Apoptosis and expression of apoptosis-related genes in mouse intestinal tissue after whole-body proton exposure.

Energetic protons are the most abundant particle type in space and can pose serious health risks to astronauts during long-duration missions. The health effects of proton exposure are also a concern for cancer patients undergoing radiation treatment with accelerated protons. To investigate the damage induced by energetic protons in vivo to radiosensitive organs, 6-week-old BALB/c male mice were subjected to 250 MeV proton radiation at whole-body doses of 0.1, 1, and 2 Gy. The gastrointestinal (GI) tract of each exposed animal was dissected 4 h post-irradiation, and the isolated small intestinal tissue was analyzed for histopathological and gene expression changes. Histopathologic observation of the tissue using standard hematoxylin and eosin (H&E) staining methods to screen for morphologic changes showed a marked increase in apoptotic lesions for even the lowest dose of 0.1 Gy, similar to X- or γ rays. The percentage of apoptotic cells increased dose-dependently, but the dose response appeared supralinear, indicating hypersensitivity at low doses. A significant decrease in surviving crypts and mucosal surface area, as well as in cell proliferation, was also observed in irradiated mice. Gene expression analysis of 84 genes involved in the apoptotic process showed that most of the genes affected by protons were common between the low (0.1 Gy) and high (1 and 2 Gy) doses. However, the genes that were distinctively responsive to the low or high doses suggest that high doses of protons may cause apoptosis in the small intestine by direct damage to the DNA, whereas low doses of protons may trigger apoptosis through a different stress response mechanism.

Whole-transcriptome RNAseq analysis from minute amount of total RNA.

RNA sequencing approaches to transcriptome analysis require a large amount of input total RNA to yield sufficient mRNA using either poly-A selection or depletion of rRNA. This feature makes it difficult to miniaturize transcriptome analysis for greater efficiency. To address this challenge, we devised and validated a simple procedure for the preparation of whole-transcriptome cDNA libraries from a minute amount (500 pg) of total RNA. We compared a single-sample library prepared by this Ovation RNA-Seq system with two available methods of mRNA enrichment (TruSeq poly-A enrichment and RiboMinus rRNA depletion). Using the Ovation preparation method for a set of eight mouse tissue samples, the RNA sequencing data obtained from two different next-generation sequencing platforms (SOLiD and Illumina Genome Analyzer IIx) yielded negligible rRNA reads (<3.5%) while retaining transcriptome sequencing fidelity. We further validated the Ovation amplification technique by examining the resulting library complexity, reproducibility, evenness of transcript coverage, 5' and 3' bias and platform-specific biases. Notably, in this side-by-side comparison, SOLiD sequencing chemistry is biased toward higher GC content of transcriptome and Illumina Genome analyzer IIx is biased away from neutral to lower GC content of the transcriptomics regions.

Reactive oxygen species mediated tissue damage in high energy proton irradiated mouse brain.

Although radiation related research has been conducted extensively, the molecular toxicology and cellular mechanisms affected by proton radiation remain poorly understood. We recently reported that the high energy protons induce cell death through activation of apoptotic signaling genes; caspase 3 and 8 (Baluchamy et al. J Biol Chem 285:24769-24774, 2010). In this study, we investigated the effect of different doses of protons in in vivo mouse system, particularly, brain tissues. A significant dose-dependent induction of reactive oxygen species and lipid peroxidation and reduction of antioxidants; glutathione and superoxide dismutase were observed in proton irradiated mouse brain as compared to control brain. Furthermore, histopathology studies on proton irradiated mouse brain showed significant tissue damage as compared to control brain. Together, our in vitro and in vivo results suggest that proton irradiation alters oxidant and antioxidant levels in the cells to cause proton mediated DNA/tissue damage followed by apoptotic cell death.

Induction of cell death through alteration of oxidants and antioxidants in lung epithelial cells exposed to high energy protons.

Radiation affects several cellular and molecular processes, including double strand breakage and modifications of sugar moieties and bases. In outer space, protons are the primary radiation source that poses a range of potential health risks to astronauts. On the other hand, the use of proton irradiation for tumor radiation therapy is increasing, as it largely spares healthy tissues while killing tumor tissues. Although radiation-related research has been conducted extensively, the molecular toxicology and cellular mechanisms affected by proton irradiation remain poorly understood. Therefore, in this study, we irradiated rat lung epithelial cells with different doses of protons and investigated their effects on cell proliferation and death. Our data show an inhibition of cell proliferation in proton-irradiated cells with a significant dose-dependent activation and repression of reactive oxygen species and antioxidants glutathione and superoxide dismutase, respectively, compared with control cells. In addition, the activities of apoptosis-related genes such as caspase-3 and -8 were induced in a dose-dependent manner with corresponding increased levels of DNA fragmentation in proton-irradiated cells compared with control cells. Together, our results show that proton irradiation alters oxidant and antioxidant levels in cells to activate the apoptotic pathway for cell death.

