Gene Expression Changes in a Prefinal Health Stage of Lethally Irradiated Male and Female Rhesus Macaques.

Radiation-induced gene expression (GE) changes can be used for early and high-throughput biodosimetry within the first three days postirradiation. However, is the method applicable in situations such as the Alexander Litvinenko case or the Goiania accident, where diagnosis occurred in a prefinal health stage? We aimed to characterize gene expression changes in a prefinal health stage of lethally irradiated male and female rhesus macaques. Peripheral blood was drawn pre-exposure and at the prefinal stage of male and female animals, which did not survive whole-body exposure with 700 cGy (LD66/60). RNA samples originated from a blinded randomized Good Laboratory Practice study comprising altogether 142 irradiated rhesus macaques of whom 60 animals and blood samples (15 samples for both time points and sexes) were used for this analysis. We evaluated GE on 34 genes widely used in biodosimetry and prediction of the hematological acute radiation syndrome severity (H-ARS) employing quantitative real-time polymerase chain reaction (qRT-PCR). These genes were run in duplicate and triplicate and altogether 96 measurements per time point and sex could be performed. In addition, 18S ribosomal RNA (rRNA) was measured to depict the ribosome/transcriptome status as well as for normalization purposes and 16S rRNA was evaluated as a surrogate for bacteremia. Mean differential gene expression (DGE) was calculated for each gene and sex including all replicate measurements and using pre-exposure samples as the reference. From 34 genes, altogether 27 genes appeared expressed. Pre-exposure samples revealed no signs of bacteremia and 18S rRNA GE was in the normal range in all 30 samples. Regarding prefinal samples, 46.7% and 40% of animals appeared infected in females and males, respectively, and for almost all males this was associated with out of normal range 18S rRNA values. The total number of detectable GE measurements was sixfold (females) and 15-fold (males) reduced in prefinal relative to pre-exposure samples and about tenfold lower in 80% of prefinal compared to pre-exposure samples (P < 0.0001). An overall 11-fold (median) downregulation in prefinal compared to pre-exposure samples was identified for most of the 27 genes and even FDXR appeared 4-14-fold downregulated in contrast to a pronounced up-regulation according to cited work. This pattern of overall downregulation of almost all genes and the rapid reduction of detectable genes at a prefinal stage was found in uninfected animals with normal range 18S rRNA as well. In conclusion, in a prefinal stage after lethal radiation exposure, the ribosome/transcriptome status remains present (based on normal range 18S rRNA values) in 60-67% of animals, but the whole transcriptome activity in general appears silenced and cannot be used for biodosimetry purposes, but probably as an indicator for an emerging prefinal health stage.

Experimental and theoretical analysis of Dy3+-doped fiber lasers for efficient yellow emission.

We report a detailed experimental and theoretical analysis of the $^4{{\rm F}_{9/2}}$ to $^6{{\rm H}_{13/2}}$ lasing transition of a dysprosium (${{\rm Dy}^{3 +}}$)-doped ZBLAN fiber, a strong candidate for future compact and highly efficient yellow laser emission. Experimentally, we used a gallium nitride laser diode emitting at 447 nm as a pump source and measured yellow laser output generated with a maximum slope efficiency of 33%, which is less than half of the Stokes limit (of ${\sim}78\%$). This result is commensurate with two other reports of yellow emission from ${{\rm Dy}^{3 +}}$. As a result, we developed a numerical model to understand and analyze the improvement potential of this fiber laser system. For reliable spectroscopic data input to the numerical model, we measured the absorption and emission cross sections from ${{\rm Dy}^{3 +}}$-doped ZBLAN glass. We investigated the potential causes of the low experimental slope efficiency and found contributions from the background loss of the fiber and excited-state absorption (ESA) of the intracavity yellow light. We estimated the signal re-absorption cross section using the emission cross section and the McCumber relation, which was subsequently used in our numerical model to compare successfully with our experimental results. We show that the ESA can be reduced for future ${{\rm Dy}^{3 +}}$-doped yellow laser systems by cascade lasing or co-doping with a suitable rare earth ion desensitizer.

Predicting the Radiation Sensitivity of Male and Female Rhesus Macaques Using Gene Expression.

