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Discovering new bacterial signaling pathways offers unique antibiotic strategies. Here, through an unbiased resistance screen of 3,884 gene knockout strains, we uncovered a previously unknown non-lytic bactericidal mechanism that sequentially couples three transporters and downstream transcription to lethally suppress respiration of the highly virulent P. aeruginosa strain PA14 - one of three species on the WHO's 'Priority 1: Critical' list. By targeting outer membrane YaiW, cationic lacritin peptide 'N-104' translocates into the periplasm where it ligates outer loops 4 and 2 of the inner membrane transporters FeoB and PotH, respectively, to suppress both ferrous iron and polyamine uptake. This broadly shuts down transcription of many biofilm-associated genes, including ferrous iron-dependent TauD and ExbB1. The mechanism is innate to the surface of the eye and is enhanced by synergistic coupling with thrombin peptide GKY20. This is the first example of an inhibitor of multiple bacterial transporters., Competing Interests: DECLARATION OF INTERESTS GWL is cofounder and CSO of TearSolutions, Inc; and cofounder and CTO of IsletRegen, LLC. Other authors declare no competing interests.

Membranes with molecular-sized, high-density nanopores, which are stable under industrially relevant conditions, are needed to decrease energy consumption for separations. Interfacial polymerization has demonstrated its potential for large-scale production of organic membranes, such as polyamide desalination membranes. We report an analogous ultrafast interfacial process to generate inorganic, nanoporous carbon-doped metal oxide (CDTO) nanofilms for precise molecular separation. For a given pore size, these nanofilms have 2 to 10 times higher pore density (assuming the same tortuosity) than reported and commercial organic solvent nanofiltration membranes, yielding ultra-high solvent permeance, even if they are thicker. Owing to exceptional mechanical, chemical, and thermal stabilities, CDTO nanofilms with designable, rigid nanopores exhibited long-term stable and efficient organic separation under harsh conditions.

Two-dimensional (2D) materials provide a great opportunity for fabricating ideal membranes with ultrathin thickness for high-throughput separation. Graphene oxide (GO), owing to its hydrophilicity and functionality, has been extensively studied for membrane applications. However, fabrication of single-layered GO-based membranes utilizing structural defects for molecular permeation is still a great challenge. Optimization of the deposition methodology of GO flakes could offer a potential solution for fabricating desired nominal single-layered (NSL) membranes that can offer a dominant and controllable flow through structural defects of GO. In this study, a sequential coating methodology was adopted for depositing a NSL GO membrane, which is expected to have no or minimum stacking of GO flakes and thus ensure GO's structural defects as the major transport pathway. We have demonstrated effective rejection of different model proteins (bovine serum albumin (BSA), lysozyme, and immunoglobulin G (IgG)) by tuning the structural defect size via oxygen plasma etching. By generating appropriate structural defects, similar-sized proteins (myoglobin and lysozyme; molecular weight ratio (MWR): ∼1.14) were effectively separated with a separation factor of ∼6 and purity of 92%. These findings may provide new opportunities of using GO flakes for fabricating NSL membranes with tunable pores for applications in the biotechnology industry.

Membrane-based separations allow energy-efficient purification of organic solvents which are typically carried out by energy-intensive distillation. Polymer membranes are inexpensive and have obtained widespread industrial acceptance for water and biotech applications but not organic solvent nanofiltration due to relatively low selectivities. In this work, a new class of polymer brush membranes was prepared with high selectivities for methanol-toluene separation. Stiffening the brush structure by cross-linking with aromatic trimesic acid and aliphatic itaconic acid resulted in an increase in selectivity from 1.4 to 6.5-11.5. This was achieved by graft polymerization of a primary amine monomer (aminoethyl methacrylate) using single electron transfer-living radical polymerization (SET-LRP) followed by cross-linking. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and captive bubble contact angle measurements were used to characterize these membranes. The stiffness of the brush membranes was measured using a quartz crystal microbalance-dissipation (QCM-D) and correlated positively with selectivity for separating organic feed mixtures. This new class of membranes offers a tunable and scalable method for purification of organics.