Voltage-controlled metal binding on polyelectrolyte-functionalized nanopores.

Most of the research in the field of nanopore-based platforms is focused on monitoring ion currents and forces as individual molecules translocate through the nanopore. Molecular gating, however, can occur when target analytes interact with receptors appended to the nanopore surface. Here we show that a solid state nanopore functionalized with polyelectrolytes can reversibly bind metal ions, resulting in a reversible, real-time signal that is concentration dependent. Functionalization of the sensor is based on electrostatic interactions, requires no covalent bond formation, and can be monitored in real time. Furthermore, we demonstrate how the applied voltage can be employed to tune the binding properties of the sensor. The sensor has wide-ranging applications and, its simplest incarnation can be used to study binding thermodynamics using purely electrical measurements with no need for labeling.

The effect of acute dose charge particle radiation on expression of DNA repair genes in mice.

The space radiation environment consists of trapped particle radiation, solar particle radiation, and galactic cosmic radiation (GCR), in which protons are the most abundant particle type. During missions to the moon or to Mars, the constant exposure to GCR and occasional exposure to particles emitted from solar particle events (SPE) are major health concerns for astronauts. Therefore, in order to determine health risks during space missions, an understanding of cellular responses to proton exposure is of primary importance. The expression of DNA repair genes in response to ionizing radiation (X-rays and gamma rays) has been studied, but data on DNA repair in response to protons is lacking. Using qPCR analysis, we investigated changes in gene expression induced by positively charged particles (protons) in four categories (0, 0.1, 1.0, and 2.0 Gy) in nine different DNA repair genes isolated from the testes of irradiated mice. DNA repair genes were selected on the basis of their known functions. These genes include ERCC1 (5' incision subunit, DNA strand break repair), ERCC2/NER (opening DNA around the damage, Nucleotide Excision Repair), XRCC1 (5' incision subunit, DNA strand break repair), XRCC3 (DNA break and cross-link repair), XPA (binds damaged DNA in preincision complex), XPC (damage recognition), ATA or ATM (activates checkpoint signaling upon double strand breaks), MLH1 (post-replicative DNA mismatch repair), and PARP1 (base excision repair). Our results demonstrate that ERCC1, PARP1, and XPA genes showed no change at 0.1 Gy radiation, up-regulation at 1.0 Gy radiation (1.09 fold, 7.32 fold, 0.75 fold, respectively), and a remarkable increase in gene expression at 2.0 Gy radiation (4.83 fold, 57.58 fold and 87.58 fold, respectively). Expression of other genes, including ATM and XRCC3, was unchanged at 0.1 and 1.0 Gy radiation but showed up-regulation at 2.0 Gy radiation (2.64 fold and 2.86 fold, respectively). We were unable to detect gene expression for the remaining four genes (XPC, ERCC2, XRCC1, and MLH1) in either the experimental or control animals.

Progress on plague vaccine development.

Yersinia pestis (YP), the gram-negative plague bacterium, has shaped human history unlike any other pathogen known to mankind. YP (transmitted by the bite of an infected flea) diverged only recently from the related enteric pathogen Yersinia pseudotuberculosis but causes radically different diseases. Three forms of plague exist in humans: bubonic (swollen lymph nodes or bubos), septicemic (spread of YP through the lymphatics or bloodstream from the bubos to other organs), and contagious, pneumonic plague which can be communicated via YP-charged respiratory droplets resulting in person-person transmission and rapid death if left untreated (50-90% mortality). Despite the potential threat of weaponized YP being employed in bioterrorism and YP infections remaining prevalent in endemic regions of the world where rodent populations are high (including the four corner regions of the USA), an efficacious vaccine that confers immunoprotection has yet to be developed. This review article will describe the current vaccine candidates being evaluated in various model systems and provide an overall summary on the progress of this important endeavor.

Expression profile of DNA damage signaling genes in 2 Gy proton exposed mouse brain.

