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Haptic control interfaces can significantly improve the quality of teleoperation in robotic and mechatronic systems. However, the required force feedback introduces new challenges when highly constrained environments and systems hinder the use of conventional force sensors. In this article, we propose a haptic control method that provides feedback to the operator when force data can be extracted from a sound signal. Since sound can be acquired by a microphone deployed outside the workspace, this method enables remote feedback in any process that can be characterized through acoustic emissions (e.g. machining), without the need for sensors embedded in the system and deployed in the work environment. The performance of the method is verified by an example application with a teleoperated robot that executes a machining process (milling and grinding), whose sound is acquired with a microphone and processed to successfully estimate the machining force for haptic feedback.

The maintenance of critical industrial components is often hindered by limited access, tortuous passages, and complex geometries. In highly constrained environments, inspection tasks are currently performed with borescopes, but even skilled operators struggle with hard-to-reach targets, and the limited mobility prevents in situ repair when defects are identified. Thanks to an active shape control, snake-like and continuum robots can outperform borescopes for short-range inspection as well as enable intervention. However, their actuation technology limits their scalability in length, as longer bodies pose control challenges due to their intrinsically low stiffness and space constraints. To overcome the limitations of both borescopes and continuum robots, here, we propose a modular design at their intersection, with both active tendon-driven and passively flexible segments. The main elements of the novel design, including the actuation and control interface, are described, and the system is demonstrated in scenarios for aerospace assets, nuclear installations, and robot-assisted surgery.

The many degrees of freedom of continuum robots (CRs) enable unique applications in the search and rescue, medical and aerospace industries. However, the many motors required result in unwieldy external actuation packs and additional weight. Underactuation can achieve any pose with four motors by locking sections through clutching of cables. Existing underactuated (UA) solutions are characterized by lengthy joint locking times, add significant weight and are not scalable. In this article, a novel design and motion strategy is introduced, with shape memory alloy wires to control clutches that lock cables for section locking. In addition to using conventional smooth cables, beaded cables are proposed for improved clutching strength. The workspace of the design is analyzed, and design tools are proposed to optimize bead pitch. Finally, the operation of the proposed UA CR is demonstrated on a prototype that achieves a mean repeatability of 1.41mm, equivalent to 0.43% of the backbone length. Section locking is achieved in 1 s and section unlocking is achieved in 5 s.

Current research on walking robots strives to achieve a higher efficiency, a better load capacity, and an increased adaptability. Parallel kinematic manipulators (PKMs) are characterized by high payload and accuracy, but conventional PKMs with fixed configurations are limited to constrained workspaces in known structured environments. In this article, we propose a parallel reconfigurable walking machine tool that overcomes these limits by adapting its configuration and gaits to different scenarios. A lightweight and compact positioning system with shape memory alloy actuation is presented to achieve reconfiguration capabilities. Furthermore, kinematic, stability, and force analyses are reported to determine the optimal walking gaits in three different scenarios (with inclined slopes at different angles) and four robot configurations. Finally, a set of experiments with the physical prototype validates the proposed models. The results show that symmetric configurations present a better performance at lower ground inclinations (0.5% error), whereas asymmetric configurations can climb on slope conditions that would prevent the use of conventional PKMs (18% or 10°).

In this paper, the grasping operation of CubeSat microsatellites is analyzed with a topological study of grasping strategies as functions of CubeSat geometry. Grasping conditions and limitations are introduced for the square-profiled bodies of CubeSats of 1U and 12U sizes. A topology search defines fingertip forms and configurations to fulfill requirements, and operational limitations are presented in terms of geometry and dynamic parameters. The grasping performance is then analyzed in the side grasp and corner grasp cases and validated with a numerical case study.

Developing a robotic torso mechanism is crucial in replicating human mobility in humanoid robots. Previous research has presented the LARMbot humanoid's torso as a potential solution, which has now been improved with a novel design proposed in this paper. We conduct a kinematic analysis on the proposed LARMbot torso design, which is developed through an analysis of the human spine's structure. A kinematic model of the novel design is proposed using piecewise constant curvature to capture the relationship between input and output parameters and to represent the workspace. A prototype is utilized for conducting experiments to test the human-like movements of the novel mechanism. The outcomes show that the proposed LARMbot torso architecture can enhance performance. The generated humanoid torso prototype has the capacity to bend approximately 30 deg, and as such, it can be expected to make humanoid robots achieve human-like motion and tasks.