The production of the biofuel, isobutanol, in E. coli faces limitations due to alcohol toxicity, product inhibition, product recovery, and long-term industrial feasibility. Here we demonstrate an approach of combining both in vivo with in vitro metabolic engineering to produce isobutanol. The in vivo production of α-ketoisovalerate (KIV) was conducted through CRISPR mediated integration of the KIV pathway in bicistronic design (BCD) in E. coli and inhibition of competitive valine pathway using CRISPRi technology. The subsequent in vitro conversion to isobutanol was carried out with engineered enzymes for 2-ketoacid decarboxylase (KIVD) and alcohol dehydrogenase (ADH). For the in vivo production of KIV and subsequent in vitro production of isobutanol, this two-step serial approach resulted in yields of 56% and 93%, productivities of 0.62 and 0.074 g L -1 h -1 , and titers of 5.6 and 1.78 g L -1 , respectively. Thus, this combined biosynthetic system can be used as a modular approach for producing important metabolites, like isobutanol, without the limitations associated with in vivo production using a consolidated bioprocess., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

There is an increasing demand for bioproducts produced by metabolically engineered microbes, such as pharmaceuticals, biofuels, biochemicals and other high value compounds. In order to meet this demand, modular optimization, the optimizing of subsections instead of the whole system, has been adopted to engineer cells to overproduce products. Research into modularity has focused on traditional approaches such as DNA, RNA, and protein-level modularity of intercellular machinery, by optimizing metabolic pathways for enhanced production. While research into these traditional approaches continues, limitations such as scale-up and time cost hold them back from wider use, while at the same time there is a shift to more novel methods, such as moving from episomal expression to chromosomal integration. Recently, nontraditional approaches such as co-culture systems and cell-free metabolic engineering (CFME) are being investigated for modular optimization. Co-culture modularity looks to optimally divide the metabolic burden between different hosts. CFME seeks to modularly optimize metabolic pathways in vitro , both speeding up the design of such systems and eliminating the issues associated with live hosts. In this review we will examine both traditional and nontraditional approaches for modular optimization, examining recent developments and discussing issues and emerging solutions for future research in metabolic engineering.

A completely immobilized cell-free enzyme reaction system was used to convert ketoisovaleric acid to isobutanol, a desirable biofuel, with a molar yield of 43% and a titer of 2 g/L, which are comparable to high performing in vivo systems (e.g. 41% and 5.4 g/L, respectively, for Clostridium thermocellum). The approach utilizes, for the first time, a series of previously reported enzyme mutants that either overproduce the product or are more stable when compared with their wild type. The selected enzyme variants include keto-acid decarboxylase attached to a maltose binding protein, alcohol dehydrogenase, and formate dehydrogenase. These enzymes were screened for thermal, pH, and product stability to choose optima for this system which were pH 7.4 and 35 °C. This system is designed to address well-known limitations of in vivo systems such as low product concentrations due to product feedback inhibition, instability of cells, and lack of economic product recovery., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Piscidins are histidine-enriched antimicrobial peptides that interact with lipid bilayers as amphipathic α-helices. Their activity at acidic and basic pH in vivo makes them promising templates for biomedical applications. This study focuses on p1 and p3, both 22-residue-long piscidins with 68% sequence identity. They share three histidines (H3, H4, and H11), but p1, which is significantly more permeabilizing, has a fourth histidine (H17). This study investigates how variations in amphipathic character associated with histidines affect the permeabilization properties of p1 and p3. First, we show that the permeabilization ability of p3, but not p1, is strongly inhibited at pH 6.0 when the conserved histidines are partially charged and H17 is predominantly neutral. Second, our neutron diffraction measurements performed at low water content and neutral pH indicate that the average conformation of p1 is highly tilted, with its C-terminus extending into the opposite leaflet. In contrast, p3 is surface bound with its N-terminal end tilted toward the bilayer interior. The deeper membrane insertion of p1 correlates with its behavior at full hydration: an enhanced ability to tilt, bury its histidines and C-terminus, induce membrane thinning and defects, and alter membrane conductance and viscoelastic properties. Furthermore, its pH-resiliency relates to the neutral state favored by H17. Overall, these results provide mechanistic insights into how differences in the histidine content and amphipathicity of peptides can elicit different directionality of membrane insertion and pH-dependent permeabilization. This work features complementary methods, including dye leakage assays, NMR-monitored titrations, X-ray and neutron diffraction, oriented CD, molecular dynamics, electrochemical impedance spectroscopy, surface plasmon resonance, and quartz crystal microbalance with dissipation.

