Impact of inherent biases built into proteomic techniques: Proximity labeling and affinity capture compared.

The characterization of protein-protein interactions (PPIs) is of high value for understanding protein function. Two strategies are popular for identification of PPIs direct from the cellular environment: affinity capture (pulldown) isolates the protein of interest with an immobilized matrix that specifically captures the target and potential partners, whereas in BioID, genetic fusion of biotin ligase facilitates proximity biotinylation, and labeled proteins are isolated with streptavidin. Whilst both methods provide valuable insights, they can reveal distinct PPIs, but the basis for these differences is less obvious. Here, we compare both methods using four different trypanosome proteins as baits: poly(A)-binding proteins PABP1 and PABP2, mRNA export receptor MEX67, and the nucleoporin NUP158. With BioID, we found that the population of candidate interacting proteins decreases with more confined bait protein localization, but the candidate population is less variable with affinity capture. BioID returned more likely false positives, in particular for proteins with less confined localization, and identified low molecular weight proteins less efficiently. Surprisingly, BioID for MEX67 identified exclusively proteins lining the inner channel of the nuclear pore complex (NPC), consistent with the function of MEX67, whereas the entire NPC was isolated by pulldown. Similarly, for NUP158, BioID returned surprisingly few PPIs within NPC outer rings that were by contrast detected with pulldown but instead returned a larger cohort of nuclear proteins. These rather significant differences highlight a clear issue with reliance on a single method to identify PPIs and suggest that BioID and affinity capture are complementary rather than alternative approaches.

Nucleoside analogue activators of cyclic AMP-independent protein kinase A of Trypanosoma.

Protein kinase A (PKA), the main effector of cAMP in eukaryotes, is a paradigm for the mechanisms of ligand-dependent and allosteric regulation in signalling. Here we report the orthologous but cAMP-independent PKA of the protozoan Trypanosoma and identify 7-deaza-nucleosides as potent activators (EC50 ≥ 6.5 nM) and high affinity ligands (KD ≥ 8 nM). A co-crystal structure of trypanosome PKA with 7-cyano-7-deazainosine and molecular docking show how substitution of key amino acids in both CNB domains of the regulatory subunit and its unique C-terminal αD helix account for this ligand swap between trypanosome PKA and canonical cAMP-dependent PKAs. We propose nucleoside-related endogenous activators of Trypanosoma brucei PKA (TbPKA). The existence of eukaryotic CNB domains not associated with binding of cyclic nucleotides suggests that orphan CNB domains in other eukaryotes may bind undiscovered signalling molecules. Phosphoproteome analysis validates 7-cyano-7-deazainosine as powerful cell-permeable inducer to explore cAMP-independent PKA signalling in medically important neglected pathogens.

Oral health care for patients with epidermolysis bullosa--best clinical practice guidelines.

OBJECTIVE: To provide the users with information on the current best practices for managing the oral health care of people living with EB. METHODS: A systematic literature search, in which the main topic is dental care in patients with Epidermolysis Bullosa, was performed. Consulted sources, ranging from 1970 to 2010, included MEDLINE, EMBASE, CINAHL, The Cochrane Library, DARE, and the Cochrane controlled trials register (CENTRAL). In order to formulate the recommendations of the selected studies the SIGN system was used. The first draft was analysed and discussed by clinical experts, methodologists and patients representatives on a two days consensus meeting. The resulting document went through an external review process by a panel of experts, other health care professionals, patient representatives and lay reviewers. The final document was piloted in three different centres in United Kingdom, Czech Republic and Argentina. RESULTS: The guideline is composed of 93 recommendations divided into 3 main areas: 1) Oral Care--access issues, early referral, preventative strategies, management of microstomia, prescriptions and review appointments 2) Dental treatment: general treatment modifications, radiographs, restorations, endodontics, oral rehabilitation, periodontal treatment, oral surgery and orthodontics, and 3) Anaesthetic management of dental treatment. CONCLUSIONS: A preventive protocol is today's dental management approach of choice.

Preclinical evaluation of the breast cancer cell-binding peptide, p160.

PURPOSE: Selective delivery of drugs into the target tissue is expected to result in high drug concentrations in the tissue of interest and therefore enhanced drug efficacy. To develop a peptide-based radiopharmaceutical, we investigated the properties of a peptide with affinity for human breast cancer, which has been selected through phage display. EXPERIMENTAL DESIGN: The bioactivity of the p160 peptide (VPWMEPAYQRFL) was evaluated in vitro and in vivo. The specific binding to human breast cancer MDA-MB-435 cells was confirmed in competition experiments. Internalization of the peptide was investigated with confocal microscopy. Furthermore, the biodistribution of (131)I-labeled p160 was studied in tumor-bearing mice. In vivo stability was evaluated at different periods after tracer administration using high-performance liquid chromatography analysis. RESULTS: The binding of (125)I-labeled p160 was inhibited up to 95% by the unlabeled peptide with an IC(50) value of 0.6 micromol/L. In addition, 40% of the total bound activity was found to be internalized into the human breast cancer cells. Although a rapid degradation was seen, biodistribution studies in nude mice showed a higher uptake in tumor than in most of the organs. Perfusion of the animals caused a reduction of the radioligand accumulation in the healthy tissues, whereas the tumor uptake remained constant. A comparison of [(131)I]p160 with a (131)I-labeled Arg-Gly-Asp peptide revealed a higher tumor-to-organ ratio for [(131)I]p160. CONCLUSIONS: p160 has properties that make it an attractive carrier for tumor imaging and the intracellular delivery of isotopes or chemotherapeutic drugs.