Numeric reconstruction of cytoskeleton with finite element method and topology optimization method

The importance of mechanical aspects related to cell activity and its environment is becoming more evident due to their influence in stem cell differentiation and in the development of diseases such as atherosclerosis. The mechanical tension homeostasis is related to normal tissue behavior and its lack may be related to the formation of cancer, which shows a higher mechanical tension. Due to the complexity of cellular activity, the application of simplified models may elucidate which factors are really essential and which have a marginal effect. The development of a systematic method to reconstruct the elements involved in the perception of mechanical aspects by the cell may accelerate substantially the validation of these models. This work proposes the development of a routine capable of reconstructing the topology of focal adhesions and the actomyosin portion of the cytoskeleton from the displacement field generated by the cell on a flexible substrate. Another way to think of this problem is to develop an algorithm to reconstruct the forces applied by the cell from the measurements of the substrate displacement, which would be characterized as an inverse problem. For these kind of problems, the Topology Optimization Method (TOM) is suitable to find a solution. TOM is consisted of an iterative application of an optimization method and an analysis method to obtain an optimal distribution of material in a fixed domain. One way to experimentally obtain the substrate displacement is through Traction Force Microscopy (TFM), which also provides the forces applied by the cell. Along with systematically generating the distributions of focal adhesion and actin-myosin for the validation of simplified models, the algorithm also represents a complementary and more phenomenological approach to TFM. As a first approximation, actin fibers and flexible substrate are represented through two-dimensional linear Finite Element Method. Actin contraction is modeled as an initial stress of the FEM elements. Focal adhesions connecting actin and substrate are represented by springs. The algorithm was applied to data obtained from experiments regarding cytoskeletal prestress and micropatterning, comparing the numerical results to the experimental ones

A non-cross-bridge, static tension is present in permeabilized skeletal muscle fibers after active force inhibition or actin extraction

Cornachione AS, Rassier DE. A non-cross-bridge, static tension is present in permeabilized skeletal muscle fibers after active force inhibition or actin extraction. Am J Physiol Cell Physiol 302: C566-C574, 2012. First published November 16, 2011; doi: 10.1152/ajpcell.00355.2011.-When activated muscle fibers are stretched, there is a long-lasting increase in the force. This phenomenon, referred to as "residual force enhancement," has characteristics similar to those of the " static tension," a long-lasting increase in force observed when muscles are stretched in the presence of Ca2+ but in the absence of myosin-actin interaction. Independent studies have suggested that these two phenomena have a common mechanism and are caused either by 1) a Ca2+-induced stiffening of titin or by 2) promoting titin binding to actin. In this study, we performed two sets of experiments in which activated fibers (pCa(2+) 4.5) treated with the myosin inhibitor blebbistatin were stretched from 2.7 to 2.8 mu m at a speed of 40 L-o/s, first, after partial extraction of TnC, which inhibits myosin-actin interactions, or, second, after treatment with gelsolin, which leads to the depletion of thin (actin) filaments. We observed that the static tension, directly related with the residual force enhancement, was not changed after treatments that inhibit myosin-actin interactions or that deplete fibers from troponin C and actin filaments. The results suggest that the residual force enhancement is caused by a stiffening of titin upon muscle activation but not with titin binding to actin. This finding indicates the existence of a Ca2+-regulated, titin-based stiffness in skeletal muscles.

