Topical application of LEVORAG® as first-line treatment for chronic anal fissures: a preliminary multicentric study.

AIM: The aim of the present study was to assess the safety and efficacy of this new topical agent as a first line treatment in patients with chronic anal fissures. METHODS: Nine centres were involved in the study. Patients with chronic anal fissures were recruited and received Levorag® for 40 days. Follow-up visits were conducted at 10, 20 and 40 days from the recruitment. Primary outcome was the healing rate, secondary outcome the reduction of pain at the end of the treatment measured with a VAS scale. RESULTS: Fifty patients completed the treatment. No adverse events were recorded. 60% of patients healed completely at the end of the treatment. In those that did not heal the reduction of mean VAS values was 60%. CONCLUSION: The use of Levorag® on patients affected by chronic anal fissures achieved in the short term results similar to those experienced by more classic local treatments without any side effect.

Congenital anomalies and variations of the bile and pancreatic ducts: magnetic resonance cholangiopancreatography findings, epidemiology and clinical significance.

PURPOSE: The objective of this paper is to document the magnetic resonance cholangiopancreatography (MRCP) findings and the epidemiology of congenital anomalies and variations of the bile and pancreatic ducts and to discuss their clinical significance. MATERIALS AND METHODS: Three-hundred and fifty patients of both sexes (150 females, 200 males, age range 0-76 years, average age 38 years) underwent MRCP for clinically suspected lithiasic, neoplastic or inflammatory disease of the bile and pancreatic ducts. Patients were imaged with a 1.5-T superconductive magnet (Magnetom Vision, Siemens, Erlangen, Germany), a four-channel phased-array body coil, breath-hold technique, with multislice T2-weighted half-Fourier acquisition single-shot turbo spin echo (HASTE), MIP reconstructions, and a single-shot T2-weighted turbo-spin-echo sequence rapid acquisition with relaxation enhancement (RARE) with different slice thicknesses. Studies in oncological patients were completed with fat saturation 3D T1 gradient-echo sequences during the intravenous injection of gadolinium diethylene triamine pentaacetate acid (DTPA) (0.2 ml/kg). RESULTS: MRCP demonstrated recurrent and therefore normal bile and pancreatic ducts in 57% of patients. In the remaining 42.3%, it documented anatomical variants (41%) and congenital anomalies (1.3%). Variants of the intrahepatic bile duct were seen in 21% of cases: crossover anomaly (6.7%), anterior branch of the right hepatic duct draining the IV and VII segments that flow together with the left bile duct (3.1%) and anterior and posterior branches of the right hepatic duct that flow together with the common hepatic duct (3.3%). Variants of the extrahepatic bile ducts were present in 8.8% of patients: low insertion of the cystic duct into the common hepatic duct (4.5%), emptying of the cystic duct into the right hepatic duct (2.7%) and a second-order large branch draining into the cystic duct (1.6%). MRCP identified a double gall bladder in 3% of patients and anatomical variants of the biliopancreatic system in 8.2%: pancreas divisum (5.2%) and a long sphincter of Oddi (3%). Finally, congenital anomalies were diagnosed in 1.3% of cases: bile duct cysts (0.3%), atresia of the bile ducts (0.3%) and multiple biliary hamartomatosis (0.7%). CONCLUSIONS: The congenital anomalies and anatomical variants of the bile and pancreatic ducts present a complex spectrum of frequent alterations, which are worthy of attention in both the clinical and surgical settings and are readily identified by MRCP.

IRES interaction with translation initiation factors: functional characterization of novel RNA contacts with eIF3, eIF4B, and eIF4GII.

