Quantitative Proteomic Analysis Reveals Metabolic Alterations, Calcium Dysregulation, And Increased Expression Of Extracellular Matrix Proteins In Laminin α2 Chain-deficient Muscle.

Congenital muscular dystrophy with laminin α2 chain deficiency (MDC1A) is one of the most severe forms of muscular disease and is characterized by severe muscle weakness and delayed motor milestones. The genetic basis of MDC1A is well known, yet the secondary mechanisms ultimately leading to muscle degeneration and subsequent connective tissue infiltration are not fully understood. In order to obtain new insights into the molecular mechanisms underlying MDC1A, we performed a comparative proteomic analysis of affected muscles (diaphragm and gastrocnemius) from laminin α2 chain-deficient dy(3K)/dy(3K) mice, using multidimensional protein identification technology combined with tandem mass tags. Out of the approximately 700 identified proteins, 113 and 101 proteins, respectively, were differentially expressed in the diseased gastrocnemius and diaphragm muscles compared with normal muscles. A large portion of these proteins are involved in different metabolic processes, bind calcium, or are expressed in the extracellular matrix. Our findings suggest that metabolic alterations and calcium dysregulation could be novel mechanisms that underlie MDC1A and might be targets that should be explored for therapy. Also, detailed knowledge of the composition of fibrotic tissue, rich in extracellular matrix proteins, in laminin α2 chain-deficient muscle might help in the design of future anti-fibrotic treatments. All MS data have been deposited in the ProteomeXchange with identifier PXD000978 (http://proteomecentral.proteomexchange.org/dataset/PXD000978).

Chemo-, Regio- And Stereoselective Heck Arylation Of Allylated Malonates: Mechanistic Insights By Esi-ms And Synthetic Application Toward 5-arylmethyl-γ-lactones.

We describe herein a general method for the controlled Heck arylation of allylated malonates. Both electron-rich and electron-poor aryldiazonium salts were readily employed as the aryl-transfer agents in good yields and in high chemo-, regio-, and stereoselectivity without formation of decarboxylated byproducts. Reaction monitoring via ESI-MS was used to support the formation of chelated Pd species through the catalytic cycle. Additionally, some Heck adducts were successfully used in the total synthesis of pharmacologically active γ-lactones.

Sim-xl: A Powerful And User-friendly Tool For Peptide Cross-linking Analysis.

Chemical cross-linking has emerged as a powerful approach for the structural characterization of proteins and protein complexes. However, the correct identification of covalently linked (cross-linked or XL) peptides analyzed by tandem mass spectrometry is still an open challenge. Here we present SIM-XL, a software tool that can analyze data generated through commonly used cross-linkers (e.g., BS3/DSS). Our software introduces a new paradigm for search-space reduction, which ultimately accounts for its increase in speed and sensitivity. Moreover, our search engine is the first to capitalize on reporter ions for selecting tandem mass spectra derived from cross-linked peptides. It also makes available a 2D interaction map and a spectrum-annotation tool unmatched by any of its kind. We show SIM-XL to be more sensitive and faster than a competing tool when analyzing a data set obtained from the human HSP90. The software is freely available for academic use at http://patternlabforproteomics.org/sim-xl. A video demonstrating the tool is available at http://patternlabforproteomics.org/sim-xl/video. SIM-XL is the first tool to support XL data in the mzIdentML format; all data are thus available from the ProteomeXchange consortium (identifier PXD001677).

The Total Synthesis Of (-)-cryptocaryol A.

A stereoselective total synthesis of (-)-cryptocaryol A () is described. Key features of the 17-step route include the use of three boron-mediated aldol reaction-reduction sequences to control all stereocenters and an Ando modification of the Horner-Wadsworth-Emmons olefination that permitted the installation of the Z double bond of the α-pyrone ring.

