Jennifer A. Meyer

Topographic diversity of fungal and bacterial communities in human skin

Traditional culture-based methods have incompletely defined the microbial landscape of common recalcitrant human fungal skin diseases, including athlete’s foot and toenail infections. Skin protects humans from invasion by pathogenic microorganisms and provides a home for diverse commensal microbiota. Bacterial genomic sequence data have generated novel hypotheses about species and community structures underlying human disorders. However, microbial diversity is not limited to bacteria; microorganisms such as fungi also have major roles in microbial community stability, human health and disease. Genomic methodologies to identify fungal species and communities have been limited compared with those that are available for bacteria. Fungal evolution can be reconstructed with phylogenetic markers, including ribosomal RNA gene regions and other highly conserved genes. Here we sequenced and analysed fungal communities of 14 skin sites in 10 healthy adults. Eleven core-body and arm sites were dominated by fungi of the genus Malassezia, with only species-level classifications revealing fungal-community composition differences between sites. By contrast, three foot sites—plantar heel, toenail and toe web—showed high fungal diversity. Concurrent analysis of bacterial and fungal communities demonstrated that physiologic attributes and topography of skin differentially shape these two microbial communities. These results provide a framework for future investigation of the contribution of interactions between pathogenic and commensal fungal and bacterial communities to the maintainenace of human health and to disease pathogenesis.Traditional culture-based methods have incompletely defined the etiology of common recalcitrant human fungal skin diseases including athlete’s foot and toenail infections. Skin protects humans from invasion by pathogenic microorganisms, while providing a home for diverse commensal microbiota1. Bacterial genomic sequence data have generated novel hypotheses about species and community structures underlying human disorders2,3,4. However, microbial diversity is not limited to bacteria; microorganisms such as fungi also play major roles in microbial community stability, human health and disease5. Genomic methodologies to identify fungal species and communities have been limited compared with tools available for bacteria6. Fungal evolution can be reconstructed with phylogenetic markers, including ribosomal RNA gene regions and other highly conserved genes7. Here, we sequenced and analyzed fungal communities of 14 skin sites in 10 healthy adults. Eleven core body and arm sites were dominated by Malassezia fungi, with species-level classifications revealing greater topographical resolution between sites. By contrast, three foot sites, plantar heel, toenail, and toeweb, exhibited tremendous fungal diversity. Concurrent analysis of bacterial and fungal communities demonstrated that skin physiologic attributes and topography differentially shape these two microbial communities. These results provide a framework for future investigation of interactions between pathogenic and commensal fungal and bacterial communities in maintaining human health and contributing to disease pathogenesis.

A perspective on the role of metals in diabetes: past findings and possible future directions

In this review, the authors present a brief overview of metals and their possible roles as determinants in the pathogenesis of diabetes and complications. Of course, due to the complexity of diabetes and its far-reaching complications, it would be difficult to cover every metal that has been implicated in diabetes. Therefore, this review has two main objectives, the first of which is to educate the reader with regards to the types of diabetes and complications, especially in relation to hyperglycemia and anti-oxidant properties. Following an overview of the more cited metals in diabetes, the second objective of this review is to offer some opinions about current needs in the area of metal analysis. Specifically, the challenges for scientists to perform quantitative determinations on biological samples in near-real time with subcellular-level spatial resolution.

A Molecular Level Understanding of Zinc Activation of C-peptide and its Effects on Cellular Communication in the Bloodstream

Inspired by previous reports, our group has recently demonstrated that C-peptide exerts beneficial effects upon interactions with red blood cells (RBCs). These effects can be measured in RBCs obtained from animal models of both type 1 diabetes and type 2 diabetes, though to different extents. To date, the key metrics that have been measured involving C-peptide and RBCs include an increase in glucose uptake by these cells and a subsequent increase in adenosine triphosphate (ATP) release. Importantly, to date, our group has only been able to elicit these beneficial effects when the C-peptide is prepared in the presence of Zn2+. The C-peptide-induced release of ATP is of interest when considering that ATP is a purinergic signaling molecule known to stimulate the production of nitric oxide (NO) in the endothelium and in platelets. This NO production has been shown to participate in smooth muscle relaxation and subsequent vessel dilation. Furthermore, NO is a well-established platelet inhibitor. The objective of this review is to provide information pertaining to C-peptide activity on RBCs. Special attention is paid to the necessity of Zn2+ activation, and the origin of that activation in vivo. Finally, a mechanism is proposed that explains how C-peptide is exerting its effects on other cells in the bloodstream, particularly on endothelial cells and platelets, via its ability to stimulate the release of ATP from RBCs.

