Dose-rate effects of protons on in vivo activation of nuclear factor-kappa B and cytokines in mouse bone marrow cells

The objective of this study was to determine the kinetics of nuclear factor-kappa B (NF-κB) activation and cytokine expression in bone marrow (BM) cells of exposed mice as a function of the dose rate of protons. The cytokines included in this study are pro-inflammatory [i.e., tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), and IL-6] and anti-inflammatory cytokines (i.e., IL-4 and IL-10). We gave male BALB/cJ mice a whole-body exposure to 0 (sham-controls) or 1.0 Gy of 100 MeV protons, delivered at 5 or 10 mGy min⁻¹, the dose and dose rates found during solar particle events in space. As a reference radiation, groups of mice were exposed to 0 (sham-controls) or 1 Gy of ¹³⁷Cs γ rays (10 mGy min⁻¹). After irradiation, BM cells were collected at 1.5, 3, 24 h, and 1 month for analyses (five mice per treatment group per harvest time). The results indicated that the in vivo time course of effects induced by a single dose of 1 Gy of 100 MeV protons or ¹³⁷Cs γ rays, delivered at 10 mGy min⁻¹, was similar. Although statistically significant levels of NF-κB activation and pro-inflammatory cytokines in BM cells of exposed mice when compared to those in the corresponding sham controls (Student’s t-test, p < 0.05 or <0.01) were induced by either dose rate, these levels varied over time for each protein. Further, only a dose rate of 5 mGy min⁻¹ induced significant levels of anti-inflammatory cytokines. The results indicate dose-rate effects of protons.     

SSR and EST-SSR-based genetic linkage map of cassava (Manihot esculenta Crantz)

Simple sequence repeat (SSR) markers provide a powerful tool for genetic linkage map construction that can be applied for identification of quantitative trait loci (QTL). In this study, a total of 640 new SSR markers were developed from an enriched genomic DNA library of the cassava variety 'Huay Bong 60' and 1,500 novel expressed sequence tag-simple sequence repeat (EST-SSR) loci were developed from the Genbank database. To construct a genetic linkage map of cassava, a 100 F(1) line mapping population was developed from the cross Huay Bong 60 by 'Hanatee'. Polymorphism screening between the parental lines revealed that 199 SSRs and 168 EST-SSRs were identified as novel polymorphic markers. Combining with previously developed SSRs, we report a linkage map consisted of 510 markers encompassing 1,420.3?cM, distributed on 23 linkage groups with a mean distance between markers of 4.54?cM. Comparison analysis of the SSR order on the cassava linkage map and the cassava genome sequences allowed us to locate 284 scaffolds on the genetic map. Although the number of linkage groups reported here revealed that this F(1) genetic linkage map is not yet a saturated map, it encompassed around 88% of the cassava genome indicating that the map was almost complete. Therefore, sufficient markers now exist to encompass most of the genomes and efficiently map traits in cassava.     

Comparison of Immunogenicity and Safety of Four Doses and Four Double Doses vs. Standard Doses of Hepatitis B Vaccination in HIV-Infected Adults: A Randomized,Controlled Trial

Background

HBV vaccination is recommended in HIV-infected adults with CD4+ cell count >200/mm³ although the efficacy is only 33.3% -65%. We conducted a randomized, controlled trial to evaluate the efficacy and safety of three regimens of HBV vaccination at Chiang Mai University Hospital, Thailand.

Methods

From February 4, 2011 to May 4, 2012, 132 HIV-infected adults with CD4+ cell counts >200 cells/mm³, undetectable plasma HIV-1 RNA, and negative for all HBV markers were randomly assigned to receive one of three recombinant vaccine (Hepavax-Gene^® Berna, Korea) regimens: 20 μg IM at months 0, 1, and 6 (Standard doses group, n=44), 20 μg IM at months 0, 1, 2, 6 (four doses group, n=44), or 40 μg IM at months 0, 1, 2, and 6 (four double doses group, n=44). The primary outcomes were to compare the immunogenicity and safety between the four-doses groups with the Standard doses group.

Results

At months 7 and 12, the percentages of responders (anti-HBs ≥10 mIU/mL) were 88.6% and 70.4% in the Standard doses group, 93.2% and 86.4% in the four doses group, (P=0.713 and 0.119), and 95.4% and 88.6% in the four double doses group, (P=0.434 and 0.062), respectively. Factors associated with a high titer level (anti-HBs ≥100 mIU/mL) were vaccination schedule and younger age. The most common adverse event was pain at the injection site (42.4%); this was significantly more frequent in the four double doses group compared to the Standard doses group. No serious adverse events were observed.

