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Liddle syndrome is a classic model for hypertension from high epithelial Na+ channel (ENaC) activity. In the Liddle mouse model, vasopressin-2 receptors stimulate both ENaC and aquaporin-2, which increases Na+ and water retention to such an extent that hypertension ensues. Liddle mice will preserve plasma tonicity at the expense of a higher blood pressure; these data highlight the inherent limitation in which the kidney must use ENaC as a pathway to regulate both plasma tonicity and blood pressure.

There is a consensus that Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) originated naturally from Bat coronaviruses (CoVs), in particular RaTG13. However, the SARS-CoV-2 host tropism/adaptation pattern has significant discrepancies compared to other CoVs, raising questions concerning the proximal origin of SARS-CoV-2. This article is protected by copyright. All rights reserved.

Activity of the Epithelial Na+ Channel (ENaC) in the distal nephron fine-tunes renal sodium excretion. Appropriate sodium excretion is a key factor in the regulation of blood pressure. Consequently, abnormalities in ENaC function can cause hypertension. Casein Kinase II (CKII) phosphorylates ENaC. The CKII phosphorylation site in ENaC resides within a canonical “anchor” ankyrin binding motif. CKII-dependent phosphorylation of ENaC is necessary and sufficient to increase channel activity and is thought to influence channel trafficking in a manner that increases activity. We test here the hypothesis that phosphorylation of ENaC by CKII within an anchor motif is necessary for ankyrin-3 (Ank-3) regulation of the channel, which is required for normal channel locale and function, and the proper regulation of renal sodium excretion. This was addressed using a fluorescence imaging strategy combining total internal reflection fluorescence (TIRF) microscopy with fluorescence recovery after photobleaching (FRAP) to quantify ENaC expression in the plasma membrane in living cells; and electrophysiology to quantify ENaC activity in split-open collecting ducts from principal cell-specific Ank-3 knockout mice. Sodium excretion studies also were performed in parallel in this knockout mouse. In addition, we substituted a key serine residue in the consensus CKII site in β-ENaC with alanine to abrogate phosphorylation and disrupt the anchor motif. Findings show that disrupting CKII signaling decreases ENaC activity by decreasing expression in the plasma membrane. In the principal cell-specific Ank-3 KO mouse, ENaC activity and sodium excretion were significantly decreased and increased, respectively. These results are consistent with CKII phosphorylation of ENaC functioning as a “switch” that favors Ank-3 binding to increase channel activity.

The current Coronavirus Disease 19 (COVID-19) pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) shows similar pathology to MERS and SARS-CoV, with a current estimated fatality rate of 1.4%. Open reading frame 10 (ORF10) is a unique SARS-CoV-2 accessory protein, which contains eleven cytotoxic T lymphocyte (CTL) epitopes each of nine amino acids in length. Twenty-two unique SARS-CoV-2 ORF10 variants have been identified based on missense mutations found in sequence databases. Some of these mutations are predicted to decrease the stability of ORF10 in silico physicochemical and structural comparative analyses were carried out on SARS-CoV-2 and Pangolin-CoV ORF10 proteins, which share 97.37% amino acid (aa) homology. Though there is a high degree of ORF10 protein similarity of SARS-CoV-2 and Pangolin-CoV, there are differences of these two ORF10 proteins related to their sub-structure (loop/coil region), solubility, antigenicity and shift from strand to coil at aa position 26 (tyrosine). SARS-CoV-2 ORF10, which is apparently expressed in vivo since reactive T cell clones are found in convalescent patients should be monitored for changes which could correlate with the pathogenesis of COVID-19.

Lipopolysaccharide (LPS) is a component of gram-negative bacteria wall that elicits inflammatory response in the host through the toll-like receptor 4 (TLR4) activation. In the lower urinary tract (LUT), bacteria-derived LPS has been associated with lower urinary tract symptoms (LUTS); however, little is known about the effects of LPS in the urethral smooth muscle (USM). In the present study, we evaluated the functional and molecular effects of LPS in mouse USM in vitro, focusing on the LPS-induced TLR4-signaling pathway. Male C57BL6/JUnib and TLR4 knockout mice (TLR4 KO) were used. The USM contraction was performed in the presence of LPS (62.5–500 μg/mL), indomethacin (10 μM), L-NAME (100 μM), and TAK 242 (1 μM). The RT-PCR assay for the IL-1β, NF-kB, and COX-2 genes was also evaluated in the presence of LPS (125 μg/mL) and caspase 1 inhibitor (20 μM). Our results showed that LPS reduces mouse USM contraction elicited by phenylephrine and vasopressin. This LPS-induced urethral inhibitory effect was not reversed by the TLR4 inhibition or its absence in the TLR4 KO mice. Conversely, indomethacin (but not L-NAME) reversed the LPS-induced USM hypocontractility. Molecular protocols indicated upregulation of IL-1β, NF-kβ, and COX-2 mRNA upon LPS incubation, which were blunted by caspase 1 inhibition. Our data showed that LPS reduced mouse USM contraction independently of TLR4 activation, involving caspase 1 and IL1β, NF-kB, and COX-2 gene overexpression. Therefore, this alternative pathway might be a valuable target to reduce the LPS-induced urethral dysfunction under infection and inflammatory conditions.

