Bitter taste receptors and gastrointestinal chemosensing in mice

Il sistema digerente è un organo sensorio capace di riconoscere le sostanze presenti nel lume e di iniziare un’ adeguata risposta, che si traduce in digestione ed assorbimento per i nutrienti o eliminazione per le sostanze nocive. E’ stato dimostrato che i recettori per l’amaro (T2R), un’ampia famiglia di GPCR in grado di riconoscere composti amari nella bocca, così come le molecole coinvolte nella trasduzione del segnale, sono espressi a livello della mucosa intestinale. Poiché l’amaro si è evoluto come un meccanismo di allarme nella bocca, proponiamo che i T2R nel sistema gastrointestinale possano servire come un secondo livello di difesa verso composti nocivi. In questo studio abbiamo usato qRT-PCR per stabilire la distribuzione di due sottotipi recettoriali dei T2R (mT2R138 e mT2R108), e della loro subunità Gα-Gustducin (Gust), nel sistema digerente del topo, e varie diete sono state usate per vedere se il contenuto del lume è in grado di modulare i recettori dell’amaro. Per testare l’ipotesi che i T2R nel sistema digerente possano servire come meccanismo di difesa nei confronti di patogeni, in ulteriori studi si è misurato tramite WB il grado di fosforilazione della proteina chinasi attivata da mitogeni (MAPK p44/42) per valutare se i T2Rs rispondano ad acil-omoserin-lattoni, molecole prodotte da batteri Gram negativi. La risposta, valutata su cellule STC-1 e NCM-460, è stata confrontata con quella evocata da agonisti per i T2R_feniltiocarbamide (PTC) e denatonio benzoato (DB)_e bloccata dall’antagonista del recettore T2R138, Probenecid. Il meccanismo che segue l’ attivazione dei T2R è stato ulteriormente caratterizzato con l’uso di GF-1, un inibitore della proteina chinasi C, e di nitrendipina, un bloccante dei canali calcio. Questo studio dimostra che mT2R138, mT2R108 e Gust sono espressi lungo tutto il sistema digerente, con diversa abbondanza, e che diverse diete sono in grado di modulare specificamente alcuni recettori, in aree selezionate lungo il sistema digerente. Abbiamo anche dimostrato che sia la linea cellulare umana di colonociti NCM-460, sia la linea di cellule enteroendocrine di topo STC-1 esprimono T2R e rispondono allo stimolo del gusto amaro e ad AHL con una rapida fosforilazione della MAPK p44/42, mostrando un profilo dose-dipendente. Il segnale indotto da PTC e AHL è bloccato dal Probenecid e ridotto dal GF-1, ma non dalla nitrendipina, al contrario del segnale indotto dal DB. Inoltre, l’esposizione delle cellule NCM-460 per 4-24 ore a PTC o AHL induce un aumento significativo dell’espressione del recettore T2R38 umano, omologo del T2R138 nel topo. In conclusione, questo lavoro di tesi propone che i T2R siano coinvolti nell’abilità sensoria del sistema gastrointestinale e che possano esistere diverse funzioni a seconda del sottotipo recettoriale espresso, del sito di espressione e del meccanismo utilizzato. Abbiamo mostrato che diversi tipi di cellule di origine intestinale esprimono diversi sottotipi di T2R e che essi possano usare diversi