Mitochondrial FAD shortage in SLC25A32 deficiency affects folate-mediated one-carbon metabolism.

The SLC25A32 dysfunction is associated with neural tube defects (NTDs) and exercise intolerance, but very little is known about disease-specific mechanisms due to a paucity of animal models. Here, we generated homozygous (Slc25a32Y174C/Y174C and Slc25a32K235R/K235R) and compound heterozygous (Slc25a32Y174C/K235R) knock-in mice by mimicking the missense mutations identified from our patient. A homozygous knock-out (Slc25a32-/-) mouse was also generated. The Slc25a32K235R/K235R and Slc25a32Y174C/K235R mice presented with mild motor impairment and recapitulated the biochemical disturbances of the patient. While Slc25a32-/- mice die in utero with NTDs. None of the Slc25a32 mutations hindered the mitochondrial uptake of folate. Instead, the mitochondrial uptake of flavin adenine dinucleotide (FAD) was specifically blocked by Slc25a32Y174C/K235R, Slc25a32K235R/K235R, and Slc25a32-/- mutations. A positive correlation between SLC25A32 dysfunction and flavoenzyme deficiency was observed. Besides the flavoenzymes involved in fatty acid ß-oxidation and amino acid metabolism being impaired, Slc25a32-/- embryos also had a subunit of glycine cleavage system-dihydrolipoamide dehydrogenase damaged, resulting in glycine accumulation and glycine derived-formate reduction, which further disturbed folate-mediated one-carbon metabolism, leading to 5-methyltetrahydrofolate shortage and other folate intermediates accumulation. Maternal formate supplementation increased the 5-methyltetrahydrofolate levels and ameliorated the NTDs in Slc25a32-/- embryos. The Slc25a32K235R/K235R and Slc25a32Y174C/K235R mice had no glycine accumulation, but had another formate donor-dimethylglycine accumulated and formate deficiency. Meanwhile, they suffered from the absence of all folate intermediates in mitochondria. Formate supplementation increased the folate amounts, but this effect was not restricted to the Slc25a32 mutant mice only. In summary, we established novel animal models, which enabled us to understand the function of SLC25A32 better and to elucidate the role of SLC25A32 dysfunction in human disease development and progression.

Forskolin induced remodeling of lipid droplets in rat adipocytes.

Adipose tissue is the main energy reserve of the body. When energy is required, adipocyte triglycerides stored in lipid droplets (LDs) are broken down by lipase, and free fatty acids are released to supply the physiological need. Intracellular LDs are active metabolic organelles in mammalian cells, particularly in adipocytes. The present study was aimed to investigate the morphological changes of LDs and the alternation of LD-associated perilipin family proteins during long-term lipolysis stimulated by forskolin. Primary differentiated adipocytes derived from epididymal fat pads of Sprague-Dawley (SD) rats were incubated in the presence or absence of 1 µmol/L forskolin for 24 h. Content of glycerol released to the culture medium was determined by a colorimetric assay and served as an index of lipolysis. Morphological changes of LDs were observed by Nile red staining. The mRNA level of perilipin family genes was detected by quantitative real-time PCR. The protein level and subcellular localization were examined by immunoblotting and immunofluorescence staining, respectively. The results showed that forskolin induced sustained lipolysis in differentiated adipocytes. The morphology of LDs changed in a time-dependent manner. Large clustered LDs became gradually smaller in size and eventually disappeared; in contrast, peripheral micro-LDs increased gradually in number until the cytoplasm was filled with numerous micro-LDs. The protein level of the perilipin family proteins showed obvious alternation. Mature adipocytes physiologically expressed a very low level of Plin2 protein, whereas in adipocytes stimulated with lipolytic forskolin, the protein and mRNA levels of Plin2 were significantly increased, and the increased Plin2 was specifically bound to the surface of LDs. During chronic stimulation of forskolin, the mRNA level of Plin3 was unchanged, but the mRNA levels of Plin1, Plin4 and Plin5 were significantly decreased. These results suggest that the morphology of LDs and perilipin family proteins on the surface of LDs are significantly altered during long-term lipolysis stimulated by forskolin, representing a dynamic process of the remodeling of LDs.

Mediastinal type B3 thymoma combined with germ cell tumor: cytologic diagnosis.

