Production by Clostridium acetobutylicum ATCC 824 of CelG,a cellulosomal glycoside hydrolase belonging to family 9

The genome sequence of Clostridium acetobutylicum ATCC 824, a noncellulolytic solvent-producing strain, predicts the production of various proteins with domains typical for cellulosomal subunits. Most of the genes coding for these proteins are grouped in a cluster similar to that found in cellulolytic clostridial species, such as Clostridium cellulovorans. CAC0916, one of the open reading frames present in the putative cellulosome gene cluster, codes for CelG, a putative endoglucanase belonging to family 9, and it was cloned and overexpressed in Escherichia coli. The overproduced CelG protein was purified by making use of its high affinity for cellulose and was characterized. The biochemical properties of the purified CelG were comparable to those of other known enzymes belonging to the same family. Expression of CelG by C. acetobutylicum grown on different substrates was studied by Western blotting by using antibodies raised against the purified E. coli-produced protein. Whereas the antibodies cross-reacted with CelG-like proteins secreted by cellobiose- or cellulose-grown C. cellulovorans cultures, CelG was not detectable in extracellular medium from C. acetobutylicum grown on cellobiose or glucose. However, notably, when lichenan-grown cultures were used, several bands corresponding to CelG or CelG-like proteins were present, and there was significantly increased extracellular endoglucanase activity.     

Relationship of Psychiatric Diagnosis and Weight Loss Maintenance in Obese Breast Cancer Survivors

Objective: Obese breast cancer survivors are a unique population for weight loss counseling because both obesity and a diagnosis of breast cancer can increase the risk of depression. In this pilot study, weight loss maintenance was examined in obese breast cancer survivors with relationship to psychiatric diagnosis. Research Methods and Procedures: Forty‐eight subjects were enrolled. The intervention, which used individualized counseling for diet and exercise, lasted 24 months. After a 6‐month period of no contact with study subjects, a follow‐up body weight was obtained at 30 months. Results: The nine subjects who dropped out of the study before 12 months all failed to complete a structured psychiatric interview. Of the remaining 39 subjects, 9 had major depressive disorder, and 10 had a definable psychiatric disorder of lesser severity such as adjustment disorder. Subjects with any type of psychiatric diagnosis displayed significantly less weight loss at the 12‐month time‐point than those with no diagnosis (6.3% vs. 12.6% loss of baseline weight, respectively). At the 30‐month follow‐up visit, subjects with any psychiatric disorder had a mean weight loss of 1.2% of baseline weight compared with 7.8% weight loss in subjects with no diagnosis. Discussion: These results suggest that the presence of psychiatric disorders can interfere with weight loss. Therefore, recognition and treatment of psychiatric disorders may be important in attempts at weight reduction, and this will be especially important in populations such as cancer survivors, who seem to have higher rates of depression and other disorders than the general population.     

Western corn rootworm (Diabrotica virgifera virgifera LeConte) population dynamics

1 The western corn rootworm Diabrotica virgifera virgifera LeConte is a major insect pest of field maize, Zea mays L. Larvae can cause substantial injury by feeding on maize roots. Larval feeding may destroy individual roots or root nodes, and reduce plant growth, stability, and yield. Costs associated with managing corn rootworms in continuous maize are annually one of the largest expenditures for insect management in the United States Corn Belt.
2 Even though D. virgifera virgifera has been studied intensively for over 50 years, there is renewed interest in the biology, ecology, and genetics of this species because of its ability to rapidly adapt to management tactics, and its aggressive invasive nature.
3 This article provides a comprehensive review of D. virgifera virgifera population dynamics, specifically: diapause, larval and adult development, seasonality, spatial and temporal dynamics at local and landscape scales, invasiveness in North America and Europe, and non-trophic interactions with other arthropods.
4 Gaps in current knowledge are identified and discussed especially within the context of challenges that scientists in North America and Europe are currently facing regarding pest dynamics and the need to develop appropriate management strategies for each geographic area.     

Reagentless fluorescent biosensors from artificial families of antigen binding proteins

Antibodies and artificial families of antigen binding proteins (AgBP) are constituted by a connected set of hypervariable (or randomized) residue positions, supported by a constant polypeptide backbone. The residues that form the binding site for a given antigen, are selected among the hypervariable residues. We showed that it is possible to transform any AgBP of these families into a reagentless fluorescent biosensor, specific of the target antigen, simply by coupling a solvatochromic fluorophore to one of the hypervariable residues that have little or no importance for the interaction with the antigen, after changing this residue into cysteine by mutagenesis. We validated this approach with a DARPin (Designed Ankyrin Repeat Protein) and a Nanofitin (also known as Affitin) with high success rates. Reagentless fluorescent biosensors recognize their antigen in an immediate, quantitative, selective and specific way, without any manipulation of the sample to analyze or addition of reagent.     

The inner rod of virulence‐associated type III secretion systems constitutes a needle adapter of one helical turn that is deeply integrated into the system's export apparatus

Type III secretion injectisomes are essential virulence factors for many pathogenic bacteria by mediating the transport of effector proteins into eukaryotic host cells. The secretion conduit of injectisomes is formed by a helical assembly of three hydrophobic proteins (SctR, SctS and SctT), an inner rod (SctI) and a needle filament (SctF). SctI is thought to play a role in switching between the secretion of different substrate classes and assembly of the inner rod has been implicated in regulating the length of the needle filament. While high‐resolution structures of the hydrophobic components and of the needle filament have been solved, little is known about the structure and the assembly of the inner rod, which impedes the deeper assessment of its function. Here we show by exhaustive in vivo photocrosslinking that SctI engages in extensive interactions with SctR and SctT throughout its entire length. Our data imply that the inner rod serves as an adapter between the export apparatus and the needle filament by forming one helical turn. We show that assembly of the inner rod does not play a role in needle length control nor in substrate specificity switching. Instead, our findings imply that inner rod assembly must precede assembly of the needle filament.     

