[Association between congenital hypothyroidism and in-hospital adverse outcomes in very low birth weight infants].

Objective: To investigate the association between congenital hypothyroidism (CH) and the adverse outcomes during hospitalization in very low birth weight infants (VLBWI). Methods: This prospective, multicenter observational cohort study was conducted based on the data from the Sino-northern Neonatal Network (SNN). Data of 5 818 VLBWI with birth weight <1 500 g and gestational age between 24-<37 weeks that were admitted to the 37 neonatal intensive care units from January 1st, 2019 to December 31st, 2022 were collected and analyzed. Thyroid function was first screened at 7 to 10 days after birth, followed by weekly tests within the first 4 weeks, and retested at 36 weeks of corrected gestational age or before discharge. The VLBWI were assigned to the CH group or non-CH group. Chi-square test, Fisher exact probability method, Wilcoxon rank sum test, univariate and multivariate Logistic regression were used to analyze the relationship between CH and poor prognosis during hospitalization in VLBWI. Results: A total of 5 818 eligible VLBWI were enrolled, with 2 982 (51.3%) males and the gestational age of 30 (29, 31) weeks. The incidence of CH was 5.5% (319 VLBWI). Among the CH group, only 121 VLBWI (37.9%) were diagnosed at the first screening. Univariate Logistic regression analysis showed that CH was associated with increased incidence of extrauterine growth retardation (EUGR) (OR=1.31(1.04-1.64), P<0.05) and retinopathy of prematurity (ROP) of stage Ⅲ and above (OR=1.74(1.11-2.75), P<0.05). However, multivariate Logistic regression analysis showed no significant correlation between CH and EUGR, moderate to severe bronchopulmonary dysplasia, grade Ⅲ to Ⅳ intraventricular hemorrhage, neonatal necrotizing enterocolitis in stage Ⅱ or above, and ROP in stage Ⅲ or above (OR=1.04 (0.81-1.33), 0.79 (0.54-1.15), 1.15 (0.58-2.26), 1.43 (0.81-2.53), 1.12 (0.70-1.80), all P>0.05). Conclusion: There is no significant correlation between CH and in-hospital adverse outcomes, possibly due to timely diagnosis and active replacement therapy.

[Optic atrophy in a patient with axonal Charcot-Marie-Tooth disease 2A2A due to MFN2 gene mutations].

A 27-year-old male patient had progressive vision loss in both eyes, which was mainly manifested by impaired ganglion cells in the macular area, accompanied by systemic muscle atrophy in limbs. A complete mitochondrial exon gene detection was performed. The final diagnosis was bilateral optic atrophy and axonal Charcot-Marie-Tooth disease 2A2A caused by mutations of the MFN2 gene. There has been no effective treatment. Applications of nutrients to restore the mitochondrial function may alleviate the clinical symptoms.

[Analysis of clinicopathological characteristics of non-alcoholic fatty liver disease].

