Clinicopathological and molecular genetic analysis of 13 cases of primary retroperitoneal Ewing sarcoma.

Retroperitoneal Ewing sarcomas (RES) are very rare and mostly described in case reports. The purpose of this study was to retrospectively analyze the clinicopathology, molecular characteristics, biological behavior, and therapeutic information of 13 cases of primary RES with immunohistochemical staining, fluorescence in situ hybridization, RT-PCR and NGS sequencing detection techniques. The thirteen patients included eight males and five females with a mean age of 34 years. Morphologically, the tumors were comprised of small round or epithelial-like cells with vacuolated cytoplasm (6/13,46 %) arranged in diffuse, nested (8/13,62 %) and perivascular (7/13,54 %) patterns. Unusual morphologic patterns, such as meningioma-like swirling structures and sieve-like structures were relatively novel findings. Immunohistochemical studies showed CD99 (12/13; 92 %), CD56 (11/13; 85 %), NKX2.2 (9/13; 69 %), PAX7 (10/11;91 %) and CD117(6/9;67 %) to be positive.12 cases (92 %) demonstrated EWSR1 rearrangement and 3 cases displayed EWSR1::FLI1 fusion by FISH. ERCC4 splice-site variant, a novel pathogenic variant, was discovered for the first time via RNA sequencing. With a median follow-up duration of 14 months (6 to 79 months), 8/13 (62 %) patients died, while 5/13(38 %) survived. Three cases recurred, and five patients developed metastasis to the liver (2 cases), lung (2 cases) and bone (1 case). RES is an aggressive, high-grade tumor, prone to multiple recurrences and metastases, with distinctive morphologic, immunohistochemical, and molecular genetic features. ERCC4 splicing mutation, which is a novel pathogenic variant discovered for the first time, with possible significance for understanding the disease, as well as the development of targeted drugs.

Morphological and immunohistochemical characteristics associated with metastatic and recurrent progression in pheochromocytoma/paraganglioma: A cohort study.

This study aimed to evaluate immunohistochemical markers of pheochromocytoma/paraganglioma (PPGL) and the relationships between the grading system for adrenal pheochromocytoma and paraganglioma (GAPP) and the prognosis of PPGL in a Chinese population. A retrospective analysis was conducted on a cohort of 102 PPGL cases, from January 2012 to December 2019, with complete clinicopathological and follow-up data. Surgical pathology slides were re-reviewed. All histological parameters involved in GAPP were summarized. The relationship between clinical characteristics, expression of SDHB (succinate dehydrogenase), S-100 and Ki-67 as well as GAPP classifications and prognosis of PPGL was statistically analyzed. The 102 cases included 51 males (50%) and 51 females (50%) with a median age of 48.7 years. The median tumor size was 6.8 cm. Metastases or relapse developed in 23 (22.5%) cases. Larger tumor size, extra-adrenal location, and poorly differentiated PPGL according to GAPP were associated with metastases or relapse (P < 0.05). Histological parameters, including the appearance of large or fused cell nests, necrosis, vascular invasion, and capsular invasion, were more common in the cases with metastases or relapse (P < 0.01). Loss of SDHB or S-100 expression was more common in poorly differentiated PPGL and associated with metastases or relapse (P < 0.01). However, no significant difference in the Ki-67 index between the clinically malignant and benign group was observed. GAPP is thus helpful for evaluations of the biological behavior of PPGL.

H2 O2 induces PP2A demethylation to downregulate mTORC1 signaling in HEK293 cells.

