Protein stability governed by α1-2 helices in Pvr4 is essential for localization and cell death.

Plant nucleotide-binding domain leucine-rich-repeat receptor (NLR) confers disease resistance to various pathogens by recognizing effectors derived from the pathogen. Previous studies have shown that overexpression of the CC domain in several NLRs triggers cell death, implying that the CC domain plays an important role as a signaling module. However, how CC domain transduces immune signals remains largely unknown. A Potyvirus-resistant NLR protein, Pvr4, possesses a CC domain (CCPvr4 ) that induces cell death upon transient overexpression in Nicotiana benthamiana. In this study, loss-of-function mutants were generated by error-prone PCR-based random mutagenesis to understand the molecular mechanisms underlying CCPvr4 -mediated cell death. Cell biology and biochemical studies revealed that M16 and Q52 in the α1 and α2 helices, respectively, are crucial for protein stability, and mutation of these residues disrupts localization to the plasma membrane and oligomerization activity. The increase of the protein stability of these mutants by tagging a green fluorescent protein (GFP) variant led to restoration of cell death-inducing activity and plasma membrane localization. Another mutant, I7E in the very N-terminal region, lost cell death-inducing activity by weakening the interaction with plasma membrane H+ -ATPase compared to CCPvr4 , although the protein remained in the plasma membrane. Moreover, most of the mutated residues are on the outer surface of the funnel shape in the predicted pentameric CCPvr4 , implying that the disordered N-terminal region plays a crucial role in association with PMA as well as targeting to the plasma membrane. This work could provide insights into the molecular mechanisms of cell death induced by NLR immune receptors.

S100 Calcium-Binding Protein A9, a Potential Novel Diagnostic Biomarker for Idiopathic Pulmonary Fibrosis.

BACKGROUND: Neutrophilic inflammation is a characteristic feature of idiopathic pulmonary fibrosis (IPF). S100 calcium-binding protein A9 (S100A9) is a neutrophil-derived protein involved in the development of neutrophil-related chronic inflammatory disorders. However, the role of S100A9 in IPF remains unclear. METHODS: We used enzyme-linked immunosorbent assays to measure S100A9 levels in bronchoalveolar lavage fluid (BALF) and serum obtained from healthy controls (HCs) and patients with IPF, non-specific interstitial pneumonia, hypersensitivity pneumonitis, and sarcoidosis. RESULTS: Compared with HCs, BALF S100A9 levels were significantly higher in IPF patients (P < 0.001), patients with hypersensitivity pneumonitis (P = 0.043), and patients with nonspecific interstitial pneumonia (P < 0.001). The S100A9 level in BALF of 0.093 ng/mL could distinguish IPF patients from HCs, with a specificity of 78.8% and a sensitivity of 81.6%. Similarly, the S100A9 level in BALF of 0.239 ng/mL had a specificity of 64.7% and a sensitivity of 66.7% for distinguishing IPF patients from patients with other interstitial lung diseases. Additionally, BALF S100A9 levels were significantly correlated with neutrophil counts (r = 0.356, P < 0.001) in BALF. IPF patients with S100A9 levels in BALF > 0.533 ng/mL had lower survival rates, compared with patients who had levels ≤ 0.553 ng/mL (n = 49; hazard ratio [HR], 3.62; P = 0.021). Combination analysis revealed that IPF patients with S100A9 levels in BALF> 0.553 ng/mL or neutrophil percentages > 49.1% (n = 43) had significantly lower survival rates than patients with S100A9 levels in BALF ≤ 0.553 ng/mL and neutrophil percentages ≤ 49.1% (n = 41) (HR, 3.91; P = 0.014). Additionally, patients with serum S100A9 levels > 0.077 ng/mL (n = 29) had significantly lower survival rates than patients with levels ≤ 0.077 ng/mL (n = 53, HR, 2.52; P = 0.013). S100A9 was expressed on neutrophils and macrophages in BALF from IPF patients as well as α-smooth muscle actin positive cells in the lung tissues. CONCLUSION: S100A9 is involved in the development and progression of IPF. Moreover, S100A9 levels in BALF and serum may be surrogate markers for IPF diagnosis and survival prediction, particularly when analyzed in combination with neutrophil percentages.

