Ligation-mediated PCR (LM-PCR) is a classical method for isolating flanking sequences; however, it has a common limitation of reduced success rate owing to the circularization or multimerization of target restriction fragments including the known sequence. To address this limitation, we developed a novel LM-PCR method, termed Cyclic Digestion and Ligation-Mediated PCR (CDL-PCR). The novelty of this approach involves the design of new adapters that cannot be digested after being ligated with the restriction fragment, and cyclic digestion and ligation may be manipulated to block the circularization or multimerization of the target restriction fragments. Moreover, to improve the generality and flexibility of CDL-PCR, an adapter precursor sequence was designed, which could be digested to prepare 12 different adapters at low cost. Using this method, the flanking sequences of T-DNA insertions were obtained from transgenic rice and Arabidopsis thaliana. The experimental results demonstrated that CDL-PCR is an efficient and flexible method for identifying the flanking sequences in transgenic rice and Arabidopsis thaliana.

The Arabidopsis protein DELAY OF GERMINATION 1 (DOG1) is a key regulator of seed dormancy, which is a life history trait that determines the timing of seedling emergence. The amount of DOG1 protein in freshly harvested seeds determines their dormancy level. DOG1 has been identified as a major dormancy QTL and variation in DOG1 transcript levels between accessions contributes to natural variation for seed dormancy. The DOG1 gene is alternatively spliced. Alternative splicing increases the transcriptome and proteome diversity in higher eukaryotes by producing transcripts that encode for proteins with altered or lost function. It can also generate tissue specific transcripts or affect mRNA stability. Here we suggest a different role for alternative splicing of the DOG1 gene. DOG1 produces five transcript variants encoding three protein isoforms. Transgenic dog1 mutant seeds expressing single DOG1 transcript variants from the endogenous DOG1 promoter did not complement because they were non-dormant and lacked DOG1 protein. However, transgenic plants overexpressing single DOG1 variants from the 35S promoter could accumulate protein and showed complementation. Simultaneous expression of two or more DOG1 transcript variants from the endogenous DOG1 promoter also led to increased dormancy levels and accumulation of DOG1 protein. This suggests that single isoforms are functional, but require the presence of additional isoforms to prevent protein degradation. Subsequently, we found that the DOG1 protein can bind to itself and that this binding is required for DOG1 function but not for protein accumulation. Natural variation for DOG1 binding efficiency was observed among Arabidopsis accessions and contributes to variation in seed dormancy.

This study aimed to investigate the effect of using a deep neural network (DNN) in breast cancer (BC) detection.

Resistance to pathogens is essential for survival of wild and cultivated plants. Pathogen susceptibility causes major losses of crop yield and quality. Durable field resistance combined with high yield and other superior agronomic characters are therefore, important objectives in every crop breeding program. Precision and efficacy of resistance breeding can be enhanced by molecular diagnostic tools, which result from knowledge of the molecular basis of resistance and susceptibility. Breeding uses resistance conferred by single R genes and polygenic quantitative resistance. The latter is partial but considered more durable. Molecular mechanisms of plant pathogen interactions are elucidated mainly in experimental systems involving single R genes, whereas most genes important for quantitative resistance in crops like potato are unknown. Quantitative resistance of potato to Phytophthora infestans causing late blight is often compromised by late plant maturity, a negative agronomic character. Our objective was to identify candidate genes for quantitative resistance to late blight not compromised by late plant maturity. We used diagnostic DNA-markers to select plants with different field levels of maturity corrected resistance (MCR) to late blight and compared their leaf transcriptomes before and after infection with P. infestans using SuperSAGE (serial analysis of gene expression) technology and next generation sequencing. We identified 2034 transcripts up or down regulated upon infection, including a homolog of the kiwi fruit allergen kiwellin. 806 transcripts showed differential expression between groups of genotypes with contrasting MCR levels. The observed expression patterns suggest that MCR is in part controlled by differential transcript levels in uninfected plants. Functional annotation suggests that, besides biotic and abiotic stress responses, general cellular processes such as photosynthesis, protein biosynthesis, and degradation play a role in MCR.