Severe acute respiratory syndrome coronavirus 2 pandemic capacity is derived from the unique structural features on its spike protein: fast viral surfing over the epithelium with flat N‐terminal domain, tight binding to ACE2 entry receptor, and furin protease utilization. In addition, the possible involvement of other components such as lipid rafts, CLRs, and neuropilin is, in combination, mediating the accelerated cell entry and other critical steps in its overwhelming contagious capacity and pandemy., Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is the causative agent of the pandemic coronavirus disease 2019 (COVID‐19) that exhibits an overwhelming contagious capacity over other human coronaviruses (HCoVs). This structural snapshot describes the structural bases underlying the pandemic capacity of SARS‐CoV‐2 and explains its fast motion over respiratory epithelia that allow its rapid cellular entry. Based on notable viral spike (S) protein features, we propose that the flat sialic acid‐binding domain at the N‐terminal domain (NTD) of the S1 subunit leads to more effective first contact and interaction with the sialic acid layer over the epithelium, and this, in turn, allows faster viral ‘surfing’ of the epithelium and receptor scanning by SARS‐CoV‐2. Angiotensin‐converting enzyme 2 (ACE‐2) protein on the epithelial surface is the primary entry receptor for SARS‐CoV‐2, and protein–protein interaction assays demonstrate high‐affinity binding of the spike protein (S protein) to ACE‐2. To date, no high‐frequency mutations were detected at the C‐terminal domain of the S1 subunit in the S protein, where the receptor‐binding domain (RBD) is located. Tight binding to ACE‐2 by a conserved viral RBD suggests the ACE2‐RBD interaction is likely optimal. Moreover, the viral S subunit contains a cleavage site for furin and other proteases, which accelerates cell entry by SARS‐CoV‐2. The model proposed here describes a structural basis for the accelerated host cell entry by SARS‐CoV‐2 relative to other HCoVs and also discusses emerging hypotheses that are likely to contribute to the development of antiviral strategies to combat the pandemic capacity of SARS‐CoV‐2.

The coronavirus disease 2019 (COVID-19) is caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) which is pandemic with an estimated fatality rate less than 1% is ongoing. SARS-CoV-2 accessory proteins ORF3a, ORF6, ORF7a, ORF7b, ORF8, and ORF10 with putative functions to manipulate host immune mechanisms such as interferons, immune signaling receptor NLRP3 (NOD-, LRR-, and pyrin domain-containing 3) inflammasome, inflammatory cytokines such as interleukin 1β(IL-1β) are critical in COVID-19 pathology. Outspread variations of each of the six accessory proteins of all complete proteomes (available as of October 26, 2020, in the National Center for Biotechnology Information depository) of SARS-CoV-2, were observed across six continents. Across all continents, the decreasing order of percentage of unique variations in the accessory proteins was found to be ORF3a>ORF8>ORF7a>ORF6>ORF10>ORF7b. The highest and lowest unique variations of ORF3a were observed in South America and Oceania, respectively. This finding suggests that the wide variations of accessory proteins seem to govern the pathogenicity of SARS-CoV-2, and consequently, certain propositions and recommendations can be made in the public interest.

Angiotensin-converting enzyme 2 (ACE2) is the cellular receptor for the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that is engendering the severe coronavirus disease 2019 (COVID-19) pandemic. The spike (S) protein receptor-binding domain (RBD) of SARS-CoV-2 binds to the three sub-domains viz. amino acids (aa) 22-42, aa 79-84, and aa 330-393 of ACE2 on human cells to initiate entry. It was reported earlier that the receptor utilization capacity of ACE2 proteins from different species, such as cats, chimpanzees, dogs, and cattle, are different. A comprehensive analysis of ACE2 receptors of nineteen species was carried out in this study, and the findings propose a possible SARS-CoV-2 transmission flow across these nineteen species.

Snake venoms are complex chemical mixtures of biologically active proteins and non-protein components. Toxins have a wide range of targets and effects to include ion channels and membrane receptors, and platelet aggregation and platelet plug formation. Toxins target these effectors and effects at high affinity and selectivity. From a pharmacological perspective, snake venom compounds are a valuable resource for drug discovery and development. However, a major challenge to drug discovery using snake venoms is isolating and analyzing the bioactive proteins and peptides in these complex mixtures. Getting molecular information from complex mixtures such as snake venoms requires proteomic analyses, generally combined with transcriptomic analyses of venom glands. The present review summarizes current knowledge and highlights important recent advances in venomics with special emphasis on contemporary separation techniques and bioinformatics that have begun to elaborate the complexity of snake venoms. Several analytical techniques such as two-dimensional gel electrophoresis, RP-HPLC, size exclusion chromatography, ion exchange chromatography, MALDI-TOF-MS, and LC-ESI-QTOF-MS have been employed in this regard. The improvement of separation approaches such as multidimensional-HPLC, 2D-electrophoresis coupled to soft-ionization (MALDI and ESI) mass spectrometry has been critical to obtain an accurate picture of the startling complexity of venoms. In the case of bioinformatics, a variety of software tools such as PEAKS also has been used successfully. Such information gleaned from venomics is important to both predicting and resolving the biological activity of the active components of venoms, which in turn is key for the development of new drugs based on these venom components.