Mammalian target of rapamycin (mTOR) is a Ser/Thr protein kinase that functions as an ATP and amino acid sensor to govern cell growth and proliferation by mediating mitogen- and nutrient-dependent signal transduction. Protein phosphatase 2A (PP2A), a ubiquitously expressed serine/threonine phosphatase, negatively regulates mTOR signaling. Methylation of PP2A is catalyzed by leucine carboxyl methyltransferase-1 (LCMT1) and reversed by protein phosphatase methylesterase 1 (PME-1), which regulates PP2A activity and substrate specificity. However, whether PP2A methylation is related to mTOR signaling is still unknown. In this study, we examined the effect of PP2A methylation on mTOR signaling in HEK293 cells under oxidative stress. Our results show that oxidative stress induces PP2A demethylation and inhibits the mTORC1 signaling pathway. Next, we examined two strategies to block PP2A demethylation under oxidative stress. One strategy was to prevent PP2A demethylation using a PME-1 inhibitor; the other strategy was to activate PP2A methylation via overexpression of LCMT1. The results show that both the PME-1 inhibitor and LCMT1 overexpression prevent the mTORC1 signaling suppression induced by oxidative stress. Additionally, LCMT1 overexpression rescued cell viability and the mitochondrial membrane potential decrease in response to oxidative stress. These results demonstrate that H2 O2 induces PP2A demethylation to downregulate mTORC1 signaling. These findings provide a novel mechanism for the regulation of PP2A demethylation and mTORC1 signaling under oxidative stress.

[Significance of CD123 in histiocytic necrotizing lymphadenitis].

OBJECTIVE: To investigate the mode of presentation, cytologic features of the plasmacytoid dendritic cells (pDC), and the expression of CD123 and its significance in Kikuchi's disease. METHODS: CD123 expression was evaluated by EliVision immunohistochemical staining in formalin-fixed and paraffin-embedded tissues from 30 cases of Kikuchi's disease, 5 cases of T cell lymphoma, 10 cases of reactive lymphoid hyperplasia and 10 cases of chronic tonsillitis. RESULTS: Clusters of CD123 positive PDC were observed in Kikuchi's disease (28 of 30 cases, 93.3%) and the staining intensity was more prominent in the PDC at the periphery of the lesion and around the high endothelial venule-like vessels. CD123 showed three staining patterns: membranous (10 cases, 33.3%), cytoplasmic (10 cases, 33.3%), and membranous and cytoplasmic (8 cases, 26.7%). In the control group, CD123 showed cytoplasmic staining in reactive hyperplasia and chronic tonsillitis. Regarding the staining intensity, 12 of 28 cases (42.9%) were 3+ for CD123, 8 of 28 cases (28.6%) were 2+, and 8 of 28 cases (28.6%) were 1+. In contrast, PDC clusters with 1+ staining intensity were observed in 1 of 10 cases of reactive lymphoid hyperplasia; 2 of 10 chronic tonsillitis diseases; and much less in T cell lymphoma. CONCLUSIONS: Large cluster of PDC is detected in both proliferative and necrotizing types of Kikuchi's disease, making this a useful adjunctive diagnostic marker.

Solar-driven water evaporation using a collaborative photothermal conversion material system based on carbonized waste polyphenylene sulfide and copper sulfide.

Recently, water resources have become scarce due to the growing global population and human impact on the environment, coupled with the effects of climate change. For solving the problem of global freshwater shortage and increasing the value of discarded polyphenylene sulfide (PPS) filter bags, in this study, balsa wood was used as the base of a photothermal solar evaporator, chitosan solution was used as the binder, and the main photothermal conversion materials used were polyphenylene sulfide (CP) carbide and copper sulfide. In order to create synergistic photothermal conversion materials, freeze-drying and in situ precipitation were used to deposit the photothermal conversion materials on top of the balsa wood. The prepared CP/CuS-wood evaporator has excellent water evaporation performance and light conversion capability, with a water evaporation rate of 2.68 kg m-2 h-1 and a photothermal conversion efficiency of 93.2% under simulated one solar intensity irradiation. In addition, the evaporator can effectively remove organic dyes such as methylene blue and methyl orange. The evaporator's durability and seawater desalination capability have also been confirmed through seawater desalination experiments and outdoor tests. Studies have shown that solar interface photothermal evaporators are a viable solution for desalination and wastewater treatment. This eco-friendly, economically viable and stable photothermal evaporator mentioned in this paper has pioneering features and will be a new paradigm for desalination and wastewater treatment.

p53 aberration and TFE3 gene amplification may be predictors of adverse prognosis in epithelioid angiomyolipoma of the kidney.