BackgroundThe regulation of the chromatin state by epigenetic mechanisms plays a central role in gene expression, cell function, and maintenance of cell identity. Hereditary disorders of chromatin regulation are a group of conditions caused by abnormalities of the various components of the epigenetic machinery, namely writers, erasers, readers, and chromatin remodelers. Although neurological dysfunction is almost ubiquitous in these disorders, the constellation of additional features characterizing many of these genes and the emerging clinical overlap among them indicate the existence of a community of syndromes. The introduction of high-throughput next generation sequencing (NGS) methods for testing multiple genes simultaneously is a logical step for the implementation of diagnostics of these disorders.MethodsWe screened a heterogeneous cohort of 263 index patients by an NGS-targeted panel, containing 68 genes associated with more than 40 OMIM entries affecting chromatin function.ResultsThis strategy allowed us to identify clinically relevant variants in 87 patients (32%), including 30 for which an alternative clinical diagnosis was proposed after sequencing analysis and clinical re-evaluation.ConclusionOur findings indicate that this approach is effective not only in disorders with locus heterogeneity, but also in order to anticipate unexpected misdiagnoses due to clinical overlap among cognate disorders. Finally, this work highlights the utility of a prompt diagnosis in such a clinically and genetically heterogeneous group of disorders that we propose to group under the umbrella term of chromatinopathies.

In this paper, a novel cable-driven parallel robot, CUBE, is introduced for the assistance of patients in rehabilitation exercising of both upper and lower limbs. The system is characterized by a lightweight structure that is easy to set-up and operate, for both clinical and home usage for both pre-determined and customized exercises, with control over the position of the end-effector while locking its rotation around the horizontal axes. Its cable-driven design makes it inherently safe in human/robot interactions also due to the extremely low inertia. While a novel end-effector design makes the device wearable both on the upper and lower limbs without having to disassemble any part of the structure. The design is presented with its kinematic analysis. Then, the manufacturing through 3D-printing and commercial components of a first prototype is reported. Finally, the system is validated through motion tests along simple trajectories and two different spatial exercises.

This paper presents an analysis of a novel 3-SPR parallel manipulator with 3 degrees-of-freedom that is characterized by a mobile platform consisting of a single point where all the three open-loop kinematic chains converge. The constraint singularity problem of the mechanism is solved in closed form by computing the Jacobian of the manipulator. Then, the expression of the transmission index is obtained for the 3-SPR manipulator by using the pressure angle definition. In order to evaluate the influence of secondary parameters, an equivalent 6-SPS mechanism is analyzed and its transmission index is computed in the parameter space. Finally, the proposed manipulator is compared to a standard 3-SPR parallel mechanism to investigate the influence of the mobile platform radius over transmission index and workspace volume. The results are discussed in order to highlight the advantages and disadvantages of the proposed structure of parallel manipulator.

In this work, proper orthogonal decomposition (POD) was applied to large eddy simulations (LES) of two high-loaded low-pressure turbine cascades under unsteady inflow to investigate entropy production in different parts of the blade passage. The turbulent kinetic energy (TKE) production, diffusion, and dissipation terms from the stagnation pressure transport equation were integrated over the computational domain. The POD-based method splits the contribution of different coherent flow dynamics to TKE production and dissipation, including the migration, bowing, tilting, and reorientation of incoming wake filaments, as well as the breakup of streaky structures in the blade boundary layer and the formation of Von Karman vortices in the blade wake. This helps designers identify the dominant POD modes (turbulent flow structures) responsible for loss generation, their dynamics, and the spatial locations where they act, providing insights into the physical phenomena to be controlled. Following this optimization strategy, a new low-pressure turbine profile was designed. After performing LES on the optimized geometry, POD results indicated the possibility of designing a higher-loaded profile with a lower global loss coefficient. Thanks to the stronger acceleration imposed on the bulk flow in the former part of the blade passage, the new loading distribution is shown to be responsible for lower upstream wake migration losses, as well as for a smaller amount of TKE production in the trailing edge wake zone, as a result of the early suction side boundary layer transition.

Fish despite their low collocation in the vertebrate phylum possess a complete immune system. In teleost fish both innate and adaptive immune responses have been described with melanomacrophage centers (MMCs) equivalent to mammalian germinal centers. Primary lymphoid organs are represented by the thymus and kidney, while spleen and mucosa-associated lymphoid tissues act as secondary lymphoid organs. Functions of either innate immune cells (e.g., macrophages and dendritic cells) or adaptive immune cells (T and B lymphocytes) will be described in detail, even including their products, such as cytokines and antibodies. In spite of a robust immune arsenal, fish are very much exposed to infectious agents (marine bacteria, parasites, fungi, and viruses) and, consequentially, mortality is very much enhanced especially in farmed fish. In fact, in aquaculture stressful events (overcrowding), microbial infections very frequently lead to a high rate of mortality. With the aim to reduce mortality of farmed fish through the reinforcement of their immune status the current trend is to administer natural products together with the conventional feed. Then, in the second part of the present review emphasis will be placed on a series of products, such as prebiotics, probiotics and synbiotics, β-glucans, vitamins, fatty acids and polyphenols all used to feed farmed fish. With special reference to polyphenols, results of our group using red grape extracts to feed farmed European sea bass will be illustrated. In particular, determination of cytokine production at intestinal and splenic levels, areas of MMCs and development of hepatopancreas will represent the main biomarkers considered. All together, our own data and those of current literature suggests that natural product administration to farmed fish for their beneficial effects may, in part, solve the problem of fish mortality in aquaculture, enhancing their immune responses.

