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The family of two-pore domain potassium (K2P) channels is critically involved in central cellular functions such as ion homeostasis, cell development, and excitability. K2P channels are widely expressed in different human cell types and organs. It is therefore not surprising that aberrant expression and function of K2P channels are related to a spectrum of human diseases, including cancer, autoimmune, CNS, cardiovascular, and urinary tract disorders. Despite homologies in structure, expression, and stimulus, the functional diversity of K2P channels leads to heterogeneous influences on human diseases. The role of individual K2P channels in different disorders depends on expression patterns and modulation in cellular functions. However, an imbalance of potassium homeostasis and action potentials contributes to most disease pathologies. In this review, we provide an overview of current knowledge on the role of K2P channels in human diseases. We look at altered channel expression and function, the potential underlying molecular mechanisms, and prospective research directions in the field of K2P channels.

Background As in other types of cancers, sex is an essential factor in the origin and progression of glioblastomas. Research in the field of endocrinology and cancer suggests that gonadal steroid hormones play an important role in the progression and prevalence of glioblastomas. In the present review, we aim to discuss the actions and mechanism triggered by gonadal steroid hormones in glioblastomas. Main body Glioblastoma is the most common malignant primary brain tumor. According to the epidemiological data, glioblastomas are more frequent in men than in women in a 1.6/1 proportion both in children and adults. This evidence, and the knowledge about sex influence over the prevalence of countless diseases, suggest that male gonadal steroid hormones, such as testosterone, promote glioblastomas growth. In contrast, a protective role of female gonadal steroid hormones (estradiol and progesterone) against glioblastomas has been questioned. Several pieces of evidence demonstrate a variety of effects induced by female and male gonadal steroid hormones in glioblastomas. Several studies indicate that pregnancy, a physiological state with the highest progesterone and estradiol levels, accelerates the progression of low-grade astrocytomas to glioblastomas and increases the symptoms associated with these tumors. In vitro studies have demonstrated that progesterone has a dual role in glioblastoma cells: physiological concentrations promote cell proliferation, migration, and invasion while very high doses (out physiological range) reduce cell proliferation and increases cell death. Conclusion Gonadal steroid hormones can stimulate the progression of glioblastomas through the increase in proliferation, migration, and invasion. However, the effects mentioned above depend on the concentrations of these hormones and the receptor involved in hormone actions. Estradiol and progesterone can exert promoter or protective effects while the role of testosterone has been always associated to glioblastomas progression.

BACKGROUND/AIMS: Arginine vasopressin (AVP) neurons play an important role for sensing a change in the plasma osmolarity and thereby responding with regulated AVP secretion in order to maintain the body fluid homeostasis. The osmo-sensing processes in magnocellular neurosecretory cells (MNCs) including AVP and oxytocin (OXT) neurons of the hypothalamus were reported to be coupled to sustained osmotic shrinkage or swelling without exhibiting discernible cell volume regulation. Since increasing evidence has shown some important differences in properties between AVP and OXT neurons, osmotic volume responses are to be reexamined with distinguishing these cell types from each other. We previously reported that AVP neurons identified by transgenic expression of enhanced green fluorescence protein (eGFP) possess the ability of regulatory volume decrease (RVD) after hypoosmotic cell swelling. Thus, in the present study, we examined the ability of regulatory volume increase (RVI) after hyperosmotic cell shrinkage in AVP neurons. METHODS: Here, we used eGFP-identified AVP neurons acutely dissociated from AVP-eGFP transgenic rats. We performed single-cell size measurements, cytosolic RT-PCR analysis, AVP secretion measurements, and patch-clamp studies. RESULTS: The AVP neurons were found to respond to a hyperosmotic challenge with physiological cell shrinkage caused by massive secretion of AVP, called a secretory volume decrease (SVD), superimposed onto physical osmotic cell shrinkage, and also to exhibit the ability of RVI coping with osmotic and secretory cell shrinkage. Furthermore, our pharmacological and molecular examinations indicated that AVP secretion and its associated SVD event are triggered by activation of T-type Ca2+ channels, and the RVI event is attained by parallel operation of Na+/H+ exchanger and Cl-/HCO3- anion exchanger. CONCLUSION: Thus, it is concluded that AVP neurons respond to hyperosmotic stimulation with the regulatory volume increase and the secretory volume increase by activating ion transporters and Ca2+ channels, respectively.

Despite advances in diagnostics and therapy of non-small cell lung cancer (NSCLC), the problem of prognosis and prevention of tumor progression is still highly important. Even if NSCLC is diagnosed in the early stages, almost a quarter of patients develop relapse; most of them die from recurrent disease. A large number of different markers have been proposed to predict the risk of NSCLC progression; however, none of them are used in clinical practice. It is obvious that this situation is related to the economic and methodological complexity of the proposed markers and/or their insufficient efficiency due to a lack of effective study models and tumor heterogeneity. Another reason may be that potential markers are developed for NSCLC progression in general, which is represented by at least four pathogenetically-distinct processes: synchronous lymph node metastasis, local, regional, and distant recurrence. In this review, we summarize data from published literature on clinicopathological, genetic, and molecular factors associated with different types of NSCLC progression and emphasize challenges and approaches to developing prognostic factors. In conclusion, we highlight the importance of further studies to reveal molecular mechanisms of NSCLC progression and the need for differential analysis of markers of local, regional, and distant recurrences for disease prognosis.

Mechanobiological stimuli, such as low-intensity pulsed ultrasound (LIPUS), have been shown to promote bone regeneration and fresh fracture repair, but the fundamental biophysical mechanisms involved remain elusive. Here, we propose that a mechanosensitive ion channel of Piezo1 plays a pivotal role in the noninvasive ultrasound-induced mechanical transduction pathway to trigger downstream cellular signal processes. This study aims to investigate the expression and role of Piezo1 in MC3T3-E1 cells after LIPUS treatment. Immunofluorescence analysis shows that Piezo1 was present on MC3T3-E1 cells and could be ablated by shRNA transfection. MC3T3-E1 cell migration and proliferation were significantly increased by LIPUS stimulation, and knockdown of Piezo1 restricted the increase in cell migration and proliferation. After labeling with Fluo-8, MC3T3-E1 cells exhibited fluorescence intensity traces with several high peaks compared with the baseline during LIPUS stimulation. No obvious change in the fluorescence intensity tendency was observed after LIPUS stimulation in shRNA-Piezo1 cells, which was similar to the results in the GsMTx4-treated group. The phosphorylation ratio of ERK1/2 in MC3T3-E1 cells was significantly increased (P 2+ serves as a second messenger to activate ERK1/2 phosphorylation and perinuclear F-actin filament polymerization, which regulate the proliferation of MC3T3-E1 cells.

BackgroundThere is growing evidence of sex differences in placental vascular development. The objective of this study was to investigate the effect of fetal sex on uterine artery pulsatility index (PI) throughout gestation in a cohort of normal and complicated pregnancies.MethodsA prospective longitudinal study was conducted in 240 pregnant women. Pulsed wave Doppler ultrasound of the proximal uterine arteries was performed at a 4-weekly interval between 14 and 40 weeks of gestation. The patients were classified retrospectively as normal or complicated (one or more of maternal preeclampsia, preterm birth, or small for gestational age). To assess if the change in uterine artery PI during gestation differed between normal and complicated pregnancies and between fetal sexes, the uterine artery PI was modeled using a linear function of gestational age and the rate of change was estimated from the slope.ResultsWhile the uterine artery PI did not differ over gestation between females and males for normal pregnancies, the trajectory of this index differed by fetal sex for pregnancies complicated by either preeclampsia, preterm birth, or fetal growth restriction (p< 0.0001). The male fetuses in the complicated pregnancy group had an elevated slope compared to the other groups (p< 0.0001), suggesting a more progressive deterioration in uteroplacental perfusion over gestation.ConclusionsThe uterine artery PI is widely used to assess uteroplacental function in clinical settings. The observation that this metric changes more rapidly in complicated pregnancies where the fetus was male highlights the importance of sex when interpreting hemodynamic markers of placental maturation.

BACKGROUND/AIMS: Signaling and metabolic perturbations contribute to dysregulated skeletal muscle protein homeostasis and secondary sarcopenia in response to a number of cellular stressors including ethanol exposure. Using an innovative multiomics-based curating of unbiased data, we identified molecular and metabolic therapeutic targets and experimentally validated restoration of protein homeostasis in an ethanol-fed mouse model of liver disease. METHODS: Studies were performed in ethanol-treated differentiated C2C12 myotubes and physiological relevance established in an ethanol-fed mouse model of alcohol-related liver disease (mALD) or pair-fed control C57BL/6 mice. Transcriptome and proteome from ethanol treated-myotubes and gastrocnemius muscle from mALD and pair-fed mice were analyzed to identify target pathways and molecules. Readouts including signaling responses and autophagy markers by immunoblots, mitochondrial oxidative function and free radical generation, and metabolic studies by gas chromatography-mass spectrometry and sarcopenic phenotype by imaging. RESULTS: Multiomics analyses showed that ethanol impaired skeletal muscle mTORC1 signaling, mitochondrial oxidative pathways, including intermediary metabolite regulatory genes, interleukin-6, and amino acid degradation pathways are β-hydroxymethyl-butyrate targets. Ethanol decreased mTORC1 signaling, increased autophagy flux, impaired mitochondrial oxidative function with decreased tricarboxylic acid cycle intermediary metabolites, ATP synthesis, protein synthesis and myotube diameter that were reversed by HMB. Consistently, skeletal muscle from mALD had decreased mTORC1 signaling, reduced fractional and total muscle protein synthesis rates, increased autophagy markers, lower intermediary metabolite concentrations, and lower muscle mass and fiber diameter that were reversed by β-hydroxymethyl-butyrate treatment. CONCLUSION: An innovative multiomics approach followed by experimental validation showed that β-hydroxymethyl-butyrate restores muscle protein homeostasis in liver disease.

