How undergraduates historically underrepresented in biomedical sciences value multiple components of a research training program.

To promote diversity in the STEM workforce, undergraduate research training programs incorporating a variety of intervention strategies have been developed to support students from historically underrepresented backgrounds in overcoming numerous systemic barriers to pursuing careers in science. However, relatively little research has focused on how students experience and value these interventions and the ways in which the interventions support student success. The current study analyzed qualitative interviews from participants (n=15) in a comprehensive research training program for undergraduates historically underrepresented in biomedical research to investigate the student perspective on how specific program components address barriers and support their research training, academic progress, and career preparation. Findings indicated that students benefit from authentic research experiences, mentoring, supplemental curriculum, financial assistance, and a supportive program environment. Participants described how the program helped them address financial concerns, navigate academic and career choices, build science identity and efficacy, and feel a sense of belonging within a caring community. The study highlights how multi-faceted research training programs offering a variety of supports can contribute to student retention and development according to the needs and circumstances of individual students.

Lessons Learned from Clinical and Translational Science Faculty and Student Survey as COVID-19 Pandemic Continues to Shift Education Online.

INTRODUCTION: As the pandemic continues with new variants emerging, faculty and students require support with education's rapid shift to the virtual space. The Mayo Clinic Center for Clinical and Translational Science curriculum team works closely with faculty to support a smooth transition to offering graduate courses in a virtual learning environment. The aim of the present project was to explore faculty and student perceptions of these remote learning strategies to gain an understanding of the innovations required to improve future educational offerings. METHODS: All faculty and learners involved in nine Clinical and Translational Science courses in spring 2020 were invited to participate in a web-based questionnaire. Quantitative analysis was performed on closed-ended items, including 5-point Likert-scale questions used to assess the range of views. Qualitative free-text responses were independently analyzed for repetitive themes and summarized. Additionally, comparisons of faculty and course evaluations and student grade point averages (GPAs) from the in-person courses and their subsequent virtual course offerings were considered. RESULTS: Survey results indicated several positive impacts with moving courses into the virtual environment, including increased accessibility as well as more student-centered education. Learners joining from sites outside of the originating campus were especially grateful for the virtual classroom because they felt newly integrated within classes. Faculty and course evaluations, as well as student GPAs, remained consistent. CONCLUSION: New COVID-19 variants continue to shift education online, and innovative ideas are required to further improve future virtual course offerings. Increased engagement is warranted, both from faculty to incorporate activities designed specifically for a virtual classroom, and from students to increase participation by activating their microphones and webcams. Greater opportunities for global involvement and connectedness arise. Finally, this project advocates for adequate eLearning staffing to support quality online education as the need for pedagogical and technical provision continues.

Isavuconazonium Sulfate Use in Multi-Modal Management of Invasive Mucormycosis in Four Pediatric Allogeneic Hematopoietic Cell Transplant Patients.

Prolonged neutropenia increases the risk for lethal invasive fungal infections (IFIs) such as those caused by Rhizopus species. Isavuconazonium sulfate is a new triazole that lacks pediatric dosing recommendations. Clinical courses of 4 pediatric patients with IFIs in the peri-allogeneic hematopoietic cell transplantation (alloHCT) period between 2015 and 2017 were reviewed. The reviews included previously unreported pharmacokinetic and safety data, and the IFIs included Rhizopus. Isavuconazonium sulfate was initiated with a loading dose followed by daily dosing, adjusted to a goal trough concentration of >3 mg/L based on adult literature. This target was achieved at a median of 7 days, demonstrating varying rates of metabolism. Renal insufficiency, electrolyte disturbances, and transaminitis were noted, although attribution was confounded by other alloHCT complications. One patient survived infection-free to hospital discharge and 1 of 3 deceased patients had evidence of an unresolved IFI (case 2). Case 2 was subtherapeutic for 39% of the duration of treatment, compared with others at an average of 29%, suggesting this target trough to be clinically relevant because case 2 demonstrated positive sinus and nasal cultures for Rhizopus on autopsy. We recommend initiation of isavuconazonium 10 mg/kg with a maximum dose of 372 mg. A loading dose of 10 mg/kg is used every 8 hours for 6 doses followed by 10 mg/kg dosing every 24 hours. Monitoring must continue beyond steady state. If early monitoring is not possible, we recommend a first drug level at week 3. If dose increases are required, a partial reload has been more successful instead of increasing daily doses. Further larger studies are needed to demonstrate optimum dosing in pediatric patients.