Primary mediastinal thymoma combined with germ cell tumor (GCT) is extremely rare, and is likely to be misdiagnosed. Here we report a case of mediastinal type B3 thymoma combined with seminoma in which the seminoma component was missed by histologic examination and initially diagnosed by using a pleural effusion sample. The patient was a 46 year old male with chest distress, cough, and supraclavicular lymph node enlargement. A large anterior mediastinal mass was revealed by diagnostic imaging. The tumor was completely removed by thoracotomy. Grossly, a solid mass about 10 cm × 8 cm × 5 cm with cystic degeneration was found. Histologic examination revealed Type B3 thymoma accompanying with multiple lymph node metastases. One year later, CT scan found an irregular mass on the right side of anterior-superior mediastinum with a large amount of effusion in the right side pleural cavity. Cytologic examination and immunostains of the pleural effusion sample revealed metastatic seminoma. Then the original surgical sample was reviewed and the seminoma component also was found besides the thymoma. To the best of our knowledge, this is the first description of type B3 thymoma combined with seminoma, diagnosed by histology and pleural effusion together. We also present a literature review.

[Impact of C/N Ratio on Nitrogen Removal Performance and N2O Release of Granular Sludge CANON Reactor].

To Explore a suitable C/N ratio for efficient nitrogen removal and simultaneously achieving N2O release reduction, ammonia-rich wastewater with sodium acetate as an organic carbon source in a granular sludge completely autotrophic nitrogen removal over nitrite (CANON) reactor under different C/N water conditions were studied to determine the reactor's nitrogen removal performance and N2O release. The results showed that the total nitrogen (TN) removal rate and the removal load tended to increase gradually with the increase of C/N, ranging from 0 to 2.0. When C/N=0, the TN removal rate was 56.50 mg·L-1 in 7 h; the highest TN removal efficiency was 49%. When C/N=2.0, the highest TN removal rate was 71.42 mg·L-1 in 7 h; the highest TN removal efficiency was 59.52%, and the contribution of CANON to nitrogen removal gradually decreased, whereas the denitrification contribution gradually increased. When △NO3--N/△TN=0.086, the contribution of CANON nitrogen removal was only 51.48% and that of denitrification was 48.52%. The N2O release volume and release ratio decreased with increasing C/N. When C/N=0, the N2O release volume and rate were the highest, namely 3.60 mg and 2.13%, respectively. The lowest N2O release volume and rate were 1.61 mg and 0.75%, respectively, when C/N=2.0.

[Short-cut Nitrification Recovery and Its Transformation into CANON Process in a Biofilm Reactor].

A short-cut nitrification process with modified polyethylene as carrier was operated to investigate the biofilm short-cut nitrification recovery using synthetic inorganic ammonia-rich wastewater as influent at 30℃ ±1℃. The short-cut nitrification was destroyed first by excessive aeration, and it was not built in 83 days under the condition of continuous aeration with DO less than 0.5 mg·L-1 and free ammonia (FA) more than 1.5 mg·L-1, which are very beneficial to short-cut nitrification. However,short-cut nitrification was realized by changing continuous aeration to intermittent aeration on 84th day, and it was proved again on 142nd day. After that, biofilm system provided a living environment for ANAMMOX bacteria, anaerobic ammonia oxidation occurred, and the biofilm short-cut nitrification process was gradually transformed into CANON process. As the load of influent and aeration increased, the total nitrogen removal efficiency and removal load increased, and the total nitrogen removal load could reach up to 2.52 kg·(m3·d)-1. Finally, in the 3rd stage, ΔNO3--N/ΔTN was 0.10 on average, which means stabe short-cut nitrification in the CANON process. Therefore, once NOB was adapted to FA, it would be not very easy to recover short-cut nitrification, while intermittent aeration was an effective way, and the nitriation process would be finally transformed into CANON process, which would further improve the short-cut nitrification stability.

EGFR mutations in non-small cell lung cancer: an audit from West China Hospital.