Objective: To investigate the clinical and pathologic characteristics of obese adults with nonalcoholic fatty liver disease (NAFLD) and to aid the diagnosis of nonalcoholic steatohepatitis (NASH). Methods: A total of 262 patients eligible for inclusion who received volume reduction metabolism surgery and liver biopsy in the First Affiliated Hospital of Nanjing Medical University from June 2018 to September 2019 were selected. HE staining, reticular fiber staining and Masson staining were performed. Statistical analysis was performed using SPSS 20.0. Results: The patients ranged in age from 18 to 66 years. Among the 262 cases, 65 cases (65/262, 24.8%) were male and 197 cases (197/262, 75.2%) were female. Sixty-one cases (61/262, 23.3%) were non-NAFLD, 201 cases (201/262, 76.7%) were NAFLD including 27 cases (27/201, 13.4%) of nonalcoholic fatty live (NAFL) and 174 cases (174/201, 86.6%) of NASH. The main lesions of NAFLD were in hepatic acinus zone 3. There were significant differences in age, alanine aminotransferase (ALT), glutamic oxaloacetic transaminase (AST), body mass index (BMI), fasting blood-glucose (FPG) and apolipoprotein A (APOA) levels among the non-NAFLD group, NAFL group and NASH group (P<0.05). Patients with BMI≥35 m/kg2 combined with type 2 diabetes had a higher prevalence of NASH. Multiple logistic regression showed that ALT and APOA were independent predictors of NASH (P<0.001, OR=1.05, 95%CI: 1.020-1.082; P=0.027, OR=0.916, 95%CI: 0.878-0.941). Total cholesterol (CHO) and high-density lipoprotein (HDL) were independent predictors of lobular inflammation (P=0.043, 95%CI: 0.010-0.634; P=0.024, 95%CI:-3.068--0.216). AST and HDL were independent predictors of fibrosis stage (P=0.029, 95%CI: 0.001-0.021; P<0.001, 95%CI:-2.670--0.645). Conclusions: Biochemical indicators of NAFLD are closely related to its pathology. The histological lesions of NAFLD are mainly present in hepatic acinar area 3. The diagnosis of NASH is supported by extensive steatosis and high levels of CHO, ALT, AST and BMI, low levels of HDL and ApoA in biochemical markers, but pathological examination is still the gold standard for it.

[Myelin oligodendrocyte glycoprotein antibody positive optic neuritis with anti-N-methyl-D-aspartate receptor encephalitis: a case report].

A 6-year-old girl had binocular vision loss with pain for one week. The patient presented with symptoms such as non-communication, language deterioration, dysphonia, and choking when drinking and eating during the course. The serum myelin oligodendrocyte glycoprotein antibody was positive. Both the serum and cerebrospinal fluid anti-N-methyl-D-aspartate receptor antibody were also positive. The diagnoses were myelin oligodendrocyte glycoprotein antibody positive optic neuritis and anti-N-methyl-D-aspartate receptor encephalitis. High-dose intravenous glucocorticoids were given. Recurrence was not observed during the 15-month clinical follow-up.

[Changes of immunohistochemical biomarkers before and after neoadjuvant chemotherapy in breast cancer and their prognosis].

Objective: To investigate changes in the expression of immunohistochemical (IHC) markers and factors associated with the effect of chemotherapy before and after neoadjuvant chemotherapy (NAC). Methods: A retrospective study included 200 breast cancer patients treated with NAC between January 2016 and December 2018. We analyzed the changes in the expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and Ki-67 in pre- and post-treated samples and the predictive factors of NAC. Results: Among the 200 cases, 16 cases were luminal A, 108 cases were luminal B, 36 cases were HER2+subtype, and 40 cases were basal-like. Twenty-five patients (12.50%) achieved pathological complete remission (PCR).There were significant differences in PR and Ki-67 before and after NAC but there were no differences in ER and HER2.In univariate analysis, factors associated with PCR were tumor less than 5 cm(P=0.009), non-luminal breast cancer (P=0.001), ER negative(P=0.001), PR negative (P=0.029) and HER2 positive(P=0.001). Tumor less than 5 cm [P=0.020, OR=2.581, 95%CI (1.207, 5.753)], ER negative [P=0.011, OR=2.264, 95%CI (1.207, 4.248)] and HER2 positive[P=0.007, OR=2.412, 95%CI (1.275, 4.561)] remained predictive variables in multivariate analysis after correction for the other variables. Conclusions: The expression of Ki-67 decreases after NAC. Negative PR and ER and positive HER2 status are related to the efficacy of pCR for breast cancer, and have guiding significance for the prognosis evaluation of NAC.

Effect of RhoC silencing on multiple myeloma xenografts and angiogenesis in nude mice.