Protein-protein interactions are central to biological processes. In vitro methods to examine protein-protein interactions are generally categorized into two classes: in-solution and surface-based methods. Here, using the multivalent interactions between nucleocytoplasmic transport factors and intrinsically disordered FG repeat containing nuclear pore complex proteins as a model system, we examined the utility of three surface-based methods: atomic force microscopy, quartz crystal microbalance with dissipation, and surface plasmon resonance. Although results were comparable to those of previous reports, the apparent effect of mass transport limitations was demonstrated. Additional experiments with a loss-of-interaction FG repeat mutant variant demonstrated that the binding events that take place on surfaces can be unexpectedly complex, suggesting particular care must be exercised in interpretation of such data., Competing Interests: The authors have declared that no competing interests exist.

We have engineered an intein which spontaneously and reversibly forms a thiazoline ring at the native N-terminal Lys-Cys splice junction. We identified conditions to stablize the thiazoline ring and provided the first crystallographic evidence, at 1.54 Å resolution, for its existence at an intein active site. The finding bolsters evidence for a tetrahedral oxythiazolidine splicing intermediate. In addition, the pivotal mutation maps to a highly conserved B-block threonine, which is now seen to play a causative role not only in ground-state destabilization of the scissile N-terminal peptide bond, but also in steering the tetrahedral intermediate toward thioester formation, giving new insight into the splicing mechanism. We demonstrated the stability of the thiazoline ring at neutral pH as well as sensitivity to hydrolytic ring opening under acidic conditions. A pH cycling strategy to control N-terminal cleavage is proposed, which may be of interest for biotechnological applications requiring a splicing activity switch, such as for protein recovery in bioprocessing., (© 2018 Wiley Periodicals, Inc.)

After 70 years, modern pressure-driven polymer membrane processes with liquids are mature and accepted in many industries due to their good performance, ease of scale-up, low energy consumption, modular compact construction, and low operating costs compared with thermal systems. Successful isothermal operation of synthetic membranes with liquids requires consideration of three critical aspects or "legs" in order of relevance: selectivity, capacity (i.e. permeation flow rate per unit area) and transport of mass and momentum comprising concentration polarization (CP) and fouling (F). Major challenges remain with respect to increasing selectivity and controlling mass transport in, to and away from membranes. Thus, prediction and control of membrane morphology and a deep understanding of the mechanism of dissolved and suspended solute transport near and in the membrane (i.e. diffusional and convective mass transport) is essential. Here, we focus on materials development to address the relatively poor selectivity of liquid membrane filtration with polymers and discuss the critical aspects of transport limitations. Machine learning could help optimize membrane structure design and transport conditions for improved membrane filtration performance., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Amyloid β (Aβ) peptide accumulation in the brain is considered to be one of the hallmarks of Alzheimer's disease. Here, we compare two analytical techniques for detecting neurotoxic Aβ 1-42 oligomers - Quartz Crystal Microbalance with Dissipation (QCM-D) and Single Molecule Array (Simoa). Both detection methods exploit a feature of the monoclonal antibody bapineuzumab, which targets N-terminal residues 1-5 of Aβ with high affinity and use it as both a capture and detection reagent. Assays developed with the two methods allow us to specifically recognize neurotoxic Aβ 1-42 oligomers and higher aggregates such as fibrils but discriminate against Aβ 1-42 monomer species. We find that for detection of Aβ 1-42 oligomers, Simoa was roughly 500 times more sensitive than the QCM-D technique with limits of detection of 0.22 nM and 125 nM, respectively., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Knowing that abeta amyloid peptide (Aβ 42 ) dimers are the smallest and most abundant neurotoxic oligomers for Alzheimer's disease (AD), we used molecular simulations with advanced sampling methods (replica-exchange) to characterize and compare interactions between the N-termini (residues 1-16) of wild type (WT-WT) and five mutant dimers under constrained and unconstrained conditions. The number of contacts and distances between the N-termini, and contact maps of their conformational landscape illustrate substantial differences for a single residue change. The N-terminal contacts are significantly diminished for the dimers containing the monomers that protect against (WT-A2T) as compared with those that predispose toward (A2V-A2V) AD and for the control WT-WT dimers. The reduced number of N-terminal contacts not only occurs at or near the second residue mutations but also is distributed through to the 10th residue. These findings provide added support to the accumulating evidence for the "N-terminal hypothesis of AD" and offer an alternate mechanism for the cause of protection from the A2T mutant.