BACKGROUND: Although epithelioid angiomyolipoma of the kidney has been studied by several groups, the reported prevalence of malignant behavior remains uncertain and there are not yet definitive predictive biomarkers. We evaluated the behavior of renal epithelioid angiomyolipoma in a consecutive series in a single institution and investigated the prognostic value of aberrant p53 expression and TFE3 gene abnormality. METHODS: We retrospectively reviewed 14 epithelioid angiomyolipomas, most with pure or close to pure epithelioid components, comprising 12 consecutive cases who had attended our institution and two consultation cases. Fluorescence in situ hybridization with TFE3 break-apart probe was performed on 14 cases. The 14 cases were also labeled for p53 and TFE3 by immunohistochemistry. All cases were followed up. RESULTS: Three of the epithelioid angiomyolipomas were strongly positive for TFE3 and two had a mutant expression of p53. Although no TFE3 gene rearrangement was found, the two tumors with strong TFE3 expression showed TFE3 gene amplification. Follow-up details were available for seven of the 12 consecutive cases: two of them had developed metastases and died (29%), their mean overall survival was 41 months, and both had mutant p53 expression. The two consultation cases with TFE3 gene amplification developed recurrence/metastasis within 1 year after surgery. CONCLUSIONS: Our series study from a single institution presented the prevalence of malignant behavior in pure epithelioid angiomyolipomas, although the small number of cases with follow-up data greatly reduced the accuracy. p53 may be a prognostic marker for epithelioid angiomyolipoma. Cases with TFE3 gene amplification had poor prognoses.

[Clinicopathologic analysis of intestinal follicular lymphoma first presenting with gastrointestinal symptoms].

OBJECTIVE: To study the clinical features, endoscopic findings, pathologic diagnosis and treatment options of intestinal follicular lymphoma first presenting with gastrointestinal symptoms. METHODS: The clinical features, pathologic findings and follow-up data were retrospectively studied in 9 cases of intestinal follicular lymphoma. Immunohistochemical study for CD3, CD5, CD20, CD21, Ki-67, bcl-2, bcl-6, CD10 and cyclin D1 was carried out. RESULTS: Seven of the 9 patients were females and two were males. The age of patients ranged from 5 to 60 years (mean = 44 years). The clinical manifestations included abdominal pain (5 cases), blood in stool (3 cases) and abdominal distension (1 case). The commonest site of involvement was ileocecal region (6/9). Endoscopic examination had been carried out in 6 patients and all showed the presence of multiple polyps. Five cases had undergone endoscopic biopsy. Histologic examination of the endoscopic biopsies showed lymphoma cells located mainly in mucosal layer, forming vague nodules with ill-defined boundaries. Plasma cells and eosinophils were not conspicuous. Immunohistochemically, the tumor cells in all cases diffusely expressed CD20, CD10 and bcl-2. The staining for CD3, CD5 and cyclin D1 was negative. Lymphoid cells with weak CD10-positivity were identified in the interfollicular regions. Four cases were treated with surgical resection and chemotherapy. The other 3 cases received chemotherapy only and the remaining cases were treated conservatively. All of them were still alive on follow up. CONCLUSIONS: Primary intestinal follicular lymphoma affects predominantly elderly patients and has a female predilection. The commonest site of involvement is ileocecal region. Endoscopic examination shows polypoid changes. The disease often runs a relatively indolent clinical course. The prognosis is better than that of primary nodal follicular lymphoma.

Case report: NUT carcinoma in an elderly woman with unique morphology and immunophenotype highlights a diagnostic pitfall.

