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Two controversial tenets of metapopulation biology are whether patch quality and the surrounding matrix are more important to turnover (colonisation and extinction) than biogeography (patch area and isolation) and whether factors governing turnover during equilibrium also dominate nonequilibrium dynamics. We tested both tenets using 18 years of surveys for two secretive wetland birds, black and Virginia rails, during (1) a period of equilibrium with stable occupancy and (2) after drought and arrival of West Nile Virus (WNV), which resulted in WNV infections in rails, increased extinction and decreased colonisation probabilities modified by WNV, nonequilibrium dynamics for both species and occupancy decline for black rails. Area (primarily) and isolation (secondarily) drove turnover during both stable and unstable metapopulation dynamics, greatly exceeding the effects of patch quality and matrix conditions. Moreover, slopes between turnover and patch characteristics changed little between equilibrium and nonequilibrium, confirming the overriding influences of biogeographic factors on turnover.

The randomization and screening of combinatorial DNA libraries is a powerful technique for understanding sequence-function relationships and optimizing biosynthetic pathways. Although it can be difficult to predict a priori which sequence combinations encode functional units, it is often possible to omit undesired combinations that inflate library size and screening effort. However, defined library generation is difficult when a complex scan through sequence space is needed. To overcome this challenge, we designed a hybrid valve- and droplet-based microfluidic system that deterministically assembles DNA parts in picoliter droplets, reducing reagent consumption and bias. Using this system, we built a combinatorial library encoding an engineered histidine kinase (HK) based on bacterial CpxA. Our library encodes designed transmembrane (TM) domains that modulate the activity of the cytoplasmic domain of CpxA and variants of the structurally distant "S helix" located near the catalytic domain. We find that the S helix sets a basal activity further modulated by the TM domain. Surprisingly, we also find that a given TM motif can elicit opposing effects on the catalytic activity of different S-helix variants. We conclude that the intervening HAMP domain passively transmits signals and shapes the signaling response depending on subtle changes in neighboring domains. This flexibility engenders a richness in functional outputs as HKs vary in response to changing evolutionary pressures.

We describe the de novo design of an allosterically regulated protein, which comprises two tightly coupled domains. One domain is based on the DF (Due Ferri in Italian or two-iron in English) family of de novo proteins, which have a diiron cofactor that catalyzes a phenol oxidase reaction, while the second domain is based on PS1 (Porphyrin-binding Sequence), which binds a synthetic Zn-porphyrin (ZnP). The binding of ZnP to the original PS1 protein induces changes in structure and dynamics, which we expected to influence the catalytic rate of a fused DF domain when appropriately coupled. Both DF and PS1 are four-helix bundles, but they have distinct bundle architectures. To achieve tight coupling between the domains, they were connected by four helical linkers using a computational method to discover the most designable connections capable of spanning the two architectures. The resulting protein, DFP1 (Due Ferri Porphyrin), bound the two cofactors in the expected manner. The crystal structure of fully reconstituted DFP1 was also in excellent agreement with the design, and it showed the ZnP cofactor bound over 12 Å from the dimetal center. Next, a substrate-binding cleft leading to the diiron center was introduced into DFP1. The resulting protein acts as an allosterically modulated phenol oxidase. Its Michaelis-Menten parameters were strongly affected by the binding of ZnP, resulting in a fourfold tighter K m and a 7-fold decrease in k cat These studies establish the feasibility of designing allosterically regulated catalytic proteins, entirely from scratch.

Site-specific protein cleavage is essential for many protein-production protocols and typically requires proteases. We report the development of a chemical protein-cleavage method that is achieved through the use of a sequence-specific nickel-assisted cleavage (SNAC)-tag. We demonstrate that the SNAC-tag can be inserted before both water-soluble and membrane proteins to achieve fusion protein cleavage under biocompatible conditions with efficiency comparable to that of enzymes, and that the method works even when enzymatic cleavages fail.

