A decade of crystallization drops: Crystallization of the cbb3 cytochrome c oxidase from Pseudomonas stutzeri

The cbb₃ cytochrome c oxidases are distant members of the superfamily of heme copper oxidases. These terminal oxidases couple O₂ reduction with proton transport across the plasma membrane and, as a part of the respiratory chain, contribute to the generation of an electrochemical proton gradient. Compared with other structurally characterized members of the heme copper oxidases, the recently determined cbb₃ oxidase structure at 3.2 Å resolution revealed significant differences in the electron supply system, the proton conducting pathways and the coupling of O₂ reduction to proton translocation. In this paper, we present a detailed report on the key steps for structure determination. Improvement of the protein quality was achieved by optimization of the number of lipids attached to the protein as well as the separation of two cbb₃ oxidase isoenzymes. The exchange of n‐dodecyl‐β‐d ‐maltoside for a precisely defined mixture of two α‐maltosides and decanoylsucrose as well as the choice of the crystallization method had a most profound impact on crystal quality. This report highlights problems frequently encountered in membrane protein crystallization and offers meaningful approaches to improve crystal quality.     

Validation of a fluorescence-based screening concept to identify ribosome assembly defects in Escherichia coli

While the structure of mature ribosomes is analyzed in atomic detail considerably less is known about their assembly process in living cells. This is mainly due to technical and conceptual hurdles. To analyze ribosome assembly in vivo, we designed and engineered an Escherichiacoli strain—using chromosomal gene knock-in techniques—that harbors large and small ribosomal subunits labeled with the fluorescent proteins EGFP and mCherry, respectively. A thorough characterization of this reporter strain revealed that its growth properties and translation apparatus were wild-type like. Alterations in the ratio of EGFP over mCherry fluorescence are supposed to indicate ribosome assembly defects. To provide proof of principle, subunit specific assembly defects were provoked and could be identified by both manual and fully automated fluorometric in vivo assays. This is to our knowledge the first methodology that directly detects ribosome assembly defects in vivo in a high-throughput compatible format. Screening of knock-out collections and small molecule libraries will allow identification of new ribosome assembly factors and possible inhibitors.     

Observations of neutral buoyancy in diapausing copepods Calanoides acutus during Antarctic winter

The herbivorous Antarctic copepod Calanoides acutus overwinters inactively in a resting stage (diapause) at depths below 500 m. It is assumed that during diapause C. acutus is neutrally buoyant in order to retain energy reserves otherwise depleted by swimming activities. However, so far, no experimental observations on its buoyancy have been reported and our knowledge of buoyancy regulation mechanisms is incomplete. In the present study, species-specific differences in buoyancy were assessed visually. Observations were made of specimens from the diapausing cohort of C. acutus and compared to another herbivorous copepod Calanus propinquus, which overwinters actively feeding in the upper water layers. Freshly caught copepods were anaesthetized in a 3-amino-benzoic acid ethyl ester (MS222) in seawater solution in order to exclude the influence of swimming movements on buoyancy control. It was shown that C. propinquus was negatively buoyant, whereas diapausing C. acutus remained neutrally buoyant. This is the first record that neutral buoyancy in diapausing copepods is maintained by the biochemical body composition without the additional need of swimming movements.     

The CDK4/CDK6 inhibitor PD0332991 paradoxically stabilizes activated cyclin D3-CDK4/6 complexes

