On the Flow of a Cement Suspension: The Effects of Nano-Silica and Fly Ash Particles.

Additives such as nano-silica and fly ash are widely used in cement and concrete materials to improve the rheology of fresh cement and concrete and the performance of hardened materials and increase the sustainability of the cement and concrete industry by reducing the usage of Portland cement. Therefore, it is important to study the effect of these additives on the rheological behavior of fresh cement. In this paper, we study the pulsating Poiseuille flow of fresh cement in a horizontal pipe by considering two different additives and when they are combined (nano-silica, fly ash, combined nano-silica, and fly ash). To model the fresh cement suspension, we used a modified form of the power-law model to demonstrate the dependency of the cement viscosity on the shear rate and volume fraction of cement and the additive particles. The convection-diffusion equation was used to solve for the volume fraction. After solving the equations in the dimensionless forms, we conducted a parametric study to analyze the effects of nano-silica, fly ash, and combined nano-silica and fly ash additives on the velocity and volume fraction profiles of the cement suspension. According to the parametric study presented here, larger nano-silica content results in lower centerline velocity of the cement suspension and larger non-uniformity of the volume fraction. Compared to nano-silica, fly ash exhibits an opposite effect on the velocity. Larger fly ash content results in higher centerline velocity, while the effect of the fly ash on the volume fraction is not obvious. For cement suspension containing combined nano-silica and fly ash additives, nano-silica plays a dominant role in the flow behavior of the suspension. The findings of the study can help the design and operation of the pulsating flow of fresh cement mortars and concrete in the 3D printing industry.

Macrophage-membrane-coated hybrid nanoparticles with self-supplied hydrogen peroxide for enhanced chemodynamic tumor therapy.

Addressing the challenges of chemodynamic therapies (CDTs) relying on Fenton reactions in malignant tumors is an active research area. Here, we report a method to develop pH-responsive hybrid nanoparticles for enhanced chemodynamic tumor treatment. Reactive CaO2 nanoparticles (core) are isolated by biocompatible ZIF-8 doped with Fe2+ (shell), and then encapsulated by macrophage membranes (symbolized as CaO2@Fe-ZIF-8@macrophage membrane or CFZM), thus endowed with high stability under normal physiological conditions. Our design features active tumor-homing by the macrophage-membrane coating, tumor microenvironment (TME)-responsive cargo release, and self-supplied hydrogen peroxide for promotion of the Fenton reaction. We demonstrate the improved delivery/tumor cell uptake of CFZM, the efficient production of toxic ˙OH with self-supplied H2O2 in CFZM, and high-efficacy tumor ablation on BALB/c mice bearing CT26 tumor cells. This offers a translational strategy to develop active tumor-targeting and TME-responsive nanotherapeutics with enhanced CDT against malignant tumors.

Hypoxia-targeted and spatial-selective tumor suppression by near infrared nanoantenna sensitized engineered bacteria.

It is an active research area in the development of engineered bacteria to address the bottleneck issue of hypoxic tumors, which otherwisely possess resistance to chemotherapies, radiotherapies, and photodynamic therapies. Here we report a new method to ablate hypoxic tumors with NIR-nanoantenna sensitized engineered bacteria (NASEB) in a highly effective and dual selective manner. It features engineered E. coli MG1655 (EB) with coatings of lanthanide upconversion nanoparticles (UCNPs) as external antennas on bacterial surface (MG1655/HlyE-sfGFP@UCNP@PEG), enabling NIR laser-switchable generation/secretion of HlyE perforin to kill cancer cells. We have demonstrated that NASEB enrichment on hypoxic tumor sites via their innate chemotactic tendency, in assistance of localized NIR laser irradiation, can suppress tumors with improved efficacy and selectivity, thus minimizing potential side effects in cancer treatment. The NIR-responsive nanoantenna sensitized switching in engineering bacteria is distinct from the previous reports, promising conceptually new development of therapeutics against hypoxic tumors. STATEMENT OF SIGNIFICANCE: Tumor hypoxia exacerbates tumor progression, but also reduces the efficacy of conventional chemotherapies, radiotherapies, or photodynamic therapies. Here we develop near infrared Nano Antenna Sensitized Engineered Bacteria (NASEB) to treat hypoxic tumors. NASEB can accumulate and proliferate on hypoxic tumor sites via their innate chemotactic tendency. After receiving NIR laser signals, the upconversion nanoparticles on NASEB surface as antennas can transduce them to blue light for activation of HlyE perforin in the protein factory of EB. Our method features dual selectivity on the tumor sites, contributed by hypoxic tumor homing of anaerobic bacteria and spatial confinement through selective NIR laser irradiation. The concept of NASEB promises to address the challenges of tumor hypoxia for cancer therapies.

