Rapid fabrication of precise high-throughput filters from membrane protein nanosheets.

Biological membranes are ideal for separations as they provide high permeability while maintaining high solute selectivity due to the presence of specialized membrane protein (MP) channels. However, successful integration of MPs into manufactured membranes has remained a significant challenge. Here, we demonstrate a two-hour organic solvent method to develop 2D crystals and nanosheets of highly packed pore-forming MPs in block copolymers (BCPs). We then integrate these hybrid materials into scalable MP-BCP biomimetic membranes. These MP-BCP nanosheet membranes maintain the molecular selectivity of the three types of ß-barrel MP channels used, with pore sizes of 0.8 nm, 1.3 nm, and 1.5 nm. These biomimetic membranes demonstrate water permeability that is 20-1,000 times greater than that of commercial membranes and 1.5-45 times greater than that of the latest research membranes with comparable molecular exclusion ratings. This approach could provide high performance alternatives in the challenging sub-nanometre to few-nanometre size range.

Distribution of α-Gustducin and Vimentin in premature and mature taste buds in chickens.

The sensory organs for taste in chickens (Gallus sp.) are taste buds in the oral epithelium of the palate, base of the oral cavity, and posterior tongue. Although there is not a pan-taste cell marker that labels all chicken taste bud cells, α-Gustducin and Vimentin each label a subpopulation of taste bud cells. In the present study, we used both α-Gustducin and Vimentin to further characterize chicken taste buds at the embryonic and post-hatching stages (E17-P5). We found that both α-Gustducin and Vimentin label distinct and overlapping populations of, but not all, taste bud cells. A-Gustducin immunosignals were observed as early as E18 and were consistently distributed in early and mature taste buds in embryos and hatchlings. Vimentin immunoreactivity was initially sparse at the embryonic stages then became apparent in taste buds after hatch. In hatchlings, α-Gustducin and Vimentin immunosignals largely co-localized in taste buds. A small subset of taste bud cells were labeled by either α-Gustducin or Vimentin or were not labeled. Importantly, each of the markers was observed in all of the examined taste buds. Our data suggest that the early onset of α-Gustducin in taste buds might be important for enabling chickens to respond to taste stimuli immediately after hatch and that distinctive population of taste bud cells that are labeled by different molecular markers might represent different cell types or different phases of taste bud cells. Additionally, α-Gustducin and Vimentin can potentially be used as molecular markers of all chicken taste buds in whole mount tissue.

Bulk phase biochemistry of PIF1 and RecQ4 family helicases.

DNA helicases are involved in nearly all facets of genome integrity, and in humans, mutations in helicase-encoding genes are often linked to diseases of genomic instability. Two highly studied and evolutionarily conserved helicase families are the PIF1 and RecQ helicases. Enzymes in these families have known roles in DNA replication, recombination, and repair, as well as telomere maintenance, DNA recombination, and transcription. Although genetics, structural biology, and a variety of other techniques have been used to study these helicases, ensemble analyses of their basic biochemical activities such as DNA binding, ATP hydrolysis, and DNA unwinding have made significant contributions to our understanding of their physiological roles. Here, we present general methods to generate recombinant proteins from both helicase families, as well as standard biochemical assays to investigate their activities on DNA.

Insight into the AcrAB-TolC Complex Assembly Process Learned from Competition Studies.

The RND family efflux pump AcrAB-TolC in E. coli and its homologs in other Gram-negative bacteria are major players in conferring multidrug resistance to the cells. While the structure of the pump complex has been elucidated with ever-increasing resolution through crystallography and Cryo-EM efforts, the dynamic assembly process remains poorly understood. Here, we tested the effect of overexpressing functionally defective pump components in wild type E. coli cells to probe the pump assembly process. Incorporation of a defective component is expected to reduce the efflux efficiency of the complex, leading to the so called "dominant negative" effect. Being one of the most intensively studied bacterial multidrug efflux pumps, many AcrA and AcrB mutations have been reported that disrupt efflux through different mechanisms. We examined five groups of AcrB and AcrA mutants, defective in different aspects of assembly and substrate efflux. We found that none of them demonstrated the expected dominant negative effect, even when expressed at concentrations many folds higher than their genomic counterpart. The assembly of the AcrAB-TolC complex appears to have a proof-read mechanism that effectively eliminated the formation of futile pump complex.

Probing the Dynamics of AcrB Through Disulfide Bond Formation.

The resistant-nodulation-division (RND) superfamily member tripartite AcrA-AcrB-TolC efflux pump is a major contributor to the multidrug resistance in Escherichia coli. AcrB is the inner membrane protein of the efflux complex and is responsible for the recognition and binding of compounds before their transportation out of the cell. Understanding the dynamics of AcrB during functional rotation in the process of drug efflux is the focus of this study. For this purpose, we introduced six inter-subunit disulfide bonds into the periplasmic domain of AcrB using site-directed mutagenesis to study the importance of the relative flexibility at the inter-subunit interface. Western blot analysis revealed the formation of disulfide bond-linked AcrB oligomers, which were reduced into monomers under reducing conditions. The impact of mutation and formation of disulfide bond on efflux were evaluated via comparison of the minimum inhibitory concentration (MIC) of an acrB knockout strain expressing different mutants. The double Cys mutants tested led to equal or higher susceptibility to AcrB substrates compared to their corresponding single mutants. To determine if the reduction of activity in a double mutant is due to restriction on conformational changes by the disulfide bond formation, ethidium bromide accumulation assays were conducted utilizing dithiothreitol (DTT) as the reducing agent. In two cases, the activities of the double Cys mutants were partially restored by DTT reduction, confirming the importance of relative movement in the respective location for function. These findings provide new insights into the dynamics of the AcrAB-TolC efflux pump in E. coli.

