Few existing protein-protein interface design methods allow for extensive backbone rearrangements during the design process. There is also a dichotomy between redesign methods, which take advantage ...of the native interface, and de novo methods, which produce novel binders.
Here, we propose a new method for designing novel protein reagents that combines advantages of redesign and de novo methods and allows for extensive backbone motion. This method requires a bound structure of a target and one of its natural binding partners. A key interaction in this interface, the anchor, is computationally grafted out of the partner and into a surface loop on the design scaffold. The design scaffold's surface is then redesigned with backbone flexibility to create a new binding partner for the target. Careful choice of a scaffold will bring experimentally desirable characteristics into the new complex. The use of an anchor both expedites the design process and ensures that binding proceeds against a known location on the target. The use of surface loops on the scaffold allows for flexible-backbone redesign to properly search conformational space.
This protocol was implemented within the Rosetta3 software suite. To demonstrate and evaluate this protocol, we have developed a benchmarking set of structures from the PDB with loop-mediated interfaces. This protocol can recover the correct loop-mediated interface in 15 out of 16 tested structures, using only a single residue as an anchor.
Pigment patterns are useful for elucidating fundamental mechanisms of pattern formation and how these mechanisms evolve. In zebrafish, several pigment cell classes interact to generate stripes, yet ...the developmental requirements and origins of these cells remain poorly understood. Using zebrafish and a related species, we identified roles for thyroid hormone (TH) in pigment cell development and patterning, and in postembryonic development more generally. We show that adult pigment cells arise from distinct lineages having distinct requirements for TH and that differential TH dependence can evolve within lineages. Our findings demonstrate critical functions for TH in determining pigment pattern phenotype and highlight the potential for evolutionary diversification at the intersection of developmental and endocrine mechanisms.
Objectives: The aim of this study was to evaluate the minimum inhibitory concentration (MIC) of their antibacterial properties against Gram-positive and Gram-negative bacteria.
Methods: The ...antimicrobial activity of ethanolic and aqueous extracts of 141 plant species was determined using a deep-well broth microdilution method on commercially available bacterial strains.
Results: The ethanolic extracts of 51 species inhibited
Escherichia coli, and 114 ethanolic extracts inhibited
Staphylococcus aureus. In contrast, only 30 aqueous extracts showed activity against
Escherichia coli and 38 extracts against
Staphylococcus aureus. The MIC concentrations ranged from 0.008 to 256
mg/ml.
Conclusions: The presence of antibacterial activity could be confirmed in medicinal plants used Peru for the treatment of bacterial infections. The MIC for most species employed showed a large range. Traditional knowledge provides leads to elucidate candidates for future antibiotic agent development.
Study area: Peruvian Departments of Amazonas, Piura, Lambayeque, La Libertad, Cajamarca, San Martin, and the Ecuadorian Province of Loja.
The plant species reported here are traditionally used in Northern Peru to treat bacterial infections, often addressed by the local healers as “inflammation”. The aim of this study was to evaluate the minimum inhibitory concentration (MIC) of their antibacterial properties against Gram-positive and Gram-negative bacteria.
The antimicrobial activity of ethanolic and water extracts of 141 plant species was determined using a deep-well broth microdilution method on commercially available bacterial strains.
The ethanolic extracts of 51 species inhibited
Escherichia coli, and 114 ethanolic extracts inhibited
Staphylococcus aureus. In contrast, only 30 aqueous extracts showed activity against
Escherichia coli and 38 extracts against
Staphylococcus aureus. The MIC concentrations were mostly very high and ranged from 0.008 to 256
mg/ml, with only 36 species showing inhibitory concentrations of <4
mg/ml. The ethanolic extracts exhibited stronger activity and a much broader spectrum of action than the aqueous extracts.
Hypericum laricifolium,
Hura crepitans,
Caesalpinia paipai,
Cassia fistula,
Hyptis sidifolia,
Salvia sp.,
Banisteriopsis caapi,
Miconia salicifolia and
Polygonum hydropiperoides showed the lowest MIC values and would be interesting candidates for future research.
The presence of antibacterial activity could be confirmed in most species used in traditional medicine in Peru which were assayed in this study. However, the MIC for the species employed showed a very large range, and were mostly very high. Nevertheless, traditional knowledge might provide some leads to elucidate potential candidates for future development of new antibiotic agents.
Viral fusion proteins undergo dramatic conformational transitions during membrane fusion. For viruses that enter through the endosome, these conformational rearrangements are typically pH sensitive. ...Here, we provide a comprehensive review of the molecular interactions that govern pH-dependent rearrangements and introduce a paradigm for electrostatic residue pairings that regulate progress through the viral fusion coordinate. Analysis of structural data demonstrates a significant role for side-chain protonation in triggering conformational change. To characterize this behavior, we identify two distinct residue pairings, which we define as Histidine-Cation (HisCat) and Anion-Anion (AniAni) interactions. These side-chain pairings destabilize a particular conformation via electrostatic repulsion through side-chain protonation. Furthermore, two energetic control mechanisms, thermodynamic and kinetic, regulate these structural transitions. This review expands on the current literature by identification of these residue clusters, discussion of data demonstrating their function, and speculation of how these residue pairings contribute to the energetic controls.
