Clear zone formation in microdroplets for high-throughput screening for lactic acid bacteria.

Droplet microfluidic-based technology is a powerful tool for biotechnology, and it is also expected that it will be applied to culturing and screening methods. Using this technology, a new high-throughput screening method for lactic acid bacteria was developed. In this study, the conventional culture of lactic acid bacteria that form clear zones on an agar medium was reproduced in water-in-oil droplets, and only the droplets in which lactic acid bacteria grew were collected one by one. Using this method, the specific recovery of Lactiplantibacillus plantarum from a mixture of L. plantarum and Escherichia coli and the acquirement of lactic acid bacteria from an environmental sample were successful. This method could be applied to various conventional screening methods using the clear zone as a microbial growth indicator. This has expanded the possibilities of applying droplet microfluidic-based technology to microbial cultivations.

Transcriptome and Deletion Mutant Analyses Revealed that an RpoH Family Sigma Factor Is Essential for Photosystem Production in Roseateles depolymerans under Carbon Starvation.

Roseateles depolymerans is an obligately aerobic bacterium that produces a photosynthetic apparatus only under the scarcity of carbon substrates. We herein examined changes in the transcriptomes of R. depolymerans cells to clarify the expression of photosynthesis genes and their upstream regulatory factors under carbon starvation. Transcriptomes 0, 1, and 6| |h after the depletion of a carbon substrate indicated that transcripts showing the greatest variations (a 500-fold increase [6 h/0 h]) were light-harvesting proteins (PufA and PufB). Moreover, loci with more than 50-fold increases (6 h/0| |h) were fully related to the photosynthetic gene cluster. Among 13 sigma factor genes, the transcripts of a sigma 70 family sigma factor related to RpoH (SP70) increased along photosynthesis genes under starvation; therefore, a knockout experiment of SP70 was performed. ΔSP70 mutants were found to lack photosynthetic pigments (carotenoids and bacteriochlo-rophyll a) regardless of carbon starvation. We also examined the effects of heat stress on ΔSP70 mutants, and found that SP70 was also related to heat stress tolerance, similar to other RpoH sigma factors (while heat stress did not trigger photosystem production). The deficient accumulation of photosynthetic pigments and the heat stress tolerance of ΔSP70 mutants were both complemented by the introduction of an intact SP70 gene. Furthermore, the transcription of photosynthetic gene operons (puf, puh, and bch) was markedly reduced in the ΔSP70 mutant. The RpoH homologue SP70 was concluded to be a sigma factor that is essential for the transcription of photosynthetic gene operons in R. depolymerans.

A transchromosomic rat model with human chromosome 21 shows robust Down syndrome features.

Progress in earlier detection and clinical management has increased life expectancy and quality of life in people with Down syndrome (DS). However, no drug has been approved to help individuals with DS live independently and fully. Although rat models could support more robust physiological, behavioral, and toxicology analysis than mouse models during preclinical validation, no DS rat model is available as a result of technical challenges. We developed a transchromosomic rat model of DS, TcHSA21rat, which contains a freely segregating, EGFP-inserted, human chromosome 21 (HSA21) with >93% of its protein-coding genes. RNA-seq of neonatal forebrains demonstrates that TcHSA21rat expresses HSA21 genes and has an imbalance in global gene expression. Using EGFP as a marker for trisomic cells, flow cytometry analyses of peripheral blood cells from 361 adult TcHSA21rat animals show that 81% of animals retain HSA21 in >80% of cells, the criterion for a "Down syndrome karyotype" in people. TcHSA21rat exhibits learning and memory deficits and shows increased anxiety and hyperactivity. TcHSA21rat recapitulates well-characterized DS brain morphology, including smaller brain volume and reduced cerebellar size. In addition, the rat model shows reduced cerebellar foliation, which is not observed in DS mouse models. Moreover, TcHSA21rat exhibits anomalies in craniofacial morphology, heart development, husbandry, and stature. TcHSA21rat is a robust DS animal model that can facilitate DS basic research and provide a unique tool for preclinical validation to accelerate DS drug development.

Microdroplet-based system for culturing of environmental microorganisms using FNAP-sort.

