Characterization of levansucrase produced by novel Bacillus siamensis and optimization of culture condition for levan biosynthesis.

Levan has attracted interest due to the potential health benefits associated with its prebiotic, biological, and functional properties. However, the production of levan is expensive due to its high resource requirements. With the growing demand for levan, it is vital to determine suitable cultivation condition for its production and reduce costs accordingly. The present study characterized the enzyme levansucrase produced by a novel strain of Bacillus siamensis and optimized the conditions for the biosynthesis of levansucrase and levan. The crude levansucrase enzyme production by B. siamensis was induced at a specific temperature in a medium containing different concentrations of sucrose, fructose, and glucose to evaluate transfructosylation and hydrolysis activities. Crude levansucrase significantly increased transfructosylation relative to hydrolysis activity at 37 °C in a medium containing 20% (w/v) sucrose. Both transfructosylation and hydrolysis activities were inhibited in glucose and fructose containing medium. Purification and characterization of the levansucrase were performed by precipitating the enzyme with ammonium sulfate solution, purified anion-exchange chromatography, and analyzed by Sodium Dodecyl Sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed the molecular weight of the enzyme to be approximately 30 kDa with specific activity at 15.95 U/mg, corresponding to a protein purification efficiency of 11.47 and a yield of 78.75%. The optimal culture condition for the purified-levansucrase activity for levan biosynthesis was obtained at 37 °C after 48 h, at pH 6.0 in 50 mM phosphate buffer and 20% (w/v) sucrose. The study demonstrated the optimized condition for levan biosynthesis utilizing the B. siamensis that can serve as a model for various commercial and industrial applications for efficient levan production.

Early- and late-occurring damage in bone marrow cells of male CBA/Ca mice exposed whole-body to 1 GeV/n 48Ti ions.

PURPOSE: To determine the early- and late-occurring damage in the bone marrow (BM) and peripheral blood cells of male CBA/Ca mice after exposure to 0, 0.1, 0.25, or 0.5 Gy of 1 GeV/n titanium (48Ti) ions (one type of space radiation). METHOD: We used the mouse in vivo blood-erythrocyte micronucleus (MN) assay for evaluating the cytogenetic effects of various doses of 1 GeV/n 48Ti ions. The MN assay was coupled with the characterization of epigenetic alterations (the levels of global 5-methylcytosine and 5-hydroxymethylcytosine) in DNA samples isolated from BM cells. These analyses were performed in samples collected at an early time-point (1 week) and a late time-point (6 months) post-irradiation. RESULTS: Our results showed that 48Ti ions induced genomic instability in exposed mice. Significant dose-dependent loss of global 5-hydroxymethylcytosine was found but there were no changes in global 5-methylcytosine levels. CONCLUSION: Since persistent genomic instability and loss of global 5-hydroxymethylcytosine are linked to cancer, our findings suggest that exposure to 48Ti ions may pose health risks.

Persistent depletion of plasma gelsolin (pGSN) after exposure of mice to heavy silicon ions.

Little is known about plasma proteins that can be used as biomarkers for early and late responses to radiation. The purpose of this study was to determine a link between depletion of plasma gelsolin (pGSN) and cell-death as well as inflammatory responses in the lung (one of the tissues known to be radiosensitive) of the same exposed CBA/CaJ mice after exposure to heavy silicon (28Si) ions. To prevent the development of multiple organ dysfunctions, pGSN (an important component of the extracellular actin-scavenging system) is responsible for the removal of actin that is released into the circulation during inflammation and from dying cells. We evaluated the levels of pGSN in plasma collected from groups of mice (5 mice in each) at 1 week (wk) and 1 month (1 mo) after exposure whole body to different doses of 28Si ions, i.e. 0, 0.1, 0.25, or 0.5 Gy (2 fractionated exposures, 15 days apart that totaled each selected dose). In the same mouse, the measurements of pGSN levels were coupled with the quantitation of injuries in the lung, determined by (a) the levels of cleaved poly (ADP-ribose) polymerase (cleaved-PARP), a marker of apoptotic cell-death, (b) the levels of activated nuclear factor-kappa B (NF-κB) and selected cytokines, i.e. tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), and IL-6, from tissue-lysates of the lung. Further, the ratio of neutrophils and lymphocytes (N/L) was determined in the same mouse. Our data indicated: (i) the magnitude of pGSN depletion was dependent to radiation dose at both harvest times, (ii) a persistent depletion of pGSN up to 1 mo post-exposure to 0.25 or 0.5 Gy of 28Si ions, (iii) an inverse-correlation between pGSN depletion and increased levels of cleaved-PARP, including activated NF-κB/pro-inflammatory cytokines in the lung, and (iv) at both harvest times, statistically significant increases in the N/L ratio in groups of mice exposed to 0.5 Gy only. Our findings suggested that depletion in pGSN levels reflects not only the responses to 28Si-ion exposure at both harvest times but also early and late-occurring damage.

