102,059 research outputs found
Stapled anopexy versus transanal haemorrhoidal dearterialization for haemorrhoidal disease: a three-year follow-up from a randomized study
No full textBACKGROUND: This randomized study compared the medium-term results of stapled anopexy (SA) and transanal hemorrhoidal dearterialization with anopexy (THD) in 4 homogeneous groups of patients, 2 with third- and 2 with fourth-degree hemorrhoids.
METHODS: Forty patients with third-degree and 30 with fourth-degree hemorrhoids were randomly submitted to SA (N.=20+15) and THD (N.=20+15), respectively. Clinical controls were done every 6 months from 1 to 42 months after the operation, with incidence of recurrent hemorrhoids as primary outcome measure. Operative time, complications, pain, time to return to normal activity, costs, Short Form-36, and overall patient satisfaction were also evaluated.
RESULTS: Frequencies of preoperative obstructed defecation symptoms and prolapse recurrence were higher in patients with fourth-degree hemorrhoids, and SA was more effective than THD in reducing the risk of recurrence at 36±6 months follow-up (P=0.049). Operative time, complications, pain, and time of return to normal activity were similar in the 4 groups. Costs were significantly higher for SA in patients with fourth-degree hemorrhoids (P>0.01). A significant improvement of quality of life was observed in all groups, and no significant difference was found in overall patient satisfaction.
CONCLUSIONS: Both techniques are safe and effective in the mid-term period. SA is more effective in reducing prolapse and obstructed defecation symptoms in fourth-degree hemorrhoids, with the disadvantage of higher costs. Prolapse size and presence of obstructed defecation symptoms could be predictive criteria for choice of the best surgical technique
(Cite this article as: Venturi M, Salamina G, Vergani C. Stapled anopexy versus transanal hemorrhoidal dearterialization for hemorrhoidal disease: a three-year follow-up from a randomized study. Minerva Chir 2016;71:365-71
Helicobacter pylori Pathogenic Factors
No full textFrom 1994, Helicobacter pylori was classified by WHO (World Health Organization) as a class I carcinogen and its infection has been associated to gastroduodenal disease. It colonizes more than half of worldwide population, with a prevalent infection rate in developed countries. In spite of the majority of infected people are asymptomatic, around 20% develop severe pathologies like peptic ulcers and the 1% lymphoma of the mucosa-associated lymphoid tissue (MALT) and stomach cancer. This significant epidemiological study both of the unique characteristics of H. pylori inspired many scientists, as bacteriologist, gastroenterologists, cancer and pharmaceutical scientists to understand physio-pathological aspects of this bacterium, and also microbiologist, taxonomist, microbial ecologist and molecular biologist, for a more detailed molecular approach.
H. pylori, a Gram negative, microaerophilic bacteria that colonize human gastric mucosa. It is not an acidophilus bacterium and even if the stomach lumen presents inhospitable condition for most microbes, it is able to survive for a short period, sufficient to enter in the highly viscous mucosa, reach gastric epithelium, and colonize the gastro-enteric tract. H. pylori colonization is mediated by a predominant virulence factor, the flagellar motility associated to chemotaxis. To avoid its discharge in the intestinal tract by peristalsis, the bacteria establish a persistent infection inside the viscous gastric mucus film that covers the gastric epithelium. A nickel containing enzyme, the urease, hydrolyzes the urea present in the stomach to ammonia and CO2, buffering the pH of the periplasm. The most severe clinical outcomes are always associated to cag+ strains. cag-PAI is defined as the “Cytotoxic Associated Genes Pathogenicity Island” and it consists of a characteristic chromosome, flanked by transposable elements. Another important virulent factor is the vacuolating cytotoxin A, known as VacA, which induces the formation of large cytoplasmic vacuoles in gastric cultured cell lines. Moreover the iron and nickel acquisition is essential grow factors and a large number of genes are responsible of this mechanism.
While the development of an efficient vaccine against H. pylori is now the aim of many researchers, the search for new specific antibiotics as a new pharmaceutical target is required for the complete eradication of H. pylori.