Aquaporins (AQPs) feature highly selective water transport through cell membranes, where the dipolar orientation of structured water wires spanning the AQP pore is of considerable importance for the selective translocation of water over ions. We recently discovered that water permeability through artificial water channels formed by stacked imidazole I-quartet superstructures increases when the channel water molecules are highly organized. Correlating water structure with molecular transport is essential for understanding the underlying mechanisms of (fast) water translocation and channel selectivity. Chirality adds another factor enabling unique dipolar oriented water structures. We show that water molecules exhibit a dipolar oriented wire structure within chiral I-quartet water channels both in the solid state and embedded in supported lipid bilayer membranes (SLBs). X-ray single-crystal structures show that crystallographic water wires exhibit dipolar orientation, which is unique for chiral I-quartets. The integration of I-quartets into SLBs was monitored with a quartz crystal microbalance with dissipation, quantizing the amount of channel water molecules. Nonlinear sum-frequency generation vibrational spectroscopy demonstrates the first experimental observation of dipolar oriented water structures within artificial water channels inserted in bilayer membranes. Confirmation of the ordered confined water is obtained via molecular simulations, which provide quantitative measures of hydrogen bond strength, connectivity, and the stability of their dipolar alignment in a membrane environment. Together, uncovering the interplay between the dipolar aligned water structure and water transport through the self-assembled I-quartets is critical to understanding the behavior of natural membrane channels and will accelerate the systematic discovery for developing artificial water channels for water desalting.

In this work, quartz crystal microbalance with dissipation (QCM-D) was employed to study the kinetic processes involved in the interaction of proteins with self-assembled monolayers (SAMs) of multimodal (MM) ligands. SAMs were fabricated to mimic two chromatographic multimodal resins with varying accessibility of the aromatic moiety to provide a well-defined model system. Kinetic parameters were determined for two different proteins in the presence of the arginine and guanidine and a comparison was made with chromatographic retention data. The results indicated that the accessibility of the ligand's aromatic moiety can have an important impact on the kinetics and chromatographic retention behavior. Interestingly, arginine and guanidine had very different effects on the protein adsorption and desorption kinetics in these MM systems. For cytochrome C, arginine resulted in a significant decrease and increase in the adsorption and desorption rates, respectively, while guanidine produced a dramatic increase in the desorption rate, with minimal effect on the adsorption rate. In addition, at different concentrations of arginine, two distinct kinetic scenarios were observed. For α-chymotrypsin, the presence of 0.1 M guanidine in the aromatic exposed ligand system produced an increase in the adsorption rate and only a moderate increase in the desorption rate, which helped to explain the surprising increase in the chromatographic salt elution concentration. These results demonstrate that protein adsorption kinetics in the presence of different mobile phase modifiers and MM ligand chemistries can play an important role in contributing to selectivity in MM chromatography.

