Blast induces oxidative stress, inflammation, neuronal loss and subsequent short-term memory impairment in rats.

Molecular and cellular mechanisms of brain injury after exposure to blast overpressure (BOP) are not clearly known. The present study hypothesizes that pro-oxidative and pro-inflammatory pathways in the brain may be responsible for neuronal loss and behavioral deficits following BOP exposure. Male Sprague-Dawley rats were anesthetized and exposed to calibrated BOP of 129.23±3.01kPa while controls received only anesthesia. In situ dihydroethidium fluorescence staining revealed that BOP significantly increased the production of reactive oxygen species in the brain. In addition, real-time reverse transcriptase-polymerase chain reaction, immunofluorescence staining and enzyme-linked immunosorbent assay demonstrated a significant up-regulation of mRNA and protein expressions of pro-inflammatory mediators, such as interferon-γ and monocyte chemoattractant protein-1, in brains collected from BOP-exposed animals compared with the controls. Furthermore, immunoreactivity of neuronal nuclei in brains indicated that fewer neurons were present following BOP exposure. Moreover, novel object recognition paradigm showed a significant impairment in the short-term memory at 2weeks following BOP exposure. These results suggest that pro-oxidative and pro-inflammatory environments in the brain could play a potential role in BOP-induced neuronal loss and behavioral deficits. It may provide a foundation for defining a molecular and cellular basis of the pathophysiology of blast-induced neurotrauma (BINT). It will also contribute to the development of new therapeutic approaches selectively targeting these pathways, which have great potential in the diagnosis and therapy of BINT.

A new model for mild blast injury utilizing Drosophila melanogaster - biomed 2013.

Current models for blast injury involve the use of mammalian species, which are costly and require extensive monitoring and housing, making it difficult to generate large numbers of injuries. The fruit fly, Drosophila melanogaster, has been utilized for many models of human disease including neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases. In this study, a model of blast injury was designed based on Drosophila, to provide a mechanism to investigate blast injury in large numbers and assess biochemical mechanisms of brain injury. Such studies may be used to identify specific pathways involved in blast-associated neurodegeneration, allowing more effective use of mammalian models. A custom-built blast wave simulator (ORA Inc.), comprised of a driver, test section, and wave eliminator, was used to create a blast wave. An acetate membrane was placed between the driver and the rectangular test section before compressed helium caused the membrane to rupture creating the blast wave. Membrane thickness correlates with the blast wave magnitude, which averaged 120 kPa for this experiment. Pressure sensors were inserted into the side of the tube in order to quantify the level of overpressure that the flies were exposed to. Five day old flies were held in a rectangular enclosed mesh fixture (10 flies per enclosure) which was placed in the center of the test section for blast delivery. Sham controls were exposed to same conditions with exception of blast. Lifespan and negative geotaxis, a measurement of motor function, was measured in flies after blast injury. Mild blast resulted in death of 28% of the flies. In surviving flies, motor function was initially reduced, but flies regained normal function by 8 days after injury. Although surviving flies regained normal motor function, flies subjected to mild blast died earlier than uninjured controls, with a 15.4% reduction in maximum lifespan and a 17% reduction in average lifespan, mimicking the scenario observed in humans exposed to mild blast. Although further work is needed, results suggest that utilizing Drosophila as a blast model may provide a rapid, effective means of assessing physiological and biochemical changes induced by mild blast.

A modular approach for the generation, storage, mixing, and detection of droplet libraries for high throughput screening.