OBJECTIVES: To discover the incidence and characteristics of EGFR mutations in non-small cell lung cancer (NSCLC) in a single, large cohort as a part of routine diagnostic investigations. METHODS: We reviewed EGFR mutations investigated by Amplification Refractory Mutation System (ARMS) PCR (covering 29 known mutations) using DNA samples from FFPE tissue or cell clot specimens in a total of 3894 cases of NSCLC analysed between 2012-2014. RESULTS: EGFR mutations are preferentially associated with adenocarcinomaand adenosquamous histology, particularly those well to moderately differentiated, and were significantly more common in female than male patients irrespective of histological subtypes. Exon 19 deletion (45.7%) and exon 21 L858R (45.6%) accounted for the vast majority of the EGFR mutations detected, with the remaining mutations being infrequent (<2%). Compound mutations were seen in 51 (3%) of the mutant cases, the combination of these compound mutations could be classified into three subgroups according to the potential impact of individual mutations on EGFR TKI therapy. Accordingly, 7 cases had both sensitive mutations, 4 cases harboured one sensitive and one less responsive /uncertain mutation, 19 cases contained one sensitive and one resistant change, and a further 21 cases had two less responsive /uncertain mutations. CONCLUSION: Our data represents the largest EGFR mutation survey based on routine clinical diagnostic laboratory data from a single institution, it confirms the incidence and characteristics of EGFR mutations in NSCLC seen in Asian patients, and also unravels the combinatorial nature of rare compound EGFR mutations.

[Effect of Initial pH on Nitrogen Removal Performance and N2O Emission of a Sequencing Batch CANON Reactor].

A completely autotrophic nitrogen removal over nitrite (CANON) reactor with haydite as carrier was operated in a sequencing batch biofilm reactor. The effect of different initial pH on nitrogen removal performance and N2O emission was investigated using synthetic inorganic ammonia-rich wastewater as influent at 30℃±1℃. During the experiment, the pH of influent was controlled at 6.64, 6.98, 7.15, 7.88 and 7.95 under the same influent ammonia concentration condition, with hydraulic retention time of 5 hours and aeration rate of 6 m3·(m3·h)-1. The results showed that, when the initial pH was between 6.64 and 7.95, the performance of autotrophic nitrogen removal over nitrite was basically stable. The total nitrogen removal efficiencies were 81.38%, 87.32%, 92.12%, 88.21% and 86.84%, respectively. And the total nitrogen removal loads were all higher than 1.56 kg·(m3·d)-1. Initial N2O emission rates were basically equal and decreased after rising to a peak value. Besides, the lower the initial pH was, the higher the maximum N2O emission rate was. In addition, N2O emissions and ratios decreased with rising initial pH. Initial pH between 6.64 and 7.95 had little influence on nitrogen removal but N2O emissions. Initial pH should be kept at about 7.90 to achieve high efficient nitrogen removal and reduction of N2O emission synchronously.

Immunohistochemical differentiation between type B3 thymomas and thymic squamous cell carcinomas.

Type B3 thymomas and thymic squamous cell carcinomas have some overlapping histological features, so it is difficult to make the differential diagnosis between these two entities, especially when the biopsy specimen is small. Only a few markers such as CD5 and CD 117 were applied to the differential diagnosis, the purpose of this study is to identify other diagnostic markers to help making the differential diagnosis more accurate. GLUT-1, MUC-1, CD117, CD5, CEA, P63, CK19, CK5/6, CD1a and TdT were evaluated using 16 cases of type B3 thymoma and 20 cases of thymic squamous cell carcinoma. Staining scores were obtained by calculating the percentage of positive cells. The sensitivity of GLUT-1 or MUC-1 for thymic squamous cell carcinomas was highest (100%), followed by CK5/6 (95%), CD117 (90%), P63 (85%), CD5 (80%) and CEA (75%). The specificities of CD5, CD117 and CEA for thymic squamous cell carcinomas all were 100%, next was MUC-1 (56.3%), followed by GLUT-1 (50%), P63 (25%), CK5/6 (12.5%). The sensitivities of CK19, TdT, and CD1a for type B3 thymomas were 100%, 93.8% and 87.5%, respectively. The specificity of CD1a for type B3 thymomas was highest (100%), followed by TdT (95%), CK19 (10%). The reactivity of GLUT-1, MUC-1, CD117, CD5, CEA, CD1a and TdT in thymic squamous cell carcinomas and type B3 thymomas had significant difference. Usually a panel of markers is needed, if we combine GLUT-1 or MUC-1 which sensitivity for thymic squamous cell carcinomas is highest with CD5, CD117, CEA, CD1a or TdT which have high specificity, we can make the differential diagnosis effectively.