Radiosensitivity differs in humans and likely among closely-related primates. Reasons for variation in radiosensitivity are not well known. We examined preirradiation gene expression in peripheral blood among male and female rhesus macaques which did or did not survive (up to 60 days) after whole-body irradiation with 700 cGy (LD66/60). RNA samples originated from a blinded randomized Good Laboratory Practice study in 142 irradiated rhesus macaques. Animals were untreated (placebo), or treated using recombinant human IL-12, G-CSF or combination of the two. We evaluated gene expression in a two-phase study design where phase I was a whole genome screen [next generation sequencing (NGS)] for mRNAs (RNA-seq) using five RNA samples from untreated male and female animals per group of survivor and non-survivor (total n = 20). Differential gene expression (DGE) was defined as a statistically significant and ≥2-fold up- or downregulation of mRNA species and was calculated between groups of survivors and non-survivors (reference) and by gender. Altogether 659 genes were identified, but the overlapping number of differentially expressed genes (DGE) observed in both genders was small (n = 36). Fifty-eight candidate mRNAs were chosen for independent validation in phase II using the remaining samples (n = 122) evaluated with qRT-PCR. Among the 58 candidates, 16 were of significance or borderline significance (t test) by DGE. Univariate and multivariate logistic regression analysis and receiver operating characteristic (ROC) curve analysis further refined and identified the most outstanding validated genes and gene combinations. For untreated male macaques, we identified EPX (P = 0.005, ROC=1.0), IGF2BP1 (P = 0.05, ROC=0.74) and the combination of EPX with SLC22A4 (P = 0.03, ROC=0.85) which appeared most predictive for the clinical outcome for treated and combined (untreated and treated) male macaque groups, respectively. For untreated, treated and both combined female macaque groups the same gene (MBOAT4, P = 0.0004, ROC = 0.81) was most predictive. Based on the probability function of the ROC curves, up to 74% of preirradiation RNA measurements predicted survival with a positive and negative predictive value ranging between 85-100% and associated odds ratios reflecting a 2-3-fold elevated risk for surviving per unit change (cycle threshold value) in gene expression. In conclusion, we identified gender-dependent genes and gene combinations in preirradiation blood samples for survival prediction after irradiation in rhesus macaques.

Differentiating Total- or Partial-Body Irradiation in Baboons Using mRNA Expression Patterns: A Proof of Concept.

To better predict clinical outcome after radiation exposure, it is very important to know the absorbed dose and body areas exposed. Previously we found that 22 miRNAs appeared to predict total- and partial-body irradiation (TBI and PBI, respectively) patterns and were suggestive of the percentage of the body exposed in a baboon model. Motivated by these results, we performed a similar analysis on the transcriptional level (mRNAs) using whole genome microarrays. From 17 irradiated baboons, blood samples were taken before, and at 1, 2, 7, 28 and 75-106 days postirradiation to an equivalent TBI dose of 2.5 or 5 Gy applied either to the total body or to different parts of the body such as the upper body (UBE) or left hemibody (LHB). We compared quantile normalized log2-transformed gene expression values with three exposure pattern comparisons, namely TBI vs. PBI, TBI vs. LHB and UBE vs. LHB using Kruskal-Wallis and logistic regression analysis for receiver-operator characteristic (ROC) calculation. We found several hundred significantly (P < 0.05) and ≥2-fold deregulated mRNAs per exposure pattern comparison with a peak of 163-860 mRNAs at day 28. Lower numbers on day 2 (60 mRNAs) and day 7 (91-162 mRNAs) were observed, with the lowest number of deregulated mRNAs at day 75-106 (22-58 mRNAs). The 14 most promising mRNAs (e.g., LTF, DEFA3, OLFM4) appeared 10.1-46.2-fold upregulated and the exposure groups were completely or almost completely discriminated (ROC between 0.8-1.0). Several of the mRNA gene expression changes were significantly associated with the percentage of the body exposed. The numbers of overlapping genes used for diagnosis on consecutive days postirradiation were mostly 0 or less than 10. Bioinformatic analysis confirmed that at each time point different biological processes predominated. Our results suggest mRNA changes over time may be used to retrospectively determine radiation exposure patterns as partial or total body. mRNA gene expression changes likely could be applied over a longer time frame (2-75 days postirradiation) than miRNA, but due to the transient gene expression changes a different set of candidate mRNAs appears to be required at each day after irradiation.

Role of integrons in the proliferation of multiple drug resistance in selected bacteria occurring in poultry production.