The global public health is endangered due to COVID-19 pandemic, which is caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Despite having similar pathology to MERS and SARS-CoV, the infection fatality rate of SARS-CoV-2 is likely lower than 1%. SARS-CoV-2 has been reported to be uniquely characterized by the accessory protein ORF10, which contains eleven cytotoxic T lymphocyte (CTL) epitopes of nine amino acids length each, across various human leukocyte antigen (HLA) subtypes. In this study, all missense mutations found in sequence databases were examined across twnety-two unique SARS-CoV-2 ORF10 variants that could possibly alter viral pathogenicity. Some of these mutations decrease the stability of ORF10, e.g. I4L and V6I were found in the MoRF region of ORF10 which may also possibly contribute to Intrinsic protein disorder. Furthermore, a physicochemical and structural comparative analysis was carried out on SARS-CoV-2 and Pangolin-CoV ORF10 proteins, which share 97.37% amino acid homology. The high degree of physicochemical and structural similarity of ORF10 proteins of SARS-CoV-2 and Pangolin-CoV open questions about the architecture of SARS-CoV-2 due to the disagreement of these two ORF10 proteins over their sub-structure (loop/coil region), solubility, antigenicity and change from the strand to coil at amino acid position 26, where tyrosine is present. Altogether, SARS-CoV-2 ORF10 is a promising pharmaceutical target and a protein which should be monitored for changes which correlate to change pathogenesis and clinical course of COVID-19 infection.

Immune evasion is one of the unique characteristics of COVID-19 attributed to the ORF8 protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This protein is involved in modulating the host adaptive immunity through downregulating MHC (Major Histocompatibility Complex) molecules and innate immune responses by surpassing the interferon mediated antiviral response of the host. To understand the immune perspective of the host with respect to the ORF8 protein, a comprehensive study of the ORF8 protein as well as mutations possessed by it, is performed. Chemical and structural properties of ORF8 proteins from different hosts, that is human, bat and pangolin, suggests that the ORF8 of SARS-CoV-2 and Bat RaTG13-CoV are very much closer related than that of Pangolin-CoV. Eighty-seven mutations across unique variants of ORF8 (SARS-CoV-2) are grouped into four classes based on their predicted effects. Based on geolocations and timescale of collection, a possible flow of mutations was built. Furthermore, conclusive flows of amalgamation of mutations were endorsed upon sequence similarity and amino acid conservation phylogenies. Therefore, this study seeks to highlight the uniqueness of rapid evolving SARS-CoV-2 through the ORF8.

There is a consensus that SARS-CoV-2 originated naturally from BatCoVs. Although based on phylogenetic analysis SARS-CoV-2 seems to be related to BatCoVs RaTG13 or RmYN02, its exact origin of lineage is not clear. In fact, the flat and non-sunken surface of the sialic acid-binding domain of SARS-CoV-2 spike protein conflicts with the general adaptation and survival pattern observed for all other coronaviruses (CoVs). Unlike RaTG13, for SARS-CoV-2 a recombination apparently occured between the S1/S2 domains of spike protein that enabled furin protease utilization. Interestingly, the clinical strains of SARS-CoV-2 do not have any further recombination that would be in conflict with the recombination models of CoVs. Despite millions of SARS-CoV-2 infections, the spike protein receptor-binding domain (RBD) has not accumulated a high-frequency substitution, differentiating SARS-CoV-2 from other CoVs that have positive selection sites on their RBDs, calling into question the sources of the origin of the apparently optimized angiotensin-converting enzyme 2 (ACE2) binding of SARS-CoV-2. The SARS-CoV-2 host tropism pattern has these major discrepancies compared to other CoVs, raising questions concerning the proximal origin of SARS-CoV-2.

Immune evasion is one of the unique characteristics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) attributed to its ORF8 protein. This protein modulates the adaptive host immunity through down-regulation of MHC-1 (Major Histocompatibility Complex) molecules and innate immune responses by surpassing the host's interferon-mediated antiviral response. To understand the host's immune perspective concerning the ORF8 protein, a comprehensive study of the ORF8 protein and mutations possessed by it have been performed. Chemical and structural properties of ORF8 proteins from different hosts, such as human, bat, and pangolin, suggest that the ORF8 of SARS-CoV-2 is much closer to ORF8 of Bat RaTG13-CoV than to that of Pangolin-CoV. Eighty-seven mutations across unique variants of ORF8 in SARS-CoV-2 can be grouped into four classes based on their predicted effects (Hussain et al., 2021) [1]. Based on the geo-locations and timescale of sample collection, a possible flow of mutations was built. Furthermore, conclusive flows of amalgamation of mutations were found upon sequence similarity analyses and consideration of the amino acid conservation phylogenies. Therefore, this study seeks to highlight the uniqueness of the rapidly evolving SARS-CoV-2 through the ORF8., Graphical abstract Image 1

The recently described ‘gasomediator’ hydrogen sulfide (H2S) has been involved in pain mechanisms, but its effect on pruritus, a sensory modality that similarly to pain acts as a protective mechanism, is poorly known and controversial. The effects of the slow-releasing (GYY4137) and spontaneous H2S donors (Na2S and Lawesson’s reagent, LR) were evaluated in histamine and compound 48/80 (C48/80)-dependent dorsal skin pruritus and inflammation in male BALB/c mice. Animals were intradermally (i.d.) injected with C48/80 (3 μg/site) or histamine (1 μmol/site) alone or co-injected with Na2S, LR or GYY4137 (within the 0.3–100 nmol range). The involvement of endogenous H2S and KATP channel-dependent mechanism were also evaluated. Pruritus was assessed by the number of scratching bouts, whilst skin inflammation was evaluated by the extravascular accumulation of intravenously injected 125I-albumin (plasma extravasation) and myeloperoxidase (MPO) activity (neutrophil recruitment). Histamine or C48/80 significantly evoked itching behavior paralleled by plasma extravasation and increased MPO activity. Na2S and LR significantly ameliorated histamine or C48/80-induced pruritus and inflammation, although these effects were less pronounced or absent with GYY4137. Inhibition of endogenous H2S synthesis increased both Tyrode and C48/80-induced responses in the skin, whereas the blockade of KATP channels by glibenclamide did not. H2S-releasing donors significantly attenuate C48/80-induced mast cell degranulation either in vivo or in vitro. We provide first evidences that H2S donors confer protective effect against histamine-mediated acute pruritus and cutaneous inflammation. These effects can be mediated, at least in part, by stabilizing mast cells, known to contain multiple mediators and to be primary initiators of allergic processes, thus making of H2S donors a potential alternative/complementary therapy for treating inflammatory allergic skin diseases and related pruritus.

