Implication of an Outer Surface Lipoprotein in Adhesion of Bifidobacterium bifidum to Caco-2 Cells

We found that the human intestinal isolate Bifidobacterium bifidum MIMBb75 strongly adhered to Caco-2 cells. Proteinase K and lithium chloride treatments showed that proteins play a key role in MIMBb75 adhesion to Caco-2 cells. By studying the cell wall-associated proteins, we identified a surface protein, which we labeled BopA. We purified the protein chromatographically and found that it functioned as an adhesion promoter on Caco-2 cells. In silico analysis of the gene coding for this protein and globomycin experiments showed that BopA is a cysteine-anchored lipoprotein expressed as a precursor polypeptide. A database search indicated that BopA appears to function biologically as an oligopeptide/tripeptide-solute-binding protein in the ABC transport system. We discovered a protein corresponding to BopA and its gene in eight other highly adherent B. bifidum strains. Finally, we found that B. bifidum MIMBb75 and BopA affected the production of interleukin-8 in Caco-2 epithelial cells. BopA is the first protein described to date to be directly involved in the adhesion of bifidobacteria to Caco-2 cells and to show immunomodulatory activity.     

Generation and Comparison of Bioluminescent and Fluorescent Bacillus licheniformis

The environmental bacterium Bacillus licheniformis was transformed with two different shuttle-vectors (pCSS810 and pGFPratiometric) containing insect luciferase and green fluorescent protein genes, respectively. The cells were treated with various antimicrobial agents and the emitted bioluminescence and fluorescence were measured. Plasmid harboring the green fluorescent protein gene was totally segregated without selective pressure, and fluorescent B. licheniformis showed a slower growth rate than the wild-type strain; those cells were bright green as visualized by epifluorescent microscopy. However, fluorescence was not correlated to the growth state of cells or affected by the antibiotic treatments. To the contrary, luminescent transformant was found to be stable without antibiotic selection and showed analogous growth behavior compared to non-plasmid-bearing cells. The luminescent strain functioned as a biosensor for the antibiotics employed. Bioluminescence measurements allowed one to determine the viability of the recombinant cells and the kinetics of the antibacterial action could be followed. Thus, the light emission was found to be a reliable, sensitive, and real-time indicator of the "well-being" of cells, whereas fluorescence allowed one to visualize both metabolically active and inactive cells.     

Response of calcareous nannofossils to the Paleocene–Eocene Thermal Maximum: Observations on composition,preservation and calcification in sediments from ODP Site 1263 (Walvis Ridge — SW Atlantic)

In this study we present the results of a detailed analysis on calcareous nannofossil assemblages from sediment cores of ODP Site 1263 (Southern East Atlantic, Walvis Ridge). This section represents one of the few complete deep-sea sections that document the Paleocene–Eocene Thermal Maximum (PETM) in the pelagic realm. The PETM transient event was characterized by a brief, but intense interval of global warming, a global negative carbon isotope excursion (CIE), and widespread dissolution of seafloor carbonate sediments. Paired analysis at polarizing light microscope (LM) and Scanning Electron Microscope (SEM) documents the different “behavior” of nannofossils through the different phases of the PETM, at the onset of CIE, within the CIE, and during the recovery interval. The presence of anomalous specimens and morphotypes within some nannofossil taxa, recorded during previous LM high resolution analyses, has been further investigated in selected samples at the SEM. Besides the known representatives of the CIE-PETM “excursion nanno-flora”, as Rhomboaster calcitrapa group and Discoaster anartios, the analysis revealed the presence of peculiar morphotypes of Fasciculithus and deformed specimens of Discoaster nobilis group, Discoaster mediosus and Discoaster multiradiatus that are considered related to the anomalous amount of CO₂ in the ocean-atmosphere system during the early phase of PETM. Comparative analyses were performed in few selected samples from other PETM sections located at different latitudes in the Atlantic and Pacific oceans. Although the anomalous geochemical conditions during the PETM-CIE interval seem to have had some influence on the nannofossil production, calcification and assemblage composition, it results that local productivity together with post depositional (diagenetic) conditions were additional important controlling factors on nannofossil assemblages. Preliminary data from Eocene Thermal Maximum 2 (ETM2 or Elmo) suggest that nannofossil malformations are not exclusive of the PETM, and are associated to other episodes of perturbation of the C cycle.     