Thymic epithelial tumors: a clinicopathologic study of 249 cases from a single institution.

The WHO histological classification for thymic epithelial tumors of 2004 edition is widely used, but its prognostic value is still controversial. In the present study we collected 249 Chinese patients with thymic epithelial tumors from West China Hospital of Sichuan University since 1999-2009 to assess the prognosis relating to tumor stages, histological classifications, MG and adjuvant therapy. There were 18 cases of type A (7.2%), 97 of type AB (39.0%), 22 of type B1 (8.8%), 63 of type B2 (25.3%), 16 of type B3 (6.5%) thymomas and 33 of thymic carcinomas (13.3%). According to the Massaoka staging, there were 107 patients in stage I (43%), 73 patients in stage II (29.3%), 50 patients in stage III (20.1%) and 19 in stage IV (7.6%). 101 patients (40.6%) complicated with MG, the incidence of MG was highest in type B3 thymomas, then in B2, none of thymic carcinomas complicated with MG. Cox regression analysis showed the Masaoka stage was the most important prognostic factor. Besides of staging, WHO histological classification was also an independent prognostic factor. The age, gender, MG and adjuvant therapy have no significant influence to the prognosis of the patients.

[The clinical value of combining multiple features of CT in discriminating between benign and malignant lung lesions].

OBJECTIVE: To develop a set of combined criteria of multiple features of chest CT for discriminating between benign and malignant lung lesions. METHODS: Patients whose chest CT showed abnormalities were recruited from the West China Hospital in March and April 2010. The patients were examined with bronchoscopy and the results of CT and pathology were compared. RESULTS: A total of 105 patients participated in this study and 85 had confirmed pathological results. The CT identified 27 cases of malignant lesions, 22 of which were confirmed by the pathology. The CT identified 58 cases of benign lesions, 55 of which were confirmed by the pathology. The set of combined criteria of multiple features of chest CT had an accuracy of 90.59%, a sensitivity of 88.00%, and a specificity of 91.67% in diagnosing benign and malignant lung lesions. CONCLUSION: The combined criteria of multiple imaging signs of CT have good clinical values for diagnosing malignant lung lesions.

Cytological differential diagnosis among adenocarcinoma, epithelial mesothelioma, and reactive mesothelial cells in serous effusions by immunocytochemistry.

The objective of the study is to estimate the expression of some antibodies in the metastatic adenocarcinomas, malignant epithelial mesotheliomas, and reactive mesothelial cells in serous effusions and to choose effective panel to the differential diagnosis. Totally 113 effusion cytology samples (80 pleural fluid, 30 ascitic, and 3 pericardial fluid) from 60 cases of metastatic adenocarcinoma (ACA), 18 cases of malignant epithelial mesothelioma (MM), and 35 cases of reactive mesothelium (RM) were included in this study. The cytological diagnoses of these cases were confirmed by histopathology or clinical datas. Smears and cell blocks were prepared for each case. Immunocytochemical study was performed on the cell block sections. The sensitivity of E-cadherin, CEA, MOC-31, and Ber-EP4 for adenocarcinoma was 86.7%, 80%, 70%, and 76.4%, respectively. The specificity was 98.1%, 96.2%, 92.5%, and 86.8%, respectively. The sensitivity of calretinin, HBME-1, and thrombomodulin for RM/MM was 83%, 79.2%, and 47.2% respectively. The specificity was 88.3%, 21.7%, and 70%, respectively. The expression of E-cadherin, CEA, MOC-31, Ber-EP4, calretinin, and thrombomodulin showed significant difference between ACA and RM/MM (P < 0.01). The reactivity of EMA and Des showed significant difference between RM and MM (P < 0.01). In our opinion, the antibody panel that consists of E-cadherin, CEA, calretinin, and thrombomodulin should be the best for differential diagnosis between metastatic adenocarcinomas and RM/MM in serous effusions. EMA and Des should be used to differentiate malignant epithelial mesothelioma and reactive mesothelial cells. EMA positive and Des negative favor MM, while Des positive and EMA negative favor RM.

Adrenal sarcomatoid carcinoma: a rare case depicted on multi-detector row computed tomography.