Municipal wastewater is an attractive alternative to freshwater sources to meet the cooling water needs of thermal power plants. Here we offer an energy-efficient integrated microbial fuel cell (MFC)/ultrafiltration (UF) process to purify primary clarifier effluent from a municipal wastewater treatment plant for use as cooling water. The microbial fuel cell was shown to significantly reduce chemical oxygen demand (COD) in the primary settled wastewater effluent upstream of the UF module, while eliminating the energy demand required to deliver dissolved oxygen in conventional aerobic treatment. We investigated surface modification of the UF membranes to control fouling. Two promising hydrophilic monomers were identified in a high-throughput search: zwitterion (2-(Methacryloyloxy)-ethyl-dimethyl-(3-sulfopropyl ammoniumhydroxide, abbreviated BET SO 3 - ), and amine (2-(Methacryloyloxy) ethyl trimethylammonium chloride, abbreviated N(CH 3 ) 3 + ). Monomers were grafted using UV-induced polymerization on commercial poly (ether sulfone) membranes. Filtration of MFC effluent by membranes modified with BET SO 3 - and N(CH 3 ) 3 + exhibited a lower rate of resistance increase and lower energy consumption than the commercially available membrane. The MFC/UF process produced high quality cooling water that meets the Electrical Power Research Institute (EPRI) recommendations for COD, a suite of metals (Fe, Al, Cu, Zn, Si, Mn, S, Ca and Mg), and offered extremely low corrosion rates (<0.05 mm/yr). A series of AC and DC diagnostic tests were used to evaluate the MFC performance., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Cell attachment is essential for the growth and polarization of retinal pigment epithelial (RPE) cells. Currently, surface coatings derived from biological proteins are used as the gold standard for cell culture. However, downstream processing and purification of these biological products can be cumbersome and expensive. In this study, we constructed a library of chemically modified nanofibers to mimic the Bruch's membrane of the retinal pigment epithelium. Using atmospheric-pressure plasma-induced graft polymerization with a high-throughput screening platform to modify the nanofibers, we identified three polyethylene glycol (PEG)-grafted nanofiber surfaces (PEG methyl ether methacrylate, n = 4, 8, and 45) from a library of 62 different surfaces as favorable for RPE cell attachment, proliferation, and maturation in vitro with cobblestone morphology. Compared with the biologically derived culture matrices such as vitronectin-based peptide Synthemax, our newly discovered synthetic PEG surfaces exhibit similar growth and polarization of retinal pigment epithelial (RPE) cells. However, they are chemically defined, are easy to synthesize on a large scale, are cost-effective, are stable with long-term storage capability, and provide a more physiologically accurate environment for RPE cell culture. To our knowledge, no one has reported that PEG derivatives directly support attachment and growth of RPE cells with cobblestone morphology. This study offers a unique PEG-modified 3D cell culture system that supports RPE proliferation, differentiation, and maturation with cobblestone morphology, providing a new avenue for RPE cell culture, disease modeling, and cell replacement therapy.

Although the amyloid (abeta peptide, Aβ) hypothesis is 25 years old, is the dominant model of Alzheimer's disease (AD) pathogenesis, and guides the development of potential treatments, it is still controversial. One possible reason is a lack of a mechanistic path from the cleavage products of the amyloid precursor protein (APP) such as soluble Aβ monomer and soluble molecular fragments to the deleterious effects on synaptic form and function. From a review of the recent literature and our own published work including aggregation kinetics and structural morphology, Aβ clearance, molecular simulations, long-term potentiation measurements with inhibition binding, and the binding of a commercial monoclonal antibody, aducanumab, we hypothesize that the N-terminal domains of neurotoxic Aβ oligomers are implicated in causing the disease.

Whole genome sequencing has recently revealed the protective effect of a single A2T mutation in heterozygous carriers against Alzheimer's disease (AD) and age-related cognitive decline. The impact of the protective cross-interaction between the wild-type (WT) and A2T variants on the dimer structure is therefore of high interest, as the Aβ dimers are the smallest known neurotoxic species. Toward this goal, extensive atomistic replica exchange molecular dynamics simulations of the solvated WT homo- and A2T hetero- Aβ 1-42 dimers have been performed, resulting into a total of 51 μs of sampling for each system. Weakening of a set of transient, intrachain contacts formed between the central and C-terminal hydrophobic residues is observed in the heterodimeric system. The majority of the heterodimers with reduced interaction between central and C-terminal regions lack any significant secondary structure and display a weak interchain interface. Interestingly, the A2T N-terminus, particularly residue F4, is frequently engaged in tertiary and quaternary interactions with central and C-terminal hydrophobic residues in those distinct structures, leading to hydrophobic burial. This atypical involvement of the N-terminus within A2T heterodimer revealed in our simulations implies possible interference on Aβ 42 aggregation and toxic oligomer formation, which is consistent with experiments. In conclusion, the present study provides detailed structural insights onto A2T Aβ 42 heterodimer, which might provide molecular insights onto the AD protective effect of the A2T mutation in the heterozygous state.

