Morphological, transcriptomic and metabolomic analyses of Sophora davidii mutants for plant height.

Sophora davidii is an important plant resource in the karst region of Southwest China, but S. davidii plant-height mutants are rarely reported. Therefore, we performed phenotypic, anatomic structural, transcriptomic and metabolomic analyses to study the mechanisms responsible for S. davidii plant-height mutants. Phenotypic and anatomical observations showed that compared to the wild type, the dwarf mutant displayed a significant decrease in plant height, while the tall mutant displayed a significant increase in plant height. The dwarf mutant cells were smaller and more densely arranged, while those of the wild type and the tall mutant were larger and loosely arranged. Transcriptomic analysis revealed that differentially expressed genes (DEGs) involved in cell wall biosynthesis, expansion, phytohormone biosynthesis, signal transduction pathways, flavonoid biosynthesis and phenylpropanoid biosynthesis were significantly enriched in the S. davidii plant-height mutants. Metabolomic analysis revealed 57 significantly differential metabolites screened from both the dwarf and tall mutants. A total of 8 significantly different flavonoid compounds were annotated to LIPID MAPS, and three metabolites (chlorogenic acid, kaempferol and scopoletin) were involved in phenylpropanoid biosynthesis and flavonoid biosynthesis. These results shed light on the molecular mechanisms of plant height in S. davidii mutants and provide insight for further molecular breeding programs.

Genetic polymorphisms of 12 X-chromosomal STR loci in Taiwanese individuals and likelihood ratio calculations applied to case studies of blood relationships.

Allele frequencies for the 12 short tandem repeat loci of the Investigator Argus X-12 kit were obtained from 514 unrelated Taiwanese individuals (327 males and 187 females). Hardy-Weinberg equilibrium tests with samples demonstrated no significant deviation from expected values for all 12 loci (p > 0.05). The linkage disequilibrium for the 12 loci in the female samples was identical to what was observed in other Han Chinese populations, with only the DXS10103 and DXS10101 loci showing significant linkage disequilibrium after corrected by Bonferroni's correction for multiple testing (p < 0.05/66). No significant differences were observed by population pairwise genetic distance analysis between Taiwanese and other Han Chinese populations. When compared with other Asian, European, and African populations, however, significant differences were observed at more than one locus. The combined mean exclusion chance was 0.99999 in duo cases and 0.99999999 in trio cases. This study used mathematical logic inferred likelihood ratio calculation formulas for full-sister, half-sister from the same father, and paternal grandmother-granddaughter relationships. The results for these three real familial cases suggest that these 12 short tandem repeat loci may appropriate for forensic relationship testing.

Transcriptome analysis provides insights into the response of Lotus corniculatus roots to low-phosphorus stress.

Introduction: A lack of soil phosphorus (P) is a principal factor restricting the normal growth of Lotus corniculatus in the karst area of Guizhou Province, China, but the response mechanism of L. corniculatus under low-phosphorus stress remains unclear. Methods: Therefore, we treated two selected L. corniculatus lines (low-P-intolerant line 08518 and low-P-tolerant line 01549) from 13 L. corniculatus lines with normal phosphorus (0.5 mmol/L KH2PO4, NP) and low phosphorus (0.005 mmol/L KH2PO4, LP) concentrations to study changes in morphological, physiological and transcriptome data under low-phosphorus stress. Results: The low-P-tolerant line 01549 exhibited better performance under low-phosphorus stress. Compared with the NP treatment, all root morphological indicators of the low-P-tolerant line 01549 increased, and those of the low-P-intolerant line 08518 decreased under low-P stress. Compared with the NP treatment, acid phosphatase (ACP), catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) activities, and the malondialdehyde (MDA), soluble sugar (SS), soluble protein (SP) and proline (Pro) contents of the two L. corniculatus lines increased under low-P stress. A transcriptome analysis of L. corniculatus showed that a total of 656 and 2243 differentially expressed genes (DEGs) were identified in line 01549 and line 08518, respectively. Meanwhile, the main pathways, such as carbohydrate metabolism, acid phosphatases, phosphate transporters and biosynthesis of secondary metabolites, as well as related genes were also screened by performing a KEGG enrichment analysis. Discussion: The findings provide an essential point of reference for studying the physiological and molecular mechanism of the response to low-P stress in L. corniculatus.

