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Biochar amendment of soil is well known to improve soil fertility and microbial function. However, little is known about the effect of biochar addition to reclaimed soil in coal mining subsidence area on microbial community. A plant soil cultivation experiment was conducted with wheat grown and four treatments were included: P and K fertilizer (CK); NPK inorganic fertilizer (NPK); NPK inorganic fertilizer and straw (NPKS); and NPK inorganic fertilizer and biochar (NPKB). The results indicated that biochar amendment significantly increased the concentrations of NH4+-N, total N, and available P and K compared with the NPK. Biochar addition also significantly increased the grain yield and total biomass of wheat. Furthermore, biochar amendment treatment increased the absolute abundance and altered the community structure of soil bacteria and fungi in the reclaimed soil. Illumina MiSeq sequencing showed that the addition of biochar increased α-diversity of bacteria and relative abundances of Proteobacteria, Actinobacteria, and Bacteroidetes, whereas the relative abundance of Firmicutes were decreased by 61%. However, biochar addition did not change the relative abundance of dominant fungal phyla. Redundancy analysis (RDA) suggested that total N, available P, and K contents were the key factors correlated with changes in microbial community structure. Overall, our results suggest that biochar amendment in reclaimed soil in coal mine subsidence area could increase wheat yield and abundance and alter microbial community compositions.

Biochar as a soil amendment has been regarded as a promising way to improve soil fertility. However, the response of microbial community after biochar and biochar compound fertilizer (BCF) application has not been thoroughly elucidated. This study evaluated the changes in abundance and composition of bacterial and fungal communities using quantitative real-time PCR (qPCR) and Illumina MiSeq amplicon sequencing. The field experiment ran for 3 years and comprised five treatments: chemical fertilizer as control (CK), straw-returning combined with chemical fertilizer (CS), low biochar application combined with chemical fertilizer (LB), high biochar application combined with chemical fertilizer (HB) and BCF. The results showed that biochar amendment results no changes in the abundance and diversity of bacteria in the bulk and rhizosphere soils. However, the abundance of soil fungi was significantly increased by biochar amendment (LB and HB). LB treatment significantly increased the fungal alpha diversity, while there was no significant change under HB. Furthermore, the dominant bacterial phyla found in the samples were Proteobacteria, Actinobacteria, and Acidobacteria. Biochar addition increased the relative abundance of Actinobacteria in both bulk and rhizosphere soils. The dominant fungal phyla were Ascomycota, Mortierellomycota, and Basidiomycota. The relative abundance of Ascomycota significantly decreased, but Mortierellomycota significantly increased in LB and HB. In addition, redundancy analysis indicated that the changes in bacterial and fungal communities are associated with soil properties such as SOC and TN, which are crucial contributors in regulating the community composition. This study is expected to provide significant theoretical and practical knowledge for the application of biochar in agricultural ecosystem.

The circadian clock is an endogenous timekeeper system that controls and optimizes biological processes, which are consistent with a master circadian clock and peripheral clocks and are controlled by various genes. Notably, the disruption of circadian clock genes has been identified to affect a wide range of ailments, including cancers. The cancer-immunity cycle is composed of seven major steps, namely cancer cell antigen release and presentation, priming and activation of effector immunity cells, trafficking, and infiltration of immunity to tumors, and elimination of cancer cells. Existing evidence indicates that the circadian clock functions as a gate that govern many aspects of the cancer-immunity cycle. In this review, we highlight the importance of the circadian clock during tumorigenesis, and discuss the potential role of the circadian clock in the cancer-immunity cycle. A comprehensive understanding of the regulatory function of the circadian clock in the cancer-immunity cycle holds promise in developing new strategies for the treatment of cancer. Video Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12964-021-00721-2.