This study evaluated cellular and molecular effects of radicicol, a heat shock protein (HSP) inducer, on the regeneration of skeletal muscle injured by crotoxin, the main toxin isolated from Crotalus durissus terrificus venom. Regenerating muscles treated with radicicol had decreased NF-kB activation. Differentiating myoblasts treated with radicicol showed reduced number of NF-kB positive nuclei and increased fusion index. The results suggest that radicicol enhances regeneration of muscle by attenuating NF-kB activation and increasing myogenic differentiation.

Angiotensin-converting enzyme 2 (ACE2) is the cellular receptor for the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that is engendering the severe coronavirus disease 2019 (COVID-19) pandemic. The spike (S) protein receptor-binding domain (RBD) of SARS-CoV-2 binds to the three sub-domains viz. amino acids (aa) 22&ndash, 42, aa 79&ndash, 84, and aa 330&ndash, 393 of ACE2 on human cells to initiate entry. It was reported earlier that the receptor utilization capacity of ACE2 proteins from different species, such as cats, chimpanzees, dogs, and cattle, are different. A comprehensive analysis of ACE2 receptors of nineteen species was carried out in this study, and the findings propose a possible SARS-CoV-2 transmission flow across these nineteen species.

Acute pancreatitis (AP) may be severe and cause hospitalization or death, and the available treatment is insufficient to control pancreatic inflammation and pain. Rutin is a natural flavonoid with the potential to treat AP via anti-inflammatory, antinociceptive, and antioxidant activities.This study investigated the beneficial effects of rutin on experimental AP induced by l-arginine administration in mice.The l-arginine-induced AP model was used in Swiss mice (n = 6-8). Mice submitted to AP induction were treated with rutin (37.5, 75, or 150 mg kgMice submitted to l-arginine injections developed abdominal hyperalgesia and increased serum amylase, lipase, C-reactive protein and IL-6 concentrations; and increased pancreatic myeloperoxidase activity, edema index, MDA, and 3-nitrotyrosine contents. A marked decrease in catalase activity was observed in the pancreas without alterations of superoxide dismutase (SOD) activity compared with the control group. Rutin treatment significantly impaired all the parameters that were altered by AP induction, but increased catalase and SOD activities in the pancreas compared with the vehicle-treated group.Rutin treatment exerted a protective effect on l-arginine-induced AP by mechanisms involving the reduction of oxidative stress, which suggests that this flavonoid has a potential for future approaches designed for the management of AP.

Hydrogen sulfide (H2S) has been highlighted as an endogenous signaling molecule and we have previously found that it can inhibit histamine-mediated itching. Pruritus is the most common symptom of cutaneous diseases and anti-histamines are the usual treatment; however, anti-histamine-resistant pruritus is common in some clinical settings. In this way, the involvement of mediators other than histamine in the context of pruritus requires new therapeutic targets. Considering that the activation of proteinase-activated receptor 2 (PAR-2) is involved in pruritus both in rodents and humans, in this study we investigated the effect of H2S donors on the acute scratching behavior mediated by PAR-2 activation in mice, as well as some of the possible pharmacological mechanisms involved. The intradermal injection of the PAR-2 peptide agonist SLIGRL-NH2 (8-80nmol) caused a dose-dependent scratching that was unaffected by intraperitoneal pre-treatment with the histamine H1 antagonist pyrilamine (30mg/kg). Co-injection of SLIGRL-NH2 (40nmol) with either the slow-release H2S donor GYY4137 (1 and 3nmol) or the spontaneous donor NaHS (1 and 0.3nmol) significantly reduced pruritus. Co-treatment with the KATP channel blocker glibenclamide (200nmol) or the nitric oxide (NO) donor sodium nitroprusside (10nmol) abolished the antipruritic effects of NaHS; however, the specific soluble guanylyl cyclase inhibitor ODQ (30μg) had no significant effects. The transient receptor potential ankyrin type 1 (TRPA1) antagonist HC-030031 (20μg) significantly reduced SLIGRL-NH2-induced pruritus; however pruritus induced by the TRPA1 agonist AITC (1000nmol) was unaffected by NaHS. Based on these data, we conclude that pruritus secondary to PAR-2 activation can be reduced by H2S, which acts through KATP channel opening and involves NO in a cyclic guanosine monophosphate (cGMP)-independent manner. Furthermore, TRPA1 receptors mediate the pruritus induced by activation of PAR-2, but H2S does not interfere with this pathway. These results provide additional support for the development of new therapeutical alternatives, mainly intended for treatment of pruritus in patients unresponsive to anti-histamines.

