Recent Updates on the Therapeutic Prospects of Reversion-Inducing Cysteine-Rich Protein with Kazal Motifs (RECK) in Liver Injuries.

The reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a membrane-anchored glycoprotein, negatively regulates various membrane proteins involved in the tissue governing extracellular matrix (ECM) remodeling such as metalloproteases (MMPs) and the sheddases ADAM10 and ADAM17. The significance of the present review is to summarize the current understanding of the pathophysiological role of RECK, a newly discovered signaling pathway associated with different liver injuries. Specifically, this review analyzes published data on the downregulation of RECK expression in hepatic ischemia/reperfusion (I/R) injury, liver-related cancers, including hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), as well as in the progression of nonalcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis (NASH). In addition, this review discusses the regulation of RECK by inducers, such as FXR agonists. The RECK protein has also been suggested as a potential diagnostic and prognostic marker for liver injury or as a biomarker with predictive value for drug treatment efficacy.

Developmental and Nutritional Dynamics of Malpighian Tubule Autofluorescence in the Asian Tiger Mosquito Aedes albopictus.

Malpighian tubules (MTs) are arthropod excretory organs crucial for the osmoregulation, detoxification and excretion of xenobiotics and metabolic wastes, which include tryptophan degradation products along the kynurenine (KYN) pathway. Specifically, the toxic intermediate 3-hydroxy kynurenine (3-HK) is metabolized through transamination to xanthurenic acid or in the synthesis of ommochrome pigments. Early investigations in Drosophila larval fat bodies revealed an intracellular autofluorescence (AF) that depended on tryptophan administration. Subsequent observations documented AF changes in the MTs of Drosophila eye-color mutants genetically affecting the conversion of tryptophan to KYN or 3-HK and the intracellular availability of zinc ions. In the present study, the AF properties of the MTs in the Asian tiger mosquito, Aedes albopictus, were characterized in different stages of the insect's life cycle, tryptophan-administered larvae and blood-fed adult females. Confocal imaging and microspectroscopy showed AF changes in the distribution of intracellular, brilliant granules and in the emission spectral shape and amplitude between the proximal and distal segments of MTs across the different samples. The findings suggest AF can serve as a promising marker for investigating the functional status of MTs in response to metabolic alterations, contributing to the use of MTs as a potential research model in biomedicine.

MCD Diet Modulates HuR and Oxidative Stress-Related HuR Targets in Rats.

The endogenous antioxidant defense plays a big part in the pathogenesis of non-alcoholic fatty liver disease (NAFLD), a common metabolic disorder that can lead to serious complications such as cirrhosis and cancer. HuR, an RNA-binding protein of the ELAV family, controls, among others, the stability of MnSOD and HO-1 mRNA. These two enzymes protect the liver cells from oxidative damage caused by excessive fat accumulation. Our aim was to investigate the expression of HuR and its targets in a methionine-choline deficient (MCD) model of NAFLD. To this aim, we fed male Wistar rats with an MCD diet for 3 and 6 weeks to induce NAFLD; then, we evaluated the expression of HuR, MnSOD, and HO-1. The MCD diet induced fat accumulation, hepatic injury, oxidative stress, and mitochondrial dysfunction. A HuR downregulation was also observed in association with a reduced expression of MnSOD and HO-1. Moreover, the changes in the expression of HuR and its targets were significantly correlated with oxidative stress and mitochondrial injury. Since HuR plays a protective role against oxidative stress, targeting this protein could be a therapeutic strategy to both prevent and counteract NAFLD.

Three-dimensional spheroid cell culture of human MSC-derived neuron-like cells: New in vitro model to assess magnetite nanoparticle-induced neurotoxicity effects.