Breast cancer is the most common malignant tumor in women and one of the three most common cancers worldwide. It is a life-threatening disease among women and the leading cause of death among women. New drugs or new drug translations and laboratory clinical studies are ongoing. A new antitumor protein (PNAP) purified from edible fungus Pholiota nameko has potential for treating breast cancer. We have previously found that PNAP exhibits anti-proliferative and apoptosis-inducing activities in a human breast cancer cell line (MCF-7). In this study, we constructed a BALB/c mouse model of MCF-7 tumor xenografts. In vivo experiments show that PNAP can effectively inhibit the malignant proliferation of MCF-7 solid tumors. This is because PNAP can successfully activate the death receptor pathway and mitochondrial apoptosis pathway of MCF-7 tumor cells in vivo, and induce tumor cells to wither. It is estimated that PNAP may also have an immunoregulatory ability to indirectly inhibit malignant proliferation of tumors. We also found that PNAP may also have the immunomodulatory ability to indirectly inhibit the malignant proliferation of tumors, which can shift the balance of Th1/Th2 to Th1 and eventually inhibit the growth of tumors. The study reveals a new therapeutic approach for breast cancer patient.

Aim and objective: To study the effect of Gupi Xiaoji Prescription (GXP) on hepatitis B virus(HBV)-related liver cancer through network pharmacology coupled with in vitro experiments and explore their related mechanisms. Materials and methods: Gupi Xiaoji Prescription’s chemical constituents and the action targets of its six medicinal components were identified using several databases. These included the Traditional Chinese Medicine Systems Pharmacology Database （TCMSP), the Bioinformatics Analysis Tool for Molecular mechANism of TCM (BATMAN-TCM), and the Traditional Chinese Medicine Integrated Database (TCMID), while GeneCards and OMIM were used to compile relevant liver cancer disease targets. Pathway enrichment of gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), analysis of potential targets, and analysis of the enriched pathways in literature were executed in R. The Hepatocellular carcinoma (HCC)-derived HepG2.2.15 cell line stably expresses and replicates HBV. In vitro experiments with HepG2.2.15 were used to verify GXP’s effects on HBV-related liver cancer, while the human liver cancer cell line HepG2 was used as the control. Results: 171 active ingredients and 259 potential drug targets were screened from GXP, involving 181 pathways in vitro. These assays identified Polyphyllin I as an effective GXP component. Notably, GXP inhibited cell proliferation and metastasis in a concentration-dependent manner (P

This study is performed to examine the impacts of microRNA-218 (miR-218) on cardiac microvascular endothelial cells (CMECs) injury induced by coronary artery disease (CAD). Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) was applied for detecting miR-218 expression in serum of patients with CAD and healthy controls, and the correlation between miR-218 expression and the clinical indexes such as creatine kinase, creatine kinase-myocardial band, cardiac troponin I, and coronary Gensini score was analyzed. CMECs were coincubated with homocysteine for 24 hr for CMECs injury, and the cells were transfected with miR-218 mimics or miR-218 inhibitors. Besides, we used oxidized low density lipoprotein as an inducer to incubate with CMECs for 24 hr, and the model of CMECs injury was established to be transfected with miR-218 mimics. RT-qPCR and western blot analysis were used to detect miR-218 and HMGB1 expression in CMECs. A series of experiments were used to determine cell proliferation, apoptosis, migration, and angiogenesis ability of CMECs. Vascular endothelial growth factor expression and inflammatory factor contents were measured. The obtained results suggested that miR-218 expression in peripheral blood of patients with CAD descended substantially versus that of healthy controls. Low miR-218 expression was found in CAD-induced CMECs injury. Overexpressed miR-218 promoted the proliferation, migration, angiogenesis ability, induced apoptosis, and alleviated the inflammatory injury of CAD-induced CMECs. miR-218 may negatively regulate the expression of HMGB1 in CAD. This study demonstrates that upregulation of miR-218 reduces CMECs injury induced by CAD through the inhibition of HMGB1.