Binding citrate/DNA in presence of divalent cations – Potential mimicry of acidic peptides/DNA interactions

Citric acid whose structure is comparable to that of small acidic peptides, can bind to DNA in the presence of divalent cations (Cu²⁺, Fe²⁺, Zn²⁺, Mg²⁺). Citrate-DNA interaction occurs also in a cell homogenate and in this experimental model too requires the presence of natural divalent cations. In fact the addition of 2 mM EDTA to cell homogenate strongly decreases the DNA-citrate binding. The results demonstrate that divalent cations can act as bridges between two acidic molecules and that citric acid can mimic the structure of acidic peptides.     

Aggregation of small peptides studied by molecular dynamics simulations

Peptides and proteins tend to aggregate under appropriate conditions. The amyloid fibrils that are ubiquitously found among these structures are associated with major human diseases like Alzheimer's disease, type II diabetes, and various prion diseases. Lately, it has been observed that even very short peptides like tetra and pentapeptides can form ordered amyloid structures. Here, we present aggregation studies of three such small polypeptide systems, namely, the two amyloidogenic peptides DFNKF and FF, and a control (nonamyloidogenic) one, the AGAIL. The respective aggregation process is studied by all-atom Molecular Dynamics simulations, which allow to shed light on the fine details of the association and aggregation process. Our analysis suggests that naturally aggregating systems exhibit significantly diverse overall cluster shape properties and specific intermolecular interactions. Additional analysis was also performed on the previously studied NFGAIL system.     

Effects of sterol biosynthesis inhibitors on endosymbiont-bearing trypanosomatids

Some protozoa of the Trypanosomatidae family have a close relationship with an endosymbiotic bacterium. As the prokaryote envelope has a controversial origin, a sterol 24-methyltransferase inhibitor (20-piperidin-2-yl-5alpha-pregnan-3beta,20-diol; 22,26-azasterol) was used as a tool to investigate lipid biosynthetic pathways in Crithidia deanei, an endosymbiont-bearing trypanosomatid. Apart from antiproliferative effects, this drug induced ultrastructural alterations, consisting of myelin-like figures in the cytoplasm and endosymbiont envelope vesiculation. Concurrently, a dramatic reduction of 24-alkyl sterols was observed after 22,26-azasterol treatment, both in whole cell homogenates, as well as in isolated mitochondria. These effects were associated with changes of phospholipid composition, in particular a reduction of the phosphatidylcholine content and a concomitant increase in phosphatidylethanolamine levels. Lipid analyses of purified endosymbionts indicated a complete absence of sterols, and their phospholipid composition was different from that of mitochondria or whole protozoa, being similar to eubacteria closely associated with eukaryotes.     

Meaning of DNA detection during the follow-up of HIV-1 infected patients: a brief review

A growing body of evidence indicates that proviral DNA load quantitation is an important parameter in establishing the dynamics of HIV infection. Proviral DNA load can be determined during the follow-up of infected individuals to evaluate reservoir status in addition to viral replication. Hence, the study of viral reservoirs, represented by HIV-1 latently infected cells, including resting memory CD4+ T cells, monocytes and macrophages, by which HIV-1 can be reactivated, opens new perspectives in the assessment and the comprehension of HIV-1 infection. However, the identification of viral reservoirs, that can store both wild and drug resistance viruses, is one of the most important steps in developing treatment strategies because it is now clear that viral reservoirs not only prevent sterilizing immunity but also represent a major obstacle to curing the infection with the potent antiretroviral drugs currently in use. Even if only careful evaluation of virological and immunological markers is necessary to fully characterize the course of HIV-1 infection and to provide a more complete laboratory-based assessment of disease progression, the availability of a new standardized assay such as DNA proviral load will be important to assess the true extent of virological suppression in treated patients and to verify the efficacy of new immune-based therapies aimed at purging HIV-1 DNA reservoirs. Several studies demonstrate, in fact, that HIV-1 cellular DNA load may be an indicator of spread of infection whereas the plasma RNA load is indicates active infection. This article will review the importance of monitoring HIV-1 proviral load DNA during the follow-up of HIV-1 infected subjects, suggesting that additional information complementing HIV RNA load could provide crucial information to monitor viral replication and the effectiveness of HAART therapy.     