Droplet microfluidics has emerged as a powerful technology for improving the culturing efficiency of environmental microorganisms. However, its widespread adoption has been limited due to considerable technical challenges, especially related to identification and manipulation of individual growth-positive droplets. Here, we combined microfluidic droplet technology with on-chip "fluorescent nucleic acid probe in droplets for bacterial sorting" (FNAP-sort) for recovery of growth-positive droplets and droplet microdispensing to establish an end-to-end workflow for isolation and culturing of environmental microbes. As a proof-of-concept, we demonstrate the ability of our technique to yield high-purity cultures of rare microorganisms from a representative complex environmental microbiome. As our system employs off-the-shelf commercially available equipment, we believe that it can be readily adopted by others and may thus find widespread use toward culturing the high proportion of as-of-yet uncultured microorganisms in different biomes.

A non-mosaic transchromosomic mouse model of down syndrome carrying the long arm of human chromosome 21.

Animal models of Down syndrome (DS), trisomic for human chromosome 21 (HSA21) genes or orthologs, provide insights into better understanding and treatment options. The only existing transchromosomic (Tc) mouse DS model, Tc1, carries a HSA21 with over 50 protein coding genes (PCGs) disrupted. Tc1 is mosaic, compromising interpretation of results. Here, we "clone" the 34 MB long arm of HSA21 (HSA21q) as a mouse artificial chromosome (MAC). Through multiple steps of microcell-mediated chromosome transfer, we created a new Tc DS mouse model, Tc(HSA21q;MAC)1Yakaz ("TcMAC21"). TcMAC21 is not mosaic and contains 93% of HSA21q PCGs that are expressed and regulatable. TcMAC21 recapitulates many DS phenotypes including anomalies in heart, craniofacial skeleton and brain, molecular/cellular pathologies, and impairments in learning, memory and synaptic plasticity. TcMAC21 is the most complete genetic mouse model of DS extant and has potential for supporting a wide range of basic and preclinical research.

Development of certified reference material NMIJ CRM 6205-a for the validation of DNA quantification methods: accurate mass concentrations of 600-bp DNA solutions having artificial sequences.

Two 600-bp DNA solutions (DNA600-G and DNA600-T) were developed as certified reference material, NMIJ CRM 6205-a, for the validation of DNA quantification methods. Both DNA600-G and DNA600-T are ideal as "spike-in control" because these materials have artificial nucleic acid sequences. The certified values were determined as the mass concentration of total DNA (whole DNA materials in sample solution regardless of sequence) at 25 °C by formic acid hydrolysis/liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) and inductively coupled plasma-mass spectrometry (ICP-MS) based on the amount of phosphorus. DNAs were synthesized, and plasmids including the synthesized DNAs were cloned into Escherichia coli DH5α. The amplified plasmids were digested with a restriction enzyme and highly purified. Then, the purified DNAs were diluted with water to approximately 1 ng/µL. By using the CRM-validated methods in fields where DNA quantification is required, the reliability of DNA quantification could be improved. Graphical abstract.

Fluorescent nucleic acid probe in droplets for bacterial sorting (FNAP-sort) as a high-throughput screening method for environmental bacteria with various growth rates.

We have developed a new method for selectively sorting droplets containing growing bacteria using a fluorescence resonance energy transfer (FRET)-based RNA probe. Bacteria and the FRET-based RNA probe are encapsulated into nanoliter-scale droplets, which are incubated to allow for cell growth. The FRET-based RNA probe is cleaved by RNase derived from the bacteria propagated in the droplets, resulting in an increase in fluorescence intensity. The fluorescent droplets containing growing bacteria are distinguishable from quenching droplets, which contain no cells. We named this method FNAP-sort based on the use of a fluorescent nucleic acid probe in droplets for bacterial sorting. Droplets containing the FRET-based RNA probe and four species of pure cultures, which grew in the droplets, were selectively enriched on the basis of fluorescence emission. Furthermore, fluorescent droplets were sorted from more than 500,000 droplets generated using environmental soil bacteria and the FRET-based RNA probe on days 1, 3, and 7 with repeated incubation and sorting. The bacterial compositions of sorted droplets differed on days 1, 3, and 7; moreover, on day 7, the bacterial composition of the fluorescent droplets was drastically different from that of the quenching droplets. We believe that FNAP-sort is useful for high-throughput cultivation and sorting of environmental samples containing bacteria with various growth rates, including slow-growing microbes that require long incubation times.