Efficient clearance of periodontitis pathogens by S. gordonii membrane-coated H2O2 self-supplied nanocomposites in a "Jenga" style.

As a key pathogen of periodontitis, P. gingivalis requires support of the initial colonizing bacterium (S. gordonii preferably) to form symbiotic biofilms on gingival tissues with enhanced antibiotic resistance. Here, we report a new strategy to treat periodontitis biofilms with S. gordonii membrane-coated H2O2 self-supplied nanocomposites (ZnO2/Fe3O4@MV NPs) in a "Jenga" style. Integration of our special MV coatings enables selectively enhanced internalization of the cargos in S. gordonii, thus inducing severe damage to the foundational bacterial layer and collapse/clearance of symbiotic biofilms consequently. This strategy allows us to clear the symbiotic biofilms of S. gordonii and P. gingivalis with active hydroxyl radicals (˙OH) derived from ZnO2-Fe3O4@MV NPs in a H2O2 self-supplied, nanocatalyst-assisted manner. This "Jenga-style" treatment provides a cutting-edge proof of concept for the removal of otherwise robust symbiotic biofilms of periodontitis where the critical pathogens are difficult to target and have antibiotic resistance.

Single-Cell Enzymatic Screening for Epithelial Mesenchymal Transition with an Ultrasensitive Superwetting Droplet-Array Microchip.

The phenotypic changes of circulating tumor cells (CTCs) during the epithelial-mesenchymal transition (EMT) have been a hot topic in tumor biology and cancer therapeutic development. Here, an integrated platform of single-cell fluorescent enzymatic assays with superwetting droplet-array microchips (SDAM) for ultrasensitive functional screening of epithelial-mesenchymal sub-phenotypes of CTCs is reported. The SDAM can generate high-density, volume well-defined droplet (0.66 nL per droplet) arrays isolating single tumor cells via a discontinuous dewetting effect. It enables sensitive detection of MMP9 enzyme activities secreted by single tumor cells, correlating to their epithelial-mesenchymal sub-phenotypes. In the pilot clinical double-blind tests, the authors have demonstrated that SDAM assays allow for rapid identification and functional screening of CTCs with different epithelial-mesenchymal properties. The consistency with the clinical outcomes validates the usefulness of single-cell secreted MMP9 as a biomarker for selective CTC screening and tumor metastasis monitoring. Convenient addressing and recovery of individual CTCs from SDAM have been demonstrated for gene mutation sequencing, immunostaining, and transcriptome analysis, revealing new understandings of the signaling pathways between MMP9 secretion and the EMT regulation of CTCs. The SDAM approach combined with sequencing technologies promises to explore the dynamic EMT plasticity of tumors at the single-cell level.

A super water-resistant MXene sponge flexible sensor for bifunctional sensing of physical and chemical stimuli.