The origin of the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) virus causing the COVID-19 pandemic has not yet been fully determined. Despite the consensus about the SARS-CoV-2 origin from bat CoV RaTG13, discrepancy to host tropism to other human Coronaviruses exist. SARS-CoV-2 also possesses some differences in its S protein receptor-binding domain, glycan-binding N-terminal domain and the surface of the sialic acid-binding domain. Despite similarities based on cryo-EM and biochemical studies, the SARS-CoV-2 shows higher stability and binding affinity to the ACE2 receptor. The SARS-CoV-2 does not appear to present a mutational “hot spot” as only the D614G mutation has been identified from clinical isolates. As laboratory manipulation is highly unlikely for the origin of SARS-CoV-2, the current possibilities comprise either natural selection in animal host before zoonotic transfer or natural selection in humans following zoonotic transfer. In the former case, despite SARS-CoV-2 and bat RaTG13 showing 96% identity some pangolin Coronaviruses exhibit very high similarity to particularly the receptor-binding domain of SARS-CoV-2. In the latter case, it can be hypothesized that the SARS-CoV-2 genome has adapted during human-to-human transmission and based on available data, the isolated SARS-CoV-2 genomes derive from a common origin. Before the origin of SARS-CoV-2 can be confirmed additional research is required

Animal venoms are used as defense mechanisms or to immobilize and digest prey. In fact, venoms are complex mixtures of enzymatic and non-enzymatic components with specific pathophysiological functions. Peptide toxins isolated from animal venoms target mainly ion channels, membrane receptors and components of the hemostatic system with high selectivity and affinity. The present review shows an up-to-date survey on the pharmacology of snake-venom bioactive components and evaluates their therapeutic perspectives against a wide range of pathophysiological conditions. Snake venoms have also been used as medical tools for thousands of years especially in tradition Chinese medicine. Consequently, snake venoms can be considered as mini-drug libraries in which each drug is pharmacologically active. However, less than 0.01% of these toxins have been identified and characterized. For instance, Captopril® (Enalapril), Integrilin® (Eptifibatide) and Aggrastat® (Tirofiban) are drugs based on snake venoms, which have been approved by the FDA. In addition to these approved drugs, many other snake venom components are now involved in preclinical or clinical trials for a variety of therapeutic applications. These examples show that snake venoms can be a valuable source of new principle components in drug discovery.

A significant number of experimental and clinical studies published in peer-reviewed journals have demonstrated promising pharmacological properties of capsaicin in relieving signs and symptoms of non-communicable diseases (chronic diseases). This chapter provides an overview made from basic and clinical research studies of the potential therapeutic effects of capsaicin, loaded in different application forms, such as solution and cream, on chronic diseases (e.g. arthritis, chronic pain, functional gastrointestinal disorders and cancer). In addition to the anti-inflammatory and analgesic properties of capsaicin largely recognized via, mainly, interaction with the TRPV1, the effects of capsaicin on different cell signalling pathways will be further discussed here. The analgesic, anti-inflammatory or apoptotic effects of capsaicin show promising results in arthritis, neuropathic pain, gastrointestinal disorders or cancer, since evidence demonstrates that the oral or local application of capsaicin reduce inflammation and pain in rheumatoid arthritis, promotes gastric protection against ulcer and induces apoptosis of the tumour cells. Sadly, these results have been paralleled by conflicting studies, which indicate that high concentrations of capsaicin are likely to evoke deleterious effects, thus suggesting that capsaicin activates different pathways at different concentrations in both human and rodent tissues. Thus, to establish effective capsaicin doses for chronic conditions, which can be benefited from capsaicin therapeutic effects, is a real challenge that must be pursued.

In this study we investigated the gene expression of proteins related to myostatin (MSTN) signaling during skeletal muscle longitudinal growth. To promote muscle growth, Wistar male rats were submitted to a stretching protocol for different durations (12, 24, 48, and 96 hours). Following this protocol, soleus weight and length and sarcomere number were determined. In addition, expression levels of the genes that encode MSTN, follistatin isoforms 288 and 315 (FLST288 and FLST315), follistatin-like 3 protein (FLST-L3), growth and differentiation factor-associated protein-1 (GASP-1), activin IIB receptor (ActIIB), and SMAD-7 were determined by real-time polymerase chain reaction. Prolonged stretching increased soleus weight, length, and sarcomere number. In addition, MSTN gene expression was increased at 12-24 hours, followed by a decrease at 96 hours when compared with baseline values. FLST isoforms, FLST-L3, and GASP-1 mRNA levels increased significantly over all time-points. ActIIB gene expression decreased quickly at 12-24 hours. SMAD-7 mRNA levels showed a late increase at 48 hours, which peaked at 96 hours. The gene expression pattern of inhibitory proteins related to MSTN signaling suggests a strong downregulation of this pathway in response to prolonged stretching.

In addition to reducing blood pressure, hydralazine can reduce the production of inflammatory cytokines and reduce the expression of leukocyte adhesion molecules. Differences in leukocyte behavior and leukocyte adhesion molecule expression in spontaneously hypertensive rats (SHR) compared to normotensive rats have been reported. However, whether hydralazine can reduce leukocyte migration in vivo in hypertension and in normotension remains unknown. To address this question, male SHR and Wistar rats were treated for 15 days with hydralazine at a dose of ~3.5 mg/kg or ~14 mg/kg in their drinking water. The numbers of rollers and adherent and migrated cells were determined by direct vital microscopy, and blood pressure was assessed by tail plethysmography. In addition, following treatment with the higher dose, immunohistochemistry was used to measure the expression of intercellular adhesion molecule-1 (ICAM-1), P-selectin, and platelet-endothelial cell adhesion molecule-1 (PECAM-1) in endothelial cells, while flow cytometry was used to evaluate the expression of leukocyte CD18 and L-selectin. Hydralazine reduced leukocyte adherence and migration in SHR either at the higher, that reduced blood pressure levels, or lower dose, which did not reduce it. Reduced ICAM-1 expression might be involved in the reduced migration observed in SHR. In Wistar rats, only at the higher dose hydralazine reduced blood pressure levels and leukocyte migration. Reduced P-selectin expression might be involved. We therefore conclude that hydralazine reduces leukocyte migration by different mechanisms in SHR and Wistar rats, specifically by reducing ICAM-1 expression in the former and P-selectin expression in the latter.

