Determination of the Course of Cyan Fluorescence of Pseudomonas aeruginosa with a Handheld Bacterial Imaging Device.

Chronic wound infections are of clinical concern as they often lead to high rates of mortality and morbidity. A point-of-care handheld bacterial fluorescence imaging has been designed to detect the auto-fluorescent characteristics of most clinically relevant species of bacteria. This device causes most species of bacteria to exhibit red fluorescence due to the production of exoproduct porphyrins. One of the most significant contributors to the pathogenicity of chronic wounds is the pathogen Pseudomonas aeruginosa, and interestingly, this organism exhibits an additional unique cyan fluorescence signature. There is an over 90% positive predictive value that, when a chronic wound exhibits cyan fluorescence with the bacterial fluorescence imaging device, the wound will harbor P. aeruginosa. This project seeks to understand what genetic factor(s) contribute to the cyan phenotype observed.

Modular Multiwell Viscoelastic Hydrogel Platform for Two- and Three-Dimensional Cell Culture Applications.

Hydrogels have gained significant popularity as model platforms to study reciprocal interactions between cells and their microenvironment. While hydrogel tools to probe many characteristics of the extracellular space have been developed, fabrication approaches remain challenging and time-consuming, limiting multiplexing or widespread adoption. Thus, we have developed a modular fabrication approach to generate distinct hydrogel microenvironments within the same 96-well plate for increased throughput of fabrication as well as integration with existing high-throughput assay technologies. This approach enables in situ hydrogel mechanical characterization and is used to generate both elastic and viscoelastic hydrogels across a range of stiffnesses. Additionally, this fabrication method enabled a 3-fold reduction in polymer and up to an 8-fold reduction in fabrication time required per hydrogel replicate. The feasibility of this platform for two-dimensional (2D) cell culture applications was demonstrated by measuring both population-level and single-cell-level metrics via microplate reader and high-content imaging. Finally, a 96-well hydrogel array was utilized for three-dimensional (3D) cell culture, demonstrating the ability to support high cell viability. Together, this work demonstrates a versatile and easily adaptable fabrication approach that can support the ever-expanding tool kit of hydrogel technologies for cell culture applications.

Biodentine as a pulpotomy medicament for primary molars: a retrospective chart review.

This retrospective chart review study investigates the long-term clinical outcome of Biodentine® (Tricalcium silicate) as a medicament for pulpotomy in primary molars. Data in this retrospective study was collected from the dental records of all patients that had at least one primary molar receive pulpotomy treatment (CDT code: D3221) between 01 July 2012 and 01 July 2015. This data includes child's age, medical history, dental history, dental radiographs, pulpotomy procedure details and follow-up clinical notes. Kaplan-Meier Estimate was used to measure the fraction of successful pulpotomy procedures for up to 24 months. A total of 1758 pulpotomy procedures were performed on 1032 patients in our institute in the three-year period and 21.4% of them (N = 376) had follow-up dental records that qualified for the study. Eleven teeth out of 376 teeth were excluded from the statistical analysis due to loss of/broken stainless steel crowns (3.1%). Seventeen pulpotomy failures were identified out of the remaining 365 procedures. The survival probablity of using Biodentine® as a pulpotomy medicament is 96.3% for 18-month follow-up and 95.4% for 24-month follow-up. Biodentine®, a tricalcium silicate formulation, used as a pulpotomy medicament demonstrates a high clinical success rate (95.4%) over a 24-month peroid in primary molars.

Modular multiwell viscoelastic hydrogel platform for two- and three-dimensional cell culture applications.

