The comparison of the process of manual and robotic positioning of the electrode performing radiofrequency ablation under the control of a surgical navigation system.

This study is aimed at the comparison of the process of manual and robotic positioning of the electrode performing radiofrequency ablation under the control of a surgical navigation system. The main hypothesis of this experiment was that the use of a collaborative manipulator (KUKA iiwa) will allow to position the active part of the electrode relative to the center of the tumor more accurately and from the first attempt. We also monitor the stability of the electrode's velocity during insertion and consider some advantages in ergonomics using the robotic manipulator. We use three more criteria to compare the surgeon's and robotic performance, unlike other studies, where only the target point's accuracy criterion is observed. The main idea is to examine the movement parameters of the electrode that can lead to potential patient trauma. Sphere-shaped tumor phantoms measuring 8 mm in diameter were filled with contrast and inserted in bovine livers. 10 livers were used for the robotic experiment and an equal quantity for manual surgery. The livers were encased in silicone phantoms designed to imitate the liver position in a real patient's abdominal cavity. Analysis of CT data gave the opportunity to find the entry and the target point for each tumor phantom. This data was loaded into a surgical navigation system that was used to track and record the position of the RF-electrode during the operation for further analysis. The standard deviation of points from the programmed linear trajectory totaled in the average 0.3 mm for the robotic experiment and 2.33 mm for the manual operation with a maximum deviation of 0.55 mm and 7.99 mm respectively. Standard deviation from the target point was 2.69 mm for the collaborative method and 2.49 mm for the manual method. The average velocity was 2.97 mm/s for the manipulator and 3.12 mm/s for the manual method, but the standard deviation of the velocity relative to the value of the average velocity was 0.66 mm/s and 3.05 mm/s respectively. Thus, in two criteria out of three, the manipulator is superior to the surgeon, and equality is established in one. Surgeons also noticed advantages in ergonomics performing the procedure using the manipulator. This experiment was produced as part of the work on the developing of a robotic multifunctional surgical complex. We can confirm the potential advantages of using collaborative robotic manipulators for minimally invasive surgery in case of practice for cancer treatment.

Latent Inflammation and Defect in Adipocyte Renewal as a Mechanism of Obesity-Associated Insulin Resistance.

Obesity is a major risk factor for type 2 diabetes and metabolic syndrome and an essential medical and social problem. In the first part of the review, we briefly highlight the biochemical basis of metabolic disbalance in obesity and evolution of our views on the mechanisms of insulin resistance development in insulin-sensitive tissues. Because obesity relates to the disturbance in the normal physiology of fat tissue, the second part of the review focuses on latent inflammation that develops in obesity and is supported by immune cells. Finally, the problem of adipocyte hypertrophy, reduced regenerative potential of fat progenitor cells, and impaired renewal of fat depots is discussed in the context of type 2 diabetes pathogenesis.

Chemical Inducers of Obesity-Associated Metabolic Stress Activate Inflammation and Reduce Insulin Sensitivity in 3T3-L1 Adipocytes.

Obesity is accompanied by dyslipidemia, hypoxia, endoplasmic reticulum (ER) stress, and inflammation, representing the major risk factor for the development of insulin resistance (IR) and type 2 diabetes. We modeled these conditions in cultured 3T3-L1 adipocytes and studied their effect on insulin signaling, glucose uptake, and inflammatory response via activation of stress-dependent JNK1/2 kinases. Decreased insulin-induced phosphorylation of the insulin cascade components IRS, Akt, and AS160 was observed under all tested conditions (lipid overloading of cells by palmitate, acute inflammation induced by bacterial lipopolysaccharide, hypoxia induced by Co2+, and ER stress induced by brefeldin A). In all the cases, except the acute inflammation, glucose uptake by adipocytes was reduced, and the kinetics of JNK1/2 activation was bi-phasic exhibiting sustained activation for 24 h. By contrast, in acute inflammation, JNK1/2 phosphorylation increased transiently and returned to the basal level within 2-3 h of stimulation. These results suggest a critical role of sustained (latent) vs. transient (acute) inflammation in the induction of IR and impairment of glucose utilization by adipose tissue. The components of the inflammatory signaling can be promising targets in the development of new therapeutic approaches for preventing IR and type 2 diabetes.