Flexible wearable sensors with multifunctional features have attracted great interest in various applications such as disease diagnosis, environmental detection and healthcare monitoring. However, it is still a challenge to achieve a multifunctional sensor with super water resistance without compromising the overall performance of the sensing material. Here, we developed a 3D bifunctional flexible sensor based on an MXene melamine sponge (MS) through a simple and effective ultrasonic mixing process and a further vacuum annealing process. The sensor is able to show excellent response to different stimuli, including pressure and humidity. The thermal annealing treatment allows MXene to adhere more firmly to the internal skeleton of the sponge, which does not easily fall off and improves the water resistance, thus achieving wearability and high sensitivity over a wide area. The T-MXene@MS sensor has a sensitivity of 9.97 kPa-1 in the 5-15 kPa range, a fast response time (180 ms), and good stability at 4000 cycles, enabling accurate monitoring of human movement. The sensor has a rich porous structure while maintaining its inherent flexibility, which allows for long term testing of human respiration as well as the ability to respond quickly to dynamic changes in humidity, demonstrating excellent long-term stability for 40 days of humidity detection.

How Placenta Promotes the Successful Reproduction in High-Altitude Populations: A Transcriptome Comparison between Adaptation and Acclimatization.

As the best adapted high altitude population, Tibetans feature a relatively high offspring survival rate. Genome-wide studies have identified hundreds of candidate SNPs related to high altitude adaptation of Tibetans, although most of them have unknown functional relevance. To explore the mechanisms behind successful reproduction at high altitudes, we compared the placental transcriptomes of Tibetans, sea level Hans (SLHan), and Han immigrants (ImHan). Among the three populations, placentas from ImHan showed a hyperactive gene expression pattern. Their increased activation demonstrates a hypoxic stress response similar to sea level individuals experiencing hypoxic conditions. Unlike ImHan, Tibetan placentas were characterized by the significant up-regulation of placenta-specific genes, and the activation of autophagy and the tricarboxylic acid (TCA) cycle. Certain conserved hypoxia response functions, including the antioxidant system and angiogenesis, were activated in both ImHan and Tibetans, but mediated by different genes. The coherence of specific transcriptome features linked to possible genetic contribution was observed in Tibetans. Furthermore, we identified a novel Tibetan-specific EPAS1 isoform with a partial deletion at exon six, which may be involved in the adaption to hypoxia through the EPAS1-centred gene network in the placenta. Overall, our results show that the placenta grants successful pregnancies in Tibetans by strengthening the natural functions of the placenta itself. On the other hand, the placenta of ImHan was in an inhabiting time-dependent acclimatization process representing a common hypoxic stress response pattern.

The Antitumor Effects of Icaritin Against Breast Cancer is Related to Estrogen Receptors.

OBJECTIVE: We aim to investigate the anticancer effects and mechanisms of icaritin against breast cancer. MATERIALS AND METHODS: Both estrogen receptor (ER) positive breast cancer cells MCF- 7 and ER-negative MDA-MB-231 cells were employed. We examined the effects of icaritin on the proliferation and migration by wound healing assay and transwell assay. Cell apoptosis and cell cycle of MCF-7 and MDA-MB-231 cells were analyzed using Flow cytometry. Cell autophagy of MCF-7 and MDA-MB-231 cells was assessed by western blotting, acridine orange staining and confocal microscopy. We also detected the expression of apoptosis-related genes by western blotting. In addition, an autophagy inhibitor was used to investigate whether cytoprotective autophagy was induced. Meanwhile, an ER inhibitor was utilized to explore whether ER was involved in autophagy. RESULTS: Icaritin inhibited the proliferation and migration, and induced cell cycle arrest of both MDA-MB-231 and MCF-7 cells. Icaritin significantly induced apoptosis of MDA-MB- 231 cells by activating caspase-3. And icaritin stimulated autophagy in MCF-7 cells, as evidenced by increased LC3II/LC3I, enhanced p62 degradation, the accumulation of endogenous LC3 puncta formation, and the increased autophagy flux. Icaritin induced autophagy through upregulating the phosphorylation of AMPK and ULK1. Chloroquine, an autophagy inhibitor, increased icaritin-induced apoptosis and proliferation inhibition of MCF-7 cells. Meanwhile, tamoxifen, an ER inhibitor, reversed icaritin-induced autophagy and proliferation inhibition of MCF-7 cells. CONCLUSION: Our study demonstrated that the antitumor effects of icaritin against breast cancer are related to ER, which suggested that the status of ER should be considered in the clinical application of icaritin.