In this thesis has been investigate the structural and function role of different pathogenic proteins involved in the H. pylori colonization of human gastric mucosa. These potential drug targets have been cloned, 8 out of 11 were expressed in a heterologous expression system, after purification, 2 of them generate protein crystals and only one was possible to characterize the molecular structure. In particular it has been elucidated a possible physiological role of CeuE (HP1561), a Class III SPB (Substrate Binding Protein), crystalized with Ni(His)2 complex and it was determined its affinity to the complex by an in vitro approach. The H. pylori flagella play a key role during infection allowing the bacterium to move through the mucous layer. The H. pylori hook scaffolding protein FlgD were cloned, expressed, purified and crystalized. A study of other purified pathogenic H. pylori factors belonging to flagellar component apparatus and transcriptional factors involved in cellular stress response has been reported.
To obtain these results, different experimental approaches has been used. Bioinformatics analysis of target proteins has been performed to predict the best candidates for a crystallographic study and for genetic construction design. Molecular cloning in plasmid vectors has been performed from PCR amplification. The expression conditions were optimized and performed in E. coli, a heterologous system. The solubility of recombinant proteins were checked and obtained also with protein refolding methods. Different purification techniques were used in order to obtain pure protein. Target characterization was performed due analytical gel filtration, UV spectroscopy, DLS (Dynamic Light Scattering) and CD (Circular Dichroism). The proteins were concentrated to crystallization trials. The protein crystals obtained were analyzed at ESRF synchrotron (Grenoble, France). Functional in vitro approaches were performed using fluorescence spectroscopy, SPR (Surface Plasmon Resonance) and Mass spectroscopy.
In the second chapter is described the three dimensional structure of a H. pylori pathogenic protein crystalized in presence with its possible physiological substrate. HP1561 (CeuE) is a H. pylori protein predicted to be an ABC transporter component, periplasmic iron-bind transporter. Recently it was published that CeuE and fecDE genes of H. mustelae encode for a nickel and cobalt acquisition system. In Gram-negative bacteria, nickel uptake is guaranteed by multiple and complex systems that operate at the membrane and periplasmic level. H. pylori employs other yet uncharacterized systems to import the nickel required for the maturation of key enzymes, such as urease and hydrogenase. To understand this contradiction of the data about Ni2+ acquisition system in H. pylori CeuE was cloned, expressed, purified, crystallized and its structure determined. Identity between the sequences of the two Helicobacter is 44%. The two Histidine residues (H103 and H197), potentially involved in Siderophores/Ni2+ binding coordination in H. pylori CeuE, are partially conserved. The His corresponding to H. pylori position 103 is conserved, whilst His197 is replaced by a Leucine. In order to check, if this substitution influence the binding of siderophores/Ni2+, the mutant of H. pylori CeuE H197L was than produced and purified. The crystal structure of H. pylori CeuE has been determined at 1.65Å resolution using the SAD method, in Apo-form and in complex with Ni(His)2. It comprises two structurally similar globular domains, each consisting of a central five-stranded β-sheet surrounded by α-helices, an arrangement commonly classified as a Rossmann-like fold. Structurally, H. pylori CeuE belongs to the class III periplasmic substrate-binding protein. Crystallographic data, fluorescence binding assays and SPR analysis allow to exclude a role of the protein in the transport of VitB12, heme, enterobactin and isolated Ni2+ ions. On the contrary, the crystal structure of the protein/Ni(L-His)2 complex and dissociation constant obtained by SPR technique suggests that H. pylori CeuE binds and transport nickel in vivo thanks to the formation of a Ni2+/histidine complex or to some ligand that mimics it.