Terminal lakes are declining globally because of human water demands, drought, and climate change. Through literature synthesis and feedback from the resource and conservation community, we review the state of research for terminal lakes in the Great Basin of the United States, which support millions of waterbirds annually, to prioritize ecological and hydrologic information needs. From an ecological perspective, research priorities include measuring the underlying differences in waterbird resource selection and distribution, migratory connectivity, abiotic factors that interact with prey densities to affect prey availability, and waterbird fitness or demography. Integrated links between water availability, water quality, and food webs are lacking in the literature. Scarce water availability data hinder the current knowledge of water extraction and evapotranspiration rates. Research that can address these priorities would help advance our understanding of how the Great Basin terminal lakes function as an interrelated system and support conservation efforts to reverse the decline of these critical lakes. [ABSTRACT FROM AUTHOR]

Context Obesity is prevalent in type 1 diabetes (T1D) and is problematic with higher risk for diabetes complications. It is unknown to what extent gut microbiome changes are associated with obesity and T1D. Objective This work aimed to describe the gut microbiome and microbial metabolite changes associated with obesity in T1D. We hypothesized statistically significant gut microbial and metabolite differences in lean T1D youth (body mass index [BMI]: 5%-<85%) vs those with obesity (BMI: ≥95%). Methods We analyzed stool samples for gut microbial (using metagenomic shotgun sequencing) and short-chain fatty acid (SCFA) differences in lean (n = 27) and obese (n = 21) T1D youth in a pilot study. The mean ± SD age was 15.3 ± 2.2 years, glycated hemoglobin A1c 7.8 ± 1.3%, diabetes duration 5.1 ± 4.4 years, 42.0% female, and 94.0% were White. Results Bacterial community composition showed between sample diversity differences (β-diversity) by BMI group (P =.013). There was a higher ratio of Prevotella to Bacteroides in the obese group (P =.0058). There was a differential distribution of significantly abundant taxa in either the lean or obese groups, including increased relative abundance of Prevotella copri , among other taxa in the obese group. Functional profiling showed an upregulation of branched-chain amino acid (BCAA) biosynthesis in the obese group and upregulation of BCAA degradation, tyrosine metabolism, and secondary bile acid biosynthesis in the lean group. Stool SCFAs were higher in the obese vs the lean group (P <.05 for all). Conclusion Our findings identify a gut microbiome and microbial metabolite signature associated with obesity in T1D. These findings could help identify gut microbiome–targeted therapies to manage obesity in T1D. [ABSTRACT FROM AUTHOR]

Background:The microbiome influences malaria parasite fitness and transmission efficiency in mosquitoes and appears to affect malaria dynamics in mammalian hosts as well. Nascent research examining the interrelationship of malaria and the mammalian microbiome has yielded interesting insights inviting further study. Methods:We conducted a systematic review of the literature examining associations between the microbiome and malaria in mammalian hosts. An electronic search algorithm was adapted to PubMed, MEDLINE, Scopus, Embase, and Web of Science, and reference lists of relevant sources were manually searched. Identified studies were screened and assessed independently by 2 authors, and results were compiled in a qualitative synthesis of the evidence. Results:Ten relevant studies were identified. They demonstrate associations between certain intestinal communities and protection against Plasmodium infection and modulation of disease severity. Plasmodium infection acutely and reversibly reshapes gut microbial composition in mice. The makeup of human skin microbial communities may influence mosquito attraction and thus disease transmission. Conclusions:Early research supports a relationship between malaria and the microbiome. The evidence is incomplete, but the observed associations are evocative and signal a promising avenue of inquiry. Microbiome-based studies of malaria can be readily integrated into field-based research.

The hydrophobic Aβ peptide is highly aggregation prone; it first forms soluble oligomers, which then convert into the amyloid fibrils found in the cerebral plaques of Alzheimer's disease. It is generally understood that as the peptide concentration of Aβ increases, the fibrillization process is accelerated, but we examine the limits on this phenomenon. We found that once a threshold concentration of Aβ is exceeded, a stable oligomer is formed at the expense of fibril formation. The suppression of fibril formation was observed by amyloid-binding dye Thioflavin T and solution nuclear magnetic resonance (NMR). Small-angle X-ray scattering, size exclusion chromatography, and analytical ultracentrifugation demonstrated that Aβ peptides form a range of compact species, with a dimer being an early highly populated oligomer. Solution NMR allowed us to define the secondary structure of this Aβ dimer, which shows interlocking contacts between C-terminal peptide strands. Thus, we present a novel Aβ oligomer that resists conversion to fibrils and remains stable for more than one year.