A transcriptome and proteome of the tick Rhipicephalus microplus shaped by the genetic composition of its hosts and developmental stage.

The cattle tick, Rhipicephalus microplus, is a monoxenous tick that co-evolved with indicine cattle on the Indian subcontinent. It causes massive damage to livestock worldwide. Cattle breeds present heritable, contrasting phenotypes of tick loads, taurine breeds carrying higher loads of the parasite than indicine breeds. Thus, a useful model is available to analyze mechanisms that determine outcomes of parasitism. We sought to gain insights on these mechanisms and used RNA sequencing and Multidimensional Protein Identification Technology (MudPIT) to generate a transcriptome from whole larvae and salivary glands from nymphs, males and females feeding on genetically susceptible and resistant bovine hosts and their corresponding proteomes. 931,698 reads were annotated into 11,676 coding sequences (CDS), which were manually curated into 116 different protein families. Male ticks presented the most diverse armamentarium of mediators of parasitism. In addition, levels of expression of many genes encoding mediators of parasitism were significantly associated with the level and stage of host immunity and/or were temporally restricted to developmental stages of the tick. These insights should assist in developing novel, sustainable technologies for tick control.

Proteome Wide Profiling of N-ε-Lysine Acetylation Reveals a Novel Mechanism of Regulation of the Chitinase Activity in Francisella novicida.

Francisella tularensis is a Gram-negative bacterium that causes the zoonotic disease tularemia. The historical development of tularemia as a biological weapon has led to it being characterized by the CDC as a category A biothreat agent. Neither posttranslational modification (PTM) of proteins, in particular lysine acetylation, in Francisella nor its subsequent regulation of the protein activity has been well studied. In this work, we analyze N-ε-lysine acetylation of the F. tularensis ssp. novicida proteome by mass spectrometry for the first time. To create a comprehensive acetylation profile, we enriched protein acetylation using two approaches: (1) the addition of glucose or acetate into the culture medium and (2) direct chemical acetylation of N-ε-lysines with acetyl phosphate. We discovered 280 acetylated proteins with 1178 acetylation sites in the F. tularensis ssp. novicida strain U112. Lysine acetylation is an important PTM that regulates multiple cellular processes in bacteria, including metabolism, transcription, translation, stress response, and protein folding. We discovered that Francisella chitinases A and B are acetylated naturally and when chemically induced by acetyl phosphate. Moreover, chemical overacetylation of chitinases results in silencing of the enzymatic activity. Our findings suggest a novel mechanism of posttranslational regulation of the chitinase activity and that acetylation may play a role in Francisella's regulation of the protein activity.

Proteomic Approach for Extracting Cytoplasmic Proteins from Streptococcus sanguinis using Mass Spectrometry.

Streptococcus sanguinis is a commensal and early colonizer of oral cavity as well as an opportunistic pathogen of infectious endocarditis. Extracting the soluble proteome of this bacterium provides deep insights about the physiological dynamic changes under different growth and stress conditions, thus defining "proteomic signatures" as targets for therapeutic intervention. In this protocol, we describe an experimentally verified approach to extract maximal cytoplasmic proteins from Streptococcus sanguinis SK36 strain. A combination of procedures was adopted that broke the thick cell wall barrier and minimized denaturation of the intracellular proteome, using optimized buffers and a sonication step. Extracted proteome was quantitated using Pierce BCA Protein Quantitation assay and protein bands were macroscopically assessed by Coomassie Blue staining. Finally, a high resolution detection of the extracted proteins was conducted through Synapt G2Si mass spectrometer, followed by label-free relative quantification via Progenesis QI. In conclusion, this pipeline for proteomic extraction and analysis of soluble proteins provides a fundamental tool in deciphering the biological complexity of Streptococcus sanguinis.