PGbEXPA2 (Promoter of GbEXPA2 ) was preferentially and strongly expressed during cotton fiber development, and the 461-bp PGbEXPA2 fragment was essential for responding to exogenous GA and ABA in Arabidopsis. Cotton fibers are highly elongated single-cell, unbranched and non-glandular seed trichomes. Previous studies have reported that the transcript level of GbEXPA2 is significantly up-regulated during fiber cell elongation, suggesting that GbEXPA2 has an important function in fiber development. In this study, the promoter of GbEXPA2 (839 bp) from the DT sub-genome was isolated from Gossypium barbadense 3–79. Consistent with the expression pattern of GbEXPA2, the promoter PGbEXPA2 was able to express GUS to high levels in elongating fibers, but not in the root, stem, or leaf. In Arabidopsis, GUS activity was only found in the rosette leaf trichomes and rosette leaf vascular tissue, indicating that the transcription factors which bind to PGbEXPA2 in the leaf trichomes of transgenic Arabidopsis were similar to those found in cotton fiber. A deletion analysis of PGbEXPA2 revealed that a 461-bp fragment was sufficient to drive GUS expression in cotton fibers and Arabidopsis rosette leaf trichomes. Exogenous phytohormonal treatments on transgenic Arabidopsis with different promoter lengths (P-839, P-705, P-588 and P-461) showed that GUS activity in Arabidopsis trichomes could be strongly up-regulated by GA and, in contrast, down-regulated by ABA.

Long noncoding RNAs (lncRNAs) are transcripts of at least 200 bp in length, possess no apparent coding capacity and are involved in various biological regulatory processes. Until now, no systematic identification of lncRNAs has been reported in cotton (Gossypium spp.). Here, we describe the identification of 30 550 long intergenic noncoding RNA (lincRNA) loci (50 566 transcripts) and 4718 long noncoding natural antisense transcript (lncNAT) loci (5826 transcripts). LncRNAs are rich in repetitive sequences and preferentially expressed in a tissue-specific manner. The detection of abundant genome-specific and/or lineage-specific lncRNAs indicated their weak evolutionary conservation. Approximately 76% of homoeologous lncRNAs exhibit biased expression patterns towards the At or Dt subgenomes. Compared with protein-coding genes, lncRNAs showed overall higher methylation levels and their expression was less affected by gene body methylation. Expression validation in different cotton accessions and coexpression network construction helped to identify several functional lncRNA candidates involved in cotton fibre initiation and elongation. Analysis of integrated expression from the subgenomes of lncRNAs generating miR397 and its targets as a result of genome polyploidization indicated their pivotal functions in regulating lignin metabolism in domesticated tetraploid cotton fibres. This study provides the first comprehensive identification of lncRNAs in Gossypium.

EST–SSRs of Gossypium barbadense are mainly developed using traditional Sanger sequencing. However, due to the high cost and low throughput of Sanger sequencing, it is necessary to use high throughput sequencing technology for the development of more ESTs to more effectively analyze the structure and function of this species. In this study, a G. barbadense acc. 3–79 unnormalized fiber cDNA library (219.63 Mb) and a G. barbadense cv. Hai7124 normalized root cDNA library (204.61 Mb) were obtained by 454 sequencing. EST–SSRs were identified from the two libraries, and only 7,255 SSRs were obtained from the unnormalized library, with an average frequency of 1/31.00 kb. In contrast, 16,087 SSRs were obtained from the normalized library, with an average frequency of 1/13.02 kb. The frequencies of dinucleotides and tetranucleotides in the two libraries were very different. Comparing the two libraries, we found that a normalized cDNA library is more efficient for mining SSRs. Integrating the two libraries allowed the development of 1,129 EST–SSR markers, and 311 polymorphic loci were integrated into our interspecific BC1 genetic linkage map. The mapping results showed that the distribution of EST–SSRs on sub-genomes and chromosomes was uneven; however, the distribution of the mapped G. barbadense EST–SSRs on homologous chromosomes was similar, with the exception of Chr05 versus Chr19 and Chr12 versus Chr26. This study provided new EST–SSR markers that will facilitate studies on cotton genetics and breeding.

