Separating positional noise from neutral alignment in multicomponent statistical shape models.

Given sufficient training samples, statistical shape models can provide detailed population representations for use in anthropological and computational genetic studies, injury biomechanics, musculoskeletal disease models or implant design optimization. While the technique has become extremely popular for the description of isolated anatomical structures, it suffers from positional interference when applied to coupled or articulated input data. In the present manuscript we describe and validate a novel approach to extract positional noise from such coupled data. The technique was first validated and then implemented in a multicomponent model of the lower limb. The impact of noise on the model itself as well as on the description of sexual dimorphism was evaluated. The novelty of our methodology lies in the fact that no rigid transformations are calculated or imposed on the data by means of idealized joint definitions and by extension the models obtained from them.

Cerebral venous sinuses thrombosis in both transverse sinus and torcula: Multistep endovascular treatment and stenting.

Purpose Cerebral venous sinus thrombosis (CVST) is an unusual and potentially life-threatening condition with variable and nonspecific clinical symptoms and high morbimortality rates. Standard therapy consists of systemic anticoagulation; although there is no clear evidence about the best choice for treatment, intravenous heparin is used as the first-line treatment modality. Intravenous sinus thrombolysis can be an effective and relatively safe treatment for acutely deteriorating patients who have not responded to conventional therapy. This case report presents the possibility of endovascular treatment in multiple steps with mechanical thrombolysis with balloon, local pharmacological thrombolysis and stenting, in a patient with a severe form of CVST. Case summary A 67-year-old woman presented severe headache, agitation and confusion with diagnosis of venous sinus dural thrombosis in both lateral sinus and torcula. After 24 h there was neurological worsening evolving with seizures and numbness even after starting heparin, without sinus recanalization; CT scan showed left temporal intracerebral hemorrhage. We decided to take an endovascular approach in multiple steps. The first step was mechanical static thrombolysis with balloon; the second step was dynamic mechanical thrombolysis with a balloon partially deflated and "pulled"; the third step was local thrombolysis with Actilyse™; finally, the fourth step was angioplasty and reconstruction of the sinuses using multiple carotid stents and complete angiographic recanalization of both sinuses and torcula. After 24 h of endovascular treatment there was full clinical recovery and no tomographic complications. Conclusion This result shows that mechanical clot disruption, intrasinus thrombolysis and reconstruction of wall sinuses with stenting can be an endovascular option in the severe form of CVST with intracerebral hemorrhage and rapid worsening of neurological symptoms. Although this type of treatment can re-channel the occluded sinuses, further comparative and randomized studies are needed to clarify its efficacy versus other therapeutic modalities.

Causes of organ donation failure in Brazil.

INTRODUCTION: There has been a great improvement in transplantation medicine in Brazil in the last 2 decades. However, there remain several barriers regarding notification of brain and cardiac death as well as completion of the donation process. METHODS: This retrospective study was performed between January 2008 and December 2010. We reviewed all deaths in a University Hospital, observing the causes of non-notification to the State Transplantation Authority and non-donations. RESULTS: There were 41 notifications of brain death resulting in donation in only 19.5% of those cases. Cardiac death was diagnosed in 21 patients, resulting in 52.4% donations. The main cause for non-donation were family refusal (37.2%), infectious diseases (30.2%), and clinical contraindications (32.6%). Most of the missed possible donors occurred during the night (54.8%) and in the emergency room (80.9%). CONCLUSION: There is an urgent need for better education of the Brazilian population about organ donation and brain death definitions. Other identified problems include lack of uniformity in brain death determinations among hospitals, rigid contraindications to donation in the State of Parana, physician unawareness or disbelief about brain death diagnostic criteria, and lack of structure of our Hospital.

Subacute inflammatory demyelinating polyneuropathy.