Cloning short DNA into plasmids by one-step PCR.

BACKGROUND: Plasmid construction of small fragments of interest (such as insertion of small fragment marker genes, expression of shRNA, siRNA, etc) is the basis of many biomolecular experiments. Here, we describe a method to clone short DNA into vectors by polymerase chain reaction (PCR), named one-step PCR cloning. Our method uses PCR to amplify the entire circular plasmid. The PCR was performed by the primers containing the gene of short DNA with overlapping sequences between 10-15 bp. The PCR products were then transformed into E. coli and cyclized by homologous recombination in vivo. METHODS: The pEGFP-N1-HA plasmid was constructed by one-step PCR and transformation. Cells were transfected with pEGFP-N1-HA and pEGFP-N1 plasmid using TurboFect transfection reagent. Protein expression was detected by western blotting and the HA-GFP fusion protein was detected by confocal microscopy. RESULTS: The pEGFP-N1-HA plasmid was successfully constructed and HA expression in cells. CONCLUSIONS: Free from the limitations of restriction enzyme sites and omitting the ligation process, our method offers a flexible and economical option of plasmid construction. KEY POINTS: Significant findings of the study A method to clone short DNA into plasmids was found. What this study adds Our study provides a flexible and economical option to clone short DNA into plasmids.

Hypomethylation in HBV integration regions aids non-invasive surveillance to hepatocellular carcinoma by low-pass genome-wide bisulfite sequencing.

BACKGROUND: Circulating cell-free DNA (cfDNA) methylation has been demonstrated to be a promising approach for non-invasive cancer diagnosis. However, the high cost of whole genome bisulfite sequencing (WGBS) hinders the clinical implementation of a methylation-based cfDNA early detection biomarker. We proposed a novel strategy in low-pass WGBS (~ 5 million reads) to detect methylation changes in circulating cell-free DNA (cfDNA) from patients with liver diseases and hepatocellular carcinoma (HCC). METHODS: The effective small sequencing depth were determined by 5 pilot cfDNA samples with relative high-depth WGBS. CfDNA of 51 patients with hepatitis, cirrhosis, and HCC were conducted using low-pass WGBS. The strategy was validated in an independent WGBS cohort of 32 healthy individuals and 26 early-stage HCC patients. Fifteen paired tumor tissue and buffy coat samples were used to characterize the methylation of hepatitis B virus (HBV) integration regions and genome distribution of cfDNA. RESULTS: A significant enrichment of cfDNA in intergenic and repeat regions, especially in previously reported HBV integration sites were observed, as a feature of cfDNA and the bias of cfDNA release. Methylation profiles nearby HBV integration sites were a better indicator for hypomethylation of tumor genome comparing to Alu and LINE (long interspersed nuclear element) repeats, and were able to facilitate the cfDNA-based HCC prediction. Hypomethylation nearby HBV integration sites (5 kb flanking) was detected in HCC patients, but not in patients with hepatitis and cirrhosis (MethylHBV5k, median:0.61 vs 0.72, P = 0.0003). Methylation levels of integration sites certain candidate regions exhibited an area under the receiver operation curve (AUC) value > 0.85 to discriminate HCC from non-HCC samples. The validation cohort achieved the prediction performance with an AUC of 0.954. CONCLUSIONS: Hypomethylation around viral integration sites aids low-pass cfDNA WGBS to serve as a non-invasive approach for early HCC detection, and inspire future efforts on tumor surveillance for oncovirus with integration activity.

Metagenomic Profiling of Ocular Surface Microbiome Changes in Meibomian Gland Dysfunction.

