RNA Quality Control Using External Standard RNA

In this paper, we propose a new evaluation method using external standard RNA for quality control of the extracted RNA. RNA Integrity Number and UV absorption are generally used as a basis for RNA quality control; however, these methods do not always reflect the quality of mRNA. While standard RNA is supposedly designed on the basis of mRNA, it has the potential to be used to evaluate the quality of the mRNA. In this study, we took into consideration the three essential factors, viz., yield of mRNA, inhibition to DNA polymerase, and degradation of mRNA for determining the RNA quality using standard RNA. It would be possible to know yield of mRNA and inhibition of the enzyme reaction by adding standard RNA before RNA extraction and looking at standard RNA loss. Degradation was evaluated by comparing the differences in the 3’ and 5’ regions of the RNA. In our study, it was demonstrated that in the crude extract of Saccharomyces cerevisiae, degradation was comparatively higher at the 3’ end of RNA than at the 5’ end. Hence, the degree of RNA degradation can be evaluated by comparing the ratio of degradation from the 3’ and 5’ end

Characterization of Plantaricin Genes and Lactic Acid Production by Lactiplantibacillus plantarum strains Isolated from Ishizuchi-Kurocha

Background and Objective: Ishizuchi-kurocha is a post-fermented tea that involved two main kinds of microorganisms, namely fungi and lactic acid bacteria, which are primary and secondary fermentation, respectively. Therefore, this research aimed to confirm the role of Lactiplantibacillus plantarum during secondary fermentation of Ishizuchi-kurocha and the anti-bacterial effect due to lactic acid production and genes detection of plantaricin. Material and Methods: Antimicrobial were estimated using well diffusion method. Lactic acid was determined with spectrophotometric method. Detection of plantaricin genes were confirmed by Real-Time qPCR. The genes were sequenced through DNA Sequencing Analytical service by the Division of Genomic Research, Gifu University using the Multi-capillary DNA Sequencer ABI Prism 3100/3130 Genetic Analyzer and the data analyzed by the CLC Sequence Viewer 8.0 and BioEdit 7.2. Statistical analysis was evaluated by one-way of variance followed Tukey’s post hoc test using RStudio version 4.1.3. Results and Conclusion: L. plantarum strain IYO1511 has higher antibacterial activities than strain IYO1501. In addition, L. plantarum strain IYO1511 produced higher lactic acid than strain IYO1501 and has plantaricin genes, plnA, plnEF, plnN, plnJ and plnK. However, L. plantarum strain IYO1501 only encoded plnEF, plnN, and plnJ. The plantaricin genes from the strains IYO1501 and IYO1511 were sequenced to identify the heterologous gene clusters of each species. It was discovered that plnA, plnEF, and plnJ of L. plantarum IYO1511 showed 100% similarity homology toward GenBank. The plnN of strain IYO1511 and plnEF of IYO1501 present extra base pairs inserted into the DNA. L. plantarum strains can be used as food preservative for artificial fermentation to control the safety and quality of the product of Ishizuchi-kurocha.The lactic acid and plantaricin were expected to inhibit pathogenically and spoilage bacteria to produce a unique acidic flavor as well as fragrance to Ishizuchi‐kurocha. Conflict of interest: The authors declare no conflict of interest

DNA Strand Break Properties of Protoporphyrin IX by X-ray Irradiation against Melanoma

Recent reports have suggested that 5-aminolevulinic acid (5-ALA), which is a precursor to protoporphyrin IX (PpIX), leads to selective accumulation of PpIX in tumor cells and acts as a radiation sensitizer in vitro and in vivo in mouse models of melanoma, glioma, and colon cancer. In this study, we investigated the effect of PpIX under X-ray irradiation through ROS generation and DNA damage. ROS generation by the interaction between PpIX and X-ray was evaluated by two kinds of probes, 3′-(p-aminophenyl) fluorescein (APF) for hydroxyl radical (•OH) detection and dihydroethidium (DHE) for superoxide (O2•-). •OH showed an increase, regardless of the dissolved oxygen. Meanwhile, the increase in O2•- was proportional to the dissolved oxygen. Strand breaks (SBs) of DNA molecule were evaluated by gel electrophoresis, and the enhancement of SBs was observed by PpIX treatment. We also studied the effect of PpIX for DNA damage in cells by X-ray irradiation using a B16 melanoma culture. X-ray irradiation induced γH2AX, DNA double-strand breaks (DSBs) in the context of chromatin, and affected cell survival. Since PpIX can enhance ROS generation even in a hypoxic state and induce DNA damage, combined radiotherapy treatment with 5-ALA is expected to improve therapeutic efficacy for radioresistant tumors

An ancient but promiscuous host–symbiont association between Burkholderia gut symbionts and their heteropteran hosts

Here, we investigated 124 stinkbug species representing 20 families and 5 superfamilies for their Burkholderia gut symbionts, of which 39 species representing 6 families of the superfamilies Lygaeoidea and Coreoidea were Burkholderia-positive. Diagnostic PCR surveys revealed high frequencies of Burkholderia infection in natural populations of the stinkbugs, and substantial absence of vertical transmission of Burkholderia infection to their eggs. In situ hybridization confirmed localization of the Burkholderia in their midgut crypts. In the lygaeoid and coreoid stinkbugs, development of midgut crypts in their alimentary tract was coincident with the Burkholderia infection, suggesting that the specialized morphological configuration is pivotal for establishment and maintenance of the symbiotic association. The Burkholderia symbionts were easily isolated as pure culture on standard microbiological media, indicating the ability of the gut symbionts to survive outside the host insects. Molecular phylogenetic analysis showed that the gut symbionts of the lygaeoid and coreoid stinkbugs belong to a β-proteobacterial clade together with Burkholderia isolates from soil environments and Burkholderia species that induce plant galls. On the phylogeny, the stinkbug-associated, environmental and gall-forming Burkholderia strains did not form coherent groups, indicating host–symbiont promiscuity among these stinkbugs. Symbiont culturing revealed that slightly different Burkholderia genotypes often coexist in the same insects, which is also suggestive of host–symbiont promiscuity. All these results strongly suggest an ancient but promiscuous host–symbiont relationship between the lygaeoid/coreoid stinkbugs and the Burkholderia gut symbionts. Possible mechanisms as to how the environmentally transmitted promiscuous symbiotic association has been stably maintained in the evolutionary course are discussed

Reproducibility of fluorescent expression from engineered biological constructs in E. coli

We present results of the first large-scale interlaboratory study carried out in synthetic biology, as part of the 2014 and 2015 International Genetically Engineered Machine (iGEM) competitions. Participants at 88 institutions around the world measured fluorescence from three engineered constitutive constructs in E. coli. Few participants were able to measure absolute fluorescence, so data was analyzed in terms of ratios. Precision was strongly related to fluorescent strength, ranging from 1.54-fold standard deviation for the ratio between strong promoters to 5.75-fold for the ratio between the strongest and weakest promoter, and while host strain did not affect expression ratios, choice of instrument did. This result shows that high quantitative precision and reproducibility of results is possible, while at the same time indicating areas needing improved laboratory practices.Peer reviewe