Pulmonary fibrosis is characterized by lung fibroblast proliferation and collagen secretion. In lipopolysaccharide (LPS)-induced acute lung injury (ALI), aberrant proliferation of lung fibroblasts is initiated in early disease stages, but the underlying mechanism remains unknown. In this study, we knocked down Toll-like receptor 4 (TLR4) expression in cultured mouse lung fibroblasts using TLR4-siRNA-lentivirus in order to investigate the effects of LPS challenge on lung fibroblast proliferation, phosphoinositide3-kinase (PI3K)-Akt pathway activation, and phosphatase and tensin homolog (PTEN) expression. Lung fibroblast proliferation, detected by BrdU assay, was unaffected by 1 mug/mL LPS challenge up to 24 hours, but at 72 hours, cell proliferation increased significantly. This proliferation was inhibited by siRNA-mediated TLR4 knockdown or treatment with the PI3K inhibitor, Ly294002. In addition, siRNA-mediated knockdown of TLR4 inhibited the LPS-induced up-regulation of TLR4, down-regulation of PTEN, and activation of the PI3K-Akt pathway (overexpression of phospho-Akt) at 72 hours, as detected by real-time PCR and Western blot analysis. Treatment with the PTEN inhibitor, bpV(phen), led to activation of the PI3K-Akt pathway. Neither the baseline expression nor LPS-induced down-regulation of PTEN in lung fibroblasts was influenced by PI3K activation state. PTEN inhibition was sufficient to exert the LPS effect on lung fibroblast proliferation, and PI3K-Akt pathway inhibition could reverse this process. Collectively, these results indicate that LPS can promote lung fibroblast proliferation via a TLR4 signaling mechanism that involves PTEN expression down-regulation and PI3K-Akt pathway activation. Moreover, PI3K-Akt pathway activation is a downstream effect of PTEN inhibition and plays a critical role in lung fibroblast proliferation. This mechanism could contribute to, and possibly accelerate, pulmonary fibrosis in the early stages of ALI/ARDS.

Tuber yield and starch content of the cultivated potato are complex traits of decisive importance for breeding improved varieties. Natural variation of tuber yield and starch content depends on the environment and on multiple, mostly unknown genetic factors. Dissection and molecular identification of the genes and their natural allelic variants controlling these complex traits will lead to the development of diagnostic DNA-based markers, by which precision and efficiency of selection can be increased (precision breeding).

A systematic review and meta-analysis was made to see whether extracorporeal membrane oxygenation (ECMO) in liver transplantation could improve non-heart-beating donors (NHBDs) recipients' outcomes compared with donors after brain death (DBDs) recipients.

Bacillus utilize preferred sugars such as glucose over other carbon sources due to carbon catabolite repression (CCR). Surfactin is a small signal molecule to regulate the quorum-sensing (QS) response such as biofilm formation and sporulation in B. subtilis. Here, the srfA operon for synthesis of surfactin was mutated for disrupting the production of surfactin in B. amyloliquefaciens. The srfA-mutant strain showed a defective biofilm and sporulation but could be restored by addition with surfactin, indicating that surfactin is a QS signal molecule in B. amyloliquefaciens. Unexpectedly, mutation of srfA also led to the cells' death although nutrients were still enough to support the bacterial growth during this period. Analysis of transcriptomes found that the srfA-mutant strain could not relieve CCR to use non-preferred carbon sources after glucose exhaustion due to deficiency of surfactin. This was further verified by the fact that addition with glucose could dramatically restore the growth, and addition with surfactin could improve the enzymes' activity (e.g., glucanase and α-amylase) to use non-preferred carbon sources in the srfA-mutant strain. After glucose exhaustion, the cells produce surfactin to relieve CCR for utilizing non-preferred sugars. As a signal molecule to regulate QS, surfactin also directly or indirectly relieves the CcpA-mediated CCR to utilize non-preferred carbon sources countering nutrient limitation (e.g., glucose deprivation) in the environment. In conclusion, our findings provide the first evidence that the QS signal molecule of surfactin is also involved in relieving the CcpA-mediated CCR in B. amyloliquefaciens.

