RNAi-directed downregulation of OsBADH2 results in aroma (2-acetyl-1-pyrroline) production in rice (Oryza sativa L.)

<p>Abstract</p> <p>Background</p> <p>Aromatic rice is popular worldwide because of its characteristic fragrance. Genetic studies and physical fine mapping reveal that a candidate gene (<it>fgr</it>/<it>OsBADH2</it>) homologous to <it>betaine aldehyde dehydrogenase </it>is responsible for aroma metabolism in fragrant rice varieties, but the direct evidence demonstrating the functions of <it>OsBADH2 </it>is lacking. To elucidate the physiological roles of <it>OsBADH2</it>, sequencing approach and RNA interference (RNAi) technique were employed to analyze allelic variation and functions of <it>OsBADH2 </it>gene in aroma production. Semi-quantitative, real-time reverse transcription-polymerase chain reaction (RT-PCR), as well as gas chromatography-mass spectrometry (GC-MS) were conducted to determine the expression levels of <it>OsBADH2 </it>and the fragrant compound in wild type and transgenic <it>OsBADH2</it>-RNAi repression lines, respectively.</p> <p>Results</p> <p>The results showed that multiple mutations identical to <it>fgr </it>allele occur in the 13 fragrant rice accessions across China; <it>OsBADH2 </it>is expressed constitutively, with less expression abundance in mature roots; the disrupted <it>OsBADH2 </it>by RNA interference leads to significantly increased 2-acetyl-1-pyrroline production.</p> <p>Conclusion</p> <p>We have found that the altered expression levels of <it>OsBADH2 </it>gene influence aroma accumulation, and the prevalent aromatic allele probably has a single evolutionary origin.</p

Comprehensive analysis of codon bias in 13 Ganoderma mitochondrial genomes

IntroductionCodon usage bias is a prevalent phenomenon observed across various species and genes. However, the specific attributes of codon usage in the mitochondrial genome of Ganoderma species remain unknown.MethodsIn this study, we investigated the codon bias of 12 mitochondrial core protein-coding genes (PCGs) in 9 Ganoderma species, including 13 Ganoderma strains.ResultsThe codons of all Ganoderma strains showed a preference for ending in A/T. Additionally, correlations between codon base composition and the codon adaptation index (CAI), codon bias index (CBI) and frequency of optimal codons (FOP) were identified, demonstrating the impact of base composition on codon bias. Various base bias indicators were found to vary between or within Ganoderma strains, including GC3s, the CAI, the CBI, and the FOP. The results also revealed that the mitochondrial core PCGs of Ganoderma have an average effective number of codons (ENC) lower than 35, indicating strong bias toward certain codons. Evidence from neutrality plot and PR2-bias plot analysis indicates that natural selection is a major factor affecting codon bias in Ganoderma. Additionally, 11 to 22 optimal codons (ΔRSCU&gt;0.08 and RSCU&gt;1) were identified in 13 Ganoderma strains, with GCA, AUC, and UUC being the most widely used optimal codons in Ganoderma. By analyzing the combined mitochondrial sequences and relative synonymous codon usage (RSCU) values, the genetic relationships between or within Ganoderma strains were determined, indicating variations between them. Nevertheless, RSCU-based analysis illustrated the intra- and interspecies relationships of certain Ganoderma species.DiscussionThis study deepens our insight into the synonymous codon usage characteristics, genetics, and evolution of this important fungal group

Using Phenol Formaldehyde Resin, Hexamethylenetetramine and Matrix Asphalt to Synthesize Hard-Grade Asphalts for High-Modulus Asphalt Concrete

Traditional hard-grade asphalts for high-modulus asphalt concrete (HMAC) are produced by using natural hard-grade asphalt to modify matrix asphalts. However, natural hard-grade asphalts are scarce and expensive. To find a sustainable alternative, this study presented a method to synthesize hard-grade asphalts using phenol formaldehyde resin (PFR), hexamethylenetetramine (HMTA) and matrix asphalts. Infrared radiation (IR) spectra analysis and fraction analysis for the modifiers and synthesize asphalts show that asphalt molecules can be cross-linked into larger polymeric groups by the thermosetting phenol formaldehyde resin (TPFR) which is the reaction product of PFR and HMTA. This process increased the asphaltene and resin fraction in asphalt, thus transforming a matrix asphalt into hard grade. With the dosing combinations of 4% PFR/15~20% HMTA, 6% PFR/8~10% HMTA and 8% PFR/5~5.7% HMTA, dynamic modules of HMAC were 14,000~16,000 MPa, which satisfied the basic application requirements for HMAC. The rutting resistance of the new hard-grade asphalts with the above dosage combinations completely exceeds the traditional product using the Trinidad Lake asphalt as the raw material. Increasing the amount of PFR/HMTA can further improve the rutting resistance. However, to ensure the fatigue and cracking resistance of the HMAC can get a level like the traditional product, the dosages of HMTA should be controlled below 15%

Prognostic significance of CDK6 amplification in esophageal squamous cell carcinoma

