Microtubule and auditory function - an underestimated connection.

The organ of Corti, located in the cochlea within the inner ear is the receptor organ for hearing. It converts auditory signals into neuronal action potentials that are transmitted to the brain for further processing. The mature organ of Corti consists of a variety of highly differentiated sensory cells that fulfil unique tasks in the processing of auditory signals. The actin and microtubule cytoskeleton play essential function in hearing, however so far, more attention has been paid to the role of actin. Microtubules play important roles in maintaining cellular structure and intracellular transport in virtually all eukaryotic cells. Their functions are controlled by interactions with a large variety of microtubule-associated proteins (MAPs) and molecular motors. Current advances show that tubulin posttranslational modifications, as well as tubulin isotypes could play key roles in modulating microtubule properties and functions in cells. These mechanisms could have various effects on the stability and functions of microtubules in the highly specialised cells of the cochlea. Here, we review the current understanding of the role of microtubule-regulating mechanisms in the function of the cochlea and their implications for hearing, which highlights the importance of microtubules in the field of hearing research.

A family of protein-deglutamylating enzymes associated with neurodegeneration.

Polyglutamylation is a posttranslational modification that generates glutamate side chains on tubulins and other proteins. Although this modification has been shown to be reversible, little is known about the enzymes catalyzing deglutamylation. Here we describe the enzymatic mechanism of protein deglutamylation by members of the cytosolic carboxypeptidase (CCP) family. Three enzymes (CCP1, CCP4, and CCP6) catalyze the shortening of polyglutamate chains and a fourth (CCP5) specifically removes the branching point glutamates. In addition, CCP1, CCP4, and CCP6 also remove gene-encoded glutamates from the carboxyl termini of proteins. Accordingly, we show that these enzymes convert detyrosinated tubulin into Δ2-tubulin and also modify other substrates, including myosin light chain kinase 1. We further analyze Purkinje cell degeneration (pcd) mice that lack functional CCP1 and show that microtubule hyperglutamylation is directly linked to neurodegeneration. Taken together, our results reveal that controlling the length of the polyglutamate side chains on tubulin is critical for neuronal survival.

Tryptophan Modulation in Cancer-Associated Cachexia Mouse Models.

Cancer cachexia is a multifactorial syndrome that interferes with treatment and reduces the quality of life and survival of patients. Currently, there is no effective treatment or biomarkers, and pathophysiology is not clear. Our group reported alterations on tryptophan metabolites in cachectic patients, so we aim to investigate the role of tryptophan using two cancer-associated cachexia syngeneic murine models, melanoma B16F10, and pancreatic adenocarcinoma that is KPC-based. Injected mice showed signs of cancer-associated cachexia as reduction in body weight and raised spleen weight, MCP1, and carbonilated proteins in plasma. CRP and Myostatin also increased in B16F10 mice. Skeletal muscle showed a decrease in quadriceps weight and cross-sectional area (especially in B16F10). Higher expression of atrophy genes, mainly Atrogin1, was also observed. Plasmatic tryptophan levels in B16F10 tumor-bearing mice decreased even at early steps of tumorigenesis. In KPC-injected mice, tryptophan fluctuated but were also reduced and in cachectic patients were significantly lower. Treatment with 1-methyl-tryptophan, an inhibitor of tryptophan degradation, in the murine models resulted in the restoration of plasmatic tryptophan levels and an improvement on splenomegaly and carbonilated proteins levels, while changes in plasmatic inflammatory markers were mild. After the treatment, CCR2 expression in monocytes diminished and lymphocytes, Tregs, and CD8+, were activated (seen by increased in CD127 and CD25 expression, respectively). These immune cell changes pointed to an improvement in systemic inflammation. While treatment with 1-MT did not show benefits in terms of muscle wasting and atrophy in our experimental setting, muscle functionality was not affected and central nuclei fibers appeared, being a feature of regeneration. Therefore, tryptophan metabolism pathway is a promising target for inflammation modulation in cancer-associated cachexia.

Implementation of a peroral endoscopic myotomy program.