Differential Physiological, Transcriptomic, and Metabolomic Responses of Paspalum wettsteinii Under High-Temperature Stress.

Global warming has far-reaching effects on plant growth and development. As a warm-season forage grass, Paspalum wettsteinii is highly adaptable to high temperatures. However, the response mechanism of P. wettsteinii under high-temperature stress is still unclear. Therefore, we investigated the physiological indicators, transcriptome and metabolome of P. wettsteinii under different heat stress treatments. Plant height, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), and the contents of soluble sugar, proline, chlorophyll a, and chlorophyll b increased and then decreased, while the malondialdehyde (MDA) content decreased and then increased with increasing heat stress. Transcriptomic analysis revealed that genes related to energy and carbohydrate metabolism, heat shock proteins (HSPs), and transcription factors (TFs), secondary metabolite biosynthesis and the antioxidant system significantly changed to varying degrees. Metabolomic analysis showed that only free fatty acids were downregulated, while amino acids and their derivatives, organic acids, flavonoids, and sugars were both up- and downregulated under heat stress. These combined analyses revealed that growth was promoted at 25-40°C, while at 45°C, excess reactive oxygen species (ROS) damage reduced antioxidant and osmoregulatory effects and inactivated genes associated with the light and electron transport chains (ETCs), as well as damaged the PS II system and inhibited photosynthesis. A small number of genes and metabolites were upregulated to maintain the basic growth of P. wettsteinii. The physiological and biochemical changes in response to high-temperature stress were revealed, and the important metabolites and key genes involved in the response to high temperature were identified, providing an important reference for the physiological and molecular regulation of high-temperature stress in plants.

Transcriptomic and Metabolomic Analyses Reveal Key Metabolites, Pathways and Candidate Genes in Sophora davidii (Franch.) Skeels Seedlings Under Drought Stress.

Soil aridification and desertification are particularly prominent in China's karst areas, severely limiting crop yields and vegetation restoration. Therefore, it is very important to identify naturally drought-tolerant plant species. Sophora davidii (Franch.) Skeels is resistant to drought and soil infertility, is deeply rooted and is an excellent plant material for soil and water conservation. We studied the transcriptomic and metabolomic changes in S. davidii in response to drought stress (CK, control; LD, mild drought stress; MD, moderate drought stress; and SD, severe drought stress). Sophora davidii grew normally under LD and MD stress but was inhibited under SD stress; the malondialdehyde (MDA), hydrogen peroxide (H2O2), soluble sugar, proline, chlorophyll a, chlorophyll b and carotenoid contents and ascorbate peroxidase (APX) activity significantly increased, while the superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities and soluble protein content significantly decreased. In the LD/CK, MD/CK and SD/CK comparison groups, there were 318, 734 and 1779 DEGs, respectively, and 100, 168 and 281 differentially accumulated metabolites, respectively. Combined analysis of the transcriptomic and metabolomic data revealed the metabolic regulation of S. davidii in response to drought stress. First, key candidate genes such as PRR7, PRR5, GI, ELF3, PsbQ, PsaK, INV, AMY, E2.4.1.13, E3.2.1.2, NCED, PP2C, PYL, ABF, WRKY33, P5CS, PRODH, AOC3, HPD, GPX, GST, CAT and SOD1 may govern the drought resistance of S. davidii. Second, three metabolites (oxidised glutathione, abscisic acid and phenylalanine) were found to be related to drought tolerance. Third, several key candidate genes and metabolites involved in 10 metabolic pathways were identified, indicating that these metabolic pathways play an important role in the response to drought in S. davidii and possibly other plant species.

Bacillus amyloliquefaciens GB03 augmented tall fescue growth by regulating phytohormone and nutrient homeostasis under nitrogen deficiency.

