ZMAT3 hypomethylation contributes to early senescence of preadipocytes from healthy first‐degree relatives of type 2 diabetics

Senescence of adipose precursor cells (APC) impairs adipogenesis, contributes to the age‐related subcutaneous adipose tissue (SAT) dysfunction, and increases risk of type 2 diabetes (T2D). First‐degree relatives of T2D individuals (FDR) feature restricted adipogenesis, reflecting the detrimental effects of APC senescence earlier in life and rendering FDR more vulnerable to T2D. Epigenetics may contribute to these abnormalities but the underlying mechanisms remain unclear. In previous methylome comparison in APC from FDR and individuals with no diabetes familiarity (CTRL), ZMAT3 emerged as one of the top‐ranked senescence‐related genes featuring hypomethylation in FDR and associated with T2D risk. Here, we investigated whether and how DNA methylation changes at ZMAT3 promote early APC senescence. APC from FDR individuals revealed increases in multiple senescence markers compared to CTRL. Senescence in these cells was accompanied by ZMAT3 hypomethylation, which caused ZMAT3 upregulation. Demethylation at this gene in CTRL APC led to increased ZMAT3 expression and premature senescence, which were reverted by ZMAT3 siRNA. Furthermore, ZMAT3 overexpression in APC determined senescence and activation of the p53/p21 pathway, as observed in FDR APC. Adipogenesis was also inhibited in ZMAT3‐overexpressing APC. In FDR APC, rescue of ZMAT3 methylation through senolytic exposure simultaneously downregulated ZMAT3 expression and improved adipogenesis. Interestingly, in human SAT, aging and T2D were associated with significantly increased expression of both ZMAT3 and the P53 senescence marker. Thus, DNA hypomethylation causes ZMAT3 upregulation in FDR APC accompanied by acquisition of the senescence phenotype and impaired adipogenesis, which may contribute to FDR predisposition for T2D.     

Indole derivatives as dual-effective agents for the treatment of neurodegenerative diseases: Synthesis,biological evaluation,and molecular modeling studies

Several indole derivatives, that were highly potent ligands of GluN2B-subunit-containing N-methyl-d-aspartate (NMDA) receptor, also demonstrated antioxidant properties in ABTS method. In particular, the 2-(4-benzylpiperidin-1-yl)-1-(5-hydroxy-1H-indol-3-yl)ethanone (1) proved to be a dual-effective neuroprotective agent. With the aim to increase the antioxidant properties we added a catechol moiety onto piperidine moiety. The designed hybrid derivative 3,4-dihydroxy-N-[1-[2-(5-hydroxy-1H-indol-3-yl)-2-oxoethyl]piperidin-4-yl]benzamide (10) was the most effective antioxidant agent (>94.1 ± 0.1% of inhibition at 17 μM) and showed GluN2B/NMDA receptor affinity at low micromolar concentration (IC₅₀ 0.66 μM). By means of computational studies we explored the effect of the presence of this antioxidant fragment during the recognition process to binding pocket.     

Influence measures based on the volume of confidence ellipsoids for GEE

The generalized estimating equations (GEE) derived by Liang and Zeger to analyze longitudinal data have been used in a wide range of medical and biological applications. To make regression a useful and meaningful statistical tool, emphasis should be placed not only on inference or fitting, but also on diagnosing potential data problems. Most of the usual diagnostics for linear regression models have been generalized for GEE. However, global influence measures based on the volume of confidence ellipsoids are not available for GEE analysis. This article presents an extension of these measures that is valid for correlated‐measures regression analysis using GEEs. The proposed measures are illustrated by an analysis of epileptic seizure count data arising from a study of prograbide as an adjuvant therapy for partial seizures and some simulated data sets.     

Fine needle aspiration biopsy of three cases of squamous cell carcinoma presenting as a thyroid mass: cytological findings and differential diagnosis

M. Rosa and K. Toronczyk Fine needle aspiration biopsy of three cases of squamous cell carcinoma presenting as a thyroid mass: cytological findings and differential diagnosis Objective: Primary squamous cell carcinomas of the thyroid gland are extremely rare, comprising about 1% of thyroid malignancies. Although squamous cell carcinomas are readily identified as such on aspiration cytology in the majority of cases, the differentiation of primary versus metastatic tumour might not always be easy. Herein, we report three cases of squamous cell carcinomas involving the thyroid gland. Methods: Fine needle aspiration cytology (FNAC) was performed in three patients with a thyroid mass using standard guidelines. Smears were stained with Diff‐Quik and Papanicolaou stains. Results: Two patients were male and one was female, aged 59, 45 and 35 years, respectively. In all three patients a thyroid mass was present. FNAC smears in all cases showed cytological features of squamous cell carcinoma including keratinization and necrosis. After clinical and cytological correlation, one case appeared to be primary, one case metastatic, and in the third case no additional clinical information or biopsy follow‐up was available for further characterization. Conclusions: Because primary squamous cell carcinoma of the thyroid is a rare finding, metastatic squamous cell carcinoma should always be excluded first. Metastatic disease usually presents in the setting of widespread malignancy, therefore a dedicated clinical and radiological investigation is necessary in these cases. In both clinical scenarios the patient’s prognosis is poor.     