Exposure of living systems to radiation results in a wide assortment of lesions, the most significant of is damage to genomic DNA which alter specific cell functions including cell proliferation. The radiation induced DNA damage investigation is one of the important area in biology, but still the information available regarding the effects of proton is very limited. In this report, we investigated the differential gene expression pattern of DNA damage signaling genes such as damaged DNA binding, repair, cell cycle arrest, checkpoints and apoptosis using quantitative real-time RT-PCR in proton exposed mouse brain tissues. The expression profiles showed significant changes in DNA damage related genes in 2 Gy proton exposed mouse brain tissues as compared to control brain tissues. Furthermore, we also show that significantly increased levels of apoptotic related genes, caspase-3 and 8 activities in these cells, suggesting that in addition to differential expression of DNA damage genes, the alteration of apoptosis related genes may also contribute to the radiation induced DNA damage followed by programmed cell death. In summary, our findings suggest that proton exposed cells undergo severe DNA damage which in turn destabilize the chromatin stability.

Spaceflight and modeled microgravity effects on microbial growth and virulence.

For unsuspecting bacteria, the difference between life and death depends upon efficient and specific responses to various stressors. Facing a much larger world, microbes are invariably challenged with ever-changing environments where temperature, pH, chemicals, and nutrients are in a constant state of flux. Only those that are able to rapidly reprogram themselves and express subsets of genes needed to overcome the stress will survive and outcompete neighboring microbes. Recently, low shear stress, emulating microgravity (MG) experienced in space, has been characterized in a number of microorganisms including fungi and prokaryotes ranging from harmless surrogate organisms to bona fide pathogens. Interestingly, MG appears to induce a plethora of effects ranging from enhanced pathogenicity in several Gram-negative enterics to enhanced biofilm formation. Furthermore, MG-exposed bacteria appeared better able to handle subsequent stressors including: osmolarity, pH, temperature, and antimicrobial challenge while yeast exhibited aberrant budding post-MG-exposure. This review will focus on MG-induced alterations of virulence in various microbes with the emphasis placed on bacteria.

Induction of apoptosis in rat lung epithelial cells by multiwalled carbon nanotubes.

Carbon nanotubes (CNTs), the most promising material with unique characteristics, find its application in different fields ranging from composite materials to medicine and from electronics to energy storage. However, little is known about the mechanism behind the interaction of these particles with cells and their toxicity. So, here we investigated the adverse effects of multiwalled CNTs (MWCNTs) in rat lung epithelial (LE) cells. The results showed that the incubation of LE cells with 0.5-10 microg/mL of MWCNTs caused a dose- and time-dependent increase in the formation of free radicals, the accumulation of peroxidative products, the loss of cell viability, and antioxidant depletion. The significant amount of incorporation of dUTPs in the nucleus after 24 h confirms the induction of apoptosis. It was also observed that there is an increase in the activity of both caspases-3 and caspase-8 in cells, with increases in time and the concentration of MWCNTs. No significant incorporation of dUTPs was observed in cells, incubated with z-VAD-fmk, which confirmed the role of caspases in DNA fragmentation. The present study reveals that MWCNTs induced oxidative stress and stimulated apoptosis signaling pathway through caspase activation in rat LE cell lines.

Analysis of stress responsive genes induced by single-walled carbon nanotubes in BJ Foreskin cells.

Nanotechnology is finding its use as a potential technology in consumer products, defense, electronics, and medical applications by exploiting the properties of nanomaterials. Single-walled carbon nanotubes are novel forms of these nanomaterials with potential for large applications. However, the toxicity studies on this material are not explored in detail and therefore limiting its use. It has been earlier reported that single-walled carbon nanotubes induces oxidative stress and also dictates activation of specific signaling pathway in keratinocytes. The present study explores the effect of single-walled carbon nanotubes on stress genes in human BJ Foreskin cells. The results show induction of oxidative stress in BJ Foreskin cells by single-walled carbon nanotubes and increase in stress responsive genes. The genes included inducible genes like HMOX1, HMOX2, and Cyp1B1. In addition we validated increase for four genes by SWCNT, namely ATM, CCNC, DNAJB4, and GADD45A by RT-PCR. Moreover results of the altered stress related genes have been discussed and that partially explains some of the toxic responses induced by single-walled carbon nanotubes.

Ribonucleases and bacterial virulence.