In this study, we investigated the expression of RhoC in the multiple myeloma (MM) cell line RPMI- 8226, as well as the effects of silencing RhoC on the growth of tumor xenografts and tumor-induced angiogenesis in nude mice with MM. For this purpose, we transduced RPMI-8226 cells with lentiviral particles overexpressing short hairpin RNAs (shRNA) targeting RhoC. Tumor xenografts were generated by subcutaneously injecting nude mice with RPMI-8226 cells overexpressing control shRNA [negative control (NC) group] or the RhoC shRNA [the experimental (S) group], respectively. RhoC protein and mRNA levels in the tumor xenografts were measured. Nude mice were also subcutaneously inoculated with Matrigel mixed with vascular endothelial growth factor, and CD31 and KI67 levels in the tumor xenografts were measured by immunohistochemistry. Similarly, we assessed tumor xenograft growth and angiogenesis in Matrigel implants in the mice of both groups. We found that RhoC levels, microvessel density, and CD31 labeling index were more reduced in the S group than in the NC group. However, there was no significant difference in the size of tumor xenografts between the 2 groups. The number of new vessels and the neovascular length in the Matrigel implants were significantly lower in the S group than in the NC group. Therefore, we concluded that RhoC expression in myeloma xenografts has important effects on the induction of angiogenesis.

[HER2 status in gastric adenocarcinoma of Chinese: a multicenter study of 40 842 patients].

Objective: To investigation HER2 status in gastric adenocarcinoma of Chinese and contributing factors to the HER2 expression. Methods: HER2 status of 40 842 gastric adenocarcinomas and clinical data were retrospectively collected from 23 hospitals dated from 2013 to 2016. The association between HER2 positivity and clinicopathologic features was analyzed. Results: Of the 40 842 patients the median age was 62 years, the male female ratio was 2.6∶1.0. The rate of HER2 positivity was 8.8% (3 577/40 842). HER2 expression was related to the tissue type, tumor location, Lauren classification and tumor differentiation (P values: 0.009, 0.001, <0.01 and <0.01, respectively). Different HER2 expression status was observed between primary and recurrent tumors in 7.6% (48/635) cases. The rates of HER2 positivity ranged from 2% to 10% among different institutions. The rates of HER2 FISH amplification were dramatically different among the 23 hospitals (0-100%) with an average rate of 10% (810/8 156) in patients with HER2 IHC 2+ . Conclusions: HER2 expression is associated with clinicopathologic characteristics. HER2 re-assessment of tumor tissue and use of in situ hybridization techniques increase HER2 positivity. The current retrospective study should reflect the HER2 status in gastric adenocarcinoma of Chinese patients.

Effects of tumor-associated macrophages on the proliferation and migration of esophageal cancer-associated lymphatic endothelial cells.

The aim of this study was to explore whether M2 macrophages can be transformed into M1 macrophages, and to investigate the effect of different types of macrophages on the proliferation, migration and ring-forming ability of esophageal cancer-related lymphatic endothelial cell (LEC). Human monocytic leukemia cell line (THP-1 cell) was induced to differentiate to M1 macrophages (M1 group) and M2 macrophages (M2 group), and co-cultured with esophageal cancer-associated LEC. The individual esophageal cancer co-cultured with LEC was used as control. Different types of macrophages were observed by Cell counting kit-8 (CCK-8). Enzyme-linked immunosorbent assay (ELISA) was used to detect the VEGF-C concentration; the expression of VEGFR-3 protein and its mRNA was detected by Western blot and qRT-PCR, respectively. The positive rate of the M1 group induced by IFN-γ and LPS was significantly higher than that of M2 macrophages (48.57%5.98% vs 25.83%1.95%). The expression of VEGF-C in the supernatant of the M2 group was higher than that in the control group, but no significant differences regarding the expression of VEGF-C between M1 and control groups were found. In addition, the expression of VEGFR-3 on both mRNA and protein in esophageal cancer-related LEC of the M2 group was significantly higher than those in the control group; however, the M1 group had a significantly lower VEGFR-3 level on both mRNA and protein than the control group. Human M2 macrophages can be transformed into M1 macrophages, and can promote the proliferation, migration and ring-forming ability of esophageal cancer-associated LEC.

Study on correlation between PKIB and pAkt expression in breast cancer tissues.