Antimicrobial peptides (AMPs) are phylogenetically ancient substances released by living organisms for self protection against a broad variety of microbes. Moreover, AMPs are endowed with immune modulatory activities, linking innate and adaptive immunity together. Lantibiotics are AMPs of bacterial origin currently investigated for the generation of a new class of anti-infective compounds, owing to the phenomenon of antibiotic resistance against a broad variety of bacteria. Also, plants and marine AMPs are screened as novel drugs against human pathogens. Human AMPs encompass defensins and cathelicidins produced by various cell types mostly at mucosal sites. Besides their antimicrobial activity, both AMPs have been shown to trigger either inflammatory or anti-inflammatory pathways. Food-derived AMPs are mostly represented by lactoferrin and lysozyme both present in secretions, e.g., milk, and appear to be very exploitable for the generation of functional foods. Finally, the role of natural products ingested with food or administered as supplements on induction and production of AMPs will be discussed.

Antimicrobial Peptides (AMPs) are produced by a variety of human immune and non immune cells in health and disease. In virtue of their antimicrobial activity, AMPs have been exploited in human disease and here this aspect will extensively be described. AMPs in comparison to antibiotics possess a larger spectrum of antimicrobial activity without inducing microbial resistance. Therefore, their use in the course of antibiotic-resistant infections is justified. AMP activity in early life, in the airways, in the oral and gastro-enteric system, in the skin and in the female reproductive tract, respectively, will be elucidated. In addition, the use of AMPs in sepsis will be discussed due to the frequency of this pathological condition characterized by multiple organ dysfunctions. Finally, the evidence that AMPs represent valid substitutes of antibiotics will be provided and a series of novel substances able to reinforce the innate immune response in different clinical settings will be discussed.

The sirtuin family comprises seven NAD+-dependent deacetylases which control the overall health of organisms through the regulation of pleiotropic metabolic pathways. Sirtuins are important modulators of adipose tissue metabolism and their expression is higher in lean than obese subjects. At present, the role of sirtuins in adipose-derived stem cells has not been investigated yet. Therefore, in this study, we evaluated the expression of the complete panel of sirtuins in adipose-derived stem cells isolated from both subcutaneous and visceral fat of non-obese and obese subjects. We aimed at investigating the influence of obesity on sirtuins’ levels, their role in obesity-associated inflammation, and the relationship with the peroxisome proliferator-activated receptor delta, which also plays functions in adipose tissue metabolism. The mRNA levels in the four types of adipose-derived stem cells were evaluated by quantitative polymerase chain reaction, in untreated cells and also after 8 h of hypoxia exposure. Correlations among sirtuins’ expression and clinical and molecular parameters were also analyzed. We found that sirtuin1–6 exhibited significant higher mRNA expression in visceral adipose-derived stem cells compared to subcutaneous adipose-derived stem cells of non-obese subjects. Sirtuin1–6 levels were markedly reduced in visceral adipose-derived stem cells of obese patients. Sirtuins’ expression in visceral adipose-derived stem cells correlated negatively with body mass index and C-reactive protein and positively with peroxisome proliferator-activated receptor delta. Finally, only in the visceral adipose-derived stem cells of obese patients hypoxia-induced mRNA expression of all of the sirtuins. Our results highlight that sirtuins’ levels in adipose-derived stem cells are consistent with protective effects against visceral obesity and inflammation, and suggest a transcriptional mechanism through which acute hypoxia up-regulates sirtuins in the visceral adipose-derived stem cells of obese patients.

Immune cells actively participate to the central nervous system (CNS) injury either damaging or protecting neural tissue with release of various mediators. Residential microglia and monocyte-derived macrophages play a fundamental role within the injured CNS and, here, special emphasis will be placed on M1 and M2 macrophages for their different functional activities. On the other hand, peripheral T regulatory (Treg) cells exert antiinflammatory activities in the diseased host. In this respect, activation of Treg cells by nutraceuticals may represent a novel approach to treat neuroinflammation. Omega-3 fatty acids and polyphenols will be described as substances endowed with antioxidant and anti-inflammatory activities. However, taking into account that Treg cells act in the later phase of CNS injury, favoring immune suppression, manipulation of host immune system with both substances requires caution to avoid undesired side effects.

Immune cells actively participate to the central nervous system (CNS) injury either damaging or protecting neural tissue with release of various mediators. Residential microglia and monocyte-derived macrophages play a fundamental role within the injured CNS and, here, special emphasis will be placed on M1 and M2 macrophages for their different functional activities. On the other hand, peripheral T regulatory (Treg) cells exert antiinflammatory activities in the diseased host. In this respect, activation of Treg cells by nutraceuticals may represent a novel approach to treat neuroinflammation. Omega-3 fatty acids and polyphenols will be described as substances endowed with antioxidant and anti-inflammatory activities. However, taking into account that Treg cells act in the later phase of CNS injury, favoring immune suppression, manipulation of host immune system with both substances requires caution to avoid undesired side effects.