1. The increase in microbial resistance, and in particular multiple drug resistance (MDR), is an increasing threat to public health. The uncontrolled use of antibiotics and antibacterial chemotherapeutics in the poultry industry, especially in concentrations too low to cause inhibition, and the occurrence of residues in feed and in the environment play a significant role in the development of resistance among zoonotic food-borne microorganisms.2. Determining the presence and transmission methods of resistance in bacteria is crucial for tracking and preventing antibiotic resistance. Horizontal transfer of genetic elements responsible for drug resistance is considered to be the main mechanism for the spread of antibiotic resistance.3. Of the many well-known genetic elements responsible for horizontal gene transfer, integrons are among the most important factors contributing to multiple drug resistance. The mechanism of bacterial drug resistance acquisition through integrons is one of the essential elements of MDR prevention in animal production.

miRNA Expression Patterns Differ by Total- or Partial-Body Radiation Exposure in Baboons.

In a radiation exposure event, a likely scenario may include either total-body irradiation (TBI) or different partial-body irradiation (PBI) patterns. Knowledge of the exposure pattern is expected to improve prediction of clinical outcome. We examined miRNA species in 17 irradiated baboons receiving an upper-body, left hemibody or total-body irradiation of 2.5 or 5 Gy. Blood samples were taken before irradiation and at 1, 2, 7, 28 and 75-106 days after irradiation. Using a qRT-PCR platform for simultaneous detection of 667 miRNAs, we identified 55 miRNAs over all time points. Candidate miRNAs, such as miR-17, miR-128 or miR-15b, significantly discriminated TBI from different PBI exposure patterns, and 5-to-10-fold changes in gene expression were observed among the groups. A total of 22 miRNAs (including miR-17) revealed significant linear associations of gene expression changes with the percentage of the exposed body area (P < 0.0001). All these changes were primarily observed at day 7 postirradiation and almost no miRNAs were detected either before or after 7 days. A significant association in the reduction of lymphocyte counts in TBI compared to PBI animals corresponded with the number of miRNA candidates. This finding suggests that our target miRNAs predominantly originated from irradiated lymphocytes. In summary, gene expression changes in the peripheral blood provided indications of the exposure pattern and a suggestion of the percentage of the exposed body area.

Q-switched Dy:ZBLAN fiber lasers beyond 3 µm: comparison of pulse generation using acousto-optic modulation and inkjet-printed black phosphorus.

We report high-energy mid-infrared pulse generation by Q-switching of dysprosium-doped fiber lasers for the first time. Two different modulation techniques are demonstrated. Firstly, using active acousto-optic modulation, pulses are produced with up to 12 µJ energy and durations as short as 270 ns, with variable repetition rates from 100 Hz to 20 kHz and central wavelengths tunable from 2.97 to 3.23 µm. Experiments are supported by numerical modeling, identifying routes for improved pulse energies and to avoid multi-pulsing by careful choice of modulator parameters. Secondly, we demonstrate passive Q-switching by fabricating an inkjet-printed black phosphorus saturable absorber, simplifying the cavity and generating 1.0 µJ pulses with 740 ns duration. The performance and relative merits of each modulation approach are then critically discussed. These demonstrations highlight the potential of dysprosium as a versatile gain medium for high-performance pulsed sources beyond 3 µm.

Impact of Inter-Individual Variance in the Expression of a Radiation-Responsive Gene Panel Used for Triage.

In previous studies we determined a gene expression signature in baboons for predicting the severity of hematological acute radiation syndrome. We subsequently validated a set of eight of these genes in leukemia patients undergoing total-body irradiation. In the current study, we addressed the effect of intra-individual variability on the basal level of expression of those eight radiation-responsive genes identified previously, by examining baseline levels in 200 unexposed healthy human donors (122 males and 88 females with an average age of 46 years) using real-time PCR. In addition to the eight candidate genes ( DAGLA, WNT3, CD177, PLA2G16, WLS, POU2AF1, STAT4 and PRF1), we examined two more genes ( FDXR and DDB2) widely used in ex vivo whole blood experiments. Although significant sex- (seven genes) and age-dependent (two genes) differences in expression were found, the fold changes ranged only between 1.1-1.6. These were well within the twofold differences in gene expression generally considered to represent control values. Age and sex contributed less than 20-30% to the complete inter-individual variance, which is calculated as the fold change between the lowest (reference) and the highest Ct value minimum-maximum fold change (min-max FC). Min-max FCs ranging between 10-17 were observed for most genes; however, for three genes, min-max FCs of complete inter-individual variance were found to be 37.1 ( WNT3), 51.4 ( WLS) and 1,627.8 ( CD177). In addition, to determine whether discrimination between healthy and diseased baboons might be altered by replacing the published gene expression data of the 18 healthy baboons with that of the 200 healthy humans, we employed logistic regression analysis and calculated the area under the receiver operating characteristic (ROC) curve. The additional inter-individual variance of the human data set had either no impact or marginal impact on the ROC area, since up to 32-fold change gene expression differences between healthy and diseased baboons were observed.