Zinc-activated C-peptide resistance to the type 2 diabetic erythrocyte is associated with hyperglycemia-induced phosphatidylserine externalization and reversed by metformin

Insulin resistance can broadly be defined as the diminished ability of cells to respond to the action of insulin in transporting glucose from the bloodstream into cells and tissues. Here, we report that erythrocytes (ERYs) obtained from type 2 diabetic rats display an apparent resistance to Zn(2+)-activated C-peptide. Thus, the aims of this study were to demonstrate that Zn(2+)-activated C-peptide exerts potentially beneficial effects on healthy ERYs and that these same effects on type 2 diabetic ERYs are enhanced in the presence of metformin. Incubation of ERYs (obtained from type 2 diabetic BBZDR/Wor-rats) with Zn(2+)-activated C-peptide followed by chemiluminescence measurements of ATP resulted in a 31.2 +/- 4.0% increase in ATP release from these ERYs compared to a 78.4 +/- 4.9% increase from control ERYs. Glucose accumulation in diabetic ERYs, measured by scintillation counting of (14)C-labeled glucose, increased by 35.8 +/- 1.3% in the presence of the Zn(2+)-activated C-peptide, a value significantly lower than results obtained from control ERYs (64.3 +/- 5.1%). When Zn(2+)-activated C-peptide was exogenously added to diabetic ERYs, immunoassays revealed a 32.5 +/- 8.2% increase in C-peptide absorbance compared to a 64.4 +/- 10.3% increase in control ERYs. Phosphatidylserine (PS) externalization and metformin sensitization of Zn(2+)-activated C-peptide were examined spectrofluorometrically by measuring the binding of FITC-labeled annexin to PS. The incubation of diabetic ERYs with metformin prior to the addition of Zn(2+)-activated C-peptide resulted in values that were statistically equivalent to those of controls. Summarily, data obtained here demonstrate an apparent resistance to Zn(2+)-activated C-peptide by the ERY that is corrected by metformin.

Pan-PCR, a computational method for designing bacterium-typing assays based on whole-genome sequence data.

ABSTRACT With increasing rates of antibiotic resistance, bacterial infections have become more difficult to treat, elevating the importance of surveillance and prevention. Effective surveillance relies on the availability of rapid, cost-effective, and informative typing methods to monitor bacterial isolates. PCR-based typing assays are fast and inexpensive, but their utility is limited by the lack of targets which are capable of distinguishing between strains within a species. To identify highly informative PCR targets from the growing base of publicly available bacterial genome sequences, we developed pan-PCR. This computer algorithm uses existing genome sequences for isolates of a species of interest and identifies a set of genes whose patterns of presence or absence provide the best discrimination between strains in this species. A set of PCR primers targeting the identified genes is then designed, with each PCR product being of a different size to allow multiplexing. These target DNA regions and PCR primers can then be utilized to type bacterial isolates. To evaluate pan-PCR, we designed an assay for the emerging pathogen Acinetobacter baumannii. Taking as input a set of 29 previously sequenced genomes, pan-PCR identified 6 genetic loci whose presence or absence was capable of distinguishing all the input strains. This assay was applied to a set of patient isolates, and its discriminatory power was compared to that of multilocus sequence typing (MLST) and whole-genome optical maps. We found that the pan-PCR assay was capable of making clinically relevant distinctions between strains with identical MLST profiles and showed a discriminatory power similar to that of optical maps. Pan-PCR represents a tool capable of exploiting available genome sequence data to design highly discriminatory PCR assays. The ease of design and implementation makes this approach feasible for diagnostic facilities of all sizes.