In the third chapter is presented the study of FlgD, a flagellar component involved in the formation the extracellular complex, the flagellar hook. The motility of H. pylori is considered a colonization factor, due the fact that less motile strains are less able to colonize or survive in the host than full motile strains. In the flagellum machinery are involved more than 50 genomic genes for regulation and assembly. The three major components are the filament, the hook and the basal body. FlgD is not present when the flagellum is completed, but plays a key role during the assembly. Therefore, it has been classified as the hook-scaffolding protein, considering it also as the hook capping protein, interacting with FlgL and FlgK and the basal body rod – modification protein. In H. pylori G27 strain FlgD correspond to the gene hp0858 that was amplified from purified genomic DNA and cloned in an expression plasmid vector. The protein was produced in E. coli BL21 in reach medium ad it resulted to a soluble protein. DLS and analytical gel filtration confirm the oligomeric state of FlgD that resulted to be a tetramer in solution. The protein was concentrated to 30g/l and crystalized after a couple of month of incubation. The crystals had diffracted at 2.7Å of maximum resolution. For molecular replacement approach was used homology modeling. Different molecular models were built to fit experimental diffraction data. The secondary structure of the generated models was fitted with experimental CD spectra, where FlgD resulted to have around 12% of helices and 45% of β-sheets (190-260nm). Crystallographic statistics do not properly converged to a positive molecular refinement with the tested models. To solve FlgD structure are necessary crystals of recombinant Selenomethionine FlgD that was expressed, purified and crystalized.
In the fourth chapter are reported H. pylori pathogenic proteins that had been characterized. These proteins could be divided in two groups, the first one of flagellar proteins and the second of cellular stress response factors, in collaboration with Professor V. Scarlato of the department of Biology of Bologna University.
FliN is a cytosolic protein, localized in the C ring of the flagellar basal body. It interacts with the other two components FliM and FliG. Missense or mutation of fliN had been associated to non-motile strains. It has been reported that regulates the clockwise/counterclockwise switching of flagella. H. pylori FliN was cloned, expressed and purified from the inclusion body after refolding. Oligomerization after refolding was tested by DLS and analytical gel filtration. The protein resulted to be poly-disperse in solution and no protein crystals have been obtained.
FliD is the filament capping protein and it was observed that interact with FliT that is not only a flagellar type III substrate specific export chaperone but also inhibits the expression of fliD thought its specific interaction with the master regulator FlhD4C2 complex. In order to analyze possible structure of the co-crystalized FliD-FliT, it was plan to co-express these proteins. Both were cloned with a different affinity purification system, but only FliT was possible to express and purify from inclusion bodies. The CD spectra presented a strong β-sheet component in the secondary structure. DLS and analytical gel filtration revealed that this protein is poly-disperse in solution and no protein crystals were be obtained.
FlgN is a type III secretion chaperone and it has been reported to interact with the two hook junction protein FlgK and FlgL preventing the protein proteolysis when the flagellum is not assembled. These proteins have been cloned in different type of plasmid vectors for a co-expression experiment, but only FlgN was properly expressed in E. coli. Recombinant FlgN was purified by Ni-IMAC and resulted to be soluble in solution. The protein was characterized by analytical gel filtration, DLS and CD. The protein resulted to be a monomer in solution with a 30% of not defined secondary structure (190-260nm). FlgN was concentrated and different crystallization conditions were tested.
In the latter group there are three proteins related to Heat shock response, produced when bacteria encounter stress such as the elevated temperatures, ethanol, H2O2 and acid. It was demonstrated that H. pylori Hsps play an important role during the host infection. HrcA and HspR are negative repressor of groESL and dnaK machinery. HrcA activity depends by the presence of HspR, because it is demonstrated that HrcA is not able to bind DNA in absence of HspR. These two proteins were expressed in E. coli and purified by Ni-IMAC affinity. During the concentration step, these proteins present a solubility limit influenced by the concentration. Mutagenesis of a Cys in HspR and detergent solubility screening with HrcA has been performed, but no suitable protein for crystallization trials has been obtained.
Hp1026 is a gene present in the same operon of HspR (hp1025). The function of this gene has not been reported. From sequence homology was possible to identify a helicase domain and ATP-binding domain. This protein, ORF, has been expressed in E. coli and purified by Ni-IMAC affinity. Analytical gel filtration and CD has been performed to characterize this protein. The protein was a dimer in solution with a 35% of α-helices component. Crystallization trials have been performed at different protein concentrations and also in presence of its possible cofactor, ATPγS. No crystals have been obtained in tested condition.
