Mapping the protein interaction network in methicillin-resistant Staphylococcus aureus

Mortality attributable to infection with methicillin-resistant Staphylococcus aureus (MRSA) has now overtaken the death rate for AIDS in the United States, and advances in research are urgently needed to address this challenge. We report the results of the systematic identification of protein-protein interactions for the hospital-acquired strain MRSA-252. Using a high-throughput pull-down strategy combined with quantitative proteomics to distinguish specific from nonspecific interactors, we identified 13,219 interactions involving 608 MRSA proteins. Consecutive analyses revealed that this protein interaction network (PIN) exhibits scale-free organization with the characteristic presence of highly connected hub proteins. When clinical and experimental antimicrobial targets were queried in the network, they were generally found to occupy peripheral positions in the PIN with relatively few interacting partners. In contrast, the hub proteins identified in this MRSA PIN that are essential for network integrity and stability have largely been overlooked as drug targets. Thus, this empirical MRSA-252 PIN provides a rich source for identifying critical proteins essential for network stability, many of which can be considered as prospective antimicrobial drug targets.     

Development of p97 AAA ATPase inhibitors

Specific p97 inhibitors are valuable research tools to carry out mechanistic and cellular investigations of p97 biology. p97 is an abundant, ubiquitin-selective chaperone that has multiple functions and is essential for life. Therefore, genetic methods that require long incubations like siRNA or expression of dominant-negative p97 mutants are likely to generate complicated outcomes due to secondary consequences that arise upon slow depletion of p97 activity. We recently identified a small molecule p97 inhibitor, N ( 2) ,N ( 4) -dibenzylquinazoline-2,4-diamine (DBeQ), and documented its effects on blocking autophagic degradation of LC3-II and proteasomal degradation of a p97-dependent ubiquitin-proteasome system (UPS) substrate. What distinguishes DBeQ from conventional proteasome inhibitors is that DBeQ affects both the UPS and autophagic protein degradation pathways and rapidly activates cell death. Whether DBeQ activates autophagic and/or apoptotic cell death will require further work to evaluate its detailed mechanism of action. An exciting goal for the future will be to generate p97 inhibitors that affect one or the other pathway. We propose that generation of 'separation of function' inhibitors will be a challenging adventure for chemical biologists but will yield extremely powerful tools to study p97 and enable evaluation of the therapeutic potential of targeting distinct p97 complexes.     

Disentangling the roles of approach, activation and valence in instrumental and pavlovian responding

Hard-wired, Pavlovian, responses elicited by predictions of rewards and punishments exert significant benevolent and malevolent influences over instrumentally-appropriate actions. These influences come in two main groups, defined along anatomical, pharmacological, behavioural and functional lines. Investigations of the influences have so far concentrated on the groups as a whole; here we take the critical step of looking inside each group, using a detailed reinforcement learning model to distinguish effects to do with value, specific actions, and general activation or inhibition. We show a high degree of sophistication in Pavlovian influences, with appetitive Pavlovian stimuli specifically promoting approach and inhibiting withdrawal, and aversive Pavlovian stimuli promoting withdrawal and inhibiting approach. These influences account for differences in the instrumental performance of approach and withdrawal behaviours. Finally, although losses are as informative as gains, we find that subjects neglect losses in their instrumental learning. Our findings argue for a view of the Pavlovian system as a constraint or prior, facilitating learning by alleviating computational costs that come with increased flexibility.     

Chikungunya virus neutralization antigens and direct cell-to-cell transmission are revealed by human antibody-escape mutants

Chikungunya virus (CHIKV) is an alphavirus responsible for numerous epidemics throughout Africa and Asia, causing infectious arthritis and reportedly linked with fatal infections in newborns and elderly. Previous studies in animal models indicate that humoral immunity can protect against CHIKV infection, but despite the potential efficacy of B-cell-driven intervention strategies, there are no virus-specific vaccines or therapies currently available. In addition, CHIKV has been reported to elicit long-lasting virus-specific IgM in humans, and to establish long-term persistence in non-human primates, suggesting that the virus might evade immune defenses to establish chronic infections in man. However, the mechanisms of immune evasion potentially employed by CHIKV remain uncharacterized. We previously described two human monoclonal antibodies that potently neutralize CHIKV infection. In the current report, we have characterized CHIKV mutants that escape antibody-dependent neutralization to identify the CHIKV E2 domain B and fusion loop "groove" as the primary determinants of CHIKV interaction with these antibodies. Furthermore, for the first time, we have also demonstrated direct CHIKV cell-to-cell transmission, as a mechanism that involves the E2 domain A and that is associated with viral resistance to antibody-dependent neutralization. Identification of CHIKV sub-domains that are associated with human protective immunity, will pave the way for the development of CHIKV-specific sub-domain vaccination strategies. Moreover, the clear demonstration of CHIKV cell-to-cell transmission and its possible role in the establishment of CHIKV persistence, will also inform the development of future anti-viral interventions. These data shed new light on CHIKV-host interactions that will help to combat human CHIKV infection and inform future studies of CHIKV pathogenesis.     

