We report improved whole-genome shotgun sequences for the genomes of indica and japonica rice, both with multimegabase contiguity, or almost 1,000-fold improvement over the drafts of 2002. Tested against a nonredundant collection of 19,079 full-length cDNAs, 97.7% of the genes are aligned, without fragmentation, to the mapped super-scaffolds of one or the other genome. We introduce a gene identification procedure for plants that does not rely on similarity to known genes to remove erroneous predictions resulting from transposable elements. Using the available EST data to adjust for residual errors in the predictions, the estimated gene count is at least 38,000–40,000. Only 2%–3% of the genes are unique to any one subspecies, comparable to the amount of sequence that might still be missing. Despite this lack of variation in gene content, there is enormous variation in the intergenic regions. At least a quarter of the two sequences could not be aligned, and where they could be aligned, single nucleotide polymorphism (SNP) rates varied from as little as 3.0 SNP/kb in the coding regions to 27.6 SNP/kb in the transposable elements. A more inclusive new approach for analyzing duplication history is introduced here. It reveals an ancient whole-genome duplication, a recent segmental duplication on Chromosomes 11 and 12, and massive ongoing individual gene duplications. We find 18 distinct pairs of duplicated segments that cover 65.7% of the genome; 17 of these pairs date back to a common time before the divergence of the grasses. More important, ongoing individual gene duplications provide a never-ending source of raw material for gene genesis and are major contributors to the differences between members of the grass family.
Comparative genome sequencing of indica and japonica rice reveals that duplication of genes and genomic regions has played a major part in the evolution of grass genomes
The wild species of the genus Oryza contain a largely untapped reservoir of agronomically important genes for rice improvement. Here we report the 261-Mb de novo assembled genome sequence of Oryza brachyantha. Low activity of long-terminal repeat retrotransposons and massive internal deletions of ancient long-terminal repeat elements lead to the compact genome of Oryza brachyantha. We model 32,038 protein-coding genes in the Oryza brachyantha genome, of which only 70% are located in collinear positions in comparison with the rice genome. Analysing breakpoints of non-collinear genes suggests that double-strand break repair through non-homologous end joining has an important role in gene movement and erosion of collinearity in the Oryza genomes. Transition of euchromatin to heterochromatin in the rice genome is accompanied by segmental and tandem duplications, further expanded by transposable element insertions. The high-quality reference genome sequence of Oryza brachyantha provides an important resource for functional and evolutionary studies in the genus Oryza.
The wild rice species can be used as germplasm resources for this crop’s genetic improvement. Here Chen and colleagues report the de novo sequencing of the O. brachyantha genome, and identify the origin of genome size variation, the role of gene movement and its implications on heterochromatin evolution in the rice genome.
Tanshinone I (Tan-I) is a diterpene quinone extracted from the traditional herbal medicine Salvia miltiorrhiza Bunge. Recently, Tan-I has been reported to have anti-tumor effects. In this study, we investigated the growth inhibition and apoptosis inducing effects of Tan-I on three kinds of monocytic leukemia cells (U937, THP-1 and SHI 1). Cell viability was measured by MTT assay. Cell apoptosis was assessed by flow cytometry (FCM) and AnnexinV/PI staining. Reverse transcriptase polymerase chain reaction (RT-PCR) and PCR–enzyme-linked immunosorbent assay (ELISA) were used to detect human telomerase reverse transcriptase (hTERT) expression and telomerase activity before and after apoptosis. The activity of caspase-3 was determined by Caspase colorimetric assay kit and Western blot analysis. Expression of the anti-apoptotic gene Survivin was assayed by Western blot and Real-time RT-PCR using the ABI PRISM 7500 Sequence Detection System. The results revealed that Tan-I could inhibit the growth of these three kinds of leukemia cells and cause apoptosis in a time- and dose-dependent manner. After treatment by Tan-I for 48 h, Western blotting showed cleavage of the caspase-3 zymogen protein with the appearance of its 17-kD subunit, and a 89-kD cleavage product of poly (ADP-ribose) polymerase (PARP), a known substrate of caspase-3, was also found clearly. The expression of hTERT mRNA as well as activity of telomerase were decreased concurrently in a dose-dependent manner. Moreover, Real-time RT-PCR and Western blot revealed a significant down-regulation of Survivin. We therefore conclude that the induction of apoptosis by Tan-I in monocytic leukemia U937 THP-1 and SHI 1 cells is highly correlated with activation of caspase-3 and decreasing of hTERT mRNA expression and telomerase activity as well as down-regulation of Survivin expression. To our knowledge, this is the first report about the effects of Tan-I on monocytic leukemia cells.