The vast majority of patients with advanced breast cancer present skeletal complications that severely compromise their quality of life. Breast cancer cells are characterized by a strong tropism to the bone niche. After engraftment and colonization of bone, breast cancer cells interact with native bone cells to hinder the normal bone remodeling process and establish an osteolytic “metastatic vicious cycle”. The sympathetic nervous system has emerged in recent years as an important modulator of breast cancer progression and metastasis, potentiating and accelerating the onset of the vicious cycle and leading to extensive bone degradation. Furthermore, sympathetic neurotransmitters and their cognate receptors have been shown to promote several hallmarks of breast cancer, such as proliferation, angiogenesis, immune escape, and invasion of the extracellular matrix. In this review, we assembled the current knowledge concerning the complex interactions that take place in the tumor microenvironment, with a special emphasis on sympathetic modulation of breast cancer cells and stromal cells. Notably, the differential action of epinephrine and norepinephrine, through either a- or ß-adrenergic receptors, on breast cancer progression prompts careful consideration when designing new therapeutic options. In addition, the contribution of sympathetic innervation to the formation of bone metastatic foci is highlighted. In particular, we address the remarkable ability of adrenergic signaling to condition the native bone remodeling process and modulate the bone vasculature, driving breast cancer cell engraftment in the bone niche. Finally, clinical perspectives and developments on the use of ß-adrenergic receptor inhibitors for breast cancer management and treatment are discussed. This work was financed by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and Portuguese funds through FCT/ MCTES in the framework of the project “SproutOC” (POCI-01-0145-FEDER-030158, PTDC/MED-PAT/30158/2017). F.C. is a recipient of the Ph.D. fellowship SFRH/BD/ 128771/2017. D.M.S. is a recipient of the postdoctoral fellowship SFRH/BPD/115341/ 2016.

High density of intracellular macromolecules creates a special condition known as macromolecular crowding (MC). One well-established consequence of MC is that only a slight change in the concentration of macromolecules (e.g., proteins) results in a shift of chemical equilibria towards the formation of macromolecular complexes and oligomers. This suggests a physiological mechanism of converting cell density changes into cellular responses. In this review, we start by providing a general overview of MC; then we examine the available experimental evidence that MC may act as a direct signaling factor in several types of cellular activities: mechano- and osmosensing, cell volume recovery in anisosmotic solutions, and apoptotic shrinkage. The latter phenomenon is analyzed in particular detail, as persistent shrinkage is known both to cause apoptosis and to occur during apoptosis resulting from other stimuli. We point to specific apoptotic reactions that involve formation of macromolecular complexes and, therefore, may provide a link between shrinkage and downstream responses.

BACKGROUND/AIMS: Tea, produced from the evergreen Camellia sinensis, has reported therapeutic properties against multiple pathologies, including hypertension. Although some studies validate the health benefits of tea, few have investigated the molecular mechanisms of action. The KCNQ5 voltage-gated potassium channel contributes to vascular smooth muscle tone and neuronal M-current regulation.METHODS: We applied electrophysiology, myography, mass spectrometry and in silico docking to determine effects and their underlying molecular mechanisms of tea and its components on KCNQ channels and arterial tone.RESULTS: A 1% green tea extract (GTE) hyperpolarized cells by augmenting KCNQ5 activity >20-fold at resting potential; similar effects of black tea were inhibited by milk. In contrast, GTE had lesser effects on KCNQ2/Q3 and inhibited KCNQ1/E1. Tea polyphenols epicatechin gallate (ECG) and epigallocatechin-3-gallate (EGCG), but not epicatechin or epigallocatechin, isoform-selectively hyperpolarized KCNQ5 activation voltage dependence. In silico docking and mutagenesis revealed that activation by ECG requires KCNQ5-R212, at the voltage sensor foot. Strikingly, ECG and EGCG but not epicatechin KCNQ-dependently relaxed rat mesenteric arteries.CONCLUSION: KCNQ5 activation contributes to vasodilation by tea; ECG and EGCG are candidates for future anti-hypertensive drug development.

Bone remodeling is precisely coordinated by bone resorption and formation. Apoptotic osteoclasts generate large amounts of apoptotic bodies (ABs) marking the end of the bone resorption phase, whereas the functions of osteoclast-derived ABs remain largely unknown. Here, we identified the molecular profile of ABs derived from osteoclasts at distinct differentiation stages and investigated their corresponding functions. ABs were isolated from apoptotic bone marrow macrophages, preosteoclasts, and mature osteoclasts induced by staurosporine. Proteomic signature analysis with liquid chromatography-tandem mass spectrometry suggested marked protein cargo differences among the different ABs. Further bioinformatic analysis showed that the proteomic signatures of the ABs were highly similar to those of their parental cells. Functionally, pOC-ABs induced endothelial progenitor cell differentiation and increased CD31hiEmcnhi endothelial cell formation in a murine bone defect model via their PDGF-BB cargo. mOC-ABs induced osteogenic differentiation of mesenchymal stem cells and facilitated osteogenesis via RANKL reverse signaling. In summary, we mapped the detailed proteomic landscapes of ABs derived from osteoclasts and showed that their potential biological roles are important in coupling bone formation with resorption during bone remodeling.

The distinguished feature of neutron as a scattering probe is an isotope effect, especially the large difference in neutron scattering length between hydrogen and deuterium. The difference renders the different visibility between hydrogenated and deuterated proteins. Therefore, the combination of deuterated protein and neutron scattering enables the selective visualization of a target domain in the complex or a target protein in the multi-component system. Despite of this fascinating character, there exist several problems for the general use of this method: difficulty and high cost for protein deuteration, and control and determination of deuteration ratio of the sample. To resolve them, the protocol of protein deuteration techniques is presented in this report. It is strongly expected that this protocol will offer more opportunity for conducting the neutron scattering studies with deuterated proteins.

The snake cube puzzle made of a linear array of 27 cubes and its modified and extended versions are used as theoretical models to study the mechanism of folding of proteins into their sequence-specific native three-dimensional structures. Each of the three versions is characterized by the respective set of characteristics attributed to each of its constituent cubes and an array is characterized by its specific sequence of the cube characteristics. The aim of the puzzles is to fold the cube array into a compact 3×3×3 cubic structure. In all three versions, out of all possible sequences, only a limited fraction of sequences are found foldable into the compact cube. Even among foldable sequences, the structures folded into the compact 3×3×3 cube are found often not uniquely determined from the sequence. By comparing the results obtained for the three versions of models, we conclude that the power of the hydrophobic interactions to make the folded structure unique to the sequence is much weaker than the geometrical varieties of constituent cubes as modelled in the original snake cube puzzle. However, when this weak cube attribute is compounded to that of the original snake cube puzzle, the power is enhanced very effectively. This is a strong manifestation of the consistency principle: The sequence-specific native structure of protein is realized as a result of consistency of various types of interactions working in protein.

Background Sex differences in experimental stroke outcomes are well documented, such that adult males have a greater infarct volume, increased stroke-induced mortality, and more severe sensory-motor impairment. Based on recent evidence that the gut is an early responder to stroke, the present study tested the hypothesis that sex differences in stroke severity will be accompanied by rapid and greater permeability of the gut-blood barrier and gut dysbiosis in males as compared to females. Method Male and female Sprague-Dawley rats (5–7 months of age) were subject to endothelin (ET)-1-induced middle cerebral artery occlusion (MCAo). Sensory-motor tests were conducted pre- and 2 days after MCAo. Gut permeability was assessed in serum samples using biomarkers of gut permeability as well as functional assays using size-graded dextrans. Histological analysis of the gut was performed with H&E staining, periodic acid-Schiff for mucus, and immunohistochemistry for the tight junction protein, ZO-1. Fecal samples obtained pre- and post-stroke were analyzed for bacterial taxa and short-chain fatty acids (SCFAs). Results After stroke, males displayed greater mortality, worse sensory-motor deficit, and higher serum levels of proinflammatory cytokines IL-17A, MCP-1, and IL-5 as compared to females. MCAo-induced gut permeability was rapid and severe in males as indicated by dextran extravasation from the gut to the blood in the hyperacute (< 2 h) and early acute (2 days) phase of stroke. This was accompanied by dysmorphology of the gut villi and dysregulation of the tight junction protein ZO-1 in the acute phase. Fecal 16s sequencing showed no differences in bacterial diversity in the acute phase of stroke. Predictive modeling indicated that markers of gut permeability were associated with acute sensory-motor impairment and infarct volume. Conclusions These data show that extensive leakiness of the gut barrier is associated with severe post-stroke disability and suggest that reinforcing this barrier may improve stroke outcomes.

Background Pioglitazone is a promising therapeutic method for nonalcoholic fatty liver disease (NAFLD) patients with or without type 2 diabetes. However, there is remarkable variability in treatment response. We analyzed our previous randomized controlled trial to examine the effects of gender and other factors on the efficacy of pioglitazone in treating Chinese nonalcoholic fatty liver disease (NAFLD) patients with abnormal glucose metabolism. Methods This is a post hoc analysis of a previous randomized, parallel controlled, open-label clinical trial (RCT) with an original purpose of evaluating the efficacy of berberine and pioglitazone on NAFLD. The total population (n = 185) was randomly divided into three groups: lifestyle intervention (LSI), LSI + pioglitazone (PGZ) 15 mg qd, and LSI + berberine (BBR) 0.5 g tid, respectively, for 16 weeks. The study used proton magnetic resonance spectroscopy (1H-MRS) to assess liver fat content. Results As compared with LSI, PGZ + LSI treatment further decreased liver fat content in women (− 15.24% ± 14.54% vs. − 8.76% ± 13.49%, p = 0.025), but less decreased liver fat content in men (− 9.95% ± 15.18% vs. − 12.64% ± 17.78%, p = 0.046). There was a significant interaction between gender and efficacy of pioglitazone before and after adjustment for age, smoking, drinking, baseline BMI, BMI change, treatment adherence, baseline liver fat content, and glucose metabolism. Conclusion The study recommends pioglitazone plus lifestyle intervention for Chinese NAFLD female patients with abnormal glucose metabolism. Trial registration Role of Pioglitazone and Berberine in Treatment of Non-Alcoholic Fatty Liver Disease, NCT00633282. Registered on 3 March 2008, https://register.clinicaltrials.gov.