Background: NUT carcinoma (NC) is a rapidly progressing and rare neoplasm that primarily affects younger patients and has a survival time of about 1 year. Most of these neoplasms express epithelial markers with no neuroendocrine markers observed. Retrospective studies have shown that pathologists and clinicians do not have a sufficient understanding of the disease due to the lack of common clinical manifestations, imaging, and morphological features. Case Description: A 60-year-old female presented at Beijing Friendship Hospital, showing repetitive epistaxis, nasal pain, and nasal congestion with obvious masses in the right nasal sinus and frontal sinus. Microscope analysis revealed two unique morphological changes which have not been previously reported in the existing literature: (I) small spindle cells with sparse cytoplasm and densely stained nuclei and (II) large tumor cells with abundant cytoplasm, some cells resembling plasma cells. The sudden appearance of keratinization was also a prominent feature. Immunohistochemical staining showed differences between the two cell morphologies. Small spindle cells simultaneously expressed CK5/6 and P40, and the Ki67 proliferation index was 40%. The large round cells did not express CK5/6 and P40 but were focal positive for synaptophysin and the Ki67 index was 10%. NUT and P63 were strongly expressed in both cell types and fluorescence in situ hybridization (FISH) revealed BRD4-NUTM1 translocation. Following 20 rounds of postoperative radiation treatment, the patient was alive and no recurrence or metastasis was observed during a 5-month follow-up. Conclusions: We present novel information from the oldest known and surviving patient of NC originating in the nasal cavity with unique morphological features and different immunohistochemical results. NUT antibody testing should be performed in undifferentiated or poorly differentiated malignancies, particularly those with either or both cytoplasmic vacuolation of medium-sized cells and abrupt keratinization, irrespective of patient age.

High-grade B-cell lymphoma with gastroduodenal involvement showing paraneoplastic cerebellar degeneration: a case report.

Background: Paraneoplastic cerebellar degeneration (PCD), which displays ataxia and other cerebellar symptoms, is the most common paraneoplastic neurological syndrome (PNS). PCD is more likely to occur in individuals with small cell lung cancer (SCLC), gynecological malignancies, and Hodgkin disease, but it is rarely associated with non-Hodgkin lymphoma (NHL). Case Description: We report a case of PCD accompanying high-grade B-cell lymphoma embedded in an individual's stomach and duodenum, who also presented with acute onset of gait ataxia and slurred speech. The results of the common laboratory tests for neurological disorders, including the paraneoplastic antibody test, were negative. The key to the accurate diagnosis was the positron emission tomography/computed tomography findings. The final diagnosis of high-grade B-cell lymphoma was unclear until the performance of repeated esophagogastroscopy with multipoint deep excavation biopsies. After standard chemotherapy, the patient's gastric tumor was significantly alleviated and cerebellar syndrome was significantly improved. Conclusions: This case highlights the challenges of diagnosing PNS associated with occult malignancy. PNS patients may present with a variety of neurological disorders; Thus, if any unexplained neurological symptoms appear after a series of specific laboratory and imaging tests, a diagnosis of PNS should be taken into consideration in the differential diagnosis list, as it may help clinicians identify asymptomatic malignancies and ensure patients receive correct treatments in a timely manner. A high-quality endoscopic biopsy is essential, as it helps hematologists make an accurate diagnosis of lymphoma with gastroduodenal involvement based on pathology.

Pulmonary mucoepidermoid carcinoma in the Chinese population: A clinical characteristic and prognostic analysis.