OBJECTIVE: This study is to explore the expression of cyclic AMP (cAMP) dependent protein kinase inhibitor (PKIB) in human breast cancer and the correlation with phosphorylated protein kinase B (pAkt) expression in the tumor tissues. MATERIALS AND METHODS: Surgical removal of the tissue samples from 148 patients with primary breast cancer from 2011-2015 were selected, and then we detected the PKIB, estrogen receptor (ER), progesterone receptor (PR) and proto oncogene (HER2) by using immunohistochemical technique and the Allred score classification standard. The clinical pathological factors such as tumor diameter, lymph node metastasis and tumor stage, etc. were analyzed statistically. Then we detected that the PKIB and pAkt respectively of immunohistochemical expression and cellular localization of four subtypes in patients which were luminal A, luminal B, HER2+/ER-type and triple negative breast cancer type. RESULTS: Immunohistochemistry staining showed when pAkt was positive and there were significant correlations with the expression of PKIB (p<0.05). Both positive staining reactions occurred in the cytoplasm of the tumor. Histopathological type, tumor diameter, lymph node metastasis, tumor stage and other clinical pathological factors were not significantly associated with the expression of PKIB. In addition, the expression of PKIB was also significantly associated with the triple negative breast cancer in the four subtypes (p<0.05). CONCLUSIONS: The expression of PKIB in the cytoplasm of tumor is closely related to pAkt and the triple negative breast cancer. It was concluded that the PKIB promoted the occurrence and development of breast tumors by regulating the Akt signaling pathway. PKIB will be a potential therapeutic target for breast cancer, especially in the diagnosis and treatment of triple negative breast cancer.

[Predictive value of liver pathologic changes in determining the effectiveness of entecavir treatment for chronic hepatitis B-An analysis of 1 366 cases].

Objective: To analyze the relationship between baseline liver pathologic changes and the effectiveness of entecavir(ETV) and investigate the predictive value of baseline liver pathologic changes in determining the effectiveness of ETV, to provide reliable basis for precision medicine in patients with chronic hepatitis B(CHB). Methods: A total of 1 366 cases with CHB were retrospectively recruited who underwent liver biopsy between January 2006 to June 2016 and were treated with ETV over 96 weeks.The relationship between baseline liver pathologic changes and the antiviral responses to ETV at 48, 96 weeks were compared. Results: Liver pathology was employed to make the definite inflammation grade and the fibrosis stage.According to the liver inflammation and fibrosis, patients were divided into 4 groups(G1, G2, G3, G4 and S1, S2, S3, S4 respectively). The complete response rate of G1, G2, G3 and G4 after 48 weeks ETV treatment was 26.3%(10/38), 30.9%(121/391), 35.3%(101/286), 44.4%(52/117) respectively in HBeAg positive patients and was 61.5%(24/39), 80.4%(148/184), 82.4%(201/244), 88.1%(59/67) respectively in HBeAg negative patients.There was statistical difference in the complete response rates among liver inflammation grades both in HBeAg positive patients(χ(2)=8.510, P<0.05) and in HBeAg negative patients(χ(2)=12.054, P<0.05)respectively.The differences were still statistical significant after 96 weeks ETV treatment (P<0.05). The complete response rates of S1, S2, S3 and S4 after 48 weeks ETV treatment were 39.0%(41/105), 37.8%(127/336), 30.9%(97/314), 24.7%(19/77), respectively in HBeAg positive patients and was 85.7%(30/35), 84.4%(92/109), 83.9%(162/193), 75.1%(148/197) respectively in HBeAg negative patients. Whether HBeAg was positive or not, the rates were in decline but there was no statistical difference in the complete response rates among liver fibrosis stages(χ(2)=7.765, P>0.05; χ(2)=6.729, P>0.05). The differences were still not statistical significant after 96 weeks ETV treatment (P>0.05). But after further grouping, whether HBeAg was positive or not, as the degree of fibrosis stage was aggravating, the complete response rate of G2, G3 and G4 after 48 weeks ETV treatment decreased at the same degree of inflammation grade and the differences were statistically significant (P<0.05). The differences were still statistical significant after 96 weeks ETV treatment (P<0.05). Conclusions: The responses to ETV treatment are closely related with baseline liver pathology.The CHB patients with higher score of inflammation and lower score of fibrosis will have a good response to ETV treatment.The degree of inflammation grades and fibrosis stages can be used as early predictors of ETV treatment for CHB.