As nanoparticles (NPs) can access the brain and impact on CNS function, novel in vitro models for the evaluation of NPs-induced neurotoxicity are advocated. Three-dimensional spheroids of primary neuron-like cells (hNLCs) of human origin have been generated, from differentiation of human umbilical cord mesenchymal stem cells (MSCs). The study evaluated Fe3 O4 NP impact on the differentiation process by applying the challenge at complete 3D hNLC spheroid formation (after 4 days, T4) or at beginning of neurogenic induction/simultaneously 3D forming (T0). Different endpoints were monitored over time (up to 10 days): spheroid growth, size, morphology, ATP, cell death, neuronal markers (ß-Tub III, MAP-2, and NSE), NP uptake. At T0 application, a marked concentration- and time-dependent cell mortality occurred: effect started early (day 2) and low concentration (1 µg/ml) and exacerbated (80% mortality) after prolonged time (day 6) and increased concentrations (50 µg/ml). ATP was strikingly affected. All neuronal markers were downregulated, and spheroid morphology altered in a concentration-dependent manner (from ≥5 µg/ml) after day 2. Fe3 O4 NPs applied at complete 3D formation (T4) still induced adverse effects although less severe: cell mortality (20-60%) and ATP content decrease (10-40%) were observed in a concentration-dependent manner (from ≥ 5 µg/ml). A neuronal-specific marker effect and spheroid size reduction from 25 µg/ml without morphology alteration were evidenced. This finding provides additional information on neurotoxic effects of Fe3 O4 NPs in a new 3D hNLC spheroid model derived from MSCs that could find a consistent application as in a testing strategy serving in first step hazard identification for correct risk assessment.

MCD Diet Rat Model Induces Alterations in Zinc and Iron during NAFLD Progression from Steatosis to Steatohepatitis.

We evaluate the effects of the methionine-choline-deficient (MCD) diet on serum and hepatic zinc (Zn) and iron (Fe) and their relationships with matrix metalloproteinases (MMPs) and their modulators (TIMPs and RECK) as well as hepatic fatty acids using male Wistar rats fed 2-, 4- and 8-week MCD diets. Serum and hepatic Zn decrease after an 8-week MCD diet. Serum Fe increases after an 8-week MCD diet and the same occurs for hepatic Fe. An increase in hepatic MMP activity, associated with a decrease in RECK and TIMPs, is found in the MCD 8-week group. Liver Fe shows a positive correlation versus MMPs and RECK, and an inverse correlation versus TIMPs. A positive correlation is found comparing liver Zn with stearic, vaccenic and arachidonic acids, and an inverse correlation is found with linolenic and docosatetraenoic acids. An opposite trend is found between liver Fe versus these fatty acids. During NAFLD progression from steatosis to steatohepatitis, MCD rats exhibit an increase in Zn and a decrease in Fe levels both in serum and tissue associated with alterations in hepatic MMPs and their inhibitors, and fatty acids. The correlations detected between Zn and Fe versus extracellular matrix modulators and fatty acids support their potential role as therapeutic targets.

Long-term cold storage preservation does not affect fatty livers from rats fed with a methionine and choline deficient diet.

BACKGROUND: Waiting lists that continue to grow and the lack of organs available for transplantation necessitate the use of marginal livers, such as fatty livers. Since steatotic livers are more susceptible to damage from ischemia and reperfusion, it was investigated whether fatty livers with different lipidomic profiles show a different outcome when subjected to long-term cold storage preservation. METHODS: Eight-week-old male Wistar rats fed for 2 weeks by a methionine-choline-deficient (MCD) diet or control diet were employed in this study. Livers were preserved in a University of Wisconsin (UW) solution at 4 °C for 6, 12 or 24 h and, after washout, reperfused for 2 h with a Krebs-Henseleit buffer at 37 °C. Hepatic enzyme release, bile production, O2-uptake, and portal venous pressure (PVP) were evaluated. The liver fatty acid profile was evaluated by a gas chromatography-mass spectrometry (GC/MS). RESULTS: MCD rats showed higher LDH and AST levels with respect to the control group. When comparing MCD livers preserved for 6, 12 or 24 h, no differences in enzyme release were found during both the washout or the reperfusion period. The same trend occurred for O2-uptake, PVP, and bile flow. A general decrease in SFA and MUFA, except for oleic acid, and a decrease in PUFA, except for arachidonic, eicosadienoic, and docosahexanaeoic acids, were found in MCD rats when compared with control rats. Moreover, the ratio between SFA and the various types of unsaturated fatty acids (UFA) was significantly lower in MCD rats. CONCLUSIONS: Although prolonged cold ischemia negatively affects the graft outcome, our data suggest that the quality of lipid constituents could influence liver injury during cold storage: the lack of an increased hepatic injury in MCD may be justified by low SFA, which likely reduces the deleterious tendency toward lipid crystallization occurring under cold ischemia.