In liver the mitochondrial sirtuin, SIRT5, controls ammonia detoxification by regulating CPS1, the first enzyme of the urea cycle. However, while SIRT5 is ubiquitously expressed, urea cycle and CPS1 are only present in the liver and, to a minor extent, in the kidney. To address the possibility that SIRT5 is involved in ammonia production also in nonliver cells, clones of human breast cancer cell lines MDA-MB-231 and mouse myoblast C2C12, overexpressing or silenced for SIRT5 were produced. Our results show that ammonia production increased in SIRT5-silenced and decreased in SIRT5-overexpressing cells. We also obtained the same ammonia increase when using a new specific inhibitor of SIRT5 called MC3482. SIRT5 regulates ammonia production by controlling glutamine metabolism. In fact, in the mitochondria, glutamine is transformed in glutamate by the enzyme glutaminase, a reaction producing ammonia. We found that SIRT5 and glutaminase coimmunoprecipitated and that SIRT5 inhibition resulted in an increased succinylation of glutaminase. We next determined that autophagy and mitophagy were increased by ammonia by measuring autophagic proteolysis of long-lived proteins, increase of autophagy markers MAP1LC3B, GABARAP, and GABARAPL2, mitophagy markers BNIP3 and the PINK1-PARK2 system as well as mitochondrial morphology and dynamics. We observed that autophagy and mitophagy increased in SIRT5-silenced cells and in WT cells treated with MC3482 and decreased in SIRT5-overexpressing cells. Moreover, glutaminase inhibition or glutamine withdrawal completely prevented autophagy. In conclusion we propose that the role of SIRT5 in nonliver cells is to regulate ammonia production and ammonia-induced autophagy by regulating glutamine metabolism.

Elevated intraocular pressure is a crucial pathologic event for the development of glaucoma (GL). We have reported that nerve growth factor (NGF) reaches retinal cells and the optic nerve (ON) when applied to the eye. Whether ocular application of NGF prevents or reduces damage to retinal ganglion cell (RGC) is not known.

NG108-15 cells differentiate into neurons by 1 mM sodium butyrate (NaB) treatment. Differentiated cells resulted more resistant to staurosporine (STS) than proliferating cells. In particular, STS treatment decreased Bcl-2 and Bcl-xL content in mitochondria of proliferating cells, but not in mitochondria of differentiated cells. Bad was phosphorylated and down-regulated only in differentiated cells. Bax accumulated in the mitochondria of proliferating but not differentiated cells. Mitochondrial release of cytochrome c was observed in proliferating cells, whereas mitochondria of differentiated cells retained cytochrome c. Proliferating cells treated with STS accumulated Endo G and AIF in the nucleus. By contrast, differentiated cells did not show such nuclear accumulation. Treatment of differentiated cells with Insulin-like Growth Factor-1 (IGF-1) and STS resulted in a 17,1% increase of cell viability. The survival role of IGF-1 was demonstrated by treating differentiated cells with an anti-IGF-1 neutralizing antibody. Such treatment significantly increased STS-induced cell death. Electrophysiology studies showed that in STS-treated cells membrane potential oscillations were reduced in amplitude without giving rise to spontaneous action potentials (APs). However, the percentage of cells yielding overshooting APs returned to 100% after STS removal. It is concluded that neuronal differentiation of NG108-15 cells induces resistance to apoptotic cell death and that IGF-1 plays a central role in sustaining this mechanism.

Adaptation to hypoxia through activation of the hypoxia inducible factor-1 (HIF-1) is crucial for tumor cells survival. Here we describe the antitumoral effects of the new molecule CR 3294 on tumor cells in the presence of hypoxia. Treatment of the breast carcinoma cell line MDA-MB-231 with CR 3294 in 1% O2 resulted in an in vivo and in vitro inhibition of tumor growth. CR 3294 induced accumulation of autophagosomes in hypoxic MDA-MB-231 cells as assessed by both transmission electron microscopy (TEM) and the autophagic marker LC3-II. TEM analysis revealed the presence of invaginations of the cytoplasm into the nucleus. Autophagosomes were present in such invaginations. Moreover, CR 3294 inhibited both the DNA binding of HIF-1α and VEGF mRNA synthesis. Immunoprecipitation and immunofluorescence studies showed an interaction between LC3 and HIF-1α. We next detailed the effect of inhibitors and activators of autophagy on both HIF-1α and LC3. In particular, 3 methyladenine (3MA) and wortmannin, two macroautophagic inhibitors, prevented both the decrease of HIF-1α protein levels and LC3 processing in cells treated with CR 3294. Bafilomycin and leupeptin, inhibitors of lysosomes, prevented HIF-1α decrease without affecting LC3 processing. By contrast, treating hypoxic MDA-MB-231 cells with trifluoperazine (TFP) or serum withdrawal (SW), two activators of autophagy, diminished HIF-1α levels and stimulated LC3 processing. These results indicate that activation of the autophagic pathway in hypoxic cells by the new molecule CR 3294, as well as by TFP or SW, can have potentially important implications for cancer treatment.