Plastias de estenoses de intestino delgado na doença de Crohn: resultados imediatos e tardios

BACKGROUND: Strictureplasty is an alternative surgical procedure for Crohn?s disease, particulary in patients with previous resections or many intestinal stenosis. AIM: To analyze surgical complications and clinical follow-up in patients submitted to strictureplasty secondary to Crohn?s disease. METHODS: Twenty-eight patients (57.1% male, mean age 33.3 years, range 16-54 years) with Crohn?s disease and intestinal stenosis (small bowel, ileocecal region and ileocolic anastomosis) were submitted to strictureplasty, at one institution, between September 1991 and May 2004. Thirteen patients had previous intestinal resections. The mean follow-up was 58.1 months. A total of 116 strictureplasties were done (94 Heineke-Mikulicz - 81%, 15 Finney - 13%, seven side-to-side ileocolic strictureplasty - 6%). Three patients were submitted to strictureplasty at two different surgical procedures and two in three procedures. RESULTS: Regarding to strictureplasty, postoperative complication rate was 25% and mortality was 3.6%. Early local complication rate was 57.1%, with three suture leaks (10.7%) and late complication was present in two patients, both with incisional hernial and enterocutaneous fistulas (28.6%). Patients remained hospitalized during a medium time of 12.4 days. Clinical and surgical recurrence rates were 63% and 41%, respectively. Among the patients submitted to another surgery, two patients had two more operations and one had three. Recurrence rate at strictureplasty site was observed in 3.5%, being Finney technique the commonest one. Presently, 19 patients had been asymptomatic with the majority of them under medical therapy. CONCLUSION: Strictureplasties have low complication rates, in spite of having been done at compromised site, with long term pain relief. Considering the clinical course of Crohn?s disease, with many patients being submitted to intestinal resections, strictureplasties should be considered as an effective surgical treatment to spare long intestinal resections.

Complicações imediatas e tardias após cirurgia de reservatório ileal na polipose adenomatosa familiar

BACKGROUND: Restorative proctocolectomy is the procedure of choice to treat familial adenomatous polyposis, however it can be associated to short-term and long-term postoperative complications. AIM: To evaluate the occurrence of complications related to the surgical treatment of familial adenomatous polyposis with ileal pouch technique. METHODS: Retrospective study of 69 patients with familial adenomatous polyposis after rectocolectomy with ileal reservoir between 1984 and 2006, operated on Coloproctology Group, Medical Sciences Faculty, State University of Campinas, Campinas, SP, Brazil. The median follow-up period was 82 (2-280) months. Data obtained were surgical techniques and postoperative complications. RESULTS: The morbidity and mortality were 63.8% and 2.9%, respectively. The most frequent complications were small-bowel obstruction (17.4%), anastomotic stricture (15.9%) and pelvic sepsis (10.1%). Acute ischemia of the ileal pouch (4.3%), pouchitis (2.9%) and ileal pouch-related fistula (2.9%) had poorer frequency than others. CONCLUSIONS: The morbid-mortality was similar to the literature?s data and it is acceptable for a complex surgery in two terms like the ileal reservoir-anal anastomosis. The small-bowel obstruction was the most frequent complication. However, ischemia of the reservoir, pouchitis and pelvic sepsis were important complications and was related to the failure of the ileal reservoir.

Resultados da cirurgia de reservatórios ileais em pacientes com doença de Crohn

BACKGROUND: Total rectocolectomy and ileal pouch-anal anastomosis is the choice surgical procedure for patients with ulcerative colitis. In cases of Crohn's disease post-operative diagnosis, it can be followed by pouch failure. AIM: To evaluate ileal pouch-anal anastomosis long-term outcome in patients with Crohn's disease. METHODS: Between February 1983 and March 2007, 151 patients were submitted to ileal pouch-anal anastomosis by Campinas State University Colorectal Unit, Campinas, SP, Brazil, 76 had pre-operative ulcerative colitis diagnosis and 11 had post-operative Crohn's disease diagnosis. Crohn's disease diagnosis was made by histopathological biopsies in nine cases, being one in surgical specimen, two cases in rectal stump, small bowel in two cases, ileal pouch in three and in perianal abscess in one of them. The median age was 30.6 years and eight (72.7%) were female. RESULTS: All patients had previous ulcerative colitis diagnosis and in five cases emergency colectomy was done by toxic megacolon. The mean time until of Crohn's disease diagnosis was 30.6 (6-80) months after ileal pouch-anal anastomosis. Ileostomy closure was possible in 10 cases except in one that had ileal pouch fistula, perianal disease and small bowel involvement. In the long-term follow-up, three patients had perineal fistulas and one had also a pouch-vaginal fistula. All of them were submitted to a new ileostomy and one had the pouch excised. Another patient presented pouch-vaginal fistula which was successfully treated by mucosal flap. Three patients had small bowel involvement and three others, pouch involvement. All improved with medical treatment. Presently, the mean follow-up is 76.5 months and all patients are in clinical remission, and four have fecal diversion. The remaining patients have good functional results with 6-10 bowel movements/day. CONCLUSION: Crohn's disease diagnosis after ileal pouch-anal anastomosis for ulcerative colitis may be usual and later complications such fistulas and stenosis are common. However, when left in situ ileal pouch is associated with good function.