Verticillium wilt causes massive annual losses of cotton yield, but the mechanism of cotton resistance to Verticillium dahliae is complex and poorly understood. In this study, a comparative proteomic analysis was performed in resistant cotton (Gossypium barbadense cv7124) on infection with V. dahliae. A total of 188 differentially expressed proteins were identified by mass spectrometry (MALDI-TOF/TOF) analysis and could be classified into 17 biological processes based on Gene Ontology annotation. Most of these proteins were implicated in stimulus response, cellular processes and metabolic processes. Based on the proteomic analysis, several genes involved in secondary metabolism, reactive oxygen burst and phytohormone signaling pathways were identified for further physiological and molecular analysis. The roles of the corresponding genes were further characterized by employing virus-induced gene silencing (VIGS). Based on the results, we suggest that the production of gossypol is sufficient to affect the cotton resistance to V. dahliae. Silencing of GbCAD1, a key enzyme involving in gossypol biosynthesis, compromised cotton resistance to V. dahliae. Reactive oxygen species and salicylic acid signaling may be also implicated as regulators in cotton responsive to V. dahliae according to the analysis of GbSSI2, an important regulator in the crosstalk between salicylic acid and jasmonic acid signal pathways. Moreover, brassinosteroids and jasmonic acid signaling may play essential roles in the cotton disease resistance to V. dahliae. The brassinosteroids signaling was activated in cotton on inoculation with V. dahliae and the disease resistance of cotton was enhanced after exogenous application of brassinolide. Meanwhile, jasmonic acid signaling was also activated in cotton after inoculation with V. dahliae and brassinolide application. These data provide highlights in the molecular basis of cotton resistance to V. dahliae.

Transforming growth factor beta1- (TGF-β1-) induced p21-dependent mesangial cell (MC) hypertrophy plays a key role in the pathogenesis of chronic renal diseases including diabetic nephropathy (DN). Increasing evidence demonstrated the role of posttranscriptional modifications (PTMs) in the event; however, the precise regulatory mechanism of histone lysine methylation remains largely unknown. Here, we examined the roles of both histone H3 lysine 4 and lysine 9 methylations (H3K4me/H3K9me) in TGF-β1 induced p21 gene expression in rat mesangial cells (RMCs). Our results indicated that TGF-β1 upregulated the expression of p21 gene in RMCs, which was positively correlated with the increased chromatin marks associated with active genes (H3K4me1/H3K4me2/H3K4me3) and negatively correlated with the decreased levels of repressive marks (H3K9me2/H3K9me3) at p21 gene promoter. TGF-β1 also elevated the recruitment of the H3K4 methyltransferase (HMT) SET7/9 to the p21 gene promoter. SET7/9 gene silencing with small interfering RNAs (siRNAs) significantly abolished the TGF-β1 induced p21 gene expression. Taken together, these results reveal the key role of histone H3Kme in TGF-β1 mediated p21 gene expression in RMC, partly through HMT SET7/9 occupancy, suggesting H3Kme and SET7/9 may be potential renoprotective agents in managing chronic renal diseases.

Gossypium hirsutum contributes the most production of cotton fibre, but G. barbadense is valued for its better comprehensive resistance and superior fibre properties. However, the allotetraploid genome of G. barbadense has not been comprehensively analysed. Here we present a high-quality assembly of the 2.57 gigabase genome of G. barbadense, including 80,876 protein-coding genes. The double-sized genome of the A (or At) (1.50 Gb) against D (or Dt) (853 Mb) primarily resulted from the expansion of Gypsy elements, including Peabody and Retrosat2 subclades in the Del clade and the Athila subclade in the Athila/Tat clade. Substantial gene expansion and contraction were observed and rich homoeologous gene pairs with biased expression patterns were identified, suggesting abundant gene sub-functionalization occurred by allopolyploidization. More specifically, the CesA gene family has adapted differentially temporal expression patterns, suggesting an integrated regulatory mechanism of CesA genes from At and Dt subgenomes for the primary and secondary cellulose biosynthesis of cotton fibre in a “relay race”-like fashion. We anticipate that the G. barbadense genome sequence will advance our understanding the mechanism of genome polyploidization and underpin genome-wide comparison research in this genus.

