Novel isoforms of the giant protein Titin in the regulation of lymphocyte trafficking

Titin (TTN) is the largest protein in the human genome and is mainly known as the third component of the sarcomere together with actin and myosin. In this context, TTN is responsible for maintaining muscle passive stiffness providing structural, scaffolding, and mechano-signaling properties. Here, we describe unexpected role of TTN in the regulation of human lymphocyte physiology. Human T lymphocytes express three novel TTN isoforms exhibiting cell-specific expression and different distribution to subcellular compartments. By performing fluorescence microscopy 3D imaging, we found that the LTTN1 and the LTTN2 isoforms span cytosolic as well as nuclear compartments, whereas the smallest LTTN3 isoform seems to be restricted to the cytosol. Recent data from our laboratory showed that LTTN1 covers a pivotal role in immune system physiology. Indeed, LTTN1 controls microvilli stricture and, thus, selectin-mediated tethering and rolling, chemokinetriggered integrin activation, chemotaxis, and in vitro cell deformability underflow. The new in vivo data confirmed LTTN1 putative role in T lymphocytes resilience to mechanical stress induced by passive deformation in the microcirculation. This makes LTTN1 a crucial player not only in multiple steps of T lymphocyte trafficking but, and importantly, in T lymphocyte survival. Besides LTTN1, we demonstrated that also LTTN3 is involved in the inside-out pathway of chemokinetriggered integrin activation. LTTN3 facilitates chemotaxis and controls chemokine-triggered integrin-mediated adhesion. Accordingly, it mediates RhoA and Rac1 small GTPases activation. Thus, while LTTN1 is a critical housekeeping regulator of T lymphocyte physiology, our results also suggest a possible involvement of LTTN3 as a central modulator of chemokine-induced signal transduction in T lymphocytes

Capillary electrochromatography : a versatile instrumental technique for nanodomain interaction studies

This doctoral thesis describes the development of a miniaturized capillary electrochromatography (CEC) technique suitable for the study of interactions between various nanodomains of biological importance. The particular focus of the study was low-density lipoprotein (LDL) particles and their interaction with components of the extracellular matrix (ECM). LDL transports cholesterol to the tissues through the blood circulation, but when the LDL level becomes too high the particles begin to permeate and accumulate in the arteries. Through binding sites on apolipoprotein B-100 (apoB-100), LDL interacts with components of the ECM, such as proteoglycans (PGs) and collagen, in what is considered the key mechanism in the retention of lipoproteins and onset of atherosclerosis. Hydrolytic enzymes and oxidizing agents in the ECM may later successively degrade the LDL surface. Metabolic diseases such as diabetes may provoke damage of the ECM structure through the non-enzymatic reaction of glucose with collagen. In this work, fused silica capillaries of 50 micrometer i.d. were successfully coated with LDL and collagen, and steroids and apoB-100 peptide fragments were introduced as model compounds for interaction studies. The LDL coating was modified with copper sulphate or hydrolytic enzymes, and the interactions of steroids with the native and oxidized lipoproteins were studied. Lipids were also removed from the LDL particle coating leaving behind an apoB-100 surface for further studies. The development of collagen and collagen decorin coatings was helpful in the elucidation of the interactions of apoB-100 peptide fragments with the primary ECM component, collagen. Furthermore, the collagen I coating provided a good platform for glycation studies and for clarification of LDL interactions with native and modified collagen. All methods developed are inexpensive, requiring just small amounts of biomaterial. Moreover, the experimental conditions in CEC are easily modified, and the analyses can be carried out in a reasonable time frame. Other techniques were employed to support and complement the CEC studies. Scanning electron microscopy and atomic force microscopy provided crucial visual information about the native and modified coatings. Asymmetrical flow field-flow fractionation enabled size measurements of the modified lipoproteins. Finally, the CEC results were exploited to develop new sensor chips for a continuous flow quartz crystal microbalance technique, which provided complementary information about LDL ECM interactions. This thesis demonstrates the potential of CEC as a valuable and flexible technique for surface interaction studies. Further, CEC can serve as a novel microreactor for the in situ modification of LDL and collagen coatings. The coatings developed in this study provide useful platforms for a diversity of future investigations on biological nanodomains.Not availabl

Influence of environmental and anthropogenic parameters on thallium oxidation state in natural waters