BLCA-4 and UBC combined detection for early diagnosis of bladder cancer.

The objective of the present study was to report the clinical significance of bladder cancer specific nuclear matrix protein 4 (BLCA-4) and urinary bladder cancer (UBC) on early diagnosis of bladder cancers. Enzyme-linked immunosorbent assay (ELISA) was used to detect BLCA-4 and UBC of 56 bladder cancer patients and 26 patients with urinary tract benign diseases, serving as controls. Urine exfoliated cell test was performed, and then the significance of BLCA-4 and UBC on the diagnosis of bladder cancers was analyzed. The sensitivity of BLCA-4 and UBC of the bladder cancer patients was significantly higher than that of the urine exfoliated cell test (P less than 0.05). The difference of BLCA-4 and UBC was not significant (P >0.05). The difference of BLCA-4 and UBC in the tumors with different gradings and stagings was not significant (P >0.05). Combined detection of BLCA-4 and UBC could improve the diagnosis sensitivity and specificity of bladder cancers with the advantages of high maneuverability, repeatability and objective results.

Mechanism of action of EBV, Bcl-2, p53, c-Myc and Rb in non-Hodgkin's lymphoma.

OBJECTIVE: The aim of the present study is to explore the mechanism of action of several proteins, including Epstein-Barr virus (EBV), B-cell lymphoma (Bcl)-2, p53, c-Myc and retinoblastoma (Rb), in Non-Hodgkin's lymphoma (NHL). PATIENTS AND METHODS: Between July 2010 and July 2015, samples of 142 patients with pathologically confirmed NHL which presented at our institution were included in the observation group. In addition, samples from 55 patients with hyperplastic lymphadenitis presented during the same period were enrolled as control group. The expressions of EBV (+), p53(+), Bcl-2(+), Rb(-) and c-Myc(+) were determined and compared PATIENTS AND METHODS: Between July 2010 and July 2015, samples of 142 patients with pathologically confirmed NHL which presented at our institution were included in the observation group. In addition, samples from 55 patients with hyperplastic lymphadenitis presented during the same period were enrolled as control group. The expressions of EBV (+), p53(+), Bcl-2(+), Rb(-) and c-Myc(+) were determined and compared among different subtypes and stages of NHLs of observation group. Besides, the correlation of EBV with p53, Bcl-2, Rb and c-Myc were investigated in NHLs of observation group. RESULTS: In the observation group, the expression rates of EBV(+), p53(+), Bcl-2(+), Rb(-), and c-Myc(+) were significantly higher than those, respectively, in the control group (p < 0.05). No significant correlation was observed between EBV expression and the expressions of p53, Bcl-2, Rb and c-Myc in the observation group (p > 0.05). The expression rates of p53(+) and Bcl-2(+) were significantly higher in aggressive and highly-aggressive NHLs than in indolent NHLs of the observation group (p < 0.05). The expressions of EBV(+), p53(+), Bcl-2(+), Rb(-), and c-Myc(+) were significantly higher in stage III-IV NHLs than in stage I-II NHLs (p < 0.05). CONCLUSIONS: The expressions of EBV(+), p53(+), Bcl-2(+), Rb(-), and c-Myc(+) are closely associated with NHL pathogenesis. Expressions of these proteins are higher in later stages of NHLs, and expressions of p53(+) and Bcl-2(+) are higher in more aggressive NHLs.

Liver X Receptor activation delays chondrocyte hypertrophy during endochondral bone growth.