Many factors including growth factors (GF), scaffold materials, and chemical and physical cues determine the cell behaviors. For many years, growth factors have been considered as the pivotal cell behavior regulators, whereas recent studies emphasize also the key role of physical factors such as mechanical forces, cell shape, surface topographies, and extracellular matrix (ECM) in regulating the cell proliferation, apoptosis, differentiation, etc. through mechanotransduction pathways. In this process, the cell morphology and mechanical properties of the cell's micro/ nano-environments and ECM can be conveyed to the nucleus by regulating transcriptional factors such as Yes-associated protein and transcriptional coactivator with PDZ-binding motif (TAZ). Generally, YAP/TAZ activity is considered as the key factor for the growth of whole organs, however, recent studies have also repeatedly addressed the role of YAP/TAZ in mechanotransduction. In this review, the biological functions of the YAP/TAZ pathway and its contribution to the mechanotransduction and cell behavior regulation in response to the mechanical cues have been summarized. Also, the role of key mechanical checkpoints in the cell including focal adhesions, cytoskeletal tension, Rho small GTPases, and nuclear membrane protein elements involved in the transfer of environmental mechanical cues from the cell surface to the nucleus and their effect in regulating the YAP/TAZ activity are discussed.

BACKGROUND/AIMS: Compelling evidence indicates that CK2α, which is one of the two catalytic isoforms of protein kinase CK2, is required for cell viability and plays an important role in cell proliferation and differentiation. While much is known on CK2 in the context of disease states, particularly cancer, its critical role in non-cancerous cell growth has not been extensively investigated. METHODS: In the present study, we have employed a cell line derived from rat heart with inducible down-regulation of CK2α and CK2α-knockout mouse tissue to identify CK2-mediated molecular mechanisms regulating cell growth. For this, we have performed Incucyte® live-cell analysis and applied flow cytometry, western blot, immunoprecipitation, immunohistochemistry, RT-qPCR and luciferase-based methods. RESULTS: Here, we show that lack of CK2α results in significantly delayed cell cycle progression through G1, inhibition of cyclin E-CDK2 complex, decreased phosphorylation of Rb protein at S795, and inactivation of E2F transcription factor. These events are accompanied by nuclear accumulation and up-regulation of the cyclin-dependent kinase inhibitor p27KIP1 in cells and CK2α-knockout mouse tissues. We found that increased levels of p27KIP1 are mainly attributable to post-translational modifications, namely phosphorylation at S10 and T197 amino acid residues catalyzed by Dyrk1B and AMPK, respectively, as silencing of FoxO3A transcription factor, which activates CDKN1B the gene coding for p27KIP1, does not result in markedly decreased expression levels of the corresponding protein. Interestingly, simultaneous silencing of CK2α and p27KIP1 significantly impairs cell cycle progression without increasing cell death. CONCLUSION: Taken together, our study sheds light on the molecular mechanisms controlling cell cycle progression through G1 phase when myoblasts proliferation potential is impaired by CK2α depletion. Our results suggest that elevated levels of p27KIP1,which follows CK2α depletion, contribute to delay the G1-to-S phase transition. Effects seen when p27KIP1 is down-regulated are independent of CK2α and reflect the protective role exerted by p27KIP1 under unfavorable cell growth conditions.

Background/aims The disruption of redox equilibrium by oxidative stress, which is characterized by an overproduction of reactive oxygen species (ROS), is considered to be associated with fibroblast death in severe lung diseases. Indole-6-carboxaldehyde (I6CA) is a natural indole derivative isolated from Sargassum thunbergii, which a type of brown algae. However, the antioxidative effects of I6CA, and their mechanisms, have not been identified. This study was conducted to investigate the potential protective effects of I6CA against oxidative stress in V79-4 Chinese hamster lung fibroblasts. Methods Cell viability and mechanisms related to antioxidant activity of I6CA (ROS production, cell cycle, DNA damage, mitochondrial membrane potential (MMP) and apoptosis) were studied. Western blot analysis was carried out to understand the involvement of various genes at protein level. Results Our results demonstrated that I6CA inhibited hydrogen peroxide (H2O2)-induced cytotoxicity by blocking abnormal ROS accumulation. H2O2 treatment of V79-4 fibroblasts caused cell cycle arrest at the G2/M phase, which was accompanied by increased expression of the cyclin-dependent kinase (Cdk) inhibitor p21WAF1/CIP1 and decreased expression of cyclin B1 and cyclin A. However, these effects were attenuated by treatment with I6CA. I6CA also effectively protected V79-4 cells against H2O2-induced apoptosis by increasing the Bcl-2/Bax ratio and suppressing the loss of MMP and the cytosolic release of cytochrome c. In addition, the activation of nuclear factor-erythroid-2-related factor 2 (Nrf2) was markedly promoted by I6CA, which was associated with enhanced expression and activity of heme oxygenase-1 (HO-1). However, inhibiting the activity of HO-1 by zinc protoporphyrin IX, a potent inhibitor of HO-1, eliminated the ROS scavenging and anti-apoptotic effects of I6CA, indicating that I6CA was able to protect V79-4 lung fibroblasts from H2O2-induced oxidative stress by activating the Nrf2 signaling pathway. Conclusion We suggest that I6CA may be useful as a candidate therapeutic agent for the treatment of oxidative stress-related lung diseases.

Made available in DSpace on 2021-06-25T10:13:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Background/Aims: Considering the importance of inflammation on obesity-related disorders pathogenesis, including cardiac dysfunction, the interest in natural anti-inflammatory therapeutic strategies has emerged. The lycopene is a carotenoid presents in tomato and red fruits that displays anti-inflammatory properties. In this sense, we will evaluate the anti-inflammatory effect of tomato-oleoresin supplementation on obesity- related cardiac dysfunction by modulating myocardial calcium kinetic. Methods: Male Wistar rats were initially randomized into 2 experimental groups: (Control, n= 20) or high sugar- fat diet (HSF, n=20) for 20 weeks. At week 20th, once detected the cardiac dysfunction (cardiac remodeling, systolic and diastolic dysfunction) by echocardiography in HSF group, animals were randomly divided to begin the treatment with tomato-oleoresin, performing 4 groups: Control (n= 10); Control + tomato tomato-oleoresin supplementation (Control + Ly, n= 10); HSF (n= 10) or HSF + tomato tomato-oleoresin supplementation (HSF + Ly, n= 10). Tomato oleoresin was mixed with maize oil equivalent to 10mg lycopene/kg body weight (BW) per day and given orally, by gavage, every morning for a 10-week period. It was analyzed cardiac inflammatory parameters by the enzyme-linked immunosorbent assay (ELISA) and in vivo (echocardiography) and in vitro (studying isolated papillary muscles from the left ventricle) cardiac function. The groups were compared by Two-Way analysis of variance (ANOVA). Results: The HSF diet induced cardiac dysfunction (FS(%) C: 60.4±1.3; C+Ly: 60.9±1.3; HSF: 51.7±1.3; HSF+Ly: 59.4±1.4) and inflammation (TNF-a: C: 1.88±0.41; C+Ly: 1.93±1.01; HSF: 4.58±1.99; HSF+Ly: 2.03±0.55; IL-6: C: 0.58±0.16; C+Ly: 0.40±0.16; HSF: 2.00±0.45; HSF+Ly: 0.53±0.26; MCP-1: C: 0.31±0.08; C+Ly: 0.43±0.22; HSF: 1.54±0.32; HSF+Ly: 0.50±0.16). Tomato-oleoresin supplementation improved cardiac remodeling and dysfunction, cardiac inflammation and myocardial calcium kinetic. Conclusion: the anti-inflammatory effect of tomato-oleoresin supplementation treated the obesity-induced cardiac dysfunction by modulating myocardial calcium handling. Medical School Sao Paulo State University (UNESP) Institute of Biosciences Sao Paulo State University (UNESP) Medical School Sao Paulo State University (UNESP) Institute of Biosciences Sao Paulo State University (UNESP) FAPESP: 2020/12578-0

Background/aims We recently described a novel regulatory role for histone deacetylase 2 (HDAC2) in protecting endothelial cells from oxidized low-density lipoprotein (OxLDL)-induced injury. In this study, we examined the effects of endothelial-specific HDAC2 overexpression on endothelial-dependent vasorelaxation and atherogenesis in vivo. Methods Endothelial-specific HDAC2-overexpressing transgenic mice (HDAC2-Tg) were generated under control of the Tie2 promoter. An atherosclerosis model was produced by injecting HDAC2-Tg and wild-type (WT) mice with adeno-associated virus encoding a PCSK9 gain-of-function mutant under control of a liver-specific promoter and feeding them a high-fat diet for 12 weeks. Aortic stiffness in vivo was determined by measuring pulse wave velocity. Wire myography was used to measure endothelium dependent (acetylcholine) and independent (sodium nitroprusside) relaxation in isolated mice aortas. Atherosclerotic plaque burden in aortas was determined by Oil Red O staining and protein expression was determined by western blotting. Results At baseline, HDAC2-Tg mice had normal mean arterial blood pressure (MAP) and body weight, but pulse wave velocity (PWV), an inverse measure of vascular health and stiffness, was decreased, suggesting that their vessels were more compliant. Moreover, basal nitric oxide production was enhanced in the vessels of HDAC2-Tg mice as compared to that in WT controls, although no significant differences in acetylcholine (endothelial component)- or sodium nitroprusside (non-endothelial component)-mediated relaxation were observed. However, after exposure to OxLDL, aortas from HDAC2-Tg mice exhibited greater acetylcholine-induced relaxation than did those from WT mice. Thus, endothelial-specific vasodilator production was enhanced despite oxidative injury. Atherosclerosis induction in WT mice led to a significant increase in PWV, but in HDAC2-Tg mice, PWV and MAP remained unchanged. Further, aortic rings from HDAC2-Tg exhibited better endothelial-dependent vascular relaxation than did those from WT mice, but not when treated with nitric oxide synthase inhibitor L-NAME. Finally, plaque burden, determined by Oil red O staining, was significantly increased in WT, but not HDAC2-Tg mice, subjected to the atherogenic model. Deletion of endothelial HDAC2 led to impaired endothelial cell-dependent vascular relaxation and increased PWV, compared with those in littermate controls. Conclusion HDAC2 protects against endothelial dysfunction and atherogenesis induced by oxidized lipids. Hence, overexpression or activation of HDAC2 represents a novel therapy for endothelial dysfunction and atherosclerosis. HDAC2-Tg mice provide an opportunity to determine the role of endothelial HDAC2 in vascular endothelial homeostasis.