Determination of uremic solutes in biological fluids of chronic kidney disease patients by HPLC assay

During chronic kidney disease (CKD), solutes called uremic solutes, accumulate in blood and tissues of patients. We developed an HPLC method for the simultaneous determination of several uremic solutes of clinical interest in biological fluids: phenol (Pol), indole-3-acetic acid (3-IAA), p-cresol (p-C), indoxyl sulfate (3-INDS) and p-cresol sulfate (p-CS). These solutes were separated by ion-pairing HPLC using an isocratic flow and quantified with a fluorescence detection. The mean serum concentrations of 3-IAA, 3-INDS and p-CS were 2.12, 1.03 and 13.03 μM respectively in healthy subjects, 3.21, 17.45 and 73.47 μM in non hemodialyzed stage 3-5 CKD patients and 5.9, 81.04 and 120.54 μM in hemodialyzed patients (stage 5D). We found no Pol and no p-C in any population. The limits of quantification for 3-IAA, 3-INDS, and p-CS were 0.83, 0.72, and 3.2 μM respectively. The within-day CVs were between 1.23 and 3.12% for 3-IAA, 0.98 and 2% for 3-INDS, and 1.25 and 3.01% for p-CS. The between-day CVs were between 1.78 and 5.48% for 3-IAA, 1.45 and 4.54% for 3-INDS, and 1.19 and 6.36% for p-CS. This HPLC method permits the simultaneous and quick quantification of several uremic solutes for daily analysis of large numbers of samples.     

Structural basis for regulation of the Crk signaling protein by a proline switch

Proline switches, controlled by cis-trans isomerization, have emerged as a particularly effective regulatory mechanism in a wide range of biological processes. Here we report the structures of both the cis and trans conformers of a proline switch in the Crk signaling protein. Proline isomerization toggles Crk between two conformations: an autoinhibitory conformation, stabilized by the intramolecular association of two tandem SH3 domains in the cis form, and an uninhibited, activated conformation promoted by the trans form. In addition to acting as a structural switch, the heterogeneous proline recruits cyclophilin A, which accelerates the interconversion rate between the isomers, thereby regulating the kinetics of Crk activation. The data provide atomic insight into the mechanisms that underpin the functionality of this binary switch and elucidate its remarkable efficiency. The results also reveal new SH3 binding surfaces, highlighting the binding versatility and expanding the noncanonical ligand repertoire of this important signaling domain.     

Genotype by environment interactions for Pinus radiata in New South Wales, Australia

Historical data from 26 progeny trials in the NSW breeding program was analyzed to determine the extent and practical importance of genotype by environment (G×E) interactions for a range of traits. Significant G×E interaction was present for diameter (DBH) with 75% of the 35 estimated between-site genetic correlations falling below the threshold value of 0.6 where regionalization should be considered. Estimated genetic correlations for stem straightness, branch quality, and outerwood density were much higher, indicating these traits are not as affected by G×E. Levels of G×E for DBH are sufficient to have serious impacts on the expression of genetic gain in deployed material. For DBH, altitude differences between sites appear to be the key factor driving the G×E with a difference of greater than 280 m in altitude leading to a breakdown in correlations. Two groups of sites were identified as having limited G×E within each group: one for sites above 900 m elevation plus a lower-altitude group. Sites included in the higher-altitude group were located across the entire north–south geographic range of NSW. Equations for prediction of site mean DBH indicate that altitude, prior land use, and underlying geology are key driving variables. A more complex model was developed for predicting the between-site genetic correlations for DBH with the model accounting for approximately 50% of the observed variation.     

Low‐generation asymmetric dendrimers exhibit minimal toxicity and effectively complex DNA

Conventional dendrimers are spherical symmetrically branched polymers ending with active surface functional groups. Polyamidoamine (PAMAM) dendrimers have been widely studied as gene delivery vectors and have proven effective at delivering DNA to cells in vitro. However, higher‐generation (G4‐G8) PAMAM dendrimers exhibit toxicity due to their high cationic charge density and this has limited their application in vitro and in vivo. Another limitation arises when attempts are made to functionalize spherical dendrimers as targeting moieties cannot be site‐specifically attached. Therefore, we propose that lower‐generation asymmetric dendrimers, which are likely devoid of toxicity and to which site‐specific attachment of targeting ligands can be achieved, would be a viable alternative to currently available dendrimers. We synthesized and characterized a series of peptide‐based asymmetric dendrimers and compared their toxicity profile and ability to condense DNA to spherical PAMAM G1 dendrimers. We show that asymmetric dendrimers are minimally toxic and condense DNA into stable toroids which have been reported necessary for efficient cell transfection. This paves the way for these systems to be conjugated with targeting ligands for gene delivery in vitro and in vivo. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.