The desire to make microfluidic technology more accessible to the biological research community has led to the notion of "modular microfluidics", where users can build a fluidic system using a toolkit of building blocks. This paper applies a modular approach for performing droplet-based screening, including the four integral steps of library generation, storage, mixing, and optical interrogation. Commercially available cross-junctions are used for drop generation, flexible capillary tubing for storage, and tee-junctions for serial mixing. Optical interrogation of the drops is achieved using fiber-optic detection modules which can be incorporated inline at one or more points in the system. Modularity enables the user to hand-assemble systems for functional assays or applications. Three examples are shown: (1) a "mix and read" assay commonly used in high throughput screening (HTS); (2) generation of chemically distinct droplets using microfractionation in droplets (microFD); and (3) in situ encapsulation and culture of eukaryotes. Using components with IDs ranging from 150 microm to 1.5 mm, this approach can accommodate drop assays with volumes ranging from 2 nL to 2 microL, and storage densities ranging from 300 to 3000 drops per metre tubing. Generation rates are up to 200 drops per second and merging rates are up to 10 drops per second. The impact of length scale, carrier fluid viscosity, and flow rates on system performance is considered theoretically and illustratively using 2D CFD simulations. Due to its flexibility, the widespread availability of components, and some favorable material properties compared to PDMS, this approach can be a useful part of a researcher's toolkit for prototyping droplet-based assays.

Autoimmunity to the M(r) 32,000 subunit of replication protein A in breast cancer.

PURPOSE: We sought to identify autoantigens recognized by antibodies in breast cancer patient sera with potential diagnostic or prognostic significance. EXPERIMENTAL DESIGN: Serum from a female breast cancer patient exhibiting a high titer antinuclear antibody was used to screen a HeLa cDNA expression library, leading to the cloning of a cDNA for the M(r) 32,000 subunit of replication protein A (RPA32). RPA32 expression and localization were assayed in autologous tumor by monoclonal antibody staining. A specific ELISA using recombinant protein was used to screen sera from 801 breast cancer patients and 65 controls. RESULTS: A relationship between anti-replication protein A (RPA) antibodies and the ductal breast carcinoma of the proband was suggested by overexpression and aberrant localization of RPA32 in tumor cells as compared with surrounding normal ductal tissue and by the presence of anti-RPA32 antibodies before the diagnosis. The prevalence of anti-RPA32 antibodies was significantly higher (P < 0.01) among breast cancer patients (87 of 801 patients) than among noncancer controls (0 of 65 controls). Similarly, anti-RPA32 antibodies were present in 4 of 39 patients with intraductal in situ carcinoma. No associations were found between anti-RPA antibodies and survival, occurrence of a second tumor, metastases, or antibodies to p53. Reactivity to RPA32 also was detected in sera from 3 of 47 patients with other cancers. CONCLUSIONS: In view of the central role of RPA in DNA replication, recombination, and repair, we suggest that autoimmunity to RPA32 may reflect molecular changes involved in the process of tumorigenesis. The finding of antibodies to RPA32 before diagnosis and their prevalence in in situ carcinoma suggest that they are potentially useful markers of early disease.

Antinuclear antibodies as potential markers of lung cancer.

There are multiple case reports of antinuclear antibodies (ANAs) in patients with malignancies, yet to date there has not been a systematic survey of ANAs in lung cancer. We have previously reported that autoantibodies to collagen antigens resembling those found in the connective tissue diseases are consistently detected in the sera from lung cancer patients. In this work, we looked for the presence of ANAs in the sera from these same patients. Sera from 64 patients with lung cancer and 64 subjects without a history of cancer were retrospectively tested for reactivity on immunoblots of nuclear extracts of HeLa, small cell carcinoma, squamous cell carcinoma, adenocarcinoma, large cell carcinoma of the lung, and of normal lung cells. Associations were sought between the reactivities on immunoblots and lung cancer cell type, diagnosis, and progression-free survival by the method of classification and regression trees (CARTs). Cross-validated CART analyses indicated that reactivities to certain bands in immunoblots are associated with different types of lung cancer. Some of these autoantibodies were associated with a prolonged survival without disease progression. Our data suggest that autoimmunity is often a prominent feature of lung cancer and that molecular characterization of these antigens may lead to the discovery of proteins with diagnostic and prognostic value.