Low proliferative potential of adipose-derived stromal cells associates with hypertrophy and inflammation in subcutaneous and omental adipose tissue of patients with type 2 diabetes mellitus.

BACKGROUND: Obesity and type 2 diabetes mellitus (T2DM) are among the most important morbidity factors. In this study we tested the hypothesis that low proliferative potential of adipose derived stromal cells (ADSC) associates with reduced formation of new fat depots, excess accumulation of fat in the functional adipocytes and their hypertrophy, resulting in fat inflammation and insulin resistance. METHODS: We screened two groups of obese patients with or without T2DM, matched for BMI, age, and duration of obesity to test the hypothesis that hypertrophy and decreased renewal of adipocytes may underlie transition from obesity to T2DM. All patients were matched for carbohydrate metabolism (fasting blood glucose level, glycated hemoglobin, HOMA-IR index and M-index). The subcutaneous and omental fat tissue biopsies were obtained during bariatric surgery from obese individuals with or without T2DM. The morphology and immunophenotype of subcutaneous and omental fat was assessed in frozen tissue sections. ADSC were isolated from both types of fat tissue biopsies and screened for morphology, proliferative potential and inflammatory status. RESULTS: The non-diabetic patients had normal carbohydrate metabolism and moderate insulin resistance measured by HOMA-IR and hyperinsulinemic clamp (M-index), while T2DM patients were extremely insulin resistant by both indexes. The average size of diabetic adipocytes was higher than that of non-diabetic in both subcutaneous and omental fat tissues, indicating adipocyte hypertrophy in T2DM. Both these tissues contained higher level of macrophage infiltration and increased M1-like to M2-like ratio of macrophage subpopulations, suggesting increased fat inflammation in T2DM. This was confirmed by increased activatory phosphorylation of stress-induced JNK1/2 in diabetic ADSC. CONCLUSION: These results suggest that blunted proliferation and increased hypertrophy of diabetic ADSC may lead to reduced insulin sensitivity via increased inflammation mediated by M1 macrophages and JNK1/2 pathway.

Interleukin-4 Restores Insulin Sensitivity in Lipid-Induced Insulin-Resistant Adipocytes.

Obesity and latent inflammation in adipose tissue significantly contribute to the development of insulin resistance (IR) and type 2 diabetes. Here we studied whether the antiinflammatory interleukin-4 (IL-4) can restore insulin sensitivity in cultured 3T3-L1 adipocytes. The activity of key components of the insulin signaling cascade was assessed by immunoblotting using phospho-specific antibodies to insulin receptor substrate IRS1 (Tyr612), Akt (Thr308 and Ser473), and AS160 (Ser318) protein that regulates translocation of the GLUT4 glucose transporter to the plasma membrane. IR was induced in mature adipocytes with albumin-conjugated palmitate. IR significantly reduced phosphorylation levels of all the above-mentioned proteins. Addition of IL-4 to the culturing medium during IR induction led to a dose-dependent stimulation of the insulin-promoted phosphorylation of IRS1, Akt, and AS160. At the optimal concentration of 50 ng/ml, IL-4 fully restored activation of the insulin cascade in IR cells, but it did not affect insulin signaling activation in the control cells. IL-4 neither upregulated expression of key adipogenesis markers GLUT4 and PPARγ nor caused lipid accumulation in the adipocytes. These results demonstrate that IL-4 can restore insulin sensitivity in adipocytes via mechanisms not associated with induced adipogenesis or de novo formation of lipid depots.

Higher Ca2+ -sensitivity of arterial contraction in 1-week-old rats is due to a greater Rho-kinase activity.

