Self-Assembly of a Confined Rhodium Catalyst for Asymmetric Hydroformylation of Unfunctionalized Internal Alkenes

A chiral supramolecular ligand has been assembled and applied to the rhodium-catalyzed asymmetric hydroformylation of unfunctionalized internal alkenes. Spatial confinement of the metal center within a chiral pocket results in reversed regioselectivity and remarkable enantioselectivities

Temperature Dependence of the Proteome Profile of the Psychrotolerant Pathogenic Food Spoiler <i>Bacillus weihenstephanensis</i> Type Strain WSBC 10204

Bacillus weihenstephanensis is a subspecies of the Bacillus cereus <i>sensu lato</i> group of spore-forming bacteria known to cause food spoilage or food poisoning. The key distinguishing phenotype of B. weihenstephanensis is its ability to grow below 7 °C or, from a food safety perspective, to grow and potentially produce toxins in a refrigerated environment. Comparison of the proteome profile of B. weihenstephanensis upon its exposure to different culturing conditions can reveal clues to the mechanistic basis of its psychrotolerant phenotype as well as elucidate relevant aspects of its toxigenic profile. To this end, the genome of the type strain B. weihenstephanensis WSBC 10204 was sequenced and annotated. Subsequently, the proteome profiles of cells grown at either 6 or 30 °C were compared, which revealed considerable differences and indicated several hundred (uncharacterized) proteins as being subproteome- and/or temperature-specific. In this manner, several processes were newly indicated to be dependent on growth temperature, such as varying carbon flux routes and a different role for the urea cycle. Furthermore, a possible post-translational regulatory function for acetylation was suggested. Toxin production was determined to be largely independent of growth temperature

In Pursuit of Protein Targets: Proteomic Characterization of Bacterial Spore Outer Layers

<i>Bacillus cereus</i>, responsible for food poisoning, and <i>Clostridium difficile</i>, the causative agent of <i>Clostridium difficile</i>-associated diarrhea (CDAD), are both spore-forming pathogens involved in food spoilage, food intoxication, and other infections in humans and animals. The proteinaceous coat and the exosporium layers from spores are important for their resistance and pathogenicity characteristics. The exosporium additionally provides an ability to adhere to surfaces eventually leading to spore survival in food. Thus, studying these layers and identifying suitable protein targets for rapid detection and removal of spores is of the utmost importance. In this study, we identified 100 proteins from <i>B. cereus</i> spore coat, exosporium and 54 proteins from the <i>C. difficile</i> coat insoluble protein fraction. In an attempt to define a universal set of spore outer layer proteins, we identified 11 superfamily domains common to the identified proteins from two <i>Bacilli</i> and one <i>Clostridium</i> species. The evaluated orthologue relationships of identified proteins across different spore formers resulted in a set of 13 coat proteins conserved across the spore formers and 12 exosporium proteins conserved in the <i>B. cereus</i> group, which could be tested for quick and easy detection or targeted in strategies aimed at removal of spores from surfaces

CM inhibits HIV-1 <i>trans</i>-infection by iDCs.

<p>(A) iDCs were pre-incubated with 100 fold diluted CM, 20μg/ml AZN-D1 (DC-SIGN blocking antibody), 50μg/ml mannan or medium (control) before addition of LAI. Using flow cytometry viral outgrowth in iDC – CD4 T-lymphocyte co-cultures was measured by intracellular staining for p24. Depicted is the number of p24⁺ cells per 1x10⁵ CD3⁺ T cells. (B) Shown are representative dot plots of the data depict in (A). Data points were performed in triplicate.</p

DC-SIGN binding capacity of CM varies greatly between individuals.

<p>(A) Serial dilutions of the 21 CM samples were coated onto an ELISA plate and the end-point dilution showing binding of DC-SIGN-Fc is depicted. For all samples maximal binding was achieved when 11.7μg/ml CM was coated. A large variation between donors was observed ranging from high to no DC-SIGN binding and where the samples can be divided into three groups as indicated with dotted lines (B). The ability of samples to prevent DC-SIGN-Fc binding to trimeric gp140 was determined with a blocking ELISA. 11.7μg/ml CM was pre-incubated with DC-SIGN-Fc before addition to a gp140 coated plate. Next, DC-SIGN binding to gp140 was correlated with the OD found in the DC-SIGN binding ELISA where 11.7μg/ml CM was coated (P<0.01). As a control 5μg/ml mannan was included, depicted as an open square.</p

Mass spectrometry indicates that human CM lactoferrin binds DC-SIGN.