Hydrogels have gained significant popularity as model platforms to study the reciprocal interactions between cells and their microenvironment. While hydrogel tools to probe many characteristics of the extracellular space have been developed, fabrication approaches remain challenging and time-consuming, limiting multiplexing or widespread adoption. Thus, we have developed a modular fabrication approach to generate distinct hydrogel microenvironments within 96-well plates for increased throughput of fabrication as well as integration with existing high-throughput assay technologies. This approach enables in situ hydrogel mechanical characterization and was used to generate both elastic and viscoelastic hydrogels across a range of stiffnesses. Additionally, this fabrication method enabled a 3-fold reduction in polymer and up to an 8-fold reduction in fabrication time required per hydrogel replicate. The feasibility of this platform for cell culture applications was demonstrated by measuring both population-level and single cell-level metrics via microplate reader and high-content imaging. Finally, the 96-well hydrogel array was utilized for 3D cell culture, demonstrating the ability to support high cell viability. Together, this work demonstrates a versatile and easily adoptable fabrication approach that can support the ever-expanding tool kit of hydrogel technologies for cell culture applications.

A Dual Enzyme-Based Biochemical Test Rapidly Detects Third-Generation Cephalosporin-Resistant CTX-M-Producing Uropathogens in Clinical Urine Samples.

Extended-spectrum ß-lactamase (ESBL)-producing Gram-negative bacteria (GNB) are increasingly identified as the cause of both community and healthcare-associated urinary tract infections (UTIs), with CTX-Ms being the most common ESBLs identified. CTX-M-producing GNB are resistant to most ß-lactam antibiotics and are frequently multidrug-resistant, which limits treatment options. Rapid diagnostic tests that can detect ESBL-producing GNB, particularly CTX-M producers, in the urine of patients with UTIs are needed. Results from such a test could direct the selection of appropriate antimicrobial therapy at the point-of-care (POC). In this study, we show that a chromogenic, dual enzyme-mediated amplification system (termed DETECT [dual-enzyme trigger-enabled cascade technology]) can identify CTX-M-producing GNB from unprocessed urine samples in 30 minutes. We first tested DETECT against a diverse set of recombinant ß-lactamases and ß-lactamase-producing clinical isolates to elucidate its selectivity. We then tested DETECT with 472 prospectively collected clinical urine samples submitted for urine culture to a hospital clinical microbiology laboratory. Of these, 118 (25%) were consistent with UTI, 13 (11%) of which contained ESBL-producing GNB. We compared DETECT results in urine against a standard phenotypic method to detect ESBLs, and polymerase chain reaction and sequencing for CTX-M genes. DETECT demonstrated 90.9% sensitivity and 97.6% specificity (AUC, 0.937; 95% confidence interval, 0.822-1.000), correctly identifying 10 of 11 urine samples containing a clinically significant concentration of CTX-M-producing GNB (including Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis). Our results demonstrate the clinical potential of DETECT to deliver diagnostic information at the POC, which could improve initial antibiotic selection.

TOM40 mediates mitochondrial dysfunction induced by α-synuclein accumulation in Parkinson's disease.

Alpha-synuclein (α-Syn) accumulation/aggregation and mitochondrial dysfunction play prominent roles in the pathology of Parkinson's disease. We have previously shown that postmortem human dopaminergic neurons from PD brains accumulate high levels of mitochondrial DNA (mtDNA) deletions. We now addressed the question, whether alterations in a component of the mitochondrial import machinery--TOM40--might contribute to the mitochondrial dysfunction and damage in PD. For this purpose, we studied levels of TOM40, mtDNA deletions, oxidative damage, energy production, and complexes of the respiratory chain in brain homogenates as well as in single neurons, using laser-capture-microdissection in transgenic mice overexpressing human wildtype α-Syn. Additionally, we used lentivirus-mediated stereotactic delivery of a component of this import machinery into mouse brain as a novel therapeutic strategy. We report here that TOM40 is significantly reduced in the brain of PD patients and in α-Syn transgenic mice. TOM40 deficits were associated with increased mtDNA deletions and oxidative DNA damage, and with decreased energy production and altered levels of complex I proteins in α-Syn transgenic mice. Lentiviral-mediated overexpression of Tom40 in α-Syn-transgenic mice brains ameliorated energy deficits as well as oxidative burden. Our results suggest that alterations in the mitochondrial protein transport machinery might contribute to mitochondrial impairment in α-Synucleinopathies.