Conclusions

In Northern Thailand, the standard three-doses HBV vaccination in HIV-infected adults with CD4+ cell counts >200 cells/mm³ and undetectable plasma HIV-1 RNA is highly effective. Although regimens of four injections of either standard or double doses could not significantly increase the response rate, these regimens may induce higher levels of antibody to the virus.Trial registration information: ClinicalTrials.gov; NCT1289106; http://clinicaltrials.gov/ct2/show/NCT01289106     

Persistence of apoptosis and inflammatory responses in the heart and bone marrow of mice following whole-body exposure to 28Silicon (28Si) ions

It has been well established that the bone marrow (BM) is a radiosensitive tissue, but the radiosensitivity of the heart is poorly understood. In this study, we investigated the comparative effects of ²⁸Silicon (²⁸Si) ions (one type of heavy ion found in space) on tissue from the heart and the BM of exposed mice. 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 300 MeV/nucleon (n) ²⁸Si ions, using a fractionated schedule (two exposures, 15 days apart that totaled each selected dose). The heart and BM were collected from 5 mice per treatment group at various times up to 6 months post-irradiation. In each mouse, we obtained tissue lysates from the heart and from the total population of BM cells for measuring the levels of cleaved poly (ADP-ribose) polymerase (cleaved PARP, a marker of apoptotic cell death) and the levels of activated nuclear factor-kappa B (NF-κB) and selected NF-κB-regulated cytokines known to be involved in inflammatory responses. Our data showed that, up to 6 months post-irradiation, the levels of apoptotic cell death and inflammatory responses in tissues from the heart and BM collected from exposed mice were statistically higher than those in sham controls. Hence, these findings are suggestive of chronic apoptotic cell death and inflammation in both tissues after exposure to ²⁸Si ions. In summary, our data are indicative of a possible association between exposure to ²⁸Si ions during space flight and long-term health risk.     

Crystal structure of BinB: A receptor binding component of the binary toxin from Lysinibacillus sphaericus

The binary toxin (Bin), produced by Lysinibacillus sphaericus, is composed of BinA (42 kDa) and BinB (51 kDa) proteins, which are both required for full toxicity against Culex and Anopheles mosquito larvae. Specificity of Bin toxin is determined by the binding of BinB component to a receptor present on the midgut epithelial membranes, while BinA is proposed to be a toxic component. Here, we determined the first crystal structure of the active form of BinB at a resolution of 1.75 Å. BinB possesses two distinct structural domains in its N‐ and C‐termini. The globular N‐terminal domain has a β‐trefoil scaffold which is a highly conserved architecture of some sugar binding proteins or lectins, suggesting a role of this domain in receptor‐binding. The BinB β‐rich C‐terminal domain shares similar three‐dimensional folding with aerolysin type β‐pore forming toxins, despite a low sequence identity. The BinB structure, therefore, is a new member of the aerolysin‐like toxin family, with probably similarities in the cytolytic mechanism that takes place via pore formation. Proteins 2014; 82:2703–2712. © 2014 Wiley Periodicals, Inc.     

Loop‐mediated isothermal amplification assay targeting the blaCTX‐M9 gene for detection of extended spectrum β‐lactamase‐producing Escherichia coli and Klebsiella pneumoniae

Extended‐spectrum β‐lactamases (ESBLs) produced by Enterobacteriaceae are one of the resistance mechanisms to most β‐lactam antibiotics. ESBLs are currently a major problem in both hospitals and community settings worldwide. Rapid and reliable means of detecting ESBL‐producing bacteria is necessary for identification, prevention and treatment. Loop‐mediated isothermal amplification (LAMP) is a technique that rapidly amplifies DNA with high specificity and sensitivity under isothermal conditions. This study was aimed to develop a convenient, accurate and inexpensive method for detecting ESBL‐producing bacteria by a LAMP technique. ESBLs‐producing Escherichia coli and Klebsiella pneumoniae were isolated from a tertiary hospital in Bangkok, Thailand and reconfirmed by double‐disk synergy test. A set of four specific oligonucleotide primers of LAMP for detection of bla_CTX‐M9 gene was designed based on bla_CTX‐M9 from E. coli (GenBank Accession No. AJ416345). The LAMP reaction was amplified under isothermal temperature at 63°C for 60 min. Ladder‐like patterns of band sizes from 226 bp of the bla_CTX‐M9 DNA target was observed. The LAMP product was further analyzed by restriction digestion with MboI and TaqI endonucleases. The fragments generated were approximately 168, 177 and 250 bp in size for MboI digestion and 165, 193, 229, 281 and 314 bp for TaqI digestion, which is in agreement with the predicted sizes. The sensitivity of the LAMP technique to bla_CTX‐M9 was greater than that of the PCR method by at least 10,000‐fold. These results showed that the LAMP primers specifically amplified only the bla_CTX‐M9 gene. Moreover, the presence of LAMP amplicon was simply determined by adding SYBR Green I in the reaction. In conclusion, this technique for detection of ESBLs is convenient, reliable and easy to perform routinely in hospitals or laboratory units in developing countries.     

Development of simple sequence repeat markers from expressed sequence tags of the black tiger shrimp (Penaeus monodon)

In this study, we describe the development of expressed sequence tag-simple sequence repeat markers from expressed sequence tags of the black tiger shrimp (Penaeus monodon) deposited in public sequence databases. A total of 46 primer pairs were designed and screened on 26 individuals of P. monodon from a natural population. Of these, 16 primer pairs showed polymorphic profiles with between two and five alleles per locus. The average unbiased and direct count heterozygosities were 0.4662 and 0.3516, respectively. Cross-amplification was tested with five individuals of Penaeus vannamei and polymorphic products were detected at five loci.