Bisphenol AF (4,4'-hexafluoroisopropylidene-2-diphenol, BPAF), an endocrine disruptor, has been shown to stimulate the proliferation of human breast cancer cells. However, the underlying mechanism has not been fully elucidated. We found that BPAF promoted the in vitro proliferation of estrogen receptor α (ERα)-positive breast cancer cells (T47D and MCF7), but not ERα-negative cells (MDA-MB-231 and MDA-MB-435s). BPAF significantly stimulated the proliferation of cultured T47D cell in a dose-dependent manner, and the half-maximal effective concentration (EC50) was approximately 123 nM. We employed lentivirus-mediated short hairpin RNA (shRNA) to knockdown ERα and ER antagonist ICI 182780 to inhibit ER activation, which resulted in the repression of BPAF-induced proliferation of T47D and MCF7 cells. We observed that C-X-C chemokine ligand 12 (CXCL12) was up-regulated in T47D cells under treatment with BPAF. Quantitative real-time PCR results showed that BPAF caused a time and dose dependent increase in mRNA level of CXCL12. Furthermore, treatment of T47D cells with BPAF increased CXCL12 secretion according to ELISA assay. BPAF-induced CXCL12 transcription and secretion was significantly attenuated by small interfering RNA (siRNA) targeting ERα and ICI 182780, indicating BPAF-induced CXCL12 expression is mediated through ERα. Notably, knockdown CXCL12 in T47D cells significantly attenuated BPAF-induced cell proliferation. We also observed that inhibition of CXCL12 binding to its receptors CXCR4 and CXCR7 by chalcone 4 blocked BPAF-induced cell growth. Our results indicated that CXCL12 facilitated BPAF-induced proliferation of T47D cells. Taken together, our data provided support that BPAF stimulated transcription and secretion of CXCL12 depending on ERα, and ERα/CXCL12 signaling positively regulated BPAF-induced proliferation of cultured T47D breast cancer cells.

microRNAs (miRNAs) are 20-24 nucleotide non-coding small RNAs that play important roles in plant development. The stages of cotton fiber development include initiation, elongation, secondary wall thickening (SWT) and maturation. We constructed seven fiber RNA libraries representing the initiation, elongation and SWT stages. In total, 47 conserved miRNA families and seven candidate miRNAs were profiled using small RNA sequencing. Northern blotting and real-time polymerase chain reaction (PCR) analyses revealed the dynamic expression of miRNAs during fiber development. In addition, 140 targets of 30 conserved miRNAs and 38 targets of five candidate miRNAs were identified through degradome sequencing. Analysis of correlated expression between miRNAs and their targets demonstrated that specific miRNAs suppressed the expression of transcription factors, SBP and MYB, a leucine-rich receptor-like protein kinase, a pectate lyase, α-tubulin, a UDP-glucuronic acid decarboxylase and cytochrome C oxidase subunit 1 to affect fiber development. Histochemical analyses detected the biological activity of miRNA156/157 in ovule and fiber development. Suppressing miRNA156/157 function resulted in the reduction of mature fiber length, illustrating that miRNA156/157 plays an essential role in fiber elongation.