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•Viral fusion proteins undergo pH-dependent conformational changes•Electrostatic repulsion between side chains mediates structural transitions•Histidine-cation (HisCat) and anion-anion (AniAni) interactions are common•Ability of the virus to sense pH changes as it enters the cell plays an important role during the infection
Harrison et al. review this problem and point to specific side chain-side chain interactions that regulate envelope glycoprotein conformational changes and allow viral entry.
The marine polychaete annelid Platynereis dumerilii has recently emerged as a prominent organism for the study of development, evolution, stem cells, regeneration, marine ecology, chronobiology and ...neurobiology within metazoans. Its phylogenetic position within the spiralian/ lophotrochozoan clade, the comparatively high conservation of ancestral features in the Platynereis genome, and experimental access to any stage within its life cycle, make Platynereis an important model for elucidating the complex regulatory and functional molecular mechanisms governing early development, later organogenesis, and various features of its larval and adult life. High resolution RNA-seq gene expression data obtained from specific developmental stages can be used to dissect early developmental mechanisms. However, the potential for discovery of these mechanisms relies on tools to search, retrieve, and compare genome-wide information within Platynereis, and across other metazoan taxa.
To facilitate exploration and discovery by the broader scientific community, we have developed a web-based, searchable online research tool, PdumBase, featuring the first comprehensive transcriptome database for Platynereis dumerilii during early stages of development (2 h ~ 14 h). Our database also includes additional stages over the P. dumerilii life cycle and provides access to the expression data of 17,213 genes (31,806 transcripts) along with annotation information sourced from Swiss-Prot, Gene Ontology, KEGG pathways, Pfam domains, TmHMM, SingleP, and EggNOG orthology. Expression data for each gene includes the stage, the normalized FPKM, the raw read counts, and information that can be leveraged for statistical analyses of differential gene expression and the construction of genome-wide co-expression networks. In addition, PdumBase offers early stage transcriptome expression data from five further species as a valuable resource for investigators interested in comparing early development in different organisms. To understand conservation of Platynereis gene models and to validate gene annotation, most Platynereis gene models include a comprehensive phylogenetic analysis across 18 species representing diverse metazoan taxa.
PdumBase represents the first online resource for the early developmental transcriptome of Platynereis dumerilii. It serves as a research platform for discovery and exploration of gene expression during early stages, throughout the Platynereis life cycle, and enables comparison to other model organisms. PdumBase is freely available at http://pdumbase.gdcb.iastate.edu .
Motility and the coordination of moving food through the gastrointestinal tract rely on a complex network of neurons known as the enteric nervous system (ENS). Despite its critical function, many of ...the molecular mechanisms that direct the development of the ENS and the elaboration of neural network connections remain unknown. The goal of this study was to transcriptionally identify molecular pathways and candidate genes that drive specification, differentiation and the neural circuitry of specific neural progenitors, the phox2b expressing ENS cell lineage, during normal enteric nervous system development. Because ENS development is tightly linked to its environment, the transcriptional landscape of the cellular environment of the intestine was also analyzed.
Thousands of zebrafish intestines were manually dissected from a transgenic line expressing green fluorescent protein under the phox2b regulatory elements Tg(phox2b:EGFP)
. Fluorescence-activated cell sorting was used to separate GFP-positive phox2b expressing ENS progenitor and derivatives from GFP-negative intestinal cells. RNA-seq was performed to obtain accurate, reproducible transcriptional profiles and the unbiased detection of low level transcripts. Analysis revealed genes and pathways that may function in ENS cell determination, genes that may be identifiers of different ENS subtypes, and genes that define the non-neural cellular microenvironment of the ENS. Differential expression analysis between the two cell populations revealed the expected neuronal nature of the phox2b expressing lineage including the enrichment for genes required for neurogenesis and synaptogenesis, and identified many novel genes not previously associated with ENS development. Pathway analysis pointed to a high level of G-protein coupled pathway activation, and identified novel roles for candidate pathways such as the Nogo/Reticulon axon guidance pathway in ENS development.
We report the comprehensive gene expression profiles of a lineage-specific population of enteric progenitors, their derivatives, and their microenvironment during normal enteric nervous system development. Our results confirm previously implicated genes and pathways required for ENS development, and also identify scores of novel candidate genes and pathways. Thus, our dataset suggests various potential mechanisms that drive ENS development facilitating characterization and discovery of novel therapeutic strategies to improve gastrointestinal disorders.
Traditional preparation methods take different toxicity levels in aqueous and ethanol extracts into account when choosing the appropriate solvent for the preparation of a remedy.
The plant species ...reported here are traditionally used in Northern Peru for a wide range of illnesses. Most remedies are prepared as ethanol or aqueous extracts and then ingested. The aim of this study was to evaluate the potential toxicity of these extracts.
The toxicity of ethanolic and water extracts of 341 plant species was determined using a brine-shrimp assay.