INTRODUCTION: the aim of our study was to develop a peroral endoscopic myotomy (POEM) program in our Unit following a two-step sequence: training on animal models and supervision by an experienced endoscopist during the first human cases. METHODS: a single endoscopist experienced in advanced endoscopy was trained in POEM. After observing POEM in referral centers, training was implemented on swine models (preclinical phase). Technical aspects and adverse events were prospectively recorded. A first subset of cases (group A) was compared to a second one (group B) to assess our progression. Finally, POEM was implemented in humans under the supervision of an experienced endoscopist (clinical phase). The outcomes and adverse events were prospectively recorded. RESULTS: during the preclinical phase, 15 POEM procedures were performed on live pigs. Severe adverse events (AE) were less frequent in group B than in group A (12 % vs 57 %, p = 0.07). After nine cases, a plateau of adverse events was reached. During the clinical phase, eleven POEM procedures were performed in patients under expert supervision. Technical and clinical (Eckardt score ≤ 3) success were 100 % and 91 %, respectively (follow-up 3-21 months). In two cases, intervention of an experienced endoscopist was required (cases 2 and 3) because of a difficult orientation at the esophagogastric junction. One mild pneumoperitoneum occurred, with no severe adverse events reported. CONCLUSIONS: training in animal models and supervision by an experienced endoscopist during the first cases could provide the necessary skills to perform POEM safely and effectively.

Alterations in the balance of tubulin glycylation and glutamylation in photoreceptors leads to retinal degeneration.

Tubulin is subject to a wide variety of posttranslational modifications, which, as part of the tubulin code, are involved in the regulation of microtubule functions. Glycylation has so far predominantly been found in motile cilia and flagella, and absence of this modification leads to ciliary disassembly. Here, we demonstrate that the correct functioning of connecting cilia of photoreceptors, which are non-motile sensory cilia, is also dependent on glycylation. In contrast to many other tissues, only one glycylase, TTLL3, is expressed in retina. Ttll3-/- mice lack glycylation in photoreceptors, which results in shortening of connecting cilia and slow retinal degeneration. Moreover, absence of glycylation results in increased levels of tubulin glutamylation in photoreceptors, and inversely, the hyperglutamylation observed in the Purkinje cell degeneration (pcd) mouse abolishes glycylation. This suggests that both posttranslational modifications compete for modification sites, and that unbalancing the glutamylation-glycylation equilibrium on axonemes of connecting cilia, regardless of the enzymatic mechanism, invariably leads to retinal degeneration.

Biodegradable polydioxanone stents: histologic and structural effects in an experimental model of tracheal stenosis.

OBJECTIVES: To evaluate the biologic impact of polydioxanone (PDO) stenting in an animal model of inflammatory tracheal stenosis (TS). Additionally, to compare these results with those obtained in the same model without a stent and after placing one PDO stent in a healthy trachea. METHODS: 40 adult NZ rabbits were distributed into 3 groups: Group A, 8 animals with a healthy trachea and a PDO stent; group B, 17 rabbits with a TS and no stent; and group C, 15 animals with TS and a PDO stent. Histopathological studies included Masson's trichrome staining for submucosal fibrosis and Safranin O to assess structural integrity of cartilage. Morphometric analyses were performed in the 3 groups. RESULTS: Stent placement was successful in every case. Histological studies did not show a significant increase in tracheal wall collagen area and cartilage structure was not modified in those rabbits with a PDO stent, even in a TS scenario. Stent implantation permitted recovery of normal tracheal lumen levels in the TS model. CONCLUSIONS: PDO stenting in the normal trachea and in a model of TS neither caused increase in the collagen matrix nor modification of the cartilaginous support. Additionally, radial force exhibited by PDO stents was effective in restoring normal tracheal lumen when placed in a stenotic lesion. These findings suggest that they may be safe and useful in the setting of an acquired TS.

Histological and structural effects of biodegradable polydioxanone stents in the rabbit trachea.