Appendix:
Structural and functional study on a human protease S1P/ SKI1
The study of human S1P/SKI1 protease was performed in collaboration with Professor S. Kunz of the Institute of Microbiology, University Hospital Center and university of Lausanne, Switzerland. S1P/SKI-1 is a serine protease that belongs to the mammalian family of Proprotein Convertases (PC). The aim of this family member is to mediate the activation of different important substrates for cell live. Among these proteases, S1P has been shown to have unique substrate specificity, preferring cleavage after non-basic amino acids. Known S1P cellular targets are SREBP-2, involved in the biosynthesis and uptake of lipids and cholesterol, BDNF, ATF-6 and the surface glycoprotein of viruses belonging to the family of Arenaviridae. S1P is 118 kDa multi-domain protein; two regions of S1P have been investigated, the "Prodomain", involved in the regulation of S1P catalytic activity, and the so called "catalytic domain", which include the residues responsible for the cleavage reaction itself. Moreover it was analyzed an inactive mutant of cS1P: H249A. Also for ProD was chosen one constructs (ProD_AB and ProD_AC) involved in the affinity of the protease substrate. Hence, the sequences corresponding to the domains were synthesized as optimized genes for the expression in E. coli and sub-cloned in expression plasmids in order to obtain C-term His-tagged fusion proteins. These constructs have been expressed in E. coli, purified by Ni-IMAC and positive fractions have been collected and concentrated in order to perform crystallization trials. Unfortunately no protein crystals have been obtained in tested condition. To elucidate the role of a mutated variant of the cleavage site “C” of Pro Domain, it was performed a mass spectrometry analysis. Secreted S1P/SKI1 mutant C was purified from culture medium of HEK293 cell line was isolated by IMAC-Co. The sample, loaded in RP-HPLC, was denatured in 6 M Guanidine-HCl. The chromatographic fractions corresponding to the major HPLC peaks were dried out in a speed-vac concentrator and directly injected in the ESI source. Mass measurements were performed with a quadrupole-TOF spectrometer. Analysis of mass spectra, compared with wild-type form of S1P, allows generating a preliminary Pro Domain auto-processing profile.Dal 1994 il batterio Helicobacter pylori è stato classificato come organismo cancerogeneno di prima classe e la sua infezione è associata a patologie gastroduodenali. Più di metà della popolazione mondiale ne è infettata con una maggiore prevalenza nei paesi sviluppati. Nonostante la maggior parte dei casi le infezioni sono asintomatiche, il 20% sviluppa gravi patologie come ulcere peptiche e nell’1% dei casi genera linfomi e gastro carcinomi. L’incidenza e le caratteristiche di questo batterio hanno ispirato batteriologi, gastroenterologi, oncologi e farmacologi per indagare gli aspetti fisiopatologici legati all’infezione, così come microbiologi, ecologi, biologi molecolari hanno cercato i fattori di virulenza coinvolti in nell’infezione.
H. pylori è un batterio microaerofilico Gram negativo che colonizza la mucosa gastrica. Non è un batterio acidofilo, anche se è in grado di sopravvivere nel lume dello stomaco per un breve periodo necessario per raggiungere le cellule epiteliali spostandosi attraverso la mucosa gastrica. La colonizzazione è mediata da fattori di virulenza predominanti come la motilità flagellare associata alla chemiotassi. Per evitare che sia espulso dal tratto intestinale dalla peristalsi, il batterio H. pylori stabilisce un’infezione cronica. L’ureasi, che è un enzima nickel dipendente, che idrolizza l’urea presente in ammoniaca e CO2 tamponando il pH acido dello stomaco. I casi più gravi sono associati ai ceppi che esprimono l’isola di patogenicità cag-PAI, che consiste in un cromosoma delimitato da elementi trasponibili. Un altro importante fattore di virulenza è la tossina vacuolizzante VacA, che induce la formazione di vacuoli citoplasmatici. Anche il meccanismo di acquisizione di ferro e nickel è fondamentale per la colonizzazione batterica e dunque finemente regolata da un gran numero di geni.