Tanshinone I (Tan-I); telomerase; survivin; leukemia
Macrophages and dendritic cells are recognized as key players in the defense against mycobacterial infection. Recent research has confirmed that alveolar epithelial cells (AECs) also play important roles against mycobacterium infections. Thus, establishing a stable cattle AEC line for future endogenous immune research on bacterial invasion is necessary. In the present study, we first purified and immortalized type II AECs (AEC II cells) by transfecting them with a plasmid containing the human telomerase reverse trancriptase gene. We then tested whether or not the immortalized cells retained the basic physiological properties of primary AECs by reverse-transcription polymerase chain reaction and Western blot. Finally, we tested the secretion capacity of immortalized AEC II cells upon stimulation by bacterial invasion. The cattle type II alveolar epithelial cell line (HTERT-AEC II) that we established retained lung epithelial cell characteristics: the cells were positive for surfactants A and B, and they secreted tumor necrosis factor-α and interleukin-6 in response to bacterial invasion. Thus, the cell line we established is a potential tool for research on the relationship between AECs and Mycobacterium tuberculosis.
Residents of the Tibetan Plateau show heritable adaptations to extreme altitude. We sequenced 50 exomes of ethnic Tibetans, encompassing coding sequences of 92% of human genes, with an average coverage of 18X per individual. Genes showing population-specific allele frequency changes, which represent strong candidates for altitude adaptation, were identified. The strongest signal of natural selection came from EPAS1, a transcription factor involved in response to hypoxia. One SNP at EPAS1 shows a 78% frequency difference between Tibetan and Han samples, representing the fastest allele frequency change observed at any human gene to date. This SNP’s association with erythrocyte abundance supports the role of EPAS1 in adaptation to hypoxia. Thus, a population genomic survey has revealed a functionally important locus in genetic adaptation to high altitude.
Summary: Transcription and chromatin regulators, and histone modifications play essential roles in gene expression regulation. We have created CistromeMap as a web server to provide a comprehensive knowledgebase of all of the publicly available ChIP-Seq and DNase-Seq data in mouse and human. We have also manually curated metadata to ensure annotation consistency, and developed a user-friendly display matrix for quick navigation and retrieval of data for specific factors, cells and papers. Finally, we provide users with summary statistics of ChIP-Seq and DNase-Seq studies.
Availability: Freely available on the web at http://cistrome.dfci.harvard.edu/pc/
Nuclear receptors (NRs) comprise a superfamily of ligand-activated transcription factors that play important roles in both physiology and diseases including cancer. The technologies of Chromatin ImmunoPrecipitation followed by array hybridization (ChIP-chip) or massively parallel sequencing (ChIP-seq) has been used to map, at an unprecedented rate, the in vivo genome-wide binding (cistrome) of NRs in both normal and cancer cells. We developed a curated database of 88 NR cistrome datasets and other associated high-throughput datasets, including 121 collaborating factor cistromes, 94 epigenomes and 319 transcriptomes. Through integrative analysis of the curated NR ChIP-chip/seq datasets, we discovered novel factor-specific noncanonical motifs that may have important regulatory roles. We also revealed a common feature of NR pioneering factors to recognize relatively short and AT-rich motifs. Most NRs bind predominantly to introns and distal intergenetic regions, and binding sites closer to transcription start sites (TSSs) were found to be neither stronger nor more evolutionarily conserved. Interestingly, while most NRs appear to be predominantly transcriptional activators, our analysis suggests that the binding of ESR1, RARA and RARG has both activating and repressive effects. Through meta-analysis of different omic data of the same cancer cell line model from multiple studies, we generated consensus cistrome and expression profiles. We further made probabilistic predictions of the NR target genes by integrating cistrome and transcriptome data, and validated the predictions using expression data from tumor samples. The final database, with comprehensive cistrome, epigenome, transcriptome datasets, and downstream analysis results, constitutes a valuable resource for the nuclear receptor and cancer community.