Autofluorescence-based optical biopsy: An effective diagnostic tool in hepatology.

Autofluorescence emission of liver tissue depends on the presence of endogenous biomolecules able to fluoresce under suitable light excitation. Overall autofluorescence emission contains much information of diagnostic value because it is the sum of individual autofluorescence contributions from fluorophores involved in metabolism, for example, NAD(P)H, flavins, lipofuscins, retinoids, porphyrins, bilirubin and lipids, or in structural architecture, for example, fibrous proteins, in close relationship with normal, altered or diseased conditions of the liver. Since the 1950s, hepatocytes and liver have been historical models to study NAD(P)H and flavins as in situ, real-time autofluorescence biomarkers of energy metabolism and redox state. Later investigations designed to monitor organ responses to ischaemia/reperfusion were able to predict the risk of dysfunction in surgery and transplantation or support the development of procedures to ameliorate the liver outcome. Subsequently, fluorescent fatty acids, lipofuscin-like lipopigments and collagen were characterized as optical biomarkers of liver steatosis, oxidative stress damage, fibrosis and disease progression. Currently, serum AF is being investigated to improve non-invasive optical diagnosis of liver disease. Validation of endogenous fluorophores and in situ discrimination of cancerous from non-cancerous tissue belong to the few studies on liver in human subjects. These reports along with other optical techniques and the huge work performed on animal models suggest many optically based applications in hepatology. Optical diagnosis is currently offering beneficial outcomes in clinical fields ranging from the respiratory and gastrointestinal tracts, to dermatology and ophthalmology. Accordingly, this review aims to promote an effective bench to bedside transfer in hepatology.

Fatty liver oxidative events monitored by autofluorescence optical diagnosis: Comparison between subnormothermic machine perfusion and conventional cold storage preservation.

AIMS: Livers with moderate steatosis are currently recruited as marginal organs to face donor shortage in transplantation, even though lipid excess and oxidative stress increase preservation injury risk. Sensitive, real-time detection of liver metabolism engagement could help donor selection and preservation procedures, ameliorating the graft outcome. Hence, we investigated endogenous biomolecules with autofluorescence (AF) properties as biomarkers supporting the detection of liver oxidative events and the assessment of metabolic responses to external stimuli. METHODS: Livers from male Wistar rats fed a 12-day methionine/choline-deficient (MCD) diet were subjected to AF spectrofluorometric analysis (fiber-optic probe, 366-nm excitation) before and after organ isolation, and following preservation (cold storage or 20°C machine perfusion) and reperfusion. RESULTS: Innovative dynamic AF results on lipid oxidation to lipofuscin-like lipopigments, correlating with biochemical oxidative damage (thiobarbituric acid reactive substances) and antioxidant defense (glutathione) parameters, suggested lipid engagement in MCD livers counteracting reactive oxidizing species. The maintained MCD liver functionality was supported by limited changes in bilirubin AF spectral profile, reflecting bile composition balance, despite their intrinsic mitochondrial weakness, confirmed by adenosine 5'-triphosphate levels, and regardless of different preservation effects on energy metabolism revealed by conventional reduced forms of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate and flavin AF data. CONCLUSION: Autofluorescence showed that, after a relatively short time on an MCD diet, livers are still able to face oxidizing events and maintain a functional balance. These results strengthen AF as a supportive diagnostic tool in experimental hepatology, to characterize marginal livers in real time, monitor their response to ischemia/reperfusion, and investigate protective therapeutic agents.