Background/aims Trace amines (TA) are small organic compounds that have neuromodulator activity due to their interaction with some neuron-related receptors, such as trace amine associated receptors (TAARs), α2-adrenergic receptor (α2-AR) and s-adrenergic receptor (s-AR). However, there is little information on whether TA and dopamine (DOP) can interact with other adrenergic receptors (ARs) such as the mammalian α1-AR and the bacterial counterpart QseC, which is involved in quorum sensing of some Gram-negative pathogens. The aim of this study was to investigate the interaction of TA and DOP with α1-AR and QseC. Methods We performed an in silico study using 3D structure from SWISS MODEL and analyzed the protein interaction via molecular docking using PyMol, PoseView and PyRX 8.0. For the in vitro study, we investigated the QseC kinase activity by measuring the remaining ATP in a reaction containing QseC-enriched membrane incubated together with purified QseB and EPI, TA, DOP, or PTL respectively. We also measured the intracellular Ca++ levels, which represents the α1-AR activation, in LNCAP (pancreatic cell line) cells treated with EPI, TA, DOP and PTL respectively using a fluorescence-based assay. The LNCAP cell proliferation was measured using an MTT-based assay. Results Our in silico analysis revealed that TAs and DOP have high binding affinity to the human α1-AR and the bacterial adrenergic receptor (QseC), comparable to epinephrine (EPI). Both are membrane-bound kinases. Experimental studies with pancreatic cell line (LNCAP) showed that the TAs and DOP act as α1-AR antagonist by counteracting the effect of EPI. In the presence of EPI, TA and DOP trigger an increase of the intracellular Ca++ levels in the LNCAP cells leading to an inhibition of cell proliferation. Although in silico data suggest an interaction of TA and DOP with QseC, they do not inhibit the kinase activity of QseC, a histidine kinase receptor involved in quorum sensing which is also sensitive to EPI. Conclusion Our study showed that the TAs and DOP act as α1-AR antagonist but no effect was observed for QseC.

Background/aims The role of VDAC1, the most abundant mitochondrial outer membrane protein, in cell death depends on cell types and stimuli. Both silencing and upregulation of VDAC1 in various type of cancer cell lines can stimulate apoptosis. In contrast, in mouse embryonic stem (MES) cells and mouse embryonic fibroblasts (MEFs), the roles of VDAC1 knockout (VDAC1-/-) in apoptotic cell death are contradictory. The contribution and underlying mechanism of VDAC1-/- in oxidative stress-induced cell death in cardiac cells has not been established. We hypothesized that VDAC1 is an essential regulator of oxidative stress-induced cell death in H9c2 cells. Methods We knocked out VDAC1 in this rat cardiomyoblast cell line with CRISPR-Cas9 genome editing technique to produce VDAC1-/- H9c2 cells, and determined if VDAC1 is critical in promoting cell death via oxidative stress induced by tert-butylhydroperoxide (tBHP), an organic peroxide, or rotenone (ROT), an inhibitor of mitochondrial complex I by measuring cell viability with MTT assay, cell death with TUNEL stain and LDH release. The mitochondrial and glycolytic stress were examined by measuring O2 consumption rate (OCR) and extracellular acidification rate (ECAR) with a Seahorse XFp analyzer. Results We found that under control conditions, VDAC1-/- did not affect H9c2 cell proliferation or mitochondrial respiration. However, compared to the wildtype (WT) cells, exposure to either tBHP or ROT enhanced the production of ROS, ECAR, and the proton (H+) production rate (PPR) from glycolysis, as well as promoted apoptotic cell death in VDAC1-/- H9c2 cells. VDAC1-/- H9c2 cells also exhibited markedly reduced mitochondria-bound hexokinase II (HKII) and Bax. Restoration of VDAC1 in VDAC1-/- H9c2 cells reinstated mitochondria-bound HKII and concomitantly decreased tBHP and ROT-induced ROS production and cell death. Interestingly, mitochondrial respiration remained the same after tBHP treatment in VDAC1-/- and WT H9c2 cells. Conclusion Our results suggest that VDAC1-/- in H9c2 cells enhances oxidative stress-mediated cell apoptosis that is directly linked to the reduction of mitochondria-bound HKII and concomitantly associated with enhanced ROS production, ECAR, and PPR.

We previously reported 18FPRGD2 uptake by the coxofemoral lining, intervertebral discs and facet joint osteophytes in OA using PET/SCAN imaging. However, the molecular mechanism by which the PRGD2 tracer interacts with joint tissues and osteophytes in OA remains unclear. As PRGD2 ligands are expected to belong to the RGD-specific integrin family, the purpose of this study was (i) to determine which integrin complexes display the highest affinity for PRGD2-based ligands, (ii) to analyze integrin expression in relevant tissues, and (iii) to test integrin regulation in chondrocytes using OA-related stimuli to increase the levels of fibrosis and ossification markers. To this end, the affinity of PRGD2-based ligands for five heterodimeric integrins was measured by competition with 125I-echistatin. In situ analyses were performed in human normal vs. OA cartilage and spinal osteophytes. Osteophytes were characterized by (immuno-)histological staining. Integrin subunit expression was tested in chondrocytes undergoing dedifferentiation, osteogenic differentiation, and inflammatory stimulation. The integrins αVβ5, αVβ3, and αVβ6 presented the highest affinity for PRGD2-based ligands. In situ, the expression of these integrins was significantly increased in OA compared to normal cartilage. Within osteophytes, the mean integrin expression score was significantly higher in blood vessels, fibrous areas, and cells from the bone lining than in osteocytes and cartilaginous zones. In vitro, the levels of integrin subunits were significantly increased during chondrocyte dedifferentiation (except for β6), fibrosis, and osteogenic differentiation as well as under inflammatory stimuli. In conclusion, anatomical zones (such as OA cartilage, intervertebral discs, and facet joint osteophytes) previously reported to show PRGD2 ligand uptake in vivo expressed increased levels of αVβ5, αVβ3, and β6 integrins, whose subunits are modulated in vitro by OA-associated conditions that increase fibrosis, inflammation, and osteogenic differentiation. These results suggest that the increased levels of integrins in OA compared to normal tissues favor PRGD2 uptake and might explain the molecular mechanism of OA imaging using the PRGD2-based ligand PET/CT.

Background/aims Light-induced control of the cell membrane potential has enabled important advances in the study of biological processes involving the nervous system and muscle activity. The use of these light-induced modifications is expected in various medical applications, including the control of physiological responses and the recovery of lost functions by regulating nerve activity. In particular, charge-separating linkage molecules (Charge-Separation (CS) molecules) can depolarize cells by photoexcitation without genetic processing. However, the molecular mechanisms underlying cell membrane depolarization are unknown and have hindered its application. Here, we show that CS molecules localized in the cell membrane of PC12 cells using a high-density lipoprotein (HDL)-based drug carrier can excite the cells through a novel membrane current regulation mechanism by light irradiation. Methods Membrane potential, channel activity, and membrane capacitance were measured by patch clamp method in rat adrenal gland pheochromocytoma (PC12) cells and KV-overexpressing PC12 cells. CS molecules localized in the cell membrane of PC12 cells using HDL-based drug carrier. The localization of CS molecule was measured by a confocal microscopy. The mRNA expression was tested by RT-PCR. Results Current clamp measurements revealed that the photo-activated CS molecule causes a sharp depolarization of about 15 mV. Furthermore, it was shown by voltage clamp measurement that this mechanism inactivates the voltage-dependent potassium current and simultaneously generates photo-activated CS molecule induced (PACS) current owing to the loss of the cell membrane capacitance. This activity continues the depolarization of the target cell, but is reversible via a regenerative mechanism such as endocytosis and exocytosis because the cell membrane is intact. Conclusion Thus, the mechanism of photo-induced depolarization concludes that photo-activated TC1 causes depolarization by generating PACS current in parallel with the suppression of the K+ current. Moreover, the depolarization slowly restores by internalization of TC1 from the membrane and insertion of new lipids into the cell membrane, resulting in the restoration of KV to normal activity and eliminating PACS currents, without cell damage. These results suggest the possibility of medical application that can safely control membrane excitation.

Background/aims GnIH receptors (GnIHRs) belong to the family of G-protein coupled receptors (GPCRs) and play a key role in the regulation of reproduction from fishes to mammals, either by inhibiting or stimulating the expression of gonadotropins. The aim of this study was to characterize GnIH receptor (GnIHR2) from Indian Major Carp, Labeo catla and its docking and simulation with GnIH antagonist RF313. Methods The full length sequence of GnIHR2 was obtained with RACE PCR. The docking analysis of RF313 with GnIHR2 receptor was performed with AutoDock v. 4.2.6 and molecular dynamics (MD) simulation with GROMACS 5.0. Results In the present study, we cloned full-length cDNA (1733 bp) of GnIHR2 from the brain of L. catla. The phylogenetic analysis showed clustering of catla GnIHR2 with goldfish and zebrafish in the GPR147 group. L. catla GnIHR2 receptor comprised seven transmembrane domains and the 3D-structure was predicted by I-TASSER tool. The docking analysis revealed high binding affinity (-11.6 kcal/mol) of GnIH antagonist, RF313 towards GnIHR2 receptor. The primary bonds involved were alkyl and hydrogen bonds while the amino acids participated were proline 43, 210, 339, cysteine 214, leucine 211, serine 213 and phenylalanine 338. The MD simulation analysis of docked complex for 100 nano-seconds (ns) in the lipid membrane environment showed the stability of the complex with time. Conclusion Our study showed that GnIH antagonist, RF313 interact tightly with the GnIH receptor, GnIHR2 of L. catla. To the best of our knowledge, this is the first report on computational modelling and MD simulation of GnIH receptor in fishes. This will help in functional characterization studies of GnIH/GnIHR system in vertebrates.