NsaS is one of four intramembrane histidine kinases (HKs) in Staphylococcus aureus that mediate the pathogen's response to membrane active antimicrobials and human innate immunity. We describe the first integrative structural study of NsaS using a combination of solution state NMR spectroscopy, chemical-cross-linking, molecular modeling and dynamics. Three key structural features emerge: First, NsaS has a short N-terminal amphiphilic helix that anchors its transmembrane (TM) bundle into the inner leaflet of the membrane such that it might sense neighboring proteins or membrane deformations. Second, the transmembrane domain of NsaS is a 4-helix bundle with significant dynamics and structural deformations at the membrane interface. Third, the intracellular linker connecting the TM domain to the cytoplasmic catalytic domains of NsaS is a marginally stable helical dimer, with one state likely to be a coiled-coil. Data from chemical shifts, heteronuclear NOE, H/D exchange measurements and molecular modeling suggest that this linker might adopt different conformations during antibiotic induced signaling.

Peptide-based supramolecular assemblies are a promising class of nanomaterials with important biomedical applications, specifically in drug delivery and tissue regeneration. However, the intrinsic antibacterial capabilities of these assemblies have been largely overlooked. The recent identification of common characteristics shared by antibacterial and self-assembling peptides provides a paradigm shift towards development of antibacterial agents. Here we present the antibacterial activity of self-assembled diphenylalanine, which emerges as the minimal model for antibacterial supramolecular polymers. The diphenylalanine nano-assemblies completely inhibit bacterial growth, trigger upregulation of stress-response regulons, induce substantial disruption to bacterial morphology, and cause membrane permeation and depolarization. We demonstrate the specificity of these membrane interactions and the development of antibacterial materials by integration of the peptide assemblies into tissue scaffolds. This study provides important insights into the significance of the interplay between self-assembly and antimicrobial activity and establishes innovative design principles toward the development of antimicrobial agents and materials.

Coiled-coils are essential components of many protein complexes. First discovered in structural proteins such as keratins, they have since been found to figure largely in the assembly and dynamics required for diverse functions, including membrane fusion, signal transduction and motors. Coiled-coils have a characteristic repeating seven-residue geometric and sequence motif, which is sometimes interrupted by the insertion of one or more residues. Such insertions are often highly conserved and critical to interdomain communication in signaling proteins such as bacterial histidine kinases. Here we develop the "accommodation index" as a parameter that allows automatic detection and classification of insertions based on the three dimensional structure of a protein. This method allows precise identification of the type of insertion and the "accommodation length" over which the insertion is structurally accommodated. A simple theory is presented that predicts the structural perturbations of 1, 3, 4 residue insertions as a function of the length over which the insertion is accommodated. Analysis of experimental structures is in good agreement with theory, and shows that short accommodation lengths give rise to greater perturbation of helix packing angles, changes in local helical phase, and increased structural asymmetry relative to long accommodation lengths. Cytoplasmic domains of histidine kinases in different signaling states display large changes in their accommodation lengths, which can now be seen to underlie diverse structural transitions including symmetry/asymmetry and local variations in helical phase that accompany signal transduction.

Triggering antimicrobial mechanisms in macrophages infected with intracellular pathogens, such as mycobacteria, is critical to host defense against the infection. To uncover the unique and shared antimicrobial networks induced by the innate and adaptive immune systems, gene expression profiles generated by RNA sequencing (RNAseq) from human monocyte-derived macrophages (MDMs) activated with TLR2/1 ligand (TLR2/1L) or IFN-γ were analyzed. Weighed gene correlation network analysis identified modules of genes strongly correlated with TLR2/1L or IFN-γ that were linked by the "defense response" gene ontology term. The common TLR2/1L and IFN-γ inducible human macrophage host defense network contained 16 antimicrobial response genes, including S100A12, which was one of the most highly induced genes by TLR2/1L. There is limited information on the role of S100A12 in infectious disease, leading us to test the hypothesis that S100A12 contributes to host defense against mycobacterial infection in humans. We show that S100A12 is sufficient to directly kill Mycobacterium tuberculosis and Mycobacterium leprae. We also demonstrate that S100A12 is required for TLR2/1L and IFN-γ induced antimicrobial activity against M. leprae in infected macrophages. At the site of disease in leprosy, we found that S100A12 was more strongly expressed in skin lesions from tuberculoid leprosy (T-lep), the self-limiting form of the disease, compared to lepromatous leprosy (L-lep), the progressive form of the disease. These data suggest that S100A12 is part of an innate and adaptive inducible antimicrobial network that contributes to host defense against mycobacteria in infected macrophages.