Early detection of myocardial dysfunction in Fabry disease (FD) cardiomyopathy suggests the contribution of myofilament structural alterations. Six males with untreated FD cardiomyopathy submitted to cardiac studies, including tissue Doppler imaging and left ventricular endomyocardial biopsy. Active and resting tensions before and after treatment with protein kinase A (PKA) were determined in isolated Triton-permeabilized cardiomyocytes. Cardiomyocyte cross-sectional area, glycosphingolipid vacuole area, myofibrillolysis, and extent of fibrosis were also determined. Biopsies of mitral stenosis in patients with normal left ventricles served as controls. Active tension was four times lower in FD cardiomyocytes and correlated with extent of myofibrillolysis. Resting tension was six times higher in FD cardiomyocytes than in controls. PKA treatment decreased resting tension but did not affect active force. Protein analysis revealed troponin I and desmin degradation products. FD cardiomyocytes were significantly larger and filled with glycosphingolipids. Fibrosis was mildly increased compared with controls. Tissue Doppler imaging lengthening and shortening velocities were reduced in FD cardiomyocytes compared with controls, correlating with resting and active tensions, respectively, but not with cardiomyocyte area, percentage of glycosphingolipids, or extent of fibrosis. In conclusion, myofilament degradation and dysfunction contribute to FD cardiomyopathy. Partial reversal of high resting tension after pharmacological PKA treatment of cardiomyocytes suggests potential benefits from enzyme replacement therapy and/or energy-releasing agents.

Pyrrolo1,2-b1,2,5benzothiadiazepine 5,5-dioxides (PBTDs) induced apoptosis in human BCR-ABL-expressing leukemia cells. The apoptotic activity was also observed in primary leukemic blasts, obtained from chronic myelogenous leukemia (CML) patients at onset or from patients in blast crisis and who were imatinib-resistant. Compounds 5and14 induced apoptosis before BCR-ABL protein expression and tyrosin phosphorylation were affected and activated different caspases in the apoptotic pathway. PBTDs are a new class of valid candidates for the treatment of CML.

Objectives:Macrophage migration inhibitory factor (MIF), a multifunctional proinflammatory cytokine, has been recently involved in many aspects of reeproduction including pregnancy. However, no evidence is available on the role of MIF in gestational tissues nor on factors regulating MIF production. This study, conducted on explants of human fetal membranes at term gestation, has been undertaken to investigate whether: (1) MIF is produced by fetal membranes; (2) nitric oxide (NO) can regulate local MIF production; and (3) MIF, in turn, can influence NO release in these tissues.Methods:Tissues were obtained from 56 healthy women who underwent elective cesarean delivery. Fetal membranes have been incubated with either sodium nitroprusside (NP), a NO donor, or recombinant MIF (r-MIF), or a specific anti-MIF antibody (MIF-Ab). Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, enzyme-linked immunosorbent assay (ELISA), and colorimetric assay have been used to detect MIF mRNA and protein, inducible nitric oxide synthase (iNOS), and NO metabolites.Results:Fetal membranes basally express MIF mRNA and protein and release MIF. Exposing tissues to NP results in an increase of MIF mRNA expression and protein release. Conversely, treatment of tissues with MIF is followed by a reduction in iNOS mRNA and protein expression as well as in NO release. These effects are reversed by adding MIF-Ab.Conclusions:MIF is generated and released by human fetal membranes at term. MIF mRNA and protein expression and release are modulated by NO. MIF, in turn, can reduce iNOS expression and NO release by these tissues. NO could be a regulator of MIF production in pregnancy and labor.

Objectives: Macrophage migration inhibitory factor (MIF), a multifunctional proinflammatory cytokine, has been recently involved in many aspects of reproduction including pregnancy. However, no evidence is available on the role of MIF in gestational tissues nor on factors regulating MIF production. This study, conducted on expiants of human fetal membranes at term gestation, has been undertaken to investigate whether: (1) MIF is produced by fetal membranes; (2) nitric oxide (NO) can regulate local MIF production; and (3) MIF, in turn, can influence NO release in these tissues. Methods: Tissues were obtained from 56 healthy women who underwent elective cesarean delivery. Fetal membranes have been incubated with either sodium nitroprusside (NP), a NO donor, or recombinant MIF (r-MIF), or a specific anti-MIF antibody (MIF-Ab). Reverse transcriptase—polymerase chain reaction (RT-PCR), Western blot, enzyme-linked immunosorbent assay (ELISA), and colorimetric assay have been used to detect MIF mRNA and protein, inducible nitric oxide synthase (iNOS), and NO metabolites. Results: Fetal membranes basally express MIF mRNA and protein and release MIF. Exposing tissues to NP results in an increase of MIF mRNA expression and protein release. Conversely, treatment of tissues with MIF is followed by a reduction in iNOS mRNA and protein expression as well as in NO release. These effects are reversed by adding MIF-Ab. Conclusions: MIF is generated and released by human fetal membranes at term. MIF mRNA and protein expression and release are modulated by NO. MIF, in turn, can reduce iNOS expression and NO release by these tissues. NO could be a regulator of MIF production in pregnancy and labor.