A microfluidic technique for monitoring bloodstream analytes indicative of C-peptide resistance in type 2 diabetes

A simple poly(dimethylsiloxane) (PDMS) microchip was employed to establish a relationship between red blood cell (RBC) antioxidant status and the ability of RBCs to interact with metal-activated C-peptide, a bio-active peptide reported to reduce some complications often associated with diabetes. It is known that the reduced form of glutathione (GSH) levels in the RBCs obtained from people with type 2 diabetes are lower in comparison to those RBCs obtained from healthy controls and accordingly, this correlation has the potential to implicate type 2 diabetes in high-risk individuals. A parallel channel microfluidic device for the quantification of GSH in age-based fractions, along with control and diabetic RBCs is described. Important to the fluorescence-based measurement is the simultaneous determination of the antioxidant without prior separation in either a six- or twelve-channel microchip. Here, we separated the RBCs using a density-based Percoll solution and quantitatively determined the concentration of GSH in younger, less dense RBCs to be increased more than 2-fold (336.7 +/- 29.6 amol/RBC) than older, more dense RBCs (137.0 +/- 25.3 amol/RBC). The ability of C-peptide to interact with the RBC membrane of the separated fractions was determined by immunoassay and it was found that the recovery of the C-peptide added to the younger RBCs increased by more than 40.6 +/- 12.7% above basal levels while with the older cells C-peptide increased by only 9.18 +/- 4.60%. These results suggest that GSH concentrations in the RBC may be useful in screening for resistance to C-peptide in vivo.

Kinetic analysis of PCNA clamp binding and release in the clamp loading reaction catalyzed by Saccharomyces cerevisiae replication factor C

DNA polymerases require a sliding clamp to achieve processive DNA synthesis. The toroidal clamps are loaded onto DNA by clamp loaders, members of the AAA+family of ATPases. These enzymes utilize the energy of ATP binding and hydrolysis to perform a variety of cellular functions. In this study, a clamp loader-clamp binding assay was developed to measure the rates of ATP-dependent clamp binding and ATP-hydrolysis-dependent clamp release for the Saccharomyces cerevisiae clamp loader (RFC) and clamp (PCNA). Pre-steady-state kinetics of PCNA binding showed that although ATP binding to RFC increases affinity for PCNA, ATP binding rates and ATP-dependent conformational changes in RFC are fast relative to PCNA binding rates. Interestingly, RFC binds PCNA faster than the Escherichia coli γ complex clamp loader binds the β-clamp. In the process of loading clamps on DNA, RFC maintains contact with PCNA while PCNA closes, as the observed rate of PCNA closing is faster than the rate of PCNA release, precluding the possibility of an open clamp dissociating from DNA. Rates of clamp closing and release are not dependent on the rate of the DNA binding step and are also slower than reported rates of ATP hydrolysis, showing that these rates reflect unique intramolecular reaction steps in the clamp loading pathway.

Use of the red blood cell as a simple drug target and diagnostic by manipulating and monitoring its ability to release adenosine triphosphate (ATP)

Without question, one of the main tasks of the red blood cell (RBC) is to deliver oxygen to various tissues and organs in vivo. However, due to the lack of a nucleus and mito-chondria, the RBC is typically not thought to be a determinant in many diseases or abnormal physiological conditions. Recent efforts by many labs world-wide are resulting in a body of evidence, suggesting that the RBC may serve many other roles in vivo besides that of an oxygen carrier. If so, the RBC may eventually emerge as one of the simplest drug targets and diagnostic tools available. Here, molecular evidence is provided, suggesting that the RBC, via its ability (or inability) to maintain proper levels of adenosine triphosphate (ATP) release in the circulation, may be a major factor in vascular regulation. Moreover, due to the RBC’s response to slight modifications in its normal environment, the use of the RBC as an important diagnostic for early prediction of disease onset is discussed.