Improved tendon healing using bFGF, BMP-12 and TGFß1 in a rat model.

Several growth factors (GFs) are expressed as tendons heal, but it remains unknown whether their combined application enhances the healing process. This matter was addressed by applying a combination of basic fibroblast growth factor (bFGF), bone morphogenetic protein 12 (BMP-12) and transforming growth factor beta 1 (TGFß1) in a rat Achilles tendon transection model. GFs were applied in one of the three following ways: i) direct application of all three factors at the time of surgery; ii) sequential, tiered percutaneous injection of individual factors immediately after surgery, 48 h and 96 h later; iii) load of all three factors onto a collagen sponge implanted at the time of surgery. After 1, 2, 4 and 8 weeks, healing was assessed based on tendon length and thickness, mechanical strength, stiffness and histology. Best results were achieved when GFs were loaded onto a collagen sponge - with a rapid increase in mechanical strength (load to failure, 71.2 N vs. 7.7 N in controls), consistent tendon length over time (9.9 mm vs. 16.2 mm in controls) and faster tendon remodelling, as measured by histology - followed by tiered injection therapy over 96 h. In conclusion, implantation of a GF-loaded collagen sponge could provide a promising treatment, especially in high-performance athletes and revision cases prone to re-rupture. For conservative treatment, tiered percutaneous GF application could be an option for improving clinical outcome.

Analysis of Gene Expression Changes in PHA-M Stimulated Lymphocytes - Unraveling PHA Activity as Prerequisite for Dicentric Chromosome Analysis.

Dicentric chromosome analysis (DCA) is the gold standard for individual radiation dose assessment. However, DCA is limited by the time-consuming phytohemagglutinin (PHA)-mediated lymphocyte activation. In this study using human peripheral blood lymphocytes, we investigated PHA-associated whole genome gene expression changes to elucidate this process and sought to identify suitable gene targets as a means of meeting our long-term objective of accelerating cell cycle kinetics to reduce DCA culture time. Human peripheral whole blood from three healthy donors was separately cultured in RPMI/FCS/antibiotics with BrdU and PHA-M. Diluted whole blood samples were transferred into PAXgene tubes at 0, 12, 24 and 36 h culture time. RNA was isolated and aliquots were used for whole genome gene expression screening. Microarray results were validated using qRT-PCR and differentially expressed genes [significantly (FDR corrected) twofold different from the 0 h value reference] were analyzed using several bioinformatic tools. The cell cycle positions and DNA-synthetic activities of lymphocytes were determined by analyzing the correlated total DNA content and incorporated BrdU level with flow cytometry after continued BrdU incubation. From 42,545 transcripts of the whole genome microarray 47.6%, on average, appeared expressed. The number of differentially expressed genes increased linearly from 855 to 2,858 and 4,607 at 12, 24 and 36 h after PHA addition, respectively. Approximately 2-3 times more up- than downregulated genes were observed with several hundred genes differentially expressed at each time point. Earliest enrichment was observed for gene sets related to the nucleus (12 h) followed by genes assigned to intracellular structures such as organelles (24 h) and finally genes related to the membrane and the extracellular matrix were enriched (36 h). Early gene expression changes at 12 h, in particular, were associated with protein classes such as chemokines/cytokines (e.g., CXCL1, CXCL2) and chaperones. Genes coding for biological processes involved in cell cycle control (e.g., MYBL2, RBL1, CCNA, CCNE) and DNA replication (e.g., POLA, POLE, MCM) appeared enriched at 24 h and later, but many more biological processes (42 altogether) showed enrichment as well. Flow cytometry data fit together with gene expression and bioinformatic analyses as cell cycle transition into S phase was observed with interindividual differences from 12 h onward, whereas progression into G2 as well as into the second G1 occurred from 36 h onward after activation. Gene set enrichment analysis over time identifies, in particular, two molecular categories of PHA-responsive gene targets (cytokine and cell cycle control genes). Based on that analysis target genes for cell cycle acceleration in lymphocytes have been identified ( CDKN1A/B/C, RBL-1/RBL-2, E2F2, Deaf-1), and it remains undetermined whether the time expenditure for DCA can be reduced by influencing gene expression involved in the regulatory circuits controlling PHA-associated cell cycle entry and/or progression at a specific early cell cycle phase.

Correlation of Radiation Dose Estimates by DIC with the METREPOL Hematological Classes of Disease Severity.