Most of colorectal adenocarcinomas are believed to arise from adenomas, which are premalignant lesions. Sequencing the whole exome of the adenoma will help identifying molecular biomarkers that can predict the occurrence of adenocarcinoma more precisely and help understanding the molecular pathways underlying the initial stage of colorectal tumorigenesis. We performed the exome capture sequencing of the normal mucosa, adenoma and adenocarcinoma tissues from the same patient and sequenced the identified mutations in additional 73 adenomas and 288 adenocarcinomas. Somatic single nucleotide variations (SNVs) were identified in both the adenoma and adenocarcinoma by comparing with the normal control from the same patient. We identified 12 nonsynonymous somatic SNVs in the adenoma and 42 nonsynonymous somatic SNVs in the adenocarcinoma. Most of these mutations including OR6X1, SLC15A3, KRTHB4, RBFOX1, LAMA3, CDH20, BIRC6, NMBR, GLCCI1, EFR3A, and FTHL17 were newly reported in colorectal adenomas. Functional annotation of these mutated genes showed that multiple cellular pathways including Wnt, cell adhesion and ubiquitin mediated proteolysis pathways were altered genetically in the adenoma and that the genetic alterations in the same pathways persist in the adenocarcinoma. CDH20 and LAMA3 were mutated in the adenoma while NRXN3 and COL4A6 were mutated in the adenocarcinoma from the same patient, suggesting for the first time that genetic alterations in the cell adhesion pathway occur as early as in the adenoma. Thus, the comparison of genomic mutations between adenoma and adenocarcinoma provides us a new insight into the molecular events governing the early step of colorectal tumorigenesis.
The mechanisms involved in diabetic neuropathic pain are complex and involve peripheral and central pathophysiological phenomena. Proinflammatory tumour necrosis factor α (TNF-α) and TNF-α receptor 1, which are markers of inflammation, contribute to neuropathic pain. The purpose of this experimental study was to evaluate the effect of curcumin on diabetic pain in rats. We tested 24 rats with diabetes induced by a single intraperitoneal injection of streptozotocin and 24 healthy control rats. Twelve rats in each group received 60 mg/kg oral curcumin daily for 28 days, and the other 12 received vehicle. On days 7, 14, 21, and 28, we tested mechanical allodynia with von Frey hairs and thermal hyperalgesia with radiant heat. Markers of inflammation in the spinal cord dorsal horn on day 28 were estimated with a commercial assay and Western blot analysis. Compared to control rats, diabetic rats exhibited increased mean plasma glucose concentration, decreased mean body weight, and significant pain hypersensitivity, as evidenced by decreased paw withdrawal threshold to von Frey hairs and decreased paw withdrawal latency to heat. Curcumin significantly attenuated the diabetes-induced allodynia and hyperalgesia and reduced the expression of both TNF-α and TNF-α receptor 1. Curcumin seems to relieve diabetic hyperalgesia, possibly through an inhibitory action on TNF-α and TNF-α receptor 1.
diabetic neuropathic pain; hyperalgesia; curcumin; tumour necrosis factor α; tumour necrosis factor α receptor 1.
