👤 Manuel Schiff

Regardless of its mechanism, hyperammonemia can cause coma and death, and requires urgent management. This study aims at describing the landscape of causes of hyperammonemia in adults and at evaluating the performance of targeted next-generation sequencing (NGS) in this setting. We analyzed two cohorts. The first included patients aged ≥15 years presenting with hyperammonemia ≥100 μmol/L at Necker-Enfants Malades (NEM) University Hospital for 10 years and at Toulouse University Hospital for 1.5 years. The second cohort included patients who underwent genetic testing for inherited metabolic disease (IMD) via targeted NGS at NEM hospital over a 5 year-period, regardless of their inclusion in the first cohort, all with hyperammonemia ≥100 μmol/L after age 15. We included 184 patients in the first cohort, with a median peak ammonia concentration of 155 μmol/L. Among them, 61 patients (33 %) presented with coma. Non-genetic liver failure or portosystemic shunt was present in 133 patients. Twenty-three patients had received asparaginase treatment (none with coma despite a median ammonia level of 257 μmol/L), 7 had received valproic acid, 3 had undergone surgical ureterorectal anastomosis, 2 had multiple myeloma, 1 was receiving 5-Fluorouracil (5FU) for metastatic gastrointestinal cancer, 1 had disseminated atypical mycobacteriosis with Mycobacterium genavense (urease-producing bacteria) in a renal transplant setting and 13 had a genetically confirmed IMD diagnosed in adulthood. In the second cohort of 17 patients, genetic testing was positive in 5 of 6 patients with IMD-suggestive biochemical profiles (2 CPS1 deficiencies, 1 OTC deficiency, 1 multiple acyl-coA dehydrogenase deficiency, and 1 lysinuric protein intolerance), and negative in patients without biochemical profile suggesting an IMD. Among them, four patients suffered from protein malnutrition related to various severe conditions (gastric bypass, metastatic colorectal adenocarcinoma, Duchenne muscular dystrophy, and short bowel syndrome). The causes of hyperammonemia in adults are varied. In cases of acute episodes without unequivocal metabolic profiles (when unwell) and with an acquired identified cause of hyperammonemia, genetic investigations had a low yield. Show less

Despite intensive study of the mechanisms of chemotherapeutic drug resistance in human breast cancer, few reports have systematically investigated the mechanisms that underlie resistance to the chemotherapy-sensitizing agent tumor necrosis factor (TNF)-alpha. Additionally, the relationship between TNF-alpha resistance mediated by MEK5/Erk5 signaling and epithelial-mesenchymal transition (EMT), a process associated with promotion of invasion, metastasis, and recurrence in breast cancer, has not previously been investigated. To compare differences in the proteome of the TNF-alpha resistant MCF-7 breast cancer cell line MCF-7-MEK5 (in which TNF-alpha resistance is mediated by MEK5/Erk5 signaling) and its parental TNF-a sensitive MCF-7 cell line MCF-7-VEC, two-dimensional gel electrophoresis and high performance capillary liquid chromatography coupled with tandem mass spectrometry approaches were used. Differential protein expression was verified at the transcriptional level using RT-PCR assays. An EMT phenotype was confirmed using immunofluorescence staining and gene expression analyses. A short hairpin RNA strategy targeting Erk5 was utilized to investigate the requirement for the MEK/Erk5 pathway in EMT. Proteomic analyses and PCR assays were used to identify and confirm differential expression of proteins. In MCF-7-MEK5 versus MCF-7-VEC cells, vimentin (VIM), glutathione-S-transferase P (GSTP1), and creatine kinase B-type (CKB) were upregulated, and keratin 8 (KRT8), keratin 19 (KRT19) and glutathione-S-transferase Mu 3 (GSTM3) were downregulated. Morphology and immunofluorescence staining for E-cadherin and vimentin revealed an EMT phenotype in the MCF-7-MEK5 cells. Furthermore, EMT regulatory genes SNAI2 (slug), ZEB1 (delta-EF1), and N-cadherin (CDH2) were upregulated, whereas E-cadherin (CDH1) was downregulated in MCF-7-MEK5 cells versus MCF-7-VEC cells. RNA interference targeting of Erk5 reversed MEK5-mediated EMT gene expression. This study demonstrates that MEK5 over-expression promotes a TNF-alpha resistance phenotype associated with distinct proteomic changes (upregulation of VIM/vim, GSTP1/gstp1, and CKB/ckb; and downregulation of KRT8/krt8, KRT19/krt19, and GSTM3/gstm3). We further demonstrate that MEK5-mediated progression to an EMT phenotype is dependent upon intact Erk5 and associated with upregulation of SNAI2 and ZEB1 expression. Show less

A rat model to study the local effects of testicular vein ligation is described. One hour after unilateral testicular vein ligation, the testicular concentration of testosterone was significantly greater (P less than 0.01) on the side that was ligated than in the contralateral testis (177.1 +/- 19.7 [SEM] ng/gm of tissue as compared with 108.8 +/- 11.8 ng/gm of tissue, respectively). This effect was not seen 1 week after the testicular vein ligation. The testosterone concentration in the ligated testis was also higher than that in sham-operated animals. These differences in testicular testosterone concentration were not associated with changes in peripheral serum testosterone levels. ligation of the testicular vein causes an acute rise in the testicular concentration of testosterone and may thus mediate changes in testicular function. Show less