Liver Graft Susceptibility during Static Cold Storage and Dynamic Machine Perfusion: DCD versus Fatty Livers.

We compared static preservation (cold storage, CS, 4 °C) with dynamic preservation (machine perfusion, MP, 20 °C) followed by reperfusion using marginal livers: a model of donation after cardiac death (DCD) livers and two models of fatty livers, the methionine-choline deficient (MCD) diet model, and obese Zucker (fa/fa) rats. CS injury in DCD livers was reversed by an oxygenated washout (OW): hepatic damage, bile flow, and the ATP/ADP ratio in the OW + CS group was comparable with the ratio obtained with MP. Using fatty livers, CS preservation induced a marked release in hepatic and biliary enzymes in obese Zucker rats when compared with the MCD group. The same trend occurred for bile flow. No difference was found when comparing MP in MCD and obese Zucker rats. Fatty acid analysis demonstrated that the total saturated (SFA)/polyunsaturated fatty acid (PUFA) ratio was, respectively, 1.5 and 0.71 in obese Zucker and MCD rats. While preservation damage in DCD livers is associated with the ATP/ADP recovered with OW, injury in fatty livers is linked to fatty acid constituents: livers from obese. Zucker rats, with greater content in saturated FA, might be more prone to CS injury. On the contrary, MCD livers with elevated PUFA content might be less susceptible to hypothermia.

MCD diet-induced steatohepatitis is associated with alterations in asymmetric dimethylarginine (ADMA) and its transporters.

Using an experimental model of NASH induced by a methionine-choline-deficient (MCD) diet, we investigated whether changes occur in serum and tissue levels of asymmetric dimethylarginine (ADMA). Male Wistar rats underwent NASH induced by 8-week feeding with an MCD diet. Serum and hepatic biopsies at 2, 4 and 8 weeks were taken, and serum enzymes, ADMA and nitrate/nitrite (NOx), were evaluated. Hepatic biopsies were used for mRNA and protein expression analysis of dimethylarginine dimethylaminohydrolase-1 (DDAH-1) and protein methyltransferases (PRMT-1), enzymes involved in ADMA metabolism and synthesis, respectively, and ADMA transporters (CAT-1, CAT-2A and CAT-2B). Lipid peroxides (TBARS), glutathione, ATP/ADP and DDAH activity were quantified. An increase in serum AST and ALT was detected in MCD animals. A time-dependent decrease in serum and tissue ADMA and increase in mRNA expression of DDAH-1 and PRMT-1 as well as higher rates of mRNA expression of CAT-1 and lower rates of CAT-2A and CAT-2B were found after 8-week MCD diet. An increase in serum NOx and no changes in protein expression in DDAH-1 and CAT-1 and higher content in CAT-2 and PRMT-1 were found at 8 weeks. Hepatic DDAH activity decreased with a concomitant increase in oxidative stress, as demonstrated by high TBARS levels and low glutathione content. In conclusion, a decrease in serum and tissue ADMA levels in the MCD rats was found associated with a reduction in DDAH activity due to the marked oxidative stress observed. Changes in ADMA levels and its transporters are innovative factors in the onset and progression of hepatic alterations correlated with MCD diet-induced NASH.

Histochemical and ultrastructural localization of triterpene saponins in Medicago truncatula.

In the Medicago genus, saponins are complex mixtures of triterpene pentacyclic glycosides extensively studied for their different and economically relevant biological and pharmaceutical properties. This research is aimed at determining for the first time the tissue and cellular localization of triterpene saponins in vegetative organs of Medicago truncatula, a model plant species for legumes, by histochemistry and transmission electron microscopy. The results showed that saponins are present mainly in the palisade mesophyll layer of leaves, whereas in stems they are mostly located in the primary phloem and the subepidermal cells of cortical parenchyma. In root tissue, saponins occur in the secondary phloem region. Transmission electron microscopy revealed prominent saponin accumulation within the leaf and stem chloroplasts, while in the roots the saponins are found in the vesicular structures. Our results demonstrate the feasibility of using histochemistry and transmission electron microscopy to localize M. truncatula saponins at tissue and cellular levels and provide important information for further studies on biosynthesis and regulation of valuable bioactive saponins on agronomic relevant Medicago spp., such as alfalfa (Medicago sativa L.). RESEARCH HIGHLIGHTS: The Medicago genus represents a valuable rich source of saponins, one of the most interesting groups of secondary plant metabolites, which possess relevant biological and pharmacological properties. Plant tissue and cellular localization of saponins is of great importance to better understand their biological functions, biosynthetic pathway, and regulatory mechanisms. We elucidate the localization of saponins in Medicago truncatula with histochemical and transmission electron microscopy studies.