In order to gain insight into intracellular mechanisms involved in longitudinal growth of skeletal muscle, we determined gene expression of ubiquitin-ligases (MAFbx/atrogin-1, E3 alpha, and MuRF-1) and deubiquitinating enzymes (UBP45, UBP69, and USP28) at different time-points (24, 48, and 96 h) of continuous stretch of the soleus and tibialis anterior (TA) muscles. In the soleus, real-time polymerase chain reaction (PCR) showed that MAFbx/atrogin-1, E3 alpha, and MuRF-1 gene expression was downregulated, peaking at 24-48 h. Gene expression of all deubiquitinating enzymes increased with continuous stretch of soleus. In the TA, gene expression of the ubiquitin-ligases MAFbx/atrogin-1 and MuRF-1 was elevated, whereas expression of UBP45 and UBP69 was downregulated. Western blot analysis showed that the overall ubiquitination level decreased in the soleus and increased in the TA during stretch. These results suggest that ubiquitin-ligases and deubiquitinating enzymes are involved in longitudinal growth induced by continuous muscle stretch.

The aim of the present study was to investigate the effect of different resistance-training regimens (S or P) on the expression of genes related to the MSTN signaling pathway in physically-active men. 29 male subjects with at least 2 years of experience in strength training were assigned to either a strength-training group (S; n=11) or a power-training group (P; n=11). The control group (C; n=7) was composed of healthy physically-active males. The S and the P groups performed high- and low-intensity squats, respectively, 3 times per week, for 8 weeks. Muscle biopsies from the vastus lateralis muscle were collected before and after the training period. No change was observed in MSTN, ACTIIB, GASP-1 and FOXO-3 A gene expression after the training period. A similar increase in the gene expression of the inhibitory proteins of the MSTN signaling pathway, FLST (S: 4.2 fold induction and P: 3.7 fold induction, p0.01) and FL-3 (S: 5.6 fold induction and P: 5.6 fold induction, p0.01), was detected after the training period. SMAD-7 gene expression was similarly augmented after both training protocols (S: 2.5 fold induction; P: 2.8 fold induction; p0.05). In conclusion, the resistance-training regimens (S and P) activated the expression of inhibitors of the MSTN signaling pathway in a similar manner.

Cyclosporin-A (CsA) is an immunosuppressive drug that acts as an inhibitor of calcineurin, a calcium phosphatase that has been suggested to play a role in skeletal muscle hypertrophy. The aim of the present study was to determine the effect of CsA administration (25 mg kg -1 day -1 ) on skeletal muscle mass and phenotype during disuse and recovery. Male Wistar rats received vehicle (N = 8) or CsA (N = 8) during hind limb immobilization (N = 8) and recovery (N = 8). Muscle weight (dry/wet) and cross-sectional area were evaluated to verify the effect of CsA treatment on muscle mass. Muscle phenotype was assessed by histochemistry of myosin ATPase. CsA administration during immobilization and recovery did not change muscle/body weight ratio in the soleus (SOL) or plantaris (PL). Regarding muscle phenotype, we observed a consistent slow-to-fast shift in all experimental groups (immobilized only, receiving CsA only, and immobilized receiving CsA) as compared to control in both SOL and PL (P < 0.05). During recovery, no difference was observed in SOL or PL fiber type composition between the experimental recovered group and recovered group receiving CsA compared to their respective controls. Considering the muscle/body weight ratio, CsA administration does not maximize muscle mass loss induced by immobilization. Our results also indicate that CsA fails to block skeletal muscle regrowth after disuse. The present data suggest that calcineurin inhibition by CsA modulates muscle phenotype rather than muscle mass.

Triiodothyronine (T3) is known to play a key role in the function of several tissues/organs via the thyroid hormone receptor isoforms alpha (TRalpha) and beta (TRbeta). We have investigated the effects of GC-24, a novel synthetic TRbeta-selective compound, on skeletal muscle fiber-type determination, cross-sectional area, and gene expression in rat skeletal muscles. For fiber typing, cross sections of soleus and extensor digitorum longus (EDL) muscles were stained for myosin ATPase activity at various pHs. Serum T3, T4, and cholesterol levels were also determined. Analysis of highly T3-responsive genes, viz., myosin heavy chain IIa (MHCIIa) and sarcoendoplasmic reticulum adenosine triphosphatase (SERCA1), was performed by quantitative real-time polymerase chain reaction. Equimolar doses of T3 and GC-24 had a similar cholesterol-lowering effect. T3, but not GC-24, decreased fiber type I and increased fiber type II abundance in soleus and EDL muscles. Conversely, in EDL, both T3 and GC-24 decreased the mean cross-sectional area of type I fibers. MHCIIa gene expression was reduced (approximately 50%) by T3 and unchanged by GC-24. SERCA1 gene expression was strongly induced by T3 (approximately 20-fold) and mildly induced by GC-24 (approximately two-fold). These results show that GC-24 does not significantly alter the composition of skeletal muscle fiber type and further strengthens the putative use of GC compounds as therapeutic agents.