Male reproduction in flowering plants is highly sensitive to high temperature (HT). To investigate molecular mechanisms of the response of cotton (Gossypium hirsutum) anthers to HT, a relatively complete comparative transcriptome analysis was performed during anther development of cotton lines 84021 and H05 under normal temperature and HT conditions. In total, 4,599 differentially expressed genes were screened; the differentially expressed genes were mainly related to epigenetic modifications, carbohydrate metabolism, and plant hormone signaling. Detailed studies showed that the deficiency in S-ADENOSYL-l-HOMOCYSTEINE HYDROLASE1 and the inhibition of methyltransferases contributed to genome-wide hypomethylation in H05, and the increased expression of histone constitution genes contributed to DNA stability in 84021. Furthermore, HT induced the expression of CASEIN KINASEI (GhCKI) in H05, coupled with the suppression of starch synthase activity, decreases in glucose level during anther development, and increases in indole-3-acetic acid (IAA) level in late-stage anthers. The same changes also were observed in Arabidopsis (Arabidopsis thaliana) GhCKI overexpression lines. These results suggest that GhCKI, sugar, and auxin may be key regulators of the anther response to HT stress. Moreover, PHYTOCHROME-INTERACTING FACTOR genes (PIFs), which are involved in linking sugar and auxin and are regulated by sugar, might positively regulate IAA biosynthesis in the cotton anther response to HT. Additionally, exogenous IAA application revealed that high background IAA may be a disadvantage for late-stage cotton anthers during HT stress. Overall, the linking of HT, sugar, PIFs, and IAA, together with our previously reported data on GhCKI, may provide dynamic coordination of plant anther responses to HT stress.

Bisphenol AF (BPAF)-induced transcriptional activity has been evaluated by luciferase reporter assay. However, the molecular mechanism of BPAF-induced endogenous transcription in human breast cancer cells has not been fully elucidated. In the present study, we investigated the effect and mechanism of BPAF-induced endogenous transcription detected by real-time PCR in human breast cancer cells. We found that BPAF stimulated transcription of estrogen responsive genes, such as trefoil factor 1 (TFF1), growth regulation by estrogen in breast cancer 1 (GREB1) and cathepsin D (CTSD), through dose-dependent and time-dependent manners in T47D and MCF7 cells. Gene-silencing of ERα, ERβ and G protein-coupled estrogen receptor 1 (GPER) by small interfering RNA revealed that BPAF-induced endogenous transcription was dependent on ERα and GPER, implying both genomic and nongenomic pathways might be involved in the endogenous transcription induced by BPAF. ERα-mediated gene transcription was further confirmed by inhibition of ER activity using ICI 182780 in ERα-positive T47D and MCF7 cells as well as overexpression of ERα in ERα-negative MDA-MB-231 breast cancer cells. Moreover, we utilized Src tyrosine kinase inhibitor PP2 and two MEK inhibitors PD98059 and U0126 to elucidate the rapid nongenomic activation of Src/MEK/ERK1/2 cascade on endogenous transcription. Our data showed that BPAF-induced transcription could be significantly blocked by PP2, PD98059 and U0126, suggesting activation of ERK1/2 was also required to regulate endogenous transcription. Taken together, these results indicate that BPAF-induced endogenous transcription of estrogen responsive genes is mediated through both genomic and nongenomic pathways involving the ERα and ERK1/2 activation in human breast cancer cells.

Background MicroRNAs (miRNAs) are small, endogenously expressed, non-coding RNA molecules involved in gene transcription and expression that combine with specific mRNA site of target genes to inhibit protein synthesis or degrade mRNA. Since the first plant miRNA was reported in 2002, numerous new miRNAs and their targets have been discovered via high-throughput sequencing and computational approaches. However, the genetic variation of miRNA genes is poorly understood due to the lack of miRNA-specific DNA markers. Results To study the genetic variation and map miRNAs and their putative target genes in cotton, we designed specific primers based on pre-miRNAs and published putative target genes. A total of 83 pre-miRNA primers and 1,255 putative target gene primers were surveyed, and 9 pre-miRNA polymorphic loci were mapped on 7 of the 26 tetraploid cotton chromosomes. Furthermore, 156 polymorphic loci of the target genes were mapped on the cotton genome. To map more miRNA loci, miRNA-based SRAP (sequence-related amplified polymorphism) markers were used to map an additional 54 polymorphic loci on the cotton genome with the exception of Chr01, Chr22, and Chr24. Finally, a network between miRNAs and their targets was constructed. All pre-miRNAs and 98 putative target genes were selected for RT-PCR analysis, revealing unique expression patterns across different fiber development stages between the mapping parents. Conclusions Our data provide an overview of miRNAs, their putative targets, and their network in cotton as well as comparative expression analyses between Gossypium hirsutum and G. barbadense. These data provide a foundation for understanding miRNA regulation during cotton fiber development.