Laser microdissection (LMD) is an accurate and fast method to procure pure populations of cells from complex, heterogeneous tissues under direct microscopic visualization. It can be applied to a wide range of cell preparation, including paraffin-embedded material. The morphology of the captured cells is retained, and DNA, RNA, and proteins can be extracted for molecular analysis. The potential applications of LMD to human cardiovascular research are multiple, including viral/autoimmune myocarditis and arteritis, atherosclerotic lesions, and myocardial and vascular cell proliferation and death. Molecular and genetic analysis of LMD-procured cells in cardiac and vascular tissues may provide a better understanding of several cardiac diseases.

The development of effective enzyme replacement/enhancement therapy makes of clinical relevance considering Fabry disease in the differential diagnosis of patients with hypertrophic cardiomyopathy. In particular the opportunity to significantly modify the clinical progression of the disease has reinforced the need for early diagnosis of Fabry cardiomyopathy.

Induction of apoptosis in HeLa cells with staurosporine produced a rise in the intracellular pH (pHi). Intracellular alkalinization was accompanied by translocation of Bax to the mitochondria, cytochromecrelease, and cell death. The chloride channel inhibitor furosemide prevented intracellular alkalinization, Bax translocation, cytochrome crelease, and cell death. Translocation of full-length Bid to the mitochondria was also prevented by furosemide. The cleavage product of Bid degradation (truncated Bid, tBid) was not detectable in the mitochondria. Its accumulation in the cytosol was prevented by furosemide. Apoptosis induced by tumor necrosis factor-α (TNF) lowered pHi, an effect also accompanied by Bax translocation, cytochrome crelease, and cell killing. Furosemide prevented all of these events. TNF induced a depletion of full-length Bid from the mitochondria and the cytosol but induced an accumulation of mitochondrial tBid. Furosemide only delayed full-length Bid depletion and tBid accumulation. The caspase 8 inhibitor IETD did not prevent the translocation of Bax. Although IETD did inhibit the cleavage of Bid and the accumulation of tBid, cell killing was reduced only slightly. It is concluded that with either staurosporine or TNF a furosemide-sensitive change in pHiis linked to Bax translocation, cytochrome crelease, and cell killing. With TNF Bax translocation occurs as Bid is depleted and can be dissociated from the accumulation of tBid. With staurosporine a role for full-length Bid in Bax translocation cannot be excluded but is not necessary as evidenced by the data with TNF.

Treatment of L929 fibroblasts by the topoisomerase II inhibitor etoposide killed 50% of the cells within 72 h. The cell killing was preceded by the release of cytochromecfrom the mitochondria. Simultaneous treatment of the cells with wortmannin, cycloheximide, furosemide, cyclosporin A, or decylubiquinone prevented the release of cytochrome cand significantly reduced the loss of viability. Etoposide caused the phosphorylation of p53 within 6 h, an effect prevented by wortmannin, an inhibitor of DNA-dependent protein kinase (DNA-PK). The activation of p53 by etoposide resulted in the up-regulation of the pro-apoptotic protein Bax, a result that was prevented by the protein synthesis inhibitor cycloheximide. The increase in the content of Bax was followed by the translocation of this protein from the cytosol to the mitochondria, an event that was inhibited by furosemide, a chloride channel inhibitor. Stably transfected L929 fibroblasts that overexpress Akt were resistant to etoposide and did not translocate Bax to the mitochondria or release cytochrome c. Bax levels in these transfected cells were comparable with the wild-type cells. The release of cytochromecupon translocation of Bax has been attributed to induction of the mitochondrial permeability transition (MPT). Cyclosporin A and decylubiquinone, inhibitors of MPT, prevented the release of cytochrome cwithout affecting Bax translocation. These data define a sequence of biochemical events that mediates the apoptosis induced by etoposide. This cascade proceeds by coupling DNA damage to p53 phosphorylation through the action of DNA-PK. The activation of p53 increases Bax synthesis. The translocation of Bax to the mitochondria induces the MPT, the event that releases cytochrome cand culminates in the death of the cells.