Translation initiation promoted by picornavirus internal ribosome entry site (IRES) elements is dependent on the association of specific IRES sequences to the initiation factor eIF4G. However the RNA determinants interacting with other components of the translational machinery are still unknown. In this study, we have identified novel RNA-protein interactions between the foot-and-mouth disease virus (FMDV) IRES and three translation initiation factors. A doublet of 116/110 kDa that crosslinked to the FMDV IRES is a component of eIF3. We show here that domain 5 holds the preferential binding site for eIF3, although this complex initiation factor can establish multiple contacts with the IRES structure. We have also identified the phylogenetically conserved hairpin of domain 5 as the RNA motif responsible for eIF4B interaction. Mutation of this stem-loop structure abrogated eIF4B, but not eIF3, binding to the IRES. Remarkably, IRES mutants severely affected in their interaction with eIF4B showed a mild reduction in IRES activity when tested in the context of a bicistronic expression vector in transfected cells. Finally, we provide evidence of the interaction of eIF4GII with FMDV IRES, the RNA determinants for this interaction being shared with its functional homolog eIF4GI. The FMDV Lb protease generated a C-terminal fragment of eIF4GII that binds to the IRES as efficiently as the intact protein. Competition experiments showed that titration of eIF4B or p110/116 interaction with the FMDV IRES required a large excess of competitor relative to eIF4G, strongly suggesting that eIF4G-IRES interaction is a limiting factor to titrate the IRES. Comparative analysis of the activity of IRES mutants affected in domains 4 and 5 regarding their pattern of RNA-protein complex formation demonstrates that while binding of eIF4B with the FMDV IRES is dispensable, interaction of eIF4G is a central feature of the activity of this element.

Interaction of the eIF4G initiation factor with the aphthovirus IRES is essential for internal translation initiation in vivo.

The strategies developed by internal ribosome entry site (IRES) elements to recruit the translational machinery are poorly understood. In this study we show that protein-RNA interaction of the eIF4G translation initiation factor with sequences of the foot-and-mouth disease virus (FMDV) IRES is a key determinant of internal translation initiation in living cells. Moreover, we have identified the nucleotides required for eIF4G-RNA functional interaction, using native proteins from FMDV-susceptible cell extracts. Substitutions in the conserved internal AA loop of the base of domain 4 led to strong impairment of both eIF4G-RNA interaction in vitro and IRES-dependent translation initiation in vivo. Conversely, substitutions in the vicinity of the internal AA loop that did not impair IRES activity retained their ability to interact with eIF4G. Direct UV-crosslinking as well as competition assays indicated that domains 1-2, 3, and 5 of the IRES did not contribute to this interaction. In agreement with this, binding to domain 4 alone was as efficient as to the full-length IRES. The C-terminal fragment of eIF4G, proteolytically processed by the FMDV Lb protease, was sufficient to interact with the IRES or to its domain 4 alone. Additionally, we show here that binding of the eIF4B initiation factor to the IRES required domain 5 sequences. Moreover, eIF4G-IRES interaction was detected in the absence of eIF4B-IRES binding, suggesting that both initiation factors interact with the 3' region of the IRES but use different residues. The strong correlation found between eIF4G-RNA interaction and IRES activity in transfected cells suggests that eIF4G acts as a linker to recruit the translational machinery in IRES-dependent initiation.

Internal initiation of translation efficiency in different hepatitis C genotypes isolated from interferon treated patients.

Initiation of translation of hepatitis C viral RNA occurs internally and it is mediated by a segment of about 330 nucleotides termed Internal Ribosome Entry Site (IRES) located in the 5' end region. While being the most conserved part of the genome, this region also accumulates nucleotide substitutions which are often covariant. In this study we have examined the activity and sequence variation of IRES elements belonging to genotypes 1b, 2a/2c and 3a in patients that responded or not to interferon therapy. The substitutions found in the IRES region analyzed were predicted to maintain the secondary structure of the RNA. Comparison of their efficiency to promote internal initiation of translation in bicistronic constructs supported the conclusion that for both 1b and 3a genotypes, response to interferon therapy and IRES activity are unrelated, although sequence homology was not always found among isolates from patients with different type of response. IRES activity of the studied genotypes varied about 4-fold under the conditions used in our in vivo assays depending on the cell line used for transfection. Such differences were not evidenced in vitro suggesting that the differences observed depend on trans-acting factors present in the transfected cell.

Involvement of the aphthovirus RNA region located between the two functional AUGs in start codon selection.

