Background: Staphylococcus aureus and S. epidermidis biofilms differ in structure, growth and regulation, and thus the high-throughput method of evaluating biofilm susceptibility that has been published for S. epidermidis cannot be applied to S. aureus without first evaluating the assay's reproducibility and reliability with S. aureus biofilms.

Methods: Staphylococcus aureus biofilms were treated with eleven approved antibiotics, lysostaphin, or Conflikt®, exposed to the oxidation reduction indicator Alamar blue, and reduction relative to untreated controls was determined visually and spectrophotometrically. The minimum biofilm inhibitory concentration (MBIC) was defined as ≤ 50% Alamar blue reduction and a purple/blue well 60 min after the addition of Alamar blue. Because all of the approved antibiotics had MBICs >128 μg/ml (most >2048 μg/ml), lysostaphin and Conflikt®, with relatively low MBICs, were used to correlate Alamar blue reduction with 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction and viable counts (CFU/ml) for S. aureus ATCC 29213 and three clinical isolates. Alamar blue's stability and lack of toxicity allowed CFU/ml to be determined from the same wells as Alamar blue absorbances.

Results: Overall, Alamar blue reduction had excellent correlation with XTT reduction and with CFU/ml. For ATCC 29213 and two clinical isolates treated with lysostaphin or Conflikt®, Alamar blue reduction had excellent correlation with XTT reduction (r = 0.93-0.99) and with CFU/ml (r = 0.92-0.98). For one of the clinical isolates, the results were moderately correlated for Conflikt® (r = 0.76, Alamar blue vs. XTT; r = 0.81, Alamar blue vs. CFU/ml) and had excellent correlation for lysostaphin (r = 0.95, Alamar blue vs. XTT; r = 0.97, Alamar blue vs. CFU/ml).

Conclusion: A reliable, reproducible method for evaluating biofilm susceptibility was successfully applied to S. aureus biofilms. The described method provides researchers with a simple, nontoxic, relatively inexpensive, high throughput measure of viability after drug treatment. A standardized biofilm Alamar blue assay should greatly increase the rate of discovery of S. aureus biofilm specific agents.

We present an analysis of the stellar populations of 102 visually selected early-type galaxies (ETGs) with spectroscopic redshifts (0.35 ≲ z ≲ 1.5) from observations in the Early Release Science program with the Wide Field Camera 3 (WFC3) on the Hubble Space Telescope (HST). We fit one- and two-component synthetic stellar models to the ETGs UV-optical-near-IR spectral energy distributions and find that a large fraction (∼40%) are likely to have experienced a minor (fYC ≲ 10% of stellar mass) burst of recent (tYC ≲ 1 Gyr) star formation. The measured age and mass fraction of the young stellar populations do not strongly trend with measurements of galaxy morphology. We note that massive (M > 1010.5M☼) recent star-forming ETGs appear to have larger sizes. Furthermore, high-mass, quiescent ETGs identified with likely companions populate a distinct region in the size-mass parameter space, in comparison with the distribution of massive ETGs with evidence of recent star formation (RSF). We conclude that both mechanisms of quenching star formation in disk-like ETGs and (gas-rich, minor) merger activity contribute to the formation of young stars and the size-mass evolution of intermediate redshift ETGs. The number of ETGs for which we have both HST WFC3 panchromatic (especially UV) imaging and spectroscopically confirmed redshifts is relatively small, therefore, a conclusion about the relative roles of both of these mechanisms remains an open question.

The membrane proximal region (MPR, residues 649–683) and transmembrane domain (TMD, residues 684–705) of the gp41 subunit of HIV-1’s envelope protein are highly conserved and are important in viral mucosal transmission, virus attachment and membrane fusion with target cells. Several structures of the trimeric membrane proximal external region (residues 662–683) of MPR have been reported at the atomic level; however, the atomic structure of the TMD still remains unknown. To elucidate the structure of both MPR and TMD, we expressed the region spanning both domains, MPR-TM (residues 649–705), in Escherichia coli as a fusion protein with maltose binding protein (MBP). MPR-TM was initially fused to the C-terminus of MBP via a 42 aa-long linker containing a TEV protease recognition site (MBP-linker-MPR-TM).

Biophysical characterization indicated that the purified MBP-linker-MPR-TM protein was a monodisperse and stable candidate for crystallization. However, crystals of the MBP-linker-MPR-TM protein could not be obtained in extensive crystallization screens. It is possible that the 42 residue-long linker between MBP and MPR-TM was interfering with crystal formation. To test this hypothesis, the 42 residue-long linker was replaced with three alanine residues. The fusion protein, MBP-AAA-MPR-TM, was similarly purified and characterized. Significantly, both the MBP-linker-MPR-TM and MBP-AAA-MPR-TM proteins strongly interacted with broadly neutralizing monoclonal antibodies 2F5 and 4E10. With epitopes accessible to the broadly neutralizing antibodies, these MBP/MPR-TM recombinant proteins may be in immunologically relevant conformations that mimic a pre-hairpin intermediate of gp41.