Background: Hepatic fibrosis is a premalignant lesion, and how injured hepatocytes transform into malignancy in a fibrotic microenvironment is poorly understood. Senescence is one of major fates of activated hepatic stellate cells (HSCs). Paucity of literature is available regarding the influence of senescent HSCs on behavior of steatotic hepatocytes. Methods: Senescent HSCs were identified in a murine model of nonalcoholic steatohepatitis (NASH)-fibrosis-hepatocellular carcinoma (HCC) and human NASH-HCC specimens. Secretome of senescent HSCs was analyzed by label-free mass-spectrum (NanoRPLC-MS/MS) and verified quantitatively. Results: Senescent HSCs were increased along with the progression from nonalcoholic fatty liver (NAFL), NASH to NASH-fibrosis, and reached a peak at the stage of advanced fibrosis and then decreased when hepatocellular dysplasia or HCC was developed. Critical components affecting proliferation, epithelial-mesenchymal transition (EMT) or migration were identified from secretome of senescent HSCs, and may activate morphogenic hedgehog or oncogenic Wnt signaling pathways to accelerate malignant transformation of steatotic or dysplastic hepatocytes. Primary hepatocytes stimulated with conditioned medium from senescent HSCs, in co-culture or co-cultured in 3D spheroids with senescent HSCs exhibited an enhanced proliferating or EMT profile. Conclusion: Senescent HSCs secreted a characterized protein profile favoring malignant transformation of steatotic or dysplastic hepatocytes through activating morphogenic hedgehog or oncogenic Wnt signaling pathways in the progression from NASH to malignancy.

Potassium-sulfur batteries attract tremendous attention as high-energy and low-cost energy storage system, but achieving high utilization and long-term cycling of sulfur remains challenging. Here we show a strategy of optimizing potassium polysulfides for building high-performance potassium-sulfur batteries. We design the composite of tungsten single atom and tungsten carbide possessing potassium polysulfide migration/conversion bi-functionality by theoretical screening. We create two ligand environments for tungsten in the metal-organic framework, which respectively transmute into tungsten single atom and tungsten carbide nanocrystals during pyrolysis. Tungsten carbide provide catalytic sites for potassium polysulfides conversion, while tungsten single atoms facilitate sulfides migration thereby significantly alleviating the insulating sulfides accumulation and the associated catalytic poisoning. Resultantly, highly efficient potassium-sulfur electrochemistry is achieved under high-rate and long-cycling conditions. The batteries deliver 89.8% sulfur utilization (1504 mAh g-1), superior rate capability (1059 mAh g-1 at 1675 mA g-1) and long lifespan of 200 cycles at 25 °C. These advances enlighten direction for future KSBs development.

Panicle traits are among the most important agronomic characters which directly relate to yield in rice. Grain number (GN), panicle length (PL), primary branch number (PBN), and secondary branch number (SBN) are the major components of rice panicle structure, and are all controlled by quantitative trait loci (QTLs). In our research, four advanced backcross overlapping populations (BIL152, BIL196a, BIL196b, and BIL196b-156) carrying introgressed segments from chromosome 6 were derived from an indica/japonica cross that used the super-hybrid rice restorer line HR1128 and the international sequenced japonica cultivar 'Nipponbare' as the donor and recurrent parents, respectively. The four panicle traits, GN, PL, PBN, and SBN, were evaluated for QTL effects using the inclusive composite interval mapping (ICIM) method in populations over two years at two sites. Results showed that a total of twelve QTLs for GN, PL, PBN, and SBN were detected on chromosome 6. Based on marker loci physical positions, the QTLs were found to be tightly linked to three important chromosomal intervals described as RM7213 to RM19962, RM20000 to RM20210, and RM412 to RM20595. Three QTLs identified in this study, PL6-5, PBN6-1, and PBN6-2, were found to be novel compared with previous studies. A major QTL (PL6-5) for panicle length was detected in all four populations at two locations, and its position was narrowed down to a 1.3Mb region on chromosome 6. Near isogenic lines (NILs) carrying PL6-5 will be developed for fine mapping of the QTL, and our results will provide referable information for gene excavation of panicle components in rice.