Nucleotide-binding leucine-rich repeat network underlies nonhost resistance of pepper against the Irish potato famine pathogen Phytophthora infestans.

Nonhost resistance (NHR) is a robust plant immune response against non-adapted pathogens. A number of nucleotide-binding leucine-rich repeat (NLR) proteins that recognize non-adapted pathogens have been identified, although the underlying molecular mechanisms driving robustness of NHR are still unknown. Here, we screened 57 effectors of the potato late blight pathogen Phytophthora infestans in nonhost pepper (Capsicum annuum) to identify avirulence effector candidates. Selected effectors were tested against 436 genome-wide cloned pepper NLRs, and we identified multiple functional NLRs that recognize P. infestans effectors and confer disease resistance in the Nicotiana benthamiana as a surrogate system. The identified NLRs were homologous to known NLRs derived from wild potatoes that recognize P. infestans effectors such as Avr2, Avrblb1, Avrblb2, and Avrvnt1. The identified CaRpi-blb2 is a homologue of Rpi-blb2, recognizes Avrblb2 family effectors, exhibits feature of lineage-specifically evolved gene in microsynteny and phylogenetic analyses, and requires pepper-specific NRC (NLR required for cell death)-type helper NLR for proper function. Moreover, CaRpi-blb2-mediated hypersensitive response and blight resistance were more tolerant to suppression by the PITG_15 278 than those mediated by Rpi-blb2. Combined results indicate that pepper has stacked multiple NLRs recognizing effectors of non-adapted P. infestans, and these NLRs could be more tolerant to pathogen-mediated immune suppression than NLRs derived from the host plants. Our study suggests that NLRs derived from nonhost plants have potential as untapped resources to develop crops with durable resistance against fast-evolving pathogens by stacking the network of nonhost NLRs into susceptible host plants.

The Phytophthora capsici RxLR effector CRISIS2 triggers cell death via suppressing plasma membrane H+-ATPase in the host plant.

Pathogen effectors can suppress various plant immune responses, suggesting that they have multiple targets in the host. To understand the mechanisms underlying plasma membrane-associated and effector-mediated immunity, we screened the Phytophthora capsici RxLR cell death-inducer suppressing immune system (CRISIS). We found that the cell death induced by the CRISIS2 effector in Nicotiana benthamiana was inhibited by the irreversible plasma membrane H+-ATPase (PMA) activator fusicoccin. Biochemical and gene-silencing analyses revealed that CRISIS2 physically and functionally associated with PMAs and induced host cell death independent of immune receptors. CRISIS2 induced apoplastic alkalization by suppressing PMA activity via its association with the C-terminal regulatory domain. In planta expression of CRISIS2 significantly enhanced the virulence of P. capsici, whereas host-induced gene-silencing of CRISIS2 compromised the disease symptoms and the biomass of the pathogen. Thus, our study has identified a novel RxLR effector that plays multiple roles in the suppression of plant defense and in the induction of cell death to support the pathogen hemibiotrophic life cycle in the host plant.

Comparative analysis of de novo genomes reveals dynamic intra-species divergence of NLRs in pepper.

BACKGROUND: Peppers (Capsicum annuum L.) containing distinct capsaicinoids are the most widely cultivated spices in the world. However, extreme genomic diversity among species represents an obstacle to breeding pepper. RESULTS: Here, we report de novo genome assemblies of Capsicum annuum 'Early Calwonder (non-pungent, ECW)' and 'Small Fruit (pungent, SF)' along with their annotations. In total, we assembled 2.9 Gb of ECW and SF genome sequences, representing over 91% of the estimated genome sizes. Structural and functional annotation of the two pepper genomes generated about 35,000 protein-coding genes each, of which 93% were assigned putative functions. Comparison between newly and publicly available pepper gene annotations revealed both shared and specific gene content. In addition, a comprehensive analysis of nucleotide-binding and leucine-rich repeat (NLR) genes through whole-genome alignment identified five significant regions of NLR copy number variation (CNV). Detailed comparisons of those regions revealed that these CNVs were generated by intra-specific genomic variations that accelerated diversification of NLRs among peppers. CONCLUSIONS: Our analyses unveil an evolutionary mechanism responsible for generating CNVs of NLRs among pepper accessions, and provide novel genomic resources for functional genomics and molecular breeding of disease resistance in Capsicum species.

