The Role of the Insulin/Glucose Ratio in the Regulation of Pathogen Biofilm Formation.

During the management of patients in acute trauma the resulting transient hyperglycemia is treated by administration of insulin. Since the effect of insulin, a quorum sensing compound, together with glucose affects biofilm formation in a concentration-specific manner, we hypothesize that the insulin/glucose ratio over the physiologic range modulates biofilm formation potentially influencing the establishment of infection through biofilm formation. METHODS: A variety of Gram-positive and Gram-negative bacteria were grown in peptone (1%) yeast nitrogen base broth overnight in 96-well plates with various concentrations of glucose and insulin. Biofilm formation was determined by the crystal violet staining procedure. Expression of insulin binding was determined by fluorescent microscopy (FITC-insulin). Controls were buffer alone, insulin alone, and glucose alone. RESULTS: Overall, maximal biofilm levels were measured at 220 mg/dL of glucose, regardless of insulin concentration (10, 100, 200 µU/mL) of the organism tested. In general, insulin with glucose over the range of 160-180 mg/dL exhibited a pattern of biofilm suppression. However, either above or below this range, the presence of insulin in combination with glucose significantly modulated (increase or decrease) biofilm formation in a microbe-specific pattern. This modulation appears for some organisms to be reflective of the glucose-regulated intrinsic expression of bacterial insulin receptor expression. CONCLUSION: Insulin at physiologic levels (normal and hyperinsulinemic) in combination with glucose can affect biofilm formation in a concentration-specific and microbe-specific manner. These findings may provide insight into the importance of co-regulation of the insulin/glucose ratio in patient management.

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi.

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with respect to how viral members of the microbiome affect subsequent microbe interactions with these virus-infected host cells. The co-habitation of virus with bacteria and fungi is principally present on the mucosal surfaces of the oral cavity and genital tract. Mucosal cells, particularly those with persistent chronic or persistent latent viral infections, could have a significant impact on members of the microbiome through virus alteration in number and type of receptors expressed. Modification in host cell membrane architecture would result in altered ability of subsequent members of the normal flora and opportunistic pathogens to initiate the first step in biofilm formation, i.e., adherence. This study describes a method for quantitation and visual examination of HSV's effect on the initiation of biofilm formation (adherence) of S. aureus and C. albicans.

Herpes Simplex Virus (HSV) Modulation of Staphylococcus aureus and Candida albicans Initiation of HeLa 299 Cell-Associated Biofilm.

Although herpes simplex virus type-1 (HSV-1), and type-2 (HSV-2), Staphylococcus aureus and Candida albicans co-habit the oral and genital mucosa, their interaction is poorly understood. We determined the effect HSV has on bacterial and/or fungal adherence, the initial step in biofilm formation. HeLa229 cells were infected with HSV-1 (KOS) gL86 or HSV-2 (KOS) 333gJ (-) at a multiplicity of infection (MOI) of 50 and 10. S. aureus (ATCC 25923) and/or C. albicans (yeast forms or germ tube forms) were co-incubated for 30 min (37 °C; 5 % CO2; 5:1 organism: HeLa cell ratio; n = 16) with virus-infected HeLa cells or uninfected HeLa cell controls. Post-incubation, the monolayers were washed (3x; PBS), lysed (RIPA), and the lysate plated onto Fungisel and/or mannitol salts agar for standard colony count. The level of HeLa-associated S. aureus was significantly decreased (P < 0.05) for both HSV-1- and HSV-2-infected cells, as compared to virus-free HeLa cell controls (38 and 59 % of control, respectively). In contrast, HSV-1 and HSV-2 significantly (P < 0.05) enhanced HeLa cell association of C. albicans yeast forms and germ tube approximately two-fold, respectively. The effect of S. aureus on germ tube and yeast form adherence to HSV-1- and HSV-2-infected cells was specific for the Candida phenotype tested. Our study suggests that HSV, while antagonist towards S. aureus adherence enhances Candida adherence. Furthermore, the combination of the three pathogens results in S. aureus adherence that is either unaffected, or partially restored depending on both the herpes viral species and the fungal phenotype present.

Bone-remodeling transcript levels are independent of perching in end-of-lay white leghorn chickens.

Osteoporosis is a bone disease that commonly results in a 30% incidence of fracture in hens used to produce eggs for human consumption. One of the causes of osteoporosis is the lack of mechanical strain placed on weight-bearing bones. In conventionally-caged hens, there is inadequate space for chickens to exercise and induce mechanical strain on their bones. One approach is to encourage mechanical stress on bones by the addition of perches to conventional cages. Our study focuses on the molecular mechanism of bone remodeling in end-of-lay hens (71 weeks) with access to perches. We examined bone-specific transcripts that are actively involved during development and remodeling. Using real-time quantitative PCR, we examined seven transcripts (COL2A1 (collagen, type II, alpha 1), RANKL (receptor activator of nuclear factor kappa-B ligand), OPG (osteoprotegerin), PTHLH (PTH-like hormone), PTH1R (PTH/PTHLH type-1 receptor), PTH3R (PTH/PTHLH type-3 receptor), and SOX9 (Sry-related high mobility group box)) in phalange, tibia and femur. Our results indicate that the only significant effect was a difference among bones for COL2A1 (femur > phalange). Therefore, we conclude that access to a perch did not alter transcript expression. Furthermore, because hens have been used as a model for human bone metabolism and osteoporosis, the results indicate that bone remodeling due to mechanical loading in chickens may be a product of different pathways than those involved in the mammalian model.

OrbId: Origin-based identification of microRNA targets.

MicroRNAs coordinate networks of mRNAs, but predicting specific sites of interactions is complicated by the very few bases of complementarity needed for regulation. Although efforts to characterize the specific requirements for microRNA (miR) regulation have made some advances, no general model of target recognition has been widely accepted. In this work, we describe an entirely novel approach to miR target identification. The genomic events responsible for the creation of individual miR loci have now been described with many miRs now known to have been initially formed from transposable element (TE) sequences. In light of this, we propose that limiting miR target searches to transcripts containing a miR's progenitor TE can facilitate accurate target identification. In this report we outline the methodology behind OrbId (Origin-based identification of microRNA targets). In stark contrast to the principal miR target algorithms (which rely heavily on target site conservation across species and are therefore most effective at predicting targets for older miRs), we find OrbId is particularly efficacious at predicting the mRNA targets of miRs formed more recently in evolutionary time. After defining the TE origins of > 200 human miRs, OrbId successfully generated likely target sets for 191 predominately primate-specific human miR loci. While only a handful of the loci examined were well enough conserved to have been previously evaluated by existing algorithms, we find ~80% of the targets for the oldest miR (miR-28) in our analysis contained within the principal Diana and TargetScan prediction sets. More importantly, four of the 15 OrbId miR-28 putative targets have been previously verified experimentally. In light of OrbId proving best-suited for predicting targets for more recently formed miRs, we suggest OrbId makes a logical complement to existing, conservation based, miR target algorithms.