meccanismi. Inoltre, suggeriamo che i T2R possano interagire con batteri nel sistema gastrointestinale, a supporto dell’ipotesi per cui i recettori dell’amaro costituiscono un secondo livello di difesa nel sistema gastrointestinale, in grado di iniziare una risposta infiammatoria per combattere i batteri patogeni presenti nel lume.The gastrointestinal (GI) tract is a chemosensory organ that detects nutrients in the lumen to initiate an appropriate response of digestion and absorption of nutrients or elimination of harmful substances. It has been shown that bitter taste receptors (T2Rs), a large family of GPCRs detecting bitter compounds in the mouth, and their signaling molecules, are also expressed in the GI tract mucosa. Because bitter taste has evolved as a warning mechanism in the mouth, we hypothesize that T2Rs in the GI tract might serve as a second level of defense towards harmful compounds. In this study, we used qRT-PCR to investigate the distribution of two T2Rs subtypes (mT2R138 and mT2R108), and their signaling molecule Gα-Gustducin (Gust), in the mouse GI tract, and different diets to see whether they are modulated by luminal content. To test the hypothesis that T2Rs in the gut might serve as a mechanism of defense against pathogens, additional studies measured by WB the phosphorylation of mitogen activated protein kinase (MAPK) to evaluate whether T2Rs respond to Acyl Homoserine Lactone (AHL), quorum sensing molecule for Gram negative bacteria. The response on STC-1 and NCM-460 cells was compared to the ones elicited by T2Rs agonists_phenylthiocarbamide (PTC) and denatonuim benzoate (DB)_and blocked by the T2R138 antagonist Probenecid. The pathway following T2Rs activation was further characterized using GF-1, a protein kinase C inhibitor, and nitrendipine, a Ca++ channel blocker. We found that mT2R138, mT2R108 and Gust are expressed throughout the entire mouse GI tract, with different levels of expression, and that different diets selectively modulate T2Rs in specific GI regions. Also, we showed that both NCM-460 human colonocytes and STC-1 mouse enteroendocrine cells express T2Rs and respond to bitter stimuli and AHL with rapid dose-dependent phosphorylation of MAPK p44/42. PTC and AHL-induced signal was blocked by Probenecid and reduced by GF-1, but not by nitrendipine, in contrast with DB-induced MAPK phosphorylation. Furthermore, exposure of NCM-460 cells to PTC or AHL for 4-24 h induced a significant increase in hT2R38 mRNA, the homologus of mT2R138. In summary, these data suggest that T2Rs are involved in chemosensing in the GI tract and that different functions might exist depending upon receptor subtype, site of expression and molecular mechanism. We showed that different T2R subtypes are expressed in different GI cell types and that they might use different pathways. Also, we suggest that T2Rs might detect bacterial stimuli in GI cells, supporting the hypothesis that activation of these receptors might provide a second level of defense in the GI mucosa to initiate an inflammatory process in response to bacteria in the gut lumen

Cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS): genetic and clinical aspects

Cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) typically presents in middle life with a combination of neuropathy, ataxia and vestibular disease, with patients reporting progressive imbalance, oscillopsia, sensory disturbance and a dry cough. Examination identifies a sensory neuropathy or neuronopathy and bilaterally impaired vestibulo-ocular reflex. The underlying genetic basis is of biallelic AAGGG expansions in the second intron of replication factor complex subunit 1 (RFC1). The frequency and phenotype spectrum of RFC1 disease is expanding, ranging from typical CANVAS to site-restricted variants affecting the sensory nerves, cerebellum and/or the vestibular system. Given the wide phenotype spectrum of RFC1, the differential diagnosis is broad. RFC1 disease due to biallelic AAGGG expansions is probably the most common cause of recessive ataxia. The key to suspecting the disease (and prompt genetic testing) is a thorough clinical examination assessing the three affected systems and noting the presence of chronic cough

Mortality attributable to COVID-19 and air pollution. An italian case study

University of Pavia, ItalyIntroduction. Several evidences suggest an association between the air pollution and COVID-19 mortality. However, other factors might impact on COVID-19 mortality. The aim of present work is to explore association between air pollution and COVID-19 mortality in Emilia-Romagna region one of the Italian Region strongly involved in the initial phase of COVID-19 pandemic, taking into account other exogenous factors. Material and methods. An ecological study is used: the official open data are aggregated at the municipality level, for a total of 328 municipalities in the Emilia-Romagna region (https://www.istat.it/). The cumulative-period COVID-19 deaths were the outcome of interest. The deaths attributable to COVID-19 occurred in the pandemic period January 1 – August 31, 2020 were estimated as the excess of all deaths registered in the pandemic period with respect to the average number of all deaths in the same period of previous 5 years. As main predictor of COVID-19 mortality the particulate matter (PM) with diameter less than 10 µm was chosen. As proxy of air pollution at municipalities level, PM10 emissions in tons/year was used (https://dati.arpae.it/dataset/inventario-emissioni-aria-inemar). Several potential determinants of COVID-19 mortality or confounding for the association between PM10 exposure and mortality were also investigated: Degree of urbanization index (DEGURBA), Italian multiple deprivation index (IMD), Ecoregions index (https://www.isprambiente.gov.it/it/attivita/suolo-e-territorio/) and the amount of soil consumed. Geographic distribution of COVID-19 deaths, air pollution and predictors were plotted in maps to describe graphically the spatial distribution. After assigning zero to response variable for those municipalities with a no COVID-9 attributable deaths, negative binomial regression was applied. Results. In the 328 municipalities of Emilia-Romagna, the estimated number of deaths attributable to COVID-19 during the first 8 months of 2020 was in median around 6 deaths (IQR: 15.3). Nevertheless, 5 municipalities showed a number of deaths attributable to COVID-19 over 75th, ranging from 160 to 540; for 75 municipalities no death attributable to COVID-19 was estimated. The median PM10 emissions was around 23 tons/year (IQR: 20.7): 15 municipalities showed a PM10 over 75th. The great majority of municipalities had a thin population (about 60%) and were prevalently rich or very rich: only 7% deprived. The predominant ecoregion was that of central Tuscan and Marche Apennines (about 48%). In median the percentage of soil consumed was 8.5%. The municipalities with the highest number of COVID-19 attributable deaths had a quite high level of PM10 emissions. These were all rich or very rich urban or semi-urban municipalities. Conversely, the municipalities with the lowest tons by year of PM10 emission showed lower COVID-19 attributable mortality. These areas were also characterized by increasing level of deprivation and were mainly rural. The effect of PM10 emission on the COVID-19 attributable deaths estimated in the study period was confirmed by multivariate analysis. Conclusions. The main findings from this work seems to confirm the effect of PM10 on the COVID-19 mortality, in agreement with published evidences. But, given the limit of the ecological study our results may not be conclusive. The latter should be integrated with further studies that analyse the single individual

Enteric dysfunctions in experimental Parkinson's disease: alterations of excitatory cholinergic neurotransmission regulating colonic motility in rats

Parkinson's disease (PD) is frequently associated with gastrointestinal symptoms, mostly represented by constipation and defecatory dysfunctions. This study examined the impact of central dopaminergic denervation, induced by injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle, on distal colonic excitatory cholinergic neuromotor activity in rats. Animals were euthanized 4 and 8 weeks after 6-OHDA injection. In vivo colonic transit was evaluated by radiological assay. Electrically and carbachol-induced cholinergic contractions were recorded in vitro from longitudinal and circular muscle colonic preparations, while acetylcholine levels were assayed in their incubation media. Choline acetyltransferase (ChAT), HuC/D (pan-neuronal marker), muscarinic M2 and M3 receptors. As compared with control rats, at week 4 6-OHDA-treated animals displayed the following changes: decreased in vivo colonic transit rate; impaired electrically evoked neurogenic cholinergic contractions; enhanced carbachol-induced contractions; decreased basal and electrically stimulated acetylcholine release from colonic tissues; decreased ChAT immunopositivity in the neuromuscular layer; unchanged density of HuC/D immunoreactive myenteric neurons; increased expression of colonic muscarinic M2 and M3 receptors. The majority of such alterations were detected also at week 8 post-6-OHDA injection. These findings indicate that central nigrostriatal dopaminergic denervation is associated with an impaired excitatory neurotransmission characterized by a loss of myenteric neuronal ChAT positivity and decrease in acetylcholine release, resulting in a dysregulated smooth muscle motor activity, which likely contributes to the concomitant decrease in colonic transit rate

Role of Arginine 285 in the Active Site of Rhodotorula gracilis d-Amino Acid Oxidase A SITE-DIRECTED MUTAGENESIS STUDY