Lipopolysaccharide (LPS)-induced pulmonary fibrosis is characterized by aberrant proliferation and activation of lung fibroblasts. Epigenetic regulation of thymocyte differentiation antigen 1 (Thy-1) is associated with lung fibroblast phenotype transformation that results in aberrant cell proliferation. However, it is not clear whether the epigenetic regulation of Thy-1 expression is required for LPS-induced lung fibroblast proliferation. To address this issue and better understand the relative underlying mechanisms, we used mouse lung fibroblasts as model to observe the changes of Thy-1 expression and histone deacetylation after LPS challenge. The results showed that cellular DNA synthesis, measured by BrdU incorporation, was impacted less in the early stage (24 hrs) after the challenge of LPS, but significantly increased at 48 or 72 hrs after the challenge of LPS. Meanwhile, Thy-1 expression, which was detected by real-time PCR and Western blot, in lung fibroblasts decreased with increased time after LPS challenge and diminished at 72 hrs. We also found that the acetylation of either histone H3 or H4 decreased in the LPS-challenged lung fibroblasts. ChIP assay revealed that the acetylation of histone H4 (Ace-H4) decreased in the Thy-1 promoter region in response to LPS. In addition, all the above changes could be attenuated by depletion of TLR4 gene. Our studies indicate that epigenetic regulation of Thy-1 gene expression by histone modification is involved in LPS-induced lung fibroblast proliferation.

Abnormal and uncontrolled proliferation of lung fibroblasts may contribute to pulmonary fibrosis. Lipopolysaccharide (LPS) can induce fibroblast proliferation and differentiation through activation of phosphoinositide3-Kinase (PI3-K) pathway. However, the detail mechanism by which LPS contributes to the development of lung fibrosis is not clearly understood. To investigate the role of phosphatase and tensin homolog (PTEN), a PI3-K pathway suppressor, on LPS-induced lung fibroblast proliferation, differentiation, collagen secretion and activation of PI3-K, we transfected PTEN overexpression lentivirus into cultured mouse lung fibroblasts with or without LPS treatment to evaluate proliferation by MTT and Flow cytometry assays. Expression of PTEN, alpha-smooth muscle actin (alpha-SMA), glycogen synthase kinase 3 beta (GSK3beta) and phosphorylation of Akt were determined by Western-blot or real-time RT-PCR assays. The PTEN phosphorylation activity was measured by a malachite green-based assay. The content of C-terminal propeptide of type I procollagen (PICP) in cell culture supernatants was examined by ELISA.

To evaluate the diagnostic performance of radiologists on breast cancer with or without artificial intelligence (AI) support.

Most patients with gastric cancer (GCa) are diagnosed at an advanced stage. We aimed to investigate novel fecal signatures for clinical application in early diagnosis of GCa.

Knowledge of the exact distribution of meiotic crossovers (COs) and gene conversions (GCs) is essential for understanding many aspects of population genetics and evolution, from haplotype structure and long-distance genetic linkage to the generation of new allelic variants of genes. To this end, we resequenced the four products of 13 meiotic tetrads along with 10 doubled haploids derived from Arabidopsis thaliana hybrids. GC detection through short reads has previously been confounded by genomic rearrangements. Rigid filtering for misaligned reads allowed GC identification at high accuracy and revealed an ∼80-kb transposition, which undergoes copy-number changes mediated by meiotic recombination. Non-crossover associated GCs were extremely rare most likely due to their short average length of ∼25-50 bp, which is significantly shorter than the length of CO-associated GCs. Overall, recombination preferentially targeted non-methylated nucleosome-free regions at gene promoters, which showed significant enrichment of two sequence motifs. DOI: http://dx.doi.org/10.7554/eLife.01426.001.