An exploratory study of two Caco-2 cell models for oral absorption: a report on their within-laboratory and between-laboratory variability, and their predictive capacity

In 2005, the European Centre for the Validation of Alternative Methods (ECVAM) sponsored a study aimed at evaluating the reproducibility (between-laboratory and within-laboratory variability) and the predictive capacity of two in vitro cellular systems--the Caco-2/ATCC parental cell line and the Caco- 2/TC7 clone--for estimating the oral fraction absorbed (Fa) in humans. Two laboratories, both of which had experience with Caco-2 cultures, participated in the study. Ten test chemicals with documented in vivo oral absorption data were selected. Atenolol, cimetidine and propranolol were included as reference compounds for low, medium and high intestinal absorption, respectively. Transport experiments were independently carried out in the two laboratories, according to an agreed protocol. The apparent permeability coefficient (Papp) was calculated in either the apical to basolateral (absorption) or the basolateral to apical (efflux) direction. To investigate the involvement of possible active transport processes, experiments were also performed in the presence of sodium azide plus 2-deoxy-D-glucose in the donor compartment. Before performing the permeability experiments, the highest concentration that did not impair barrier integrity was identified for each test chemical in both cell models, by applying the chemicals together with a marker of the paracellular pathway. In addition, barrier integrity was assessed by measuring the trans-epithelial electrical resistance. All the permeability data obtained were independently analysed. Reproducibility was assessed for the seven substances for which sufficient data were available. Within-laboratory variability was based on coefficient of variation (CV) values. Median CV values of 10.4% and 14.7% were found for the two laboratories. Concerning between-laboratory reproducibility, comparable response levels were obtained for the three reference compounds and for paracetamol, while, for the other chemicals, the results were less reproducible--in particular, for compounds known to be actively transported. The Papp values obtained for both cell lines were comparable for identical experimental conditions. Despite the limited number of substances tested, the predictive capacity was investigated by using two mathematical models available in the literature. Good estimations of the human Fa were obtained for five well-absorbed compounds, while moderately and poorly absorbed compounds were overestimated. It is proposed that a confirmatory study addressing the main results, including power considerations, would now be useful.     

Evolutionary Dynamics of Multiple Sublineages of H5N1 Influenza Viruses in Nigeria from 2006 to 2008