OBJECTIVE: To report the clinical, electrophysiologic, and histologic characteristics of subacute inflammatory demyelinating polyneuropathy (SIDP) and to present the diagnostic criteria of this disease. METHODS: For a diagnosis of "definite SIDP," there were four mandatory criteria: 1) progressive motor and/or sensory dysfunction consistent with neuropathy in more than one limb with time to nadir between 4 and 8 weeks, 2) electrophysiologic evidence of demyelination in at least two nerves, 3) no other etiology of neuropathy, and 4) no relapse on adequate follow-up. Supportive criteria included high spinal fluid protein level (>55 mg/dL) and inflammatory cells in the nerve biopsy. A diagnosis of "probable SIDP" required progression of demyelinating neuropathy over a 4- to 8-week period. RESULTS: Sixteen definite SIDP patients were identified among 29 probable SIDP patients. An antecedent infection was found in 38% of cases. The two most common neuropathy types were a symmetric motor-sensory neuropathy and a pure motor neuropathy. Cranial nerve deficits and respiratory failure were rare. Spinal fluid protein was high in 93% of cases. Demyelination was documented by the motor nerve conduction in 88% of cases and by the near-nerve needle sensory nerve conduction in two cases. Almost all patients were treated with prednisone and some with additional immunotherapies. Complete recovery was achieved in 69% of cases and partial recovery in others. Definite SIDP had all the characteristics of CIDP with three exceptions: a higher rate of antecedent infection, no relapse rate, and a high rate of recovery to normal. CONCLUSION: Subacute inflammatory demyelinating polyneuropathy is a definite entity bridging the gap between Guillain-Barré syndrome and chronic inflammatory demyelinating polyneuropathy.

Adjuvant activity of a nontoxic mutant of Escherichia coli heat-labile enterotoxin on systemic and mucosal immune responses elicited against a heterologous antigen carried by a live Salmonella enterica serovar Typhimurium vaccine strain.

Systemic and mucosal antibody responses against both the major subunit of colonization factor antigen I (CFA/I) of enterotoxigenic Escherichia coli (ETEC) and the somatic lipopolysaccharide expressed by recombinant bivalent Salmonella vaccine strains were significantly enhanced by coadministration of a detoxified derivative with preserved adjuvant effects of the ETEC heat-labile toxin, LT((R192G)). The results further support the adjuvant effects of LT((R192G)) and represent a simple alternative to improve responses against passenger antigens expressed by orally delivered Salmonella vaccine strains.

Short interrupted palindromes on the extragenic DNA of Escherichia coli K-12, Haemophilus influenzae and Neisseria meningitidis.

MOTIVATION: The importance of the various kinds of repetitive nucleotide sequences for the workings of bacterial DNA has been widely recognized. This work is concerned with the distribution of a particular group of repetitive sequences, the short-sequenced interrupted extragenic palindromes, on the genetic maps of Escherichia coli K-12, Haemophilus influenzae Rd and Neisseria meningitidis Z2491 and MC58. A tool has been developed based upon a statistical hypothesis test taking into account the markovian structure of random sequences in order to determine the non-random character of extragenic palindromes. RESULTS: Totals of 7631, 12904, 4722 and 5477 non-random short interrupted palindromes have been found on the E.coli, H.influenzae, and N.meningitidis serogroup A and serogroup B genomes, respectively. Their distribution patterns on the respective genomes vary according to the bacterial species considered. Based on their position on the genome, palindromes could be distinguished as those which integrate longer, repetitive sequences; those which stand in isolation, and still others are associated to specific genome sites. AVAILABILITY: The complete list of the observed palindromes is available at the site http://www/lncc.br/~atrv. CONTACT: atrv@lncc.br

Antibody response in mice immunized with a plasmid DNA encoding the colonization factor antigen I of enterotoxigenic Escherichia coli.