Ecological impact assessment of a bioaugmentation site on remediation of chlorinated ethylenes by multi-omics analysis.

Bioremediation may affect the ecological system around bioremediation sites. However, little is known about how microbial community structures change over time after the initial injection of degraders. In this study, we have assessed the ecological impact of bioaugmentation using metagenomic and metatranscriptomic approaches to remove trichlorinated ethylene/cis-dichloroethylene (TCE/cDCE) by Rhodococcus jostii strain RHA1 as an aerobic chemical compound degrader. Metagenomic analysis showed that the number of organisms belonging to the genus Rhodococcus, including strain RHA1, increased from 0.1% to 76.6% of the total microbial community on day 0 at the injection site. Subsequently, the populations of strain RHA1 and other TCE/cDCE-degrading bacteria gradually decreased over time, whereas the populations of the anaerobic dechlorinators Geobacter and Dehalococcoides increased at later stages. Metatranscriptomic analysis revealed a high expression of aromatic compound-degrading genes (bphA1-A4) in strain RHA1 after RHA1 injection. From these results, we concluded that the key dechlorinators of TCE/cDCE were mainly aerobic bacteria, such as RHA1, until day 1, after which the key dechlorinators changed to anaerobic bacteria, such as Geobacter and Dehalococcocides, after day 6 at the injection well. Based on the α-diversity, the richness levels of the microbial community were increased after injection of strain RHA1, and the microbial community composition had not been restored to that of the original composition during the 19 days after treatment. These results provide insights into the assessment of the ecological impact and bioaugmentation process of RHA1 at bioremediation sites.

Effects of endothelin-related gene polymorphisms and aerobic exercise habit on age-related arterial stiffening: a 10-yr longitudinal study.

Increased arterial stiffness has emerged as a strong predictor of future cardiovascular events and all-cause mortality. The aim of this study was to elucidate influences of endothelin (ET)-related genetic polymorphisms and regular physical activity on age-related arterial stiffening through a 10-yr longitudinal study. A decadal change in brachial-ankle pulse wave velocity (baPWV), an index of arterial stiffness, was evaluated retrospectively among 92 volunteers (63 ± 14 yr, 51 men). The targeted single-nucleotide polymorphisms were ET-A receptor SNP rs5333 (ET-A) and ET-B receptor SNP rs5351 (ET-B). Subjects with either ET-A TC or CC genotypes exhibited significantly greater increases in baPWV (+15.3 ± 11.7 and +16.6 ± 15.7%/dec, respectively) than ET-A TT genotype holders (+9.2 ± 9.0%/dec), whereas subjects with the ET-B GG genotype showed a significantly greater increase in baPWV (+17.7 ± 14.1%/dec) than other ET-B genotype holders (AA: +9.5 ± 10.0%/dec; AG: +11.2 ± 9.6%/dec). The combination of these ET-related genetic risks was associated with a 2.4 times greater decadal increase in baPWV compared with no genetic risk (+8.1 ± 8.4 vs. 19.5 ± 16.0%/dec). In contrast, individuals engaging in >15 METs·h/wk of aerobic exercise showed substantially smaller increases in baPWV (+5.0 ± 9.7%/dec) compared with their physically inactive peers (approximately +13%/dec). These differences remained significant after adjusting for confounding factors, including baseline baPWV and ET-related genotype risk. Our current longitudinal study found that ET-related gene polymorphisms contribute to diverse age-related changes in arterial stiffness, and that regular sufficient aerobic exercise attenuates the age-related arterial stiffening independently of ET-related gene polymorphisms. NEW & NOTEWORTHY This 10-yr longitudinal study suggests that endothelin-related gene polymorphisms contribute to divergent increases in arterial stiffness with advancing age, whereas regular sufficient aerobic exercise attenuates age-related arterial stiffening independently of ET-related gene polymorphisms. This notion partly supports prevailing evidence that regular aerobic exercise contributes to a lower incidence of cardiovascular disease.