OBJECTIVE: Activation of the Liver X Receptor (LXR) has recently been identified as a therapeutic strategy for osteoarthritis (OA). Human OA articular cartilage explants show decreased LXR expression, and LXRß-null mice display OA-like symptoms. LXR agonist administration to OA articular cartilage explants suppresses proteoglycan degradation and restores LXR-activated transcription. We aimed to investigate the effect of LXR activation on chondrocyte differentiation to elucidate the molecular mechanisms behind its protection against OA. METHOD: The specific LXR agonist, GW3965, was used to examine the effect of LXR activation on chondrocyte differentiation. Tibia organ cultures were used to examine the effect of LXR activation on bone growth and growth plate morphology, followed by immunohistochemical analysis. In ATDC5 and micromass cultures, chondrocyte differentiation was examined through cellular staining and proliferation assays. Various chondrogenic markers were analyzed by real-time reverse-transcription polymerase chain reaction (qRT-PCR) in micromass RNA. RESULTS: Chondrocyte hypertrophy was suppressed by GW3965 treatment, as shown by decreased hypertrophic zone length in the tibial growth plate, decreased alkaline phosphatase staining in ATDC5 and micromass cultures, and down regulation of Col10a1, Mmp13 and Runx2 expression. Increased proliferation in treated ATDC5 cells and up-regulation of Col2a1 expression in treated micromass cultures suggest hypertrophy is suppressed secondary to prolonged proliferation. Decreased p57 levels in treated growth plates suggest this to be due to cell-cycle exit delay. CONCLUSION: Our findings regarding LXR's role in cartilage development provide insight into how LXR activation prevents cartilage breakdown, further solidifying its potential as a therapeutic target of OA.

TGF-ß1 suppression of microRNA-450b-5p expression: a novel mechanism for blocking myogenic differentiation of rhabdomyosarcoma.

Transforming growth factor beta 1 (TGF-ß1) is the most potent inhibitor of myogenic differentiation (MyoD) of rhabdomyosarcoma (RMS); however, the underlying mechanisms of this inhibition remain unclear. In this study, we identified novel TGF-ß1-related microRNAs (miRNAs); among these, miR-450b-5p is significantly regulated by TGF-ß1. We provide evidence that TGF-ß1 exerts it function by suppressing miR-450b-5p. Both in cultured cells and tumor implants, miR-450b-5p significantly arrested the growth of RMS and promoted its MyoD. Utilizing a bioinformatics approach, we identified miR-450b-5p target mRNAs. Among these candidates, only the expression of ecto-NOX disulfide-thiol exchanger 2 (ENOX2) and paired box 9 (PAX9) was augmented by miR-450b-5p knockdown examined by western blot; the engineered inhibition antagonized TGF-ß1-mediated differentiation inhibition. Furthermore, we found that the Smad3 and Smad4 pathways, but not Smad2, are the principal mediator of TGF-ß1 suppression of miR-450b-5p. Taken together, these results suggest that disrupting the TGF-ß1 suppression of miR-450b-5p, or knockdown of ENOX2 and PAX9, are effective approaches in inducing RMS MyoD.

Mechanistic target of rapamycin small interfering RNA and rapamycin synergistically inhibit tumour growth in a mouse xenograft model of human oesophageal carcinoma.

OBJECTIVES: To investigate the effect of mechanistic target of rapamycin (mTOR)-specific small interfering RNA (siRNA) and rapamycin on tumour size and levels of hypoxia inducible factor 1α(HIF-1α), vascular endothelial growth factor (VEGF) and mTOR proteins, and mTOR mRNA, in a mouse xenograft model of human oesophageal carcinoma. METHODS: Tumours were induced in BALB/c nude mice using the human oesophageal squamous cell carcinoma cell line, EC1, injected subcutaneously. Animals were divided into four treatment groups (n = 5 per group) after 7 days: control (phosphate buffered saline, daily intraperitoneal [i.p.] injection); 50 µg/kg rapamycin, daily i.p. injection; 3 µg/kg mTOR siRNA, daily i.p. injection; combined mTOR siRNA and rapamycin, daily i.p. injections. Tumour volume was measured 21 days after xenograft. Levels of mTOR, VEGF and HIF-1α were assessed via immunohistochemistry and in situ hybridization. RESULTS: mTOR siRNA and/or rapamycin significantly decreased tumour volume and levels of HIF-1α, VEGF and mTOR protein, and mTOR mRNA. Combination treatment was significantly more effective than either treatment alone. CONCLUSIONS: mTOR siRNA and/or rapamycin inhibited the growth of oesophageal carcinoma in vivo. This may represent a novel and effective treatment strategy for oesophageal carcinoma.