Abstract Arg285, one of the very few conserved residues in the active site of d-amino acid oxidases, has been mutated to lysine, glutamine, aspartate, and alanine in the enzyme from the yeast Rhodotorula gracilis (RgDAAO). The mutated proteins are all catalytically competent. Mutations of Arg285 result in an increase (≈300-fold) ofK m for the d-amino acid and in a large decrease (≈500-fold) of turnover number. Stopped-flow analysis shows that the decrease in turnover is paralleled by a similar decrease in the rate of flavin reduction (k 2), the latter still being the rate-limiting step of the reaction. In agreement with data from the protein crystal structure, loss of the guanidinium group of Arg285 in the mutated DAAOs drastically reduces the binding of several carboxylic acids (e.g. benzoate). These results highlight the importance of this active site residue in the precise substrate orientation, a main factor in this redox reaction. Furthermore, Arg285 DAAO mutants have spectral properties similar to those of the wild-type enzyme, but show a low degree of stabilization of the flavin semiquinone and a change in the redox properties of the free enzyme. From this, we can unexpectedly conclude that Arg285 in the free enzyme form is involved in the stabilization of the negative charge on the N(1)-C(2)=O locus of the isoalloxazine ring of the flavin. We also suggest that the residue undergoes a conformational change in order to bind the carboxylate portion of the substrate/ligand in the complexed enzyme

Effect of monovalency on anti-contactin-1 IgG4

Altres ajuts: Agence Nationale pour le Développement de la Recherche en Santé ; Association Française contre les Myopathies ; ArgenxAutoimmune nodopathies (AN) have been diagnosed in a subset of patients fulfilling criteria for chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) who display no or poor response to intravenous immunoglobulins. Biomarkers of AN are autoantibodies, mainly IgG4, directed against the ternary paranodal complex composed by neurofascin-155, contactin-1 (CNTN1), and Contactin-associated-protein-1 (CASPR1) or against the nodal isoforms of neurofascin. IgG4 can undergo a Fab-arm exchange (FAE) which results in functionally monovalent antibody. This phenomenon differentially affects the pathogenicity of IgG4 depending on the target of autoantibodies. Here, we have evaluated this issue by examining the impact of valency on anti-CNTN1 IgG4 which induces paranodal destruction through a function blocking activity. Sera were obtained from 20 patients with AN associated with anti-CNTN1 antibodies. The proportion of monospecific/bispecific anti-CNTN1 antibodies was estimated in each patient by ELISA by examining the ability of serum antibodies to cross-link untagged CNTN1 with biotinylated CNTN1. To determine the impact of monovalency, anti-CNTN1 IgG4 were enzymatically digested into monovalent Fab and tested in vitro on cell aggregation assay. Also, intraneural injections were performed to determine whether monovalent Fab and native IgG4 may penetrate paranode, and antibody infiltration was monitored 1- and 3-days post injection. We found that the percentage of monospecific antibodies were lower than 5% in 14 out of 20 patients (70%), suggesting that IgG4 have undergone extensive FAE in situ. The levels of monospecific antibodies correlated with the titers of anti-CNTN1 antibodies. However, no correlation was found with clinical severity, and patients with low or high percentage of monospecific antibodies similarly showed a severe phenotype. Native anti-CNTN1 IgG4 were shown to inhibit the interaction between cells expressing CNTN1/CASPR1 and cells expressing neurofascin-155 using an in vitro aggregation assay. Similarly, monovalent Fab significantly inhibited the interaction between CNTN1/CASPR1 and neurofascin-155. Intraneural injections of Fab and native anti-CNTN1 IgG4 indicated that both mono- and bivalent anti-CNTN1 IgG4 potently penetrated the paranodal regions and completely invaded this region by day 3. Altogether, these data indicate anti-CNTN1 IgG4 are mostly bispecific in patients, and that functionally monovalent anti-CNTN1 antibodies have the pathogenic potency to alter paranode

Variable echo time imaging for detecting the short T2* components of the sciatic nerve: a validation study