Coronavirus disease 2019 (COVID-19) has shown high infection and mortality rates all over the world, and despite the global efforts, there is so far no specific therapy available for COVID-19. Interestingly, while the severity and mortality of COVID-19 are higher in males than in females, the underlying molecular mechanisms are unclear. In this review, we explore sex-related differences that may be contributing factors to the observed male-biased mortality from COVID-19. Males are considered the weaker sex in aspects related to endurance and infection control. Studies show that viral RNA clearance is delayed in males with COVID-19. A recent study has indicated that the testis can harbor coronavirus, and consequently, males show delayed viral clearance. However, the role of testis involvement in COVID-19 severity and mortality needs further research. Males and females show a distinct difference in immune system responses with females eliciting stronger immune responses to pathogens. This difference in immune system responses may be a major contributing factor to viral load, disease severity, and mortality. In addition, differences in sex hormone milieus could also be a determinant of viral infections as estrogen has immunoenhancing effects while testosterone has immunosuppressive effects. The sex-specific severity of COVID-19 infections indicates that further research on understanding the sex differences is needed. Inclusion of both males and females in basic research and clinical trials is required to provide critical information on sex-related differences that may help to better understand disease outcome and therapy.

Background/Aims: High-molecular-weight advanced glycation end-products (HMW-AGEs) are abundantly present in our Western diet. There is growing evidence reporting that HMW-AGEs contribute to the development of cardiovascular dysfunction in vivo, next to the well-known low-molecular-weight AGEs. The goal of our study is to assess the ultrastructure and function of cardiomyocytes after chronic exposure to HMW-AGEs. A better understanding of underlying mechanisms is essential to create new opportunities for further research on the specific role of HMW-AGEs in the development and progression of cardiovascular diseases. Methods: Adult male rats were randomly assigned to daily intraperitoneal injection for six weeks with either HMW-AGEs (20 mg/kg/day) or a control solution. Hemodynamic measurements were performed at sacrifice. Single cardiomyocytes from the left ventricle were obtained by enzymatic dissociation through retrograde perfusion of the aorta. Unloaded cell shortening, time to peak and time to 50% relaxation were measured during field stimulation and normalized to diastolic length. L-type Ca 2+ current density (I CaL) and steady-state inactiva-tion of I CaL were measured during whole-cell ruptured patch clamp. Myofilament functional properties were measured in membrane-permeabilized cardiomyocytes. Ultrastructural examination of cardiac tissue was performed using electron microscopy. Results: Rats injected with HMW-AGEs displayed in vivo cardiac dysfunction, characterized by significant changes in Background/Aims: High-molecular-weight advanced glycation end-products (HMW-AGEs) are abundantly present in our Western diet. There is growing evidence reporting that HMWAGEs contribute to the development of cardiovascular dysfunction in vivo, next to the wellknown low-molecular-weight AGEs. The goal of our study is to assess the ultrastructure and function of cardiomyocytes after chronic exposure to HMW-AGEs. A better understanding of underlying mechanisms is essential to create new opportunities for further research on the specific role of HMW-AGEs in the development and progression of cardiovascular diseases. Methods: Adult male rats were randomly assigned to daily intraperitoneal injection for six weeks with either HMW-AGEs (20 mg/kg/day) or a control solution. Hemodynamic measurements were performed at sacrifice. Single cardiomyocytes from the left ventricle were obtained by enzymatic dissociation through retrograde perfusion of the aorta. Unloaded cell shortening, time to peak and time to 50% relaxation were measured during field stimulation and normalized to diastolic length. L-type Ca2+ current density (ICaL) and steady-state inactivation of ICaL were measured during whole-cell ruptured patch clamp. Myofilament functional properties were measured in membrane-permeabilized cardiomyocytes. Ultrastructural examination of cardiac tissue was performed using electron microscopy. Results: Rats injected with HMW-AGEs displayed in vivo cardiac dysfunction, characterized by significant changes in eft ventricular peak rate pressure rise and decline accompanied with an increased heart mass. Single cardiomyocytes isolated from the left ventricle revealed concentric hypertrophy, indicated by the increase in cellular width. Unloaded fractional cell shortening was significantly reduced in cells derived from the HMW-AGEs group and was associated with slower kinetics. Peak L-type Ca2+ current density was significantly decreased in the HMW-AGEs group. L-type Ca2+ channel availability was significantly shifted towards more negative potentials after HMW-AGEs injection. The impact of HMW-AGEs on myofilament function was measured in membrane-permeabilized cardiomyocytes showing a reduction in passive force, maximal Ca2+ activated force and rate of force development. Ultrastructural examination of cardiac tissue demonstrated adverse structural remodeling in HMW-AGEs group characterized by a disruption of the cyto-architecture, a decreased mitochondrial density and altered mitochondrial function. Conclusion: Our data indicate that HMW-AGEs induce structural and functional cellular remodeling via a different working mechanism as the well-known LMW-AGEs. Results of our research open the door for new strategies targeting HMW-AGEs to improve cardiac outcome. Acknowledgements We thank Marc Jans for assisting with the EM. The authors also thank Petra Bex, Rosette Beenaerts and Kanigula Mubagwa for their skillful technical assistance. The graphical abstract was created using images from Servier Medical Art Commons Attribution 3.0 Unported License (http://smart.servier.com). Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License. Funding This work was supported by a Bijzonder onderzoeksfonds (BOF) grant from Hasselt University (15NI06-BOF). Statement of Ethics The animal protocol was approved by the Local Ethical Committee (Ethical Commission for Animal Experimentation, UHasselt, Diepenbeek, Belgium). All animal procedures were performed conforming to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. Only trained researchers, certified with a Laboratory Animal Science course according to the Federation of European Laboratory Animal Science Associations, performed animal handling and procedures.

Background Women are more susceptible to multiple sclerosis (MS) than men by a ratio of approximately 3:1. However, being male is a risk factor for worse disability progression. Inflammatory genes have been linked to susceptibility, while neurodegeneration underlies disability progression. Thus, there appears to be a differential effect of sex on inflammation versus neurodegeneration. Further, gray matter (GM) atrophy is not uniform across the brain in MS, but instead shows regional variation. Here, we study sex differences in neurodegeneration by comparing regional GM atrophy in a cohort of men and women with MS versus their respective age- and sex-matched healthy controls. Methods Voxel-based morphometry (VBM), deep GM substructure volumetry, and cortical thinning were used to examine regional GM atrophy. Results VBM analysis showed deep GM atrophy in the thalamic area in both men and women with MS, whereas men had additional atrophy in the putamen as well as in localized cortical regions. Volumetry confirmed deep GM loss, while localized cortical thinning confirmed GM loss in the cerebral cortex. Further, MS males exhibited worse performance on the 9-hole peg test (9HPT) than MS females. We observed a strong correlation between thalamic volume and 9HPT performance in MS males, but not in MS females. Conclusion More regional GM atrophy was observed in men with MS than women with MS, consistent with previous observations that male sex is a risk factor for worse disease progression.

Background/aims The study of the effects of simulated microgravity on primary cultures of human satellite cells represents a reliable model for identifying the biomolecular processes involved in mechanic load-related muscle mass loss. Therefore, this study aims to investigate the role of myostatin and Bone Morphogenetic Protein-2 in human satellite cells response to simulated microgravity condition. Methods In order to identify the main molecules involved in the phenomena of degeneration/regeneration of muscle tissue related to the alteration of mechanic load, we performed a morphological and immunohistochemical study on 27 muscle biopsies taken from control, osteoporotic and osteoarthritic patients, underwent hip arthroplasty. For each patient, we set up primary satellite cell cultures subjected to normogravity and simulated microgravity (110h) regimens. Cellular functionality has been studied through a morphological evaluation performed by optical microscopy, and an ultrastructural evaluation carried out by transmission electron microscopy. Furthermore, we evaluated the expression of Bone Morphogenetic Protein-2 and myostatin through immunocytochemical reactions. Results Our results showed that in the very early phases of simulated microgravity condition the satellite cells are more active than those subjected to the normogravity regime, as demonstrated by both the increase in the number of myotubes and the significant increase in the expression of Bone Morphogenetic Protein-2 in all experimental groups. However, with prolongated exposure to simulated microgravity regime (>72h), satellite cells and new formed myotubes underwent to cell death. It is important to note that, in early phases, simulated microgravity can stimulate the formation of new myotubes from satellite cells derived by osteoporotic patients. Furthermore, we observed that simulated microgravity can induce changes in myostatin expression levels by group-dependent variations. Conclusion The results obtained allowed us to hypothesize a possible molecular mechanism of response to simulated microgravity, confirming the importance of Bone Morphogenetic Protein-2 and myostatin in the physio-pathogenesis of muscle tissue. In addition, these data can lay the foundation for new therapeutic approached in the prevention/cure of osteoporosis and sarcopenia.