Characterization of Spontaneous Melanization by Fluorescence Spectroscopy: A Basis for Analytical Application to Biological Substrates.

Melanin is present in various biological substrates where it may participate in several processes, from innate immunity to the still-unsolved opposite roles in antioxidant protection, including photoprotection and the related ability to interact with light. Melanin-light interaction has also been an important source of inspiration for the development of innovative bioengineering applications. These are based on melanin's light-energy-absorption ability of its chemically and structurally complex components and precursors, and on the improvement in analytical and diagnostic procedures in biomedicine. In this regard, here, we characterized the fluorescence spectral properties of melanin and of its precursor L-tyrosine in an aqueous solution during spontaneous melanization. Besides the confirmation of the typical fluorescence-emission signature of melanin and L-tyrosine, we provide additional insights on both emission and excitation spectra recorded during melanization. On these bases, we performed a subsequent characterization on the aqueous extracts from two different melanin-containing biological substrates, namely hairs from a domestic black cat and eggs from the Asian tiger mosquito. The results from the mild extraction procedure, purposely applied to obtain only the soluble components, combined with fluorescence spectral analysis are expected to promote further investigation of the melanization processes, particularly in insects.

Pathogen-Mediated Alterations of Insect Chemical Communication: From Pheromones to Behavior.

Pathogens can influence the physiology and behavior of both animal and plant hosts in a manner that promotes their own transmission and dispersal. Recent research focusing on insects has revealed that these manipulations can extend to the production of pheromones, which are pivotal in chemical communication. This review provides an overview of the current state of research and available data concerning the impacts of bacterial, viral, fungal, and eukaryotic pathogens on chemical communication across different insect orders. While our understanding of the influence of pathogenic bacteria on host chemical profiles is still limited, viral infections have been shown to induce behavioral changes in the host, such as altered pheromone production, olfaction, and locomotion. Entomopathogenic fungi affect host chemical communication by manipulating cuticular hydrocarbons and pheromone production, while various eukaryotic parasites have been observed to influence insect behavior by affecting the production of pheromones and other chemical cues. The effects induced by these infections are explored in the context of the evolutionary advantages they confer to the pathogen. The molecular mechanisms governing the observed pathogen-mediated behavioral changes, as well as the dynamic and mutually influential relationships between the pathogen and its host, are still poorly understood. A deeper comprehension of these mechanisms will prove invaluable in identifying novel targets in the perspective of practical applications aimed at controlling detrimental insect species.

Imaging and spectral analysis of autofluorescence patterns in larval head structures of mosquito vectors.

Autofluorescence (AF) in mosquitoes is currently poorly explored, despite its great potential as a marker of body structures and biological functions. Here, for the first time AF in larval heads of two mosquitoes of key public health importance, Aedes albopictus and Culex pipiens, is studied using fluorescence imaging and spectrofluorometry, similarly to a label-free histochemical approach. In generally conserved distribution patterns, AF shows differences between mouth brushes and antennae of the two species. The blue AF ascribable to resilin at the antennal bases, more extended in Cx. pipiens, suggests a potential need to support different antennal movements. The AF spectra larger in Cx. pipiens indicate a variability in material composition and properties likely relatable to mosquito biology, including diverse feeding and locomotion behaviours with implications for vector control.

Obeticholic Acid Reduces Kidney Matrix Metalloproteinase Activation Following Partial Hepatic Ischemia/Reperfusion Injury in Rats.

