Insulin secretory granules labelled with phogrin-fluorescent proteins show alterations in size, mobility and responsiveness to glucose stimulation in living β-cells

The intracellular life of insulin secretory granules (ISGs) from biogenesis to secretion depends on their structural (e.g. size) and dynamic (e.g. diffusivity, mode of motion) properties. Thus, it would be useful to have rapid and robust measurements of such parameters in living β-cells. To provide such measurements, we have developed a fast spatiotemporal fluctuation spectroscopy. We calculate an imaging-derived Mean Squared Displacement (iMSD), which simultaneously provides the size, average diffusivity, and anomalous coefficient of ISGs, without the need to extract individual trajectories. Clustering of structural and dynamic quantities in a multidimensional parametric space defines the ISGs’ properties for different conditions. First, we create a reference using INS-1E cells expressing proinsulin fused to a fluorescent protein (FP) under basal culture conditions and validate our analysis by testing well-established stimuli, such as glucose intake, cytoskeleton disruption, or cholesterol overload. After, we investigate the effect of FP-tagged ISG protein markers on the structural and dynamic properties of the granule. While iMSD analysis produces similar results for most of the lumenal markers, the transmembrane marker phogrin-FP shows a clearly altered result. Phogrin overexpression induces a substantial granule enlargement and higher mobility, together with a partial de-polymerization of the actin cytoskeleton, and reduced cell responsiveness to glucose stimulation. Our data suggest a more careful interpretation of many previous ISG-based reports in living β-cells. The presented data pave the way to high-throughput cell-based screening of ISG structure and dynamics under various physiological and pathological conditions

Time-dependent metabolic disorders induced by short-term exposure to polystyrene microplastics in the Mediterranean mussel Mytilus galloprovincialis.

Abstract In the modern society, plastic has achieved a crucial status in a myriad of applications because of its favourable properties. Despite the societal benefits, plastic has become a growing global concern due to it is persistence and bioavailability as microplastics (MPs) to aquatic biota. In order to provide mechanistic insights into the early toxicity effects of MPs on aquatic invertebrates, a short-term (up to 72 h) exposure to 3 µm red polystyrene MPs (50 particles/mL) was conducted on marine mussels Mytilus galloprovincialis, selected as model organism for their ability to ingest MPs and their commercial relevance. The use of protonic Nuclear Magnetic Resonance (1H NMR)-based metabolomics, combined with chemometrics, enabled a comprehensive exploration at fixed exposure time-points (T24, T48, T72) of the impact of MPs accumulated in mussel digestive glands, chosen as the major site for pollutants storage and detoxification processes. In detail, 1H NMR metabolic fingerprints of MP-treated mussels were clearly separated from control and grouped for experimental time-points by a Principal Component Analysis (PCA). Numerous metabolites, including amino acids, osmolytes, metabolites involved in energy metabolism, and antioxidants, participating in various metabolic pathways significantly changed over time in MP-exposed mussel digestive glands related to control, reflecting also the fluctuations in MPs accumulation and pointing out the occurrence of disorders in amino acid metabolism, osmotic equilibrium, antioxidant defense system and energy metabolism. Overall, the present work provides the first insights into the early mechanisms of toxicity of polystyrene MPs in marine invertebrates

Hypoxia inducible factor-1 alpha expression is increased in infected positive HPV16 DNA oral squamous cell carcinoma and positively associated with HPV16 E7 oncoprotein

<p>Abstract</p> <p>Background</p> <p>There is increasing evidence for the role of High Risk (HR) Human PapillomaVirus (HPV) in the pathogenesis of Oral Squamous Cell Carcinoma (OSCC). The E6 and E7 oncogenes from HR HPVs are responsible for the deregulation of p53 and pRB proteins involved in cell cycle and apoptotic pathways. In cell lines experiments, the HPV E7 protein seems to be able to enhance Hypoxia Inducible Factor-1 alpha (HIF-1α) activity, normally involved in the response to hypoxia and able to enhance angiogenesis.</p> <p>Results</p> <p>We studied tumor specimens from 62 OSCC; a higher prevalence of tumors in TNM stage II and also in pT2 class between OSCC infected positive HPV16 DNA than non-infected ones was observed. HIF-1α positivity was detected throughout the analysed fields, not associated with areas of necrosis and also observed in cells immediately adjacent to blood vessels. A significant increase in mean values of the HIF-1α labeling indexes was observed for pT1-T2, as well for stage I-II, in the infected positive HPV16 DNA tumors than non-infected ones. HIF-1α and HPV16 E7 labeling indexes showed a significantly positive correlation which suggested a positive association between HPV16 E7 and HIF-1α expression.</p> <p>Conclusions</p> <p>In our specimens HIF-1α immunoreactivity hints for an O₂-independent regulatory mechanism in infected positive HPV16 DNA tumors, especially for pT1-T2 and stage I-II tumors, suggesting a very early involvement in the development of HPV-induced OSCC. HIF-1α and HPV16 E7 labeling indexes suggest also a positive association between the two proteins in infected positive HPV16 DNA OSCC.</p

Increased expression of transketolase-like-1 in papillary thyroid carcinomas smaller than 1.5 cm in diameter is associated with lymph-node metastases.

