PDGF-BB Does Not Accelerate Healing in Diabetic Mice with Splinted Skin Wounds

Topical application of platelet-derived growth factor-BB (PDGF-BB) is considered to accelerate tissue repair of impaired chronic wounds. However, the vast literature is plagued with conflicting reports of its efficacy in animal models and this is often influenced by a wide array of experimental variables making it difficult to compare the results across the studies. To mitigate the confounding variables that influence the efficacy of topically applied PDGF-BB, we used a controlled full thickness splinted excisional wound model in db/db mice (type 2 diabetic mouse model) for our investigations. A carefully-defined silicone-splinted wound model, with reduced wound contraction, controlled splint and bandage maintenance, allowing for healing primarily by reepithelialization was employed. Two splinted 8 mm dorsal full thickness wounds were made in db/db mice. Wounds were topically treated once daily with either 3 µg PDGF-BB in 30 µl of 5% PEG-PBS vehicle or an equal volume of vehicle for 10 days. Body weights, wound contraction, wound closure, reepithelialization, collagen content, and wound bed inflammation were evaluated clinically and histopathologically. The bioactivity of PDGF-BB was confirmed by in vitro proliferation assay. PDGF-BB, although bioactive in vitro, failed to accelerate wound healing in vivo in the db/db mice using the splinted wound model. Considering that the predominant mechanism of wound healing in humans is by re-epeithelialization, the most appropriate model for evaluating therapeutics is one that uses splints to prevent excessive wound contraction. Here, we report that PDGF-BB does not promote wound closure by re-epithelialization in a murine splinted wound model. Our results highlight that the effects of cytoactive factors reported in vivo ought to be carefully interpreted with critical consideration of the wound model used.     

Human Systemic Immune Response to Ingestion of the Oral Probiotic Streptococcus salivarius BLIS K12

Probiotics and Antimicrobial Proteins - Streptococcus salivarius K12 is an oral probiotic known to contribute to protection against oral pathogenic bacteria in humans. Studies of immune responses...     

Identification and Molecular Mechanisms of the Rapid Tonicity-induced Relocalization of the Aquaporin 4 Channel

The aquaporin family of integral membrane proteins is composed of channels that mediate cellular water flow. Aquaporin 4 (AQP4) is highly expressed in the glial cells of the central nervous system and facilitates the osmotically driven pathological brain swelling associated with stroke and traumatic brain injury. Here we show that AQP4 cell surface expression can be rapidly and reversibly regulated in response to changes of tonicity in primary cortical rat astrocytes and in transfected HEK293 cells. The translocation mechanism involves PKA activation, influx of extracellular calcium, and activation of calmodulin. We identify five putative PKA phosphorylation sites and use site-directed mutagenesis to show that only phosphorylation at one of these sites, serine 276, is necessary for the translocation response. We discuss our findings in the context of the identification of new therapeutic approaches to treating brain edema.     

The bifurcation of the cyanogenic glucoside and glucosinolate biosynthetic pathways

The biosynthetic pathway for the cyanogenic glucoside dhurrin in sorghum has previously been shown to involve the sequential production of (E)‐ and (Z)‐p‐hydroxyphenylacetaldoxime. In this study we used microsomes prepared from wild‐type and mutant sorghum or transiently transformed Nicotiana benthamiana to demonstrate that CYP79A1 catalyzes conversion of tyrosine to (E)‐p‐hydroxyphenylacetaldoxime whereas CYP71E1 catalyzes conversion of (E)‐p‐hydroxyphenylacetaldoxime into the corresponding geometrical Z‐isomer as required for its dehydration into a nitrile, the next intermediate in cyanogenic glucoside synthesis. Glucosinolate biosynthesis is also initiated by the action of a CYP79 family enzyme, but the next enzyme involved belongs to the CYP83 family. We demonstrate that CYP83B1 from Arabidopsis thaliana cannot convert the (E)‐p‐hydroxyphenylacetaldoxime to the (Z)‐isomer, which blocks the route towards cyanogenic glucoside synthesis. Instead CYP83B1 catalyzes the conversion of the (E)‐p‐hydroxyphenylacetaldoxime into an S‐alkyl‐thiohydroximate with retention of the configuration of the E‐oxime intermediate in the final glucosinolate core structure. Numerous microbial plant pathogens are able to detoxify Z‐oximes but not E‐oximes. The CYP79‐derived E‐oximes may play an important role in plant defense.     