Purpose: Ocular surface microbiome changes can affect meibomian gland dysfunction (MGD) development. This study aimed to delineate differences among the microbiome of eyelid skin, conjunctiva, and meibum in healthy controls (HCs) and patients afflicted with MGD. Methods: Shotgun metagenomic analysis was used to determine if there are differences between the microbial communities in ocular sites surrounding the meibomian gland in healthy individuals and patients afflicted with MGD. Results: The meibum bacterial content of these microbiomes was dissimilar in these two different types of individuals. Almost all of the most significant taxonomic changes in the meibum microbiome of individuals with MGD were also present in their eyelid skin, but not in the conjunctiva. Such site-specific microbe pattern changes accompany increases in the gene expression levels controlling carbohydrate and lipid metabolism. Most of the microbiomes in patients with MGD possess a microbe population capable of metabolizing benzoate. Pathogens known to underlie ocular infection were evident in these individuals. MGD meibum contained an abundance of Campylobacter coli, Campylobacter jejuni, and Enterococcus faecium pathogens, which were almost absent from HCs. Functional annotation indicated that in the microbiomes of MGD meibum their capability to undergo chemotaxis, display immune evasive virulence, and mediate type IV secretion was different than that in the microbiomes of meibum isolated from HCs. Conclusions: MGD meibum contains distinct microbiota whose immune evasive virulence is much stronger than that in the HCs. Profiling differences between the meibum microbiome makeup in HCs and patients with MGD characterizes changes of microbial communities associated with the disease status.

Genome-wide DNA methylation profiles of low- and high-grade adenoma reveals potential biomarkers for early detection of colorectal carcinoma.

BACKGROUND: Abnormal DNA methylation is a hallmark of human cancers and may be a promising biomarker for early diagnosis of human cancers. However, the majority of DNA methylation biomarkers that have been identified are based on the hypothesis that early differential methylation regions (DMRs) are maintained throughout carcinogenesis and could be detected at all stages of cancer. METHODS: In this study, we identified potential early biomarkers of colorectal cancer (CRC) development by genome-wide DNA methylation assay (Illumina infinium450, 450 K) of normal (N = 20) and pre-colorectal cancer samples including 18 low-grade adenoma (LGA) and 22 high-grade adenoma (HGA), integrated with GEO and ArrayExpress datasets (N = 833). RESULTS: We identified 209 and 8692 CpG sites that were significantly hyper-methylated in LGA and HGA, respectively. Pathway analysis identified nervous system-related methylation changes that are significantly associated with early adenoma development. Integration analysis revealed that DNA methylation in the promoter region of ADHFE1 has the most potential for being an early diagnostic biomarker for colorectal adenoma and cancer (sensitivity = 0.96, specificity = 0.95, area under the curve = 0.97). CONCLUSIONS: Overall, we demonstrated that DNA methylation have been shown significant changes in the stage of LGA and HGA in the development of colon cancer. Genome-wide DNA methylation to LGA and HGA provided an important proxy to identify promising early diagnosis biomarkers for colorectal cancer.

Noninvasive chimeric DNA profiling identifies tumor-originated HBV integrants contributing to viral antigen expression in liver cancer.

BACKGROUND: Host genome integration of HBV sequence is considered to be significant in HBV antigen expression and the development of hepatocellular carcinoma (HCC). METHOD: We developed a probe-based capture strategy to enrich integrated HBV DNA for deep-sequencing analysis of integration sites in paired patient samples derived from tumor, liver tissue adjacent to tumor, saliva and plasma, as a platform for exploring the correlation, significance and utility of detecting integrations in these sample types. RESULTS: Most significantly, alpha fetoprotein levels significantly correlated to the amounts of integrations detected in tumor. Viral-host chimeric DNA fragments were successfully detected at high sequencing coverage in plasma rather than saliva samples from HCC patients, and each fragment of this type was only seen once in plasma from chronic hepatitis B patients. Almost all plasma chimeric fragments were derived from integrations in tumor rather than in adjacent liver tissues. Over 50% of them may produce viral-host chimeric transcripts according to deep RNA sequencing in paired tissue samples. Particularly, in patients with low HBV DNA level (< 250 UI/ml), the seemingly normal HBsAg titers may be explained by larger amounts of integrations detected. Meanwhile, we developed a strategy to predict integrants by pairing breakpoints for each integration event. Among four resolved viral patterns, the majority of Pattern I events (81.2%) retained the complete opening reading frame for HBV surface proteins. CONCLUSION: We achieve the efficient enrichment of plasma cell-free chimeric DNA from integration site, and demonstrate that chimeric DNA profiling in plasma is a promising noninvasive approach to monitor HBV integration in liver cancer development and to determine the ability of integrated sequences to express viral proteins that can be targeted, e.g. by immunotherapies.