OBJECTIVES: The aim of this study was to evaluate the potential biologic effects caused by the successive placement of biodegradable polydioxanone (PDO) stents in the rabbit trachea. PDO stents could eventually induce a fibroproliferative reaction in the submucosa that could be beneficial in the treatment of malacia due to an increase in its consistency without impairing the tracheal lumen. METHODS: Sixteen adult NZ rabbits were distributed into 3 groups with different survival times according to the number of stents placed: 1 stent (14 weeks), 2 stents (28 weeks) and 3 stents (42 weeks). Stent insertion was performed endoscopically in the cervical trachea of the animal. Histopathological studies included Masson's trichrome staining for submucosal fibrosis and Safranin O to assess the structural integrity of cartilage. Potential inflammatory changes were analysed by means of immunohistochemistry determining the number of CD45-positive cells. RESULTS: Stent placement was successful in every case. Histological studies did not show a statistically significant increase in tracheal wall collagen area and cartilage structure was not modified in those rabbits with 1 or more PDO stents inserted compared to non-stented tracheal sections. Furthermore, no statistically significant changes in the number of CD45+ cells were observed in stented tracheal segments compared to normal tracheal tissues. CONCLUSIONS: According to our data, successive PDO stenting caused mild inflammatory changes in the tracheal wall and no increase in the collagen matrix, and the cartilaginous support was not modified during a long follow-up period (up to 42 weeks). These findings suggest that they may be safe and show good biocompatibility in the long term.

Hepcidin treatment in Hfe-/- mice diminishes plasma iron without affecting erythropoiesis.

BACKGROUND: Iron is essential for mammalian metabolism and its cellular concentration is controlled by regulating its acquisition and storage. Haemochromatosis is a condition involving iron overload that is characterised by increased duodenal iron absorption and a progressive accumulation of iron in vital organs. Hepcidin is the main hormone that regulates iron homoestasis and it is secreted by the liver. MATERIALS AND METHODS: We have studied how extended hepcidin administration affects the iron load status, plasma and tissue iron concentration, erythropoiesis and the expression of proteins involved on iron homeostasis in haemochromatotic (Hfe(-/-)) and wild-type mice. RESULTS: Hepcidin reverted the high plasma iron concentrations in Hfe(-/-) mice to normal values. The high concentration of hepatic iron was not altered in the liver of these Hfe(-/-) mice. Hepcidin administration did not disturb erythropoiesis in either Hfe(-/-) or wild-type mice and likewise, hepcidin did not modify the expression of any protein analysed in the liver, duodenum or spleen of Hfe(-/-) and wild-type mice. These data confirm that hepcidin administration diminishes plasma iron concentrations. CONCLUSION: Treatment with sustained doses of hepcidin diminishes plasma iron concentrations in Hfe(-/-) mice.

In vivo effect of an luteinizing hormone-releasing hormone analog on vascular endothelial growth factor and epidermal growth factor receptor expression in mammary tumors.

BACKGROUND: The hypothalamic luteinizing hormone-releasing hormone (LHRH) is well known for its role in the control of pituitary gonadotropin secretion and it has demonstrated a direct antiproliferative effect on some cancer cell lines of LHRH and its synthetic analogs. The study was designed to assess whether administration of the LHRH analog (goserelin) has any effect on the expression of the vascular endothelial growth factor (VEGF) and the epidermal growth factor receptor (EGFR) in rats with N-nitroso-N-methylurea (NMU)-induced-mammary tumors " in vivo". MATERIALS AND METHODS: The animals with tumors were assessed after acute or chronic treatment with goserelin, and in all the animals VEGF and EGFR expression was examined both in plasma and tumor homogenates by enzyme immunoassay. RESULTS: The basal plasma values of VEGF were lower in the healthy control group than in rats with NMU-induced tumors ( P = 0.025). Following acute treatment with goserelin, VEGF expression in plasma increased above basal levels after 60 min ( P = 0.05) and dropped during chronic treatment. Likewise, in the tumor homogenate the mean VEGF expression was higher at 60 min post-goserelin administration than the basal levels, although VEGF expression then diminished at 90 min. Plasma EGFR expression was higher in rats with NMU-induced tumors than in healthy controls ( P Conclusions: The results allow us to conclude that goserelin may exert a short-term stimulatory effect on the release of VEGF, as well as a long-term inhibitory effect on VEGF but not EGFR expression.