Background/aims The beneficial effect of aerobic exercise training (ET) on cardiac remodeling caused by supravalvar aortic stenosis (AS) has been demonstrated in experimental studies; however, the mechanisms responsible for improving cardiac function are not entirely understood. We evaluated whether ET-generated cardioprotection in pressure-overloaded rats is dependent on cardiomyocyte proliferation, increased angiotensin-(1-7) (Ang-1-7) levels, and its receptor in the myocardium. Methods Eighteen weeks after ascending AS surgery, Wistar rats were randomly assigned to four groups: sedentary control (C-Sed), exercised control (C-Ex), sedentary aortic stenosis (AS-Sed) and exercised aortic stenosis (AS-Ex) groups. The moderate treadmill exercise protocol was performed for ten weeks. The functional capacity was assessed by treadmill exercise testing. Cardiac structure and function were evaluated by echocardiogram. Cardiomyocyte proliferation was evaluated by flow cytometry. Expression of cell cycle regulatory genes as CCND2, AURKB, CDK1, and MEIS1 was verified by RT-qPCR. Cardiac and plasma angiotensin I (Ang I), angiotensin II (Ang II), and Ang-(1-7) levels were analyzed by high-performance liquid chromatography (HPLC). The angiotensin-converting enzyme (ACE) activity was assessed by the fluorometric method and protein expression of AT1 and Mas receptors by Western blot. Results The AS-Ex group showed reduced left ventricular wall relative thickness and improved ejection fraction; also, it showed decreased gene expression of myocyte cell cycle regulators, ACE, Ang I, Ang II and Ang II/Ang-(1-7) ratio levels compared to AS-Sed group. However, ET did not induce alterations in Ang-(1-7) and cardiac Mas receptor expression and myocyte proliferation. Conclusion Aerobic exercise training improves systolic function regardless of myocyte proliferation and Ang-(1-7)/Mas receptor levels. However, the ET negatively modulates the vasoconstrictor/hypertrophic axis (ACE/Ang II) and decreases the expression of negative regulatory genes of the cell cycle in cardiomyocytes of rats with supravalvular aortic stenosis.

Motor proteins are essential units of life and are well-designed nanomachines working under thermal fluctuations. These proteins control moving direction by consuming chemical energy or by dissipating electrochemical potentials. Chitinase A from bacterium Serratia marcescens (SmChiA) processively moves along crystalline chitin by hydrolysis of a single polymer chain to soluble chitobiose. Recently, we directly observed the stepping motions of SmChiA labeled with a gold nanoparticle by dark-field scattering imaging to investigate the moving mechanism. Time constants analysis revealed that SmChiA moves back and forth along the chain freely, because forward and backward states have a similar free energy level. The similar probabilities of forward-step events (83.5%=69.3%+14.2%) from distributions of step sizes and chain-hydrolysis (86.3%=(1/2.9)/(1/2.9+1/18.3)×100) calculated from the ratios of time constants of hydrolysis and the backward step indicated that SmChiA moves forward as a result of shortening of the chain by a chitobiose unit, which stabilizes the backward state. Furthermore, X-ray crystal structures of sliding intermediate and molecular dynamics simulations showed that SmChiA slides forward and backward under thermal fluctuation without large conformational changes of the protein. Our results demonstrate that SmChiA is a burnt-bridge Brownian ratchet motor.

Estradiol and testosterone are powerful steroid hormones that impact brain function in numerous ways. During development, these hormones can act to program the adult brain in a male or female direction. During adulthood, gonadal steroid hormones can activate or inhibit brain regions to modulate adult functions. Sex differences in behavioral and neuroendocrine (i.e., hypothalamic pituitary adrenal (HPA) axis) responses to stress arise as a result of these organizational and activational actions. The sex differences that are present in the HPA and behavioral responses to stress are particularly important considering their role in maintaining homeostasis. Furthermore, dysregulation of these systems can underlie the sex biases in risk for complex, stress-related diseases that are found in humans. Although many studies have explored the role of estrogen and estrogen receptors in mediating sex differences in stress-related behaviors and HPA function, much less consideration has been given to the role of androgens. While circulating androgens can act by binding and activating androgen receptors, they can also act by metabolism to estrogenic molecules to impact estrogen signaling in the brain and periphery. This review focuses on androgens as an important hormone for modulating the HPA axis and behaviors throughout life and for setting up sex differences in key stress regulatory systems that could impact risk for disease in adulthood. In particular, impacts of androgens on neuropeptide systems known to play key roles in HPA and behavioral responses to stress (corticotropin-releasing factor, vasopressin, and oxytocin) are discussed. A greater knowledge of androgen action in the brain is key to understanding the neurobiology of stress in both sexes.

Background Individuals exposed to gestational stressors such as alcohol exhibit a spectrum of growth patterns, suggesting individualized responses to the stressors. We hypothesized that intrauterine growth responses to gestational alcohol are modified not only by the stressor’s severity but by fetal sex and the placenta’s adaptive capacity. Methods Pregnant C57BL/6J mice were assigned to one of three groups. Group 1 consumed a normal protein diet (18% protein by weight) and received 4.5 g alcohol/kg body weight (NP-Alc-8) or isocaloric maltodextrin (NP-MD-8) daily from embryonic day (E) 8.5–E17.5. Group 2 consumed the same diet but received alcohol (NP-Alc-13) or maltodextrin (NP-MD-13) daily from E13.5–E17.5. Group 3 consumed the same diet but containing a lower protein content (12% protein by weight) from E0.5 and also received alcohol (LP-Alc-8) or maltodextrin (LP-MD-8) daily from E8.5–E17.5. Maternal, placental, and fetal outcomes were assessed on E17.5 using 2-way ANOVA or mixed linear model. Results We found that intrauterine growth differed in the alcohol-exposed fetuses depending on sex and insult severity. Both NP-Alc-8 (vs. NP-MD-8) males and females had lower body weight and asymmetrical growth, but only NP-Alc-8 females had lower placental weight (P < 0.05). NP-Alc-13 (vs. NP-MD-13) females, but not their male littermates, had lower body weight (P = 0.019). Alcohol exposure beginning from E8.5 (vs. E13.5) decreased the ratio of fetal liver-to-body weight and increased the ratio of fetal brain-to-liver weight in both sexes (P < 0.05). LP-Alc-8 (vs. NP-MD-8) group had smaller litter size (P = 0.048), but the survivors had normal placental and body weight at E17.5. Nevertheless, LP-Alc-8 fetuses still showed asymmetrical growth. Correlation analyses reveal a relationship between litter size and placental outcomes, which were related to fetal outcomes in a sex-dependent manner, suggesting that the placenta may mediate the consequence of LP-Alc-altered litter size on fetal development. Conclusions Our data indicate that the placenta is strongly involved in the fetal stress response and adapts in a sex-dependent fashion to support fetal development under the alcohol stressor. These variables may further influence the spectrum of intrauterine growth outcomes observed in those diagnosed with fetal alcohol spectrum disorder.

Background/aims The tumour microenvironment is rich in multiple cells that influence cancer development. Among them, macrophages are the most abundant immune cells, which secrete factors involved in carcinogenesis. Since protein-bound polysaccharides (PBP) from the Coriolus versicolor fungus are believed to inhibit the growth of cancers, in the present study, we investigated whether these PBP influence crosstalk between triple-negative 4T1 breast cancer cells and RAW 264.7 macrophages. Methods 4T1 cells were cultured in conditioned media (CM) collected after: stimulation of the macrophages with PBP (CM-PBP) or incubation of non-treated macrophages (CM-NT). A co-cultured model of both cell lines was also employed to investigate the crosstalk between the cells. Cell viability was measured using the MTT assay. The levels of cytokines and chemokines were determined by ELISA methods. Commercial assay kits were used to assess the activity of both arginase 1 and inducible nitric oxide synthase (iNOS) and the level of cell migration. Results The results revealed that CM-NT promotes proliferation and migration of 4T1 cells, and increases the secretion of pro-angiogenic factors (VEGF, MCP-1) by cancer cells. In contrast, CM-PBP inhibits 4T1 cell growth and migration, decreases the secretion of pro-angiogenic factors (VEGF, MCP-1) and upregulates the production of pro-inflammatory mediators (IL-6, TNF-α) with certain anti-tumoral properties Moreover, PBP-treated CM significantly decreases the level of M2 macrophage markers (arginase 1 activity, IL-10 and TGF-β concentrations), but upregulates iNOS activity and IL-6 and TNF-α production, which are M1 cell markers. Conclusion The results suggest that PBP suppress the favourable tumour microenvironment by inhibiting the crosstalk between 4T1 cells and macrophages through the regulation of production of angiogenic and inflammatory mediators, and modulating the M1/M2 macrophage subtype.

Background/aims The induction of necroptosis, a form of caspase-independent cell death, represents one of the most promising anticancer therapeutic modalities, as necroptosis serves as an alternative way to eliminate apoptosis-resistant tumor cells. Here, we investigated whether protein-bound polysaccharides (PBPs) derived from the fungus Coriolus versicolor (CV) induce the necroptotic death pathway in breast cancer and melanoma cells. Methods MCF-7 and SKMel-188 cells were exposed to PBPs either alone or in combination with necrostatin-1 (Nec-1), GSK'872 or necrosulfonamide (NSA), pharmacological inhibitors of the kinases receptor-interacting protein 1 kinase (RIPK1), receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL), respectively, which are involved in necroptotic processes. The effects of cellular treatment with these inhibitors were quantified by measuring cell viability and reactive oxygen species (ROS) generation via 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and 2',7'-dichlorofluorescein diacetate (DCF-DA) assays, respectively. The morphological changes induced in these cells were detected using holotomographic (HT) microscopy. Activation of the TNF-α/TNFR1 pathway in the PBP-stimulated cells was evaluated using TNF-α-neutralizing antibody, qRT-PCR and immunofluorescence-based assays. Results PBPs showed effective antitumor activity against MCF-7 and SKMel-188 cells. Cotreatment of the cells with Nec-1, GSK'872 or NSA abrogated PBP-induced cell death, and the cells were protected against membrane rupture. Moreover, breast cancer cell death caused by PBPs was mediated by induced activation of the TNF-α/TNFR1 pathway. Interestingly, the melanoma cells did not express TNF-α or TNFR1 after PBP stimulation; instead, PBPs triggered intracellular ROS generation, which was partially diminished by the inhibitors Nec-1, GSK'872 and NSA. Conclusion These results suggest that PBPs from the fungus CV induce RIPK1/RIPK3/MLKL-mediated necroptosis in breast cancer and melanoma cells, providing novel insights into the molecular effects of PBPs on cancer cells.