The colonization factor antigen I (CFA/I) is one of the most epidemiologically relevant enterotoxigenic Escherichia coli (ETEC) fimbrial adhesins, which mediates the binding to human small intestine epithelium. A recombinant eukaryotic expression plasmid, pRECFA, encoding the CFA/I protein fused to the glycoprotein D of herpes simplex type 1 virus, was used to generate an antibody response in a murine model following intramuscular inoculation of purified DNA. Eukaryotic cells (BHK-21) transfected with pRECFA expressed the CFA/I protein in vitro, as revealed by Western blot and immunofluorescence microscopy. Administration of a single pRECFA 100-microg dose induced a long-term CFA/I-specific antibody response in BALB/c mice composed mainly of IgG and, to a lesser extent, IgA isotypes. The major CFA/I-specific IgG subclass was IgG2a, suggesting a Th-1-type immune response. A second dose with the same amount of purified DNA, given 2 weeks later, caused a booster effect on the immunoglobulin levels, but did not qualitatively alter the isotypes and subclasses of the induced antibody response. Immunization with different amounts of purified DNA and/or number of doses showed that maximal transient CFA/I-specific antibody levels could be obtained after two 100-microg doses of pRECFA given 2 weeks apart, but long-term antibody levels were similar.

Epitope specificity of a monoclonal antibody generated against the dissociated CFA/I fimbriae of enterotoxigenic Escherichia coli.

A monoclonal antibody (MAb 84) raised against the dissociated CFA/I fimbriae of enterotoxigenic Escherichia coli was characterized with regard to antigen binding and epitope specificity. Enzyme-linked immunosorbent assay (ELISA) showed that MAb 84 had higher affinity to CFA/I subunits than to intact CFA/I fimbriae and recognized a Salmonella flagellin carrying an insert corresponding to amino acids 32 to 45 of the CFA/I subunit. Fine epitope mapping based on the Pepscan technique showed that the peptide 39TFESY43, derived from the sequence of the mature CFA/I subunit, was specifically recognized by MAb 84. The 39TFESY43 sequence is probably not accessible on the surface of the native CFA/I fimbriae since MAb 84 did not bind to intact fimbriae as evaluated in inhibition ELISA tests. Moreover, MAb 84 did not agglutinate fimbriated ETEC cells nor inhibit CFA/I-mediated hemagglutination or the adhesion to Caco-2 cells.

Clonal nature of enterotoxigenic Escherichia coli serotype O6:H16 revealed by randomly amplified polymorphic DNA analysis.

The genetic relatedness among 29 enterotoxigenic Escherichia coli strains of serotype O6:H16 was investigated by randomly amplified polymorphic DNA (RAPD) analysis. The strains were isolated in different parts of the world, displayed CS1-CS3 or CS2-CS3 profiles, and expressed heat-labile toxin (LT) and heat-stable toxin; a single strain expressed only LT. Ten RAPD types were distinguished and showed significant similarity, having on average 82% of the amplified bands in common. These results indicated that, irrespective of the different geographical origin or virulence factors, these strains belonged to a widespread clonal group.

Identification of yebG as a DNA damage-inducible Escherichia coli gene.

The nucleotide sequence upstream of the Escherichia coli yebG gene presents features similar to those found in SOS system regulatory sites (putative SOS box, -10 and -35 promoter boxes and a ribosome binding site). Operon fusion assays demonstrate now that this region controls transcription in a recA-, lexA-dependent way and that the reporter gene expression is inducible by DNA damage consequent to mitomycin C treatment. Increased expression does not result from an increase in plasmid copy number. These results indicate that yebG is a novel SOS regulon gene. The yebG product is predicted to be a 96 amino acid residue, 10.7 kDa protein whose function is not yet known. Unlike other SOS genes, the construct carrying the yebG regulatory region is not stationary phase inducible.

Random amplification of polymorphic DNA reveals serotype-specific clonal clusters among enterotoxigenic Escherichia coli strains isolated from humans.

