Metabolic engineering of Clostridium acetobutylicum for the industrial production of 1,3-propanediol from glycerol

Clostridium butyricum is to our knowledge the best natural 1,3-propanediol producer from glycerol and the only microorganism identified so far to use a coenzyme B12-independent glycerol dehydratase. However, to develop an economical process of 1,3-propanediol production, it would be necessary to improve the strain by a metabolic engineering approach. Unfortunately, no genetic tools are currently available for C. butyricum and all our efforts to develop them have been so far unsuccessful. To obtain a better "vitamin B12-free" biological process, we developed a metabolic engineering strategy with Clostridium acetobutylicum. The 1,3-propanediol pathway from C. butyricum was introduced on a plasmid in several mutants of C. acetobutylicum altered in product formation. The DG1(pSPD5) recombinant strain was the most efficient strain and was further characterized from a physiological and biotechnological point of view. Chemostat cultures of this strain grown on glucose alone produced only acids (acetate, butyrate and lactate) and a high level of hydrogen. In contrast, when glycerol was metabolized in chemostat culture, 1,3-propanediol became the major product, the specific rate of acid formation decreased and a very low level of hydrogen was observed. In a fed-batch culture, the DG1(pSPD5) strain was able to produce 1,3-propanediol at a higher concentration (1104 mM) and productivity than the natural producer C. butyricum VPI 3266. Furthermore, this strain was also successfully used for very long term continuous production of 1,3-propanediol at high volumetric productivity (3 g L-1 h-1) and titer (788 mM).     

Characterization of monoclonal antibodies against altered (T103I) amidase from Pseudomonas aeruginosa

Monoclonal antibodies (MAbs) against mutant (T103I) amidase from Pseudomonas aeruginosa were raised by hybridoma technology. To select MAbs suitable for immunoaffinity chromatography, hybridoma clones secreting polyol-responsive MAbs (PR-MAbs) were screened that bind antigen tightly but release under mild and nondenaturing elution conditions. It was found that about 10% of enzyme-linked immunosorbent assay (ELISA)-positive hybridoma produce these MAbs as their ag-ab complex can be disrupted by propylene glycol in the presence of a suitable salt. Two of these hybridoma clones (F6G7 and E2A6) secreting PR-MAbs against mutant amidase were selected for optimization of experimental conditions for elution of amidase by using ELISA elution assay. These hybridoma cell lines secreted MAbs of IgM class that were purified in a single step by gel filtration chromatography, which revealed a single protein band on native polyacrylamide gel electrophoresis (PAGE). Specificity studies of this MAb revealed that it recognized specifically a common epitope on mutant and wild-type amidases as determined by direct ELISA. This MAb exhibited a higher affinity for denatured forms of wild-type and mutant amidases than for native forms as revealed by affinity constants (K), suggesting that it recognizes a cryptic epitope on an amidase molecule. Furthermore, MAb E2A6 inhibited about 60% of wild-type amidase activity, whereas it activated about 60% of mutant amidase (T103I) activity. The data presented in this work suggest that this MAb acts as a very useful probe to detect conformational changes in native and denatured amidases as well as to differentiate wild-type and mutant (T103I) amidases.     

Fertility and hatchability of eggs laid in the pullet-to-breeder transition period and in the initial production period

The initial eggs produced by broiler breeder hens are relatively small compared with later in the production cycle. An evaluation of indices related to hatchability is required when these eggs are to be used for the production of broiler chicks. Two experiments were conducted to evaluate characteristics related to the hatchability of eggs from pullet-to-breeder transition phase, at 25 and 27 weeks of age, and from the peak of production period and five weeks later, at 32 and 37 weeks of age. Eggs from birds 25 weeks had a lesser fertility in Experiment 1. Mortality occurred unevenly in early (1–5 days), middle (6–17 days) and late (18–21 days) incubation, and greater mortality was observed after the internal membrane was ruptured. The younger the hen, the lighter the egg, chick, and shell, and the longer the time required to complete the hatching process. In Experiment 2, greater mortalities were observed at the early period (1–5 days) and after “pipping” of the internal and external membranes. Embryos from heavy eggs of breeder hens 37 weeks of age took less time to complete the hatching process. Results indicated the larger the egg, the heavier the chick and shell, and the lesser the shell percentage. As breeder age advanced, characteristics related to egg fertility and hatchability improved.     

Functional analysis of Vif protein shows less restriction of human immunodeficiency virus type 2 by APOBEC3G

Viral infectivity factor (Vif) is one of the human immunodeficiency virus (HIV) accessory proteins and is conserved in the primate lentivirus group. This protein is essential for viral replication in vivo and for productive infection of nonpermissive cells, such as peripheral blood mononuclear cells (PBMC). Vif counteracts an antiretroviral cellular factor in nonpermissive cells named CEM15/APOBEC3G. Although HIV type 1 (HIV-1) Vif protein (Vif1) can be functionally replaced by HIV-2 Vif protein (Vif2), its identity is very small. Most of the functional studies have been carried out with Vif1. Characterization of functional domains of Vif2 may elucidate its function, as well as differences between HIV-1 and HIV-2 infectivity. Our aim was to identify the permissivity of different cell lines for HIV-2 vif-minus viruses. By mutagenesis specific conserved motifs of HIV-2 Vif protein were analyzed, as well as in conserved motifs between Vif1 and Vif2 proteins. Vif2 mutants were examined for their stability, expression, and cellular localization in order to characterize essential domains of Vif2 proteins. Viral replication in various target cells (PBMC and H9, A3.01, U38, and Jurkat cells) and infectivity in single cycle assays in the presence of APOBEC3G were also analyzed. Our results of viral replication show that only PBMC have a nonpermissive phenotype in the absence of Vif2. Moreover, the HIV-1 vif-minus nonpermissive cell line H9 does not show a similar phenotype for vif-negative HIV-2. We also report a limited effect of APOBEC3G in a single-cycle infectivity assay, where only conserved domains between HIV-1 and HIV-2 Vif proteins influence viral infectivity. Taken together, these results allow us to speculate that viral inhibition by APOBEC3G is not the sole and most important determinant of antiviral activity against HIV-2.     