In Jurkat cells Bid was cleaved upon activation of the Fas receptor with an anti-Fas antibody. The caspase-8 inhibitor benzyloxycarbonyl-Ile-Glu(OMe)-Thr-Asp(OMe)-CH2F (IETD) prevented the cleavage of Bid and the loss of viability. The nuclear enzyme poly(ADP-ribose)polymerase (PARP) was also cleaved upon the activation of caspases, and IETD similarly prevented PARP cleavage. The PARP inhibitor 3-aminobenzamide (3-AB) restored the cell killing in the presence of IETD, an effect that occurred without restoration of the cleavage of Bid or PARP. In the presence of 3-AB and IETD, translocation occurred of full-length Bid to the mitochondria. The induction of the mitochondrial permeability transition (MPT) was documented by the cyclosporin A (CyA) sensitivity of the release of cytochrome c, the release of malate dehydrogenase from the mitochondrial matrix, the loss of the mitochondrial membrane potential, and the pronounced swelling of these organelles, as assessed by electron microscopy. In addition to preventing all evidence of the MPT, CyA prevented the loss of cell viability, without effect on the cleavage of either Bid or PARP. The prevention of PARP cleavage by inhibition of caspase-3 resulted in a 10-fold activation of the enzyme and a resultant depletion of NAD and ATP. The PARP inhibitor 3-AB prevented the loss of NAD and ATP. Depletion of ATP by metabolic inhibitors similarly prevented the cell killing. It is concluded that the cleaving of PARP in Fas-mediated apoptosis allowed expression of an energy-dependent cell death program that included the translocation of full-length Bid to the mitochondria with induction of the MPT.

The expression of p53 and the retinoblastoma gene has been investigated by immunohistochemical and molecular analysis in 45 cases of nodal peripheral T-cell lymphoma with high-grade histology. Most cases (73·3 per cent) were primary nodal lymphomas without any extra-nodal site involvement. Most of them (75·6 per cent) were histologically classified as pleomorphic, small, medium, and large cell type. Immunohistochemistry detected p53 in nine cases (20 per cent). In each of these cases, the polymerase chain reaction (PCR)/heteroduplex analysis did not show the presence of mutations, this finding being consistent with an alteration of the p53 functional pathway, in the presence of a wild-type protein. The retinoblastoma gene product was detected by immunohistochemistry in 35 cases (77·8 per cent) and not detected in ten cases (22·2 per cent). In the latter cases, the reverse transcription (RT)-PCR analysis showed the presence of a specific retinoblastoma gene transcript in six cases and was negative in the remaining four cases. The immunohistochemical and molecular findings seem to be consistent with abnormalities of retinoblastoma gene expression at either the transcriptional or the post-transcriptional level. Since all nine p53-positive cases by immunohistochemical analysis were also retinoblastoma gene product-positive, and all ten retinoblastoma gene product-negative cases were also p53-negative, two different and mutually exclusive pathways of possible pathogenetic significance may be suggested, the former involving abormalities of the functional pathway of p53 in the absence of mutations and the latter abnormalities of retinoblastoma gene expression at the transcriptional and/or post-transcriptional level. Finally, the clinico-pathological correlations showed that p53 immunohistochemical expression is significantly associated with a poorer response to intensive chemotherapy. Copyright © 1999 John Wiley & Sons, Ltd.

The expression of p53 and the retinoblastoma gene has been investigated by immunohistochemical and molecular analysis in 45 cases of nodal peripheral T‐cell lymphoma with high‐grade histology. Most cases (73·3 per cent) were primary nodal lymphomas without any extra‐nodal site involvement. Most of them (75·6 per cent) were histologically classified as pleomorphic, small, medium, and large cell type. Immunohistochemistry detected p53 in nine cases (20 per cent). In each of these cases, the polymerase chain reaction (PCR)/heteroduplex analysis did not show the presence of mutations, this finding being consistent with an alteration of the p53 functional pathway, in the presence of a wild‐type protein. The retinoblastoma gene product was detected by immunohistochemistry in 35 cases (77·8 per cent) and not detected in ten cases (22·2 per cent). In the latter cases, the reverse transcription (RT)‐PCR analysis showed the presence of a specific retinoblastoma gene transcript in six cases and was negative in the remaining four cases. The immunohistochemical and molecular findings seem to be consistent with abnormalities of retinoblastoma gene expression at either the transcriptional or the post‐transcriptional level. Since all nine p53‐positive cases by immunohistochemical analysis were also retinoblastoma gene product‐positive, and all ten retinoblastoma gene product‐negative cases were also p53‐negative, two different and mutually exclusive pathways of possible pathogenetic significance may be suggested, the former involving abormalities of the functional pathway of p53 in the absence of mutations and the latter abnormalities of retinoblastoma gene expression at the transcriptional and/or post‐transcriptional level. Finally, the clinico‐pathological correlations showed that p53 immunohistochemical expression is significantly associated with a poorer response to intensive chemotherapy. Copyright © 1999 John Wiley & Sons, Ltd.

The immunological rejection of two strains of Ehrlich adenocarcinoma ascites cells and of leukemia L1210 ascites cells was studied respectively in Swiss and DBA/2 mice previously immunized with irradiated tumor cells. During the first phases of the reaction cell contacts between lymphocytes and cancer cells, lymphocytes and macrophages and also between two lymphocytes were frequently observed. Afterwards macrophages surrounding tumor cells and macrophages loaded either with whole tumor cells or with cell debris were a usual finding. During the immunological rejection 7S globulin could not be detected on cancer cells by fluoresceine isothiocyanate labelled rabbit γ-globulin anti-7S mouse globulin. Cytotoxicity test performed on 51Cr-labelled tumor cells showed that peritoneal cells are more active than spleen and lymph node cells. Some hypotheses on the observed phenomena are discussed.