Continuous Antithrombin III Administration in Pediatric Veno-Arterial Extracorporeal Membrane Oxygenation.

The purpose of this retrospective case-control study is to determine the effect of continuous antithrombin III (ATIII) infusion on extracorporeal membrane oxygenation (ECMO) coagulation. All ECMO patients within the pediatric intensive care unit from January 2012 to July 2014 were included. Comparison was made between those who received continuous infusion ATIII through a standardized replacement protocol with historic controls receiving intermittent ATIII doses. Patients receiving the continuous infusion ATIII protocol spent more time in goal ACT range (71.9% vs 52.2%, p < 0.0001). Mean daily ATIII activity was also increased in study group (77.3% versus 68.6%, p = 0.04). No statistical differences in number of heparin dose changes per day (3 versus 3.22, p = 0.90) were present between the 2 groups. Only 28% of the historic controls receiving intermittent ATIII doses achieved normal ATIII activity as compared with 80% of study patients (p = 0.24). Maximum heparin dose was also lower in continuous infusion protocol group (p < 0.01). Compared with nonprotocolized intermittent dosing, the use of a continuous infusion ATIII protocol demonstrated increased time within goal ACT range at a lower heparin dose, no increase in hemostatic complications, and trends toward fewer heparin changes and lower blood product usage.

Diversity in Gold Finger Structure Elucidated by Traveling-Wave Ion Mobility Mass Spectrometry.

Traveling wave ion mobility (TWIM) mass spectrometry (MS) is a powerful method for the structural and conformational analysis of proteins and peptides, enabling the differentiation of isomeric peptides (or proteins) that have the same sequence but are modified at different residues. In this study, the TWIM-MS technique was used to separate isomeric AuI metallopeptide ions that were formed by ZnII displacement from the parent zinc fingers (ZFs). The synthetic gold finger peptides were derived from the C-terminus of the HIV nucleocapsid p7 protein (NCp7-F2) and finger 3 of the Sp1 transcription factor (Sp1-F3). TWIM-MS enabled the acquisition of distinct product ion spectra for each isomer, clearly indicating the binding sites for the major conformers in the presence of multiple coordination possibilities. Collision cross-section measurements showed that the aurated peptide has a slightly more compact structure than the parent zinc compound NCp7-F2, which showed only one conformation.

Immune recognition of salivary proteins from the cattle tick Rhipicephalus microplus differs according to the genotype of the bovine host.