Syrian Hamster as an Advanced Experimental Model for Visceral Leishmaniasis.

Animal models are needed along the development and evaluation of potential chemotherapeutic agents against leishmaniasis. Infections of Syrian hamsters with Leishmania species causing visceral leishmaniasis (VL) closely mimic the disease in the natural hosts, including target organs, lesions, and clinical course. Therefore, despite some shortcomings (e.g., genetic background, price, and scarcity of reagents), it is probably the best laboratory rodent model of VL. However, handling of hamsters can be technically challenging because of their particular anatomy. Here, we describe in detail four different routes to establish an experimental VL in the hamster model using Leishmania promastigotes and amastigotes. Each route requires various manipulations and has different benefits and drawbacks. Choice of the most suitable route should be made by the researcher in accordance with the specific plan and purpose of the study.

Bone marrow transplantation into hemochromatotic mice decreases hepatic and duodenal iron overload.

Human hereditary hemochromatosis is a disorder of iron homeostasis characterized by increased absorption of iron and its deposition in parenchymal organs. The maintenance of iron homeostasis is regulated by molecules involved in the absorption, transport, storage and redox of iron. The potential of hematopoietic stem cell therapy for liver diseases has been studied in some experimental animal models. Our objective was to evaluate the effect of bone marrow transplantation from wild type mice on the status of iron overload in Hfe knockout hemochromatotic mice (Hfe(-/-)). The transplanted cells were detected in the liver (11% of the total cells) and characterized as hepatocytes and myofibroblasts. They were also detected in the duodenum and characterized as myofibroblasts. The iron content in the Hfe(-/-) mice descended 2.9-fold in the liver and 2.4-fold in the duodenum 6 months after transplantation. Non-significant changes of relative mRNA abundance of genes of iron metabolism were observed in the liver and duodenum of Hfe(-/-) transplanted mice. At 6 months after transplantation the proportion of Hfe mRNA in Hfe(-/-) mice reached 3.8% of the levels in wild type mice in the liver and 1.6% in the duodenum. In conclusion, adult stem cells from bone marrow transplant were able to differentiate into hepatocytes and myofibroblasts in hemochromatotic mice. Bone marrow transplantation assisted in reducing the iron overload in this murine model of hemochromatosis. These findings might contribute to the knowledge of pathways involved in the regulatory system of iron homeostasis.

Omentectomy Prevents Metabolic Syndrome By Reducing Appetite and Body Weight In A Diet-Induced Obesity Rat Model.

Visceral fat deposition is associated with impairment of glucose and lipid metabolism while leptin levels are frequently related to subcutaneous fat area. At present, there is considerable controversy regarding the role of visceral adipose tissue accumulation in the development of metabolic syndrome (MS). Here we show the effects of omentectomy on the liver and MS in a diet induced obesity rat model. Our results reveal that undergoing omentectomy previously the establishment of the diet-induced-obesity reduced significantly body weight gain and avoid the development of MS, including non-alcoholic fatty liver disease. Intriguingly, the significantly lower body weight gain was due to decreased food intake. Omentum drives obesity progression through leptin resistance mediated by C-reactive protein, Interleucin (IL)-6 and high lipolysis activity. Omentum removal reversed immediately the increased plasma levels of CRP and IL-6 and gradually food intake, weight gain, and features of MS in diet-induced-obesity. Omentectomy caused no changes in normal-weigh-rats. This report displays causal mechanism by which omentum promotes obesity and propose omentectomy as a promising procedure in MS prevention.

Role of NAD binding and catalytic residues in the C-terminal binding protein corepressor.

CtBP corepressor proteins potentiate the activity of many metazoan transcriptional repressors. These proteins are homologous to prokaryotic D-2-hydroxyacid dehydrogenases, possessing an NAD/NADH binding fold and conserved active site residues. When expressed in Drosophila, a catalytic site mutant retains biological activity, however, we find that an NAD binding mutant lacks biological activity. The NAD mutant, similar to a dimerization mutant, is expressed at low levels, indicating that binding of NAD/NADH may affect CtBP stability. These data support the idea that the ancestral dehydrogenase activity is not required for CtBP function, and NAD binding may play a regulatory, rather than catalytic, role.