Anderson (1965) has described a simple method for preparing relatively pure fields of leukocytes from peripheral blood for electron microscopy using an angular spatula to detach and remove the buffy coat from the wall of a standard conical centrifuge tube. the buffy coat is, however, fragile, especially after gentle centrifugation (1500 × g for 7-9 min) and brief (5-10 min) fixation. the spatula often breaks the buffy coat and mixes it with the red cells below. the conical centrifuge tube is also disadvantageous. Its large cross-section and capacity require relatively large amounts of blood and it gives a buffy coat which, for normal blood, is too thin to permit adequate representation in individual transverse sections of those leukocytes which occur in low percentages. the use of special tubes to circumvent these problems (Wright and Douglas 1902, Endres 1927, Bessis 1940, Butler and Cushmann 1941, Chevillard and Hamon 1943, Ottesen 1954, Bessis 1956) has been very useful for cell separation but the design of the tubes proposed is not suitable for routine electron microscopy.

A patient with acute rhabdomyolysis and absence of myoadenylate deaminase (MADA) associated with chronic licorice intoxication is presented. Clinical and laboratory examination of the patient and morphologic study over skeletal muscle were performed. The major effect of licorice intoxication is hypokalemia, which may explain most of the observed clinical symptoms and morphological changes. The absence of MADA may be a consequence of the direct toxic effect of licorice glycosides. To our knowledge, this is the first report in which a lack of MADA and chronic licorice intoxication has been shown to be associated with clinical, histochemical, biochemical, and morphological changes, which were completely reversed with potassium supplementation and licorice withdrawal.

A catecholamine-induced dilated cardiomyopathy is reported in a patient with multiple endocrine neoplasia, type 3. A histologic and ultrastructural study has been undertaken in cardiac biopsy samples, together with determination of myocardial Ca++and cellular membrane fatty acids. Contraction band necrosis of cardiocytes with supercontraction of sarcomeres progressing to myofibrolysis and increased levels of myocardial Ca++have been found as morphologic and biochemical abnormalities, respectively. No lipoperoxidation of cellular membranes or an α-adrenergic mediated reduction of coronary supply could be recognized in the study. We indicate a receptor-mediated intracellular Ca++overload as the main abnormality responsible for myocardial impairment.

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Background:Recent findings suggest that epithelial to mesenchymal transition (EMT), a key step during heart development, is involved in cardiac tissue repair following myocardial infarction (MI). In particular, epicardial cells that undergo this process, acquire stem cell-like properties, migrate and differentiate into a number of distinct cell types and release paracrine factors therefore supporting cardiac repair. MicroRNAs (miRNAs) act as key regulators in EMT processes; however, the mechanisms by which miRNAs target epicardial EMT remain largely unknown. Here, we investigated the role of miRNAs as regulators of epicardial EMT and their potential targets.Methods and Results:EMT was induced in epicardial-mesothelial cells (EMCs) through TGF?1 treatment for 48, 72 and 96 hours as indicated by the expression of EMT-related genes by q-RTPCR, WB and immunofluorescence. Further, enhanced expression of stemness genes was also detected. Among several EMT-related miRNAs, miR-200c-5p expression resulted the most strongly suppressed. Interestingly, we also found a significant uregulation of Follistatin-related protein 1 (FSTL1), already identified as a potent cardiogenic factor produced by epicardial cells that promotes regeneration following MI. These results were strengthened by the detection, in vivo, of higher FSTL1 mRNA expression in epicardial cells of 3 day-infarcted hearts compared to the underlying cardiac tissue and prompted us to verify a possible involvement of FSTL1 in epicardial EMT. Bioinformatics analysis and the dual-luciferase reporter assay demonstrated that miR-200c-5p directly targeted the 3?-untranslated region of FSTL1 in EMCs. Consistently, WB analysis showed that knockdown of miR-200c-5p significantly increased FSTL1 expression, whereas overexpression of miR-200c-5p counteracted TGF?1-mediated FSTL1 upregulation. Importantly, FSTL1 silencing maintained epithelial features in EMCs, despite EMT induction, and attenuated EMT-associated traits, including migration and stemness.Conclusions:Epicardial FSTL1 plays an important role in cardiac repair as a cardiogenic factor in its secreted form and by inducing EMT, stemness and migration of EMCs in a miR-200c-5p dependent pathway

A service robot for Cultural Heritage frames is proposed as a novel robotic platform with a modular design for both ground locomotion and flight capability. The peculiarities of the system are discussed by performance evaluation via simulation. A prototype has been built and tested both to prove the feasibility of the proposed design and to characterize its operation performance.

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