CDK4 and CDK6 bound to D-type cyclins are master integrators of G1 phase cell cycle regulations by initiating the inactivating phosphorylation of the central oncosuppressor pRb. Because of their frequent deregulation in cancer, cyclin D-CDK4/6 complexes are emerging as especially promising therapeutic targets. The specific CDK4/6 inhibitor PD0332991 is currently tested in a growing number of phase II/III clinical trials against a variety of pRb-proficient chemotherapy-resistant cancers. We have previously shown that PD0332991 inhibits not only CDK4/6 activity but also the activation by phosphorylation of the bulk of cyclin D-CDK4 complexes stabilized by p21 binding. Here we show that PD0332991 has either a positive or a negative impact on the activation of cyclin D-CDK4/6 complexes, depending on their binding to p21. Indeed, whereas PD0332991 inhibits the phosphorylation and activity of p21-bound CDK4/6, it specifically stabilized activated cyclin D3-CDK4/6 complexes devoid of p21 and p27. After elimination of PD0332991, these activated cyclin D3-CDK4/6 complexes persisted for at least 24 h, resulting in paradoxical cell cycle entry in the absence of a mitogenic stimulation. This unsuspected positive effect of PD0332991 on cyclin D3-CDK4/6 activation should be carefully assessed in the clinical evaluation of PD0332991, which until now only involves discontinuous administration protocols.     

Inactivation of Fructose-1,6-Bisphosphate Aldolase Prevents Optimal Co-catabolism of Glycolytic and Gluconeogenic Carbon Substrates in Mycobacterium tuberculosis

Metabolic pathways used by Mycobacterium tuberculosis (Mtb) to establish and maintain infections are important for our understanding of pathogenesis and the development of new chemotherapies. To investigate the role of fructose-1,6-bisphosphate aldolase (FBA), we engineered an Mtb strain in which FBA levels were regulated by anhydrotetracycline. Depletion of FBA resulted in clearance of Mtb in both the acute and chronic phases of infection in vivo, and loss of viability in vitro when cultured on single carbon sources. Consistent with prior reports of Mtb''s ability to co-catabolize multiple carbon sources, this in vitro essentiality could be overcome when cultured on mixtures of glycolytic and gluconeogenic carbon sources, enabling generation of an fba knockout (Δfba). In vitro studies of Δfba however revealed that lack of FBA could only be compensated for by a specific balance of glucose and butyrate in which growth and metabolism of butyrate were determined by Mtb''s ability to co-catabolize glucose. These data thus not only evaluate FBA as a potential drug target in both replicating and persistent Mtb, but also expand our understanding of the multiplicity of in vitro conditions that define the essentiality of Mtb''s FBA in vivo.     

Recommendations for the Empirical Treatment of Complicated Urinary Tract Infections Using Surveillance Data on Antimicrobial Resistance in the Netherlands

Background

Complicated urinary tract infections (c-UTIs) are among the most common nosocomial infections and a substantial part of the antimicrobial agents used in hospitals is for the treatment of c-UTIs. Data from surveillance can be used to guide the empirical treatment choices of clinicians when treating c-UTIs. We therefore used nation-wide surveillance data to evaluate antimicrobial coverage of agents for the treatment of c-UTI in the Netherlands.

Methods

We included the first isolate per patient of urine samples of hospitalised patients collected by the Infectious Disease Surveillance Information System for Antibiotic Resistance (ISIS-AR) in 2012, and determined the probability of inadequate coverage for antimicrobial agents based on species distribution and susceptibility. Analyses were repeated for various patient groups and hospital settings.

Results

The most prevalent bacteria in 27,922 isolates of 23,357 patients were Escherichia coli (47%), Enterococcus spp. (14%), Proteus mirabilis (8%), and Klebsiella pneumoniae (7%). For all species combined, the probability of inadequate coverage was <5% for amoxicillin or amoxicillin-clavulanic acid combined with gentamicin and the carbapenems. When including gram-negative bacteria only, the probability of inadequate coverage was 4.0%, 2.7%, 2.3% and 1.7%, respectively, for amoxicillin, amoxicillin-clavulanic acid, a second or a third generation cephalosporin in combination with gentamicin, and the carbapenems (0.4%). There were only small variations in results among different patient groups and hospital settings.

Conclusions

When excluding Enterococcus spp., considered as less virulent, and the carbapenems, considered as last-resort drugs, empirical treatment for c-UTI with the best chance of adequate coverage are one of the studied beta-lactam-gentamicin combinations. This study demonstrates the applicability of routine surveillance data for up-to-date clinical practice guidelines on empirical antimicrobial therapy, essential in patient care given the evolving bacterial susceptibility.