Comparative Proteomic Analysis of Molecular Differences between Leaves of Wild-Type Upland Cotton and Its Fuzzless-Lintless Mutant.

Fuzzless-lintless mutant (fl) ovules of upland cotton have been used to investigate cotton fiber development for decades. However, the molecular differences of green tissues between fl and wild-type (WT) cotton were barely reported. Here, we found that gossypol content, the most important secondary metabolite of cotton leaves, was higher in Gossypium hirsutum L. cv Xuzhou-142 (Xu142) WT than in fl. Then, we performed comparative proteomic analysis of the leaves from Xu142 WT and its fl. A total of 4506 proteins were identified, of which 103 and 164 appeared to be WT- and fl-specific, respectively. In the 4239 common-expressed proteins, 80 and 74 were preferentially accumulated in WT and fl, respectively. Pathway enrichment analysis and protein-protein interaction network analysis of both variety-specific and differential abundant proteins showed that secondary metabolism and chloroplast-related pathways were significantly enriched. Quantitative real-time PCR confirmed that the expression levels of 12 out of 16 selected genes from representative pathways were consistent with their protein accumulation patterns. Further analyses showed that the content of chlorophyll a in WT, but not chlorophyll b, was significantly increased compared to fl. This work provides the leaf proteome profiles of Xu142 and its fl mutant, indicating the necessity of further investigation of molecular differences between WT and fl leaves.

An improved protein extraction method applied to cotton leaves is compatible with 2-DE and LC-MS.

BACKGROUND: Two-dimensional electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are widely used in plant proteomics research. However, these two techniques cannot be simultaneously satisfied by traditional protein extraction methods when investigate cotton leaf proteome. RESULTS: Here, we evaluated the efficiency of three different protein extraction methods for 2-DE and LC-MS/MS analyses of total proteins obtained from cotton leaves. The protein yield of the borax/PVPP/phenol (BPP) method (0.14%) was significantly lower than the yields of the trichloroacetic acid/acetone (TCA) precipitation method (1.42%) and optimized TCA combined with BPP (TCA-B) method (0.47%). The BPP method was failed to get a clear 2-DE electrophoretogram. Fifty pairs of protein spots were randomly selected from the 2-DE gels of TCA- and TCA-B-extracted proteins for identification by MALDI TOF/TOF, and the results of 42 pairs were consistent. High-throughput proteomic analysis showed that 6339, 9282 and 9697 unique proteins were identified from the total cotton leaf proteins extracted by the TCA, BPP and TCA-B methods, respectively. Gene Ontology (GO) analysis revealed that the proteins specifically identified by TCA method were primarily distributed in the plasma membrane, while BPP and TCA-B methods specific proteins distributed in the cytosol, indicating the sub-cellular preference of different protein extraction methods. Further, ATP-dependent zinc metalloprotease FTSH 8 could be observed in the 2-DE gels of TCA and TCA-B methods, and could only be detected in the LC-MS/MS results of the BPP and TCA-B methods, showing that TCA-B method might be the optimized choice for both 2-DE and LC-MS/MS. CONCLUSION: Our data provided an improved TCA-B method for protein extraction that is compatible with 2-DE and LC-MS/MS for cotton leaves and similar plant tissues which is rich in polysaccharides and polyphenols.