Genome-wide functional analysis of hot pepper immune receptors reveals an autonomous NLR clade in seed plants.

Plants possess hundreds of intracellular immune receptors encoding nucleotide-binding domain leucine-rich repeat (NLR) proteins. Full-length NLRs or a specific domain of NLRs often induce plant cell death in the absence of pathogen infection. In this study we used genome-wide transient expression analysis to identify a group of NLRs (ANLs; ancient and autonomous NLRs) carrying autoactive coiled-coil (CCA ) domains in pepper (Capsicum annuum). CCA -mediated cell death mimics hypersensitive cell death triggered by the interaction between NLRs and pathogen effectors. Sequence alignment and mutagenesis analyses revealed that the intact α1 helix of CCA s is critical for both CCA - and ANL-mediated cell death. Cell death induced by CCA s does not require NRG1/ADR1 or NRC type helper NLRs, suggesting ANLs may function as singleton NLRs. We also found that CCA s localize to the plasma membrane, as demonstrated for Arabidopsis singleton NLR ZAR1. Extended studies revealed that autoactive CCA s are well conserved in other Solanaceae plants as well as in rice, a monocot plant. Further phylogenetic analyses revealed that ANLs are present in all tested seed plants (spermatophytes). Our study not only uncovers the autonomous NLR clade in plants but also provides powerful resources for dissecting the underlying molecular mechanism of NLR-mediated cell death in plants.

Elucidation of genomic organizations of transgenic soybean plants through de novo genome assembly with short paired-end reads.

Elucidation of the genomic organizations of transgene insertion sites is essential for the genetic studies of transgenic plants. Herein, we establish an analysis pipeline that identifies the transgene insertion sites as well as the presence of vector backbones, through de novo genome assembly with high-throughput sequencing data in two transgenic soybean lines, AtYUCCA6-#5 and 35S-UGT72E3/2-#7. Sequencing data of approximately 28× and 29× genome coverages for each line generated by high-throughput sequencing were de novo assembled. The databases generated from the de novo assembled sequences were used to search contigs that contained putative insertion sites and their flanking sequences (integration sites) of transgene fragments using transgenic vector sequences as queries. The predicted integration site sequences, which are located at three annotated genes that might regulate plant development or confer disease resistance, were then confirmed by local alignment against the soybean reference genome and PCR amplification. As results, we revealed the precise transgene-flanking sequences and sequence rearrangements at insertion sites in both the transgenic lines, as well as the aberrant insertion of a transgene fragment. Consequently, relative to experimental or enrichment technologies, our approach is straightforward and time-effective, providing an alternative method for the identification of insertion sites in transgenic plants.

Chromosomal features revealed by comparison of genetic maps of Glycine max and Glycine soja.

Recombination is a crucial component of evolution and breeding. New combinations of variation on chromosomes are shaped by recombination. Recombination is also involved in chromosomal rearrangements. However, recombination rates vary tremendously among chromosome segments. Genome-wide genetic maps are one of the best tools to study variation of recombination. Here, we describe high density genetic maps of Glycine max and Glycine soja constructed from four segregating populations. The maps were used to identify chromosomal rearrangements and find the highly predictable pattern of cross-overs on the broad scale in soybean. Markers on these genetic maps were used to evaluate assembly quality of the current soybean reference genome sequence. We find a strong inversion candidate larger than 3 Mb based on patterns of cross-overs. We also identify quantitative trait loci (QTL) that control number of cross-overs. This study provides fundamental insights relevant to practical strategy for breeding programs and for pan-genome researches.

Occurrence of land-plant-specific glycerol-3-phosphate acyltransferases is essential for cuticle formation and gametophore development in Physcomitrella patens.