Nitrogen is an important nutrient for plant growth and development. Soil microorganisms have been used to curb the imbalance between the limited content of natural environmental nitrogen and the pollution caused by increasing nitrogen fertilizer use in ecologically fragile areas. Bacillus amyloliquefaciens GB03 has been shown to confer growth promotion and abiotic stress tolerance in Arabidopsis thaliana. This study provided a new insight into the role of the plant growth-promoting rhizobacterium B. amyloliquefaciens GB03 as an initiator of defense against nitrogen deficiency in non-leguminous grass tall fescue (Festuca arundinacea). Two-week-old seedlings of tall fescue were grown with or without GB03 for 4 weeks under total nitrogen (3.75 mM NO3 -) or low nitrogen (0.25 mM NO3 -) treatment. Growth parameters, chlorophyll content, endogenous total nitrogen, total phosphorus content, and phytohormone content, including those of auxin indole-3-acetic acid, cytokinin, gibberellic acid, and abscisic acid, were determined at the time of harvest. Tall fescue grown in GB03-inoculated soil was more robust than the non-inoculated controls with respect to plant height, root length, plant biomass, chlorophyll concentration, and nutrient (total nitrogen and total phosphorus) contents under total nitrogen treatment. GB03 increased indole acetic acid content by 24.7%, whereas decreased cytokinin and abscisic acid contents by 28.4% and 26.9%, respectively, under a total nitrogen level. Remarkably, GB03 increased indole acetic acid content by more than 80% and inhibited abscisic acid production by nearly 70% under a low nitrogen level. These results showed, for the first time, that GB03 played a crucial role in mediating NO3 -dependent regulation of tall fescue growth and development, especially revealing the mechanism of soil bacteria improve resistance to nitrogen deficiency stress in non-nitrogen-fixing species.

SSR individual identification system construction and population genetics analysis for Chamaecyparis formosensis.

Chamaecyparis formosensis is an endemic species of Taiwan, threatened from intensive use and illegal felling. An individual identification system for C. formosensis is required to provide scientific evidence for court use and deter illegal felling. In this study, 36 polymorphic simple sequence repeat markers were developed. By applying up to 28 non-linked of the developed markers, it is calculated that the cumulative random probability of identity (CPI) is as low as 1.652 × 10-12, and the identifiable population size is up to 60 million, which is greater than the known C. formosensis population size in Taiwan. Biogeographical analysis data show that C. formosensis from four geographic areas belong to the same genetic population, which can be further divided into three clusters: SY (Eastern Taiwan), HV and GW (Northwestern Taiwan), and MM (Southwestern Taiwan). The developed system was applied to assess the provenance of samples with 88.44% accuracy rate and therefore can serve as a prescreening tool to reduce the range required for comparison. The system developed in this study is a potential crime-fighting tool against illegal felling.

Y-chromosomal STRs haplotypes in the Taiwanese Paiwan population.

The distribution of Y-chromosomal short tandem repeat (Y-STR) haplotypes was determined in a population of Taiwanese Paiwan aboriginals. Using 17 Y-STR markers, a total of 135 haplotypes were observed, 102 of which were unique. The overall haplotype diversity for the 17 Y-STR loci tested was 0.9922 and the discrimination capacity was 0.6490. In addition, three novel intermediate alleles at the DYS448 locus were also found.

Development and technical application of SSR-based individual identification system for Chamaecyparis taiwanensis against illegal logging convictions.

Chamaecyparis taiwanensis is an endemic plant suffering illegal logging in Taiwan for its high economic value. Lack of direct evidence to correlate stump and timber remains a hurdle for law enforcement. In this report, 23 polymorphic Genomic Simple Sequence Repeat (gSSR) and 12 Expressed Sequence Tag (EST)-SSR markers were developed and their transferability was assessed. The individual identification system built from selected non-linkage 30 SSR markers has a combined probability of identity as 5.596 × 10-12 equivalents to identifying an individual in a population of up to 18 million C. taiwanensis with 99.99% confidence level. We also applied the system in an actual criminal case by selecting 19 of these markers to correlate illegally felled timbers and victim trees. Our data demonstrate that molecular signals from three timbers hit with three victim trees with confidence level more than 99.99%. This is the first example of successfully applying SSR in C. taiwanensis as a court evidence for law enforcement. The identification system adapted advanced molecular technology and exhibits its great potential for natural resource management on C. taiwanensis.

Differentiation of mesenchymal stromal cells derived from umbilical cord Wharton's jelly into hepatocyte-like cells.