Synthetic spike-in standards for high-throughput 16S rRNA gene amplicon sequencing.

High-throughput sequencing of 16S rRNA gene amplicons (16S-seq) has become a widely deployed method for profiling complex microbial communities but technical pitfalls related to data reliability and quantification remain to be fully addressed. In this work, we have developed and implemented a set of synthetic 16S rRNA genes to serve as universal spike-in standards for 16S-seq experiments. The spike-ins represent full-length 16S rRNA genes containing artificial variable regions with negligible identity to known nucleotide sequences, permitting unambiguous identification of spike-in sequences in 16S-seq read data from any microbiome sample. Using defined mock communities and environmental microbiota, we characterized the performance of the spike-in standards and demonstrated their utility for evaluating data quality on a per-sample basis. Further, we showed that staggered spike-in mixtures added at the point of DNA extraction enable concurrent estimation of absolute microbial abundances suitable for comparative analysis. Results also underscored that template-specific Illumina sequencing artifacts may lead to biases in the perceived abundance of certain taxa. Taken together, the spike-in standards represent a novel bioanalytical tool that can substantially improve 16S-seq-based microbiome studies by enabling comprehensive quality control along with absolute quantification.

An international comparability study on quantification of mRNA gene expression ratios: CCQM-P103.1.

Measurement of RNA can be used to study and monitor a range of infectious and non-communicable diseases, with profiling of multiple gene expression mRNA transcripts being increasingly applied to cancer stratification and prognosis. An international comparison study (Consultative Committee for Amount of Substance (CCQM)-P103.1) was performed in order to evaluate the comparability of measurements of RNA copy number ratio for multiple gene targets between two samples. Six exogenous synthetic targets comprising of External RNA Control Consortium (ERCC) standards were measured alongside transcripts for three endogenous gene targets present in the background of human cell line RNA. The study was carried out under the auspices of the Nucleic Acids (formerly Bioanalysis) Working Group of the CCQM. It was coordinated by LGC (United Kingdom) with the support of National Institute of Standards and Technology (USA) and results were submitted from thirteen National Metrology Institutes and Designated Institutes. The majority of laboratories performed RNA measurements using RT-qPCR, with datasets also being submitted by two laboratories based on reverse transcription digital polymerase chain reaction and one laboratory using a next-generation sequencing method. In RT-qPCR analysis, the RNA copy number ratios between the two samples were quantified using either a standard curve or a relative quantification approach. In general, good agreement was observed between the reported results of ERCC RNA copy number ratio measurements. Measurements of the RNA copy number ratios for endogenous genes between the two samples were also consistent between the majority of laboratories. Some differences in the reported values and confidence intervals ('measurement uncertainties') were noted which may be attributable to choice of measurement method or quantification approach. This highlights the need for standardised practices for the calculation of fold change ratios and uncertainties in the area of gene expression profiling.

Synthetic PAMPS gel activates BMP/Smad signaling pathway in ATDC5 cells, which plays a significant role in the gel-induced chondrogenic differentiation.

The purposes of this study were to identify signaling pathways that were specifically activated in ATDC5 cells cultured on poly (2-acrylamido-2-methylpropanesulfonic acid) (PAMPS) gel in insulin-free maintenance medium and to evaluate the significance of the determined signaling pathways in the chondrogenic differentiation induced by this gel. In this study, ATDC5 cells cultured on PAMPS gel using the maintenance medium without insulin (PAMPS Culture) were compared with cells cultured on polystyrene using the differentiation medium containing insulin (PS-I Culture). The microarray analysis, Western blot analysis, and real-time PCR analysis demonstrated that the TGF-ß/BMP signaling pathway was significantly enhanced at Days 1, 2, and 3 in the PAMPS Culture when compared with the PS-I Culture. Inhibition of the BMP type-I receptor reduced the phosphorylation level of Smad1/5 and expression of type-2 collagen and aggrecan mRNA in the cells accompanied by a reduction in cell aggregation at Day 13 in the PAMPS Culture. The inhibition of the TGF-ß/BMP signaling pathway significantly inhibited the chondrogenic differentiation induced by the PAMPS gel. The present study demonstrated that synthetic PAMPS gel activates the TGF-ß/BMP/Smad signaling pathway in the ATDC5 cells in the absence of insulin, and that this activation plays a significant role in the chondrogenic differentiation induced by PAMPS gel. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 734-746, 2016.