We performed a systematic evaluation of how variations in sequencing depth and other parameters influence interpretation of Chromatin immunoprecipitation (ChIP) followed by sequencing (ChIP-seq) experiments. Using Drosophila S2 cells, we generated ChIP-seq datasets for a site-specific transcription factor (Suppressor of Hairy-wing) and a histone modification (H3K36me3). We detected a chromatin state bias, open chromatin regions yielded higher coverage, which led to false positives if not corrected and had a greater effect on detection specificity than any base-composition bias. Paired-end sequencing revealed that single-end data underestimated ChIP library complexity at high coverage. The removal of reads originating at the same base reduced false-positives while having little effect on detection sensitivity. Even at a depth of ~1 read/bp coverage of mappable genome, ~1% of the narrow peaks detected on a tiling array were missed by ChIP-seq. Evaluation of widely-used ChIP-seq analysis tools suggests that adjustments or algorithm improvements are required to handle datasets with deep coverage.
Epstein-Barr virus (EBV)-encoded molecules have been detected in the tumor tissues of several cancers, including nasopharyngeal carcinoma (NPC), suggesting that EBV plays an important role in tumorigenesis. However, the nature of EBV with respect to genome width in vivo and whether EBV undergoes clonal expansion in the tumor tissues are still poorly understood. In this study, next-generation sequencing (NGS) was used to sequence DNA extracted directly from the tumor tissue of a patient with NPC. Apart from the human sequences, a clinically isolated EBV genome 164.7 kb in size was successfully assembled and named GD2 (GenBank accession number HQ020558). Sequence and phylogenetic analyses showed that GD2 was closely related to GD1, a previously assembled variant derived from a patient with NPC. GD2 contains the most prevalent EBV variants reported in Cantonese patients with NPC, suggesting that it might be the prevalent strain in this population. Furthermore, GD2 could be grouped into a single subtype according to common classification criteria and contains only 6 heterozygous point mutations, suggesting the monoclonal expansion of GD2 in NPC. This study represents the first genome-wide analysis of a clinical isolate of EBV directly extracted from NPC tissue. Our study reveals that NGS allows the characterization of genome-wide variations of EBV in clinical tumors and provides evidence of monoclonal expansion of EBV in vivo. The pipeline could also be applied to the study of other pathogen-related malignancies. With additional NGS studies of NPC, it might be possible to uncover the potential causative EBV variant involved in NPC.
The increasing volume of ChIP-chip and ChIP-seq data being generated creates a challenge for standard, integrative and reproducible bioinformatics data analysis platforms. We developed a web-based application called Cistrome, based on the Galaxy open source framework. In addition to the standard Galaxy functions, Cistrome has 29 ChIP-chip- and ChIP-seq-specific tools in three major categories, from preliminary peak calling and correlation analyses to downstream genome feature association, gene expression analyses, and motif discovery. Cistrome is available at http://cistrome.org/ap/.
Oligodendrocyte precursor cells (OPCs) differentiate into oligodendrocytes (OLs), which are responsible for myelination. Myelin is essential for saltatory nerve conduction in the vertebrate nervous system. However, the molecular mechanisms of maturation and myelination by oligodendrocytes remain elusive.
Methods and Findings
In the present study, we showed that maturation of oligodendrocytes was attenuated by sodium orthovanadate (a comprehensive inhibitor of tyrosine phosphatases) and PTPi IV (a specific inhibitor of SHP-2). It is also found that SHP-2 was persistently expressed during maturation process of OPCs. Down-regulation of endogenous SHP-2 led to impairment of oligodendrocytes maturation and this effect was triiodo-L-thyronine (T3) dependent. Furthermore, over-expression of SHP-2 was shown to promote maturation of oligodendrocytes. Finally, it has been identified that SHP-2 was involved in activation of Akt and extracellular-regulated kinases 1 and 2 (ERK1/2) induced by T3 in oligodendrocytes.