As pathogenic bacteria become resistant to traditional antibiotics, alternate approaches such as designing and testing new potent selective antimicrobial peptides (AMP) are increasingly attractive. However, whereas much is known regarding the relationship between the AMP sequence and potency, less research has focused on developing links between AMP properties, such as design and structure, with mechanisms. Here we use four natural AMPs of varying known secondary structures and mechanisms of lipid bilayer disruption as controls to determine the mechanisms of four rationally designed AMPs with similar secondary structures and rearranged amino acid sequences. Using a Quartz Crystal Microbalance with Dissipation, we were able to differentiate between molecular models of AMP actions such as barrel-stave pore formation, toroidal pore formation, and peptide insertion mechanisms by quantifying differential frequencies throughout an oscillating supported lipid bilayer. Barrel-stave pores were identified by uniform frequency modulation, whereas toroidal pores possessed characteristic changes in oscillation frequency throughout the bilayer. The resulting modes of action demonstrate that rearrangement of an amino acid sequence of the AMP resulted in identical overall mechanisms, and that a given secondary structure did not necessarily predict mechanism. Also, increased mass addition to Gram-positive mimetic membranes from AMP disruption corresponded with lower minimum inhibitory concentrations against the Gram-positive Staphylococcus aureus., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The fundamentals and applications of polymer brush-modified membranes are reviewed. This new class of synthetic membranes is explored with an emphasis on tuning the membrane performance through polymer brush grafting. This work highlights the intriguing performance characteristics of polymer brush-modified membranes in a variety of separations. Polymer brushes are a versatile and effective means in designing membranes for applications in protein adsorption and purification, colloid stabilization, sensors, water purification, pervaporation of organic compounds, gas separations, and as stimuli responsive materials.

Inteins, self-splicing protein elements, interrupt genes and proteins in many microbes, including the human pathogen Mycobacterium tuberculosis Using conserved catalytic nucleophiles at their N- and C-terminal splice junctions, inteins are able to excise out of precursor polypeptides. The splicing of the intein in the mycobacterial recombinase RecA is specifically inhibited by the widely used cancer therapeutic cisplatin, cis-[Pt(NH 3 ) 2 Cl 2 ], and this compound inhibits mycobacterial growth. Mass spectrometric and crystallographic studies of Pt(II) binding to the RecA intein revealed a complex in which two platinum atoms bind at N- and C-terminal catalytic cysteine residues. Kinetic analyses of NMR spectroscopic data support a two-step binding mechanism in which a Pt(II) first rapidly interacts reversibly at the N terminus followed by a slower, first order irreversible binding event involving both the N and C termini. Notably, the ligands of Pt(II) compounds that are required for chemotherapeutic efficacy and toxicity are no longer bound to the metal atom in the intein adduct. The lack of ammine ligands and need for phosphine represent a springboard for future design of platinum-based compounds targeting inteins. Because the intein splicing mechanism is conserved across a range of pathogenic microbes, developing these drugs could lead to novel, broad range antimicrobial agents., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Antifouling surfaces improve function, efficiency, and safety in products such as water filtration membranes, marine vehicle coatings, and medical implants by resisting protein and biofilm adhesion. Understanding the role of water structure at these materials in preventing protein adhesion and biofilm formation is critical to designing more effective coatings. Such fouling experiments are typically performed under biological conditions using isotonic aqueous buffers. Previous studies have explored the structure of pure water at a few different antifouling surfaces, but the effect of electrolytes and ionic strength (I) on the water structure at antifouling surfaces is not well studied. Here sum frequency generation (SFG) spectroscopy is used to characterize the interfacial water structure at poly(ether sulfone) (PES) and two surface-modified PES films in contact with 0.01 M phosphate buffer with high and low salt (Ionic strength, I= 0.166 and 0.025 M, respectively). Unmodified PES, commonly used as a filtration membrane, and modified PES with a hydrophobic alkane (C18) and with a poly(ethylene glycol) (PEG) were used. In the low ionic strength phosphate buffer, water was strongly ordered near the surface of the PEG-modified PES film due to exclusion of phosphate ions and the creation of a surface potential resulting from charge separation between phosphate anions and sodium cations. However, in the high ionic strength phosphate buffer, the sodium and potassium chloride (138 and 3 mM, respectively) in the phosphate buffered saline screened this charge and substantially reduced water ordering. A much smaller water ordering and subsequent reduction upon salt addition was observed for the C18-modified PES, and little water structure change was seen for the unmodified PES. The large difference in water structuring with increasing ionic strength between widely used phosphate buffer and phosphate buffered saline at the PEG interface demonstrates the importance of studying antifouling coatings in the same aqueous environment for which they are designed. These results further suggest that strong long-range water structuring is limited in high ionic strength environments, such as within cells, facilitating chemical and biological reactions and processes.