The degree of severity of hematologic acute radiation syndrome (HARS) may vary across the range of radiation doses, such that dose alone may be a less reliable predictor of clinical course. We sought to elucidate the relationship between absorbed dose and risk of clinically relevant HARS in humans. We used the database SEARCH (System for Evaluation and Archiving of Radiation Accidents based on Case Histories), which contains the histories of radiation accident victims. From 153 cases we extracted data on dose estimates using the dicentric assay to measure individual biological dosimetry. The data were analyzed according to the corresponding hematological response categories of clinical significance (H1-4). These categories are derived from the medical treatment protocols for radiation accident victims (METREPOL) and represent the clinical outcome of HARS based on severity categories ranging from 1-4. In addition, the category H0 represents a post-exposure hematological response that is within the normal range for nonexposed individuals. Age at exposure, gender and ethnicity were considered as potential confounders in unconditional cumulative logistic regression analysis. In most cases, victims were Caucasian (82.4%) and male (92.8%), who originated from either the Chernobyl (69.3%) or Goiânia (10.5%) accident, and nearly 60% were aged 20-40 years at time of exposure. All individuals were whole-body exposed (mean 3.8 Gy, stdev ±3.1), and single exposures were predominantly reported (79%). Seventy percent of victims in category H0 were exposed to ≤1 Gy, with rapidly decreasing proportions of H0 seen at doses up to 5 Gy. There were few HARS H4 cases reported at exposed dose of 1-2 Gy, while 82% of H4 cases received doses of >5 Gy. HARS H1-3 cases varied among dose ranges from 1-5 Gy. In summary, single whole-body radiation doses <1 Gy and >5 Gy corresponded in general with H0 and H3-4, respectively, and this was consistent with medical expectations. This underlines the usefulness of dose estimates for HARS prediction. However, whole-body doses between 1-5 Gy poorly corresponded to HARS H1-3. The dose range of 1-5 Gy was of limited value for medical decision-making regarding, e.g., hospitalization for H2-3, but not H1 and treatment decisions that differ between H1-3. Also, there were some H0 cases at high doses and H2-4 cases at low doses, thereby challenging an individual recommendation based solely on dose.

Watt-level dysprosium fiber laser at 3.15 µm with 73% slope efficiency.

Rare-earth-doped fiber lasers are emerging as promising high-power mid-infrared sources for the 2.6-3.0 µm and 3.3-3.8 µm regions based on erbium and holmium ions. The intermediate wavelength range, however, remains vastly underserved, despite prospects for important manufacturing and defense applications. Here, we demonstrate the potential of dysprosium-doped fiber to solve this problem, with a simple in-band pumped grating-stabilized linear cavity generating up to 1.06 W at 3.15 µm. A slope efficiency of 73% with respect to launched power (77% relative to absorbed power) is achieved-the highest value for any mid-infrared fiber laser to date, to the best of our knowledge. Opportunities for further power and efficiency scaling are also discussed.

Validating Baboon Ex Vivo and In Vivo Radiation-Related Gene Expression with Corresponding Human Data.

The research for high-throughput diagnostic tests for victims of radio/nuclear incidents remains ongoing. In this context, we have previously identified candidate genes that predict risk of late-occurring hematologic acute radiation syndrome (HARS) in a baboon model. The goal of the current study was to validate these genes after radiation exposure in humans. We also examined ex vivo relative to in vivo measurements in both species and describe dose-response relationships. Eighteen baboons were irradiated in vivo to simulate different patterns of partial- or total-body irradiation (TBI), corresponding to an equivalent dose of 2.5 or 5 Sv. Human in vivo blood samples were obtained from patients exposed to different dose ranges: diagnostic computerized tomography (CT; 0.004-0.018 Sv); radiotherapy for prostate cancer (0.25-0.3 Sv); and TBI of leukemia patients (2 × 1.5 or 2 × 2 Sv, five patients each). Peripheral whole blood of another five baboons and human samples from five healthy donors were cultivated ex vivo and irradiated with 0-4 Sv. RNA was isolated pairwise before and 24 h after irradiation and converted into cDNA. Gene expression of six promising candidate genes found previously by us in a baboon model ( WNT3, POU2AF1, CCR7, ARG2, CD177, WLS), as well as three genes commonly used in ex vivo whole blood experiments ( FDXR, PCNA, DDB2) was measured using qRT-PCR. We confirmed the six baboon candidate genes in leukemia patients. However, expression for the candidate gene FDXR showed an inverse relationship, as it was downregulated in baboons and upregulated in human samples. Comparisons among the in vivo and ex vivo experiments revealed the same pattern in both species and indicated peripheral blood cells to represent the radiation-responsive targets causing WNT3 and POU2AF1 gene expression changes. CCR7, ARG2, CD177 and WLS appeared to be altered due to radiation-responsive targets other than the whole blood cells. Linear dose-response relationships of FDXR, WNT3 and POU2AF1 using human ex vivo samples corresponded with human in vivo samples, suggesting that ex vivo models for in vivo dose estimates can be used over a wide dose range (0.001-5 Sv for POU2AF1). In summary, we validated six baboon candidate genes in humans, but the FDXR measurements underscored the importance of independent assessments even when candidates from animal models have striking gene sequence homology to humans. Since whole blood cells represented the same radiation-responsive targets for FDXR, WNT3 and POU2AF1 gene expression changes, ex vivo cell culture models can be utilized for in vivo dose estimates over a dose range covering up to 3.5 log scales. These findings might be a step forward in the development of a gene expression-based high-throughput diagnostic test for populations involved in large-scale radio/nuclear incidents.

