoma cells provided by F127 Pluronic®-based micelles modulating hypericin delivery

Abstract Cervical cancer is a leading cause of death among women. The endocervical adenocarcinoma (ECA) represents an aggressive and metastatic type of cancer with no effective treatment options currently available. We evaluated the antitumoral and anti-migratory effects of hypericin (HYP) encapsulated on Pluronic F127 (F127/HYP) photodynamic therapy (PDT) against a human cell line derived from invasive cervical adenocarcinoma (HeLa) compared to a human epithelial cell line (HaCaT). The phototoxicity and cytotoxicity of F127/HYP were evaluated by the following assays: colorimetric assay, MTT, cellular morphological changes by microscopy and long-term cytotoxicity by clonogenic assay. In addition, we performed fluorescence microscopy to analyze cell uptake and subcellular distribution of F127/HYP, cell death pathway and reactive oxygen species (ROS) production. The PDT mechanism was determined with sodium azide and D-mannitol and cell migration by wound-healing assay. The treatment with F127/HYP promoted a phototoxic result in the HeLa cells in a dose-dependent and selective form. Internalization of F127/HYP was observed mainly in the mitochondria, causing cell death by necrosis and ROS production especially by the type II PDT mechanism. Furthermore, F127/HYP reduced the long-term proliferation and migration capacity of HeLa cells. Overall, our results indicate a potentially application of F127/HYP micelles as a novel approach for PDT with HYP delivery to more specifically treat ECA.

Mutagenicity in oral cells of individuals exposed to radiofrequency generated by different smartphones

Aim: This study aimed to investigate whether non-ionizing radiation emitted by smartphones is likely to cause genotoxic effects on oral epithelial cells. Methods: Thirty adults were distributed into two groups according to the mobile phone brand used, namely Samsung (Samsung, Seoul, South Korea) and Apple (Apple, California, USA). The material was collected with gentle swabbing of the right and left buccal mucosa using a cervical brush, then the micronucleus test was performed. Results: The Mann-Whitney test with a 5% significance level did not reveal statistically significant differences in micronuclei frequency between the exposed and non-exposed sides (p=0.251). The different brands do not seem to cause risks of inducing genetic damage because there were no statistically significant differences between them (p=0.47). Conclusion: Therefore, our results suggest no correlations of micronuclei frequency in the exposed buccal cells of mobile phone users at the exposure standard levels observed

Aislación y caracterización de las células epiteliales del amnios / Isolation and characterization of amniotic ephitelial cells

RESUMEN: Las células epiteliales del amnios (hAECs) son células madre pluripotenciales; tienen capacidad de diferenciarse en células de las tres capas embrionarias. Como tales, se utilizan en algunas terapias regenerativas en medicina. Este estudio tiene por objetivo describir un protocolo de aislación de las células epiteliales del amnios (hAECs) a partir de placentas humanas de partos por cesárea, así como su caracterización y comportamiento in vitro. Se aislaron hAECs de 20 placentas de partos por cesárea con un protocolo optimizado. Se caracterizaron las células mediante citometría de flujo, microscopia óptica y de fluorescencia, y se evaluó la proliferación de las células mediante MTT a los 1, 3, 5 y 7 días con y sin β-mercaptoetanol en el medio de cultivo. El análisis histológico del amnios mostró un desprendimiento prácticamente completo de las células después de la segunda digestión del amnios. El promedio de células obtenidas fue de 10.97 millones de células por gramo de amnios. Las hAECs mostraron una proliferación limitada, la cual no fue favorecida por la adición de β-mercaptoetanol en el cultivo. Se observó un cambio de morfología espontanea de epitelial a mesenquimal después del cuarto pasaje. Las células epiteliales del amnios pueden ser aisladas con un protocolo simple y efectivo, sin embargo, presentan escasa capacidad proliferativa. Bajo las condiciones de este estudio, la adición de β-mercaptoetanol no favorece la capacidad proliferativa de las células.