Mutation accumulation and the formation of range limits

The dynamics of range formation are important for understanding and predicting species distributions. Here, we focus on a process that has thus far been overlooked in the context of range formation; the accumulation of mutation load. We find that mutation accumulation severely reduces the extent of a range across an environmental gradient, especially when dispersal is limited, growth rate is low and mutations are of intermediate deleterious effect. Our results illustrate the important role deleterious mutations can play in range formation. We highlight this as a necessary focus for further work, noting particularly the potentially conflicting effects dispersal may have in reducing mutation load and simultaneously increasing migration load in marginal populations.     

Understanding the yeast host cell response to recombinant membrane protein production

Membrane proteins are drug targets for a wide range of diseases. Having access to appropriate samples for further research underpins the pharmaceutical industry's strategy for developing new drugs. This is typically achieved by synthesizing a protein of interest in host cells that can be cultured on a large scale, allowing the isolation of the pure protein in quantities much higher than those found in the protein's native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to that of the native human source cells of many proteins of interest, while also being quick, easy and cheap to grow and process. Even in these cells, the production of human membrane proteins can be plagued by low functional yields; we wish to understand why. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast host strains. By relieving the bottlenecks to recombinant membrane protein production in yeast, we aim to contribute to the drug discovery pipeline, while providing insight into translational processes.     

Overcoming barriers to membrane protein structure determination

After decades of slow progress, the pace of research on membrane protein structures is beginning to quicken thanks to various improvements in technology, including protein engineering and microfocus X-ray diffraction. Here we review these developments and, where possible, highlight generic new approaches to solving membrane protein structures based on recent technological advances. Rational approaches to overcoming the bottlenecks in the field are urgently required as membrane proteins, which typically comprise ~30% of the proteomes of organisms, are dramatically under-represented in the structural database of the Protein Data Bank.     

USER Friendly Cloning Coupled with Chitin-Based Natural Transformation Enables Rapid Mutagenesis of Vibrio vulnificus

