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Identification of Commercially-Available Antibodies that Block Ligand-Binding by BMPR2
No full textOsteoporosis, a disease of low bone mineral density, affects 10 million Americans and is a significant health problem and a considerable socioeconomic burden. Current treatments for osteoporosis have significant limitations, necessitating identifying new treatment strategies via building a better understanding of the endogenous mechanisms regulating bone mass. We recently demonstrated that removal of the BMP type 2 receptor, BMPR2, in skeletal progenitor cells of Bmpr2-cKO mice leads to reduced age-related bone loss due to a sustained elevation in bone formation rate. The molecular mechanism underlying this phenotype is being pursued in other work. In the present study, we sought to advance the translational potential of our genetic model by identifying antibodies that neutralize the ligand-binding function of the BMPR2 extracellular domain (BMPR2- ECD). We first established a modified, cell-free immunoprecipitation assay wherein the ligand BMP2 is pulled-down by BMPR2-ECD conjugated to Protein G beads; the unbound BMP2, which is found in the supernatant, is subsequently quantified by ELISA. This yielded a standard assay wherein approximately 2 ug BMPR2-ECD leads to 70% reduction in BMP2 signal. Next, we examined the neutralizing ability of 3F6, which is a mouse monoclonal antibody raised against the ligand-binding region of BMPR2, and found a dose-dependent inhibition of BMPR2-ECD ligand-binding. Given that the commercial availability of this antibody is as an ascites preparation, specificity of this assay was confirmed by demonstrating that ligand-binding activity of BMPR2-ECD is unchanged in the presence of non-specific, negative control ascites. Using these results as a guide, we then evaluated 1F12, which is another mouse monoclonal antibody raised against the ligand-binding region of BMPR2, and found that this antibody is also capable of neutralizing the ligand-binding function of BMPR2-ECD. These results provide proof-of-concept data for future studies evaluating inhibition of BMPR2 function in vivo as a means to reduce age-related bone loss
Boric Acid and Persisters in Candida Albicans
No full textPersisters are a special group of quiescent and phenotypically unique cells in a given microbial population that survive antimicrobial insults or stresses but retain properties of the original population. Persisters have been characterized and studied in bacteria; however, little work exists on evaluating the persister phenotype in yeast. Previous research with 12 clinical isolates of Candida albicans elucidated antifungal properties of boric acid (BA) as a potential treatment alternative for vaginal candidiasis. Objective: This project aimed to identify and quantify C. albicans persister colonies that may appear when exposed to BA, antifungals and BA-single antifungal combinations. Methods: C. albicans was grown in RPMI 1640 media, experimentally modified to 1% BA, 5μg/mL miconazole, 2μg/mL amphotericin B or 1μg/mL caspofungin. The combination conditions were at the same concentrations. Growth was quantified with flow-cytometry and CFUs counted on agar. Persisters were measured as a ratio of CFUs to flow events per μL. Results: Miconazole and amphotericin B both decreased percent persisters at 24h (0.92% and 1.88%) and 48h (3.74% and 2.37%) when compared to BA alone at 24h and 48h (33.34% and 119.11%) while caspofungin increased persisters at 24h and decreased at 48h (138.00% and 65.82%). Combination conditions decreased percent persisters when compared to BA and respective single antifungals alone at both 24h (BAMiconazole, 1.20%, BA-Amphotericin B, 0.46%, BA-Caspofungin, 17.11%) and 48h (1.60%, 0.43%, 13.30%). Conclusion: BA appears to have a synergistic effect on growth inhibition and persister colony formation. Further work is needed to elucidate if these colonies have a unique persister phenotype or are only survivors from antifungal insult. This work shows that BA may be a potential therapeutic to inhibit persister formation, a target separate from antifungal resistance
Assessing Readability of Online Patient Education Materials for Spine Surgery Procedures
Get PDFIncreased patient reliance on Internet-based health information has amplified the need for comprehensible online patient education articles. As suggested by the AMA and NIH, spine fusion articles should be between a 4th and 6th grade readability level to increase patient comprehension, which may contribute to improved postoperative outcomes. Objective: To determine the average readability level of online healthcare education information relating to anterior cervical discectomy and fusion (ACDF) and lumbar fusion procedures. Design: Online Health-Education Resource Qualitative Analysis. Setting: Rush University Medical Center - Department of Orthopaedic Surgery. Methods: Three popular search engines were utilized to access patient education articles for common