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.

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.

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.

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.

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.

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.

Environmental factors determining structural transition in herpetomonas samuelpessoai.

Transition from promastigote to paramastigote and opisthomastigote forms of Herpetomonas samuelpessoai in culture is dependent on environmental factors, among which paramount importance is attributed to the oxygen tension, which seems to play a decisive role in determining the choice between the aerobic and the anaerobic modes of respiration. A decrease of the oxygen tension and consequently of the pH, provokes the occurrence of structural transition, particularly in nonaerated cultures. In airgassed media, the changes in PO2 and pH occur at comparatively later stages of the culture and the cell volume does not change appreciably. A direct relationship has been found between the modal cell volume and the extent of structural transition as evaluated by the cell concentrations of the different forms in culture.

Morphological changes of Herpetomonas samuelpessoai.

The promastigote form of Herpetomonas samuelpessoai is modified into a paramastigote-like form during incubation at 28 C in high osmolarity complex media obtained through the addition of various concentrations of KCl, NaCl or glycerol. Morphological changes accompanying this modification include an increase in cell volume during exponential growth, a deep invagination of the cell membrane at the bottom of the flagellar pocket into the cytoplasm, and a prominent bulging at the emergence of the flagellum.

Trypanosoma cruzi: isolation of cloned strains and characterization of their infectivity.

Cloning of Trypanosoma cruzi strains Y, CL and Colombiana was achieved by plating on solid medium. Clones were obtained either from culture epimastigotes or from bloodstream trypomastigotes. In both cases the efficiency of plating was almost 100%. Clones from culture epimastigotes did not infect the albino mouse, while clones from bloodstream trypomastigotes remained infective even after several passages in a blood-agar/BHI biphasic medium, in which the amastigote-like forms prevail.

[Genetic analysis of lactose-fermenting Salmonella typhimurium isolated in Rio de Janeiro]. / Análise genética de Salmonella typhimurium fermentadoras de lactose isoladas no Rio de Janeiro.

Lactose fermenting Salmonella typhimurium are endemic in São Paulo, but not in Rio de Janeiro Two isolations are described from the latter city. These Rio de Janeiro strains have a plasmid of 7.4 megadaltons. These plasmids were not auto-transferable, were thermostable and were not eliminated by acridine orange. One of these strains arose from a plasmid that had the lactose operon repressed, leading us to speculate about the evolution of the lactose fermenting character in Brazilian Salmonella.

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.

Isolation and characterization of a new temperature-sensitive cell division mutant of Escherichia coli K-12.

A new temperature-sensitive mutant strain of Escherichia coli K-12 which forms filaments at 42 C has been described. The mutant, Y16, maintained growth and deoxyribonucleic acid synthesis at 42 C. The resulting multinucleate filaments gradually lost their viability at 42 C but could be recovered, even after 240 min of incubation, upon return to 30 C. Septation was resumed and growth was promptly re-established at normal rates. Recovery still took place in the presence of chloramphenicol added to the culture at the time of temperature shift from 42 to 30 C. A study has been made of the effects of adenine and various nucleosides on cultures of strain Y16 as compared with another filament-forming mutant, T44 tif-. Adenine (75 mug/ml), known to promote filamentation of strain T44 tif-, prevented the development of filaments and the loss of viability in cultures of Y16. Recovery of septation after temperature shift in cultures containing adenine presented a pattern similar to that found with the adenine-less cultures. Protection afforded by adenine at 42 C could be reversed by the addition of guanosine plus cytidine (100 mug/ml each). The effects of high concentrations of adenine and nucleosides on strain Y16 thus are the reverse of those observed with mutant T44 tif-. However, whereas tif-1 mutation promotes prophage induction at restrictive temperatures, no modification could be detected in the process of prophage induction in cultures of the lambda-lysogenic derivative of Y16 at 42 C, be it spontaneous or ultraviolet-mediated induction. The osmolarity increase afforded by 1% NaCl added to the medium did not alter the phenotype characteristics of strain Y16. The mutation has been mapped between argG and bgl. A close linkage has been observed between ftsH and argG, thereby locating the new mutation near 61 min on the map of E. coli chromosome, a previously undescribed region involved in cell division. The evidence reported indicates that strain Y16 differs in several respects from the already descirbed strains of the same class.

Influence of prophage on the efficiency of plating of Escherichia coli K12.

The efficiency of plating of Escherichia coli K12 and its derivatives K12 C600 and K12 C600(lambda) is diminished during the transition period from the lag of the exponential growth phase in cultures grown in tryptone broth. The phenomenon is suppressed in cultures of the strain K12 C600(lambda ind). The reduction in the efficiency of plating is transitory and not influenced by temperature between 30 and 42 degrees C.

Plasmid-mediated antibiotic resistance and colicinogeny among Salmonellae in Rio de Janeiro, Brazil.

A set of 25 multiple drug-resistant strains selected from Salmonellae isolated from sewage in Rio de Janeiro contained S. typhimurium (60%) and S. agona (20%) as the most frequent serotypes. There was resistance to ampicillin (Ap), 92%; chloramphenicol (Cm), 76%; gentamicin (Gm), 84%; kanamycin (Km), 84%; streptomycin (Sm), 96%; tetracycline (Tc), 76%; trimethoprim-sulfamethoxazole (SuTp), 84% and nalidixic acid (Na), 52%. The most frequent resistance patterns observed were Ap Cm Gm Km Sm Tc SuTp Na and Ap Cm Gm Km Sm Tc SuTp. Two strains, bearing the streptomycin, tetracycline double-resistance pattern were colicinogenic, producing type Ib colicin. The col+ character was cotransferable with the double-resistance; all these markers were associated with the presence of a single 60 Mdal plasmid.

Identification of multiple-resistance (R) and colicinogeny (Col) plasmids in an epidemic Salmonella agona serotype in Rio de Janeiro.

A Salmonella agona strain has caused a hospital outbreak of gastroenteritis in a pediatric unit in Rio de Janeiro. It bears two plasmids, a small (6.5 MDa molecular weight) plasmid coding for type B colicin production and a larger one (36 MDa molecular weight) determining resistance to ampicillin, gentamicin, kanamycin and trimethoprim-sulphamethoxazole. The R-plasmid, but not the Col-plasmid, is self-transferable to a Escherichia coli recipient strain. Curing for the R-plasmid was achieved by treatment with 0.05% SDS followed by incubation at 44 degrees C. It has not been possible to cure the S. agona strain for its Col-plasmid.

Timing of cold-sensitive stages in the cell division cycle of Escherichia coli K12.

Synchornous cultures obtained by selection at division were used to investigate the occurrence of cold-sensitive stages during the division cycle of Escherichia coli (lambdaind--). There are two such stages within the 50 min cycle: one (early) at 10 to 20 min and the other (late) at 40 to 45 min. Similar results were obtained from calculations based both on the age frequency distribution of cells in exponential growth and on the size of the populations which accumulate as a result of a single change of temperature. Times of about 17 and 44 min were found for the early and the late stages, respectively. It is concluded that the two-step doubling of E. coli K12 cultures synchronized by a single cold shock is due to two cold-sensitive stages in the division cycle.

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