The genetic diversity of 47 enterotoxigenic Escherichia coli (ETEC) strains of serotypes O6:H16, O27:H7, O29:H21, O128ac:H12, and O153:H45, previously isolated from diarrheic patients in Brazil over a period of 15 years, was investigated by random amplification of polymorphic DNA (RAPD). Informative band arrays were obtained with three 10-mer primers with G+C contents of 50, 60, and 70%. Based on the combination of the band profiles generated by the three primers 22 RAPD types were detected, and 5 major clonal clusters, each one with at least 80% identical bands, were established. The clonal clusters corresponded to strains having the same serotype which, in most cases, also had the same virulence factors (colonization factors and toxin types) and outer membrane protein and lipopolysaccharide sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles. The results suggested a correlation between phenotypic properties and genetic relatedness of ETEC isolates of human origin and indicated that a reduced number of clonally related strains are found in areas of ETEC endemicity in Brazil. Moreover, the RAPD technique revealed intraserotype-specific variations, undetectable by the combination of several phenotypic typing methods, among the ETEC strains analyzed. These results show that RAPD typing represents a useful tool for population genetics as well as for epidemiological studies of ETEC.

Clonal relationships among Escherichia coli serogroup O6 isolates based on RAPD.

The genetic diversity in a group of Escherichia coli strains belonging to serogroup O6 but expressing different H antigens was investigated by random amplification of polymorphic DNA (RAPD). Isolates of serotypes H16, H1, H31, and non-motile (NM) strains were typed using a set of 3 primers with different G + C contents. The amplified band arrays allowed the identification of 3 main clonal clusters corresponding to each O:H serotype analyzed. Based on their RAPD profiles NM strains could be assigned to either H1 or H31 serotypes. The results indicate that the flagellar antigen and the RAPD fingerprint represent reliable clonal markers in this E. coli group.

Characterization of enterotoxigenic Escherichia coli by random amplification of polymorphic DNA.

Two enterotoxigenic Escherichia coli (ETEC) strains (H10407 and 4011-1) were characterized by random amplification of polymorphic DNA (RAPD) profiles using 10-mer oligonucleotides with diverse GC content. All tested primers yielded arrays of amplified DNA products ranging in size from 200 to 3000 bp. The effects of annealing temperature, template concentration and GC content of the primers were evaluated and an optimal reaction procedure was established. Application of the RAPD analysis to ten ETEC strains belonging to five different serotypes showed that strains of the same serotype shared identical or almost identical band profiles, suggesting a similar genetic composition. The use of RAPD profiles as a tool in epidemiological analysis of ETEC is discussed.

Genomic variation in Trypanosoma cruzi clonal cultures.

Spontaneous changes in restriction DNA profiles and pulsed-field gel electrophoresis (PFGE) patterns, along with a concomitant loss of infectivity, were observed in infective clones of Trypanosoma cruzi strain Y either following a number of passages during the exponential growth phase of after subcloning in liver infusion tryptone (LIT) medium using as the probe a genomic fragment of the parasite (pMYP16), indicating naturally occurring rearrangements of DNA sequences. No variation could be detected when the genomic DNA was probed with conserved T. cruzi tubulin and actin genes. There was no correlation between such rearrangements and the life-cycle forms of the parasites, since trypomastigote forms showed the same karyotype and hybridization patterns as did epimastigote forms. The variations observed could be reverted and infectivity, recovered after inoculation of the parasites in newborn mice.

Interactions of Yersinia pestis penicillin-binding proteins with beta-lactam antibiotics.

The affinities of six major penicillin-binding proteins (PBPs) of Yersinia pestis EV76 to different beta-lactam antibiotics were determined. The results indicate that, similar to their counterparts in Escherichia coli, PBP2 and PBP3 are the lethal targets of amdinocillin and furazlocillin, respectively. The PBP contents of four additional Y. pestis strains and the morphological effects produced by some beta-lactam antibiotics are also reported.

Changes in Trypanosoma cruzi kinetoplast DNA minicircles induced by environmental conditions and subcloning.