Community analysis of picocyanobacteria in an oligotrophic lake by cloning 16S rRNA gene and 16S rRNA gene amplicon sequencing.

In this study, the picocyanobacterial species composition of Lake Miyagase was examined by analyzing the 16S rRNA gene in a clone library and by amplicon sequencing using a benchtop next-generation sequencer. Five separate samples were analyzed from different days over a ten-month period. In the picocyanobacterial lineage, 9 and 12 OTUs were identified from a clone library and by amplicon sequencing, respectively. Both analyses suggested that a picocyanobacterium related to Synechococcus sp. MW6B4 was dominant in Lake Miyagase. Our findings suggest that 16S rRNA gene amplicon sequencing enables detailed evaluation of picocyanobacteria composition. One OTU identified was found to be a novel cluster that does not group with any of the known freshwater picocyanobacteria.

Artificial conversion of the mating-type of Saccharomyces cerevisiae without autopolyploidization.

Crossbreeding is a classical yeast hybridization procedure, where the mating of haploid cells of opposite mating-type, MATa and MATα cells, produces a new heterozygous diploid. Here, we describe a method to generate haploid MATa and MATα cells using mating-type conversion caused by expression of the HO gene, which encodes an endonuclease. Importantly, our method prevents the autopolyploidization that typically arises during artificial mating-type conversion. This facilitates isolation of the desired mating-type of yeast cells with simple and easy procedure. In the current study, we designed a suitable genetic circuit for each haploid cell and converted MATα haploid cells into MATa haploid cells and vice versa, demonstrating the utility of constructed artificial regulation network to prevent autopolyploidization. Via forced expression of the a1 gene in MATα haploid cells or of α2 in MATa haploid cells, the undesirable mating ability of yeast cells was completely suppressed. We confirmed the success in prevention of autopolyploidization by ploidy analysis. This new approach provides a reliable and versatile tool for yeast crossbreeding, so that it will be useful for scientific research and industrial applications of yeast.

Rice phytochrome-interacting factor-like protein OsPIL1 functions as a key regulator of internode elongation and induces a morphological response to drought stress.

The mechanisms for plant growth restriction during stress conditions remains unclear. Here, we demonstrate that a phytochrome-interacting factor-like protein, OsPIL1/OsPIL13, acts as a key regulator of reduced internode elongation in rice under drought conditions. The level of OsPIL1 mRNA in rice seedlings grown under nonstressed conditions with light/dark cycles oscillated in a circadian manner with peaks in the middle of the light period. Under drought stress conditions, OsPIL1 expression was inhibited during the light period. We found that OsPIL1 was highly expressed in the node portions of the stem using promoter-glucuronidase analysis. Overexpression of OsPIL1 in transgenic rice plants promoted internode elongation. In contrast, transgenic rice plants with a chimeric repressor resulted in short internode sections. Alteration of internode cell size was observed in OsPIL1 transgenic plants, indicating that differences in cell size cause the change in internode length. Oligoarray analysis revealed OsPIL1 downstream genes, which were enriched for cell wall-related genes responsible for cell elongation. These data suggest that OsPIL1 functions as a key regulatory factor of reduced plant height via cell wall-related genes in response to drought stress. This regulatory system may be important for morphological stress adaptation in rice under drought conditions.

Identification of cis-acting promoter elements in cold- and dehydration-induced transcriptional pathways in Arabidopsis, rice, and soybean.