BACKGROUND AIMS: Mesenchymal stromal cells (MSC) isolated from bone marrow (BM), adipose tissue and umbilical cord blood can be induced to differentiate into hepatocyte-like cells. MSC can also be isolated from umbilical cord Wharton's jelly (UC MSC), which can be easily obtained. UC MSC are more primitive MSC than those isolated from other tissue sources and do not express the major histocompatibility complex (MHC) class II (HLA-DR) antigens. Previous studies have shown that UC MSC are still viable and not rejected 4 months after transplantation as xenografts, without the need for immune suppression, suggesting that they are a favorable cell source for transplantation. METHODS: UC MSC were induced to differentiate into hepatocyte-like cells by a simple one-step protocol with hepatotic growth factor (HGF) and fibroblast growth factor-4 (FGF-4). Differentiated cells were examined for the expression of hepatocyte-specific markers and hepatocyte functions. RESULTS: UC MSC were isolated. Flow cytometry analysis showed that they expressed the MSC-specific markers. They differentiated into osteoblast-, adipocyte- and chondrocyte-like cells, showing their multipotent differentiation potential. Immunocytochemistry, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis demonstrated that UC MSC expressed the hepatocyte-specific markers albumin (ALB), human alpha-fetoprotein (AFP) and cytokeratin 18 (CK-18) following hepatocyte induction. Periodic acid-Schiff staining showed that differentiated UC MSC could store glycogen, and an low-density lipoprotein (LDL)-uptake assay showed that they could uptake LDL. CONCLUSIONS: This study demonstrates that UC MSC can differentiate into functional hepatocyte-like cells following the induction of HGF and FGF-4. UC MSC can serve as a favorable cell source for tissue engineering in the treatment of liver disease.

Genetic polymorphisms of 17 Y-chromosomal short tandem repeat loci in Atayal population of Taiwan.

AIM: To define the Y-chromosomal genetic structure in a sample of Atayal men from Taiwan. METHODS: Buccal swab samples were collected from 170 unrelated healthy male volunteers from Taiwanese aboriginal Atayal population. Genomic DNA was extracted and 17 Y chromosome-specific short tandem repeat loci (DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385a/b, DYS393, DYS391, DYS439, DYS635, DYS392, Y GATA H4, DYS437, DYS438, and DYS448) were analyzed using the AmpFlSTR Yfiler Polymerase Chain Reaction Amplification Kit. RESULTS: A total of 99 different haplotypes were identified, 69 (69.7%) of which were unique. Total haplotype diversity was 0.9887. The most common haplotype was shared by 9 individuals in the study sample. Gene diversities ranged from 0.0574 for DYS438 to 0.6749 for DYS456. CONCLUSION: Our results will help provide the molecular genetic evidence for human settlement of the Pacific.

Characterization of metallization and amorphization for GaP under different hydrostatic environments in diamond anvil cell up to 40.0 GPa.

High-pressure phase stability of gallium phosphide was explored under different hydrostatic environments up to 40.0 GPa in a diamond anvil cell. Two irreversible phase transitions from the semiconductor to metal to an amorphous state appear at 19.8 and 31.5 GPa and as well as 22.6 and 35.3 GPa under nonhydrostatic and hydrostatic environments, respectively. Furthermore, the hysteresis effect of the high-pressure phase transition of a sphalerite-structure compound under a hydrostatic environment was disclosed. All of the obtained results can provide new insight into the underlying structural evolution and electrical transport characteristics for the semiconducting compound at different hydrostatic environments.

Pressure-induced metallization in MoSe2 under different pressure conditions.

In this study, the vibrational and electrical transport properties of molybdenum diselenide were investigated under both non-hydrostatic and hydrostatic conditions up to ∼40.2 GPa using the diamond anvil cell in conjunction with Raman spectroscopy, electrical conductivity, high-resolution transmission electron microscopy, atomic force microscopy, and first-principles theoretical calculations. The results obtained indicated that the semiconductor-to-metal electronic phase transition of MoSe2 can be extrapolated by some characteristic parameters including abrupt changes in the full width at half maximum of Raman modes, electrical conductivity and calculated bandgap. Under the non-hydrostatic condition, metallization occurred at ∼26.1 GPa and it was irreversible. However, reversible metallization occurred at ∼29.4 GPa under the hydrostatic condition. In addition, the pressure-induced metallization reversibility of MoSe2 can be revealed by high-resolution transmission electron and atomic force microscopy of the recovered samples under different hydrostatic conditions. This discrepancy in the metallization phenomenon of MoSe2 in different hydrostatic environments was attributed to the mitigated interlayer van der Waals coupling and shear stress caused by the insertion of pressure medium into the layers.