This work was undertaken to provide further insights into the expression of tropism-related genes in regenerating skeletal muscle of adult rats treated with cyclosporin-A (CsA), a calcineurin inhibitor. Rats were treated with CsA for 5 days and, on the 6th day, were submitted to cryolesion of the soleus muscles. CsA treatment continued for 1, 10, and 21 days after cryolesion. Muscles were removed, frozen, and stored in liquid nitrogen. Body and muscle weights, histological sections stained with toluidine blue, and gene expression of the regeneration molecular markers, viz., desmin and neonatal myosin heavy chain, were assessed to confirm that cryolesion and CsA treatment were effective during the allowed regeneration time. Quantitative reverse transcription/polymerase chain reaction demonstrated that myostatin gene expression was not altered by either cryolesion or CsA treatment combined with cryolesion. Calpain-3 gene expression decreased at 1 day after cryolesion and also following CsA treatment combined with cryolesion. However, calpain-3 gene expression was strongly up-regulated (approximately five-fold) 10 days after cryolesion and returned to control levels at day 21. CsA treatment blocked calpain-3 gene expression rise induced by 10 days of cryolesion. Atrogin-1 gene expression was decreased at 1 day after cryolesion and following cryolesion combined with CsA treatment, returning to control levels at day 10. These results suggest that (1) calpain-3 has a differential role in the early and late stages of regeneration in a calcineurin-dependent manner, and (2) atrogin-1 is involved in the early stages of regeneration independently of calcineurin.

Thyrotoxicosis is frequently associated with increased bone turnover and decreased bone mass. To investigate the role of thyroid hormone receptor-β (TRβ) in mediating the osteopenic effects of triiodothyronine (T3), female adult rats were treated daily (64 days) with GC-1 (1.5 μg/100 g body wt), a TRβ-selective thyromimetic compound. Bone mass was studied by dual-energy X-ray absorptiometry of several skeletal sites and histomorphometry of distal femur, and the results were compared with T3-treated (3 μg/100 g body wt) or control animals. As expected, treatment with T3significantly reduced bone mineral density (BMD) in the lumbar vertebrae (L2-L5), femur, and tibia by 10–15%. In contrast, GC-1 treatment did not affect the BMD in any of the skeletal sites studied. The efficacy of GC-1 treatment was verified by a reduction in serum TSH (–52% vs. control, P < 0.05) and cholesterol (–21% vs. control, P < 0.05). The histomorphometric analysis of the distal femur indicated that T3but not GC-1 treatment reduced the trabecular volume, thickness, and number. We conclude that chronic, selective activation of the TRβ isoform does not result in bone loss typical of T3-induced thyrotoxicosis, suggesting that the TRβ isoform is not critical in this process. In addition, our findings suggest that the development of TR-selective T3analogs that spare bone mass represents a significant improvement toward long-term TSH-suppressive therapy.

Concurrent training (CT) seems to impair training-induced muscle hypertrophy. This study compared the effects of CT, strength training (ST) and interval training (IT) on the muscle fiber cross-sectional area (CSA) response, and on the expression of selected genes involved in the myostatin (MSTN) signaling mRNA levels. Thirty-seven physically active men were randomly divided into 4 groups: CT (n = 11), ST (n = 11), IT (n = 8), and control group (C) (n = 7) and underwent an 8-week training period. Vastus lateralis biopsy muscle samples were obtained at baseline and 48 hours after the last training session. Muscle fiber CSA, selected genes expression, and maximum dynamic ST (1 repetition maximum) were evaluated before and after training. Type IIa and type I muscle fiber CSA increased from pre- to posttest only in the ST group (17.08 and 17.9%, respectively). The SMAD-7 gene expression significantly increased at the posttest in the ST (53.9%) and CT groups (39.3%). The MSTN and its regulatory genes ActIIb, FLST-3, FOXO-3a, and GASP-1 mRNA levels remained unchanged across time and groups. One repetition maximum increased from pre- to posttest in both the ST and CT groups (ST = 18.5%; CT = 17.6%). Our findings are suggestive that MSTN and their regulatory genes at transcript level cannot differentiate muscle fiber CSA responses between CT and ST regimens in humans.

Although it is well known that the thyroid hormone (T3) is an important positive regulator of cardiac function over a short term and that it also promotes deleterious effects over a long term, the molecular mechanisms for such effects are not yet well understood. Because most alterations in cardiac function are associated with changes in sarcomeric machinery, the present work was undertaken to find novel sarcomeric hot spots driven by T3 in the heart. A microarray analysis indicated that the M-band is a major hot spot, and the structural sarcomeric gene coding for the M-protein is severely down-regulated by T3. Real-time quantitative PCR-based measurements confirmed that T3 (1, 5, 50, and 100 physiological doses for 2 days) sharply decreased the M-protein gene and protein expression in vivo in a dose-dependent manner. Furthermore, the M-protein gene expression was elevated 3.4-fold in hypothyroid rats. Accordingly, T3 was able to rapidly and strongly reduce the M-protein gene expression in neonatal cardiomyocytes. Deletions at the M-protein promoter and bioinformatics approach suggested an area responsive to T3, which was confirmed by chromatin immunoprecipitation assay. Functional assays in cultured neonatal cardiomyocytes revealed that depletion of M-protein (by small interfering RNA) drives a severe decrease in speed of contraction. Interestingly, mRNA and protein levels of other M-band components, myomesin and embryonic-heart myomesin, were not altered by T3. We concluded that the M-protein expression is strongly and rapidly repressed by T3 in cardiomyocytes, which represents an important aspect for the basis of T3-dependent sarcomeric deleterious effects in the heart.