The genomes of three plants, Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and soybean (Glycine max), have been sequenced, and their many genes and promoters have been predicted. In Arabidopsis, cis-acting promoter elements involved in cold- and dehydration-responsive gene expression have been extensively analysed; however, the characteristics of such cis-acting promoter sequences in cold- and dehydration-inducible genes of rice and soybean remain to be clarified. In this study, we performed microarray analyses using the three species, and compared characteristics of identified cold- and dehydration-inducible genes. Transcription profiles of the cold- and dehydration-responsive genes were similar among these three species, showing representative upregulated (dehydrin/LEA) and downregulated (photosynthesis-related) genes. All (4(6) = 4096) hexamer sequences in the promoters of the three species were investigated, revealing the frequency of conserved sequences in cold- and dehydration-inducible promoters. A core sequence of the abscisic acid-responsive element (ABRE) was the most conserved in dehydration-inducible promoters of all three species, suggesting that transcriptional regulation for dehydration-inducible genes is similar among these three species, with the ABRE-dependent transcriptional pathway. In contrast, for cold-inducible promoters, the conserved hexamer sequences were diversified among these three species, suggesting the existence of diverse transcriptional regulatory pathways for cold-inducible genes among the species.

Comprehensive analysis of rice DREB2-type genes that encode transcription factors involved in the expression of abiotic stress-responsive genes.

DREB2s (dehydration-responsive element-binding protein 2s) are transcription factors that interact with a cis-acting DRE (dehydration-responsive element)/CRT (C-repeat) sequence and activate the expression of downstream genes involved in water- and heat-shock stress responses and tolerance in Arabidopsis thaliana. In this study, we performed a comprehensive analysis of all five DREB2-type genes in rice (OsDREB2 s: OsDREB2A, OsDREB2B, OsDREB2C, OsDREB2E and OsABI4) to determine which of them contribute to plant stress responses. We analysed the expression patterns of these genes under abiotic stress conditions, and we examined the subcellular localisation and transcriptional activation activity of their translational products in protoplasts. Only OsDREB2A and OsDREB2B showed abiotic stress-inducible gene expression. In addition, OsDREB2B showed nuclear specific localisation and the highest transactivation activity. OsDREB2B has functional and non-functional forms of its transcript similar to its orthologues in the grass family, and the functional form of its transcript was markedly increased during stress conditions. We analysed the splicing mechanism of OsDREB2B with transgenic rice that express the non-functional transcript and we found that the non-functional form is not a precursor of the functional form; thus, stress-inducible alternative splicing of pre-mRNA is an important mechanism for the regulation of OsDREB2B. Transgenic Arabidopsis plants overexpressing OsDREB2B showed enhanced expression of DREB2A target genes and improved drought and heat-shock stress tolerance. These results suggest that OsDREB2B is a key gene that encodes a stress-inducible DREB2-type transcription factor that functions in stress-responsive gene expression in rice.

Metabolic pathways involved in cold acclimation identified by integrated analysis of metabolites and transcripts regulated by DREB1A and DREB2A.

DREB1A/CBF3 and DREB2A are transcription factors that specifically interact with a cis-acting dehydration-responsive element (DRE), which is involved in cold- and dehydration-responsive gene expression in Arabidopsis (Arabidopsis thaliana). Overexpression of DREB1A improves stress tolerance to both freezing and dehydration in transgenic plants. In contrast, overexpression of an active form of DREB2A results in significant stress tolerance to dehydration but only slight tolerance to freezing in transgenic plants. The downstream gene products for DREB1A and DREB2A are reported to have similar putative functions, but downstream genes encoding enzymes for carbohydrate metabolism are very different between DREB1A and DREB2A. We demonstrate that under cold and dehydration conditions, the expression of many genes encoding starch-degrading enzymes, sucrose metabolism enzymes, and sugar alcohol synthases changes dynamically; consequently, many kinds of monosaccharides, disaccharides, trisaccharides, and sugar alcohols accumulate in Arabidopsis. We also show that DREB1A overexpression can cause almost the same changes in these metabolic processes and that these changes seem to improve freezing and dehydration stress tolerance in transgenic plants. In contrast, DREB2A overexpression did not increase the level of any of these metabolites in transgenic plants. Strong freezing stress tolerance of the transgenic plants overexpressing DREB1A may depend on accumulation of these metabolites.