Background: Mucoepidermoid carcinoma is dominant in salivary glands and rarely occurs in the lung. Primary pulmonary mucoepidermoid carcinoma is a type of non-small-cell lung cancer, but the prognostic factors in Chinese patients remain controversial. This investigation aimed to review cases of pulmonary mucoepidermoid carcinoma, analyse the prognosis of this disease. Methods: Patients with pathologically proven pulmonary mucoepidermoid carcinoma were screened at the Department of Respiratory and Critical Care Medicine at the Peking University Third Hospital, Beijing Friendship Hospital Affiliated to Capital Medical University, and Peking University Cancer Hospital for inclusion in this retrospective study. Demographic data, including age, sex, clinical symptoms, smoking, alcohol consumption, allergies, family history, imaging findings, fibrobronchoscopy findings, surgical procedures, tumour location and pathologic stage, were collected. Telephone follow-up was conducted for all patients not lost to follow-up. The associations of sex, age, smoking, tumour differentiation, tumour size, lymph node metastasis, pathologic stage, and patient survival were retrospectively analysed. Kaplan-Meier, univariate and multivariate analysis curves were used to analyse patient prognosis and prognostic factors. Results: Thirty-one patients, comprising 23 males and 8 females, were enrolled in the analysis. The mean age was 60.77 ± 11.44 years. The first symptom was nonspecific, with cough being the most common (21/31, 67.77%); smokers accounted for 16 of the 31 patients, and ten patients had a history of alcohol consumption. Overall, the tumours could occur in either lobe of the lungs; tumours occurred in the right lung in 19/31 patients, and tumours occurred in the left lung in 12/31 patients. Regarding TNM stage, 10 patients had stage I (5 with stage 1a, 5 with stage 1b), 5 had stage II (1 with stage 2a, 4 with stage 2b), 3 had stage III (1 with stage 3a, 2 with stage 3b), and 13 had stage IV (10 with stage 4a, 3 with stage 4b). In our Cox univariate survival analysis of patients with pulmonary mucoepidermoid carcinoma, we found that TNM stage IV, degree of differentiation and lymph node metastasis were risk factors for pulmonary mucoepidermoid carcinoma and that degree of differentiation was an independent risk factor. Conclusion: The clinical, radiographical and pathological features of pulmonary mucoepidermoid carcinoma were systemically analysed and summarized, and the degree of differentiation and lymph node metastasis, as well as prognostic factors in addition to clinical stage, were confirmed.

[Loss of pan-T cell antigens CD2, CD3, CD5 and CD7 in Kikuchi's disease].

OBJECTIVE: To study the possible loss of pan-T cell antigens CD2, CD3, CD5 and CD7 in Kikuchi's disease and to evaluate the role of T cell antigen loss in distinguishing benign from malignant T-cell lymphoid lesions. METHODS: Formalin-fixed and paraffin-embedded tissues of 33 cases of Kikuchi's disease and 15 cases of reactive lymphoid hyperplasia were studied by EliVision immunohistochemical staining for CD2, CD3, CD5 and CD7. RESULTS: Twenty-four of the 33 (72.7%) cases of Kikuchi's disease lost one or more of the pan-T cell antigens, including the loss of CD5 only (13 cases), CD7 only (1 case), CD2 only (1 case), CD2 and CD7 (2 cases), CD5 and CD7 (4 cases), CD2 and CD5 (2 cases), and CD2, CD7 and CD5 (1 case). Amongst these cases, the commonest antigen loss was CD5 (20 cases, 60.6%), followed by CD7 (8 cases, 24.2%) and CD2 (6 cases, 18.2%). Compared with the xanthomatous subtype of Kikuchi's disease, the loss of antigens was more commonly seen in the proliferative and necrotizing subtypes. Analysis of follow-up data showed that the loss of antigens in Kikuchi's disease was not significantly associated with the prognosis. In reactive lymphoid hyperplasia, the expression of CD2, CD3, CD5 and CD7 was seen in all cases with similar intensity, with no obvious pan-T cell antigen loss. CONCLUSION: Loss of one or more pan-T cell antigens in Kikuchi's disease is demonstrated in present study, suggesting that the immunophenotypic pattern is not unique in T cell lymphoma.

[Clinicopathologic features of systemic EBV-positive T/NK-cell lymphoproliferative disease in adults].