Lo sviluppo di un vaccino e nuovi antibiotici nutrono una costante ricerca di nuovi possibili bersagli farmacologici, necessari per completa ed efficiente eradicazione del batterio H. pylori.
In questa tesi sono stati analizzati il ruolo e la struttura di alcune proteine patogenetiche del H. pylori. Questi potenziali target farmacologici sono stati clonati, otto su undici sono stati espressi in un sistema eterologo, due proteine di quelle purificate hanno generato cristalli e di una sola ne è stata definita la struttura molecolare. In particolare è stato definito un possibile ruolo della proteina CeuE (HP1561), appartenete alla famiglia delle proteine che legano un substrato, cristallizzata in presenza del complesso Ni(His)2 e definita l’affinità con lo stesso in vitro.
Del flagello, che svolge un ruolo chiave durante l’infezione, ne è stata studiata la proteina coinvolta nella formazione dell’uncino FlgD che è stata clonata, espressa, purificata e cristallizzata.
Inoltre è stato riportato anche uno studio di altri fattori del flagello e di alcune proteine coinvolte nella risposta allo stress cellulare.
Per ottenere tali risultati sono stati utilizzati approcci differenti. Per individuare le migliori proteine candidate per uno studio cristallografico e progettare costrutti funzionali sono state effettuate predizioni bioinformatiche. Gli amplificati di PCR sono stati clonati in vettori plasmidici. Le condizioni di espressione sono state ottimizzate e fatte in E. coli, un sistema di espressione eterologo. La solubilità delle proteine ricombinanti è stata analizzata e ottenuta anche mediante refolding. Sono stati usati diversi sistemi di purificazione per ottenere un buon grado di purezza. Per la caratterizzazione proteica sono state usate come tecniche la gel filtrazione analitica, spettroscopia UV, DLS (Dynamic Light Scattering) e dicroismo circolare. Le proteine sono state concentrate e sottoposte a esperimenti di cristallizzazione. I cristalli sono stati analizzati al sincrotrone ESRF (Grenoble, France). Spettroscopia di fluorescenza, SPR (surface plasmon resonance) e spettroscopia di massa sono le tecniche utilizzate per la caratterizzazione In Vitro.
Nel secondo capitolo viene decritta la struttura tridimensionale di una proteina patogenetica di H. pylori, cristallizzata in presenza del suo possibile substrato fisiologico. HP1561 (CeuE) è una proteina di H. pylori annotata come componente periplasmatico di un trasportatore ABC che lega e trasporta il ferro. Recentemente è stato pubblicato chele ceuE e fecDE di H. mustelae codificano per proteine coinvolte nel acquisizione del nickel e cobalto. Nei Gram negativi, l’acquisizione del nickel è garantita da sistemi di proteine che operano a livello di membrana e periplasmatico. Per l’acquisizione del nickel, l’ H. pylori integra diversi sistemi non ancora caratterizzati, necessari per la maturazione di enzimi chiave come l’ureasi e l’idrogenasi. Per chiarire tale contraddizione nel sistema di acquisizione del nickel nell’H. pylori, CeuE è stata clonata, espressa, purificata, cristallizzata e la sua struttura è stata risolta. L’identità di sequenza tra i due Helicobacter (pylori e mustelae) è del 44%. Le due Istidine (H103 e H197), potenzialmente coinvolte nel legame di coordinazione del sistema sideroforo/Ni2+ nel H. pylori CeuE, risultano essere parzialmente conservate. L’His corrispondente alla His103 di H. pylori è conservata, mentre His197 è sostituita da una Leucina. Al fine d’identificare se tale mutazione possa influenzare il legame sideroforo/Ni2+, è stato prodotto e purificato il mutante H. pylori CeuE H197L. La struttura molecolare di H. pylori CeuE è stata determinata con una risoluzione di 1.65 Å mediante metodo SAD, sia nella forma apo, che in complesso col Ni(His)2. Essa è costituita da due domini globulari simili, ognuno costituito da cinque foglietti-β circondati da α-eliche, comunemente classificato come Rossman fold. Strutturalmente H. pylori CeuE appartiene alla Classe III della famiglia di proteine che legano un substrato specifico (SBPs). Dati cristallografici, saggi di fluorescenza e analisi all’SPR ci permettono di escludere il coinvolgimento della proteina nel trasporto della VitB12, eme, entrobactina, e ioni Ni2+ isolati. Al contrario la struttura della proteina/complesso Ni(His)2 e le costanti di dissociazione ottenute mediante SPR suggeriscono che H. pylori CeuE lega e trasporta il nickel in vivo mediante il complesso Ni2+/His o altro ligando che lo mima.