SHP-2 promotes oligodendrocytes maturation via Akt and ERK1/2 signaling in vitro.
AIM: To investigate the diverse characteristics of different pathological gradings of gastric adenocarcinoma (GA) using tumor-related genes.
METHODS: GA tissues in different pathological gradings and normal tissues were subjected to tissue arrays. Expressions of 15 major tumor-related genes were detected by RNA in situ hybridization along with 3’ terminal digoxin-labeled anti-sense single stranded oligonucleotide and locked nucleic acid modifying probe within the tissue array. The data obtained were processed by support vector machines by four different feature selection methods to discover the respective critical gene/gene subsets contributing to the GA activities of different pathological gradings.
RESULTS: In comparison of poorly differentiated GA with normal tissues, tumor-related gene TP53 plays a key role, although other six tumor-related genes could also achieve the Area Under Curve (AUC) of the receiver operating characteristic independently by more than 80%. Comparing the well differentiated GA with normal tissues, we found that 11 tumor-related genes could independently obtain the AUC by more than 80%, but only the gene subsets, TP53, RB and PTEN, play a key role. Only the gene subsets, Bcl10, UVRAG, APC, Beclin1, NM23, PTEN and RB could distinguish between the poorly differentiated and well differentiated GA. None of a single gene could obtain a valid distinction.
CONCLUSION: Different from the traditional point of view, the well differentiated cancer tissues have more alterations of important tumor-related genes than the poorly differentiated cancer tissues.
Pathological grading; Gastric adenocarcinoma; Tumor-related gene; Support vector machine; RNA in situ hybridization
When salt-tolerant Myxococcus cells are moved to a seawater environment, they change their growth, morphology, and developmental behavior. Outer membrane proteins and signal transduction pathways may play important roles in this shift. Chip hybridization targeting the genes predicted to encode 226 two-component signal transduction pathways and 74 outer membrane proteins of M. xanthus DK1622 revealed that the expression of 55 corresponding genes in the salt-tolerant strain M. fulvus HW-1 was significantly modified (most were downregulated) by the presence of seawater. Sequencing revealed that these seawater-regulated genes are highly homologous in both strains, suggesting that they have similar roles in the lifestyle of Myxococcus. Seven of the genes that had been reported in M. xanthus DK1622 are involved in different cellular processes, such as fruiting body development, sporulation, or motility. The outer membrane (Om) gene Om031 had the most significant change in expression (downregulated) in response to seawater, while the two-component system (Tc) gene Tc105 had the greatest increase in expression. Their homologues MXAN3106 and MXAN4042 were knocked out in DK1622 to analyze their functions in response to changes in salinity. In addition to having increased salt tolerance, sporulation of the MXAN3106 mutant was enhanced compared to that of DK1622, whereas mutating gene MXAN4042 produced contrary results. The results indicated that the genes that are involved in the cellular processes that are significantly changed in response to salinity may also be involved the salt tolerance of Myxococcus cells. Regulating the expression levels of these multifunctional genes may allow cells to quickly and efficiently respond to changing conditions in coastal environments.
MACS performs model-based analysis of ChIP-Seq data generated by short read sequencers.
We present Model-based Analysis of ChIP-Seq data, MACS, which analyzes data generated by short read sequencers such as Solexa's Genome Analyzer. MACS empirically models the shift size of ChIP-Seq tags, and uses it to improve the spatial resolution of predicted binding sites. MACS also uses a dynamic Poisson distribution to effectively capture local biases in the genome, allowing for more robust predictions. MACS compares favorably to existing ChIP-Seq peak-finding algorithms, and is freely available.