As a product of asexual reproduction in plants, the somatic embryo (SE) differentiates into a new plantlet via a zygotic embryogenesis-like process. Here, we present the phenotypic and cellular differences between SEs and zygotic embryos (ZEs) revealed by histological section scanning using three parallel development stages of the two types of embryos of cotton (Gossypium hirsutum cv. YZ1), including globular, torpedo and cotyledonary-stages. To identify the molecular characteristics of SE development in cotton, the digital gene expression system was used to profile the genes active during SE and ZE development. A total of 4242 differentially expressed genes (DEGs) were identified in at least one developmental stage. Expression pattern and functional classification analysis based on these DEGs reveals that SE development exhibits a transcriptional activation of stress responses. RT-PCR analysis further confirmed enhanced expression levels of stress-related genes in SEs than in ZEs. Experimental stress treatment, induced by NaCl and ABA, accelerated SE development and increased the transcription of genes related to stress response, in parallel with decelerated proliferation of embryogenic calluses under stress treatment. Our data reveal that SE development involves the activation of stress responses, which we suggest may regulate the balance between cell proliferation and differentiation. These results provide new insight into the molecular mechanisms of SE development and suggest strategies that can be used for regulating the developmental processes of somatic embryogenesis.

The majority of hepatic intracellular triacylglycerol (TG) is mobilized by lipolysis followed by reesterification to reassemble TG before incorporation into a very-low-density lipoprotein (VLDL) particle. Triacylglycerol hydrolase (TGH) is a lipase that hydrolyzes TG within hepatocytes. Immunogold electron microscopy in transfected cells revealed a disparate distribution of this enzyme within the endoplasmic reticulum (ER), with particularly intense localization in regions surrounding mitochondria. TGH is localized to the lumen of the ER by the C-terminal tetrapeptide sequence HIEL functioning as an ER retention signal. Deletion of HIEL resulted in secretion of catalytically active TGH. Mutation of HIEL to KDEL, which is the consensus ER retrieval sequence in animal cells, also resulted in ER retention and conservation of lipolytic activity. However, KDEL-TGH was not as efficient at mobilizing lipids for VLDL secretion and exhibited an altered distribution within the ER. TGH is a glycoprotein, but glycosylation is not required for catalytic activity. TGH does not hydrolyze apolipoprotein B–associated lipids. This suggests a mechanism for vectored movement of TGs onto developing VLDL in the ER as TGH may mobilize TG for VLDL assembly, but will not access this lipid once it is associated with VLDL.