The influence of doxazosin on the contractility of the urinary bladder in female pigs with experimentally induced cystitis.

The present in vitro study investigated the influence of doxazosin on the contractility of the urinary bladder in female pigs with experimentally induced cystitis. Fifteen juvenile female piglets (18-20 kg body weight) were randomly assigned into three groups (n=5 animals each): i) control (clinically healthy animals, without doxazosin treatment), ii) animals with induced inflammation of the urinary bladder, but without doxazosin treatment (experimental group I) and iii) animals with inflamed bladder, treated orally with doxazosin (0.1 mg/kg body weight for 30 days; experimental group II). Thereafter, the pigs were sacrificed and strips of the bladder trigone were suspended in organ baths. The tension and amplitude of the smooth muscles was measured before and after exposition to 5-hydroxytryptamine (5-HT; 10-6-10-4 M), acetylocholine (ACh; 10-5-10-3 M) and norepinephrine (NE; 10-9-10-7 M). 5-HT caused an increase in the tension of contractions in all the groups and the amplitude in the experimental groups, however, the effect was higher in the experimental group I than in group II as compared to that found in the pre-treatment period. ACh caused an increase in the tension in the control group and a decrease in the amplitude in both experimental groups; these changes significantly differed between the control and doxazosin-treated group. NE caused a decrease in the tension in both experimental groups and amplitude in all the groups, however, the effect was most strongly expressed in doxazosine-treated group. The present study has revealed that long-term administration of doxazosin causes a desensitization of the detrusor smooth muscle to in vitro applied mediators in the autonomic nervous system.

Using Clinical Signs and Symptoms for Medical Management of Radiation Casualties - 2015 NATO Exercise.

The utility of early-phase (≤5 days) radiation-induced clinical signs and symptoms (e.g., vomiting, diarrhea, erythema and changes in blood cell counts) was examined for the prediction of later occurring acute radiation syndrome (ARS) severity and the development of medical management strategies. Medical treatment protocols for radiation accident victims (METREPOL) was used to grade ARS severities, which were assigned response categories (RCs). Data on individuals (n = 191) with mild (RC1, n = 45), moderate (RC2, n = 19), severe (RC3, n = 20) and fatal (RC4, n = 18) ARS, as well as nonexposed individuals (RC0, n = 89) were generated using either METREPOL (n = 167) or the system for evaluation and archiving of radiation accidents based on case histories (SEARCH) database (n = 24), the latter comprised of real-case descriptions. These data were converted into tables reflecting clinical signs and symptoms, and submitted to eight teams representing five participating countries. The teams were comprised of medical doctors, biologists and pharmacists with subject matter expertise. The tables comprised cumulated clinical data from day 1-3 and day 1-5 postirradiation. While it would have reflected a more realistic scenario to provide the data to the teams over the course of a 3- or 5-day period, the logistics of doing so proved too challenging. In addition, the team members participating in this exercise chose to receive the cumulated reports of day 1-3 and 1-5. The teams were tasked with predicting ARS incidence, ARS severity and the requirement for hospitalization for multiple cases, as well as providing the certainty of their diagnosis. Five of the teams also performed dose estimates. The teams did not employ harmonized methodologies, and the expertise among the members varied, as did the tools used and the means of analyzing the clinical data. The earliest report time was 3 h after the tables were sent to the team members. The majority of cases developing ARS (89.6% ± 3.3 SD) and requiring hospitalization (88.8% ± 4.6 SD) were correctly identified by all teams. Determination of ARS severity was particularly challenging for RC2-3, which was systematically overestimated. However, RC4 was correctly predicted at 94-100% by all teams. RC0 and RC1 ARS severities were more difficult to discriminate. When reported RCs (0-1 and 3-4) were merged, on average 89.6% (±3.3 SD) of all cases could be correctly classified. Comparisons on frequency distributions revealed no statistically significant differences among the following: 1. reported ARS from different teams (P > 0.2); 2. cases generated based on METREPOL or SEARCH (P > 0.5); or 3. results reported at day 3 and 5 postirradiation (P > 0.1). Dose estimates of all teams increased significantly along with ARS severity (P < 0.0001) as well as with dose estimates generated from dicentric chromosomal-aberration measurements available for SEARCH cases (P < 0.0001). In summary, early-phase radiation-induced clinical signs and symptoms proved to be useful for rapid and accurate assessment, with minor limitations, toward predicting life-threatening ARS severity and developing treatment management strategies.