<p>(A) 4–12% SDS PAGE gel loaded (from left to right) with a 250kD protein marker, agarose beads, DC-SIGN-Fc, the supernatant from the first wash, supernatant from the second wash, and the DC-SIGN-Fc coated agarose beads loaded with the DC-SIGN binding component from CM. Band #2, 3 and 4 potentially contain the DC-SIGN binding component of CM. Ion trap mass spectrometry of in gel digests identified human lactoferrin fragments (highly abundant in band #3) and immunoglobulins in all three bands and intelectin-1 in band #4 (with trace amounts in the other bands). (B) CM representing a high DC-SIGN binder, an intermediate DC-SIGN binder and a low/no DC-SIGN binder were coated on an ELISA plate and were tested for DC-SIGN and lactoferrin binding. The first graph (left) confirms the DC-SIGN binding status while the second graph (right) shows the binding capacity of polyclonal anti-lactoferrin, which is high for CM from a DC-SIGN high binder, intermediate for an intermediate DC-SIGN binder and not present in CM from a low/no DC-SIGN binder.</p

Biochemical analysis of the DC-SIGN binding component in CM.

<p>(A) Unfractionated CM (input) or <30, 30–100 or >100kDa CM fractions were coated to an ELISA plate and DC-SIGN-Fc binding was determined. Binding of unfractionated CM is set to 100% and binding of the fractions is expressed as a relative percentage. The <30kDa fraction shows no DC-SIGN binding, between 30–100kDa shows limited binding whilst >100kDa shows stronger binding. (B) DC-SIGN-Fc was incubated with untreated, heated (10 min at 95°C), proteinase K treated CM or medium (negative control) prior to addition to a gp140 coated plate. Compared to the media alone control incubating DC-SIGN-Fc with treated or untreated CM led to similar reductions in gp140 binding. (C) DC-SIGN-Fc was incubated with CM, BSA or mannan prior to addition to a gp140 coated plate. Untreated, both CM and mannan inhibit DC-SIGN-Fc from binding gp140 compared to BSA (negative control). Depletion of mannose structures from CM, BSA and mannan by a pull-down with <i>Galanthus Nivalis</i> lectin does not alter the DC-SIGN-Fc binding capacity of CM while mannan loses its ability to prevent gp140 binding by DC-SIGN-Fc. Data points were performed in triplicate.</p

CM does not inhibit HIV-1 direct infection but does <i>trans</i>-infection.

<p>(A) NSI-18 (R5) or LAI (X4) (5ng/ml p24) was pre-incubated with medium (control) or 100 fold and 1000 fold diluted CM after which the mixture was added to TZM-bl cells. Two days post infection the cells were lysed and the luciferase activity was measured demonstrating that the level of infection was similar whether CM was present or not. (B) CD4⁺ T-lymphocytes were incubated with medium (control) or 100 fold and 1000 fold diluted CM prior to addition of LAI. Viral outgrowth, supernatant p24, was measured over several days, with no difference observed. (C) Raji DC-SIGN cells were incubated with medium (negative control) or 100 fold diluted CM or 300 fold diluted CM prior to addition of either NSI-18 or LAI virus, washed and added to CD4⁺ T-lymphocytes. Viral outgrowth, determined by capsid p24 ELISA, is depicted in Raji DC-SIGN cell—CD4⁺ T lymphocyte co-cultures. For both NSI-18 and LAI inhibition is observed with 100 fold diluted CM and less with 300 fold diluted CM. Data points were performed in triplicate.</p

CM binds DC-SIGN thereby preventing gp140 binding.

<p>(A) DC-SIGN binding ELISA with 300 fold diluted CM coated on an ELISA plate and DC-SIGN-Fc as detection antibody, demonstrates DC-SIGN-Fc binds CM compared to EGTA treated product (negative control) (p<0.0001). (B) DC-SIGN-Fc was pre-incubated with mannan (positive control) and CM dilutions before being added to a gp140 coated plate. Depicted is the percentage by which DC-SIGN-Fc binding to gp140 is blocked, pre-incubation with mannan was set to 100% blocking and pre-incubation with medium to 0%. Pre-incubating DC-SIGN-Fc with up to a 1000 fold diluted CM inhibits HIV-1 envelope gp140 trimer binding. Data points were performed in triplicate.</p