During the evolution of land plants from aquatic to terrestrial environments, their aerial surfaces were surrounded by cuticle composed of cutin and cuticular waxes to protect them from environmental stresses. Glycerol-3-phosphate acyltransferase (GPAT) harboring bifunctional sn-2 acyltransferase/phosphatase activity produces 2-monoacylglycerol, a precursor for cutin synthesis. Here, we report that bifunctional sn-2 GPATs play roles in cuticle biosynthesis and gametophore development of Physcomitrella patens. Land plant-type cuticle was observed in gametophores but not in protonema. The expression of endoplasmic reticulum-localized PpGPATs was significantly upregulated in gametophores compared with protonema. Floral organ fusion and permeable cuticle phenotypes of Arabidopsis gpat6-2 petals were rescued to the wild type (WT) by the expression of PpGPAT2 or PpGPAT4. Disruption of PpGPAT2 and PpGPAT4 caused a significant reduction of total cutin loads, and a prominent decrease in the levels of palmitic and 10,16-dihydroxydecanoic acids, which are major cutin monomers in gametophores. Δppgpat2 mutants displayed growth retardation, delayed gametophore development, increased cuticular permeability, and reduced tolerance to drought, osmotic and salt stresses compared to the WT. Genome-wide analysis of genes encoding acyltransferase or phosphatase domains suggested that the occurrence of sn-2 GPATs with both domains may be a key event in cuticle biogenesis of land plants.

TGFam-Finder: a novel solution for target-gene family annotation in plants.

Whole-genome annotation error that omits essential protein-coding genes hinders further research. We developed Target Gene Family Finder (TGFam-Finder), an alternative tool for the structural annotation of protein-coding genes containing target domain(s) of interest in plant genomes. TGFam-Finder took considerably reduced annotation run-time and improved accuracy compared to conventional annotation tools. Large-scale re-annotation of 50 plant genomes identified an average of 150, 166 and 86 additional far-red-impaired response 1, nucleotide-binding and leucine-rich-repeat, and cytochrome P450 genes, respectively, that were missed in previous annotations. We detected significantly higher number of translated genes in the new annotations using mass spectrometry data from seven plant species compared to previous annotations. TGFam-Finder along with the new gene models can provide an optimized platform for comprehensive functional, comparative, and evolutionary studies in plants.

Genetic diversity patterns and domestication origin of soybean.

KEY MESSAGE: Genotyping data of a comprehensive Korean soybean collection obtained using a large SNP array were used to clarify global distribution patterns of soybean and address the evolutionary history of soybean. Understanding diversity and evolution of a crop is an essential step to implement a strategy to expand its germplasm base for crop improvement research. Accessions intensively collected from Korea, which is a small but central region in the distribution geography of soybean, were genotyped to provide sufficient data to underpin population genetic questions. After removing natural hybrids and duplicated or redundant accessions, we obtained a non-redundant set comprising 1957 domesticated and 1079 wild accessions to perform population structure analyses. Our analysis demonstrates that while wild soybean germplasm will require additional sampling from diverse indigenous areas to expand the germplasm base, the current domesticated soybean germplasm is saturated in terms of genetic diversity. We then showed that our genome-wide polymorphism map enabled us to detect genetic loci underlying flower color, seed-coat color, and domestication syndrome. A representative soybean set consisting of 194 accessions was divided into one domesticated subpopulation and four wild subpopulations that could be traced back to their geographic collection areas. Population genomics analyses suggested that the monophyletic group of domesticated soybeans was likely originated at a Japanese region. The results were further substantiated by a phylogenetic tree constructed from domestication-associated single nucleotide polymorphisms identified in this study.

Molecular marker development and genetic diversity exploration by RNA-seq in Platycodon grandiflorum.

Platycodon grandiflorum, generally known as the bellflower or balloon flower, is the only species in the genus Platycodon of the family Campanulaceae. Platycodon plants have been traditionally used as a medicinal crop in East Asia for their antiphlogistic, antitussive, and expectorant properties. Despite these practical uses, marker-assisted selection and molecular breeding in platycodons have lagged due to the lack of genetic information on this genus. In this study, we performed RNA-seq analysis of three platycodon accessions to develop molecular markers and explore genetic diversity. First, genic simple sequence repeats (SSRs) were retrieved and compared; dinucleotide motifs were the most abundant repeats (39%-40%) followed by trinucleotide (25%-31%), tetranucleotide (1.5%-1.9%), and pentanucleotide (0.3%-1.0%) repeats. The result of in silico SSR analysis, three SSR markers were detected and showed possibility to distinguish three platycodon accessions. After several filtering procedures, 180 single nucleotide polymorphisms (SNPs) were used to design 40 cleaved amplified polymorphic sequence (CAPS) markers. Twelve of these PCR-based markers were validated as highly polymorphic and utilized to investigate genetic diversity in 21 platycodon accessions collected from various regions of South Korea. Collectively, the 12 markers yielded 35 alleles, with an average of 3 alleles per locus. Polymorphism information content (PIC) values ranged from 0.087 to 0.693, averaging 0.373 per locus. Since platycodon genetics have not been actively studied, the sequence information and the DNA markers generated from our research have the potential to contribute to further genetic improvements, genomic studies, and gene discovery in this genus.

