Periplasmic delivery of biologically active human interleukin-10 in Escherichia coli via a sec-dependent signal peptide.

Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine, with therapeutic applications in inflammatory bowel disease. For the in situ delivery of IL-10 by Escherichia coli as carrier chassis, a modified transporter was designed with the ability to secrete biologically active IL-10. De novo DNA synthesis comprised a 561-bp fragment encoding the signal sequence of the E. coli outer membrane protein F fused in frame to an E. coli codon-optimized mature human IL-10 gene under control of a T7 promoter. The construct was overexpressed in E. coli laboratory strains, E. coli BL21 (DE3) and E. coli MDS42:T7. The mean concentrations of human IL-10 in the periplasm and culture supernatant of E. coli BL21 (DE3) were 355.8 ± 86.3 and 5.7 ± 1.7 ng/ml, respectively. The molecular mass of the recombinant E. coli-derived human IL-10 was 19 kDa, while under non-reducing conditions the native IL-10 dimer could be demonstrated. Reduction of tumor necrosis factor-α secretion in lipopolysaccharide-stimulated mouse macrophages and detection of the activated form of the transcription factor signal transducer and activator of transcription protein 3 proved the biological activity of the bacteria-produced human IL-10.

Expression of two recombinant chloramphenicol acetyltransferase variants in highly reduced genome Escherichia coli strains.

Highly reduced E. coli strains, MDS40, MDS41, and MDS42, lacking approximately 15% of the genome, were grown to high cell densities to test their ability to produce a recombinant protein with high yields. These strains lack all transposons and insertion sequences, cryptic prophage and many genes of unknown function. In addition to improving genetic stability, these deletions may reduce the biosynthetic requirements of the cell potentially allowing more efficient production of recombinant protein. Basic growth parameters and the ability of the strains to produce chloramphenicol acetyltransferase (CAT) under high cell density, batch cultivation were assessed. Although growth rate and recombinant protein production of the reduced genome strains are comparable to the parental MG1655 strain, the reduced genome strains were found to accumulate significant amounts of acetate in the medium at the expense of additional biomass. A number of hypotheses were examined to explain the accumulation of acetate, including oxygen limitation, carbon flux imbalance, and metabolic activity of the recombinant protein. Use of a non-catalytic CAT variant identified the recombinant protein activity as the source of this phenomenon; implications for the metabolic efficiency of the reduced genome strains are discussed.

Emergent properties of reduced-genome Escherichia coli.

With the use of synthetic biology, we reduced the Escherichia coli K-12 genome by making planned, precise deletions. The multiple-deletion series (MDS) strains, with genome reductions up to 15%, were designed by identifying nonessential genes and sequences for elimination, including recombinogenic or mobile DNA and cryptic virulence genes, while preserving good growth profiles and protein production. Genome reduction also led to unanticipated beneficial properties: high electroporation efficiency and accurate propagation of recombinant genes and plasmids that were unstable in other strains. Eradication of stress-induced transposition evidently stabilized the MDS genomes and provided some of the new properties.

Functional genomics of cell elongation in developing cotton fibers.

Cotton fibers are single-celled seed trichomes of major economic importance. Factors that regulate the rate and duration of cell expansion control fiber morphology and important agronomic traits. For genetic characterization of rapid cell elongation in cotton fibers, approximately 14,000 unique genes were assembled from 46,603 expressed sequence tags (ESTs) from developmentally staged fiber cDNAs of a cultivated diploid species ( Gossypium arboreum L.). Conservatively, the fiber transcriptome represents 35-40% of the genes in the cotton genome. In silico expression analysis revealed that rapidly elongating fiber cells exhibit significant metabolic activity, with the bulk of gene transcripts, represented by three major functional groups - cell wall structure and biogenesis, the cytoskeleton and energy/carbohydrate metabolism. Oligonucleotide microarrays revealed dynamic changes in gene expression between primary and secondary cell wall biogenesis showing that fiber genes in the dbEST are highly stage-specific for cell expansion - a conclusion supported by the absence of known secondary cell wall-specific genes from our fiber dbEST. During the developmental switch from primary to secondary cell wall syntheses, 2553 "expansion-associated" fiber genes are significantly down regulated. Genes (81) significantly up-regulated during secondary cell wall synthesis are involved in cell wall biogenesis and energy/carbohydrate metabolism, which is consistent with the stage of cellulose synthesis during secondary cell wall modification in developing fibers. This work provides the first in-depth view of the genetic complexity of the transcriptome of an expanding cell, and lays the groundwork for studying fundamental biological processes in plant biology with applications in agricultural biotechnology.

Systematic mutagenesis of the Escherichia coli genome.