Aplicação do escore MELD em pacientes submetidos a transplante de fígado: análise retrospectiva da sobrevida e dos fatores preditivos a curto e longo prazo

BACKGROUND: The model for end-stage liver disease (MELD) was developed to predict short-term mortality in patients with cirrhosis. There are few reports studying the correlation between MELD and long-term posttransplantation survival. AIM: To assess the value of pretransplant MELD in the prediction of posttransplant survival. METHODS: The adult patients (age >18 years) who underwent liver transplantation were examined in a retrospective longitudinal cohort of patients, through the prospective data base. We excluded acute liver failure, retransplantation and reduced or split-livers. The liver donors were evaluated according to: age, sex, weight, creatinine, bilirubin, sodium, aspartate aminotransferase, personal antecedents, brain death cause, steatosis, expanded criteria donor number and index donor risk. The recipients' data were: sex, age, weight, chronic hepatic disease, Child-Turcotte-Pugh points, pretransplant and initial MELD score, pretransplant creatinine clearance, sodium, cold and warm ischemia times, hospital length of stay, blood requirements, and alanine aminotransferase (ALT >1,000 UI/L = liver dysfunction). The Kaplan-Meier method with the log-rank test was used for the univariable analyses of posttransplant patient survival. For the multivariable analyses the Cox proportional hazard regression method with the stepwise procedure was used with stratifying sodium and MELD as variables. ROC curve was used to define area under the curve for MELD and Child-Turcotte-Pugh. RESULTS: A total of 232 patients with 10 years follow up were available. The MELD cutoff was 20 and Child-Turcotte-Pugh cutoff was 11.5. For MELD score > 20, the risk factors for death were: red cell requirements, liver dysfunction and donor's sodium. For the patients with hyponatremia the risk factors were: negative delta-MELD score, red cell requirements, liver dysfunction and donor's sodium. The regression univariated analyses came up with the following risk factors for death: score MELD > 25, blood requirements, recipient creatinine clearance pretransplant and age donor >50. After stepwise analyses, only red cell requirement was predictive. Patients with MELD score < 25 had a 68.86%, 50,44% and 41,50% chance for 1, 5 and 10-year survival and > 25 were 39.13%, 29.81% and 22.36% respectively. Patients without hyponatremia were 65.16%, 50.28% and 41,98% and with hyponatremia 44.44%, 34.28% and 28.57% respectively. Patients with IDR > 1.7 showed 53.7%, 27.71% and 13.85% and index donor risk <1.7 was 63.62%, 51.4% and 44.08%, respectively. Age donor > 50 years showed 38.4%, 26.21% and 13.1% and age donor <50 years showed 65.58%, 26.21% and 13.1%. Association with delta-MELD score did not show any significant difference. Expanded criteria donors were associated with primary non-function and severe liver dysfunction. Predictive factors for death were blood requirements, hyponatremia, liver dysfunction and donor's sodium. CONCLUSION: In conclusion MELD over 25, recipient's hyponatremia, blood requirements, donor's sodium were associated with poor survival.