Although thermal desalination technology provides potable water in arid regions (e.g., Israel and the Gulf), its relatively high cost has limited application to less arid regions with large populations (e.g., California). Energy-intensive distillation is currently being replaced with less costly pressure- and electrically driven membrane-based processes. Reverse osmosis (RO) is a preferred membrane technology owing to process and pre- and posttreatment improvements that have significantly reduced energy requirements and cost. Further technical advances will require a deeper understanding of the fundamental science underlying RO. This includes determining the mechanism for water selectivity; elucidating the behavior of molecular water near polar and apolar surfaces, as well as the advantages and limitations of hydrophobic versus hydrophilic pores; learning the rules of selective water transport from nature; and designing synthetic analogs for selective water transport. Molecular dynamics simulations supporting experiments will play an important role in advancing these efforts. Finally, future improvements in RO are limited by inherent technical mass transfer limitations.

Drug-resistant pathogens are a growing problem, and novel strategies are needed to combat this threat. Among the most significant of these resistant pathogens is Mycobacterium tuberculosis, which is an unusually difficult microbial target due to its complex membrane. Here, we design peptides for specific activity against M. tuberculosis using a combination of "database filtering" bioinformatics, protein engineering, and de novo design. Several variants of these peptides are structurally characterized to validate the design process. The designed peptides exhibit potent activity (MIC values as low as 4 μM) against M. tuberculosis and also exhibit broad activity against a host of other clinically relevant pathogenic bacteria such as Gram-positive bacteria (Streptococcus) and Gram-negative bacteria (Escherichia coli). They also display excellent selectivity, with low cytotoxicity against cultured macrophages and lung epithelial cells. These first-generation antimicrobial peptides serve as a platform for the design of antibiotics and for investigating structure-activity relationships in the context of the M. tuberculosis membrane. The antimicrobial peptide design strategy is expected to be generalizable for any pathogen for which an activity database can be created., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Amyloid-beta peptides have long been implicated in the pathology of Alzheimer's disease. Bexarotene, a drug approved by the U.S. Food and Drug Administration for treating a class of non-Hodgkin's lymphoma, has been reported to facilitate the removal of amyloid-beta. We have developed a mathematical model to explore the efficacy of bexarotene treatment in reducing amyloid-beta load, and simulate amyloid-beta production throughout the lifespan of diseased mice. Both aspects of the model are based on and consistent with previous experimental results. Beyond what is known empirically, our model shows that low dosages of bexarotene are unable to reverse symptoms in diseased mice, but dosages at and above an age-dependent critical concentration can recover healthy brain cells. Further, early treatment was shown to have significantly improved efficacy versus treatment in older mice. Relevance with respect to bexarotene-based amyloid-beta-clearance mechanism and direct treatment for Alzheimer's disease is emphasized.