Initiation of translation in picornavirus RNAs occurs internally, mediated by an element termed internal ribosome entry site (IRES). In the aphthovirus RNA, the IRES element directs translation initiation at two in-frame AUGs separated by 84 nucleotides. We have found that bicistronic constructs that contained the IRES element followed by the fragment including the aphthovirus start codons in front of the second gene mimicked the translation initiation pattern of viral RNA observed in infected cells. In those constructs, the frequency of initiation at the first AUG was increased by a sequence context that resembled the favorable consensus for cap-dependent translation, although initiation at the second site was always preferred. In addition, we have found that initiation at the second start codon was not diminished under conditions in which the first initiation codon was blocked by antisense oligonucleotide interference. Interestingly, mutations that positioned the second AUG out-of-frame with the first AUG did not interfere with the frequency of initiation at the second one. On the contrary, IRES-dependent translation initiation in bicistronic constructs lacking the sequences present between functional AUGs in the viral RNA was sensitive to the presence of out-of-frame initiator codons and hairpins in the spacer region. This remarkable difference in start codon recognition was due to the nucleotide composition of the RNA that separated the IRES from the initiator codon. Thus our results indicate that the region located in the aphthovirus RNA between functional AUGs is involved in start codon recognition, strongly favoring selection of the second start AUG as the main initiator codon.

Parameters influencing translational efficiency in aphthovirus IRES-based bicistronic expression vectors.

Initiation of translation in picornavirus RNAs occurs internally, mediated by an internal ribosome entry site (IRES) element. This property has been exploited to coexpress proteins from a single bicistronic transcription unit in eukaryotic cells. The region that separates the IRES element from the authentic initiator codon of the second gene plays an important role in the translation efficiency of this cistron. In the present report, we have analyzed the effect of sequence modifications in this region on the translation efficiency directed by the foot-and-mouth disease (FMDV) IRES in bicistronic expression vectors. Insertion of various sequences, which contained additional start codons and/or the capacity to form hairpins immediately downstream of the 3' border of the IRES, strongly reduced the translation efficiency of the second gene in bicistronic RNAs. Interestingly, an increase of distance per se did not have a deleterious effect on translation efficiency. The bicistronic vector studied here tolerated 95 nucleotides between the 3' border of the IRES and the authentic start codon, provided that out-of-frame AUG codons or hairpins were not present in this RNA segment. These results indicate that FMDV-derived bicistronic constructs are extremely well suited for use in eukaryotic expression vectors.

Conserved structural motifs located in distal loops of aphthovirus internal ribosome entry site domain 3 are required for internal initiation of translation.

A comparison of picornavirus internal ribosome entry site (IRES) secondary structures revealed the existence of conserved motifs located on loops. We have carried out a mutational analysis to test their requirement for IRES-driven translation. The GUAA sequence, located in the aphthovirus 3A loop, did not tolerate substitutions that disrupt the GNRA motif. Interestingly, this motif was found at similar positions in all picornavirus IRESs, suggesting that it may form part of a tertiary-structure element. The RAAA tetranucleotide located in the 3B loop was conserved only in cardiovirus and aphthovirus. A mutational analysis of the RAAA motif revealed that activities of 3B loop mutants correlated with both the presence of a sequence close to CAAA at the new 3B loop and the absence of reorganization of the 3B and 3C stem-loops. In support of this conclusion, insertion of a large number of nucleotides close to the 3B loop, which was predicted to reorganize the 3B-3C stem-loop structure, led to defective IRES elements. We conclude that the aphthovirus IRES loops located at the most distal part of domain 3, which carries GNRA and RAAA motifs, are essential for IRES function.

Esboco de uma tecnica de atendimento para pacientes psiquiatricos de baixa renda. / Outline of a technic for psychiatric patients care with low income

Os autores, a partir da experiencia pessoal com clientela de baixa renda, tracam alguns pressupostos teoricos e apresentam uma tecnica adaptada as situacoes-limite deste grupo. Estabelecem que o psiquiatra deve assumir uma atitude ativa e dirigir seus esforcos para as expectativas dos pacientes que relatam seus sofrimentos em termos de realidade externa, e que nao se encontram habituados ao processo autocritico, o que se evidencia no carater pratico e imediato de seus atos. O psiquiatra deve ser sensivel as necessidades dos pacientes e buscar informacoes simples relacionadas com a situacao atual, evitando investigacao intensa de problemas anteriores. Muito embora nao apresentem disposicao para pensar introspectivamente desejam falar de suas dificuldades da vida diaria, quando se percebe que predominam problemas interpessoais e dificuldades financeiras.Atraves de exemplos, os autores ilustram a aplicacao do apoio, orientacao, esclarecimento e solucao concreta. Finalizam comunicando que estao concluindo um projeto de pesquisa experimental que procurara testar a hipotese da intervencao ativa com vistas a colher dados mais consistentes quanto a satisfacao do paciente com o tratamento e os efeitos sobre a sintomarologia apresentada