Sub-femtomole detection of 16s rRNA from Legionella pneumophila using surface plasmon resonance imaging.

Legionellosis has been and continues to be a life-threatening disease worldwide, even in developed countries. Given the severity and unpredictability of Legionellosis outbreaks, developing a rapid, highly specific, and sensitive detection method is thus of great pertinence. In this paper, we demonstrate that sub-femtomole levels of 16s rRNA from pathogenic Legionella pneumophila can be timely and effectively detected using an appropriate designed capture, detector probes, and a QD SPRi signal amplification strategy. To achieve specific and sensitive detection, optimal hybridization conditions and parameters were implemented. Among these parameters, fragmentation of the 16s rRNA and further signal amplification by QDs were found to be the main parameters contributing to signal enhancement. The appropriate design of the detector probes also increased the sensitivity of the detection system, mainly due to secondary structure of 16s rRNA. The use of 16s rRNA from L. pneumophila allowed for the detection of metabolically active pathogens with high sensitivity. Detection of 16s rRNA in solutions as diluted as 1 pM at 450 µL (0.45 femtomole) was achieved in less than 3h, making our approach suitable for the direct, timely, and effective detection of L. pneumophila within man-made water systems.

Novel probiotic dissolvable carboxymethyl cellulose films as oral health biotherapeutics: in vitro preparation and characterization.

OBJECTIVES: Oral health is influenced by the mouth's resident microorganisms. Dental caries and periodontitis are oral disorders caused by imbalances in the oral microbiota. Probiotics have potential for the prevention and treatment of oral disorders. Current formulations, including supplements and foods, have limitations for oral delivery including short storage time, low residence time in the mouth, effects on food consistency, and low patient compliance. Oral thin films (OTFs) may be efficient in delivering probiotics to the mouth. This research aims to develop a novel carboxymethyl cellulose (CMC)-probiotic-OTF to deliver probiotics for the treatment/prevention of oral disorders. METHODS: CMC-OTFs were developed with varying CMC concentration (1.25 - 10 mg/mL), weight (5 - 40 g), thickness (16 - 262 µm), hygroscopicity (30.8 - 78.9 mg/cm(2) film), and dissolving time (135 - 600 s). The 10 g 5 mg/mL CMC-OTF was selected and used to incorporate Lactobacillus fermentum NCIMB 5221 (6.75 × 10(8) cells/film), a probiotic with anti-inflammatory potential for periodontitis treatment and capable of inhibiting microorganisms responsible for dental caries and oral candidiasis. RESULTS: The CMC-OTF maintained probiotic viability and antioxidant activity following 150 days of storage with a production of 549.52 ± 26.08 µM Trolox equivalents. CONCLUSION: This research shows the successful development and characterization of a novel probiotic-CMC-OTF with potential as an oral health biotherapeutic.

Long-term in vitro human pancreatic islet culture using three-dimensional microfabricated scaffolds.

Human pancreatic islet in vitro culture is very challenging and requires the presence of various extra cellular matrix (ECM) components in a three-dimensional environment, which provides mechanical and biological support. The development of such an environment is vital in providing favourable conditions to preserve human islets in long-term culture. In this study, we investigated the effects of human islet culture within various three-dimensional environments; collagen I gel, collagen I gel supplemented with ECM components fibronectin and collagen IV, and microfabricated scaffold with ECM-supplemented gel. The cultured human islets were analyzed for functionality, gene expression and hormone content following long-term in vitro culture. It was clear the incorporation of ECM components within the three-dimensional support improved prolonged culture. However, long-term and highly uniform human islet culture within a microfabricated scaffold, with controlled pore structures, coupled with the presence of ECM components, displayed an insulin release profile similar to freshly isolated islets, yielding a stimulation index of approximately 1.8. Moreover, gene expression was markedly increased for all pancreatic genes, giving a approximately 50-fold elevation of insulin gene expression with respect to suspension culture. The distribution and presence of pancreatic hormones was also highly elevated. These findings provide a platform for the long-term maintenance and preservation of human pancreatic islets in vitro.