CtBP contributes quantitatively to Knirps repression activity in an NAD binding-dependent manner.

Transcriptional repressors often employ multiple activities, but the molecular mechanisms and physiological relevance of this functional diversity remain obscure. The Drosophila melanogaster Knirps repressor uses CtBP corepressor-dependent and -independent pathways. To separately analyze the components of Knirps repression activity, we elucidated the specific repression properties of CtBP and of Knirps subdomains. Like Knirps, CtBP represses adjacent transcriptional activators; but unlike Knirps, CtBP is unable to repress basal promoter elements. We determined that the ability of CtBP to recapitulate only a subset of Knirps activities is due to a quantitative, rather than qualitative, deficiency in repression activity. The CtBP-dependent portion of Knirps synergizes with the CtBP-independent repression activity to potently repress promoter elements from enhancer- or promoter-proximal positions. This result indicates that multiple repression activities are combined to exceed critical thresholds on target genes. CtBP mutant proteins unable to bind NAD fail to interact with DNA-bound factors. We show that DNA-binding Gal4-CtBP fusion proteins also require NAD binding for activity, indicating that NAD plays a role in repression at a step subsequent to CtBP recruitment to the promoter.

TGF-ß antagonist attenuates fibrosis but not luminal narrowing in experimental tracheal stenosis.

OBJECTIVE: Acquired tracheal stenosis (ATS) is an unusual disease often secondary to prolonged mechanical trauma. Acquired tracheal stenosis pathogenesis involves inflammation and subsequent fibrosis with narrowing of the tracheal lumen. Transforming growth factor-ß1 (TGF-ß) represents a pivotal factor in most fibrotic processes, and therefore a potential target in this context. The aim of this study is to analyze the role of TGF-ß as a target for anti-fibrotic interventions in tracheal stenosis. METHODS: Human stenotic tracheobronchial tissues from patients with benign airway stenosis and normal controls from pneumonectomy specimens were analyzed. Tracheal stenosis was induced in adult NZ rabbits by a circumferential thermal injury to the mucosa during open surgery and re-anastomosis. Rabbits were treated postoperatively with a peritracheal collagen sponge containing a TGF-ß peptide antagonist (p17) or vehicle. Fibrosis was determined by Masson's trichrome staining, and smooth muscle cell α-actin+ (α-SMA+ Confirm accuracy.) myofibroblasts, connective tissue growth factor (CTGF), and p-Smad2/3 expression by immunohistochemistry. RESULTS: Human and rabbit stenotic tissues showed extensive submucosal fibrosis, characterized by significantly increased α-SMA+ myofibroblasts and CTGF expression. In human stenotic lesions, increased p-Smad2/3+ nuclei were also observed. p17 treatment significantly reduced the fibrotic thickness, as well as the density of α-SMA+ myofibroblasts and CTGF+ cells in rabbit stenotic lesions, but failed to improve the luminal area. CONCLUSION: ATS is characterized by a TGF-ß dependent fibrotic process, but reduction of the fibrotic component by TGF-ß1 antagonist therapy was not sufficient to improve tracheal narrowing, suggesting that fibrosis may not be the main contributor to luminal stenosis. LEVEL OF EVIDENCE: NA. Laryngoscope, 127:561-567, 2017.

Vectorization of ultrasound-responsive nanoparticles in placental mesenchymal stem cells for cancer therapy.

A new platform constituted by engineered responsive nanoparticles transported by human mesenchymal stem cells is here presented as a proof of concept. Ultrasound-responsive mesoporous silica nanoparticles are coated with polyethylenimine to favor their effective uptake by decidua-derived mesenchymal stem cells. The responsive-release ability of the designed nanoparticles is confirmed, both in vial and in vivo. In addition, this capability is maintained inside the cells used as carriers. The migration capacity of the nanoparticle-cell platform towards mammary tumors is assessed in vitro. The efficacy of this platform for anticancer therapy is shown against mammary tumor cells by inducing the release of doxorubicin only when the cell vehicles are exposed to ultrasound.