PTEN Directly Activates the Actin Depolymerization Factor Cofilin-1 During PGE(2)-Mediated Inhibition of Phagocytosis of Fungi

Macrophage ingestion of the yeast Candida albicans requires its recognition by multiple receptors and the activation of diverse signaling programs. Synthesis of the lipid mediator prostaglandin E-2 (PGE(2)) and generation of cyclic adenosine monophosphate (cAMP) also accompany this process. Here, we characterized the mechanisms underlying PGE(2)-mediated inhibition of phagocytosis and filamentous actin (F-actin) polymerization in response to ingestion of C. albicans by alveolar macrophages. PGE(2) suppressed phagocytosis and F-actin formation through the PGE(2) receptors EP2 and EP4, cAMP, and activation of types I and II protein kinase A. Dephosphorylation and activation of the actin depolymerizing factor cofilin-1 were necessary for these inhibitory effects of PGE(2). PGE(2)-dependent activation of cofilin-1 was mediated by the protein phosphatase activity of PTEN (phosphatase and tensin homolog deleted on chromosome 10), with which it directly associated. Because enhanced production of PGE(2) accompanies many immunosuppressed states, the PTEN-dependent pathway described here may contribute to impaired antifungal defenses.

Real-time PCR assay targeting the actin gene for the detection of Cryptosporidium parvum in calf fecal samples

Cryptosporidium parvum infection is very important with respect to public health, owing to foodborne and waterborne outbreaks and gastrointestinal illness in immunocompetent and immunocompromised persons. In cattle, infection with this species manifests either as a subclinical disease or with diarrheal illness, which occurs more often in the presence of other infectious agents than when alone. The aim of this study was to develop a real-time polymerase chain reaction (PCR) assay for the detection of C. parvum in calf fecal samples and to compare the results of this assay with those of the method routinely used for the diagnosis of Cryptosporidium spp., nested PCR targeting the 18S rRNA gene. Two hundred and nine fecal samples from calves ranging in age from 1 day to 6 months were examined using real-time PCR specific for the actin gene of C. parvum and by a nested PCR targeting the 18S rRNA gene of Cryptosporidium spp. Using real-time PCR detection, 73.2% (153 out of 209) of the samples were positive for C. parvum, while 56.5% (118 out of 209) of the samples were positive for Cryptosporidium spp. when the nested PCR amplification method was used for the detection. The analytical sensitivity of the real-time PCR was approximately one C. parvum oocyst. There was no significant nonspecific DNA amplification of any of the following species and genotype: Cryptosporidium andersoni, Cryptosporidium baileyi, Cryptosporidium bovis, Cryptosporidium canis, Cryptosporidium galli, Cryptosporidium ryanae, Cryptosporidium serpentis, or avian genotype II. Thus, we conclude that real-time PCR targeting the actin gene is a sensitive and specific method for the detection of C. parvum in calf fecal samples.

Free Rhodium (II) citrate and rhodium (II) citrate magnetic carriers as potential strategies for breast cancer therapy

Abstract Background Rhodium (II) citrate (Rh2(H2cit)4) has significant antitumor, cytotoxic, and cytostatic activity on Ehrlich ascite tumor. Although toxic to normal cells, its lower toxicity when compared to carboxylate analogues of rhodium (II) indicates Rh2(H2cit)4 as a promising agent for chemotherapy. Nevertheless, few studies have been performed to explore this potential. Superparamagnetic particles of iron oxide (SPIOs) represent an attractive platform as carriers in drug delivery systems (DDS) because they can present greater specificity to tumor cells than normal cells. Thus, the association between Rh2(H2cit)4 and SPIOs can represent a strategy to enhance the former's therapeutic action. In this work, we report the cytotoxicity of free rhodium (II) citrate (Rh2(H2cit)4) and rhodium (II) citrate-loaded maghemite nanoparticles or magnetoliposomes, used as drug delivery systems, on both normal and carcinoma breast cell cultures. Results Treatment with free Rh2(H2cit)4 induced cytotoxicity that was dependent on dose, time, and cell line. The IC50 values showed that this effect was more intense on breast normal cells (MCF-10A) than on breast carcinoma cells (MCF-7 and 4T1). However, the treatment with 50 μM Rh2(H2cit)4-loaded maghemite nanoparticles (Magh-Rh2(H2cit)4) and Rh2(H2cit)4-loaded magnetoliposomes (Lip-Magh-Rh2(H2cit)4) induced a higher cytotoxicity on MCF-7 and 4T1 than on MCF-10A (p < 0.05). These treatments enhanced cytotoxicity up to 4.6 times. These cytotoxic effects, induced by free Rh2(H2cit)4, were evidenced by morphological alterations such as nuclear fragmentation, membrane blebbing and phosphatidylserine exposure, reduction of actin filaments, mitochondrial condensation and an increase in number of vacuoles, suggesting that Rh2(H2cit)4 induces cell death by apoptosis. Conclusions The treatment with rhodium (II) citrate-loaded maghemite nanoparticles and magnetoliposomes induced more specific cytotoxicity on breast carcinoma cells than on breast normal cells, which is the opposite of the results observed with free Rh2(H2cit)4 treatment. Thus, magnetic nanoparticles represent an attractive platform as carriers in Rh2(H2cit)4 delivery systems, since they can act preferentially in tumor cells. Therefore, these nanopaticulate systems may be explored as a potential tool for chemotherapy drug development.