We have previously demonstrated that the farnesoid X receptor (FXR) agonist obeticholic acid (OCA) protects the liver via downregulation of hepatic matrix metalloproteinases (MMPs) after ischemia/reperfusion (I/R), which can lead to multiorgan dysfunction. The present study investigated the capacity of OCA to modulate MMPs in distant organs such as the kidney. Male Wistar rats were dosed orally with 10 mg/kg/day of OCA (5 days) and were subjected to 60-min partial hepatic ischemia. After 120-min reperfusion, kidney biopsies (cortex and medulla) and blood samples were collected. Serum creatinine, kidney MMP-2, and MMP-9-dimer, tissue inhibitors of MMPs (TIMP-1, TIMP-2), RECK, TNF-alpha, and IL-6 were monitored. MMP-9-dimer activity in the kidney cortex and medulla increased after hepatic I/R and a reduction was detected in OCA-treated I/R rats. Although not significantly, MMP-2 activity decreased in the cortex of OCA-treated I/R rats. TIMPs and RECK levels showed no significant differences among all groups considered. Serum creatinine increased after I/R and a reduction was detected in OCA-treated I/R rats. The same trend occurred for tissue TNF-alpha and IL-6. Although the underlying mechanisms need further investigation, this is the first study showing, in the kidney, beneficial effects of OCA by reducing TNF-alpha-mediated expression of MMPs after liver I/R.

Naturally-occurring porphyrins in a spontaneous-tumour bearing mouse model.

An increase in naturally-occurring porphyrins has been described in the blood of subjects bearing different kinds of tumours, that has been proposed as an additional parameter for the diagnosis of occult cancer, although at present the reason for the phenomenon is not exactly defined. In this work the increase of porphyrins in plasma of tumour-bearing subjects has been investigated in parallel with their occurrence in other tissues, considering the systemic iron homeostasis subversion taking place in the presence of cancer. The transgenic female MMTV-neu mouse-developing spontaneous mammary adenocarcinoma has been used as an experimental model, in comparison to non-transgenic C1 mouse as a control. The spleen, accomplishing both hemocatheretic and hemopoietic functions in rodents, and the liver have been considered because of their deep engagement in heme metabolism, entailing both the fate of protoporphyrin IX (PpIX) as its ultimate precursor, and iron homeostasis. Investigations have been performed by means of microspectrofluorometric and image analysis of tissue autofluorescence (AF), and histochemical detection of non-heme iron. In tumour-bearing mouse, along with a marked PpIX presence in tumour, a PpIX enhancement in spleen and liver is observed, that is accompanied by a significant increase in plasma. The phenomenon can be related to a systemic alteration of heme metabolism induced by tumour cells to face their survival and proliferation requirements.

Enzyme-assisted photosensitization activates different apoptotic pathways in Rose Bengal acetate treated HeLa cells.

Photosensitization of tumor cells after incubation with Rose Bengal acetate (RB-Ac) induces multiple organelle photodamage followed by apoptotic cell death. We used immunocytochemical techniques in multicolor fluorescence microscopy to elucidate whether this occurs through the simultaneous activation of different apoptotic pathways, in HeLa cells. We detected in situ the activated forms of caspases 9 and 3, and the translocation from the mitochondria to the nucleus of the apoptosis inducing factor; DNA electrophoretic techniques were also used to assess the occurrence of nuclear DNA cleavage into either high- or low-molecular-weight fragments. Both the caspase-dependent and caspase-independent apoptotic pathways are activated. The genomic DNA is degraded into high molecular weight molecules only, without the formation of oligonucleosome-sized fragments. The ability of RB-Ac to induce the simultaneous release of apoptogenic signals from different photodamaged organelles makes it an especially powerful cytotoxic agent.

Fatty Acid Desaturase Involvement in Non-Alcoholic Fatty Liver Disease Rat Models: Oxidative Stress Versus Metalloproteinases.