Dysregulation of CDK6 plays crucial roles in the carcinogenesis of many kinds of human malignancies. However, the role of CDK6 in esophageal squamous cell carcinoma (ESCC) is not well known. We investigated the frequency and prognostic value of CDK6 amplification to improve the risk stratification in patients with ESCC. Pan-cancer analysis of CDK6 was conducted on The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx) and Gene Expression Omnibus (GEO) databases. CDK6 amplification was detected in 502 ESCC samples by Fluorescence in situ hybridization (FISH) through tissue microarrays (TMA). Pan-cancer analysis revealed that CDK6 mRNA level was much higher in multiple kinds of cancers and higher CDK6 mRNA level indicated a better prognosis in ESCC. In this study, CDK6 amplification was detected in 27.5% (138/502) of patients with ESCC. CDK6 amplification was significantly correlated with tumor size (p = 0.044). Patients with CDK6 amplification tended to have a longer disease-free survival (DFS) (p = 0.228) and overall survival (OS) (p = 0.200) compared with patients without CDK6 amplification but of no significance. When further divided into I–II and III–IV stage, CDK6 amplification was significantly associated with longer DFS and OS in III-IV stage group (DFS, p = 0.036; OS, p = 0.022) rather than in I-II stage group (DFS, p = 0.776; OS, p = 0.611). On univariate and multivariate analysis of Cox hazard model, differentiation, vessel invasion, nerve invasion, invasive depth, lymph node metastasis and clinical stage were significantly associated with DFS and OS. Moreover, invasion depth was an independent factor for ESCC prognosis. Taken together, for ESCC patients in III-IV stage, CDK6 amplification indicated a better prognosis

Asymmetric Migration of Human Keratinocytes under Mechanical Stretch and Cocultured Fibroblasts in a Wound Repair Model

<div><p>Keratinocyte migration during re-epithelization is crucial in wound healing under biochemical and biomechanical microenvironment. However, little is known about the underlying mechanisms whereby mechanical tension and cocultured fibroblasts or keratinocytes modulate the migration of keratinocytes or fibroblasts. Here we applied a tensile device together with a modified transwell assay to determine the lateral and transmembrane migration dynamics of human HaCaT keratinocytes or HF fibroblasts. A novel pattern of asymmetric migration was observed for keratinocytes when they were cocultured with non-contact fibroblasts, <i>i.e.</i>, the accumulative distance of HaCaT cells was significantly higher when moving away from HF cells or migrating from down to up cross the membrane than that when moving close to HF cells or when migrating from up to down, whereas HF migration was symmetric. This asymmetric migration was mainly regulated by EGF derived from fibroblasts, but not transforming growth factor α or β1 production. Mechanical stretch subjected to fibroblasts fostered keratinocyte asymmetric migration by increasing EGF secretion, while no role of mechanical stretch was found for EGF secretion by keratinocytes. These results provided a new insight into understanding the regulating mechanisms of two- or three-dimensional migration of keratinocytes or fibroblasts along or across dermis and epidermis under biomechanical microenvironment.</p></div

Transmigration of HF cells on collagen I-coated transwell chamber.

<p><i>Up-to-down</i> (<i>A</i>) or <i>down-to-up</i> (<i>B</i>) transmigration of monoclutured or cocultured HF cells in the absence or presence of mechanical stretch. Data are presented as the fraction of transmigrated HaCaT cells at <i>t</i> = 36, 48 h normalized to that at <i>t</i> = 24 h in respective cases.</p

Comparison of content of EGF secretion in the conditional medium using 2D (<i>A</i>) or 3D (<i>B</i>) assay.

<p>Data were collected from at least triplets and presented as the average optical intensity at OD 450</p

Lateral migration set-up of HaCaT or HF cells cultured on collagen I-coated PDMS membrane in a tensile device.

<p>(<i>A</i>) An in-house developed tensile device by applying mechanical stretch, <i>via</i> the PDMS membrane, to the cells (<i>upper panel</i>). Also plotted was the tensile strain profile of PDMS membrane at a pre-set 20% strain along tensile direction (<i>x</i>-axis) (<i>lower panel</i>). (<i>B</i>) Schematic of monocultured (<i>upper panel</i>) or cocultured (<i>lower panel</i>) HaCaT or HF cells on the stretched membrane where the cells tend to migrate in two directions with a distance <i>L</i> or <i>L’</i> at a given time point. <i>Arrows</i> indicate the migration direction of the cells when they are monocultured (to leftward or rightward) or cocultured (to outward or inward). (<i>C</i>, <i>D</i>) Typical images of cocultured HaCaT cells at <i>t</i> = 0 (<i>C</i>) and 6 day (<i>D</i>) with the leading edge indicated as <i>white dashed lines</i> (<i>Bar</i> = 500 µm) with the <i>inserts</i> illustrating the magnified images (<i>Bar</i> = 100 µm). The <i>white strip</i> in (<i>D</i>) was used to disconnect the oversized image.</p

Lateral migration of HF cells on collagen I-coated PDMS membrane in the absence or presence of HaCaT cells and mechanical stretch.

<p>(<i>A, B</i>) Time course of normalized <i>instantaneous</i> or <i>accumulative</i> distance of monocultured (M/N) (<i>A</i>) or cocultured (C/N) (<i>B</i>) HF cells in the absence of mechanical stretch. (<i>C</i>, <i>D</i>) Time course of normalized <i>instantaneous</i> or <i>accumulative</i> distance of monocultured (M/S) (<i>C</i>) or cocultured (C/S) (<i>D</i>) HF cells in the presence of mechanical stretch. Data were collected and presented in the same way as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074563#pone-0074563-g002" target="_blank">Fig. 2</a>.</p