Effect of PTEN antisense oligonucleotide on oesophageal squamous cell carcinoma cell lines.

OBJECTIVE: To investigate the effects of a phosphatase and tensin homologue (PTEN) antisense oligonucleotide on PTEN and mammalian target of rapamycin (mTOR) mRNA and protein, cell proliferation and apoptosis in oesophageal squamous cell carcinoma (OCSS) cell lines. METHODS: EC9706 and EC1 cells were transfected with PTEN antisense oligonucleotide, sense oligonucleotide or nonsense oligonucleotide. Cell proliferation and apoptosis were quantified. Immuno cyto chemistry and in situ hybridization were used to determine PTEN and mTOR protein and mRNA levels, respectively. RESULTS: Transfection with PTEN antisense oligonucleotide dose- and time-dependently enhanced cell proliferation and inhibited apoptosis in both EC9706 and EC1 cells. PTEN mRNA and protein were significantly downregulated, and mTOR protein and mRNA were significantly upregulated. CONCLUSION: These data suggest that PTEN is an important tumour suppressor gene in the development of OSCC.

Prevention of glutamate excitotoxicity in motor neurons by 5,6-dihydrocyclopenta-1,2-dithiole-3-thione: implication to the development of neuroprotective drugs.

Glutamate-induced excitotoxicity and oxidative damage are believed to play an important role in the development of a number of central nerve system disorders. Nuclear-factor erythroid 2-related factor 2 (Nrf2) is a master transcriptional regulator of many cytoprotective genes. We report herein that 5,6-dihydrocyclopenta-1,2-dithiole-3-thione (CPDT), which was previously shown to induce several Nrf2 target genes in non-nervous cells and tissues, significantly activates Nrf2 and Nrf2 target genes in rat spinal cord explants. More importantly, such activation is accompanied by complete inhibition of glutamate-induced motor neuron death in these explants. Further studies show that CPDT inhibits glutamate-induced intracellular Ca(2+) rise, loss of mitochondrial transmembrane potential and depletion of tissue glutathione. CPDT did not appear to modulate glutamate transport or to interfere with glutamate interaction with postsynaptic receptors. Taken together, our studies have identified CPDT as a promising neuroprotective agent and suggest that pharmacological activation of Nrf2 signaling is an important strategy for protection against glutamate-induced excitotoxicity.

Mechanism of pressure-induced thermostabilization of proteins: studies of glutamate dehydrogenases from the hyperthermophile Thermococcus litoralis.

In this study, we investigated the effect of pressure on protein structure and stability at high temperature. Thermoinactivation experiments at 5 and 500 atm were performed using the wild-type (WT) enzyme and two single mutants (D167T and T138E) of the glutamate dehydrogenase (GDH) from the hyperthermophile Thermococcus litoralis. All three GDHs were stabilized, although to different degrees, by the application of 500 atm. Interestingly, the degree of pressure stabilization correlated with GDH stability as well as the magnitude of electrostatic repulsion created by residues at positions 138 and 167. Thermoinactivation experiments also were performed in the presence of trehalose. Addition of the sugar stabilized all three GDHs; the degree of sugar-induced thermostabilization followed the same order as pressure stabilization. Previous studies suggested a mechanism whereby the enzyme adopts a more compact and rigid structure and volume fluctuations away from the native state are diminished under pressure. The present results on the three GDHs allowed us to further confirm and refine the proposed mechanism for pressure-induced thermostabilization. In particular, we propose that pressure stabilizes against thermoinactivation by shifting the equilibrium between conformational substates of the GDH hexamer, thus inhibiting irreversible aggregation.