Multiple recognition of RXLR effectors is associated with nonhost resistance of pepper against Phytophthora infestans.

Nonhost resistance (NHR) is a plant immune response to resist most pathogens. The molecular basis of NHR is poorly understood, but recognition of pathogen effectors by immune receptors, a response known as effector-triggered immunity, has been proposed as a component of NHR. We performed transient expression of 54 Phytophthora infestansRXLR effectors in pepper (Capsicum annuum) accessions. We used optimized heterologous expression methods and analyzed the inheritance of effector-induced cell death in an F2 population derived from a cross between two pepper accessions. Pepper showed a localized cell death response upon inoculation with P. infestans, suggesting that recognition of effectors may contribute to NHR in this system. Pepper accessions recognized as many as 36 effectors. Among the effectors, PexRD8 and Avrblb2 induced cell death in a broad range of pepper accessions. Segregation of effector-induced cell death in an F2 population derived from a cross between two pepper accessions fit 15:1, 9:7 or 3:1 ratios, depending on the effector. Our genetic data suggest that a single or two independent/complementary dominant genes are involved in the recognition of RXLR effectors. Multiple loci recognizing a series of effectors may underpin NHR of pepper to P. infestans and confer resistance durability.

Mutational analysis of splicing machinery genes SF3B1, U2AF1 and SRSF2 in myelodysplasia and other common tumors.

Recurrent somatic mutations in splicing machinery components, including SF3B1, U2AF1 and SRSF2 genes have recently been reported in myelodysplastic syndromes (MDS). Such a recurrent nature strongly suggests that these mutations play important roles in tumor development. To see whether SF3B1, U2AF1 and SRSF2 mutations occur in other human tumors besides MDS, we analyzed the hotspot mutation regions of these genes in 2,345 tumor tissues from various origins (61 MDS, other 616 hematologic tumors, 1,421 epithelial tumors and 247 non-epithelial stromal tumors) by single-strand conformation polymorphism analysis. We found SF3B1, U2AF1 and SRSF2 mutations in 5 (8.2%), 12 (19.7%) and 8 (13.1%) of 61 MDS, respectively. We also confirmed these mutations in other myeloid neoplasia, including de novo acute myelogenous leukemia (AML), chronic myelomonocytic leukemia and MDS/myeloproliferative disorder. In addition, we discovered that the SRSF2 gene was mutated in two childhood acute lymphoblastic leukemias (childhood ALL) (1.5%). In solid tumors, we found SF3B1 mutations in gastric and prostate cancers, and U2AF1 mutation in a borderline mucinous tumor of ovary, but the overall incidences of the hotspot mutation regions were very low (0.2%). Our data suggest that SF3B1, U2AF1 and SRSF2 mutations occur not only in myeloid lineage tumors but also in lymphoid lineage tumors. The data suggest that the splicing gene mutations play important roles in the pathogenesis of hematologic tumors, but rarely in solid tumors.

Comparison of reminder-preference test to difference and preference tests: An affective approach yielding d-prime results for sensory difference and preference.