MiRNA-208a and miRNA-208b are triggered in thyroid hormone-induced cardiac hypertrophy - role of type 1 Angiotensin II receptor (AT1R) on miRNA-208a/α-MHC modulation

Hyperthyroidism promotes cardiac hypertrophy and the Angiotensin type 1 receptor (AT1R) has been demonstrated to mediate part of this response. Recent studies have uncovered a potentially important role for the microRNAs (miRNAs) in the control of diverse aspects of cardiac function. Then, the objective of the present study was to investigate the action promoted by hyperthyroidism on β-MHC/miR-208b expression and on α-MHC/miR-208a expression, as well as the possible contribution of the AT1R in this event. The findings of this study confirmed that AT1R is a key mediator of the cardiac hypertrophy induced by hyperthyroidism. Additionally, we demonstrated that like β-MHC, miR-208b was down-regulated in the hyperthyroid group. Similarly, like the expression of its host gene, α-MHC, miR-208a expression was up-regulated in response to hyperthyroidism. Finally, our data suggest for the first time that AT1R mediates the hyperthyroidism-induced increase on cardiac miRNA-208a/α-MHC levels, while does not influence on the reduction of miRNA-208b/β-MHC levels.

Emerging role of angiotensin type 2 receptor (AT2R)/Akt/NO pathway in vascular smooth muscle cell in the hyperthyroidism

Hyperthyroidism is characterized by increased vascular relaxation and decreased vascular contraction and is associated with augmented levels of triiodothyronine (T3) that contribute to the diminished systemic vascular resistance found in this condition. T3 leads to augmented NO production via PI3K/Akt signaling pathway, which in turn causes vascular smooth muscle cell (VSMC) relaxation; however, the underlying mechanisms involved remain largely unknown. Evidence from human and animal studies demonstrates that the renin-angiotensin system (RAS) plays a crucial role in vascular function and also mediates some of cardiovascular effects found during hyperthyroidism. Thus, in this study, we hypothesized that type 2 angiotensin II receptor (AT2R), a key component of RAS vasodilatory actions, mediates T3 induced-decreased vascular contraction. Marked induction of AT2R expression was observed in aortas from T3-induced hyperthyroid rats (Hyper). These vessels showed decreased protein levels of the contractile apparatus: α-actin, calponin and phosphorylated myosin light chain (p-MLC). Vascular reactivity studies showed that denuded aortic rings from Hyper rats exhibited decreased maximal contractile response to angiotensin II (AngII), which was attenuated in aortic rings pre-incubated with an AT2R blocker. Further study showed that cultured VSMC stimulated with T3 (0.1 µmol/L) for 24 hours had increased AT2R gene and protein expression. Augmented NO levels and decreased p-MLC levels were found in VSMC stimulated with T3, both of which were reversed by a PI3K/Akt inhibitor and AT2R blocker. These findings indicate for the first time that the AT2R/Akt/NO pathway contributes to decreased contractile responses in rat aorta, promoted by T3, and this mechanism is independent from the endothelium.