OBJECTIVE: To study the clinicopathologic features, immunophenotype, clonality and Epstein-Barr virus (EBV) status of systemic EBV-positive T/NK-cell lymphoproliferative disease in adults (ASEBV(+)T/NK-LPD). METHODS: Twenty cases of ASEBV(+)T/NK-LPD were analyzed retrospectively with histopathologic review, immunohistochemistry and in-situ hybridization for EBV-encoded RNA (EBER). The follow-up data were collected. RESULTS: There were altogether 15 males and 5 females. The median age of the patients was 34 years. The average duration from onset of symptoms to diagnosis was 8.7 months. Fever (18/20), hepatosplenomegaly (18/20) and lymphadenopathy (17/20) were the main clinical manifestations. Eleven of the 17 patients died during follow-up, with a mean survival of 2.9 months. Histologically, there was obvious expansion of T zone of the involved lymph nodes, associated with diminished lymphoid follicles. The interfollicular areas were widened and infiltrated by small to median-sized lymphoid cells which showed only mild atypia. Scattered large lymphoid cells were not uncommon. The nodal capsule was thickened in 6 cases. Focal necrosis was seen in 9 cases. Sinus histiocytic proliferation with erythrophagocytosis was observed in 3 cases. In addition, there were mild atypical lymphoid cells infiltrate into the liver, spleen, intestinal mucosa and bone marrow. Immunohistochemical study and in-situ hybridization showed that the EBER-positive cells were of T-cell lineage, with CD3 expression. They were also positive for cytotoxic molecules (granzyme B or TIA-1). Only 1 case was CD56 positive. A predominance of CD8-positive cells was demonstrated in 8 of the 14 cases studied, while CD4-positive cells predominated in the remaining 5 cases. One case showed similar proportion of CD8 and CD4-positive cells. The number of EBER-positive cells ranged from 30 to more than 300 per high-power fields. These EBER-positive cells were of small to large size and located mainly in the expanded T zone and occasionally in the germinal centers. Three of the 7 cases exhibited clonal rearrangement of T-cell receptor gamma gene, while the other 4 cases exhibited polyclonal rearrangement of T-cell receptor gamma gene. CONCLUSIONS: ASEBV(+)T/NK-LPD is a systemic disease with a subacute or chronic clinical course. Most patients suffer from relapsing fever, lymphadenopathy and hepatosplenomegaly. The disease is characterized by proliferation of EBV-infected cytotoxic T cells. The T zone of the involved lymph nodes shows expansion by mildly atypical lymphoid cells. The disease is associated with poor clinical outcome and can be life-threatening. The patients often die of multiorgan failure and bleeding.

Regulation PP2Ac methylation ameliorating autophagy dysfunction caused by Mn is associated with mTORC1/ULK1 pathway.

Manganese (Mn) exposure leads to autophagy dysfunction and causes neurodegenerative diseases such as Parkinson's syndrome and Alzheimer's disease. However, the mechanism of neurotoxicity of Mn has been less clear. The methylation of the protein phosphatase 2A catalytic subunit determines the dephosphorylation activity of protein phosphatase and plays an important role in autophagy regulation. In this investigation, we established a model of Mn (0-2000 µmol/L) exposure to N2a cells for 12 h, used the PPME-1 inhibitor ABL-127, and constructed an LCMT1-overexpressing N2a cell line. We also regulated the PP2Ac methylation level and explored the effect of PP2Ac methylation on Mn-induced (0-1000 µmol/L) N2a cellular autophagy. Our results showed that Mn > 500 µmol/L induced N2a cell damage and increased oxidative stress. Moreover, Mn modulated autophagy in N2a cells by downregulating PP2Ac methylation, which regulated mTORC1 signaling pathway activation. Both ABL-127 and LCMT1 overexpression can upregulate PP2Ac methylation in parallel with ameliorating N2a cell abnormal autophagy induced by Mn, Briefly, the upregulation of PP2Ac methylation can ameliorate the autophagy disorder of N2a by Mn and effectively alleviate Mn-induced cytotoxicity and oxidative stress, indicating that regulation of autophagy is a protective strategy against Mn-induced neurotoxicity.