Preserving the sensory characteristics of reformulated products is crucial for a successful market launch. This underscores the need for precise measurement of consumer perceptions regarding sensory differences and preferences. One approach to assess these aspects is through the utilization of the reminder-preference test, an affective difference-preference test employing the A-Not A with the reminder (A-Not AR) test design. This test measures sensory differences and preferences by employing a consolidated d' index rooted in signal detection theory and Thurstonian modeling. The aim of this study was to assess the efficacy of the reminder-preference test in measuring the degree of sensory differences and interpreting their relevance concerning consumer preference. To achieve this, the test was compared with both the A-Not AR difference test and the 2-alternative choice (2-AC) preference test. A lemon-lime-flavored carbonated drink and two types of reformulated samples were tested. Young female adult subjects were randomly divided into three groups. Each took one of the following three tests: the reminder-preference test, the reminder test of A-Not AR, or the 2-AC preference test. The reference framing in the reminder-preference and reminder tests was preceded by subjects watching the product's advertisement to assist them in framing evaluative criteria. This would also facilitate memorizing the perception of the reference product. Test sensitivity in reminder-preference matched reminder and 2-AC tests for sensory differences and preferences. Affective framing enhanced sensitivity to sensory differences resulting in a higher d' value of the sensory difference than the preference. From the results, it was possible to interpret that the measured sensory differences did not affect consumer preference. The test also illuminated brand marketing effects on preferences through branded reference comparison. Thus, the reminder-preference test shows potential as an efficient sensory method to measure sensory differences and the preference of consumers, while concurrently measuring the effects of marketing on the consumers. Therefore, it can assist in making efficient business decisions regarding product reformulations.

The Phytophthora nucleolar effector Pi23226 targets host ribosome biogenesis to induce necrotrophic cell death.

Pathogen effectors target diverse subcellular organelles to manipulate the plant immune system. Although the nucleolus has emerged as a stress marker and several effectors are localized in the nucleolus, the roles of nucleolar-targeted effectors remain elusive. In this study, we showed that Phytophthora infestans infection of Nicotiana benthamiana results in nucleolar inflation during the transition from the biotrophic to the necrotrophic phase. Multiple P. infestans effectors were localized in the nucleolus: Pi23226 induced cell death in N. benthamiana and nucleolar inflation similar to that observed in the necrotrophic stage of infection, whereas its homolog Pi23015 and a deletion mutant (Pi23226ΔC) did not induce cell death or affect nucleolar size. RNA immunoprecipitation and individual-nucleotide-resolution UV crosslinking and immunoprecipitation sequencing analysis indicated that Pi23226 bound to the 3' end of 25S rRNA precursors, resulting in accumulation of unprocessed 27S pre-rRNAs. The nucleolar stress marker NAC082 was strongly upregulated under Pi23226-expressing conditions. Pi23226 subsequently inhibited global protein translation in host cells by interacting with ribosomes. Pi23226 enhanced P. infestans pathogenicity, indicating that Pi23226-induced ribosome malfunction and cell death were beneficial for pathogenesis in the host. Our results provide evidence for the molecular mechanism underlying RNA-binding effector activity in host ribosome biogenesis and lead to new insights into the nucleolar action of effectors in pathogenesis.

Single-cell transcriptome sequencing of plant leaf expressing anti-HER2 VHH-FcK cancer therapeutic protein.

The transgenic plant is a promising strategy for the production of highly valuable biotherapeutic proteins such as recombinant vaccines and antibodies. To achieve an efficient level of protein production, codon sequences and expression cassette elements need to be optimized. However, the systematical expression of recombinant proteins in plant biomass can generally be controlled for the production of therapeutic proteins after the generation of transgenic plants. Without understanding the transgene expression patterns in plant tissue, it is difficult to enhance further production levels. In this study, single-cell RNA-sequencing (scRNA-seq) analysis of transgenic tobacco (Nicotiana tabacum) leaf, expressing an immunotherapeutic llama antibody against breast cancer, anti-HER2 VHH-Fc, was conducted to obtain data on the expression pattern of tissue-specific cells. These high-quality scRNA-seq data enabled the identification of gene expression patterns by cell types, which can be applied to select the best cell types or tissues for the high production of these recombinant antibodies. These data provide a foundation to elucidate the mechanisms that regulate the biosynthesis of recombinant proteins in N. tabacum.

Reminder-preference test, affective difference-preference test using reference framing with a brand: 1. Sensitivity comparisons with the same-different difference-preference test.