BACKGROUND: Males of the cattle tick Rhipicephalus microplus produce salivary immunoglobulin-binding proteins and allotypic variations in IgG are associated with tick loads in bovines. These findings indicate that antibody responses may be essential to control tick infestations. Infestation loads with cattle ticks are heritable: some breeds carry high loads of reproductively successful ticks, in others, few ticks feed and they reproduce inefficiently. Different patterns of humoral immunity against tick salivary proteins may explain these phenotypes. METHODS: We describe the profiles of humoral responses against tick salivary proteins elicited during repeated artificial infestations of bovines of a tick-resistant (Nelore) and a tick-susceptible (Holstein) breed. We measured serum levels of total IgG1, IgG2 and IgE immunoglobulins and of IgG1 and IgG2 antibodies specific for tick salivary proteins. With liquid chromatography followed by mass spectrometry we identified tick salivary proteins that were differentially recognized by serum antibodies from tick-resistant and tick-susceptible bovines in immunoblots of tick salivary proteins separated by two-dimensional electrophoresis. RESULTS: Baseline levels of total IgG1 and IgG2 were significantly higher in tick-susceptible Holsteins compared with resistant Nelores. Significant increases in levels of total IgG1, but not of IgG2 accompanied successive infestations in both breeds. Resistant Nelores presented with significantly higher levels of salivary-specific antibodies before and at the first challenge with tick larvae; however, by the third challenge, tick-susceptible Holsteins presented with significantly higher levels of IgG1 and IgG2 tick salivary protein-specific antibodies. Importantly, sera from tick-resistant Nelores reacted with 39 tick salivary proteins in immunoblots of salivary proteins separated in two dimensions by electrophoresis versus only 21 spots reacting with sera from tick-susceptible Holsteins. CONCLUSIONS: Levels of tick saliva-specific antibodies were not directly correlated with infestation phenotypes. However, in spite of receiving apparently lower amounts of tick saliva, tick-resistant bovines recognized more tick salivary proteins. These reactive salivary proteins are putatively involved in several functions of parasitism and blood-feeding. Our results indicate that neutralization by host antibodies of tick salivary proteins involved in parasitism is essential to control tick infestations.

Variation in Spot and Stripe Patterns in Original and Regenerated Zebrafish Caudal Fins.

Tissue regeneration requires not only the replacement of lost cells and tissues, but also the recreation of morphologies and patterns. Skin pigment pattern is a relatively simple system that can allow researchers to uncover the underlying mechanisms of pattern formation. To gain insight into how pigment patterns form, undergraduate students in the senior level course Developmental Biology designed an experiment that assayed pigment patterns in original and regenerated caudal fins of wild-type, striped, and mutant, spotted zebrafish. A majority of the WT fins regenerated with a similar striped pattern. In contrast, the pattern of spots even in the original fins of the mutants varied among individual fish. Similarly, the majority of the spots in the mutants did not regenerate with the same morphology, size, or spacing as the original fins. This was true even when only a small amount of fin was removed, leaving most of the fin to potentially reseed the pattern in the regenerating tissue. This suggests that the mechanism that creates the wild-type, striped pattern persists to recreate the pattern during regeneration. The mechanism that creates the spots in the mutants, however, must include an unknown element that introduces variability.

Effects of malted and non-malted whole-grain wheat on metabolic and inflammatory biomarkers in overweight/obese adults: a randomised crossover pilot study.

Metabolic dysfunction in obesity may be attenuated by whole-grain intake, which has been attributed to synergistic actions of bioactive compounds. Germination/malting may increase grain bioactives, including polyphenols, however biological effects compared with non-germinated grains are unclear. Polyphenols and biological effects were compared between malted-wheat (MLT) and whole-grain wheat (CON) breakfast cereals. Polyphenol content and antioxidant activity were significantly higher (P<0.01 and P<0.05, respectively) in MLT. Corresponding obesity-related biomarkers were measured in 10 overweight/obese adults in a 2×4-week double-blind, randomised, crossover trial. Following both interventions, diastolic blood pressure reduced significantly with time (P<0.05) and low-density lipoprotein increased slightly (P<0.05) over time. A significant time*cereal effect (P<0.05) was found for insulin resistance, decreasing following CON and increasing with MLT. No other significant metabolic or inflammatory differences were found. Whilst MLT contained significantly increased polyphenols, cumulative effects in attenuating obesity-related metabolic dysfunction may require increased consumption.

Germinated grains: a superior whole grain functional food?