Although antibiotics are a common treatment for acne, the difficulties inherent to effective antimicrobial penetration in sebum and selective antimicrobial action in the skin are compounded by increasing resistance of Propionibacterium acnes clinical isolates. To address these problems, we engineered Pentobra, a peptide-aminoglycoside molecule that has multiple mechanisms of antibacterial action and investigated whether it can be a potential candidate for the treatment of acne. Pentobra combines the potent ribosomal activity of aminoglycosides with the bacteria-selective membrane-permeabilizing abilities of antimicrobial peptides. Pentobra demonstrated potent and selective killing of P. acnes but not against human skin cells in vitro. In direct comparison, Pentobra demonstrated bactericidal activity and drastically outperformed free tobramycin (by 5-7 logs) against multiple P. acnes clinical strains. Moreover, electron microscopic studies showed that Pentobra had robust membrane activity, as treatment with Pentobra killed P. acnes cells and caused leakage of intracellular contents. Pentobra may also have potential anti-inflammatory effects as demonstrated by suppression of some P. acnes-induced chemokines. Importantly, the killing activity was maintained in sebaceous environments as Pentobra was bactericidal against clinical isolates in comedones extracts isolated from human donors. Our work demonstrates that equipping aminoglycosides with selective membrane activity is a viable approach for developing antibiotics against P. acnes that are effective in cutaneous environments.

Cell penetrating peptides (CPPs) are intriguing molecules that have received much attention, both in terms of mechanistic analysis and as transporters for intracellular therapeutic delivery. Most CPPs contain an abundance of cationic charged residues, typically arginine, where the amino acid compositions, rather than specific sequences, tend to determine their ability to enter cells. Hydrophobic residues are often added to cationic sequences to create efficient CPPs, but typically at the penalty of increased cytotoxicity. Here, we examined polypeptides containing glycosylated, cationic derivatives of methionine, where we found these hydrophilic polypeptides to be surprisingly effective as CPPs and to also possess low cytotoxicity. X-ray analysis of how these new polypeptides interact with lipid membranes revealed that the incorporation of sterically demanding hydrophilic cationic groups in polypeptides is an unprecedented new concept for design of potent CPPs.

Novel synthetic mimics of antimicrobial peptides have been developed to exhibit structural properties and antimicrobial activity similar to those of natural antimicrobial peptides (AMPs) of the innate immune system. These molecules have a number of potential advantages over conventional antibiotics, including reduced bacterial resistance, cost-effective preparation, and customizable designs. In this study, we investigate a family of nylon-3 polymer-based antimicrobials. By combining vesicle dye leakage, bacterial permeation, and bactericidal assays with small-angle X-ray scattering (SAXS), we find that these polymers are capable of two interdependent mechanisms of action: permeation of bacterial membranes and binding to intracellular targets such as DNA, with the latter necessarily dependent on the former. We systemically examine polymer-induced membrane deformation modes across a range of lipid compositions that mimic both bacteria and mammalian cell membranes. The results show that the polymers' ability to generate negative Gaussian curvature (NGC), a topological requirement for membrane permeation and cellular entry, in model Escherichia coli membranes correlates with their ability to permeate membranes without complete membrane disruption and kill E. coli cells. Our findings suggest that these polymers operate with a concentration-dependent mechanism of action: at low concentrations permeation and DNA binding occur without membrane disruption, while at high concentrations complete disruption of the membrane occurs. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

The M2 protein is a multifunctional protein, which plays several roles in the replication cycle of the influenza A virus. Here we focus on its ability to promote budding of the mature virus from the cell surface. Using high-resolution small-angle X-ray scattering we show that M2 can restructure lipid membranes into bicontinuous cubic phases which are rich in negative Gaussian curvature (NGC). The active generation of negative Gaussian membrane curvature by M2 is essential to influenza virus budding. M2 has been observed to colocalize with the region of high NGC at the neck of a bud. The structural requirements for scission are even more stringent than those for budding, as the neck must be considerably smaller than the virus during 'pinch off'. Consistent with this, the amount of NGC in the induced cubic phases suggests that M2 proteins can generate high curvatures comparable to those on a neck with size 10× smaller than a spherical influenza virus. Similar experiments on variant proteins containing different M2 domains show that the cytoplasmic amphipathic helix is necessary and sufficient for NGC generation. Mutations to the helix which reduce its amphiphilicity and are known to diminish budding attenuated NGC generation. An M2 construct comprising the membrane interactive domains, the transmembrane helix and the cytoplasmic helix, displayed enhanced ability to generate NGC, suggesting that other domains cooperatively promote membrane curvature. These studies establish the importance of M2-induced NGC during budding and suggest that antagonizing this curvature is a viable anti-influenza strategy.