Late Effects of Low-Dose Radiation on the Bone Marrow, Lung, and Testis Collected From the Same Exposed BALB/cJ Mice.

We used 3 biological metrics highly relevant to health risks, that is, cell death, inflammation, and global DNA methylation, to determine the late effects of low doses (0.05 or 0.1 Gy) of 137Cs γ rays on the bone marrow, lung, and testis collected at 6 months post-irradiation from the same exposed BALB/cJ mouse. This integrative approach has not been used for such a purpose. Mice exposed to 0 or 1 Gy of radiation served as a sham or positive control group, respectively. The results could deliver information for better health risk assessment across tissues, including better scientific basis for radiation protection and clinical application. We found no changes in the levels of all studied biological metrics (except a significant increase in the levels of an anti-inflammatory cytokine, ie, interleukin 10) in tissues of 0.05-Gy exposed mice, when compared to those in sham controls. In contrast, significantly increased levels of cell death and inflammation, including a significant loss of global 5-hydroxymethylcytosine, were found in all tissues of the same mice exposed to 0.1 or 1.0 Gy. Our data demonstrated not only no harm but also hormesis in the 0.05-Gy exposed mice. However, the hormetic effect appears to be dependent on biological metrics and tissue.

Induction of Chronic Inflammation and Altered Levels of DNA Hydroxymethylation in Somatic and Germinal Tissues of CBA/CaJ Mice Exposed to (48)Ti Ions.

Although the lung is one of the target organs at risk for cancer induction from exposure to heavy ions found in space, information is insufficient on cellular/molecular responses linked to increased cancer risk. Knowledge of such events may aid in the development of new preventive measures. Furthermore, although it is known that germinal cells are sensitive to X- or γ-rays, there is little information on the effects of heavy ions on germinal cells. Our goal was to investigate in vivo effects of 1 GeV/n (48)Ti ions (one of the important heavy ions found in the space environment) on somatic (lung) and germinal (testis) tissues collected at various times after a whole body irradiation of CBA/CaJ mice (0, 0.1, 0.25, or 0.5 Gy, delivered at 1 cGy/min). We hypothesized that (48)Ti-ion-exposure induced damage in both tissues. Lung tissue was collected from each mouse from each treatment group at 1 week, 1 month, and 6 months postirradiation. For the testis, we collected samples at 6 months postirradiation. Hence, only late-occurring effects of (48)Ti ions in the testis were studied. There were five mice per treatment group at each harvest time. We investigated inflammatory responses after exposure to (48)Ti ions by measuring the levels of activated nuclear factor kappa B and selected pro-inflammatory cytokines in both tissues of the same mouse. These measurements were coupled with the quantitation of the levels of global 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Our data clearly showed the induction of chronic inflammation in both tissues of exposed mice. A dose-dependent reduction in global 5hmC was found in the lung at all time-points and in testes collected at 6 months postirradiation. In contrast, significant increases in global 5mC were found only in lung and testes collected at 6 months postirradiation from mice exposed to 0.5 Gy of 1 GeV/n (48)Ti ions. Overall, our data showed that (48)Ti ions may create health risks in both lung and testicular tissues.