AIM: During early post-natal development, arterial contraction depends less on Ca2+ -signalling pathways but more on changes in Ca2+ -sensitivity compared to adult animals. Whether this difference is related to Rho-kinase, one of the major players affecting Ca2+ -sensitivity, is unknown for intact vessels. Thus, we tested the hypothesis that Rho-kinase critically contributes to the higher Ca2+ -sensitivity of contraction in intact arteries of 1-week-old rats. METHODS: We studied 1-week-old, 4- to 5-week-old and 10- to 12-week-old rats performing isometric myography, Ca2+ -fluorimetry and Western blotting using intact saphenous arteries and arterial pressure measurements under urethane anaesthesia. RESULTS: In 10- to 12-week-old rats, methoxamine (MX) produced vasoconstriction associated with an increase in [Ca2+ ]i and Ca2+ -sensitivity. In contrast, in 1-week-old rats these contractions were accompanied only by an increase in Ca2+ -sensitivity. All MX-induced effects were reduced by the Rho-kinase inhibitor Y-27632; this reduction was complete only in 1-week-old rats. The Rho-kinase specific site Thr855 on MYPT1 was increasingly phosphorylated by MX in vessels of 1-week-old, but not 10- to 12-week-old rats; this effect was also inhibited completely by Y-27632. The Rho-kinase inhibitor fasudil in a dose not affecting the pressor response to MX in 4- to 5-week-old rats reduced it considerably in 1-week-old rats. CONCLUSION: Our results suggest that the higher Ca2+ -sensitivity of arterial contraction in 1-week-old compared to 10- to 12-week-old rats is due to a greater Rho-kinase activity. Constitutively active Rho-kinase contributes to MX-induced contraction in 10- to 12-week-old rats. In 1-week-old rats, additional Rho-kinase activation is involved. This remodelling of the Rho-kinase pathway is associated with its increased contribution to adrenergic arterial pressure responses.

Latent Inflammation and Insulin Resistance in Adipose Tissue.

Obesity is a growing problem in modern society and medicine. It closely associates with metabolic disorders such as type 2 diabetes mellitus (T2DM) and hepatic and cardiovascular diseases such as nonalcoholic fatty liver disease, atherosclerosis, myocarditis, and hypertension. Obesity is often associated with latent inflammation; however, the link between inflammation, obesity, T2DM, and cardiovascular diseases is still poorly understood. Insulin resistance is the earliest feature of metabolic disorders. It mostly develops as a result of dysregulated insulin signaling in insulin-sensitive cells, as compared to inactivating mutations in insulin receptor or signaling proteins that occur relatively rare. Here, we argue that inflammatory signaling provides a link between latent inflammation, obesity, insulin resistance, and metabolic disorders. We further hypothesize that insulin-activated PI3-kinase pathway and inflammatory signaling mediated by several IκB kinases may constitute negative feedback leading to insulin resistance at least in the fat tissue. Finally, we discuss perspectives for anti-inflammatory therapies in treating the metabolic diseases.

Trophic action of sympathetic nerves reduces arterial smooth muscle Ca(2+) sensitivity during early post-natal development in rats.

AIM: A decrease in the Ca(2+) sensitivity of smooth muscle contraction is a hallmark of functional remodelling of blood vessels during development. However, the responsible factors are largely unknown. Here, we tested the hypothesis that the post-natal decline of arterial Ca(2+) sensitivity is the result of trophic effects of sympathetic nerves. METHODS: Contractile responses, intracellular Ca(2+) levels and protein expression profiles were compared in saphenous arteries from young (1- and 2-week-old) and adult rats using wire myography, Ca(2+) fluorimetry and Western blotting respectively. RESULTS: We observed a lower Ca(2+) sensitivity of contractions induced by methoxamine, an agonist of α1 -adrenoceptors, and U46619, an agonist of thromboxane A2 receptors, in arteries from adult as compared to young animals. Post-natal maturation was associated with stronger expression of regulatory proteins mediating Ca(2+) -dependent contraction (myosin light chain kinase (MLCK), myosin targeting subunit (MYPT1) and h-caldesmon) and weaker expression of proteins regulating Ca(2+) -independent contraction (Rho kinase, extracellular-regulated kinases (ERK1/2) and mitogen-activated protein kinases p38 MAPK) in vessels from adult rats. To eliminate the trophic action of sympathetic nerves, we performed lumbar sympathectomy in adult rats. This resulted in higher Ca(2+) sensitivity of agonist-induced contractions in denervated as compared to control arteries. Furthermore, denervated arteries contained less MLCK, MYPT1 and h-caldesmon and more ERK1/2 and p38 MAPK. CONCLUSIONS: Sympathetic denervation reverses developmental changes both in Ca(2+) sensitivity and in the expression of regulatory proteins back to the early post-natal phenotype in the rat saphenous artery. We conclude that trophic effects of sympathetic nerves govern functional remodelling of arteries during early post-natal development.