Vibrio vulnificus is a bacterial contaminant of shellfish and causes highly lethal sepsis and destructive wound infections. A definitive identification of virulence factors using the molecular version of Koch''s postulates has been hindered because of difficulties in performing molecular genetic analysis of this opportunistic pathogen. For example, conjugation is required to introduce plasmid DNA, and allelic exchange suicide vectors that rely on sucrose sensitivity for counterselection are not efficient. We therefore incorporated USER friendly cloning techniques into pCVD442-based allelic exchange suicide vectors and other expression vectors to enable the rapid and efficient capture of PCR amplicons. Upstream and downstream DNA sequences flanking genes targeted for deletion were cloned together in a single step. Based on results from Vibrio cholerae, we determined that V. vulnificus becomes naturally transformable with linear DNA during growth on chitin in the form of crab shells. By combining USER friendly cloning and chitin-based transformation, we rapidly and efficiently produced targeted deletions in V. vulnificus, bypassing the need for two-step, suicide vector-mediated allelic exchange. These methods were used to examine the roles of two flagellin loci (flaCDE and flaFBA), the motAB genes, and the cheY-3 gene in motility and to create deletions of rtxC, rtxA1, and fadR. Additionally, chitin-based transformation was useful in moving antibiotic resistance-labeled mutations between V. vulnificus strains by simply coculturing the strains on crab shells. The methods and genetic tools that we developed should be of general use to those performing molecular genetic analysis and manipulation of other gram-negative bacteria.Vibrio vulnificus is a halophilic bacterium present naturally in estuarine waters and often contaminates oysters and other shellfish (for a review, see reference 15). V. vulnificus is an opportunistic pathogen of humans, causing primary septicemia and wound infection in susceptible individuals, and is the leading cause of reported seafood-related deaths in the United States. In susceptible humans, V. vulnificus causes a rapid, fulminating disease process resulting in extensive tissue damage. Mortality rates for susceptible individuals who develop fulminating primary septicemia are greater than 50% (17). Skin infections can lead to severe cellulitis, necrotizing fasciitis, and myositis requiring surgical debridement of infected tissues or amputation of the limb (4, 29, 42). Therapeutic intervention is often difficult since death can occur in less than 24 h after contact with the bacteria. In a mouse model of infection, V. vulnificus replicates extremely rapidly in host tissues (40, 41) and kills host cells including neutrophils (41).Over 20 years of genetic analysis, only a few virulence factors have been identified and confirmed by using the molecular version of Koch''s postulates (15). Among the confirmed virulence factors are capsular polysaccharide (49), acquisition of iron (34, 52), type IV pilus (37), RTX toxins (21, 25, 30), and flagella (22, 26). Despite these advances, the full spectrum of virulence factors responsible for the rapid and destructive disease process has not been elucidated. A major hindrance to the molecular genetic analysis of V. vulnificus is the fact that the bacteria cannot be effectively electroporated, nor can the bacteria be chemically transformed. Therefore, the only effective means of introducing plasmid DNA is by conjugation. This limitation severely restricts the availability of plasmids for creating and complementing mutations for molecular genetic analysis.A classical method to create mutants of V. vulnificus is the use of suicide vectors containing transposons, such as TnphoA (31), mini-Tn5phoA (12), mini-Tn5Km2 (12), mini-Tn5lacZ1 (12), mini-Tn10/kan (23), and Himar (1). However, transposon mutagenesis can cause polar mutations and truncated genes. The most definitive genetic analyses can be performed by deleting the genes of interest. The standard method for deleting genes is to clone the flanking upstream and downstream sequences into an allelic exchange suicide vector and introducing the plasmid into the target strain. The suicide vector integrates into the target genome via one of the flanking DNA sequences by a single-crossover event. This process is selected using antibiotic resistance encoded on the vector. The single-crossover strain is then grown without selection to allow for a second crossover that excises the allelic exchange vector. The excision event is enriched using a counterselectable marker encoded on the allelic exchange vector. A commonly used counterselectable marker is sucrose sensitivity, encoded by the sacB gene (13). If the second crossover is in the same flanking sequence as the first, the wild-type genotype is restored; however, if the second crossover is in the opposite flanking sequence, the targeted gene is deleted.This method of mutagenesis is problematic for V. vulnificus. First, one of the easiest ways to clone PCR amplicons is by TA topoisomerase-mediated ligation (TOPO TA cloning) with commercially available vectors such as pCR2.1 TOPO (Invitrogen, Carlsbad, CA). Unfortunately, these vectors are not mobilizable; hence, they cannot be used directly with V. vulnificus. Furthermore, there are no available TA topoisomerase allelic exchange vectors. Site-specific recombination vectors such as Gateway (Invitrogen) have facilitated PCR-mediated cloning and gene expression; however, none of the relevant vectors is mobilizable. Therefore, essentially all cloning for expression in or mutagenesis of V. vulnificus requires multiple subcloning steps. Second, sucrose sensitivity counterselection is not very efficient with V. vulnificus, as it is time-consuming and involves the screening of large numbers of colonies to find truly sucrose-resistant colonies. In some cases, sacB counterselection has completely failed. Another method for introducing mutations into target strains is the lambda phage red recombinase system (11). However, this procedure involves introducing additional helper plasmids into the target strain, and mobilizable helper plasmids have not been made. Finally, no generalized transducing phages and no methods for transformation have been described for V. vulnificus. Therefore, if one desires to move mutations between strains, the mutations must be cloned into suicide vectors, and the inefficient two-step allelic exchange process must be repeated for each strain.We describe here the adaption of USER (uracil-specific excision reagent) friendly cloning (3, 14) into allelic exchange and expression vectors that are commonly used with V. vulnificus. USER friendly cloning enables the creation of 3′ overhangs in PCR amplicons by use of deoxyuridine in PCR primers and treatment with USER enzyme mix (uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII). The overhangs are complementary to overhangs created in vectors. The method is very adaptable, with immense leeway in choosing the DNA sequences of the overhangs independent of restriction enzyme cleavage sites. USER friendly cloning methods and vectors enable the rapid creation of upstream-downstream clones to delete genes or loci of interest. To alleviate inefficiencies of subcloning into V. vulnificus vectors, we created an array of USER friendly cloning allelic exchange and expression vectors that should be useful to many investigators.Meibom et al. (32) recently described the ability of Vibrio cholerae to become naturally transformable during growth in the presence of chitin, as either chitohexose or crab shell fragments. We adapted their crab shell system and determined that V. vulnificus can also become naturally transformable during growth on chitin. The bacteria take up linear DNA; hence, as long as selectable markers are used, allelic exchange mutagenesis can be accomplished without the need for inefficient counterselection involving sucrose. We used these methods to create antibiotic resistance-marked deletions in V. vulnificus of the two loci encoding flagellins, flaCDE and flaFBA, and the genes encoding the following functions: the flagellar motor motAB; a critical element of signal transduction for chemotaxis, cheY-3; an RTX toxin, rtxA1; a putative RtxA-modifying enzyme, rtxC; and a regulator of fatty acid metabolism, fadR. These genetic tools and methods will facilitate the molecular genetic analysis of V. vulnificus as well as those of other gram-negative bacteria.     

