Fare Free Public Transportation Bus Systems: An Examination of Funding Strategies to Replace Passenger Fare Collection

American public transportation systems have been moving people for more than a century. To some, the transit bus may alleviate frustrations related to finding parking or having to pay expensive parking fees. To others, public transportation may be their lifeline for all travel needs including work, school, medical appointments, and even recreation. While these direct benefits assist those citizens that do use public transportation, public transportation can also bring other benefits indirectly to society in the form of decreased congestion and pollution. As more and more people begin using public transportation systems, they will soon find that public transportation systems are unique from community to community. Some provide routes, others provide curb to curb service, and others charge passengers fares while some systems are free to ride. This study used a mixed methods approach to evaluate funding information about America’s public transportation systems that do not charge fares to their passengers. Using an online survey, transit system interviews, data from the National Transit Database, and information obtained from website examinations, six categories emerged as to how public transportation systems can forgo charging fares to passengers and instead offer a public transportation system within their community that is free to all to ride. The findings obtained from this study can aid additional public transportation systems with deploying a model that allows for fare free public transportation within their communities and may also assist current fare free transit systems with generating additional operational revenue

The Hippocampal Neuroproteome with Aging and Cognitive Decline: Past Progress and Future Directions

Although steady progress on understanding brain aging has been made over recent decades through standard anatomical, immunohistochemical, and biochemical techniques, the biological basis of non-neurodegenerative cognitive decline with aging remains to be determined. This is due in part to technical limitations of traditional approaches, in which only a small fraction of neurobiologically relevant proteins, mRNAs or metabolites can be assessed at a time. With the development and refinement of proteomic technologies that enable simultaneous quantitative assessment of hundreds to thousands of proteins, neuroproteomic studies of brain aging and cognitive decline are becoming more widespread. This review focuses on the contributions of neuroproteomic investigations to advances in our understanding of age-related deficits of hippocampus-dependent spatial learning and memory. Accumulating neuroproteomic data demonstrate that hippocampal aging involves common themes of dysregulated metabolism, increased oxidative stress, altered protein processing, and decreased synaptic function. Additionally, growing evidence suggests that cognitive decline does not represent a “more aged” phenotype, but rather is associated with specific neuroproteomic changes that occur in addition to age-related alterations. Understanding if and how age-related changes in the hippocampal neuroproteome contribute to cognitive decline and elucidating the pathways and processes that lead to cognitive decline are critical objectives that remain to be achieved. Progress in the field and challenges that remain to be addressed with regard to animal models, behavioral testing, and proteomic reporting are also discussed

Multiplex quantitative PCR for single-reaction genetically modified (GM) plant detection and identification of false-positive GM plants linked to Cauliflower mosaic virus (CaMV) infection.

BACKGROUND:Most genetically modified (GM) plants contain a promoter, P35S, from the plant virus, Cauliflower mosaic virus (CaMV), and many have a terminator, TNOS, derived from the bacterium, Agrobacterium tumefaciens. Assays designed to detect GM plants often target the P35S and/or TNOS DNA sequences. However, because the P35S promoter is derived from CaMV, these detection assays can yield false-positives from non-GM plants infected by this naturally-occurring virus. RESULTS:Here we report the development of an assay designed to distinguish CaMV-infected plants from GM plants in a single multiplexed quantitative PCR (qPCR) reaction. Following initial testing and optimization via PCR and singleplex-to-multiplex qPCR on both plasmid and plant DNA, TaqMan qPCR probes with different fluorescence wavelengths were designed to target actin (a positive-control plant gene), P35S, P3 (a CaMV-specific gene), and TNOS. We tested the specificity of our quadruplex qPCR assay using different DNA extracts from organic watercress and both organic and GM canola, all with and without CaMV infection, and by using commercial and industrial samples. The limit of detection (LOD) of each target was determined to be 1% for actin, 0.001% for P35S, and 0.01% for both P3 and TNOS. CONCLUSIONS:This assay was able to distinguish CaMV-infected plants from GM plants in a single multiplexed qPCR reaction for all samples tested in this study, suggesting that this protocol is broadly applicable and readily transferrable to any interested parties with a qPCR platform

A Java Program for LRE-Based Real-Time qPCR that Enables Large-Scale Absolute Quantification