NADPH oxidase is implicated in the pathogenesis of oxidative phosphorylation dysfunction in mice fed a high-fat diet.

The aim of this study was to evaluate the role of NADPH oxidase (NADPHox) in the pathogenesis of oxidative phosphorylation (OXPHOS) dysfunction as found in mice fed a high-fat diet (HFD). C57BL/6J mice were distributed in four groups: WT/SCD: six wild-type (WT) mice fed a standard chow diet (SCD); WT/HFD, six WT mice fed a HFD; NOX2(-/-)/SCD, six NADPHox-deficient mice on a SCD; (4) NOX2(-/-)/HFD, six NADPHox-deficient mice on a HFD. After 32 weeks, we studied the liver for: histology; OXPHOS complex activity; fully assembled OXPHOS complexes and their subunits; gene expression of OXPHOS subunits; oxidative and nitrosative stress; and oxidative DNA damage. In the liver of WT/HFD mice, we found a significant decreased in the activity of all OXPHOS complexes, in fully assembled complexes, in the amount of OXPHOS subunits, and in gene expression of mitochondrial DNA-encoded subunits. 8-hydroxy-2'-deoxyguanosine was only increased in mitochondrial DNA. The liver of NOX(-/-)/HFD mice showed mild steatosis but no non-alcoholic steatohepatitis (NASH) lesions were found. OXPHOS activity, OXPHOS subunits, and assembly of subunits into OXPHOS complexes were normal in these mice. We conclude that this study shows that NADPH deficiency protects mice from developing OXPHOS dysfunction and NASH caused by a HFD.

Blockade of cell adhesion molecules enhances cell engraftment in a murine model of liver cell transplantation.

AIM: OLT is the best alternative for patients with end-stage liver diseases. However, as the need for organs surpasses donor availability, alternatives to OLT are required. LCT could be a useful option versus OLT in several patients even though its low cell-engraftment hampers its efficiency. Endothelial cell barrier is the main obstacle for the implantation of cells into the parenchyma. Our study has focused on the modification of the endothelial barrier with monoclonal antibodies against adhesion molecules in order to increase cell engraftment in a mouse model of liver cell transplantation. METHODS: Anti-mouse CD54 and anti-mouse CD61 antibodies were administered intrasplenically to healthy mice within 60 min prior to stem cell transplantation. Animals were sacrificed either short term at 2h or middle term seven days after transplantation. Immunohistochemical techniques to detect alkaline phosphatase activity were used to identify the transplanted cells within the liver parenchyma. RESULTS: Anti-CD54 and anti-CD61 administration increases vascular patency and cell engraftment. This represents a 32% and 45% increase, respectively, of engrafted cells compared to the control (p<0.05). CONCLUSION: Modification of the vascular wall with monoclonal antibodies against endothelial adhesion molecules before cell transplantation enhances cell engraftment into the mouse liver.

Class III myosins shape the auditory hair bundles by limiting microvilli and stereocilia growth.

The precise architecture of hair bundles, the arrays of mechanosensitive microvilli-like stereocilia crowning the auditory hair cells, is essential to hearing. Myosin IIIa, defective in the late-onset deafness form DFNB30, has been proposed to transport espin-1 to the tips of stereocilia, thereby promoting their elongation. We show that Myo3a(-/-)Myo3b(-/-) mice lacking myosin IIIa and myosin IIIb are profoundly deaf, whereas Myo3a-cKO Myo3b(-/-) mice lacking myosin IIIb and losing myosin IIIa postnatally have normal hearing. Myo3a(-/-)Myo3b(-/-) cochlear hair bundles display robust mechanoelectrical transduction currents with normal kinetics but show severe embryonic abnormalities whose features rapidly change. These include abnormally tall and numerous microvilli or stereocilia, ungraded stereocilia bundles, and bundle rounding and closure. Surprisingly, espin-1 is properly targeted to Myo3a(-/-)Myo3b(-/-) stereocilia tips. Our results uncover the critical role that class III myosins play redundantly in hair-bundle morphogenesis; they unexpectedly limit the elongation of stereocilia and of subsequently regressing microvilli, thus contributing to the early hair bundle shaping.