BACKGROUND: Patients with small papillary thyroid carcinoma (PTC) may have a high incidence of regional lymph-node (LN) metastases at presentation, and these are considered to be an independent risk factor for tumor recurrence. A mutated transketolase transcript (TKTL1) has been found up-regulated in different human malignancies, and strong TKTL1 protein expression has been associated with aggressiveness and poor patient survival in several epithelial cancers. METHODS: TKTL1 protein expression was analyzed in 256 consecutive cases of PTCs <or=1.5 cm by immunohistochemistry with a specific anti-TKTL1 antibody. RNA analysis was performed by real-time polymerase chain reaction (PCR) in all cases for which frozen material was available, which resulted in 55 fragments of PTC. RESULTS: Increased levels of TKTL1 transcript were detected in 50 of 55 analyzed tumors compared with their corresponding normal tissues. Significant differences in TKTL1 transcript levels were found between cases of PTC with and without LN metastases. In primary tumors, immunoreactivity for TKTL1 was detected in the majority of cases, ranging from 0% to 95.0% (mean, 50.11% +/- 27.75%). A significant association was found between TKTL1 protein expression and the presence of multifocality, bilaterality, extrathyroidal extension, vascular invasion, sclerosis, and LN metastases. In cases with LN metastases, a positive correlation was found between the TKTL1 protein expression in primary tumors and the number of metastatic LNs as well as the diameter of the largest metastatic area in LNs. CONCLUSIONS: These findings suggest that TKTL1 overexpression in PTC <or=1.5 cm may be considered a factor that facilitates tumor growth and progression. (c) 2008 American Cancer Society

Perception of the role of food and dietary modifications in patients with inflammatory bowel disease: Impact on lifestyle

Background: Diet has a relevant role in triggering symptoms in inflammatory bowel disease (IBD) from the patients’ perspective, but there is gap the between patients’ and doctors’ perceptions. Few studies have addressed this topic. The aim of this study was to evaluate food habits and nutrition knowledge in a homogeneous cohort of patients with IBD from southern Italy. Methods: 167 consecutive patients with IBD were recruited. The survey was based on the administration of a semi-structured questionnaire assessing demographics, disease features, dietary behavior, and food intolerance. Results: The majority of patients did not consider food a cause of their disease. However more than 80% changed their diet after the diagnosis and most report an improvement in symptoms. Spiced and seasoned foods, dairy products, vegetables, and fruit were often avoided. A dairy-free diet was adopted by 33.7%. Food choices were based on self-experience and not on medical counselling. Dietary modifications deeply impact on lifestyle. Conclusions: Most of the patients with IBD set diet and lifestyle on self-experience and give up many foods. This has an impact on psychosocial functioning and can lead to nutritional deficiencies. High quality studies are warranted to assess evidence-based dietary strategies and develop patient-targeted dietary recommendations

Effects of pesticides on Chelon labrosus (Risso, 1827) evaluated by enzymatic activities along the north eastern Sicilian coastlines (Italy)

Pesticides are frequently applied to agricultural activities to improve harvest, in terms of yield and product quality. Useful tools for ecotoxicological studies of marine ecosystems are based on biomarker application on bioindicator key fish species. The main aim of the present study was to detect the potential presence of pesticides in a polluted coastal marine environment, namely Milazzo Gulf, situated in the north eastern coast of Sicily (Italy), by measuring the enzymatic activities of the ecotoxicological biomarkers acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in brain and blood samples of Chelon labrosus. Also, Marinello Reserve was selected as a reference site. The data showed a significant inhibition in AChE (81%) and BChE (71%) activities in fish from Milazzo Gulf in respect to those from the reference site. The esterase inhibition is primarily due to the presence of organophosphorus insecticides and carbamates that resulted, in Milazzo Gulf, higher in concentration in respect to the reference quality standard decree (D.M. 260, 2010). The results obtained in this study confirm the suspected presence of insecticides in waters and fish from Milazzo Gulf, which may lead to a considerable hazard to humans. This study confirms the relevant advantages of the biomarker approach on fish species in the ecotoxicological evaluation of marine environments