We investigated changes in fatty acid desaturases, D5D, D6D, D9-16D and D9-18D, and their relationship with oxidative stress, matrix metalloproteinases (MMPs) and serum TNF-alpha in two rat models of non-alcoholic fatty liver disease NAFLD. Eight-week-old male Wistar rats fed for 3 weeks with methionine-choline⁻deficient (MCD) diet and eleven-week-old Obese male Zucker rats were used. Serum levels of hepatic enzymes and TNF-alpha were quantified. Hepatic oxidative stress (ROS, TBARS and GSH content) and MMP-2 and MMP-9 (protein expression and activity) were evaluated. Liver fatty acid profiling, performed by GC-MS, was used for the quantification of desaturase activities. Higher D5D and D9-16D were found in Obese Zucker rats as well as an increase in D9-18D in MCD rats. D6D was found only in MCD rats. A negative correlation between D5D and D9-16D versus TBARS, ROS and TNF-alpha and a positive correlation with GSH were shown in fatty livers besides a positive correlation between D9-18D versus TBARS, ROS and TNF-alpha and a negative correlation with GSH. A positive correlation between D5D or D9-16D or D9-18D versus protein expression and the activity of MMP-2 were found. NAFLD animal models showed comparable serum enzymes. These results reinforce and extend findings on the identification of therapeutic targets able to counteract NAFLD disorder.

Antimetastatic effect of a small-molecule vacuolar H+-ATPase inhibitor in in vitro and in vivo preclinical studies.

On the basis of the evidence that vacuolar H(+)-ATPase is implicated in the development of the metastatic phenotype, we have explored the possibility to target the enzyme function in an attempt to control the metastatic behavior of tumor cells. In this study, we used an indole derivative, NiK-12192 [4-(5,6-dichloro-1H-indol-2-yl)-3-ethoxy-N-(2,2,6,6-tetramethyl-piperidin-4-yl)-benzamide], recently identified as an effective inhibitor of vacuolar H(+)-ATPase, as a potential antimetastatic agent in the treatment of NSCLC H460 xenograft, which is able to induce lung metastases in mice. Oral administration of NiK-12192 caused a significant inhibition of formation of spontaneous metastases. In contrast, the drug exhibited a negligible effect on the development of artificial metastases (i.e., after i.v. injection of tumor cells), thus supporting that the drug affects the early events of the metastatic process (e.g., migration and invasion). Cellular effects are consistent with this interpretation. In conclusion, the available results show for the first time that a vacuolar H(+)-ATPase inhibitor is effective in modulation of the metastatic behavior of a lung carcinoma, supporting its potential therapeutic interest as a novel treatment approach.

Lipids: Evergreen autofluorescent biomarkers for the liver functional profiling.

Depending on their chemical nature, lipids can be classified in two main categories: hydrophilic, greatly contributing to membrane composition and subcellular organelle compartmentalization, and hydrophobic, mostly triglycerides, greatly enrolled in the storage and production of energy. In both cases, some lipid molecules can be involved as signaling agents in the regulation of metabolism and protective or damaging pathways in responses to harmful stimuli. These events could affect in particular the liver, because of its central role in the maintenance of lipid homeostasis. Lipids have been demonstrated to fluoresce, contributing to the overall emission signal of the liver tissue along with other endogenous fluorophores, relatable to energy metabolism and oxidative events. The mere estimation of the fluorescing lipid fraction in parallel with the other endogenous fluorophores, and with the common biochemical and histochemical biomarkers of tissue injury has been exploited to investigate the liver morpho-functional conditions in experimental hepatology. More interestingly, the fluorescing lipid fraction is greatly relatable to free fatty acids such as arachidonic, linoleic and linolenic acid, which are deserving increasing attention as precursors of products involved in several and complex signaling pathways. On these bases, the ability of autofluorescence to detect directly arachidonic acid and its balance with other unsaturated fatty acids may be exploited in the diagnosis and follow-up of fatty livers, helping to improve the personalization of the metabolic/lipidomic profiling. This could also contribute to elucidate the role of the injuring factors in the choice of suitable donors, and in the set-up of preservation procedures in liver transplantation.