Exosomes participate in many physiological and pathological processes by regulating cell–cell communication, which are involved in numerous diseases, including osteoarthritis (OA). Exosomes are detectable in the human articular cavity and were observed to change with OA progression. Several joint cells, including chondrocytes, synovial fibroblasts, osteoblasts, and tenocytes, can produce and secrete exosomes that influence the biological effects of targeted cells. In addition, exosomes from stem cells can protect the OA joint from damage by promoting cartilage repair, inhibiting synovitis, and mediating subchondral bone remodeling. This review summarizes the roles and therapeutic potential of exosomes in OA and discusses the perspectives and challenges related to exosome-based treatment for OA patients in the future.

Neutrophils were traditionally considered as short-lived cells with abundant secretory and protein synthetic activity. Recent studies, however, indicate neutrophils are in reality a heterogeneous population of cells. Neutrophils differentiate from pluripotent stem cells in the bone marrow, and can further mature in the blood stream and can have different phenotypes in health and disease conditions. Neutrophils undergo primary functions such as phagocytosis, production of reactive oxygen species (ROS), release of lipid mediators and inflammatory proteins (mainly cytokines), and apoptosis. Neutrophils stimulate other neutrophils and trigger a cascade of immune and inflammatory responses. The underpinning intracellular metabolisms that support these neutrophil functions are herein reported. It has been known for many decades that neutrophils utilize glucose as a primary fuel and produce lactate as an end product of glycolysis. Neutrophils metabolize glucose through glycolysis and the pentose- phosphate pathway (PPP). Mitochondrial glucose oxidation is very low. The PPP provides the reduced nicotinamide adenine dinucleotide phosphate (NADPH) for the NADPH-oxidase (NOX) complex activity to produce superoxide from oxygen. These cells also utilize glutamine and fatty acids to produce the required adenosine triphosphate (ATP) and precursors for the synthesis of molecules that trigger functional outcomes. Neutrophils obtained from rat intraperitoneal cavity and incubate for 1 hour at 37°C metabolize glutamine at higher rate than that of glucose. Glutamine delays neutrophil apoptosis and maintains optimal NOX activity for superoxide production. Under limited glucose provision, neutrophils move to fatty acid oxidation (FAO) to obtain the required energy for the cell function. FAO is mainly associated with neutrophil differentiation and maturation. Hypoxia, hormonal dysfunction, and physical exercise markedly change neutrophil metabolism. It is now become clear that neutrophil metabolism underlies the heterogeneity of neutrophil phenotypes and should be intense focus of investigation.

A mathematical model of amyloid fiber formation is described that is able to simply specify different rates of fiber breakage at internal versus end regions. This Note presents the derivation of the relevant equations and provides results showing the dramatic effects of position biased fiber breakage on the kinetics of amyloid growth.

形態: カラー図版あり, Physical characteristics: Original contains color illustrations, Accepted: 2020-04-17, 資料番号: PA2110058000

Background/aims Src kinase family members, including c-Src, are involved in numerous signaling pathways and have been observed inside different cellular compartments. Notably, c-Src modulates carbohydrate and fatty acid metabolism and is involved in the metabolic rewiring of cancer cells. This kinase is found within mitochondria where it targets different proteins to impact on the organelle functions and overall metabolism. Surprisingly, no global metabolic characterization of Src has been performed although c-Src knock-out mice have been available for 30 years. Considering that c-Src is sensitive to various metabolites, c-Src might represent a crucial player in metabolic adjustments induced by nutrient stress. The aim of this work was to characterize the impact of c-Src on mitochondrial activity and overall metabolism using multi-omic characterization. Methods Src+/+ and Src-/- mice were fed ad libitum or fasted during 24h and were then analyzed using multi-omics. Results We observed that deletion of c-Src is linked to lower phosphorylation of Y412-NDUFA8, inhibition of oxygen consumption and accumulation of metabolites involved in glycolysis, TCA cycle and amino acid metabolism in mice fed ad libitum. Finally, metabolomics and (phosphotyrosine) proteomics are differently impacted by Src according to nutrient availability. Conclusion The findings presented here highlight that c-Src reduces mitochondrial metabolism and impacts the metabolic adjustment induced by nutrient stress.

Bone loss in space travelers is a major challenge for long-duration space exploration. To quantify microgravity-induced bone loss in humans, we performed a meta-analysis of studies systematically identified from searching Medline, Embase, Web of Science, BIOSIS, NASA Technical reports, and HathiTrust, with the last update in November 2019. From 25 articles selected to minimize the overlap between reported populations, we extracted post-flight bone density values for 148 individuals, and in-flight and post-flight biochemical bone marker values for 124 individuals. A percentage difference in bone density relative to pre-flight was positive in the skull, +2.2% [95% confidence interval: +1.1, +3.3]; neutral in the thorax/upper limbs, −0.7% [−1.3, −0.2]; and negative in the lumbar spine/pelvis, −6.2 [−6.7, −5.6], and lower limbs, −5.4% [−6.0, −4.9]. In the lower limb region, the rate of bone loss was −0.8% [−1.1, −0.5] per month. Bone resorption markers increased hyperbolically with a time to half-max of 11 days [9, 13] and plateaued at 113% [108, 117] above pre-flight levels. Bone formation markers remained unchanged during the first 30 days and increased thereafter at 7% [5, 10] per month. Upon landing, resorption markers decreased to pre-flight levels at an exponential rate that was faster after longer flights, while formation markers increased linearly at 84% [39, 129] per month for 3–5 months post-flight. Microgravity-induced bone changes depend on the skeletal-site position relative to the gravitational vector. Post-flight recovery depends on spaceflight duration and is limited to a short post-flight period during which bone formation exceeds resorption.

Background/aims Sodium is a key player in the fundamental cell functions. Fluorescent probes are indispensable tools for monitoring intracellular sodium levels in single living cells. Since the fluorescence of sodium-sensitive dyes in cells is significantly different from that in an aqueous solution, the fluorescence signal is calibrated in situ indirectly using ionophores for equalizing external and intracellular ion concentration. Attempts to compare data obtained using fluorescent probes and by direct flame emission analysis are sparse and results are inaccurate. Methods We determined the intracellular sodium concentration in U937 cells by flow cytometry using the Na+-sensitive probe Asante Natrium Green-2 (ANG), and by standard flame emission photometry combined with the cellular water determination by cell density in Percoll gradient. The intracellular Na+ concentrations was modified using known ionophores or, alternatively, by blocking the sodium pump with ouabain or by causing cell apoptosis with staurosporine. Results It is revealed that both methods are comparable when intracellular sodium concentration was modified by ouabain-mediated blockage of the sodium pump or staurosporine-induced apoptosis. The ANG fluorescence of cells treated with ionophores is approximately two times lower than that in cells with the same Na+ concentration but not treated with ionophores. Although the mechanism is still unknown, this effect should be taken into account when a quantitative assessment of the concentration of intracellular sodium is required. Conclusion The sodium sensitive dye ANG-2 is a sensitive and useful probe for determination changes in Na+ content and concentration both in single cells and subcellular microparticles. The ANG fluorescence determined in the studied cells in the absence of ionophores, cannot be used as a measure of the real intracellular concentration of Na+ if calibration was carried out in the presence of ionophores.

Background Alcohol misuse and post-traumatic stress disorder (PTSD) are highly comorbid, and treatment outcomes are worse in individuals with both conditions. Although more men report experiencing traumatic events than women, the lifetime prevalence of PTSD is twice as high in females. Despite these data trends in humans, preclinical studies of traumatic stress reactivity have been performed almost exclusively in male animals. Methods This study was designed to examine sex differences in traumatic stress reactivity in alcohol-naive rats (experiment 1) and rats given intermittent access to 20% ethanol in a 2-bottle choice paradigm for 5 weeks (experiment 2). Animals were exposed to predator odor (bobcat urine) and tested for contextual avoidance 24 h later; unstressed controls were never exposed to predator odor. We evaluated changes in physiological arousal using the acoustic startle response (ASR) test at day 2 post-stress and anxiety-like behavior measured in the elevated plus-maze (EPM) at day 17 post-stress. In experiment 3, time course of corticosterone response was examined in male and female rats following exposure to predator odor stress. Results Alcohol-naive males and females exposed to predator odor displayed blunted weight gain 24 h post-stress, but only a subset of stressed animals exhibited avoidance behavior. In alcohol-drinking animals, the proportion of avoiders was higher in males than females, and predator odor exposure increased ASR in males only. Stressed females exhibited blunted ASR relative to unstressed females and stressed males, regardless of alcohol drinking history. Alcohol-experienced females presented lower anxiety-like behavior and higher general activity in the EPM in comparison with alcohol-experienced males. Plasma corticosterone levels were higher in females immediately after predator odor exposure until 60 min post-stress relative to males. Conclusions We report robust sex differences in behavioral and endocrine responses to bobcat urine exposure in adult Wistar rats. Also, males with a history of chronic moderate alcohol drinking exhibited increased traumatic stress reactivity relative to alcohol-drinking females. Our findings emphasize the importance of considering sex as a biological variable in the investigation of traumatic stress effects on physiology and behavior.

Background/aims This study aimed to establish a precise and well-defined working model, assessing pharmaceutical effects on vascular smooth muscle cell monolayer in-vitro. It describes various analysis techniques to determine the most suitable to measure the biomechanical impact of vasoactive agents by using CellDrum technology. Methods The so-called CellDrum technology was applied to analyse the biomechanical properties of confluent human aorta muscle cells (haSMC) in monolayer. The cell generated tensions deviations in the range of a few N/m² are evaluated by the CellDrum technology. This study focuses on the dilative and contractive effects of L-type Ca2+ channel agonists and antagonists, respectively. We analyzed the effects of Bay K8644, nifedipine and verapamil. Three different measurement modes were developed and applied to determine the most appropriate analysis technique for the study purpose. These three operation modes are called, particular time mode" (PTM), "long term mode" (LTM) and "real-time mode" (RTM). Results It was possible to quantify the biomechanical response of haSMCs to the addition of vasoactive agents using CellDrum technology. Due to the supplementation of 100nM Bay K8644, the tension increased approximately 10.6% from initial tension maximum, whereas, the treatment with nifedipine and verapamil caused a significant decrease in cellular tension: 10nM nifedipine decreased the biomechanical stress around 6,5% and 50nM verapamil by 2,8%, compared to the initial tension maximum. Additionally, all tested measurement modes provide similar results while focusing on different analysis parameters. Conclusion The CellDrum technology allows highly sensitive biomechanical stress measurements of cultured haSMC monolayers. The mechanical stress responses evoked by the application of vasoactive calcium channel modulators were quantified functionally (N/m²). All tested operation modes resulted in equal findings, whereas each mode features operation-related data analysis.