OBJECTIVE: The aim of this study was to develop and validate an MRI protocol based on a variable echo time (vTE) sensitive to the short T2* components of the sciatic nerve. MATERIALS AND METHODS: 15 healthy subjects (M/F: 9/6; age: 21-62) were scanned at 3T targeting the sciatic nerve at the thigh bilaterally, using a dual echo variable echo time (vTE) sequence (based on a spoiled gradient echo acquisition) with echo times of 0.98/5.37 ms. Apparent T2* (aT2*) values of the sciatic nerves were calculated with a mono-exponential fit and used for data comparison. RESULTS: There were no significant differences in aT2* related to side, sex, age, and BMI, even though small differences for side were reported. Good-to-excellent repeatability and reproducibility were found for geometry of ROIs (Dice indices: intra-rater 0.68-0.7; inter-rater 0.70-0.72) and the related aT2* measures (intra-inter reader ICC 0.95-0.97; 0.66-0.85) from two different operators. Side-related signal-to-noise-ratio non-significant differences were reported, while contrast-to-noise-ratio measures were excellent both for side and echo. DISCUSSION: Our study introduces a novel MR sequence sensitive to the short T2* components of the sciatic nerve and may be used for the study of peripheral nerve disorders

Characterization of Skeletal Muscle Biopsy and Derived Myoblasts in a Patient Carrying Arg14del Mutation in Phospholamban Gene.

Phospholamban is involved in the regulation of the activity and storage of calcium in cardiac muscle. Several mutations have been identified in the PLN gene causing cardiac disease associated with arrhythmogenic and dilated cardiomyopathy. The patho-mechanism underlying PLN mutations is not fully understood and a specific therapy is not yet available. PLN mutated patients have been deeply investigated in cardiac muscle, but very little is known about the effect of PLN mutations in skeletal muscle. In this study, we investigated both histological and functional features in skeletal muscle tissue and muscle-derived myoblasts from an Italian patient carrying the Arg14del mutation in PLN. The patient has a cardiac phenotype, but he also reported lower limb fatigability, cramps and fasciculations. The evaluation of a skeletal muscle biopsy showed histological, immunohistochemical and ultrastructural alterations. In particular, we detected an increase in the number of centronucleated fibers and a reduction in the fiber cross sectional area, an alteration in p62, LC3 and VCP proteins and the formation of perinuclear aggresomes. Furthermore, the patient's myoblasts showed a greater propensity to form aggresomes, even more marked after proteasome inhibition compared with control cells. Further genetic and functional studies are necessary to understand whether a definition of PLN myopathy, or cardiomyopathy plus, can be introduced for selected cases with clinical evidence of skeletal muscle involvement. Including skeletal muscle examination in the diagnostic process of PLN-mutated patients can help clarify this issue.This work was partially supported by the Italian Ministry of Health (Ministero della Salute, Ricerca Corrente 245)S

Impact of multiple sclerosis risk loci in postinfectious neurological syndromes

Background: The genetic component of multiple sclerosis (MS) is now set to 200 autosomal common variants. However, it is unclear how genetic knowledge be clinically used in the differential diagnosis between MS and other inflammatory conditions like adult-onset postinfectious neurological syndromes (PINS). The aim of this study was to investigate whether PINS and MS have a shared genetic background using an updated polygenic risk scores. / Methods: Eighty-eight PINS patients have been consecutively recruited between 1996 and 2016 at Mondino Foundation of Pavia, diagnosed according to clinical, MRI and CSF findings and followed-up for several years. Patients were typed using Illumina array, and genotypes imputed using the 1000 Genomes Project reference panel. A weighted genetic risk score (wGRS) has been calculated based on autosomal MS risk loci derived from large-scale studies, and an HLA genetic burden (HLAGB) was also calculated on loci associated to MS. / Results: PINS occurred as an episode of myelitis in 44% of patients, encephalomyelitis in 44%, and encephalitis in remaining cases, with an involvement of peripheral nervous system in 41% of patients. Mean age of onset was 50.1 years, and female:male ratio was 1.4. Patients were followed-up for a mean of 7.2 years, and at last visit 55% had a low disability grade (mRS 0–1). Disease was monophasic in 67% of patients, relapsing in 18% and chronic-progressive in 15%. The wGRS of PINS cases was comparable to 370 healthy controls, while significantly lower compared to 907 bout-onset MS (BOMS) cases (wGRS= 20.9 vs 21.2; p<0.0001). The difference was even larger for PINS with peripheral nervous system involvement (wGRS=20.6) vs BOMS. / Conclusion: The distinction between MS and PINS is not easy to make in clinical practice. However, our study shows that the new set of MS risk alleles does not confer increased susceptibility to PINS. These data support the importance to discriminate these cases from MS with pathophysiological and therapeutic implications