The histopathological hallmark of Alzheimer's disease (AD) is the aggregation and accumulation of the amyloid beta peptide (Aβ) into misfolded oligomers and fibrils. Here we examine the biophysical properties of a protective Aβ variant against AD, A2T, and a causative mutation, A2T, along with the wild type (WT) peptide. The main finding here is that the A2V native monomer is more stable than both A2T and WT, and this manifests itself in different biophysical behaviors: the kinetics of aggregation, the initial monomer conversion to an aggregation prone state (primary nucleation), the abundances of oligomers, and extended conformations. Aggregation reaction modeling of the conversion kinetics from native monomers to fibrils predicts the enhanced stability of the A2V monomer, while ion mobility spectrometry-mass spectrometry measures this directly confirming earlier predictions. Additionally, unique morphologies of the A2T aggregates are observed using atomic force microscopy, providing a basis for the reduction in long term potentiation inhibition of hippocampal cells for A2T compared with A2V and the wild type (WT) peptide. The stability difference of the A2V monomer and the difference in aggregate morphology for A2T (both compared with WT) are offered as alternate explanations for their pathological effects., (© 2016 Wiley Periodicals, Inc.)

Background and Purpose: Select neuroactive steroids tune neural activity by modulating excitatory and inhibitory neurotransmission, including the endogenous cholesterol metabolite 24(S)-hydroxycholesterol (24(S)-HC), which is an N-methyl-d-aspartate (NMDA) receptor positive allosteric modulator (PAM). NMDA receptor PAMs are potentially an effective pharmacotherapeutic strategy to treat conditions associated with NMDA receptor hypofunction., Experimental Approach: Using in vitro and in vivo electrophysiological recording experiments and behavioural approaches, we evaluated the effect of SAGE-718, a novel neuroactive steroid NMDA receptor PAM currently in clinical development for the treatment of cognitive impairment, on NMDA receptor function and endpoints that are altered by NMDA receptor hypoactivity and assessed its safety profile., Key Results: SAGE-718 potentiated GluN1/GluN2A-D NMDA receptors with equipotency and increased NMDA receptor excitatory postsynaptic potential (EPSP) amplitude without affecting decay kinetics in striatal medium spiny neurons. SAGE-718 increased the rate of unblock of the NMDA receptor open channel blocker ketamine on GluN1/GluN2A in vitro and accelerated the rate of return on the ketamine-evoked increase in gamma frequency band power, as measured with electroencephalogram (EEG), suggesting that PAM activity is driven by increased channel open probability. SAGE-718 ameliorated deficits due to NMDA receptor hypofunction, including social deficits induced by subchronic administration of phencyclidine, and behavioural and electrophysiological deficits from cholesterol and 24(S)-HC depletion caused by 7-dehydrocholesterol reductase inhibition. Finally, SAGE-718 did not produce epileptiform activity in a seizure model or neurodegeneration following chronic dosing., Conclusions and Implications: These findings provide strong evidence that SAGE-718 is a neuroactive steroid NMDA receptor PAM with a mechanism that is well suited as a treatment for conditions associated with NMDA receptor hypofunction., (© 2023 Sage Therapeutics. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Producing fuels and chemical intermediates with cell cultures is severely limited by low product concentrations (≤0.2%(v/v)) due to feedback inhibition, cell instability, and lack of economical product recovery processes. We have developed an alternate simplified production scheme based on a cell-free immobilized enzyme system. Two immobilized enzymes (keto-acid decarboxylase (KdcA) and alcohol dehydrogenase (ADH)) and one enzyme in solution (formate dehydrogenase (FDH) for NADH recycle) produced isobutanol titers 8 to 20 times higher than the highest reported titers with S. cerevisiae on a mol/mol basis. These high conversion rates and low protein leaching were achieved by covalent immobilization of enzymes (ADH) and enzyme fusions (fKdcA) on methacrylate resin. The new enzyme system without in situ removal of isobutanol achieved a 55% conversion of ketoisovaleric acid to isobutanol at a concentration of 0.135 (mole isobutanol produced for each mole ketoisovaleric acid consumed). Further increasing titer will require continuous removal of the isobutanol using an in situ recovery system., (© 2015 American Institute of Chemical Engineers.)

The backbone resonance assignments of an engineered splicing-inactive mini-RecA intein based on triple resonance experiments with [(13)C,(15)N]-labeled protein are reported. The construct contains inactivating mutations specifically designed to retain most catalytic residues, especially those that are potentially metal-coordinating. The assignments are essential for protein structure determination of a precursor with an active N-terminal catalytic cysteine and for investigation of the atomic details of splicing.