Crystal structures of the RNA-dependent RNA polymerase genotype 2a of hepatitis C virus reveal two conformations and suggest mechanisms of inhibition by non-nucleoside inhibitors

Crystal structures of the RNA-dependent RNA polymerase genotype 2a of hepatitis C virus (HCV) from two crystal forms have been determined. Similar to the three-dimensional structures of HCV polymerase genotype 1b and other known polymerases, the structures of the HCV polymerase genotype 2a in both crystal forms can be depicted in the classical right-hand arrangement with fingers, palm, and thumb domains. The main structural differences between the molecules in the two crystal forms lie at the interface of the fingers and thumb domains. The relative orientation of the thumb domain with respect to the fingers and palm domains and the beta-flap region is altered. Structural analysis reveals that the NS5B polymerase in crystal form I adopts a "closed" conformation that is believed to be the active form, whereas NS5B in crystal form II adopts an "open" conformation and is thus in the inactive form. In addition, we have determined the structures of two NS5B polymerase/non-nucleoside inhibitor complexes. Both inhibitors bind at a common binding site, which is nearly 35 A away from the polymerase active site and is located in the thumb domain. The binding pocket is predominantly hydrophobic in nature, and the enzyme inhibitor complexes are stabilized by hydrogen bonding and van der Waals interactions. Inhibitors can only be soaked in crystal form I and not in form II; examination of the enzyme-inhibitor complex reveals that the enzyme has undergone a dramatic conformational change from the form I (active) complex to the form II (inactive).     

Cyclopeptide alkaloids from Scutia buxifolia Reiss and their antimicrobial activity

The present study reports a cyclopeptide alkaloid, scutianine M, isolated from the methanolic root bark extract of Scutia buxifolia Reiss (Rhamnaceae) along with six known compounds, scutianines-B, -C, -D, -E, -F, and scutianene D. Its structure was established on the basis of spectroscopic analyses, including application of 2D NMR spectroscopic techniques. As part of a study of the bioactive compounds of medicinal plants from southern Brazil, we also compared the antimicrobial activity of the isolated compounds towards Gram (+), Gram (-) bacteria, and yeasts.     

Socioeconomic status as a risk factor for HIV infection in women in East, Central and Southern Africa: a systematic review

This is a critical, systematic review of the relationship between socioeconomic status (SES) and HIV infection in women in Southern, Central and Eastern Africa. In light of the interest in micro-credit programmes and other HIV prevention interventions structured to empower women through increasing women's access to funds and education, this review examines the epidemiological and public health literature, which ascertains the association between low SES using different measurements of SES and risk of HIV infection in women. Also, given the focus on structural violence and poverty as factors driving the HIV epidemic at a structural/ecological level, as advocated by Paul Farmer and others, this study examines the extent to which differences in SES between individuals in areas with generalized poverty affect risk for SES. Out of 71 studies retrieved, 36 studies met the inclusion criteria including 30 cross-sectional, one case-control and five prospective cohort or nested case-control studies. Thirty-five studies used at least one measurement of female's SES and fourteen also included a measurement of partner's SES. Studies used variables measuring educational level, household income and occupation or employment status at the individual and neighbourhood level to ascertain SES. Of the 36 studies, fifteen found no association between SES and HIV infection, twelve found an association between high SES and HIV infection, eight found an association between low SES and HIV infection and one was mixed. In interpreting these results, this review examines the role of potential confounders and effect modifiers such as history of STDs, number of partners, living in urban or rural areas and time and location of study in sub-Saharan Africa. It is argued that STDs and number of partners are on the causal pathway under investigation between HIV and SES and should not be adjusted as confounders in any analysis. In conclusion, it is argued that in low-income sub-Saharan Africans countries, where poverty is widespread, increasing access to resources for women may initially increase risk of HIV or have no effect on risk-taking behaviours. In some parts of Southern Africa where per capita income is higher and within-country inequalities in wealth are greater, studies suggest that increasing SES may decrease risk. This review concludes that increased SES may have differential effects on married and unmarried women and further studies should use multiple measures of SES. Lastly, it is suggested that the partner's SES (measured by education or income/employment) may be a stronger predictor of female HIV serostatus than measures of female SES.     

Fungal keratitis in the State of Paraná-Brazil: clinical, epidemiological and diagnostic findings

Fungal keratites is more prevalent in tropical and subtropical regions, such as Brazil, and causes high morbidity. Usually, it is preceded by underlying conditions like ocular trauma or immunosuppression. The diagnosis is confirmed by the demonstration of the etiologic agent in the clinical specimen. Data were analysed from 49 patients with fungal keratitis observed in Ophthalmologic Division of Hospital de Clinicas, Federal University of Parana, from 1983 to 1997. The diagnosis was confirmed by culture and/ or direct examination. Of the cases studied, 22% were diagnosed only by direct examination; 50% by isolation in culture and 26% by the association of the both methods. The most prevalent etiologic agents were: Fusarium sp. (32%), Aspergillus sp. (16,5%) and Penicillium sp. (10%).