Grains are global dietary staples that when consumed in whole grain form, offer considerable health benefits compared with milled grain foods, including reduced body weight gain and reduced cardiovascular and diabetes risks. Dietary patterns, functional foods, and other lifestyle factors play a fundamental role in the development and management of epidemic lifestyle diseases that share risks of developing adverse metabolic outcomes, including hyperglycaemia, hypertension, dyslipidaemia, oxidative stress, and inflammation. Whole grains provide energy, nutrients, fibres, and bioactive compounds that may synergistically contribute to their protective effects. Despite their benefits, the intake of grains appears to be lower than recommended in many countries. Of emerging interest is the application of germination processes, which may significantly enhance the nutritional and bioactive content of grains, as well as improve palatability. Enhancing grain foods in a natural way using germination techniques may therefore offer a practical, natural, dietary intervention to increase the health benefits and acceptability of whole grains, with potentially widespread effects across populations in attenuating adverse lifestyle disease outcomes. Continuing to build on the growing body of in-vitro studies requires substantiation with extended in-vivo trials so that we may further develop our understanding of the potential of germinated grains as a functional food.

Mechanism of endogenous regulation of the type I interferon response by suppressor of IκB kinase epsilon (SIKE), a novel substrate of TANK-binding kinase 1 (TBK1).

TANK-binding kinase 1 (TBK1) serves as a key convergence point in multiple innate immune signaling pathways. In response to receptor-mediated pathogen detection, TBK1 phosphorylation promotes production of pro-inflammatory cytokines and type I interferons. Increasingly, TBK1 dysregulation has been linked to autoimmune disorders and cancers, heightening the need to understand the regulatory controls of TBK1 activity. Here, we describe the mechanism by which suppressor of IKKε (SIKE) inhibits TBK1-mediated phosphorylation of interferon regulatory factor 3 (IRF3), which is essential to type I interferon production. Kinetic analyses showed that SIKE not only inhibits IRF3 phosphorylation but is also a high affinity TBK1 substrate. With respect to IRF3 phosphorylation, SIKE functioned as a mixed-type inhibitor (K(i, app) = 350 nM) rather than, given its status as a TBK1 substrate, as a competitive inhibitor. TBK1 phosphorylation of IRF3 and SIKE displayed negative cooperativity. Both substrates shared a similar Km value at low substrate concentrations (∼50 nM) but deviated >8-fold at higher substrate concentrations (IRF3 = 3.5 µM; SIKE = 0.4 µM). TBK1-SIKE interactions were modulated by SIKE phosphorylation, clustered in the C-terminal portion of SIKE (Ser-133, -185, -187, -188, -190, and -198). These sites exhibited striking homology to the phosphorylation motif of IRF3. Mutagenic probing revealed that phosphorylation of Ser-185 controlled TBK1-SIKE interactions. Taken together, our studies demonstrate for the first time that SIKE functions as a TBK1 substrate and inhibits TBK1-mediated IRF3 phosphorylation by forming a high affinity TBK1-SIKE complex. These findings provide key insights into the endogenous control of a critical catalytic hub that is achieved not by direct repression of activity but by redirection of catalysis through substrate affinity.

Point-of-use water disinfection using UV light-emitting diodes to reduce bacterial contamination.

The treatment process described in this research explores the impact of exposing water samples containing fecal coliforms to the radiation produced by single ultraviolet (UV) light-emitting diodes (LEDs) operating at 265 nm. UV LEDs are long lasting, compact in size and produce more efficient light output than traditional mercury-vapour bulbs, making them ideal for application in point-of-use disinfection systems, such as in remote areas. In this study, contaminated water samples containing either a pure culture of Escherichia coli or tertiary effluent from the City of Regina Wastewater Treatment Plant were used to study the application and efficiency of using UV LEDs for water disinfection. The results indicate that bacterial inactivation was achieved in a time-dependent manner, with 1- and 2.5-log E. coli reductions in water following 20 and 50 min of UV LED exposure, respectively. Ultraviolet radiation was less effective in reducing coliform bacteria in wastewater samples due to the elevated turbidity levels. Further work remains to be completed to optimize the application of UV LEDs for point-of-use disinfection systems; however, the results from this study support that bacterial inactivation using UV LEDs is possible, meriting further future technological development of the LEDs.