Increasing Salt Rejection of Polybenzimidazole Nanofiltration Membranes via the Addition of Immobilized and Aligned Aquaporins.

Aquaporins are water channel proteins in cell membrane, highly specific for water molecules while restricting the passage of contaminants and small molecules, such as urea and boric acid. Cysteine functional groups were installed on aquaporin Z for covalent attachment to the polymer membrane matrix so that the proteins could be immobilized to the membranes and aligned in the direction of the flow. Depth profiling using x-ray photoelectron spectrometer (XPS) analysis showed the presence of functional groups corresponding to aquaporin Z modified with cysteine (Aqp-SH). Aqp-SH modified membranes showed a higher salt rejection as compared to unmodified membranes. For 2 M NaCl and CaCl2 solutions, the rejection obtained from Aqp-SH membranes was 49.3 ± 7.5% and 59.1 ± 5.1%. On the other hand, the rejections obtained for 2 M NaCl and CaCl2 solutions from unmodified membranes were 0.8 ± 0.4% and 1.3 ± 0.2% respectively. Furthermore, Aqp-SH membranes did not show a significant decrease in salt rejection with increasing feed concentrations, as was observed with other membranes. Through simulation studies, it was determined that there was approximately 24% capping of membrane pores by dispersed aquaporins.

Comparison of in vitro and in vivo oligomeric states of a wild type and mutant trimeric inner membrane multidrug transporter.

Many membrane proteins exist and function as oligomers or protein complexes. Routine analytical methods involve extraction and solubilization of the proteins with detergents, which could disturb their actual oligomeric state. AcrB is a trimeric inner membrane multidrug transporter in E. coli. In previous studies, we created a mutant AcrBP223G, which behaves like a monomer when extracted from the cell membrane. However, the actual oligomeric state of AcrBP223G in cell membranes remained unclear, which complicated the interpretation of the mechanism by which the mutation affects function. Here we used several complementary methods to determine the oligomeric state of AcrBP223G in E. coli cell membranes. Two sets of quantitative fluorescent techniques were exploited. For these, we created fluorescent tagged AcrB, AcrB-CFP and AcrB-YPet. Fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) were employed to characterize independently the efficiency of energy transfer between co-expressed AcrB-CFP and AcrB-YPet, and the diffusion coefficient of AcrB-YPet and AcrBP223G-YPet in live E. coli cells. Second, we introduced Cys pairs at the inter-subunit interface and used controlled oxidation to probe inter-subunit distances. The results from all studies converge on the conclusion that AcrBP223G exists as a trimer in cell membranes, which dissociates during the purification steps. The small change in trimer affinity and structure leads to a significant loss of AcrB activity. In addition, throughout this study we developed protocols and established benchmark values, useful for further studies on membrane protein associations in cell membranes.

An Update on the Sense of Taste in Chickens: A Better Developed System than Previously Appreciated.

Taste is important in guiding nutritive choices and motivating food intake. The sensory organs for taste are the taste buds, that transduce gustatory stimuli into neural signals. It has been reported that chickens have a low taste bud number and thus low taste acuity. However, more recent studies indicate that chickens have a well-developed taste system and the reported number and distribution of taste buds may have been significantly underestimated. Chickens, as a well-established animal model for research, are also the major species of animals in the poultry industry. Thus, a clear understanding of taste organ formation and the effects of taste sensation on nutrition and feeding practices is important for improving livestock production strategies. In this review, we provide an update on recent findings in chicken taste buds and taste sensation indicating that the chicken taste organ is better developed than previously thought and can serve as an ideal system for multidisciplinary studies including organogenesis, regenerative medicine, feeding and nutritional choices.

Labeling and analysis of chicken taste buds using molecular markers in oral epithelial sheets.

In chickens, the sensory organs for taste are the taste buds in the oral cavity, of which there are ~240-360 in total number as estimated by scanning electron microscopy (SEM). There is not an easy way to visualize all taste buds in chickens. Here, we report a highly efficient method for labeling chicken taste buds in oral epithelial sheets using the molecular markers Vimentin and α-Gustducin. Immediate tissue fixation following incubation with sub-epithelially injected proteases enabled us to peel off whole epithelial sheets, leaving the shape and integrity of the tissue intact. In the peeled epithelial sheets, taste buds labeled with antibodies against Vimentin and α-Gustducin were easily identified and counted under a light microscope and many more taste buds, patterned in rosette-like clusters, were found than previously reported with SEM. Broiler-type, female-line males have more taste buds than other groups and continue to increase the number of taste buds over stages after hatch. In addition to ovoid-shaped taste buds, big tube-shaped taste buds were observed in the chicken using 2-photon microscopy. Our protocol for labeling taste buds with molecular markers will factilitate future mechanistic studies on the development of chicken taste buds in association with their feeding behaviors.