Can genome-wide haplotyping increase success following preimplantation genetic testing for a monogenic disorder (PGT-M) by including zygotes with absence of pronuclei (0PN) or the presence of only one pronucleus (1PN)?

Surfactin, a lipopeptide produced by Bacillus species, has been used for the oral delivery of insulin. In this study, another lipopeptide of iturin was tested for its ability to orally delivery insulin alone or plus surfactin. Iturin could form co-precipitate with insulin at acidic pH values. After treatment by ultrasonification, the structure of coprecipitate was destroyed that led to a significant decrease in hypoglycemic effect after oral administration. Iturin weakly binds to (Kd = 257 μM) and induce insulin structure more compact that is favorable for insulin uptake by the intestine. After being coated with Acryl-Eze by lyophilization, the coprecipitate formed the spherical enteric-coated insulin microparticles delivered by iturin with a relative oral bioavailability of 6.84% in diabetic mice. For further improving oral hypoglycemic effect, surfactin was added to form the spherical enteric-coated insulin microparticles in a formulation containing insulin, Acryl-Eze, iturin and surfactin at a ratio of 1:1:0.5: 0.5 (w/w), with an insulin encapsulation efficiency of 66.22%. The enteric-coated insulin microparticles delivered by iturin plus surfactin showed a classical profile for controlled release in the intestine with a relative bioavailability of 7.67% after oral administration, which could effectively control the postprandial blood glucose at a level about 50% of the initial one just like the subcutaneous injection. Collectively, iturin plus surfactin is more efficient for oral delivering insulin than the sole one, and the resultant enteric-coated insulin microparticles are potential for the development of oral insulin to control postprandial blood glucose in diabetic patients.

Directly harvesting solar energy for battery charging represents an ultimate solution toward low-cost, green, efficient and sustainable electrochemical energy storage. Here, we design a sunlight promotion strategy into rechargeable zinc-air battery with significantly reduced charging potential below the theoretical cell voltage of zinc-air batteries. The sunlight-promoted zinc-air battery using BiVO4 or α-Fe2O3 air photoelectrode achieves a record-low charge potential of ~1.20 and ~1.43 V, respectively, under illumination, which is lowered by ~0.5-0.8 V compared to the typical charge voltage of ~2 V in conventional zinc-air battery. The band structure and photoelectrochemical stability of photoelectrodes are found to be key factors determining the charging performance of sunlight-promoted zinc-air batteries. The introduction of photoelectrode as an air electrode opens a facile way for developing integrated single-unit zinc-air batteries that can efficiently use solar energy to overcome the high charging overpotential of conventional zinc-air batteries.

Multidrug resistance is one of the reasons for low survival of advanced hepatocellular carcinoma (HCC). Our previous studies indicate that the hedgehog signalling is involved in hepatic carcinogenesis, metastasis and chemo-resistance. The present study aims to uncover molecular mechanisms underlying hepatoma chemo-resistance. TAP1 and GLI1/2 gene expression was assessed in both poorly differentiated hepatoma cells and HCC specimens. Potential GLI-binding site in the TAP1 promoter sequence was validated by molecular assays. Approximately 75% HCC specimens exhibited an elevated expression of hedgehog GLI1 transcription factor compared with adjacent liver tissue. Both GLI1/2 and TAP1 protein levels were significantly elevated in poorly differentiated hepatoma cells. Both Huh-7-trans and Huh-7-DN displayed more karyotypic abnormalities and differential gene expression profiles than their native Huh-7 cells. Sensitivity to Sorafenib, doxorubicin and cisplatin was remarkably improved after either GLI1 or TAP1 gene was inhibited by an RNAi approach or by a specific GLI1/2 inhibitor, GANT61. Further experiments confirmed that hedgehog transcription factor GLI1/2 binds to the TAP1 promoter, indicating that TAP1 is one of GLI1/2 target genes. In conclusion, TAP1 is under direct transcriptional control of the hedgehog signalling. Targeting hedgehog signalling confers a novel insight into alleviating drug resistance in the treatment of refractory HCC.