We developed a fast, integrative pipeline to identify cis natural antisense transcripts (cis-NATs) at genome scale. The pipeline mapped mRNAs and ESTs in UniGene to genome sequences in GoldenPath to find overlapping transcripts and combining information from coding sequence, poly(A) signal, poly(A) tail and splicing sites to deduce transcription orientation. We identified cis-NATs in 10 eukaryotic species, including 7830 candidate sense–antisense (SA) genes in 3915 SA pairs in human. The abundance of SA genes is remarkably low in worm and does not seem to be caused by the prevalence of operons. Hundreds of SA pairs are conserved across different species, even maintaining the same overlapping patterns. The convergent SA class is prevalent in fly, worm and sea squirt, but not in human or mouse as reported previously. The percentage of SA genes among imprinted genes in human and mouse is 24–47%, a range between the two previous reports. There is significant shortage of SA genes on Chromosome X in human and mouse but not in fly or worm, supporting X-inactivation in mammals as a possible cause. SA genes are over-represented in the catalytic activities and basic metabolism functions. All candidate cis-NATs can be downloaded from .
Liaoning cashmere goat is a famous goat breed for cashmere wool. In order to increase the transcriptome data and accelerate genetic improvement for this breed, we performed de
novo transcriptome sequencing to generate the first expressed sequence tag dataset for the Liaoning cashmere goat, using next-generation sequencing technology.
Transcriptome sequencing of Liaoning cashmere goat on a Roche 454 platform yielded 804,601 high-quality reads. Clustering and assembly of these reads produced a non-redundant set of 117,854 unigenes, comprising 13,194 isotigs and 104,660 singletons. Based on similarity searches with known proteins, 17,356 unigenes were assigned to 6,700 GO categories, and the terms were summarized into three main GO categories and 59 sub-categories. 3,548 and 46,778 unigenes had significant similarity to existing sequences in the KEGG and COG databases, respectively. Comparative analysis revealed that 42,254 unigenes were aligned to 17,532 different sequences in NCBI non-redundant nucleotide databases. 97,236 (82.51%) unigenes were mapped to the 30 goat chromosomes. 35,551 (30.17%) unigenes were matched to 11,438 reported goat protein-coding genes. The remaining non-matched unigenes were further compared with cattle and human reference genes, 67 putative new goat genes were discovered. Additionally, 2,781 potential simple sequence repeats were initially identified from all unigenes.
The transcriptome of Liaoning cashmere goat was deep sequenced, de
novo assembled, and annotated, providing abundant data to better understand the Liaoning cashmere goat transcriptome. The potential simple sequence repeats provide a material basis for future genetic linkage and quantitative trait loci analyses.
Borna disease virus (BDV) is a neurotropic virus that produces neuropsychiatric dysfunction in a wide range of warm-blooded species. Several studies have associated BDV with human psychiatric illness, but the findings remain controversial. Although oligodendrocytes are a major glial component of brain white matter and play a pivotal role in neuronal cell function, BDV's effects on human oligodendrocytes have not been clarified. Here, the effects of two BDV strains, Hu-H1 (isolated from a bipolar patient) and Strain V (a laboratory strain), on the proliferation and apoptosis of human oligodendrocytes were investigated. Three experimental cell lines were constructed: Hu-H1-infected oligodendroglioma (Hu-H1) cells, Strain V-infected oligodendroglioma (Strain V) cells, and non-infected oligodendroglioma (control) cells. BDV infection was assayed by BDV nucleoprotein (p40) immunofluorescence, cell proliferation was assayed by Cell Counting Kit-8 (CCK8), and cell cycle phases and apoptosis were assayed by flow cytometry. Expressions of the apoptosis-related proteins Bax and Bcl-2 were measured by Western blotting. p40 expression was confirmed in Hu-H1 and Strain V on and after day three post-infection. Strain V cells showed significantly greater cellular proliferation than Hu-H1 cells on and after day three post-infection. In Hu-H1 cells, Bax and Bcl-2 expression were significantly increased and decreased, respectively, on and after day three post-infection. In contrast, in Strain V cells, Bax and Bcl-2 expression were significantly decreased and increased, respectively, on and after day three post-infection. In conclusion, Hu-H1 inhibits cellular proliferation and promotes apoptosis in human oligodendrocytes via Bax upregulation and Bcl-2 downregulation. In contrast, Strain V promotes cellular proliferation and inhibits apoptosis in human oligodendrocytes via Bax downregulation and Bcl-2 upregulation. The effects of the Hu-H1 strain (isolated from a bipolar patient) are opposite from those of Strain V (a laboratory strain), thereby providing a proof of authenticity for both.