Overall 24% of the species in water extract and 76% of the species in alcoholic extract showed elevated toxicity levels to brine-shrimp. Although in most cases multiple extracts of the same species showed very similar toxicity values, in some cases the toxicity of different extracts of the same species varied from non-toxic to highly toxic.
Traditional preparation methods take different toxicity levels in aqueous and ethanol extracts into account when choosing the appropriate solvent for the preparation of a remedy.
During and especially after World War I, the millions of black-clad widows on the streets of Europe’s cities were a constant reminder that war caused carnage on a vast scale. But widows were far more ...than just a reminder of the war’s fallen soldiers; they were literal and figurative actresses in how nations crafted their identities in the interwar era. In this extremely original study, Erika Kuhlman compares the ways in which German and American widows experienced their postwar status, and how that played into the cultures of mourning in their two nations: one defeated, the other victorious. Each nation used widows and war dead as symbols to either uphold their victory or disengage from their defeat, but Kuhlman, parsing both German and U.S. primary sources, compares widows’ lived experiences to public memory. For some widows, government compensation in the form of military-style awards sufficed. For others, their own deprivations, combined with those suffered by widows living in other nations, became the touchstone of a transnational awareness of the absurdity of war and the need to prevent it.
Key points
This study aimed to provide molecular insight into the differential effects of age and physical inactivity on the regulation of substrate metabolism during moderate‐intensity exercise.
...Using the arteriovenous balance technique, we studied the effect of immobilization of one leg for 2 weeks on leg substrate utilization in young and older men during two‐legged dynamic knee‐extensor moderate‐intensity exercise, as well as changes in key proteins in muscle metabolism before and after exercise.
Age and immobilization did not affect relative carbohydrate and fat utilization during exercise, but the older men had higher uptake of exogenous fatty acids, whereas the young men relied more on endogenous fatty acids during exercise.
Using a combined whole‐leg and molecular approach, we provide evidence that both age and physical inactivity result in intramuscular lipid accumulation, but this occurs only in part through the same mechanisms.
Age and inactivity have been associated with intramuscular triglyceride (IMTG) accumulation. Here, we attempt to disentangle these factors by studying the effect of 2 weeks of unilateral leg immobilization on substrate utilization across the legs during moderate‐intensity exercise in young (n = 17; 23 ± 1 years old) and older men (n = 15; 68 ± 1 years old), while the contralateral leg served as the control. After immobilization, the participants performed two‐legged isolated knee‐extensor exercise at 20 ± 1 W (∼50% maximal work capacity) for 45 min with catheters inserted in the brachial artery and both femoral veins. Biopsy samples obtained from vastus lateralis muscles of both legs before and after exercise were used for analysis of substrates, protein content and enzyme activities. During exercise, leg substrate utilization (respiratory quotient) did not differ between groups or legs. Leg fatty acid uptake was greater in older than in young men, and although young men demonstrated net leg glycerol release during exercise, older men showed net glycerol uptake. At baseline, IMTG, muscle pyruvate dehydrogenase complex activity and the protein content of adipose triglyceride lipase, acetyl‐CoA carboxylase 2 and AMP‐activated protein kinase (AMPK)γ3 were higher in young than in older men. Furthermore, adipose triglyceride lipase, plasma membrane‐associated fatty acid binding protein and AMPKγ3 subunit protein contents were lower and IMTG was higher in the immobilized than the contralateral leg in young and older men. Thus, immobilization and age did not affect substrate choice (respiratory quotient) during moderate exercise, but the whole‐leg and molecular differences in fatty acid mobilization could explain the age‐ and immobilization‐induced IMTG accumulation.
Key points
This study aimed to provide molecular insight into the differential effects of age and physical inactivity on the regulation of substrate metabolism during moderate‐intensity exercise.
Using the arteriovenous balance technique, we studied the effect of immobilization of one leg for 2 weeks on leg substrate utilization in young and older men during two‐legged dynamic knee‐extensor moderate‐intensity exercise, as well as changes in key proteins in muscle metabolism before and after exercise.
Age and immobilization did not affect relative carbohydrate and fat utilization during exercise, but the older men had higher uptake of exogenous fatty acids, whereas the young men relied more on endogenous fatty acids during exercise.
Using a combined whole‐leg and molecular approach, we provide evidence that both age and physical inactivity result in intramuscular lipid accumulation, but this occurs only in part through the same mechanisms.
We describe the development of an in vitro library selection system (CIS display) that exploits the ability of a DNA replication initiator protein (RepA) to bind exclusively to the template DNA from ...which it has been expressed, a property called cis-activity. A diverse peptide library is created by ligation of DNA fragments of random sequence to a DNA fragment that encodes RepA. After in vitro transcription and translation, a pool of protein-DNA complexes is formed where each protein is stably associated with the DNA that encodes it. These complexes are amenable to the affinity selection of ligands to targets of interest. Here we show that RepA is a highly faithful cis-acting DNA-binding protein and demonstrate that libraries encoding$>10^{12}$random 18-mer peptides can be constructed and used to isolate peptides that bind specifically to disparate targets. The use of DNA to encode the displayed peptides offers advantages over in vitro peptide display systems that use mRNA.
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