Fitness Trade-offs Restrict the Evolution of Resistance to Amphotericin B

The evolution of drug resistance in microbial pathogens provides a paradigm for investigating evolutionary dynamics with important consequences for human health. Candida albicans, the leading fungal pathogen of humans, rapidly evolves resistance to two major antifungal classes, the triazoles and echinocandins. In contrast, resistance to the third major antifungal used in the clinic, amphotericin B (AmB), remains extremely rare despite 50 years of use as monotherapy. We sought to understand this long-standing evolutionary puzzle. We used whole genome sequencing of rare AmB-resistant clinical isolates as well as laboratory-evolved strains to identify and investigate mutations that confer AmB resistance in vitro. Resistance to AmB came at a great cost. Mutations that conferred resistance simultaneously created diverse stresses that required high levels of the molecular chaperone Hsp90 for survival, even in the absence of AmB. This requirement stemmed from severe internal stresses caused by the mutations, which drastically diminished tolerance to external stresses from the host. AmB-resistant mutants were hypersensitive to oxidative stress, febrile temperatures, and killing by neutrophils and also had defects in filamentation and tissue invasion. These strains were avirulent in a mouse infection model. Thus, the costs of evolving resistance to AmB limit the emergence of this phenotype in the clinic. Our work provides a vivid example of the ways in which conflicting selective pressures shape evolutionary trajectories and illustrates another mechanism by which the Hsp90 buffer potentiates the emergence of new phenotypes. Developing antibiotics that deliberately create such evolutionary constraints might offer a strategy for limiting the rapid emergence of drug resistance.     

SWIMMING EXERCISE STIMULATES NEURO-GENESIS IN THE SUBVENTRICULAR ZONE VIA INCREASE IN SYNAPSIN I AND NERVE GROWTH FACTOR LEVELS

In this study, we investigated the effects of 8-weeks of swimming exercise on neurogenesis in the subventricular zone (SVZ) and on the levels of nerve growth factor (NGF) and synapsin I protein in the olfactory bulb (OB) of adult rats at a series of relevant time points (2 days, 1 week, 2 weeks, 4 weeks, 3 months, and 6 months). Ninety-six male Sprague Dawley rats were divided into 2 groups: (1) a control group (COG; n = 48, n = 8 for each time point) and (2) a swimming exercise group (SEG; total n = 48; n = 8 for each time point). SEG performed swimming exercise for 5 days per week over a period of 8 weeks. We found that the number of 5-bromo-2’-deoxyuridine-5’-monophosphate (BrdU)- and doublecortin (DCX)-positive cells was significantly higher in SEG than in COG at all time points (Day 2, Week 1, Week 2, Week 4, Month 3, and Month 6; p < 0.001). Furthermore, NGF and synapsin I protein levels were significantly higher in SEG on Day 2, and Weeks 1, 2, and 4 than in COG (p < 0.05 for each time point). Our findings suggest that regular swimming exercise in adult rats increases neurogenesis, neuronal survival, and neuronal maintenance in the SVZ; furthermore, swimming exercise increases the levels of NGF and synapsin I in the OB.     

Analytical characterization of host-cell-protein-rich aggregates in monoclonal antibody solutions

Host-cell proteins (HCPs) and high molecular weight (HMW) species have historically been treated as independent classes of impurities in the downstream processing of monoclonal antibodies (mAbs), but recent indications suggest that they may be partially linked. We have explored this connection with a shotgun proteomic analysis of HMW impurities that were isolated from harvest cell culture fluid (HCCF) and protein A eluate using size-exclusion chromatography (SEC). As part of the proteomic analysis, a cross-digest study was performed in which samples were analyzed using both the standard and native digest techniques to enable a fair comparison between bioprocess pools. This comparison reveals that the HCP profiles of HCCF and protein A eluate overlap substantially more than previous work has suggested, because hundreds of HCPs are conserved in aggregates that may be up to ~50 nm in hydrodynamic radius and that persist through the protein A capture step. Quantitative SWATH proteomics suggests that the majority of the protein A eluate's HCP mass is found in such aggregates, and this is corroborated by ELISA measurements on SEC fractions. The SWATH data also show that intra-aggregate concentrations of individual HCPs are positively correlated between aggregates that were isolated from HCCF and protein A eluate, and species that have generally been considered difficult to remove tend to be more concentrated than their counterparts. These observations support prior hypotheses regarding aggregate-mediated HCP persistence through protein A chromatography and highlight the importance of this persistence mechanism.     

Preliminary Observations on the Characteristics of the Owned Dog Population in Roseau,Dominica

The subject of sanctuaries for chimpanzees has lately become the topic of a great deal of discussion (Brent, Butler, &; Haberstroh, 1997; Committee on Long-Term Care of Chimpanzees, 1997; Dyke, Williams-Blangero, Mamelka, &; Goodwin, 1995; Peterson &; Goodall, 1993). In the United States, laboratories that use chimpanzees in research are facing a housing crisis. An increase in captive births caused by the initiation of the National Chimpanzee Breeding and Research Program in 1986 (Hobson, Graham, &; Rowell, 1991), coupled with the diminished use of chimpanzees as experimental subjects, have led to a large population of chimpanzees considered to be surplus to demand (Blood, Wolfle, &; Whitney, 1992). These chimpanzees, as well as an unknown number from the private sector, are candidates for what is currently being called retirement.