Desmoid tumor in patients with familial adenomatous polyposis

CONTEXT: Desmoid tumors constitute one of the most important extraintestinal manifestations of familial adenomatous polyposis. The development of desmoids is responsible for increasing morbidity and mortality rates in cases of familial adenomatous polyposis. OBJECTIVES: To evaluate the occurrence of desmoid tumors in familial adenomatous polyposis cases following prophylactic colectomy and to present patient outcome. METHODS: Between 1984 and 2008, 68 patients underwent colectomy for familial adenomatous polyposis at the School of Medical Sciences Teaching Hospital, University of Campinas, SP, Brazil. Desmoid tumors were found in nine (13.2%) of these patients, who were studied retrospectively by consulting their medical charts with respect to clinical and surgical data. RESULTS: Of nine patients, seven (77.8%) were submitted to laparotomy for tumor resection. Median age at the time of surgery was 33.9 years (range 22-51 years). Desmoid tumors were found in the abdominal wall in 3/9 cases (33.3%) and in an intra-abdominal site in the remaining six cases (66.7%). Median time elapsed between ileal pouch-anal anastomosis and diagnosis of desmoid tumor was 37.5 months (range 14-60 months), while the median time between colectomy with ileorectal anastomosis and diagnosis was 63.7 months (range 25-116 months). In 6/9 (66.7%) patients with desmoid tumors, the disease was either under control or there was no evidence of tumor recurrence at a follow-up visit made a mean of 63.1 months later (range 12-240 months). CONCLUSIONS: Desmoid tumors were found in 13.2% of cases of familial adenomatous polyposis following colectomy; therefore, familial adenomatous polyposis patients should be followed-up and surveillance should include abdominal examination to detect signs and symptoms. Treatment options include surgery and clinical management with antiestrogens, antiinflammatory drugs or chemotherapy.

Variation of the anal resting pressure induced by postexpiratory apnea effort in patients with constipation

CONTEXT: Intestinal constipation - a common symptom among the general population - is more frequent in women. It may be secondary to an improper diet or organic or functional disturbances, such as dyskinesia of the pelvic floor. This is basically characterized by the absence of relaxation or paradoxical contraction of the pelvic floor and anal sphincter during evacuation. OBJECTIVE: To analyze, by manometric data, the anal pressure variation at rest, during evacuation effort by using the Valsalva maneuver and forced post-expiratory apnea in subjects with secondary constipation. METHODS: Twenty-one patients (19 females - 90.4%) with a mean age of 47.5 years old (23-72) were studied. The diagnosis was performed using anorectal manometry, with a catheter containing eight channels disposed at the axial axis, measuring the proximal (1) and distal (2) portions of the anal orifice. The elevation of the pressure values in relation to the resting with the evacuation effort was present in all patients. The Agachan score was used for clinical evaluation of constipation. The variables studied were: mean anal pressure of the anal orifice for 20 seconds at rest, the effort of evacuation using Valsalva maneuver and the effort of evacuation during apnea after forced expiration, as well as the area under the curve of the manometric tracing at moments Valsalva and apnea. RESULTS: The analysis of the mean values of the anal pressure variation at rest evidenced difference between proximal and distal channels (P = 0.007), independent of the moment and tendency to differ during moments Valsalva and apnea (P = 0.06). The mean of values of the area under the manometric tracing curve showed differences between moments Valsalva and apnea (P = 0.0008), either at the proximal portion or at the distal portion of the anal orifice. CONCLUSION: The effort of evacuation associated with postexpiratory apnea, when compared with the effort associated with the Valsalva maneuver, provides lower elevation of anal pressure at rest by the parameter area under the curve.

Dos conceitos de morte aos critérios para o diagnóstico de morte encefálica

The authors present considerations about death and brain death concepts, as well the legal aspects for its diagnosis in Brazil. They also present the UNICAMP Protocol for the Diagnosis of Brain Death, revised and according with the current law, with standard techniques for the diagnostic exam. They emphasize the importance of a mature ethical position for this frequent and challenging situation.

Edema pulmonar neurogênico: relato de dois casos

Neurogenic pulmonary edema is a rare and serious complication in patients with head injury. It also may develop after a variety of cerebral insults such as subarachnoid hemorrhage, brain tumors and after epileptic seizures. Thirty six patients with severe head injury and four patients with cerebrovascular insults treated in Intensive Care Unit of HC-UNICAMP from January to September 1995 were evaluated. In this period there were two patients with neurogenic pulmonary edema, one with head injury and other with intracerebral hemorrhage. Diagnosis was made by rapid onset of pulmonary edema, severe hypoxemia, decrease of pulmonary complacence and diffuse pulmonary infiltrations, without previous history of tracheal aspiration or any other risk factor for developement of adult respiratory distress syndrom. In the first case, with severe head trauma, neurogenic pulmonary edema was diagnosed at admission one hour after trauma, associated with severe systemic inflammatory reaction, and good outcome in three days. The second case, with hemorragic vascular insult, developed neurogenic pulmonary edema the fourth day after drenage of intracerebral hematom and died.