A high-throughput method has been developed for the systematic mutagenesis of the Escherichia coli genome. The system is based on in vitro transposition of a modified Tn5 element, the Sce-poson, into linear fragments of each open reading frame. The transposon introduces both positive (kanamycin resistance) and negative (I-SceI recognition site) selectable markers for isolation of mutants and subsequent allele replacement, respectively. Reaction products are then introduced into the genome by homologous recombination via the lambdaRed proteins. The method has yielded insertion alleles for 1976 genes during a first pass through the genome including, unexpectedly, a number of known and putative essential genes. Sce-poson insertions can be easily replaced by markerless mutations by using the I-SceI homing endonuclease to select against retention of the transposon as demonstrated by the substitution of amber and/or in-frame deletions in six different genes. This allows a Sce-poson-containing gene to be specifically targeted for either designed or random modifications, as well as permitting the stepwise engineering of strains with multiple mutations. The promiscuous nature of Tn5 transposition also enables a targeted gene to be dissected by using randomly inserted Sce-posons as shown by a lacZ allelic series. Finally, assessment of the insertion sites by an iterative weighted matrix algorithm reveals that these hyperactive Tn5 complexes generally recognize a highly degenerate asymmetric motif on one end of the target site helping to explain the randomness of Tn5 transposition.

Construction and utility of 10-kb libraries for efficient clone-gap closure for rice genome sequencing.

Rice is an important crop and a model system for monocot genomics, and is a target for whole genome sequencing by the International Rice Genome Sequencing Project (IRGSP). The IRGSP is using a clone by clone approach to sequence rice based on minimum tiles of BAC or PAC clones. For chromosomes 10 and 3 we are using an integrated physical map based on two fingerprinted and end-sequenced BAC libraries to identifying a minimum tiling path of clones. In this study we constructed and tested two rice genomic libraries with an average insert size of 10 kb (10-kb library) to support the gap closure and finishing phases of the rice genome sequencing project. The HaeIII library contains 166,752 clones covering approximately 4.6x rice genome equivalents with an average insert size of 10.5 kb. The Sau3AI library contains 138,960 clones covering 4.2x genome equivalents with an average insert size of 11.6 kb. Both libraries were gridded in duplicate onto 11 high-density filters in a 5 x 5 pattern to facilitate screening by hybridization. The libraries contain an unbiased coverage of the rice genome with less than 5% contamination by clones containing organelle DNA or no insert. An efficient method was developed, consisting of pooled overgo hybridization, the selection of 10-kb gap spanning clones using end sequences, transposon sequencing and utilization of in silico draft sequence, to close relatively small gaps between sequenced BAC clones. Using this method we were able to close a majority of the gaps (up to approximately 50 kb) identified during the finishing phase of chromosome-10 sequencing. This method represents a useful way to close clone gaps and thus to complete the entire rice genome.

Delayed retroperitoneal arterial hemorrhage after inferior vena cava (IVC) filter insertion: case report and literature review of caval perforations by IVC filters.

Transvenous placement of inferior vena cava (IVC) filters has become commonplace in selected patients with deep venous thrombosis (DVT) and pulmonary embolism (PE). IVC filters have been shown to have excellent therapeutic efficacy and low complication rates. Penetration of the IVC by filter hooks or struts has been reported and commonly noted to be inconsequential. We report a laceration of a lumbar artery by a stainless steel Greenfield (SSG) filter strut that resulted in a near fatal hemorrhage, and review the world literature on caval perforation by IVC filters.

An integrated physical and genetic map of the rice genome.

Rice was chosen as a model organism for genome sequencing because of its economic importance, small genome size, and syntenic relationship with other cereal species. We have constructed a bacterial artificial chromosome fingerprint-based physical map of the rice genome to facilitate the whole-genome sequencing of rice. Most of the rice genome ( approximately 90.6%) was anchored genetically by overgo hybridization, DNA gel blot hybridization, and in silico anchoring. Genome sequencing data also were integrated into the rice physical map. Comparison of the genetic and physical maps reveals that recombination is suppressed severely in centromeric regions as well as on the short arms of chromosomes 4 and 10. This integrated high-resolution physical map of the rice genome will greatly facilitate whole-genome sequencing by helping to identify a minimum tiling path of clones to sequence. Furthermore, the physical map will aid map-based cloning of agronomically important genes and will provide an important tool for the comparative analysis of grass genomes.

Percepciones y prácticas relacionadas con la tuberculosis y la adherencia al tratamiento en Chiapas, México / Perceptions and practices of tuberculosis patients and non-adherence to therapy in Chiapas, Mexico

Objetivo. Conocer las percepciones y prácticas que los enfermos de tuberculosis tienen sobre la enfermedad y la adherencia al tratamiento. Material y métodos. Estudio cualitativo de 11 entrevistas grupales a 62 pacientes con tuberculosis diagnosticados durante 1997 y 1998 en las regiones Centro, Los Altos y Fronteriza de Chiapas, México. Resultados. Las causas de la enfermedad referidas por los pacientes fueron el contagio por trastes, el trabajo excesivo, la alimentación, el frío y otras sin relación con la transmisión de persona a persona. La incapacidad para el trabajo se reflejó en crisis económica del paciente y su familia. El estigma social impactó emocionalmente en la vida personal, familiar, laboral y de comunidad. Conclusiones. El desconocimiento sobre la enfermedad propició la elección de diferentes alternativas para su atención. Los servicios de salud y la inadecuada relación médico-paciente influyó en el retardo en el diagnóstico y falta de adherencia al tratamiento. Se sugiere un programa de difusión sobre aspectos básicos de la enfermedad y su tratamiento.