cervical and lumbar spine procedures. Relevant articles were analyzed for readability using Readability Studio Professional Edition software (Oleander Software, Ltd). Articles were stratified by organization type as follows: General Medical Websites (GMW), Healthcare Network/Academic Institutions (HNAI), and Private Practices (PP). Thirteen common readability tests were performed with the mean grade level for each readability test compared between subgroups using ANOVA analysis. Results: 79 ACDF and 231 lumbar fusion articles were determined to have a mean readability level of 10.7 ± 1.5 and 11.3 ± 1.6, respectively. GMW, HNAI, and PP subgroups had mean readability levels of 10.9 ± 2.9, 10.7 ± 2.8, and 10.7 ± 2.5 for ACDF and 10.9 ± 3.0, 10.8 ± 2.9, and 11.6 ± 2.7 for lumbar fusion articles. Of 310 total articles, only 6 (3 ACDF and 3 lumbar fusion) were written below the 7th grade reading level. Conclusions: Current online literature from medical websites containing information regarding ACDF and lumbar fusion procedures are written at a grade level higher than the suggested guidelines. Therefore, current patient education articles should be revised to accommodate the average readability level in the United States and may result in improved patient comprehension and postoperative outcomes
Identify cmy-2 Incompatibility Groups in Salmonella Multidrug Resistance Plasmids
Get PDFMany Gram negative bacteria including Salmonella enterica species exhibit evolving multi-drug resistance coded for on their chromosome and on mobile genetic elements. Salmonella species carrying multi-drug resistance plasmids can potentially disperse the cmy-2 gene conferring β- lactam resistance to new Salmonella species as well as to other enteric bacteria commonly found in the human and animal gut. Objective: This study seeks to identify the numbers and incompatibility types of plasmids carried by six different Salmonella clinical isolates. Design: Raw whole genome and DNA fragment (contig) sequence secondary data analyses were conducted using the Center for Genomic Epidemiology (Lyngby, Denmark) open source internet databases plasmid Multilocus Sequence Typing (pMLST) and Resistance Finder (ResFinder). Method: Nucleic acid sequences were separated into files, labeled by contig, and uploaded to the consensus databases for analysis against known Salmonella resistance genes and plasmid types. Results were returned in table format and identified specific resistance, susceptibility, and incompatibility groups present with greater than 90% homology between known and analyzed DNA sequences. Results: Results of this analysis show that these bacterial strains carry plasmids which confer multi-drug resistance and that multiple incompatibility groups are present across some but not all analyzed Salmonella strains. Conclusions: The researchers continue to analyze the data and have not yet formed conclusions as to their significance. This study will advance our understanding about how bacteria develop multiple antibiotic resistances and could ultimately help design more effective drugs with greater potential to combat and prevent bacterial diseases
Sr. Rachel Lindenmaier
Get PDFIn her oral history Sr. Rachel Lindenmaier reflects on her upbringing in Indianapolis and the appeal of the Franciscans who helped in her deciding to enter religious life. Also, she describes joining religious service alongside her sister, Sister Evelyn Lindenmaier, and their close relationship over the years. Additionally, Sr. Rachel recounts her joy in spending thirty-five years in education, teaching mainly in inner-city schools both in Cincinnati and Indianapolis. Lastly, she discusses the evolution of her prayer life, heavily influenced during wartime years, as well as the concept of prayer as an embodiment of love.https://mushare.marian.edu/wrp/1009/thumbnail.jp
Mitigation of Stomach Dehiscence Risk During Gastrostomy Tube Changes – A Retrospective Analysis of Patient Outcomes
No full textStomach dehiscence from the abdominal wall is a serious and potentially life-threatening complication of gastrostomy tube changes. A prospective series of 1136 gastrostomy tube changes in 415 different patients is examined to identify high-risk patients and provide evidence to support common practice patterns. Each gastrostomy change was analyzed for type of tube changed, patient demographics, patient diagnoses, and complications resulting from changes. Complications were stratified as minor complication (bleeding, infection, pain, leaking site, loss of tube requiring dilation of tract to reinsert), moderate complications (dislodged tube requiring anesthesia to replace and major complications (intra-peritoneal insertion, intra-peritoneal leak). Percutaneous Endoscopic Gastrostomy (PEG) Tube conversion to any type of tube was the only type of tube change to have a significantly (p=0.0036) higher risk of dehiscence (6.7%) vs. non-PEG change (0.39%). Children with Failure to thrive and neurologic impairment had significantly higher overall complication rates (p\u3c0.005) but not higher gastric dehiscence. Aspiration of gastric contents through the newly placed tube as a confirmatory test for intra-gastric placement was found to have a positive predictive value of 99.3% with a sensitivity of 99.7% and specificity of 33%. We conclude that non-PEG tube changes can be performed safely at the bedside and that positive gastric aspirate is a positive predictor of adequate tube placement with high sensitivity. PEG tube changes have a significant risk for dehiscence. This evidence supports endoscopic supervision during PEG tube changes. Utilit