Bacterial stress responses provide them the opportunity to survive hostile environments, proliferate and potentially cause diseases in humans and animals. The way in which pathogenic bacteria interact with host immune cells triggers a complicated series of events that include rapid genetic re-programming in response to the various host conditions encountered. Viewed in this light, the bacterial host-cell induced stress response (HCISR) is similar to any other well-characterized environmental stress to which bacteria must respond by upregulating a group of specific stress-responsive genes. Post stress, bacteria must resume their pre-stress genetic program, and, as a consequence, must degrade unnecessary stress responsive transcripts through RNA decay mechanisms. Further, there is a well-established role for several ribonucleases in the cold shock response whereby they modulate the changing transcript landscape in response to the stress, and during acclimation and subsequent genetic re-programming post stress. Recently, ribonucleases have been implicated as virulence-associated factors in several notable Gram-negative pathogens including, the yersiniae, the salmonellae, Helicobacter pylori, Shigella flexneri and Aeromonas hydrophila. This review will focus on the roles played by ribonucleases in bacterial virulence, other bacterial stress responses, and on their novel therapeutic applications.

Reversible thrombin detection by aptamer functionalized STING sensors.

Signal Transduction by Ion NanoGating (STING) is a label-free technology based on functionalized quartz nanopipettes. The nanopipette pore can be decorated with a variety of recognition elements and the molecular interaction is transduced via a simple electrochemical system. A STING sensor can be easily and reproducibly fabricated and tailored at the bench starting from inexpensive quartz capillaries. The analytical application of this new biosensing platform, however, was limited due to the difficult correlation between the measured ionic current and the analyte concentration in solution. Here we show that STING sensors functionalized with aptamers allow the quantitative detection of thrombin. The binding of thrombin generates a signal that can be directly correlated to its concentration in the bulk solution.

Ultrasensitive mycotoxin detection by STING sensors.

Signal transduction by ion nano-gating (STING) technology is a label-free biosensor capable of identifying DNA and proteins. Based on a functionalized quartz nanopipette, the STING sensor includes specific recognition elements for analyte discrimination based on size, shape and charge density. A key feature of this technology is that it does not require any nanofabrication facility; each nanopipette can be easily, reproducibly, and inexpensively fabricated and tailored at the bench, thus reducing the cost and the turnaround time. Here, we show that STING sensors are capable of the ultrasensitive detection of HT-2 toxin with a detection limit of 100 fg/ml and compare the STING capabilities with respect to conventional sandwich assay techniques.

The effects of low-shear mechanical stress on Yersinia pestis virulence.

Manned space exploration has created a need to evaluate the effects of spacelike stress on pathogenic and opportunistic microbes astronauts could carry with them to the International Space Station and beyond. Yersinia pestis (YP) causes bubonic, septicemic, and pneumonic plague and is capable of killing infected patients within 3-7 days. In this study, low-shear modeled microgravity (LSMMG), a spacelike stress, was used to physically stress YP; and its effects on proliferation, cold growth, and type III secretion system (T3SS) function were evaluated. YP was grown to saturation in either LSMMG or normal gravity (NG) conditions prior to being used for RAW 246.7 cell infections, HeLa cell infections, and Yop secretion assays. A mutant strain of YP (ΔyopB) that lacks the ability to inject Yersinia outer membrane proteins (Yops) into the host cell was used as a negative control in cell infection experiments. Our experimental results indicate that YP cultivated under LSMMG resulted in reduced YopM production and secretion compared to its NG-grown counterpart. Similarly, NG-grown YP induced more cell rounding in HeLa cells than did the LSMMG-grown YP, which suggests that LSMMG somehow impairs T3SS optimum function. Also, LSMMG-grown YP used to infect cultured RAW 246.7 cells showed a similar pattern of dysfunction in that it proliferated less than did its NG-grown counterpart during an 8-hour infection period. This study suggests that LSMMG can attenuate bacterial virulence contrary to previously published data that have demonstrated LSMMG-induced hypervirulence of other Gram-negative enterics.

Sensitive giant magnetoresistive-based immunoassay for multiplex mycotoxin detection.

Rapid and multiplexed measurement is vital in the detection of food-borne pathogens. While highly specific and sensitive, traditional immunochemical assays such as enzyme-linked immunosorbent assays (ELISAs) often require expensive read-out equipment (e.g. fluorescent labels) and lack the capability of multiplex detection. By combining the superior specificity of immunoassays with the sensitivity and simplicity of magnetic detection, we have developed a novel multiplex magnetic nanotag-based detection platform for mycotoxins that functions on a sub-picomolar concentration level. Unlike fluorescent labels, magnetic nanotags (MNTs) can be detected with inexpensive giant magnetoresistive (GMR) sensors such as spin-valve sensors. In the system presented here, each spin-valve sensor has an active area of 90 microm x 90 microm, arranged in an 8 x 8 array. Sample is added to the antibody-immobilized sensor array prior to the addition of the biotinylated detection antibody. The sensor response is recorded in real time upon the addition of streptavidin-linked MNTs on the chip. Here we demonstrate the simultaneous detection of multiple mycotoxins (aflatoxins B(1), zearalenone and HT-2) and show that a detection limit of 50 pg/mL can be achieved.