[Molecular mechanisms of gradient sensing in mesenchymal cells].

Cell migration is an important fundamental process. It occurs over the embryonic development, immune system responses, reparation and regeneration of damaged tissues. Abnormal migration is characteristic for many disorders such as cancer metastasis. Both external and intrinsic factors control directionality of cell movement. In case the external factors are of chemical nature the directed migration is called chemotaxis. Here we review current models of directional sensing in mesenchymal cells and compare them to well studied mechanisms of amoebal chemotaxis.

Chemotaxis: movement, direction, control.

This review focuses on basic principles of motility in different cell types, formation of the specific cell structures that enable directed migration, and how external signals are transduced into cells and coupled to the motile machinery. Feedback mechanisms and their potential role in maintenance of internal chemotactic gradients and persistence of directed migration are highlighted.

[NADPH oxidase controls EGF-induced proliferation via the ERK1/2-independent mechanism].

Using HyPer, a ratiometric GFP-based biosensor, the dynamics of H2O2 in living cells has been studied. It was found that activation of the receptor of the epidermal growth factor (EGF) in epithelial cells leads to sustained generation of intracellular H2O2, which is blocked by apocynin, an inhibitor of the assembly of plasma membrane NADPH oxidase. Apocynin also blocked HeLa cell proliferation induced by EGF, indicating that NADPH oxidase should be involved in the process. However, apocynin failed to alter the kinetics of the EGF-stimulated ERK1/2 activation. It was concluded that NADPH oxidase and intracellular H2O2 are the important downstream targets of the EGF receptor, which mediate the proliferation response by mechanisms distinct from the activation of the classical ERK1/2 MAP-kinase pathway.

[Myosin phosphorylation as the main way of regulating smooth muscle contractions].

Rho-kinase is a key enzyme of the receptor-dependent signal cascades and is regarded today as the most prospective target for pharmacological therapy of smooth muscle contractility disorders.

[Caldesmon changes the structure of actin at the leading edge and suppresses cell migration].

The effect of the suppression of expression of the actin-binding protein caldesmon on the motility of nonmuscle cells has been studied. A more than fivefold decrease in the content of this protein in cells by RNA interference led to the disturbance of the formation of actin stress fibrils and acceleration of cell migration to the zone of injury of the monolayer. A stimulation of stationary cells by serum induced a more than 1.5-fold accumulation of stress fibrils only in control cells but not in caldesmon-deficient cells. Similarly, the accumulation of actin filaments was observed in actively migrating cells of only wild type but not in cells with a low caldesmon content. These changes occurred mainly at the leading edge of the migrating cell where the distinct structure of actin filaments was not seen in the absence of caldesmon. It was assumed that caldesmon inhibits cell migration due to the stabilization of actin in filaments and a decrease in the dynamics of monomeric actin at the leading edge of the migrating cell.

[The contribution of protein kinase C and Rho-kinase to the control of the receptor-dependent artery contraction decreases with age independently of sympathetic innervation].

The age-related dynamics of the activity of signalling pathways coupled to alpha1-adrenergic receptors and their dependence on the sympathetic innervation of arterial smooth muscle have been studied. The effects of the protein kinase C inhibitor (GF109203X, 10(-6) M) and the Rho-kinase inhibitor (Y27632, 10(-5) M) on the isometric contraction of the rat saphenous artery, induced by the alpha1-adrenoceptor agonist methoxamine, were examined. It was shown that the sensitivity to methoxamine of arteries from 2-week-old rats that are partially innervated was reduced as compared to adults, but the effects of both inhibitors were more prominent. The denervation induced by the excision of sympathetic ganglia increased the arterial sensitivity to methoxamine but was not accompanied by changes in sensitivity to the inhibitors. Therefore, the postnatal development of the arterial smooth muscle is characterized by a decrease in the contribution of protein kinase C and Rho-kinase to the regulation of contraction; however, these changes do not correlate with changes in the sensitivity of arteries to methoxamine and development of sympathetic innervation.