Reversible changes in kinetoplast DNA (kDNA) minicircles sequences were observed in clones of Trypanosoma cruzi strain Y, following a number of passages during exponential growth phase or after subcloning in blood-free medium. kDNA restriction patterns of clones were similar to those of the original uncloned strain, while subclones presented distinct kDNA restriction patterns. Homology experiments demonstrated strong hybridization between kDNA with the same electrophoretic mobility patterns while only weak signals were observed with kDNA of different patterns. The changes observed, which are unprecedented in T. cruzi clones, characterize transkinetoplastidy, and seem to be associated with similarly reversible changes both in zymodeme and in infectivity.

Reversible changes in the isoenzyme electrophoretic mobility pattern and infectivity in clones of Trypanosoma cruzi.

The stability of zymodemes in clonal cultures of Trypanosoma cruzi derived from strain Y was followed. Reversible changes in the isoenzyme electrophoretic mobility pattern from type A to types B and C were observed after subculturing of cloned cultures in medium of different composition or after passage in newborn mice. Type A zymodeme was observed in clones grown in blood-containing media, while types B and C were found in clones and subclones grown in media progressively less rich in nutrients (10 and 5% fetal calf serum, respectively) and containing no blood. The change in zymodeme from type A to type B or C was associated with loss of infectivity, which could be recovered by passages in newborn mice. Parasites infective for mice always showed zymodeme A. Simultaneously with zymodeme change from type A to types B and C there is a decrease in the specific activity of G6PD and 6PGD.

The N-terminal amino acid sequence is essential for foot-and-mouth disease virus replicase activity.

1. Foot-and-mouth disease virus replicase was expressed by fusing its cDNA to the OmpA signal peptide coding sequence present in the pIN-III ompA series vectors. 2. Two constructions were developed to express either a full-length or truncated enzyme lacking the 20 amino acids at the N-terminal end. Bacterial extracts expressing the recombinant proteins were submitted to SDS-PAGE and the presence of the replicase was revealed by immunoblotting. The truncated form exhibited a higher mobility and the relative positions of the proteins show that the signal peptide was removed. 3. The biological activity of these two molecules was tested using a poly(A)-dependent oligo(U)-primed poly(U)-polymerase assay. The full-length replicase is active. The aminoterminal truncated enzyme had 0.02% activity of the intact one. 4. This result indicates the importance of the twenty N-terminal amino acids for the activity of FMDV RNA-dependent RNA polymerase.

In vitro activities of a recombinant foot-and-mouth disease virus replicase expressed in Escherichia coli.

1. The replicase gene of foot-and-mouth disease virus (FMDV) was expressed in Escherichia coli under the control of a tac promoter. The recombinant enzyme was purified by inclusion body precipitation, elution, and poly(U) Sepharose chromatography. 2. The enzyme exhibits poly(A)-dependent oligo(U)-primed poly(U) polymerase activity. The specific activity of the purified replicase is 1.3 x 10(5). The recombinant replicase synthesizes RNA using FMDV RNA as template, as well as heterologous RNAs, such as globin RNA and synthetic RNAs, polyadenylated or not. In all polymerization reactions, RNA products twice the size of the template are formed, both in the presence and absence of an oligo(U) primer. The enzyme is also capable of incorporating [alpha 32P]UTP in all RNAs tested except the viral template. This activity does not seem to be related to the primer independent polymerization activity. 3. The products from polymerization reactions were characterized by hybridization. In the absence of primer they consist of the template and a complementary strand covalently attached, while in the presence of primer they consist of two complementary strands synthesized de novo. 4. We propose mechanisms of RNA synthesis by the recombinant FMDV replicase in the absence and presence of primer. These mechanisms are discussed in terms of models for in vitro RNA synthesis of other picornaviruses.

Expression of an active foot-and-mouth disease virus RNA polymerase in Escherichia coli.

The expression of a native form of the foot-and-mouth disease virus RNA polymerase was obtained. Two oligonucleotides of 66 base pairs were used to rebuild the 5' end of the gene and to introduce the first methionine codon. The expression of the active polymerase in E. coli was achieved by inserting the gene before the tac promoter of the pKK223-3 plasmid.