Enthoprotin: a novel clathrin-associated protein identified through subcellular proteomics

Despite numerous advances in the identification of the molecular machinery for clathrin-mediated budding at the plasma membrane, the mechanistic details of this process remain incomplete. Moreover, relatively little is known regarding the regulation of clathrin-mediated budding at other membrane systems. To address these issues, we have utilized the powerful new approach of subcellular proteomics to identify novel proteins present on highly enriched clathrin-coated vesicles (CCVs). Among the ten novel proteins identified is the rat homologue of a predicted gene product from human, mouse, and Drosophila genomics projects, which we named enthoprotin. Enthoprotin is highly enriched on CCVs isolated from rat brain and liver extracts. In cells, enthoprotin demonstrates a punctate staining pattern that is concentrated in a perinuclear compartment where it colocalizes with clathrin and the clathrin adaptor protein (AP)1. Enthoprotin interacts with the clathrin adaptors AP1 and with Golgi-localized, gamma-ear-containing, Arf-binding protein 2. Through its COOH-terminal domain, enthoprotin binds to the terminal domain of the clathrin heavy chain and stimulates clathrin assembly. These data suggest a role for enthoprotin in clathrin-mediated budding on internal membranes. Our study reveals the utility of proteomics in the identification of novel vesicle trafficking proteins.     

Quantifying apoprotein synthesis in rodents: coupling LC-MS/MS analyses with the administration of labeled water

Stable isotope tracer studies of apoprotein flux in rodent models present difficulties as they require working with small volumes of plasma. We demonstrate the ability to measure apoprotein flux by administering either (2)H- or (18)O-labeled water to mice and then subjecting samples to LC-MS/MS analyses; we were able to simultaneously determine the labeling of several proteolytic peptides representing multiple apoproteins. Consistent with relative differences reported in the literature regarding apoprotein flux in humans, we found that the fractional synthetic rate of apoB is greater than apoA1 in mice. In addition, the method is suitable for quantifying acute changes in protein flux: we observed a stimulation of apoB production in mice following an intravenous injection of Intralipid and a decrease in apoB production in mice treated with an inhibitor of microsomal triglyceride transfer protein. In summary, we demonstrate a high-throughput method for studying apoprotein kinetics in rodent models. Although notable differences exist between lipoprotein profiles that are observed in rodents and humans, we expect that the method reported here has merit in studies of dyslipidemia as i) rodent models can be used to probe target engagement in cases where one aims to modulate apoprotein production and ii) the approach should be adaptable to studies in humans.     

Effectiveness of cognitive behaviour therapy for the treatment of catastrophisation in patients with fibromyalgia: a randomised controlled trial

Introduction  

No randomised, controlled trials have been conducted to date on the efficacy of psychological and pharmacological treatments of pain catastrophising (PC) in patients with fibromyalgia. Our aim in this study was to assess the effectiveness of cognitive-behaviour therapy (CBT) and the recommended pharmacological treatment (RPT) compared with treatment as usual (TAU) at the primary care level for the treatment of PC in fibromyalgia patients.     

The anti-fibrotic effect of liver growth factor is associated with decreased intrahepatic levels of matrix metalloproteinases 2 and 9 and transforming growth factor beta 1 in bile duct-ligated rats

Liver growth factor (LGF), a mitogen for liver cells, behaves as an anti-fibrotic agent even in extrahepatic sites, but its mechanistic basis is unknown. We aimed to determine the intrahepatic expression pattern of key modulators of liver fibrosis in bile duct-ligated rats (BDL) after injection of LGF. BDL rats received either LGF (4.5 microg/ratXdose, two doses/week, at time 0 or 2 or 5w after operation, depending on the group (BDL+LGF groups, n=20) or saline (BDL+S groups, n=20). Groups were compared in terms of fibrosis (histomorphometry), liver function (aminopyrine breath test), matrix metalloproteinases MMP-2 and MMP-9, transforming growth factor beta 1 (TGF-beta1) and liver endoglin content (Western blotting), and serum tissue inhibitor of metalloproteinases 1 (TIMP-1) levels (ELISA). In BDL+LGF rats, the fibrotic index was significantly lower at 5w, p=0.006, and at 8w, p=0.04, than in BDL+S rats. Liver function values in BDL+LGF rats were higher than those obtained in BDL+S rats (80% at 5w and 79% at 8w, versus 38% and 29%, p<0.01, taking healthy controls as 100%). Notably, in BDL+LGF rats the intrahepatic expression levels of both MMPs were lower at 2w (MMP-2, p=0.03; MMP-9, p=0.05) and 5w (MMP-2, p=0.05, MMP-9, p=0.04). In addition, the hepatic TGF-beta1 level in BDL+LGF rats was lower at 2w (36%, p=0.008), 5w (50%) and 8wk (37%), whereas intrahepatic endoglin expression remained constant in all BDL rats studied. LGF ameliorates liver fibrosis and improves liver function in BDL rats. The LGF-induced anti-fibrotic effect is associated with a decreased hepatic level of MMP-2, MMP-9 and TGF-beta1 in fibrotic rats.     

Exopolysaccharides production in Lactobacillus bulgaricus and Lactobacillus casei exploiting microfiltration

The physiology of Lactobacillus delbrueckii ssp. bulgaricus and Lactobacillus casei, extensively used in the dairy industry, was studied in order to evaluate key parameters in the synthesis of exopolysaccharides and to improve their production through novel fermentation processes. Selected strains were studied in shake flasks and in fermentor experiments using glucose and lactose as main carbon sources and bacto casitone as the only complex component, in a temperature range between 35 and 42°C. The production of exopolysaccharides was monitored and correlated to the growth conditions using both a colorimetric assay and chromatographic methods. Fermentor experiments in batch mode yielded 100 mg l⁻¹ of EPS from L. bulgaricus and 350 mg l⁻¹ from L. casei. Moreover, the use of a microfiltration (MF) bioreactor resulted in exopolysaccharides (EPS) concentrations threefold and sixfold those of batch experiments, respectively. The monosaccharidic composition of the two analyzed polymers differed from those previously reported. The optimization of the production of EPSs using the MF fermentation strategy could permit the use of these molecules produced by generally recognised as safe (GRAS) microorganisms in the place of other polysaccharides in the food industry.