Development of an analytical microbial consortia method for enhancing performance monitoring at aerobic wastewater treatment plants.

An analytical method to produce profiles of bacterial biomass fatty acid methyl esters (FAME) was developed employing rapid agitation followed by static incubation (RASI) using selective media of wastewater microbial communities. The results were compiled to produce a unique library for comparison and performance analysis at a Wastewater Treatment Plant (WWTP). A total of 146 samples from the aerated WWTP, comprising 73 samples of each secondary and tertiary effluent, were included analyzed. For comparison purposes, all samples were evaluated via a similarity index (SI) with secondary effluents producing an SI of 0.88 with 2.7% variation and tertiary samples producing an SI 0.86 with 5.0% variation. The results also highlighted significant differences between the fatty acid profiles of the tertiary and secondary effluents indicating considerable shifts in the bacterial community profile between these treatment phases. The WWTP performance results using this method were highly replicable and reproducible indicating that the protocol has potential as a performance-monitoring tool for aerated WWTPs. The results quickly and accurately reflect shifts in dominant bacterial communities that result when processes operations and performance change.

Quantitative bacteriological assessment of aerobic wastewater treatment quality and plant performance.

The application of a novel method for measuring changes in defined bacterial populations during aerobic wastewater treatment was investigated. Changes in bacterial communities and total active cells can be used as surrogates for identifying potential WWTP treatment train efficiency and operational performance malfunctions. In this study, the quantities of active heterotrophic aerobic bacteria (HAB) in weekly wastewater samples collected from twelve locations across a WWTP were determined colorimetrically using biological activity reaction tests (BART). Samples were collected for 2 months from primary, secondary and tertiary unit processes. The results show a mean HAB population decrease of 99.8% from primary influent to tertiary effluent, with the largest reductions occurring in the secondary aerobic lagoons. The results were reproducible and robust supporting the applied BART analytical method as an indicator not only of overall efficacy of the WWTP processes but also of potential WWTP process malfunctions.

Proteomic analysis of Anaplasma phagocytophilum during infection of human myeloid cells identifies a protein that is pronouncedly upregulated on the infectious dense-cored cell.

Anaplasma phagocytophilum is an obligate intracellular bacterium that invades neutrophils to cause the emerging infectious disease human granulocytic anaplasmosis. A. phagocytophilum undergoes a biphasic developmental cycle, transitioning between an infectious dense-cored cell (DC) and a noninfectious reticulate cell (RC). To gain insights into the organism's biology and pathogenesis during human myeloid cell infection, we conducted proteomic analyses on A. phagocytophilum organisms purified from HL-60 cells. A total of 324 proteins were unambiguously identified, thereby verifying 23.7% of the predicted A. phagocytophilum proteome. Fifty-three identified proteins had been previously annotated as hypothetical or conserved hypothetical. The second most abundant gene product, after the well-studied major surface protein 2 (P44), was the hitherto hypothetical protein APH_1235. APH_1235 homologs are found in other Anaplasma and Ehrlichia species but not in other bacteria. The aph_1235 RNA level is increased 70-fold in the DC form relative to that in the RC form. Transcriptional upregulation of and our ability to detect APH_1235 correlate with RC to DC transition, DC exit from host cells, and subsequent DC binding and entry during the next round of infection. Immunoelectron microscopy pronouncedly detects APH_1235 on DC organisms, while detection on RC bacteria minimally, at best, exceeds background. This work represents an extensive study of the A. phagocytophilum proteome, discerns the complement of proteins that is generated during survival within human myeloid cells, and identifies APH_1235 as the first known protein that is pronouncedly upregulated on the infectious DC form.

Characterization of human fibroblast-derived extracellular matrix components for human pluripotent stem cell propagation.