Single-Center Denial Reasons for Potential Living Kidney Donors
Get PDFBecause of the benefits of preemptive living donor (LD) transplant, the desire for LD is rising. However, in the last decade, there has been no increase in LD in the U.S, possibility due to older donor candidate population leading to increased denial rates. We previously studied denial rates and cause for denial for donor candidates between 2009 and 2011. We herein present for comparison causes for denial for donor candidates between 8/2012 and 6/2015. During the interval, our acceptance criteria have not changed. Results: Between August 2012 and June 2015, we evaluated 644 potential living donors: 2012=88, 2013=222, 2014=220, 2015 (Jan-June) =114. Of these, 31% of candidates were denied: 26% for medical reasons, 5% for psychosocial reasons. Mean age of approved candidates = 41 (range: 18-75); average denied = 43 (range, 18-72). To compare, between 2009-2011, 36% candidates were denied (32% medical, 4% psychosocial). The mean age for approved candidates = 40, while denied candidates mean age = 47 (range, 18-64). Current candidates most common medical denials reasons were obesity and suboptimal kidney anatomy, although the distribution of reasons varies by age cohort. Historical group (2009-2011): most common medical denials were hypertension and poor kidney function. Psychosocial denial reasons included: mental illness, lack of support and substance abuse. Conclusion: Over an interval of 6.5 years, the rate of donor candidate denial at our center has not changed. Overall, there has been a change in major reasons for denial; importantly, reasons for denial vary by donor age
Therapeutic Discovery for Friedreich Ataxia Using Random shRNA Selection
Get PDFWe screened a 300,000-clone, random shRNA-expressing library and identified shRNA sequences that reverse the decreased growth/survival phenotype of primary Friedreich ataxia (FA) fibroblasts grown in mitochondrial stress media. One of the hit sequences, gFA2, increases frataxin expression ~2 fold, either as a vector-expressed shRNA or as a transfected siRNA. We randomly mutagenized gFA2 to create a gFA2 variant sub-library. We screened this sub-library in primary FA fibroblasts and identified two gFA2 variants, gFA2.8 and gFA2.10, that further increase frataxin expression. Microarray analyses of primary FA fibroblasts expressing another hit shRNA, gFA11, revealed alterations in ~350 mRNAs. Bioinformatic pathway analyses indicated significant changes in mRNAs involved in cytokine secretion; we confirmed significant changes in cytokine secretion induced by gFA11 biochemically. Ingenuity Pathway Analysis revealed that inhibition of a known transcription factor, or treatment of cells with a previously studied chemical compound, induced a statistically similar pattern of gene expression to that induced by gFA11. Inhibition of the transcription factor using a directed siRNA in primary FA fibroblasts, as well as treatment of the cells with the chemical compound, recapitulated the phenotype induced by gFA11, namely reversal of decreased growth/survival in mitochondrial stress media. We are currently planning similar microarray and bioinformatics analyses of the optimized versions of gFA2. Combined with microarray analyses and bioinformatic pattern-matching, our random, shRNA library screens potentially yield, 1) small-RNA therapeutic candidates, 2) conventional chemical-compound therapeutic candidates, 3) drug-target candidates, and 4) elucidation of disease mechanisms, which may inform additional therapeutic initiatives
Comparison of Patient Tolerance of Photodynamic Therapy with Zone vs. Full Face Treatment
Get PDFBackground: Zone based photodynamic therapy (PDT) is a promising treatment option for actinic keratosis, superficial basal cell carcinoma, and in-situ squamous cell carcinoma. PDT has similar cure rates to 5-fluorouracil and imiquimod. Objective: Determine if PDT zone treatments improve patient comfort, tolerance, retention, and offer less severe reactions as compared to full face treatments. Methods: 500 PDT patients were divided into full face (n = 250) versus zone treatments (n = 250). All subjects completed a post-treatment phone survey assessing tolerance of procedure. Retention rates and severe reactions were tracked. Results: Zone treatment resulted in a higher number of excellent responses for patient tolerance vs. full face treatment group (85 % vs. 39%,