Primary adrenal sarcomatoid carcinoma is rare malignant tumor with the characteristics of carcinoma and sarcoma. To date, only one case of primary sarcomatoid carcinoma in the adrenal gland was reported. We present here computed tomography appearance and pathological features of the case with primary adrenal sarcomatoid carcinoma confirmed by pathology. In addition, a brief review of the relevant literature is presented.

[Diagnosis of hematolymphoid malignancy by using effusion fluid cytology specimens: a study of 33 cases].

OBJECTIVE: To study the diagnostic accuracy of hematolymphoid malignancy by using effusion fluid cytology specimens and to evaluate the values of immunocytochemistry for this assay. METHODS: The cytospin preparations/smears and cell block sections of effusion cytology specimens from 33 cases of hematolymphoid malignancy were retrospectively reviewed. Immunocytochemical study was performed. In selected cases, in-situ hybridization for Epstein-Barr virus-encoded RNA and immunoglobulin and T-cell receptor gene rearrangement study were carried out as indicated. RESULTS: There were 33 cases of hematolymphoid malignancy, including 12 cases of T-lymphoblastic leukemia/lymphoma, 16 cases of mature B cell neoplasm (including 9 cases of diffuse large B-cell lymphoma, 2 cases of Burkitt lymphoma, 2 cases of plasmacytoma/multiple myeloma, 2 cases of B-small lymphocytic leukemia/lymphoma and 1 case of mantle cell lymphoma), 3 cases of mature T or NK-cell neoplasm (including 1 case of extranodal nasal NK/T-cell lymphoma, 1 case of angioimmunoblastic T-cell lymphoma and 1 case of T-cell prolymphocytic leukemia), 1 case of myeloid sarcoma and 1 case of mast cell sarcoma. Amongst the 33 cases studied, 16 represented disease relapses, including 8 cases of diffuse large B-cell lymphoma, 2 cases of plasmacytoma/multiple myeloma, 2 cases of B-small lymphocytic leukemia/lymphoma, 1 case of T-lymphoblastic leukemia/lymphoma, 1 case of angioimmunoblastic T-cell lymphoma, 1 case of mantle cell lymphoma and 1 case of mast cell sarcoma. The remaining 17 cases showed serous effusion as the primary manifestation, with the diagnosis primarily made upon cytologic examination. The cytologic findings seen in all the 33 cases studied were in agreement with the corresponding histologic diagnosis. CONCLUSIONS: Diagnosis of hematolymphoid malignancy by effusion fluid cytology specimens is possible, especially when coupled with the clinical history, immunophenotype, in-situ hybridization and gene rearrangement study findings. This is especially so for cases with disease relapses.

[Significance of combining detection of E-cadherin, carcinoembryonic antigen, and calretinin in cytological differential diagnosis of serous effusion].

BACKGROUND & OBJECTIVE: The morphological characteristics of metastatic adenocarcinoma cells,malignant mesothelioma cells,and reactive mesothelial cells in serous effusions are similar. Sometimes, it is difficult to make a correct diagnosis just based on the cytological characteristics. Although now immunocytochemistry has been widely used in this field,yet only a few antibodies were reported in China,which can't help to make a correct differential diagnosis. This study was to evaluate the value of combining detection of E-cadherin,carcinoembryonic antigen (CEA),and calretinin in cytological differential diagnosis of serous effusion. METHODS: A total of 93 effusion cytology specimens (66 pleural fluid samples, 24 ascites samples, 3 pericardial fluid samples)from 55 cases of metastatic adenocarcinoma, 6 cases of malignant mesothelioma, and 32 cases of reactive mesothelium were collected. All cases were confirmed histologically or clinically. Materials from each case were prepared in HE staining smears,and cell blocks,immunocytochemical staining was performed on cell block sections. RESULTS: In diagnosing metastatic adenocarcinoma, the sensitivities of E-cadherin, and CEA were 85.5%(47/55), and 79.6% (43/55), the specificities were 100% (38/38), and 97.4% (37/38);if E-cadherin and CEA were used together,the positive rate was 96.4%(53/55). The sensitivity of calretinin for mesothelial cells was 81.6%(31/38),and the specificity was 87.2% (48/55). CONCLUSION: The combination of E-cadherin, CEA, and calretinin is a very useful antibody panel in cytological differential diagnosis for metastatic adenocarcinoma and mesothelial cells in serous effusion.