Frequency of the different mutations causing spinocerebellar ataxia (SCA1, SCA2, MJD/SCA3 and DRPLA) in a large group of Brazilian patients

Spinocerebellar ataxia type 1 (SCA1), spinocerebellar ataxia type 2 (SCA2) and Machado-Joseph disease or spinocerebellar ataxia type 3 (MJD/SCA3) are three distinctive forms of autosomal dominant spinocerebellar ataxia (SCA) caused by expansions of an unstable CAG repeat localized in the coding region of the causative genes. Another related disease, dentatorubropallidoluysian atrophy (DRPLA) is also caused by an unstable triplet repeat and can present as SCA in late onset patients. We investigated the frequency of the SCA1, SCA2, MJD/SCA3 and DRPLA mutations in 328 Brazilian patients with SCA, belonging to 90 unrelated families with various patterns of inheritance and originating in different geographic regions of Brazil. We found mutations in 35 families (39%), 32 of them with a clear autosomal dominant inheritance. The frequency of the SCA1 mutation was 3% of all patients; and 6 % in the dominantly inherited SCAs. We identified the SCA2 mutation in 6% of all families and in 9% of the families with autosomal dominant inheritance. The MJD/SCA3 mutation was detected in 30 % of all patients; and in the 44% of the dominantly inherited cases. We found no DRPLA mutation. In addition, we observed variability in the frequency of the different mutations according to geographic origin of the patients, which is probably related to the distinct colonization of different parts of Brazil. These results suggest that SCA may be occasionally caused by the SCA1 and SCA2 mutations in the Brazilian population, and that the MJD/SCA3 mutation is the most common cause of dominantly inherited SCA in Brazil.

A pilot study of topiramate in children with Lennox-Gastaut syndrome

We conducted an open, add-on study with topiramate (TPM) as adjunctive therapy in Lennox-Gastaut syndrome (LGS), to assess the long-term efficacy and safety and to evaluate quality of life (QL) measurements in the chronic use of TPM. We studied 19 patients (11 male; age ranging from 4 to 14 years) with uncontrolled seizures receiving 2-3 anti-epileptic drugs. Patients were followed up to 36 months of treatment. A questionnaire was used to query parents about QL. Seven patients completed the study at 36 months and seizure frequency was reduced > 75% in 4, and < 50% in 3 patients. Two children became seizure free for more than 24 months. Most side effects were CNS related, with the most frequent being somnolence and anorexia. These were generally transient. One patient dropped-out due to powder in the urine. None of the patients required hospitalization. At 36 months, patients' alertness (2/7), interaction with environment (5/7), ability to perform daily activities (5/7), and verbal performance (6/7) improved on TPM. We conclude that TPM may be useful as adjunctive therapy in the treatment of LGS. The efficacy of TPM was maintained in long-term treatment in more than 40% of patients, long term safety was confirmed and QL improved on TPM.

Dyskinesia induced by phenytoin

Phenytoin is an effective antiepileptic drug, although, it can be associated with many side effects, including dyskinesia. OBJECTIVE: To describe the clinical characteristics of phenytoin induced dyskinesia. METHODS: We investigated the occurrence of involuntary movements in patients followed at our adult and pediatric epilepsy clinics during the period of one year. RESULTS: Three patients presented with phenytoin-induced dyskinesia: one adult with axial and orofacial dyskinesia, and two children with choreoathetosis. They did not have other signs of phenytoin intoxication and had complete recovery after phenytoin withdrawal. CONCLUSION: Phenytoin induced dyskinesia may occur during either chronic or initial treatment and with normal serum phenytoin levels. However, it occurs most often in patients on polytherapy, usually after increasing dosage and with toxic serum levels. Other signs of phenytoin intoxication may be present in these patients, but often the dyskinesia is the only side effect, which may delay the diagnosis and treatment. The clinical characteristics of the involuntary movements vary and may be focal or generalized, most often characterized by choreoathetosis and dyskinesias. These may last for hours, days or even years, but frequently disappear completely after phenytoin withdrawal.