[KRP/telokin differentially regulates filament assembly and phosphorylation of light chains of non-muscle myosin II in fibroblasts].

Transgenic 3T3 fibroblasts have been generated that express either the wild-type KRP or its truncated mutant lacking the C-terminal domain, which primarily contributes to myosin binding of KRP. It was found that KRP-expressing cells display a significantly increased content of myosin filaments and a reduced level of rMLC phosphorylation, whereas the mock transfected cells or cells expressing the C-terminally truncated KRP do not. Our results suggest that (1) KRP promotes the polymerization of myosin II and reduces the rMLC phosphorylation level in cells, (2) KRP acts through direct binding to myosin II, and (3) transgenic 3T3 fibroblasts stably expressing KRP represent a useful and versatile model to study the role of myosin II filament dynamics in cell motility.

[Signaling regulatory mechanisms of the contractile activity of smooth muscle]. / Signal'nye mekhanizmy reguliatsii sokratitel'noi aktivnosti gladkikh myshts.

A comparative model been designed to study a contribution of proteinkinase C-(PKC)-activated intracellular signaling pathways in generation of different contractile responses of vascular (tonic) and visceral (phasic) smooth muscles. We have determined that, in tonic smooth muscle, PKC mediates activation of MAP-kinases that phosphorylate key regulatory proteins of the contractile system, myosin light chain kinase and caldesmon, leading to upregulation of actomyosine motor activity. In contrast, the MAP-kinase activation is uncoupled from the contractile machinery in phasic smooth muscles, which also reveal high levels of myosin light chain kinase-related protein KRP that contributes to relaxation. Phosphorylation of KRP following activation of PKC or cyclic nucleotide-dependent protein kinases enhances the KRP activity and further contributes to relaxion in phasic smooth muscle. A possibility is discussed for exploitation of the comparative model described herein for investigation of specific role of other regulatory intracellular pathways in generation of vascular tonic contraction.

Novel phosphospecific antibodies for monitoring phosphorylation of proteins encoded by the myosin light chain kinase genetic locus.

Myosin light chain kinase (MLCK) and the kinase-related protein (KRP), also known as telokin, are the major independent protein products of the smooth muscle/non-muscle MLCK genetic locus. They share a common C-terminal part and major sites phosphorylated in vivo. Whereas MLCK is critically involved in myosin activation and contraction initiation in smooth muscle, KRP is thought to antagonize MLCK and to exert relaxation activity. Phosphorylation controls the MLCK and KRP activities. We generated two phosphorylation and site-specific antibodies to individually monitor levels of MLCK and KRP phosphorylation on critical sites. We quantified the level of KRP phosphorylation in smooth muscle before and after an increase in intracellular free Ca2+ and stimulation of adenylate cyclase, protein kinase C, and mitogen-activated protein kinases (MAP-kinases). Forskolin and phorbol-12,13-dibutyrate increased KRP phosphorylation at Ser13 from 25 to 100% but did not produce contraction in rat ileum. The level of Ser13 phosphorylation was not altered during Ca2+-dependent contraction evoked by KCl depolarization or carbachol, but subsequently increased to maximum during forskolin-induced relaxation. These data suggest that several intracellular signaling pathways control phosphorylation of KRP on Ser13 in smooth muscle and thus may contribute to relaxation. In contrast, phosphorylation level of Ser19 of KRP increased only slightly (from 30 to 40-45%) and only in response to MAP-kinase activation, arguing against its regulatory function in smooth muscle.

Signal transduction and protein phosphorylation in smooth muscle contraction.