Phase Transition and Metallization of Orpiment by Raman Spectroscopy, Electrical Conductivity and Theoretical Calculation under High Pressure.

The structural, vibrational, and electronic characteristics in orpiment were performed in the diamond anvil cell (DAC), combined with a series of experimental and theoretical research, including Raman spectroscopy, impedance spectroscopy, atomic force microscopy (AFM), high-resolution transmission electron microscopy (HRTEM), and first-principles theoretical calculations. The isostructural phase transition at ~25.0 GPa was manifested as noticeable changes in the compressibility, bond lengths, and slope of the conductivity, as well as in a continuous change in the pressure dependence of the unit cell volume. Furthermore, a pressure-induced metallization occurred at ~42.0 GPa, accompanied by reversible electrical conductivity. We also determined the metallicity of orpiment at 45.0 GPa by first-principles theoretical calculations, and the results were in good agreement with the results of the temperature-dependent conductivity measurements. The HRTEM and AFM images of the recovered sample confirmed that orpiment remains in the crystalline phase with an intact layered structure and available crystal-shaped clusters. These high-pressure behaviors of orpiment present some crucial information on the structural phase transition, metallization, amorphization and superconductivity for the A2B3-type of engineering materials at high pressure.

MTF2 Induces Epithelial-Mesenchymal Transition and Progression of Hepatocellular Carcinoma by Transcriptionally Activating Snail.

BACKGROUND: Metal regulatory transcription factor 2 (MTF2) has been previously reported as a protein binding to the metal response element of the mouse metallothionein promoter, which is involved in chromosome inactivation and pluripotency. However, the function of MTF2 in tumor formation and progression has not yet been completely elucidated. METHODS: The expression of MTF2 and clinicopathological characteristics were evaluated by hepatocellular carcinoma (HCC) tissue microarray of 240 specimens. The role of MTF2 on HCC progression was determined using MTT, crystal violet, and transwell assays. Tumor growth was monitored in a xenograft model, and intrahepatic metastasis models were established. RESULTS: The expression of MTF2 was increased in HCC and strongly associated with the clinical characteristics and prognosis. Forced expression of MTF2 in HCC cells significantly promoted cell growth, migration, and invasion in vitro. In contrast, downregulation of MTF2 inhibited cell growth, migration, and invasion in vitro. Moreover, knock down of MTF2 suppressed tumorigenesis and intrahepatic metastasis of HCC cells in vivo. Mechanistically, MTF2 overexpression may promote growth and epithelial-mesenchymal transition processes of HCC cells by facilitating Snail transcription. CONCLUSION: MTF2 promotes the proliferation, migration, and invasion of HCC cells by regulating Snail transcription, providing a potential therapeutic candidate for patients with HCC.

Simultaneous detection and differentiation of Newcastle disease and avian influenza viruses using oligonucleotide microarrays.

Newcastle disease (ND) and avian influenza (AI) are two of the most important zoonotic viral diseases of birds throughout the world. These two viruses often have a great impact upon the poultry industry. Both viruses are associated with transmission from wild to domestic birds, and often display similar signs that need to be differentiated. A rapid surveillance among wild and domestic birds is important for early disease detection and intervention, and is the basis for what measures should be taken. The surveillance, thus, should be able to differentiate the diseases and provide a detailed analysis of the virus strains. Here, we described a fast, simultaneous and inexpensive approach to the detection of Newcastle disease virus (NDV) and avian influenza virus (AIV) using oligonucleotide microarrays. The NDV pathotypes and the AIV haemagglutinin subtypes H5 and H7 were determined at the same time. Different probes on a microarray targeting the same gene were implemented in order to encompass the diversified virus strains or provide multiple confirmations of the genotype. This ensures good sensitivity and specificity among divergent viruses. Twenty-four virus isolates and twenty-four various combinations of the viruses were tested in this study. All viruses were successfully detected and typed. The hybridization results on microarrays were clearly identified with the naked eyes, with no further imaging equipment needed. The results demonstrate that the detection and typing of multiple viruses can be performed simultaneously and easily using oligonucleotide microarrays. The proposed method may provide potential for rapid surveillance and differential diagnosis of these two important zoonoses in both wild and domestic birds.