The influence of resiniferatoxin on the chemical coding of caudal mesenteric ganglion neurons supplying the urinary bladder in the pig.

Resiniferatoxin (RTX) is used as experimental drug therapy for a range of neurogenic urinary bladder disorders. The present study investigated the chemical coding of caudal mesenteric ganglion (CaMG) neurons supplying the porcine urinary bladder after intravesical RTX instillation. The CaMG neurons were visualized with retrograde tracer Fast Blue (FB) and their chemical profile was disclosed with double-labelling immunohistochemistry using antibodies against tyrosine hydroxylase (TH), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), calbindin (CB), galanin (GAL) and neuronal nitric oxide synthase (nNOS). It was found that in both the control (n = 6) and RTX-treated pigs (n = 6), the vast majority (92.3 ± 2.7% and 93.1 ± 1.3%, respectively) of FB-positive (FB+) nerve cells were TH+. Intravesical instillation of RTX caused a decrease in the number of FB+ / TH + neurons immunopositive to NPY (91.0 ± 2.2% in control animals vs. 58.8 ± 5.0% in RTX-treated pigs) or VIP (1.7 ± 0.5% vs. 0%) and an increase in the number of FB+ / TH+ neurons immunoreactive to SOM (3.4 ± 1.5% vs. 20.6 ± 4.3%), CB (1.8 ±0.7% vs. 13.4 ± 2.3%), GAL (1.5 ± 0.6% vs. 7.5 ± 1.0%) or nNOS (0% vs. 10.9 ± 3.4%). The present results suggest that therapeutic effects of RTX on the mammalian urinary bladder can be partly mediated by CaMG neurons.

First Generation Gene Expression Signature for Early Prediction of Late Occurring Hematological Acute Radiation Syndrome in Baboons.

We implemented a two-stage study to predict late occurring hematologic acute radiation syndrome (HARS) in a baboon model based on gene expression changes measured in peripheral blood within the first two days after irradiation. Eighteen baboons were irradiated to simulate different patterns of partial-body and total-body exposure, which corresponded to an equivalent dose of 2.5 or 5 Gy. According to changes in blood cell counts the surviving baboons (n = 17) exhibited mild (H1-2, n = 4) or more severe (H2-3, n = 13) HARS. Blood samples taken before irradiation served as unexposed control (H0, n = 17). For stage I of this study, a whole genome screen (mRNA microarrays) was performed using a portion of the samples (H0, n = 5; H1-2, n = 4; H2-3, n = 5). For stage II, using the remaining samples and the more sensitive methodology, qRT-PCR, validation was performed on candidate genes that were differentially up- or down-regulated during the first two days after irradiation. Differential gene expression was defined as significant (P < 0.05) and greater than or equal to a twofold difference above a H0 classification. From approximately 20,000 genes, on average 46% appeared to be expressed. On day 1 postirradiation for H2-3, approximately 2-3 times more genes appeared up-regulated (1,418 vs. 550) or down-regulated (1,603 vs. 735) compared to H1-2. This pattern became more pronounced at day 2 while the number of differentially expressed genes decreased. The specific genes showed an enrichment of biological processes coding for immune system processes, natural killer cell activation and immune response (P = 1 × E-06 up to 9 × E-14). Based on the P values, magnitude and sustained differential gene expression over time, we selected 89 candidate genes for validation using qRT-PCR. Ultimately, 22 genes were confirmed for identification of H1-3 classifications and seven genes for identification of H2-3 classifications using qRT-PCR. For H1-3 classifications, most genes were constantly three to fivefold down-regulated relative to H0 over both days, but some genes appeared 10.3-fold (VSIG4) or even 30.7-fold up-regulated (CD177) over H0. For H2-3, some genes appeared four to sevenfold up-regulated relative to H0 (RNASE3, DAGLA, ARG2), but other genes showed a strong 14- to 33-fold down-regulation relative to H0 (WNT3, POU2AF1, CCR7). All of these genes allowed an almost completely identifiable separation among each of the HARS categories. In summary, clinically relevant HARS can be independently predicted with all 29 irradiated genes examined in the peripheral blood of baboons within the first two days postirradiation. While further studies are needed to confirm these findings, this model shows potential relevance in the prediction of clinical outcomes in exposed humans and as an aid in the prioritizing of medical treatment.