Background: Linear regression of efficiency (LRE) introduced a new paradigm for real-time qPCR that enables large-scale absolute quantification by eliminating the need for standard curves. Developed through the application of sigmoidal mathematics to SYBR Green I-based assays, target quantity is derived directly from fluorescence readings within the central region of an amplification profile. However, a major challenge of implementing LRE quantification is the labor intensive nature of the analysis. Findings: Utilizing the extensive resources that are available for developing Java-based software, the LRE Analyzer was written using the NetBeans IDE, and is built on top of the modular architecture and windowing system provided by the NetBeans Platform. This fully featured desktop application determines the number of target molecules within a sample with little or no intervention by the user, in addition to providing extensive database capabilities. MS Excel is used to import data, allowing LRE quantification to be conducted with any real-time PCR instrument that provides access to the raw fluorescence readings. An extensive help set also provides an in-depth introduction to LRE, in addition to guidelines on how to implement LRE quantification. Conclusions: The LRE Analyzer provides the automated analysis and data storage capabilities required by large-scale qPCR projects wanting to exploit the many advantages of absolute quantification. Foremost is the universal perspective afforded by absolute quantification, which among other attributes, provides the ability to directly compare quantitative data produced by different assays and/or instruments. Furthermore, absolute quantification has important implications for gene expression profiling in that it provides the foundation for comparing transcript quantities produced by any gene with any other gene, within and between samples

Chronic insulin treatment of diabetes does not fully normalize alterations in the retinal transcriptome

<p>Abstract</p> <p>Background</p> <p>Diabetic retinopathy (DR) is a leading cause of blindness in working age adults. Approximately 95% of patients with Type 1 diabetes develop some degree of retinopathy within 25 years of diagnosis despite normalization of blood glucose by insulin therapy. The goal of this study was to identify molecular changes in the rodent retina induced by diabetes that are not normalized by insulin replacement and restoration of euglycemia.</p> <p>Methods</p> <p>The retina transcriptome (22,523 genes and transcript variants) was examined after three months of streptozotocin-induced diabetes in male Sprague Dawley rats with and without insulin replacement for the later one and a half months of diabetes. Selected gene expression changes were confirmed by qPCR, and also examined in independent control and diabetic rats at a one month time-point.</p> <p>Results</p> <p>Transcriptomic alterations in response to diabetes (1376 probes) were clustered according to insulin responsiveness. More than half (57%) of diabetes-induced mRNA changes (789 probes) observed at three months were fully normalized to control levels with insulin therapy, while 37% of probes (514) were only partially normalized. A small set of genes (5%, 65 probes) was significantly dysregulated in the insulin-treated diabetic rats. qPCR confirmation of findings and examination of a one month time point allowed genes to be further categorized as prevented or rescued with insulin therapy. A subset of genes (Ccr5, Jak3, Litaf) was confirmed at the level of protein expression, with protein levels recapitulating changes in mRNA expression.</p> <p>Conclusions</p> <p>These results provide the first genome-wide examination of the effects of insulin therapy on retinal gene expression changes with diabetes. While insulin clearly normalizes the majority of genes dysregulated in response to diabetes, a number of genes related to inflammatory processes, microvascular integrity, and neuronal function are still altered in expression in euglycemic diabetic rats. Gene expression changes not rescued or prevented by insulin treatment may be critical to the pathogenesis of diabetic retinopathy, as it occurs in diabetic patients receiving insulin replacement, and are prototypical of metabolic memory.</p

SNPs and real-time quantitative PCR method for constitutional allelic copy number determination, the VPREB1 marker case

<p>Abstract</p> <p>Background</p> <p>22q11.2 microdeletion is responsible for the DiGeorge Syndrome, characterized by heart defects, psychiatric disorders, endocrine and immune alterations and a 1 in 4000 live birth prevalence. Real-time quantitative PCR (qPCR) approaches for allelic copy number determination have recently been investigated in 22q11.2 microdeletions detection. The qPCR method was performed for 22q11.2 microdeletions detection as a first-level screening approach in a genetically unknown series of patients with congenital heart defects. A technical issue related to the <it>VPREB1 </it>qPCR marker was pointed out.</p> <p>Methods</p> <p>A set of 100 unrelated Italian patients with congenital heart defects were tested for 22q11.2 microdeletions by a qPCR method using six different markers. Fluorescence In Situ Hybridization technique (FISH) was used for confirmation.</p> <p>Results</p> <p>qPCR identified six patients harbouring the 22q11.2 microdeletion, confirmed by FISH. The <it>VPREB1 </it>gene marker presented with a pattern consistent with hemideletion in one 3 Mb deleted patient, suggestive for a long distal deletion, and in additional five non-deleted patients. The long distal 22q11.2 deletion was not confirmed by Comparative Genomic Hybridization. Indeed, the <it>VPREB1 </it>gene marker generated false positive results in association with the rs1320 G/A SNP, a polymorphism localized within the <it>VPREB1 </it>marker reverse primer sequence. Patients heterozygous for rs1320 SNP, showed a qPCR profile consistent with the presence of a hemideletion.</p> <p>Conclusions</p> <p>Though the qPCR technique showed advantages as a screening approach in terms of cost and time, the <it>VPREB1 </it>marker case revealed that single nucleotide polymorphisms can interfere with qPCR data generating erroneous allelic copy number interpretations.</p