Nel terzo capitolo viene presentato lo studio su FlgD, una proteina flagellare fondamentale nella formazione di un complesso extracellulare, l’uncino del flagello. La motilità dell’H. pylori è considerata un fattore di colonizzazione, attraverso il quale ceppi meno motili hanno minori possibilità di colonizzare e sopravvivere nell’ospite di ceppi più motili. Per la formazione del flagello sono coinvolti più di 50 geni per la regolazione e l’assemblaggio delle varie componenti. Le tre componenti principali sono il filamento, l’uncino e il corpo basale. FlgD non è presente quando il flagello è maturo, ma ha un ruolo chiave durante l’assemblaggio. Perciò, è stato classificato come proteina necessaria per l’impalcatura dell’uncino (hook scaffolding protein), consi
Archivio computerizzato per la gestione ambulatoriale ed in dayhospital dei pazienti con infezione da HIV
No full textA mathematical model for HIV and hepatitis C co-infection and its assessment from a statistical perspective
No full textThe hepatitis C virus (HCV) and the human immunodeficiency virus (HIV) are a clear threat for public health, with high prevalences especially in high risk groups such as injecting drug users. People with HIV infection who are also infected by HCV suffer from a more rapid progression to HCV-related liver disease and have an increased risk for cirrhosis and liver cancer. Quantifying the impact of HIV and HCV co-infection is therefore of great importance. We propose a new joint mathematical model accounting for co-infection with the two viruses in the context of injecting drug users (IDUs). Statistical concepts and methods are used to assess the model from a statistical perspective, in order to get further insights in: (i) the comparison and selection of optional model components, (ii) the unknown values of the numerous model parameters, (iii) the parameters to which the model is most 'sensitive' and (iv) the combinations or patterns of values in the high-dimensional parameter space which are most supported by the data. Data from a longitudinal study of heroin users in Italy are used to illustrate the application of the proposed joint model and its statistical assessment. The parameters associated with contact rates (sharing syringes) and the transmission rates per syringe-sharing event are shown to play a major role. (C) 2013 Elsevier B.V. All rights reserved
La investigacion en campos de agricultores sobre frijol en dos zonas del Distrito Salamina, Colombia
No full textThe most important characteristics of bean production systems in Aranzazu and Salamina (Caldas, Colombia) are summarized. Information collected in a survey detected the main constraints for bean cultivation in the area such as erosion, diseases, low density population, low-P and acid soils, and drought. A general plan to be developed in the area by the Instituto Colombiano Agropecuario and the Integrated Rural Development project is presented. (CIAT)Se resumen las caracteristicas importantes de los sistemas de produccion de frijol en Aranzazu y Salamina (Caldas, Colombia). Con base en la informacion recogida en una encuesta exploratoria, se detectaron los principales limitantes del cultivo del frijol en la zona como son erosion, enfermedades, baja densidad de poblacion, suelos acidos y pobres en P y sequia. Se presenta un plan general de trabajo en la zona a realizar por el Instituto Colombiano Agropecuario y el proyecto de Desarrollo Rural Integrado. (CIAT
Hepatic virological and histologic features in HIV carriers with chronic hepatitis B and Delta virus infections
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Chronic liver disease after seroconversion from HBeAg to anti-HBe in HBsAg carriers. Epidemiological and clinical feature
No full textNew trends in the surgical treatment of outlet obstruction: clinical and functional results of two transanal stapled techniques from a randomised controlled trial.
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