Recent studies from our laboratory have shown that acellular substrates generated from human fibroblasts successfully maintained human pluripotent stem cells (hPSCs) in their undifferentiated state for extended periods. Aiming at better characterization, we conducted proteomic analyses to identify the extracellular matrix (ECM) proteins in mouse embryonic- and two human fibroblast-derived acellular substrates. Our studies identified heparan sulfate proteoglycan (HSPG) as a core component of these substrates and immunocytochemical analyses confirmed the presence of HSPG as well as other ECM proteins identified through proteomic analyses. In our attempt to develop surfaces that mimic fibroblast-deposited ECM and their self-renewal capabilities, substrates comprising HSPG and other core ECM proteins were formulated and assessed for the function of hPSC self-renewal. WA09 and BG01v hPSCs maintained on these substrates exhibit multiple characteristics of pluripotency, including (i) tight colony formation with typical stem cell morphology; (ii) positive expression of alkaline phosphatase, (iii) positive expression of SSEA3, SSEA4 and Oct4 based on immunocytochemical analyses; (iv) POU5F1, NANOG and SOX2 mRNA expression; and (v) in vitro differentiation and expression of germ-layer-specific markers. Our studies also reveal that although HSPG by itself-does not support hPSC self-renewal, a substrate that combines HSPG and fibronectin is sufficient for undifferentiated propagation of hPSCs. These studies form the basis for identification of appropriate ECM components in a substrate that synergistically promotes activation of adhesion and signaling pathways responsible for hPSC self-renewal.

A rapid methodology using fatty acid methyl esters to profile bacterial community structures in microbial fuel cells.

A new methodology is presented here as an effective, preliminary technique for the identification of indigenous aerobic and facultatively anaerobic bacterial communities found within microbial fuel cells (MFCs). The dual-phased method, named Rapid Agitation Static Incubation-Microbial Identification, or RASI-MIDI, is comprised of rapidly agitating the sample within a SLYM-BART tester followed by stationary incubation which produces a biomass that is subjected to extraction of methyl ester fatty acids. These distinctive fatty acid profiles represent a bacterial community fingerprint unique to the MFC, and are stored in a library for analysis. A total of 84 samples were analyzed for bacterial community structures from seven different groups of MFCs, with each MFC group comprised of a different bacterial community. Results showed that comparisons of replicate MFCs comprising the same bacterial communities generated high similarity index (SI) numbers (SI values ranging from 0.77 to 0.97), indicating highly correlated fatty acid profiles. In contrast, comparisons of MFCs having known dissimilar community structures did not consistently generate SI values in the analysis considered to be a significant match. It was found that this protocol described herein uniquely and accurately produced MFC fatty acid profiles contained in bacterial communities and thus provides a potential method for routinely studying MFC bacterial community fingerprints.

Isolation and identification of potential urinary microparticle biomarkers of bladder cancer.

Bladder cancer leads to approximately 13,000 deaths annually in the United States. Early disease is often treated with minimal morbidity and has good prognosis, while the opposite is true for advanced disease. Currently, no tools exist for early detection of this cancer. Microparticles are small, subcellular particles released by essentially all cells upon activation and are known to be produced constitutively by cancer cells. Since most bladder cancers originate in the urothelial cells lining the lumen of the organ, we hypothesize that these cells will release microparticles into the urine. The goal of this study was to identify potential biomarkers in the urinary microparticles of individuals with bladder cancer. Urine microparticles from five healthy individuals and four individuals with bladder cancer were isolated. Samples were delipidated by PAGE and trypsin-digested, peptides were extracted, and the proteome was examined by LC-MS/MS using a Thermo Finnigan LTQ and LTQ-FT ion trap mass spectrometer. Protein identification was determined by SEQUEST and relative quantitation was assessed by comparing spectral counts. Eight proteins were elevated in the microparticles from individuals with bladder cancer. They include five proteins associated with the epidermal growth factor receptor pathway, the alpha subunit of GsGTP binding protein, resistin, and retinoic acid-induced protein 3. Further studies will be needed to validate these potential biomarkers.