Late-occurring chromosome aberrations and global DNA methylation in hematopoietic stem/progenitor cells of CBA/CaJ mice exposed to silicon ((28)Si) ions.

Although myeloid leukemia (ML) is one of the major health concerns from exposure to space radiation, the risk prediction for developing ML is unsatisfactory. To increase the reliability of predicting ML risk, a much improved understanding of space radiation-induced changes in the target cells, i.e. hematopoietic stem/progenitor cells (HSPCs), is important. We focused on the in vivo induction of late-occurring damage in HSPCs of mice exposed to (28)Si ions since such damage is associated with radiation-induced genomic instability (a key event of carcinogenesis). We gave adult male CBA/CaJ mice, known to be sensitive to radiation-induced ML, a whole-body exposure (2 fractionated exposures, 15 days apart, that totaled each selected dose, delivered at the dose-rate of 1 cGy/min) to various doses of 300 MeV/n (28)Si ions, i.e. 0 (sham controls), 0.1, 0.25, or 0.5 Gy. At 6 months post-irradiation, we collected bone marrow cells from each mouse (five mice per treatment-group) for obtaining the myeloid-lineage of HSPC-derived clones for analyses. We measured the frequencies of late-occurring chromosome aberrations (CAs), using the genome-wide multicolor fluorescence in situ hybridization method. The measurement of CAs was coupled with the characterization of the global DNA methylation patterns, i.e. 5-methylcytosine (5 mC) and 5-hydroxymethylcytosine (5 hmC). A dose-dependent increase in the frequencies of CAs was detected (Analysis of Variance or ANOVA, p<0.01), indicating the induction of genomic instability after exposure of mice to 300 MeV/n (28)Si ions. Slight increases in the levels of 5 mC were observed in all treatment groups, as compared to the sham-control level. In contrast, there was a significant reduction in levels of 5 hmC (ANOVA, p<0.01). Since these endpoints were evaluated in the same mouse, our data suggested for the first time a link between a reduction in 5 hmC and genomic instability in HSPC-derived myeloid colonies of CBA/CaJ mice exposed to 300 MeV/n (28)Si ions.

Induction of chronic oxidative stress, chronic inflammation and aberrant patterns of DNA methylation in the liver of titanium-exposed CBA/CaJ mice.

PURPOSE: To investigate the biological effects of titanium ((48)Ti, one of the important heavy ions found in space) in the liver of exposed-mice. MATERIALS AND METHODS: We gave adult male CBA/CaJ mice a whole-body exposure to a total dose of 0, 0.1, 0.25 or 0.5 Gy of (48)Ti ions. The liver was collected at 1 week, 1 month, and 6 months post-irradiation (five mice per treatment-group at each harvest-time). Three biological endpoints were used for evaluating the effects of (48)Ti ions: Oxidative-stress, inflammatory responses, and DNA-methylation (5-methylcytosine and 5-hydroxymethylcytosine). RESULTS: Our data clearly demonstrated dose-dependent increases in oxidative stress and inflammatory responses in the liver of exposed mice at all time-points (Analysis of Variance or ANOVA, p < 0.05). Significant dose-dependent increases in the levels of 5-methylcytosine were detected at 1 week and 1 month (ANOVA, p < 0.05). At 6 months post-irradiation, a significant increase in the level of 5-methylcytosine was found only in 0.5-Gy-(48)Ti-ion-exposed mice. In contrast, dose-dependent decreases in 5-hydroxymethylcytosine levels were found in the liver of exposed mice (ANOVA, p < 0.05) at all time-points. CONCLUSIONS: Chronic oxidative-stress, chronic inflammation, and persistent aberrant DNA-methylation occurred in the liver of (48)Ti-exposed mice. Hence, exposure to (48)Ti ions in space may pose health risks.