Pleomorphic lipoma is a rare neoplasm that predominantly occurs in the dermis or subcutis of the posterior neck, upper back, and shoulders. Although pleomorphic lipoma is a benign tumor, it may contain atypical cells. As a variant of spindle cell lipoma, pleomorphic lipoma clinically presents as a slow-growing and well-circumscribed subcutaneous mass. Rarely, some patients have multiple lesions. Histologically, pleomorphic lipoma is composed of mature fat, bland spindle-shaped mesenchymal cells, and coarse ‘rope-like’ collagen bands. In addition, lipoma contains multinucleated floret-like giant cells. Although spindle cell lipoma/pleomorphic lipoma with little fat was seen in the original series described by Enzinger and Harvey, cases with little to no fat remain diagnostically challenging. Herein, we report a case of ‘fat-free’ pleomorphic lipoma occurring in the upper back and axilla simultaneously. Although the lipoma was typically composed of bland spindle-shaped cells, rope-like collagen, scattered floret-like giant cells, and striking stromal myxoid change in the background, mature fat was absent. Immunohistochemical analyses showed positive staining for CD34, vimentin, and Bcl-2, and negative staining for S100, confirming the diagnosis of pleomorphic lipoma.
Spindle cell lipoma; Pleomorphic lipoma; Atypical lipomatous tumor; Myxoid liposarcoma; Pleomorphic hyalinizing angiectatic tumor
The present study was conducted to determine whether recombinant human β-defensin-3 (rHBD3) in the milk of transgenic goats has an anti-bacterial activity against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Streptococcus agalactiae (S. agalactiae) that could cause mastitis. A HBD3 mammary-specific expression vector was transfected by electroporation into goat fetal fibroblasts which were used to produce fourteen healthy transgenic goats by somatic cell nuclear transfer. The expression level of rHBD3 in the milk of the six transgenic goats ranged from 98 to 121 µg/ml at 15 days of lactation, and was maintained at 90–111 µg/ml during the following 2 months. Milk samples from transgenic goats showed an obvious inhibitory activity against E. coli, S. aureus and S. agalactiae in vitro. The minimal inhibitory concentrations of rHBD3 in milk against E. coli, S. aureus and S. agalactiae were 9.5–10.5, 21.8–23.0 and 17.3–18.5 µg/mL, respectively, which was similar to those of the HBD3 standard (P>0.05). The in vivo anti-bacterial activities of rHBD3 in milk were examined by intramammary infusion of viable bacterial inoculums. We observed that 9/10 and 8/10 glands of non-transgenic goats infused with S. aureus and E. coli became infected. The mean numbers of viable bacteria went up to 2.9×103 and 95.4×103 CFU/ml at 48 h after infusion, respectively; the mean somatic cell counts (SCC) in infected glands reached up to 260.4×105 and 622.2×105 cells/ml, which were significantly higher than the SCC in uninfected goat glands. In contrast, no bacteria was presented in glands of transgenic goats and PBS-infused controls, and the SSC did not significantly change throughout the period. Moreover, the compositions and protein profiles of milk from transgenic and non-transgenic goats were identical. The present study demonstrated that HBD3 were an effective anti-bacterial protein to enhance the mastitis resistance of dairy animals.