Sex identification of owls (family Strigidae) using oligonucleotide microarrays.

Molecular sexing of the diversified avian family Strigidae is difficult. Sex identification using the intron length difference between W and Z chromosomal CHD1 genes, as visualized by agarose gel electrophoreses, often produces ambiguous results. Here we describe a simple method for sexing a variety of Strigidae species using oligonucleotide microarrays, on which several sex-specific probes operated complementarily or in concert. The sex of 8 owl species was identified clearly on the microarrays through sequence recognition. This sequence-directed method can be easily applied to a wider range of Strigidae species.

Resolution and evaluation of 3-chlorophenyl-3-hydroxypropionylhydroxamic acid as antivirulence agent with excellent eradication efficacy in Helicobacter pylori infected mice.

The continuing emergence of drug-resistant Helicobacter pylori (HP) drives the ongoing need for the development of new and effective anti-HP drugs. Urease inhibitor has now gained strong interest as an alternative approach for HP infections. 3-Chlorophenyl-3-hydroxypropionylhydroxamic acid (CPH), a novel urease inhibitor identified in our group, shows impressive potency, which was optically separated for a further exploration. Here, we report in vitro/in vivo pharmacological evaluation of (±)-CPHs and the enantiomers. The raceme and the individual enantiomers significantly suppress gastritis at 32 mg/kg b.i.d dose with lower toxicity to mammalian cells (with CC50s ≥ 3.16 mM) and mice (LD50s ≥ 2338 mg/kg) than the clinically used agent acetohydroxamic acid. Furthermore, a significant increase of eradication of HP is observed for the combination of (±)-CPHs or the enantiomers with an antimicrobial. These studies revealed that CPH is a promising candidate for an alternative treatment of HP-dependent conditions by targeting virulence factor urease, and CPH may be used as a raceme.

CPI distribution and cutoff values for duo kinship testing.

DNA-based tests commonly use 13 STR (short tandem repeat) loci in human identification and paternity testing--the Combined DNA Index System or CODIS. Its average degree of accuracy of paternity identification is greater than 0.9999 under the circumstance of a mother, a child and a putative father. However, the possibility of false inclusions increases under circumstances such as [1] only two members of a family group are available--a duo case during determination of paternity or [2] identification of human remains while only one living relative is present. In Taiwan, the National Unidentified Human Remains Database uses the CODIS 13 STR for the identification of family members. Two or more reference samples in the DNA database have been found to share one allele at all loci tested. Then the Combined Paternity Index (CPI) is used to determine and provide an estimate of kinship in such cases. Combining 499,500 sets of DNA data for the 13 STR CODIS loci, totally 431 (0.086%) cases are false inclusions where all 13 loci shared at least one allele. Simulated partial DNA profiles (not all 13 loci yielded results) were created to mimic the mutation and degradation process. All 431 real duo cases were analyzed to evaluate sensitivity and specificity. This report provided four kinship-matching situations with CPI cutoff values when the number of allele-sharing loci exceeded 11. CPI values greater or lesser than the suggested cutoff point will provide a greater degree of confidence in determining whether two samples are derived from first-degree relatives.

Sexing a wider range of avian species based on two CHD1 introns with a unified reaction condition.

Identifying the sex of a bird is important to ensure successful breeding strategies and effective conservation programs. Sex may be identified from the intron size of the CHD1 gene located on the avian sex chromosomes Z and W. However, because of the great nucleotide diversity across different avian species, no given intron is in widespread use without ambiguous results. Complicated modifications of the reaction condition are required to suit different species. Two CHD1 introns were used with a unified reaction condition in this study to simplify the procedure. Consequently, genders of 73 avian species covering 19 families were successfully identified based on this two-intron approach. This means the ability to sex a wider range of avian species using a simplified procedure, greatly assisting in population management at zoos. Zoo Biol 26:425-431, 2007. (c) 2007 Wiley-Liss, Inc.