Overview of the role of vitamins and minerals on the kynurenine pathway in health and disease.

The kynurenine pathway (KP) of L-tryptophan metabolism produces several neuroactive metabolites with an amino acid structure. These metabolites may play an important role in the pathophysiology of irritable bowel syndrome, Alzheimer's disease, Parkinson's disease, Huntington's disease, schizophrenia, AIDS-dementia complex, depression, epilepsy and the aging process. Modulation of the KP through inhibition or stimulation of enzyme synthesis and activity can be an alternative approach to traditional therapy. Furthermore, it may be responsible for the altered functioning of the enteric nervous system and the central nervous system. There is evidence that the KP is sensitive to changes in the concentration of many vitamins and minerals that play a crucial role as coenzymes and cofactors in the de novo synthesis of nicotinamide adenine dinucleotide coenzyme. A reduction in the availability of the active form of vitamin B6 (pyridoxal 5'-phosphate, PLP) is known to affect tryptophan hydroxylase, kynurenine aminotransferase and kynureninase (KYNU). Vitamin B2 deficiencies result in a reduction in the activity of the flavin adenine dinucleotide dependent enzyme, kynurenine 3-monooxygenase. Minerals are also responsible for the proper functioning of enzymes engaged in L-tryptophan metabolism. Mn(2+), Zn(2+), Co(2+) and Cu(2+) influence KYNU activity, and Mg(2+) regulates quinolinate phosphoribosyl transferase. Fe(2+) is responsible for the proper functioning of both indoleamine 2,3-dioxygenase and 3-hydroxy-anthranilic acid dioxygenase. Changes in the concentration of KP metabolites and in enzymatic activity have been found in many pathological states. Therefore, it is justifiable to regulate the concentration of certain kynurenines or enzymes in the KP which may provide a potential therapeutic target for the treatment of various health impairments. This review demonstrates the role of vitamin and mineral activity on the KP, which may have an effect on the proper functioning of the human organism. Surplus administration of vitamins did not elicit any beneficial effects on L-tryptophan metabolism. Whether a mineral surplus influences L-tryptophan metabolism is still not established. It seems that cofactor deficiencies influence the KP far more than surpluses.

Expression of aquaporin 1 and 5 and their regulation by ovarian hormones, arachidonic acid, forskolin and cAMP during implantation in pigs.

Aquaporin proteins (AQPs) are a family of channels expressed in numerous mammalian tissues, where they play a fundamental role in regulating water transport across cell membranes. Based on reports that AQPs are present in the reproductive system and participate in reproductive processes, our aim was to investigate the effect of progesterone (P(4)), estradiol (E(2)), oxytocin (OT), arachidonic acid (AA), forskolin (FSK) and cyclic adenosine monophosphate (cAMP) on AQP1 and AQP5 expression at mRNA and protein levels in porcine uterine explants from Days 14-16 of gestation in order to determine if they play a role in implantation period in pigs. Quantitative real time PCR and Western-blot analysis revealed that the uterine explants treated with FSK and cAMP produce delayed, but long-term effects on AQP1 abundance (24 h) while AQP5 had a rapid and sustained response to FSK and cAMP in protein content (3 and 24 h). AA increases gene and protein content of AQP1 after longer exposition whereas AQP5 increases after 3 h only at the protein level. Both AQPs potentially remains under control of steroid hormones. OT has been shown to increase AQP1, and decrease AQP5 mRNA, without visible changes in protein content. P(4), E(2), AA, FSK and cAMP caused the appearance of AQP5 expression in the basolateral plasma membrane of the epithelial cells. The staining represents most likely AQP5 functioning mechanism for both absorption and reabsorption across the glandular epithelium.