Glucocorticoid-induced TNF receptor (GITR) plays a crucial role in modulating immune response and inflammation, however the role of GITR in human cancers is poorly understood. In this study, we demonstrated that GITR is inactivated during tumor progression in Multiple Myeloma (MM) through promoter CpG island methylation, mediating gene silencing in primary MM plasma cells and MM cell lines. Restoration of GITR expression in GITR deficient MM cells led to inhibition of MM proliferation in vitro and in vivo and induction of apoptosis. These findings were supported by the presence of induction of p21 and PUMA, two direct downstream targets of p53, together with modulation of NF-κB in GITR-overexpressing MM cells. Moreover, the unbalanced expression of GITR in clonal plasma cells correlated with MM disease progression, poor prognosis and survival. These findings provide novel insights into the pivotal role of GITR in MM pathogenesis and disease progression.
Two dimensional (2D) materials with a monolayer of atoms represent an ultimate control of material dimension in the vertical direction. Molybdenum sulfide (MoS2) monolayers, with a direct bandgap of 1.8 eV, offer an unprecedented prospect of miniaturizing semiconductor science and technology down to a truly atomic scale. Recent studies have indeed demonstrated the promise of 2D MoS2 in fields including field effect transistors, low power switches, optoelectronics, and spintronics. However, device development with 2D MoS2 has been delayed by the lack of capabilities to produce large-area, uniform, and high-quality MoS2 monolayers. Here we present a self-limiting approach that can grow high quality monolayer and few-layer MoS2 films over an area of centimeters with unprecedented uniformity and controllability. This approach is compatible with the standard fabrication process in semiconductor industry. It paves the way for the development of practical devices with 2D MoS2 and opens up new avenues for fundamental research.
Plasmodium falciparum infection of human erythrocytes is known to result in the modification of the host cell cytoskeleton by parasite-coded proteins. However, such modifications and corresponding implications in malaria pathogenesis have not been fully explored. Here, we probed the gradual modification of infected erythrocyte cytoskeleton with advancing stages of infection using atomic force microscopy (AFM). We reported a novel strategy to derive accurate and quantitative information on the knob structures and their connections with the spectrin network by performing AFM–based imaging analysis of the cytoplasmic surface of infected erythrocytes. Significant changes on the red cell cytoskeleton were observed from the expansion of spectrin network mesh size, extension of spectrin tetramers and the decrease of spectrin abundance with advancing stages of infection. The spectrin network appeared to aggregate around knobs but also appeared sparser at non-knob areas as the parasite matured. This dramatic modification of the erythrocyte skeleton during the advancing stage of malaria infection could contribute to the loss of deformability of the infected erythrocyte.
Dexmedetomidine has been reported to reduce mortality in septic rats. This study was designed to investigate the effects of dexmedetomidine on inflammatory reaction in lung tissues of septic rats induced by CLP. After induction of sepsis, the rats were treated with normal saline or dexmedetomidine
(5, 10, or 20 μg/kg). The survival rate of septic rats in 24 h was recorded. The inflammation of lung tissues was evaluated by HE stain. The concentrations of IL-6 and
TNF-α in BALF and plasma were measured by ELISA. The expressions of TLR4 and MyD88 were measured by western blotting. The activation of
NF-κB in rat lung tissues was assessed by western blotting and immunohistochemistry. It was found that the mortality rate and pulmonary inflammation were significantly increased in septic rats. IL-6 and
TNF-α levels in BALF and plasma, NF-κB activity, and TLR4/MyD88 expression in rat lung tissues were markedly enhanced after CLP. Dexmedetomidine (10
and 20 μg/kg) significantly decreased mortality and pulmonary inflammation of septic rats, as well as suppressed CLP-induced elevation of
TNF-α and IL-6 and inhibited TLR4/MyD88 expression and NF-κB activation. These results suggest that dexmedetomidine may decrease mortality and inhibit inflammatory reaction in lung tissues of septic rats by suppressing TLR4/MyD88/NF-κB pathway.