Purpose: The aim of the study was to determine whether the similar muscle strength and hypertrophy responses observed after either low-intensity resistance exercise associated with moderate blood flow restriction or high-intensity resistance exercise are associated with similar changes in messenger RNA (mRNA) expression of selected genes involved in myostatin (MSTN) signaling. Methods: Twenty-nine physically active male subjects were divided into three groups: low-intensity (20% one-repetition maximum (1RM)) resistance training (LI) (n = 10), low-intensity resistance exercise associated with moderate blood flow restriction (LIR) (n = 10), and high-intensity (80% 1RM) resistance exercise (HI) (n = 9). All of the groups underwent an 8-wk training program. Maximal dynamic knee extension strength (1RM), quadriceps cross-sectional area (CSA), MSTN, follistatin-like related genes (follistatin (FLST), follistatin-like 3 (FLST-3)), activin IIb, growth and differentiation factor�associated serum protein 1 (GASP-1), and MAD-related protein (SMAD-7) mRNA gene expression were assessed before and after training. Results: Knee extension 1RM significantly increased in all groups (LI = 20.7%, LIR = 40.1%, and HI = 36.2%). CSA increased in both the LIR and HI groups (6.3% and 6.1%, respectively). MSTN mRNA expression decreased in the LIR and HI groups (45% and 41%, respectively). There were no significant changes in activin IIb (P > 0.05). FLST and FLST-3 mRNA expression increased in all groups from pre- to posttest (P < 0.001). FLST-3 expression was significantly greater in the HI when compared with the LIR and LI groups at posttest (P = 0.024 and P = 0.018, respectively). GASP-1 and SMAD-7 gene expression significantly increased in both the LIR and HI groups. Conclusions: We concluded that LIR was able to induce gains in 1RM and quadriceps CSA similar to those observed after traditional HI. These responses may be related to the concomitant decrease in MSTN and increase in FLST isoforms, GASP-1, and SMAD-7 mRNA gene expression.

The Rio Grande do Sul state, in Southern Brazil, is one of the foci of human cystic echinococcosis (CE). The sheep strain (G1) of Echinococcus granulosus and Echinococcus ortleppi (also known as cattle strain G5) have been reported before to infect livestock. However, up to the present, no molecular data are available on isolates of the E. granulosus complex from humans and dogs. The present study analyzed hydatid cysts from 6 CE patients and adult worms from 12 dogs. Sequencing of the mitochondrial cox1 and 12S rRNA genes detected the E. granulosus G1 genotype from four human cases, the G3 genotype (or buffalo strain) from one human case and E. ortleppi from another human case, respectively. Ten of the twelve dogs were found infected with the G1 genotype, and one dog each harbored worms of the G3 genotype and E. ortleppi. Obvious morphological differences were recognized between the G1 and E. ortleppi adult worms from dogs in this region. The buffalo strain (G3) is for the first time reported from South America.

In this study we investigated the gene expression of proteins related to myostatin (MSTN) signaling during skeletal muscle longitudinal growth. To promote muscle growth, Wistar male rats were submitted to a stretching protocol for different durations (12, 24, 48, and 96 hours). Following this protocol, soleus weight and length and sarcomere number were determined. In addition, expression levels of the genes that encode MSTN, follistatin isoforms 288 and 315 (FLST288 and FLST315), follistatin-like 3 protein (FLST-L3), growth and differentiation factor-associated protein-1 (GASP-1), activin IIB receptor (ActIIB), and SMAD-7 were determined by real-time polymerase chain reaction. Prolonged stretching increased soleus weight, length, and sarcomere number. In addition, MSTN gene expression was increased at 12-24 hours, followed by a decrease at 96 hours when compared with baseline values. FLST isoforms, FLST-L3, and GASP-1 mRNA levels increased significantly over all time-points. ActIIB gene expression decreased quickly at 12-24 hours. SMAD-7 mRNA levels showed a late increase at 48 hours, which peaked at 96 hours. The gene expression pattern of inhibitory proteins related to MSTN signaling suggests a strong downregulation of this pathway in response to prolonged stretching.

Gomes AR, Soares AG, Peviani S, Nascimento RB, Moriscot AS, Salvini TF. The effect of 30 minutes of passive stretch of the rat soleus muscle on the myogenic differentiation, myostatin, and atrogin-1 gene expressions. Objective To evaluate the effect of passive stretch, applied for 30 minutes to the rat soleus muscle, on the myogenic differentiation (myoD), myostatin, and atrogin-1 gene expressions. Design Case-controlled study. Setting University laboratory. Animals Fifty 12-week-old male Wistar rats. Interventions Six groups of animals were given a single stretch bout and were evaluated immediately and 8, 24, 48, 72, and 168 hours later. Another 3 groups were evaluated immediately after 2, 3, and 7 stretches. An intact control group was also analyzed. Main Outcome Measures The messenger ribonucleic acid (mRNA) levels of myoD, myostatin, and atrogin-1 were assessed by real-time polymerase chain reaction. Results Twenty-four hours after a single session of stretch only, the myoD mRNA levels had increased compared with the control group, whereas an increase in the atrogin-1 expression was observed after 2, 3, and 7 stretches. Conclusions A single session of passive stretch increased the myoD gene expression, a factor related to muscle growth. Interestingly, daily stretches increased the atrogin-1 gene expression, a gene primarily associated with muscle atrophy. The results indicated that gene expression was responsive to the number of stretch sessions.