Highly pathogenic A/H5N1 avian influenza (HPAI H5N1) viruses have seriously affected the Nigerian poultry industry since early 2006. Previous studies have identified multiple introductions of the virus into Nigeria and several reassortment events between cocirculating lineages. To determine the spatial, evolutionary, and population dynamics of the multiple H5N1 lineages cocirculating in Nigeria, we conducted a phylogenetic analysis of whole-genome sequences from 106 HPAI H5N1 viruses isolated between 2006 and 2008 and representing all 25 Nigerian states and the Federal Capital Territory (FCT) reporting outbreaks. We identified a major new subclade in Nigeria that is phylogenetically distinguishable from all previously identified sublineages, as well as two novel reassortment events. A detailed analysis of viral phylogeography identified two major source populations for the HPAI H5N1 virus in Nigeria, one in a major commercial poultry area (southwest region) and one in northern Nigeria, where contact between wild birds and backyard poultry is frequent. These findings suggested that migratory birds from Eastern Europe or Russia may serve an important role in the introduction of HPAI H5N1 viruses into Nigeria, although virus spread through the movement of poultry and poultry products cannot be excluded. Our study provides new insight into the genesis and evolution of H5N1 influenza viruses in Nigeria and has important implications for targeting surveillance efforts to rapidly identify the spread of the virus into and within Nigeria.Since its emergence in 1996 in Guangdong, China, highly pathogenic avian influenza virus of the H5N1 subtype (HPAI H5N1 virus) has disseminated widely across Asia, Europe, and Africa, infecting a range of domestic and wild avian species and sporadically spilling over into humans and other mammals (4, 35). Over time, the HPAI H5N1 virus has diversified into multiple phylogenetically distinct lineages, classified as clades 0 to 9 according to the unified nomenclature system (33). The H5N1 lineage currently circulating in central Asia, the Middle East, Europe, and Africa is referred to as clade 2.2 (33) and has also been described as “EMA” or Qinghai-like in previous publications (4, 17, 27). This clade originated in April 2005 during a large outbreak of a phylogenetically distinct H5N1 virus among wild bird populations at Qinghai Lake in western China (4, 17) and rapidly spread west through central Asia and Europe, eventually reaching Africa in 2006 (27). Clade 2.2 has further diversified, forming the genetic third-order clade 2.2.1 (32) and three genetically distinct sublineages (I, II, and III) (2, 19, 28), all of which are found in Africa.Since 2006 HPAI H5N1 viruses belonging to clade 2.2 have disseminated across multiple countries in western, eastern, and northern Africa: Egypt, Niger, Cameroon, Sudan, Burkina Faso, Djibouti, Ivory Coast, Ghana, Togo, Benin, and Nigeria (2). With a large poultry industry, estimated at 140 million birds (11), Nigeria has experienced several major outbreaks of HPAI H5N1 virus, posing a serious threat to food security and public health in Africa. The first case of HPAI H5N1 virus in Nigeria (sublineage I) occurred in January 2006 in the state of Kaduna, and the virus subsequently was detected in Ghana, Burkina Faso, Ivory Coast, and Sudan (2). In February 2006 sublineage II was reported in Nigeria, and it disseminated widely across the country during 2006 and 2007, also appearing in Togo (2). Clade 2.2.1, which has been prevalent in Egypt, Israel, and the Gaza Strip from 2006 to 2008, was also detected in Nigeria in 2006 (10).By the end of 2007, outbreaks of HPAI H5N1 virus in Nigeria appeared to have been successfully controlled by measures such as “stamping out with compensation,” restrictions on movement of poultry, and enhanced surveillance (13). However, in July 2008 new cases of HPAI H5N1 from a sublineage never previously detected in Africa (sublineage III) were registered in the Nigerian states of Kano and Katsina and in live bird markets in Gombe and Kebbi states (13, 21). Hence, Nigeria is the only African country where viruses belonging to clade 2.2.1 and to three different sublineages (I, II, and III) of clade 2.2 have all been detected. At least three different reassortment events between sublineages have been documented in Nigeria. Salzberg et al. identified the first reassortant strain (which we refer to as “R1”), in which four genome segments (hemagglutinin [HA], NP, NS, and PB1) belong to sublineage I and the other four segments (NA, MP, PA, and PB2) are derived from sublineage II (27). Subsequently, phylogenetic analysis showed that a 2007 reassortant strain (which we refer to as “R3”) contained the HA and NS segments from sublineage I and the other six segments from sublineage II (19, 22). Another reassortant virus (which we refer to as “R5”) contained only the NS gene segment from sublineage I, while the other seven segments were derived from sublineage II (22).Although the genetic diversity of the Nigerian HPAI H5N1 virus population has been well characterized, including multiple introductions of the virus into Nigeria and several reassortment events, little is known about the evolutionary and population growth dynamics of the virus within Nigeria. Particularly understudied are the spatial movements of individual sublineages among Nigeria''s vast poultry population. To explore the spatial, evolutionary, and population dynamics of the multiple H5N1 lineages cocirculating in Nigeria, we conducted a phylogenetic analysis of whole-genome sequences from 106 HPAI H5N1 viruses isolated between 2006 and 2008 and representing all 25 Nigerian states and the Federal Capital Territory (FCT) reporting outbreaks. Using the exact date and location of collection for each viral isolate, we inferred from their phylogenetic relationships the directionality of viral gene flow among Nigerian states and identified critical regions that are likely to serve as key sources for the H5N1 virus in Nigeria.     

Microbial biosurfactants production, applications and future potential

Microorganisms synthesise a wide range of surface-active compounds (SAC), generally called biosurfactants. These compounds are mainly classified according to their molecular weight, physico-chemical properties and mode of action. The low-molecular-weight SACs or biosurfactants reduce the surface tension at the air/water interfaces and the interfacial tension at oil/water interfaces, whereas the high-molecular-weight SACs, also called bioemulsifiers, are more effective in stabilising oil-in-water emulsions. Biosurfactants are attracting much interest due to their potential advantages over their synthetic counterparts in many fields spanning environmental, food, biomedical, and other industrial applications. Their large-scale application and production, however, are currently limited by the high cost of production and by limited understanding of their interactions with cells and with the abiotic environment. In this paper, we review the current knowledge and the latest advances in biosurfactant applications and the biotechnological strategies being developed for improving production processes and future potential.