SUMMARY: human amnion epithelial cells (hAECs) are pluripotent stem cells; they have the ability to differentiate into cells of the three embryonic layers, and are used in various regenerative therapies in medicine. This study aims to describe a protocol for the isolation of amnion epithelial cells (hAECs) from human placentas from cesarean delivery, as well as their characterization and culture conditions in vitro. hAECs were isolated from 20 cesarean delivery placentas with an optimized protocol. The cells were characterized by flow cytometry, light and fluorescence microscopy, and the proliferation of the cells was evaluated by MTT at 1, 3, 5 and 7 days with and without β-mercaptoethanol in the culture medium. Histological analysis of the amnion showed a practically complete detachment of the cells of the underlying membrane after the second digestion. The average number of cells obtained was 10.97 million cells per amnion. The hAECs perform a limited proliferation rate, which was not favored by the addition of β-mercaptoethanol in the culture. A spontaneous morphology change from epithelial to mesenchymal morphology is exhibited after the fourth passage. The epithelial cells of the amnion can be isolated with a simple and effective protocol, however, they present little proliferative capacity. Under the conditions of this study, the addition of β-mercaptoethanol does not favor the proliferation of the cells.

Preventing Uterine Cervix Cancer: The Clinical Meaning of Atypical Glandular Cells / Prevenção do câncer do colo uterino: Significado clínico das células glandulares atípicas

Abstract Objective To determine the prevalence of the atypical glandular cells (AGCs) cytology and to analyze its clinical significance in different age ranges. Methods Retrospective observational study using computerized data from the Brazilian National Cancer Institute, including women screened between January 2002 and December 2008. The women included were those with an AGC result who were properly followed-up with colposcopy and a second cytology. Results A total of 132,147 cytopathological exams were performed during the study period. Five-hundred and thirty-three (0.4%) women with AGC cytology were identified and, of these, 69.41% (370/533) were properly referred for colposcopy and a new cytology. Most of the women (79.2%) with a 1st or 2nd AGC cytology were between the ages of 25 and 54 years. The 2nd cytology demonstrated 67.6% (250/370) of normality, 24.5% (91/370) of squamous atypia, and 6.2% (23/370) of AGC, 0.8% (3/370) adenocarcinoma in situ and 0.8% (3/370) adenocarcinoma invasor. On biopsy of the women with a second AGC cytology, 43.4% (10/23) had normal histology, 43.4% (10/23) had squamous lesions, 8.7% (2/23) had invasive adenocarcinoma, and 1.2% (1/23) had an inconclusive report. All of the women with high-grade squamous intraepithelial lesion (HSIL) or invasive adenocarcinoma (respectively 5 and 2 patients), after a 2nd AGC cytology were 25 years old or older. Conclusion The prevalence of the AGC cytology was low in the studied population. Most of the AGC cytology cases occurred in adult women between the ages of 25 and 54. Although most of the patients had normal histology after follow-up, several of them presented with squamous intraepithelial lesions or invasive adenocarcinoma.

Resumo Objetivo Determinar a prevalência de citologia com laudo de células glandulares atípicas (AGCs, na sigla em inglês) e analisar a significância clínica nas diferentes faixas etárias Métodos Estudo observacional retrospectivo, usando os dados arquivados no sistema do Instituto Nacional de Câncer no Brasil, que incluiu mulheres rastreadas entre janeiro de 2002 a dezembro de 2008. As mulheres incluídas tinham citologia com resultado de AGCs, que foram acompanhadas com colposcopia e nova citologia Resultados Um total de132,147 exames citopatológicos foram incluídos durante o período de estudo. Quinhentas e trinta e três mulheres com citologia de AGC foram identificadas e destas, 69.41% (370) foram encaminhadas para colposcopia e nova citologia. A prevalência de citologia de AGC na população estudada foi 0.4%. A maioria das mulheres (79.22%) com resultado citológico de AGC tinham idade entre 25 e 54 anos. A segunda citologia demonstrou 67.56% (250/370) de normalidade, 24.5% (91/370) de atipias escamosas, e 6.2% (23/370) de AGC. Na biopsia das mulheres com a 2ª citologia de AGC, 43.4% (10/23) tinham histologia normal, 43.4% (10/23) tinha lesões escamosas, 8.7% (2/23) tinha adenocarcinoma invasor e 1.2% (1/23) tinha laudo inconclusivo. Todas as mulheres com lesões intraepiteliais escamosas de alto grau (HSIL, na sigla em inglês) ou adenocarcinoma invasor (respectivamente 5 e 2pacientes), após a 2ª citologia com AGC, tinham 25 anos de idade ou mais. Conclusão A prevalência de citologia com AGC foi baixa na população estudada. Muitos casos de citologia com AGC apareceram em mulheres adultas, entre 25 e 54 anos de idade. Embora a maioria das pacientes tiveram histologia normal após seguimento, várias apresentaram lesões intraepiteliais escamosas ou glandulares invasoras.