[Intralymphatic accumulation of lymphocytes mimicking intravascular lymphomatosis].

OBJECTIVE: To study the significance and differential diagnosis of intralymphatic accumulation of lymphocytes. METHODS: The clinical and pathologic features of 4 cases of intralymphatic accumulation of lymphocytes were reviewed retrospectively. Immunohistochemical study was carried out and follow-up data were analyzed. RESULTS: The sites of involvement included tonsil (2 cases), pharynx (1 case) and appendix (1 case). The duration of disease ranged from 1 week to 3 months. Follow up of the patients (from 3 to 84 months) showed no evidence of disease recurrence. Gross examination of the tissues (except in the case of appendiceal involvement) showed polypoid changes. Histologically, the lymphatic channels were filled up with small lymphocytes and associated with fibrosis in the vicinity. Immunohistochemical study revealed a T-cell phenotype of the intralymphatic lymphoid cells. CONCLUSIONS: The accumulation of lymphocytes in lymphatic channels is associated with a benign clinical course. This phenomenon may be due to retention of lymphocytes secondary to the perilymphatic chronic inflammation and fibrosis. Although the lesion simulates intravascular lymphomatosis morphologically and shows a uniform T-cell phenotype, the lymphoid cells lack obvious cellular pleomorphism and mitotic activity. The solitary nature of the lesion, when coupled with the indolent clinical behavior, is also helpful in the differential diagnosis.

[Expression, purification and functional assessment of asprosin inclusion body].

OBJECTIVE: The obtain purified recombinant asprosin and test its functions. METHODS: The recombinant plasmid of pET-22b-asprosin was constructed and transformed into competent E.coli BL (DE3) strain. After IPTG-induced expression, asprosin inclusion body was renatured by gradient urea and purified by Ni-NTA affinity chromatography column followed by removal of endotoxin to obtain recombinant asprosin for use in cells and animals experiments. C57 mice were injected intraperitoneally with the recombinant asprosin and blood glucose was detected using a blood glucose meter. Alamar Blue assay was used to evaluate of the effect of the recombinant asprosin on the viability of MIHA cells, and cellular glycogen content was detected using the anthrone method. RESULTS: At the absorbance at 600 nm of 0.8, induction of the recombinant host bacteria with 1 mmol/L IPTG at 37 ℃ for 4 h optimally induced the expression of asprosin inclusion body. After purification and endotoxin removal, the purity of the recombinant asprosin exceeded 95% with the content of endotoxin below 1 EU/mg. In C57 mice, intraperitoneal injection with recombinant asprosin significantly increased blood glucose level, which reached the peak level at 60 min following the injection (P=0.021) and recovered the normal level at 120 min (P=0.03). Treatment with the recombinant asprosin for 24 h did not cause obvious adverse effect on the viability of MIHA cells but significantly lowered glycogen content in the cells (P < 0.05). CONCLUSIONS: We successfully obtained recombinant asprosin using a prokaryotic expression system. The recombinant asprosin can decrease glycogen content in MIHA cells and increase blood glucose level in mice.

Systemic toxicity evaluation of novel tobacco products in Caenorhabditis elegans.