Proteomic Profiling of Hematopoietic Stem/Progenitor Cells after a Whole Body Exposure of CBA/CaJ Mice to Titanium (48Ti) Ions.

Myeloid leukemia (ML) is one of the major health concerns from exposure to radiation. However, the risk assessment for developing ML after exposure to space radiation remains uncertain. To reduce the uncertainty in risk prediction for ML, a much increased understanding of space radiation-induced changes in the target cells, i.e., hematopoietic stem/progenitor cells (HSPCs), is critically important. We used the label-free quantitative mass spectrometry (LFQMS) proteomic approach to determine the expression of protein in HSPC-derived myeloid colonies obtained at an early time-point (one week) and a late time-point (six months) after an acute whole body exposure of CBA/CaJ mice to a total dose of 0, 0.1, 0.25, or 0.5 Gy of heavy-ion titanium (48Ti ions), which are the important component of radiation found in the space environment. Mice exposed to 0 Gy of 48Ti ions served as non-irradiated sham controls. There were five mice per treatment groups at each harvest time. The Trans-Proteomic Pipeline (TPP) was used to assign a probability of a particular protein being in the sample. A proof-of-concept based Ingenuity Pathway Analysis (IPA) was used to characterize the functions, pathways, and networks of the identified proteins. Alterations of expression levels of proteins detected in samples collected at one week (wk) post-irradiation reflects acute effects of exposure to 48Ti ions, while those detected in samples collected at six months (mos) post-irradiation represent protein expression profiles involved in the induction of late-occurring damage (normally referred to as genomic instability). Our results obtained by using the IPA analyses indicate a wide array of signaling pathways involved in response to 1 GeV/n 48Ti ions at both harvest times. Our data also demonstrate that the patterns of protein expression profiles are dose and time dependent. The majority of proteins with altered expression levels are involved in cell cycle control, cellular growth and proliferation, cell death and survival, cell-to-cell signaling and interaction. The IPA analyses indicate several important processes involved in responses to exposure to 48Ti ions. These include the proteosme/ubiquination, protein synthesis, post-translation modification, and lipid metabolism. The IPA analyses also indicate that exposure to 1 GeV/n 48Ti ions affects the development and function of hematological system, immune cell trafficking, including the cytoskeleton. Further, the IPA analyses strongly demonstrate that the NF-κB and MAPKs (ERKs, JNKs, and p38MAPK) pathways play an essential role in signal transduction after exposure to 1 GeV/n 48Ti ions. At an early time-point (1 week), the top networks identified by the IPA analyses are related to metabolic disease, lipid metabolism, small molecule biochemistry, and development disorder. In contrast, the top networks identified in samples collected at a late time-point (6 mos post-irradiation) by the IPA analyses are related to cancer, hematological disorders, and immunological diseases. In summary, the proteomic findings from our study provide a foundation to uncover compounds potentially be highly effective in radiation countermeasures.

Burkholderia pseudomallei RpoS regulates OxyR and the katG-dpsA operon under conditions of oxidative stress.

Burkholderia pseudomallei, the causative agent of the potentially fatal tropical disease melioidosis, is known to be highly resistant to oxidative stress although the mechanism of this resistance remains to be fully elucidated. Previous studies have shown that an OxyR is involved in the regulation of oxidative stress via the katG and dpsA genes encoding KatG and DpsA and that the alternative sigma factor, RpoS, plays a critical role in resistance to oxidative stress by regulating katG and katE genes. Here it is shown that RpoS is essential for expression of the oxidative stress regulator OxyR, since a mutant strain lacking RpoS failed to induce oxyR expression both during normal growth and under conditions of oxidative stress. It is further demonstrated that the RpoS acts as a positive transcriptional regulator of oxyR and dpsA expression, while OxyR acts as a negative transcriptional regulator of the katG-dpsA operon via OxyR repressor under normal growth conditions, and as a positive transcriptional regulator via OxyR under conditions of oxidative stress. Therefore both RpoS and OxyR are required to promote expression of both the katG-dpsA operon and dpsA gene.