Increased expression diversity buffers the loss of adaptive potential caused by reduction of genetic diversity in new unfavourable environments.

Mechanisms underlying adaptation to rapid environmental change are issues in evolutionary biology. It is widely accepted that reduction in genetic diversity when suddenly exposed to an unfavourable environment limits the adaptive potential of populations. With growing empirical evidence that expression diversity is likely to increase in the new environment, the role that expression diversity plays in adaptation needs to be theorized. Here, we first established a negative exponential relationship between expression diversity and genetic diversity using a phenomenological differential equation. We then derived a complex trade-off relationship between the changes of expression and genetic diversity, which followed a combination of exponential functions. Furthermore, we found the increase in expression diversity could buffer the loss of adaptive potential as genetic diversity decreased to a certain extent. These theoretical deductions were validated by transcriptomic data of Miscanthus lutarioriparius grown in two experimental fields and supported by good fit and random simulation. These results suggest that increased expression diversity may compensate the loss of genetic diversity and allow the populations to maintain a certain level of phenotypic variation to cope with sudden environmental change. This may buffer the quick diminishing of adaptive potential and consequently increases the change of adaptation to the new environment.

Coexpression network revealing the plasticity and robustness of population transcriptome during the initial stage of domesticating energy crop Miscanthus lutarioriparius.

KEY MESSAGE: Coexpression network revealing genes with Co-variation Expression pattern (CE) and those with Top rank of Expression fold change (TE) played different roles in responding to new environment of Miscanthus lutarioriparius. Variation in gene expression level, the product of genetic and/or environmental perturbation, determines the robustness-to-plasticity spectrum of a phenotype in plants. Understanding how expression variation of plant population response to a new field is crucial to domesticate energy crops. Weighted Gene Coexpression Network Analysis (WGCNA) was used to explore the patterns of expression variation based on 72 Miscanthus lutarioriparius transcriptomes from two contrasting environments, one near the native habitat and the other in one harsh domesticating region. The 932 genes with Co-variation Expression pattern (CE) and other 932 genes with Top rank of Expression fold change (TE) were identified and the former were strongly associated with the water use efficiency (r ≥ 0.55, P ≤ 10-7). Functional enrichment of CE genes were related to three organelles, which well matched the annotation of twelve motifs identified from their conserved noncoding sequence; while TE genes were mostly related to biotic and/or abiotic stress. The expression robustness of CE genes with high genetic diversity kept relatively stable between environments while the harsh environment reduced the expression robustness of TE genes with low genetic diversity. The expression plasticity of CE genes was increased less than that of TE genes. These results suggested that expression variation of CE genes and TE genes could account for the robustness and plasticity of acclimation ability of Miscanthus, respectively. The patterns of expression variation revealed by transcriptomic network would shed new light on breeding and domestication of energy crops.

Genome-wide investigation and expression profiling of APX gene family in Gossypium hirsutum provide new insights in redox homeostasis maintenance during different fiber development stages.

Ascorbate peroxidase (APX) is a member of heme-containing peroxidases which catalyze the H2O2-dependent oxidation of a wide range of substrates in plants and animals. As is known, H2O2 acts as a signaling molecule in the regulation of fiber development. Our previous work reported that ascorbate peroxidase 1 (GhAPX1) was important for cotton fiber elongation. However, knowledge about APX gene family members and their evolutionary and functional characteristics in cotton is limited. Here, we report 26 GhAPX genes by genome-wide investigation of tetraploid cotton Gossypium hirsutum. Phylogenetic and gene structure analyses classified these APX members into five clades and syntenic analysis suggested two duplication events. Expression profiling of the 26 APXs revealed that ten members are expressed in cotton fibers. Notably, GhAPX10A, GhAPX10D, GhAPX12A, and GhAPX12D showed high expression levels in 30-day fiber, while GhAPX1A/D, GhAPX3A/D, and GhAPX6A/D showed very low expression levels. The enzyme activity and H2O2 content assays revealed that cotton fiber kept high enzyme activity and the lowest H2O2 level in 30-day fibers, indicating that other than GhAPX1, the newly reported APX members are responsible for the reactive oxygen species homeostasis in the cotton fiber maturation stages. Expression profiling of ten fiber-expressed APXs after phytohormone treatments revealed their regulation patterns by different stimuli, suggesting that GhAPX1, GhAPX12A, and GhAPX12D are responsible to most phytohormone treatments. Our data provided evolutionary and functional information of GhAPX gene family members and revealed that different members are responsible to redox homeostasis during different cotton fiber development stages.