Attempts to reduce the sugar content of carbonated drinks are ongoing. Reducing the sugar content while maintaining the sensory quality requires the development of an effective sensory test method that can measure a consumers' perceived sensory difference and preference of reformulated products against the reference product. The A-Not A with reminder (A-Not AR) method (sometimes called the reminder method) allows blind sensory tests of multiple test samples vs. a reference, while for the reference, marketing information, such as brand name and advertisements emphasizing the consumer benefit, can be provided to improve the marketing effects and consumer expectation. Thus, a preference test method can also simulate branded tests using A-Not AR design with a branded reference, making them suitable for consumer sensory tests. In the present study, an affective difference-preference test method using an A-Not AR design with the reference familiarization procedure of providing marketing information was developed, called the "reminder-preference test". The "reminder-preference test" sensitivity was compared with the previously suggested version of the "same-different difference-preference test", using signal detection d' analyses. A lemon-lime flavored carbonated drink was used as the reference and compared with two types of reformulations. The consumers were divided randomly into two groups that performed one of the two difference-preference test methods. With the "reminder-preference test", the consumers watched a commercial advertisement for the reference by providing its brand name before the tests to evoke the marketing effects. The proposed sensory test of the "reminder-preference test" was more sensitive than the "same-different difference-preference test", in terms of both sensory and preference discriminations. Hence, brand effects can be incorporated into the consumer sensory evaluation to achieve the reformulation objective and the potential of the "reminder-preference test" and its signal detection d' analyses to improve the effectiveness and efficiency of consumer sensory measurements.

Characteristics of amniotic mesenchymal stromal cells derived from term and preterm labor.

OBJECTIVE: Amniotic mesenchymal stromal cells (AMSCs) can be obtained from the mesoderm of human amniotic membrane. AMSCs derived from term baby show increased expression of genes associated with apoptosis and senescence. The objective of this study was to examine gene expression profiles of AMSCs derived from preterm (preterm AMSCs) and term labors (term AMSCs) and analyze common and different mechanisms. MATERIALS AND METHODS: We isolated and cultured AMSCs from 43 placentas: 27 from term labor and 16 from preterm labor. Microarray analysis and gene network analysis were performed to compare gene expression profile (GEP) of preterm (n = 6) with term AMSCs (n = 10). Senescence-associated gene (CDKN2A and CDKN2B) expression was also measured by reverse transcription quantitative PCR. RESULTS: GEP demonstrated that preterm AMSCs showed upregulation of nicotinamide adenine dinucleotide biosynthetic process and downregulation of extracellular matrix, cholesterol import and transport, lipid storage, and maintenance of location. CDKN2A and CDKN2B genes showed similar expression levels between term and preterm AMSCs. CDKN2A gene expression was correlated with CDKN2B expression and population doubling time. Compared to term AMSCs, preterm AMSCs showed significantly different expression of genes associated with inflammatory response which could be one of the major players in labor events. CONCLUSION: Increased CDKN2A expression in AMSCs is associated with placental membrane aging which participates in both preterm and term labor. To the best of our knowledge, this is the first report to demonstrate the association of AMSCs with labor.

Disruption of bis leads to the deterioration of the vascular niche for hematopoietic stem cells.

The stem cell niche plays an important role in the microenvironmental regulation of hematopoietic stem cells, but the integration of niche activity remains poorly understood. In this study, we show that a functional deficiency of Bis/BAG-3/CAIR-1, a protein related to apoptosis and the response to cellular stress, results in perturbation of the vascular stem cell niche, causing a series of hematopoietic derangements. Mice with a targeted disruption of bis (bis(-/-)) exhibited a loss of hematopoietic stem cells and defective B-cell development. However, this hematological defect of bis(-/-) mice was not reproduced when bis(-/-) bone marrow cells were transplanted into bis(+/+) recipients. Moreover, bis(+/+) bone marrow cells, when transplanted into bis(-/-) mice, reproduced the same defect as bis(-/-) cells, pointing to the microenvironmental origin of the phenotypes. Subsequent analysis of bis(-/-) mice bone marrow revealed a characteristic defect in the vascular stem cell niche that included the defective growth of stromal progenitor cells in colony forming unit-fibroblasts, the defect in sinusoidal endothelium, and the loss of stromal cells expressing CXCL-12 or IL-7 in the bone marrow. In contrast, no abnormalities were observed in the growth and hematopoietic supporting activities of osteoblasts from bis(-/-) mice bone marrows. Collectively, these results indicate that Bis functions to mediate cellular regulation of the stem cell niche on the vascular compartment and suggest that the vascular and osteoblastic compartments of the stem cell niche can be independently regulated during the in vivo orchestration of hematopoiesis.