Differential oxidative stress gene expression profile in mouse brain after proton exposure.

Radiation is known to potentially interfere with cellular functions at all levels of cell organization. The radiation-induced stress response is very complex and involves altered expression of many genes. Identification of specific genes may allow the determination of pathways important in radiation responses. Although several radiation-related research have been studied extensively, the molecular and cellular processes affected by proton exposure remain poorly understood. Our earlier reports have shown that proton radiation induces reactive oxygen species (ROS) formation and lipid peroxidation and inhibits antioxidants, superoxide dismutase, and glutathione. Therefore, in this present study, we used quantitative real-time reverse transcription polymerase chain reaction approach and showed the modulation of several genes including oxidative stress, antioxidants defense mechanism, ROS metabolism, and oxygen transporters related genes expression in 2-Gy proton-exposed mouse brain. Literature evidences suggest that change in oxidants and antioxidants levels induce DNA damage, followed by cell death. In conclusion, changes in the gene profile of mouse brain after proton irradiation are complex and the exposed cells might undergo programmed cell death through alteration of genes responsible for oxidative stress signaling mechanism.

Suspension bead array branch migration displacement assay for rapid STR analysis.

STR analysis is commonly used in forensic and genetic studies. STRs are currently discriminated based on size, primarily by gel- and column-based approaches. Hybridization-based approaches have the potential to allow high-throughput analysis of STRs; however, development of such approaches has been limited by the difficulty in discriminating between STRs of similar length. We have recently described several innovations to enable STR analysis using an array-based hybridization approach for high- throughput STR analysis. Here we extend that approach by incorporating the array into microspheres and adding a discriminatory branch migration displacement step. This microsphere-based platform uses Luminex xMAP technology and improves the sensitivity, selectivity, and speed of the assay. We demonstrate the feasibility, speed, and reliability of the assay for STR detection by correctly analyzing two STR loci in 20 forensic DNA samples of known STR type. The multiplex, bead-based approach provides a high-throughput and more portable STR analysis.

Large-scale pyrosequencing of synthetic DNA: a comparison with results from Sanger dideoxy sequencing.

Pyrosequencing is a relatively recent method for sequencing short stretches of DNA. Because both Pyrosequencing and Sanger dideoxy sequencing were recently used to characterize and validate DNA molecular barcodes in a large yeast gene-deletion project, a meta-analysis of those data allow an excellent and timely opportunity for evaluating Pyrosequencing against the current gold standard, Sanger dideoxy sequencing. Starting with yeast genomic DNA, parallel PCR amplification methods were used to prepared 4747 short barcode-containing constructs from 6000 Saccharomyces cerevisiae gene-deletion strains. Pyrosequencing was optimized for average read lengths of 25-30 bases, which included in each case a 20-mer barcode sequence. Results were compared with sequence data obtained by the standard Sanger dideoxy chain termination method. In most cases, sequences obtained by Pyrosequencing and Sanger dideoxy sequencing were of comparable accuracy, and the overall rate of failure was similar. The DNA in the barcodes is derived from synthetic oligonucleotide sequences that were inserted into yeast-deletion-strain genomic DNA by homologous recombination and represents the most significant amount of DNA from a synthetic source that has been sequenced to date. Although more automation and quality control measures are needed, Pyrosequencing was shown to be a fast and convenient method for determining short stretches of DNA sequence.

Sentinel-base DNA genotyping using multiple sequencing primers for high-risk human papillomaviruses.

Despite the various technologies in place for genotyping human papillomaviruses (HPV), clinical use and clinical research demand a method that is fast, more reliable and cost-effective. The technology described here represents a breakthrough development in that direction. By combining the method of multiple sequencing primers with DNA sequencing, we have developed a rapid assay for genotyping HPV that relies on the identification of a single, type-specific 'sentinel' base. As described here, the prototype assay has been developed to recognize the 12 most high-risk HPV types (HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59) and is capable of recognizing and simultaneously genotyping multiple HPV co-infections. By providing sequence information on multiple HPV infections, this method eliminates the need for labor- and cost-intensive PCR cloning. These proof-of-concept studies establish the assay to be accurate, reliable, rapid, flexible, and cost-effective, providing evidence of the feasibility this technique for use in clinical settings.