Smooth muscles are important constituents of vertebrate organisms that provide for contractile activity of internal organs and blood vessels. Basic molecular mechanism of both smooth and striated muscle contractility is the force-producing ATP-dependent interaction of the major contractile proteins, actin and myosin II molecular motor, activated upon elevation of the free intracellular Ca2+ concentration ([Ca2+](i)). However, whereas striated muscles display a proportionality of generated force to the [Ca2+](i) level, smooth muscles feature molecular mechanisms that modulate sensitivity of contractile machinery to [Ca2+](i). Phosphorylation of proteins that regulate functional activity of actomyosin plays an essential role in these modulatory mechanisms. This provides an ability for smooth muscle to contract and maintain tension within a broad range of [Ca2+](i) and with a low energy cost, unavailable to a striated muscle. Detailed exploration of these mechanisms is required to understand the molecular organization and functioning of vertebrate contractile systems and for development of novel advances for treating cardiovascular and many other disorders. This review summarizes the currently known and hypothetical mechanisms involved in regulation of smooth muscle Ca2+-sensitivity with a special reference to phosphorylation of regulatory proteins of the contractile machinery as a means to modulate their activity.

Actomyosin cross-linking by caldesmon in non-muscle cells.

The role of myosin-binding in cytoskeletal arrangement of non-muscle low molecular weight caldesmon (l-caldesmon) was studied. The N-terminal myosin-binding domain of caldesmon N152 colocalized with myosin in transiently transfected chicken fibroblasts. When added exogenously to the Triton-insoluble cytoskeleton, N152 enhanced l-caldesmon displacement by exogenous C-terminal actin-binding fragment (H1). Thus, a significant fraction of l-caldesmon cross-links actin and myosin. In contrast, in epithelioid HeLa cells most of l-caldesmon was only actin-bound as H1 alone was enough for its displacement. Phosphorylation by mitogen-activated protein kinase reduced the capability of H1 to displace endogenous l-caldesmon, suggesting it may represent a regulatory mechanism for actin-caldesmon interaction in vivo.

Phosphorylation of kinase-related protein (telokin) in tonic and phasic smooth muscles.

KRP (telokin), an independently expressed C-terminal myosin-binding domain of smooth muscle myosin light chain kinase (MLCK), has been reported to have two related functions. First, KRP stabilizes myosin filaments (Shirinsky et al., 1993, J. Biol. Chem. 268, 16578-16583) in the presence of ATP. Secondly, KRP can modulate the level of myosin light chain phosphorylation. In this latter role, multiple mechanisms have been suggested. One hypothesis is that light chain phosphorylation is diminished by the direct competition of KRP and MLCK for myosin, resulting in a loss of contraction. Alternatively, KRP, through an unidentified mechanism, accelerates myosin light chain dephosphorylation in a manner possibly enhanced by KRP phosphorylation. Here, we demonstrate that KRP is a major phosphoprotein in smooth muscle, and use a comparative approach to investigate how its phosphorylation correlates with sustained contraction and forskolin-induced relaxation. Forskolin relaxation of precontracted artery strips caused little increase in KRP phosphorylation, while treatment with phorbol ester increased the level of KRP phosphorylation without a subsequent change in contractility. Although phorbol ester does not induce contraction of phasic tissues, the level of KRP phosphorylation is increased. Phosphopeptide maps of KRP from both tissues revealed multiple sites of phosphorylation within the N-terminal region of KRP. Phosphopeptide maps of KRP from gizzard were more complex than those for KRP from artery consistent with heterogeneity at the amino terminus and/or additional sites. We discovered through analysis of KRP phosphorylation in vitro that Ser12, Ser15 and Ser15 are phosphorylated by cAMP-dependent protein kinase, mitogen-activated protein (MAP) kinase and glycogen synthase kinase 3 (GSK3), respectively. Phosphorylation by GSK3 was dependent upon prephosphorylation by MAP kinase. This appears to be the first report of conditional or hierarchical phosphorylation of KRP. Peptides consistent with such multiple phosphorylations were found on the in vivo phosphopeptide maps of avian KRP. Collectively, the available data indicate that there is a complex relationship between the in vivo phosphorylation states of KRP and its effects on relaxation in smooth muscle.