[Problems of diagnosis of pulmonary tuberculosis. The case of the border region of Chiapas (Mexico)]. / Problemas de diagnóstico de tuberculosis pulmonar. El caso de la región fronteriza de Chiapas (México).

OBJECTIVE: To analyze critical factors in the diagnosis of pulmonary tuberculosis at both the primary and secondary levels, in the Border Region of Chiapas, Mexico. DESIGN: A crossover study (from March to September, 1994) PARTICIPANTS: Patients with chronic cough (n = 221) who sought care in the Out-patient Department of the only second level care hospital available in the region for the uninsured population. Each subject was interviewed, three sputum specimens were requested and the clinical charts reviewed. MEASUREMENTS AND MAIN RESULTS: Fourty-four patients were found positive for pulmonary TB of which six came to the hospital for initial care. 38 had already been seen in a primary care setting. Of those 38 only two had been diagnosed previously by acid fast smear. At the hospital level, the underdiagnosis of TBP was 9%. CONCLUSIONS: The quality of care with regard to the diagnosis of pulmonary tuberculosis needs to be improved at both the primary and hospital levels. Health workers need to be sensitized to the symptomatology and trained to request sputum smears when indicated. The general population also needs to be educated with regard to pulmonary tuberculosis so as to demand better services.

Factores de riesgo para resistencia a drogas antifímicas en Chiapas, México / Risk factors for drug-resistant tuberculosis in Chiapas, Mexico

Objetivo. Determinar los factores de riesgo para resistencia antifímica en pacientes con tuberculosis pulmonar, en cuatro jurisdicciones sanitarias del estado de Chispas, México. Material y métodos. Se realizó un estudio de casos y controles en el cual se incluyeron pacientes con tuberculosis pulmonar diagnosticados por medio de baciloscopía, notificados durante 1992. Se aplicó una encuesta que incluía variables relacionadas con el diagnóstico, tratamiento y seguimiento de los pacientes con tuberculosis pulmonar y se recolectaron muestras de expectoración para su análisis. La pruebas de sensibilidad se realizaron con el método de las proporciones. Se consideró a un paciente con infección de M. tuberculosis resistente cuando existían colonias desarrolladas en la presencia de una o más drogas antifímicas. El grupo control estuvo constituido por pacientes con resultados negativos a baciloscopías y cultivos, y en caso de ser positivo este último, con informe de M. tuberculosis sensible a las drogas estudiadas. Resultados. Del total de 18 individuos con cultivos positivos y desarrollo de M. tuberculosis, 13 (72 por ciento) fueron resistentes a una o más drogas antifímicas y 10 a dos o más drogas, de los cuales tres fueron resistentes a cinco antifímicos. La resitencia más alta fue para la isoniacida con 77 por ciento. Los factores de riesgo detectados en la población estudiada fueron la monoterapia (RM=34.2), los abandonos del tratamiento (RM=6.86), el tiempo prolongado de evolución de la enfermedad (RM=6.40) y los multitratamientos (RM=28.3). Conclusiones. La proporción tan alta de pacientes resistentes a drogas antifímicas (72 por ciento), denota un grave problema de salud pública y es de clara consecuencia de los problemas originados por el manejo inadecuado del tratamiento antituberculoso

Objectives. To determine risk factors for antibiotic resistance in patients with pulmonary tuberculosis in four Health Jurisdictions of the state of Chiapas. Material and Methods. A case-control study was carried out in patients diagnosed by acid fast smear during 1992. A questionnaire was applied which included variables related to the diagnosis, treatment and follow-up of the patients. Sputum samples were collected for culture and sensitivity tests. A case of drug-resistant pulmonary tuberculosis was defined as the presence of culture colonies showing resistance to one or more drugs. The control group was patients with negative smears and cultures or positive cultures for M. tuberculosis sensitive to the specific drugs. Results. Of the total of 18 individuals reported to have positive cultures, 13 (72.2%) were resistant to one or more drugs. Resistance to two or more drugs was found in 10 of them of which three were resistant to five antituberculosis drugs. The most frequent resistance was to isoniazid (77%). Risk factors for resistance encountered in this patient population were monotherapy (OR= 34.2), abandonment of treatment (OR= 6.86), a prolonged period of illness (OR= 6.40), delay in diagnosis and a history of prior therapy (OR= 28.3). Conclusions. The high proportion of patients resistant to antituberculosis therapy poses a public health problem and is a clear consequence of the problems arising from inadequate treatment.