The abandoned mining area of Valdicastello Carducci (Tuscany, Italy) is characterized by the massive presence of thallium in the acid mine drainages and in the valley stream crossing the region. We previously found that Tl(III), generally considered the less stable oxidation state of thallium, is present both in the stream and in tap water distributed in the area, whereas acid mine drainages only contain Tl(I). These findings posed some concern related to the reactivity and dispersion of this toxic element in the environment. Since the valence state of thallium determines its toxicity, distribution and mobility, the study of thallium redox speciation appears crucial to understand its environmental behaviour. In this work, water samples collected from the mine drainages and the contaminated stream were adopted as model to study the distribution of aqueous Tl(I)/Tl(III) as a function of light exposure and solution properties and composition. The influence of three light sources and organic acids was evaluated. Thallium speciation was also assessed in tap water after treatment with common oxidizing agents, and in the rust crust collected from the public waterworks

Human exposure to thallium through tap water: A study from Valdicastello Carducci and Pietrasanta (northern Tuscany, Italy)

A geological study evidenced the presence of thallium (Tl) at concentrations of concern in groundwaters near Valdicastello Carducci (Tuscany, Italy). The source of contamination has been identified in the Tl-bearing pyrite ores occurring in the abandoned mining sites of the area. The strongly acidic internal waters flowing in the min- ing tunnels can reach exceptional Tl concentrations, up to 9000 μg/L. In September 2014 Tl contamination was also found in the tap water distributed in the same area (from 2 to 10 μg/L). On October 3, 2014 the local authorities imposed a Do Not Drink order to the population. Here we report the results of the exposure study carried out from October 2014 to October 2015, and aimed at quantifying Tl levels in 150 urine and 318 hair samples from the population of Valdicastello Carducci and Pietrasanta. Thallium was quantified by inductively coupled plasma — mass spectrometry (ICP-MS). Urine and hair were chosen as model matrices indicative of different time periods of exposure (short-term and long- term, respectively). Thallium values found in biological samples were correlated with Tl concentrations found in tap water in the living area of each citizen, and with his/her habits. Thallium concentration range found in hair and urine was 1–498 ng/g (values in unexposed subjects 0.1–6 ng/g) and 0.046–5.44 μg/L (reference value for the European population 0.006 μg/L), respectively. Results show that Tl levels in biological samples were significantly associat- ed with residency in zones containing elevated water Tl levels. The kinetics of decay of Tl concentration in urine samples was also investigated. At the best of our knowledge, this is the first study on human contamination by Tl through water involving such a high number of samples

An isoform of the giant protein titin is a master regulator of human T lymphocyte trafficking

Response to multiple microenvironmental cues and resilience to mechanical stress are essential features of trafficking leukocytes. Here, we describe unexpected role of titin (TTN), the largest protein encoded by the human genome, in the regulation of mechanisms of lymphocyte trafficking. Human T and B lymphocytes ex-press five TTN isoforms, exhibiting cell-specific expression, distinct localization to plasma membrane micro -domains, and different distribution to cytosolic versus nuclear compartments. In T lymphocytes, the LTTN1 isoform governs the morphogenesis of plasma membrane microvilli independently of ERM protein phosphor-ylation status, thus allowing selectin-mediated capturing and rolling adhesions. Likewise, LTTN1 controls chemokine-triggered integrin activation. Accordingly, LTTN1 mediates rho and rap small GTPases activation, but not actin polymerization. In contrast, chemotaxis is facilitated by LTTN1 degradation. Finally, LTTN1 con-trols resilience to passive cell deformation and ensures T lymphocyte survival in the blood stream. LTTN1 is, thus, a critical and versatile housekeeping regulator of T lymphocyte trafficking

The evolution of selective analyses of HDL and LDL cholesterol in clinical and point of care testing

Cardiovascular disease is a leading cause of death worldwide and is caused by the build up of atherosclerotic plaques in the vasculature. It is now well established that the formation of these plaques is closely related to levels of both high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterol. Thus, the importance of the effective measurement of these is critical for the improved diagnosis and management of atherosclerosis. This review discusses the emergence of methodologies for the selective determination of both LDL and HDL cholesterol. It begins with an explanation of the first methodologies based on ultracentrifugation and precipitation techniques, the development of reference methods, through to the emergence of methodologies suitable for routine laboratory use, followed by the development of professional use, point of care technologies. Finally, the current status of selective tests for cholesterol based on biosensor methodologies is reviewed and the potential for application in consumer diagnostics is discussed. © 2013 The Royal Society of Chemistry