Low-complexity (LC) sequences, regions that are predominantly made up of limited amino acids, are often observed in eukaryotic nuclear proteins. The role of these LC sequences has remained unclear for decades. Recent studies have shown that LC sequences are important in the formation of membrane-less organelles via liquid–liquid phase separation (LLPS). The RNA binding protein, fused in sarcoma (FUS), is the most widely studied of the proteins that undergo LLPS. It forms droplets, fibers, or hydrogels using its LC sequences. The N-terminal LC sequence of FUS is made up of Ser, Tyr, Gly, and Gln, which form a labile cross-β polymer core while the C-terminal Arg-Gly-Gly repeats accelerate LLPS. Normally, FUS localizes to the nucleus via the nuclear import receptor karyopherin β2 (Kapβ2) with the help of its C-terminal proline-tyrosine nuclear localization signal (PY-NLS). Recent findings revealed that Kapβ2 blocks FUS mediated LLPS, suggesting that Kapβ2 is not only a transport protein but also a chaperone which regulates LLPS during the formation of membrane-less organelles. In this review, we discuss the effects of the nuclear import receptors on LLPS.

Background/aims Galectin 3 (GAL-3) is a beta galactoside binding lectin that has different roles in normal and pathophysiological conditions. GAL-3 has been associated with heart failure and was linked to increased risk of death in a number of studies. GAL-3 was found to be up regulated in animal models of heart failure as well as myocardial infarction (MI). The objective of his study is to test if high GAL-3 after myocardial infarction has a protective role on the heart through its anti-apoptotic and anti-necrotic functions. Methods Male C57B6/J mice and GAL-3 knockout (KO) mice were used for permanent ligation of the left anterior descending artery of the heart to create infarction in the anterior myocardium. Heart and plasma samples were collected 24 hours after the induction of MI and were used for immunohistochemistry, Tunnel procedure, electron microscopy and enzyme linked immunosorbent assay (ELISA). Results Our results show that the significant increase in GAL-3 levels in the left ventricle at 24-hour following MI is associated with significant lower levels of pro-apoptotic proteins; cytochrome c, Bax, annexin V, cleaved caspase-3 and a higher levels of anti-apoptotic protein Bcl2 in GAL-3 wild MI group than GAL-3 KO group. We also have identified the anti-apoptotic activity of GAL-3 is mediated through a significant increase in Akt-1, NF kappa-B and beta- catenin proteins. In addition, we have identified the antiapoptotic activity is mediated through a significant lower levels of cathepsin-D protein. Conclusion We conclude that the increased levels of GAL-3 at 24-hour following MI regulate antiapoptotic mechanisms in the myocardium that will shape the future course of the disease. We also identified that the anti-apoptotic mechanisms are likely mediated through interaction of GAL-3 with Akt-1, NF kappa-B, beta- catenin and cathepsin D proteins.

We now know that cancer is many different diseases, with great variation even within a single histological subtype. With the current emphasis on developing personalized approaches to cancer treatment, it is astonishing that we have not yet systematically incorporated the biology of sex differences into our paradigms for laboratory and clinical cancer research. While some sex differences in cancer arise through the actions of circulating sex hormones, other sex differences are independent of estrogen, testosterone, or progesterone levels. Instead, these differences are the result of sexual differentiation, a process that involves genetic and epigenetic mechanisms, in addition to acute sex hormone actions. Sexual differentiation begins with fertilization and continues beyond menopause. It affects virtually every body system, resulting in marked sex differences in such areas as growth, lifespan, metabolism, and immunity, all of which can impact on cancer progression, treatment response, and survival. These organismal level differences have correlates at the cellular level, and thus, males and females can fundamentally differ in their protections and vulnerabilities to cancer, from cellular transformation through all stages of progression, spread, and response to treatment. Our goal in this review is to cover some of the robust sex differences that exist in core cancer pathways and to make the case for inclusion of sex as a biological variable in all laboratory and clinical cancer research. We finish with a discussion of lab- and clinic-based experimental design that should be used when testing whether sex matters and the appropriate statistical models to apply in data analysis for rigorous evaluations of potential sex effects. It is our goal to facilitate the evaluation of sex differences in cancer in order to improve outcomes for all patients.

Many crucial cardiovascular adaptations occur in the body during pregnancy to ensure successful gestation. Maladaptation of the cardiovascular system during pregnancy can lead to complications that promote cardiac dysfunction and may lead to heart failure (HF). About 12% of pregnancy-related deaths in the USA have been attributed to HF and the detrimental effects of cardiovascular complications on the heart can be long-lasting, pre-disposing the mother to HF later in life. Indeed, cardiovascular complications such as gestational diabetes mellitus, preeclampsia, gestational hypertension, and peripartum cardiomyopathy have been shown to induce cardiac metabolic dysfunction, oxidative stress, fibrosis, apoptosis, and diastolic and systolic dysfunction in the hearts of pregnant women, all of which are hallmarks of HF. The exact etiology and cardiac pathophysiology of pregnancy-related complications is not yet fully deciphered. Furthermore, diagnosis of cardiac dysfunction in pregnancy is often made only after clinical symptoms are already present, thus necessitating the need for novel diagnostic and prognostic biomarkers. Mounting data demonstrates an altered expression of maternal circulating miRNAs during pregnancy affected by cardiovascular complications. Throughout the past decade, miRNAs have become of growing interest as modulators and biomarkers of pathophysiology, diagnosis, and prognosis in cardiac dysfunction. While the association between pregnancy-related cardiovascular complications and cardiac dysfunction or HF is becoming increasingly evident, the roles of miRNA-mediated regulation herein remain poorly understood. Therefore, this review will summarize current reports on pregnancy-related cardiovascular complications that may lead to cardiac dysfunction and HF during and after pregnancy in previously healthy women, with a focus on the pathophysiological role of miRNAs.

Background/aims Heart failure with preserved ejection fraction (HFpEF) is recognised as an important cause of cardiovascular mortality and morbidity, accounting for approximately 50% of heart failure cases. Metabolic-related complications, such as obesity, have been associated with the pathophysiology of this complex syndrome. The anatomic proximity between cardiac visceral adipose tissue (CVAT) and the myocardium has been drawing attention due to its potential pathogenic role in cardiac diseases. Thus, we aimed to characterise the phenotypic and proteomic differences between CVAT from ZSF1 lean (control) and ZSF1 obese (HFpEF) rats as well as to evaluate the myocardial impact of conditioned media derived from CVAT of these 2 groups. Methods CVAT of 20-weeks-old lean and obese ZSF1 rats was collected for: 1) 24h DMEM incubation to obtain conditioned media, 2) separation of proteins to mass spectrometry identification, 3) adipokines' expression, 4) adipocytes cross-sectional area assessment. Organotypic cultures were prepared from 7 days-old Wistar Han cardiac explants and incubated for 24h with the conditioned media. After incubation, cross-section area of cardiomyocytes and fibrosis were evaluated. Cardiomyocytes were isolated from Wistar Han and incubated with conditioned media for viability studies. Results CVAT from lean rats presented a higher expression of uncoupling protein-1 (UCP-1) protein, associated with a multilocular appearance and an increased expression of brown adipose tissue markers. Contrarily, CVAT from obese rats revealed a white adipose tissue-like phenotype accompanied by hypertrophy of adipocytes. The analysis of the CVAT proteome reinforced the phenotypic differences between lean and obese CVAT, showing enrichment of proteins involved in triglyceride metabolic processes in obese CVAT. In contrast, mitochondrial proteins were prominent in lean CVAT, further suggesting a brown adipose tissue-like phenotype. The twenty-four hours-long incubation of myocardial organo-cultures with conditioned media obtained from CVAT obese (CM-obese) rats significantly reduced cell viability, induced cardiomyocytes hypertrophy and fibrosis, in stark contrast with the incubation with the conditioned media from lean rats CVAT (CM-lean). Furthermore, the deleterious effect imposed by CM-obese was associated with a pro-inflammatory profile, characterised by an increased expression of several pro-inflammatory adipokines. Conclusion Obesity promotes alterations in CVAT proteome signature, structure, composition and secretome, translating into dramatic myocardial consequences.

Diffuse idiopathic skeletal hyperostosis (DISH) is a prevalent noninflammatory spondyloarthropathy characterized by ectopic mineral formation along the anterolateral aspect of the vertebral column, yet little is known about its underlying pathogenesis. Our objective was to evaluate the histopathological features and composition of ectopic mineral within spinal tissues affected by DISH in humans. Thoracic spine segments from six embalmed cadaveric donors (one female and five males; median age 82 years) meeting the radiographic diagnostic criteria for DISH were evaluated using radiological, histological, and physical analyses. Overall, the histological features of ectopic mineralization at individual motion segments were heterogeneous, including regions of heterotopic ossification and dystrophic calcification. Heterotopic ossifications were characterized by woven and lamellar bone, multifocal areas of metaplastic cartilage, and bony bridges along the anterior aspect of the intervertebral disc space. Dystrophic calcifications were characterized by an amorphous appearance, a high content of calcium and phosphorus, an X-ray diffraction pattern matching that of hydroxyapatite, and radiodensities exceeding that of cortical bone. Dystrophic calcifications were found within the anterior longitudinal ligament and annulus fibrosus in motion segments both meeting and not meeting the radiographic criteria for DISH. In summary, our findings indicate that in DISH, ectopic mineral forms along the anterior aspect of the spine by both heterotopic ossification and dystrophic calcification of fibrocartilaginous tissues. Although both types of ectopic mineralization are captured by current radiographic criteria for DISH, dystrophic calcification may reflect a distinct disease process or an early stage in the pathogenesis of DISH.