Is Qualitative Research Second Class Science? A Quantitative Longitudinal Examination of Qualitative Research in Medical Journals

Background

Qualitative research appears to be gaining acceptability in medical journals. Yet, little is actually known about the proportion of qualitative research and factors affecting its publication. This study describes the proportion of qualitative research over a 10 year period and correlates associated with its publication.

Design

A quantitative longitudinal examination of the proportion of original qualitative research in 67 journals of general medicine during a 10 year period (1998–2007). The proportion of qualitative research was determined by dividing original qualitative studies published (numerator) by all original research articles published (denominator). We used a generalized estimating equations approach to assess the longitudinal association between the proportion of qualitative studies and independent variables (i.e. journals'' country of publication and impact factor; editorial/methodological papers discussing qualitative research; and specific journal guidelines pertaining to qualitative research).

Findings

A 2.9% absolute increase and 3.4-fold relative increase in qualitative research publications occurred over a 10 year period (1.2% in 1998 vs. 4.1% in 2007). The proportion of original qualitative research was independently and significantly associated with the publication of editorial/methodological papers in the journal (b = 3.688, P = 0.012); and with qualitative research specifically mentioned in guidelines for authors (b = 6.847, P<0.001). Additionally, a higher proportion of qualitative research was associated only with journals published in the UK in comparison to other countries, yet with borderline statistical significance (b = 1.776, P = 0.075). The journals'' impact factor was not associated with the publication of qualitative research.

Conclusions

Despite an increase in the proportion of qualitative research in medical journals over a 10 year period, the proportion remains low. Journals'' policies pertaining to qualitative research, as expressed by the appearance of specific guidelines and editorials/methodological papers on the subject, are independently associated with the publication of original qualitative research; irrespective of the journals'' impact factor.     

Suppressive effect of bacterial polysaccharides on BAFF system is responsible for their poor immunogenicity

Capsular polysaccharides of encapsulated bacteria are weakly immunogenic T cell-independent type 2 (TI-2) Ags. Recent findings suggest that BAFF system molecules have a critical role in the development of Ab responses against TI-2 Ags. In this study, we investigated the effect of bacterial polysaccharides on B cell responses to BAFF and a proliferation-inducing ligand (APRIL). We determined that B cells exposed to meningococcal type C polysaccharide (MCPS) or group B Streptococcus serotype V (GBS-V) were unresponsive to BAFF- and APRIL-induced Ig secretion. Moreover, MCPS and GBS-V strongly downregulated transmembrane activator and calcium-modulator and cyclophilin ligand interactor, the BAFF and APRIL receptor that is responsible for Ab development against TI-2 Ags. Interestingly, (4-hydroxy-3-nitrophenyl)acetyl-Ficoll (NP-Ficoll), a prototype TI-2 Ag, did not manifest a suppressive effect on B cells. Paradoxically, whereas GBS-V and MCPS inhibited IFN-γ-induced BAFF production from dendritic cells, NP-Ficoll strongly increased BAFF secretion. TLR 9 agonist CpG deoxyoligonucleotide (ODN) was able to reverse the MCPS-mediated transmembrane activator and calcium-modulator and cyclophilin ligand interactor suppression but could not rescue the Ig secretion in BAFF- or APRIL-stimulated B cells. In support of these in vitro observations, it was observed that CpG ODN could help augment the Ab response against NP in mice immunized with a CpG ODN-containing NP-Ficoll vaccine but exhibited only marginal adjuvant activity for MCPS vaccine. Collectively, these results suggest a mechanism for the weak immunogenicity of bacterial polysaccharides and explain the previously observed differences between bacterial polysaccharide and NP-Ficoll immunogenicity.     

Human Liver Infection in a Dish: Easy-To-Build 3D Liver Models for Studying Microbial Infection

Human liver infection is a major cause of death worldwide, but fundamental studies on infectious diseases affecting humans have been hampered by the lack of robust experimental models that accurately reproduce pathogen-host interactions in an environment relevant for the human disease. In the case of liver infection, one consequence of this absence of relevant models is a lack of understanding of how pathogens cross the sinusoidal endothelial barrier and parenchyma. To fill that gap we elaborated human 3D liver in vitro models, composed of human liver sinusoidal endothelial cells (LSEC) and Huh-7 hepatoma cells as hepatocyte model, layered in a structure mimicking the hepatic sinusoid, which enable studies of key features of early steps of hepatic infection. Built with established cell lines and scaffold, these models provide a reproducible and easy-to-build cell culture approach of reduced complexity compared to animal models, while preserving higher physiological relevance compared to standard 2D systems. For proof-of-principle we challenged the models with two hepatotropic pathogens: the parasitic amoeba Entamoeba histolytica and hepatitis B virus (HBV). We constructed four distinct setups dedicated to investigating specific aspects of hepatic invasion: 1) pathogen 3D migration towards hepatocytes, 2) hepatocyte barrier crossing, 3) LSEC and subsequent hepatocyte crossing, and 4) quantification of human hepatic virus replication (HBV). Our methods comprise automated quantification of E. histolytica migration and hepatic cells layer crossing in the 3D liver models. Moreover, replication of HBV virus occurs in our virus infection 3D liver model, indicating that routine in vitro assays using HBV or others viruses can be performed in this easy-to-build but more physiological hepatic environment. These results illustrate that our new 3D liver infection models are simple but effective, enabling new investigations on infectious disease mechanisms. The better understanding of these mechanisms in a human-relevant environment could aid the discovery of drugs against pathogenic liver infection.