Novel and recurrent LDLR gene mutations in Pakistani hypercholesterolemia patients

The majority of patients with the autosomal dominant disorder familial hypercholesterolemia (FH) carry novel mutations in the low density lipoprotein receptor (LDLR) that is involved in cholesterol regulation. In different populations the spectrum of mutations identified is quite different and to date there have been only a few reports of the spectrum of mutations in FH patients from Pakistan. In order to identify the causative LDLR variants the gene was sequenced in a Pakistani FH family, while high resolution melting analysis followed by sequencing was performed in a panel of 27 unrelated sporadic hypercholesterolemia patients. In the family a novel missense variant (c.1916T > G, p.(V639G)) in exon 13 of LDLR was identified in the proband. The segregation of the identified nucleotide change in the family and carrier status screening in a group of 100 healthy subjects was done using restriction fragment length polymorphism analysis. All affected members of the FH family carried the variant and none of the non-affected members nor any of the healthy subjects. In one of the sporadic cases, two sequence changes were detected in exon 9, one of these was a recurrent missense variant (c.1211C > T; p.T404I), while the other was a novel substitution mutation (c.1214 A > C; N405T). In order to define the allelic status of this double heterozygous individual, PCR amplified fragments were cloned and sequenced, which identified that both changes occurred on the same allele. In silico tools (PolyPhen and SIFT) were used to predict the effect of the variants on the protein structure, which predicted both of these variants to have deleterious effect. These findings support the view that there will be a novel spectrum of mutations causing FH in patients with hypercholesterolaemia from Pakistan.     

L-Carnosine Affects the Growth of Saccharomyces cerevisiae in a Metabolism-Dependent Manner

The dipeptide L-carnosine (β-alanyl-L-histidine) has been described as enigmatic: it inhibits growth of cancer cells but delays senescence in cultured human fibroblasts and extends the lifespan of male fruit flies. In an attempt to understand these observations, the effects of L-carnosine on the model eukaryote, Saccharomyces cerevisiae, were examined on account of its unique metabolic properties; S. cerevisiae can respire aerobically, but like some tumor cells, it can also exhibit a metabolism in which aerobic respiration is down regulated. L-Carnosine exhibited both inhibitory and stimulatory effects on yeast cells, dependent upon the carbon source in the growth medium. When yeast cells were not reliant on oxidative phosphorylation for energy generation (e.g. when grown on a fermentable carbon source such as 2% glucose), 10–30 mM L-carnosine slowed growth rates in a dose-dependent manner and increased cell death by up to 17%. In contrast, in media containing a non-fermentable carbon source in which yeast are dependent on aerobic respiration (e.g. 2% glycerol), L-carnosine did not provoke cell death. This latter observation was confirmed in the respiratory yeast, Pichia pastoris. Moreover, when deletion strains in the yeast nutrient-sensing pathway were treated with L-carnosine, the cells showed resistance to its inhibitory effects. These findings suggest that L-carnosine affects cells in a metabolism-dependent manner and provide a rationale for its effects on different cell types.