Under strictly Framework Convention on Tobacco Control, novel tobacco products are going to be promising alterations to consumers and manufactures. Even though the novel tobacco products have been considered less harmful than traditional tobaccos, there is a few knowledges about the subsequent substances during consume and their impacts to the consumers due to short introduction into the market. Thus, the present study aims to investigate the adverse effects of novel tobacco products on Caenorhabditis elegans(C. elegans) and to provide relevant references for novel tobacco products toxicity research and assessment. C. elegans individuals at L4 stage were exposed to different kinds of novel tobacco products, including electronic cigarettes liquid (e-liquid), the extract of e-cig aerosol (e-aerosol), mint and black tea flavor snus. After specific exposure time, the multiple toxic endpoints of C. elegans were measured, including acute toxicity, locomotion behavior, body length, and life-span. The oxidative stress was tested too. According to acute toxicity assays, the half lethal dose of four novel tobacco products calculated from theoretical nicotine concentration, ranked as follows e-liquid (0.29 mg/ml) > the extract of e-cig aerosol (0.43 mg/ml) > mint flavor snus (1.20 mg/ml) > black tea flavor snus (1.50 mg/ml). The equivalent lethal rate 5%~20% of four novel tobacco products were applied to following experiments. These novel tobacco products damaged nematode's locomotion including head thrashing and body bending, the damage was most evident in two flavors of snus. The similar trends were found in reproductive performance investigation. At tested concentrations, the retardation development of C. elegans was found throughout all stages with peak blockage at adulthood. Life-span tests showed that novel tobacco products at 5% lethal rate seemed no significant effect on affected the life-span of nematodes, with snus shortened the lifespan of C. elegans at 20% lethal rate. Imaging stress response indicted four types of tobacco productions causing stress response in C. elegans. Exposed to either 5% or 20% lethal levels (5% and 20%), the percentages of worms with DAF-16 redistribution among all groups varied, with higher frequencies in both snus. Summary, novel tobacco products caused multiple adverse impacts to C. elegans, including acute toxicity, locomotion behavior disruption, brood size reduction, development retardation, and life-span reduction. The toxicity was associated with both the feature and concentration of tobacco products, and oxidative stress was the main mechanism.

Involvement of gap junctions in astrocyte impairment induced by manganese exposure.

Glutamate excitotoxicity, characterized as excessive glutamate stress, is considered to be involved in cerebral ischaemia, brain trauma, and neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. Glutamate homeostasis disruption was highlighted in Mn neurotoxicity caused by high levels of Mn. Astrocytes, accounting for approximately 50% of the neuronal cells in the central nervous system and maintain glutamate homeostasis, are sensitive to neurotoxicity induced by Mn exposure. Astrocytes are tightly coupled with gap junctions (GJ), which are comprised of connexins, mainly connexin43 (Cx43). The gap junctional intercellular communication (GJIC) pathway allows small signal molecules, such as glutamate, ATP (adenosine triphosphate, ATP) and tropic factors, etc., to transfer between adjacent cells. Evidence has shown that astrocytes execute the bystander effect during cell death through the GJIC pathway. However, the pathogenic mechanism of the gap junction underlying glutamate neurotoxicity induced by manganese exposure has not been elucidated yet. In the present study, primary astrocytes were cultured and then exposed to different levels of Mn (ranging from 0 to 1000 µM) for 4/16 h to investigate the function of the GJIC in apoptosis induced by Mn. The cellular toxicity was confirmed by cell viability and apoptotic percentage through MTT assay and flow cytometry (FC). The levels of intracellular/extracellular glutamate were measured by high-performance liquid chromatography (HPLC). The fluorescent dye, Lucifer Yellow (LY), was used to assess the status of gap junctions among astrocytes after Mn exposure. The protein/gene expression of major gap junctional forming protein, Cx43, was also investigated. Cell viability was distinctly reduced when exposed to 500 and 1000 µM MnCl2 compared with control cells at both time points. The percentage of apoptosis was significantly increased among all detected Mn levels (125, 500 and 1000 µM MnCl2) of exposure (p < 0.05) with a concentration-dependent manner at either time point. Mn administration for 4/16 h also caused a remarkable intracellular/extracellular glutamate increase in a concentration-dependent manner for extracellular glutamate levels (p < 0.01). Gap junctions were prominently inhibited by Mn with Cx43 protein shown as shortening of the LY dye transfer distance at both time points. In-cell western blot indicated that Mn caused a decrease in Cx43 protein/gene expression in a dose-dependent manner. These results suggested that the gap junction intercellular communication and its forming protein, Cx43, are likely involved in glutamate excitotoxicity induced by Mn exposure.