Expression Profiling of Mitogen-Activated Protein Kinase Genes Reveals Their Evolutionary and Functional Diversity in Different Rubber Tree (Hevea brasiliensis) Cultivars.

Rubber tree (Heveabrasiliensis) is the only commercially cultivated plant for producing natural rubber, one of the most essential industrial raw materials. Knowledge of the evolutionary and functional characteristics of kinases in H. brasiliensis is limited because of the long growth period and lack of well annotated genome information. Here, we reported mitogen-activated protein kinases in H.brasiliensis (HbMPKs) by manually checking and correcting the rubber tree genome. Of the 20 identified HbMPKs, four members were validated by proteomic data. Protein motif and phylogenetic analyses classified these members into four known groups comprising Thr-Glu-Tyr (TEY) and Thr-Asp-Tyr (TDY) domains, respectively. Evolutionary and syntenic analyses suggested four duplication events: HbMPK3/HbMPK6, HbMPK8/HbMPK9/HbMPK15, HbMPK10/HbMPK12 and HbMPK11/HbMPK16/HbMPK19. Expression profiling of the identified HbMPKs in roots, stems, leaves and latex obtained from three cultivars with different latex yield ability revealed tissue- and variety-expression specificity of HbMPK paralogues. Gene expression patterns under osmotic, oxidative, salt and cold stresses, combined with cis-element distribution analyses, indicated different regulation patterns of HbMPK paralogues. Further, Ka/Ks and Tajima analyses suggested an accelerated evolutionary rate in paralogues HbMPK10/12. These results revealed HbMPKs have diverse functions in natural rubber biosynthesis, and highlighted the potential possibility of using MPKs to improve stress tolerance in future rubber tree breeding.

iTRAQ-Based Quantitative Proteomic Analysis Reveals Cold Responsive Proteins Involved in Leaf Senescence in Upland Cotton (Gossypium hirsutum L.).

Premature leaf senescence occurs in the ultimate phase of the plant, and it occurs through a complex series of actions regulated by stress, hormones and genes. In this study, a proteomic analysis was performed to analyze the factors that could induce premature leaf senescence in two cotton cultivars. We successfully identified 443 differential abundant proteins (DAPs) from 7388 high-confidence proteins at four stages between non-premature senescence (NS) and premature senescence (PS), among which 158 proteins were over-accumulated, 238 proteins were down-accumulated at four stages, and 47 proteins displayed overlapped accumulation. All the DAPs were mapped onto 21 different categories on the basis of a Clusters of Orthologous Groups (COG) analysis, and 9 clusters were based on accumulation. Gene Ontology (GO) enrichment results show that processes related to stress responses, including responses to cold temperatures and responses to hormones, are significantly differentially accumulated. More importantly, the enriched proteins were mapped in The Arabidopsis Information Resource (TAIR), showing that 58 proteins play an active role in abiotic stress, hormone signaling and leaf senescence. Among these proteins, 26 cold-responsive proteins (CRPs) are significantly differentially accumulated. The meteorological data showed that the median temperatures declined at approximately 15 days before the onset of aging, suggesting that a decrease in temperature is tightly linked to an onset of cotton leaf senescence. Because accumulations of H2O2 and increased jasmonic acid (JA) were detected during PS, we speculate that two pathways associated with JA and H2O2 are closely related to premature leaf senescence in cotton.