Hydrolysis of triglycerides is central to energy homeostasis in white adipose tissue (WAT). Hormone-sensitive lipase (HSL) was previously felt to mediate all lipolysis in WAT. Surprisingly, HSL-deficient mice show active HSL-independent lipolysis, suggesting that other lipase(s) also mediate triglyceride hydrolysis. To clarify this, we used functional proteomics to detect non-HSL lipase(s) in mouse WAT. After cell fractionation of intraabdominal WAT, most non-HSL neutral lipase activity is localized in the 100,000 × g infranatant and fat cake fractions. By oleic acid-linked agarose chromatography of infranatant followed by elution in a 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid gradient, we identified two peaks of esterase activity using p-nitrophenyl butyrate as a substrate. One of the peaks contained most of the lipase activity. In the corresponding fractions, gel permeation chromatography and SDS-PAGE, followed by tandem mass spectrometric analysis of excised Coomassie Blue-stained peptides, revealed carboxylesterase 3 (triacylglycerol hydrolase (TGH); EC 3.1.1.1). TGH is also the principle lipase of WAT fat cake extracts. Partially purified WAT TGH had lipase activity as well as lesser but detectable neutral cholesteryl ester hydrolase activity. Western blotting of subcellular fractions of WAT and confocal microscopy of fibroblasts following in vitro adipocytic differentiation are consistent with a distribution of TGH to endoplasmic reticulum, cytosol, and the lipid droplet. TGH is responsible for a major part of non-HSL lipase activity in WAT in vitro and may mediate some or all HSL-independent lipolysis in adipocytes.

Pectins are fundamental polysaccharides in the plant primary cell wall. Pectins are synthesized and secreted to cell walls as highly methyl-esterified polymers and then demethyl-esterified by pectin methylesterases (PMEs), which are spatially regulated by pectin methylesterase inhibitors (PMEIs). Although PME and PMEI genes are pivotal in plant cell wall formation, few studies have focused on the evolutionary patterns of the PME and PMEI gene families. In this study, the gene origin, evolution, and expression diversity of these two families were systematically analyzed using 11 representative species, including algae, bryophytes, lycophytes and flowering land plants. The results show that 1) for the two subfamilies (PME and proPME) of PME, the origin of the PME subfamily is consistent with the appearance of pectins in early charophyte cell walls, 2) Whole genome duplication (WGD) and tandem duplication contribute to the expansion of proPME and PMEI families in land plants, 3) Evidence of selection pressure shows that the proPME and PMEI families have rapidly evolved, particularly the PMEI family in vascular plants, and 4) Comparative expression profile analysis of the two families indicates that the eudicot Arabidopsis and monocot rice have different expression patterns. In addition, the gene structure and sequence analyses show that the origin of the PMEI domain may be derived from the neofunctionalization of the pro domain after WGD. This study will advance the evolutionary understanding of the PME and PMEI families and plant cell wall development.

Background Availability of molecular markers has proven to be an efficient tool in facilitating progress in plant breeding, which is particularly important in the case of less researched crops such as cotton. Considering the obvious advantages of single nucleotide polymorphisms (SNPs) and insertion-deletion polymorphisms (InDels), expressed sequence tags (ESTs) were analyzed in silico to identify SNPs and InDels in this study, aiming to develop more molecular markers in cotton. Results A total of 1,349 EST-based SNP and InDel markers were developed by comparing ESTs between Gossypium hirsutum and G. barbadense, mining G. hirsutum unigenes, and analyzing 3′ untranslated region (3′UTR) sequences. The marker polymorphisms were investigated using the two parents of the mapping population based on the single-strand conformation polymorphism (SSCP) analysis. Of all the markers, 137 (10.16%) were polymorphic, and revealed 142 loci. Linkage analysis using a BC1 population mapped 133 loci on the 26 chromosomes. Statistical analysis of base variations in SNPs showed that base transitions accounted for 55.78% of the total base variations and gene ontology indicated that cotton genes varied greatly in harboring SNPs ranging from 1.00 to 24.00 SNPs per gene. Sanger sequencing of three randomly selected SNP markers revealed discrepancy between the in silico predicted sequences and the actual sequencing results. Conclusions In silico analysis is a double-edged blade to develop EST-SNP/InDel markers. On the one hand, the designed markers can be well used in tetraploid cotton genetic mapping. And it plays a certain role in revealing transition preference and SNP frequency of cotton genes. On the other hand, the developmental efficiency of markers and polymorphism of designed primers are comparatively low. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-1046) contains supplementary material, which is available to authorized users.