With the increasing stress from oil price and environmental pollution, aroused attention has been paid to the microbial production of chemicals from renewable sources. The C12/14 and C16/18 alcohols are important feedstocks for the production of surfactants and detergents, which are widely used in the most respected consumer detergents, cleaning products and personal care products worldwide. Though bioproduction of fatty alcohols has been carried out in engineered E. coli, several key problems have not been solved in earlier studies, such as the quite low production of C16/18 alcohol, the lack of optimization of the fatty alcohol biosynthesis pathway, and the uncharacterized performance of the engineered strains in scaled-up system.
We improved the fatty alcohol production by systematically optimizing the fatty alcohol biosynthesis pathway, mainly targeting three key steps from fatty acyl-acyl carrier proteins (ACPs) to fatty alcohols, which are sequentially catalyzed by thioesterase, acyl-coenzyme A (CoA) synthase and fatty acyl-CoA reductase. By coexpression of thioesterase gene BTE, acyl-CoA synthase gene fadD and fatty acyl-CoA reductase gene acr1, 210.1 mg/L C12/14 alcohol was obtained. A further optimization of expression level of BTE, fadD and acr1 increased the C12/14 alcohol production to 449.2 mg/L, accounting for 75.0% of the total fatty alcohol production (598.6 mg/L). In addition, by coexpression of thioesterase gene ‘tesA, acyl-CoA synthase gene fadD and fatty acyl-CoA reductase gene FAR, 101.5 mg/L C16/18 alcohol was obtained, with C16/18 alcohol accounting for 89.2% of the total fatty alcohol production.
To our knowledge, this is the first report on selective production of C12/14 and C16/18 alcohols by microbial fermentation. This work achieved high-specificity production of both C12/14 and C16/18 alcohols. The encouraging 598.6 mg/L of fatty alcohols represents the highest titer reported so far. In addition, the 101.5 mg/L 89.2% C16/18 alcohol suggests an important breakthrough in C16/18 alcohol production. A more detailed optimization of the expression level of fatty alcohol biosynthesis pathway may contribute to a further improvement of fatty alcohol production.
Fatty alcohol; Escherichia coli; Pathway optimization; Selective production; Fermentation
The title salt, C14H16N4
−·2H2O, was obtained by the co-crystalization of 2,2′-dithiodibenzoic acid with 1,4-bis(imidazol-1-ylmethyl)benzene. It consists of 2-[(2-carboxyphenyl)disulfanyl]benzoate anions, centrosymmetric 1,1′-(p-phenylenedimethylidene)diimidazol-3-ium cations and water molecules. O—H⋯O, O—H⋯S and N—H⋯O hydrogen-bonding interactions among the components lead to the formation of a three-dimensional network.
Mammalian Gene Collection (MGC) verified over 9000 human full-ORF genes and FLJ Program reported 21 243 cDNAs of which 14 409 were unique ones and 5416 seemed to be protein-coding. The pity is that epididymis cDNA library was missing in their sequencing target list. Epididymis is a very important male accessory sex organ for sperm maturation and storage. Fully differentiated spermatozoa left from testis acquire their motility and capacity for fertilization via interactions with the epididymal epithelium duct lumen during passage through this convoluted duct. Here, we report that 20 000 clones from a healthy male epididymis cDNA library have been sequenced. The sequencing data provided 8234 known sequences and 650 unknown cDNA fragments. Hundred and six of 650 unknown cDNA clone inserts were randomly selected for fully sequencing. There were 25 unknown unique sequences and 19 released but unreported sequences came out. By northern blot analysis, four sequences randomly selected from the 19 released sequences with no known function showed positive mRNA signals in epididymis and testis. The signals for three of six from those unknown group showed as epididymis abundant in a region-specific manner but not in the testis and other tissues tested. All the sequencing data will be available on the website www.sdscli.com.
human epididymis cDNA library; transcriptomes for human epididymis; sperm maturation
The recombinant glycosyltransferase ElaGT from the elaiophylin-producing marine Streptomyces sp. SCSIO 01934 has been overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 2.9 Å resolution.
ElaGT is a glycosyltransferase from a marine Streptomyces species that is involved in the biosynthesis of elaiophylin. Here, the molecular cloning, protein expression and purification, preliminary crystallization and crystallographic characterization of ElaGT are reported. The rod-shaped crystals belonged to space group P2122, with unit-cell parameters a = 66.7, b = 131.7, c = 224.6 Å, α = 90, β = 90, γ = 90°. Data were collected to 2.9 Å resolution. A preliminary molecular-replacement solution implied the presence of two ElaGT molecules in the asymmetric unit.
ElaGT; glycosyltransferases; Streptomyces sp. SCSIO 01934
Bub1 is a critical component of the spindle assembly checkpoint (SAC) and closely linked to cell proliferation and differentiation. We previously found that spontaneous abortion embryos contained a low level of Bub1 protein but normal mRNA level, while the knockdown of Bub1 leads to abnormal numerical chromosomes in embryonic cells. Here, we investigated the mechanism through which governs the post-transcriptional regulation of Bub1 protein expression level. We first conducted bioinformatics analysis and identified eight putative miRNAs that may target Bub1. Luciferase reporter assay confirmed that miR-450a-3p can directly regulate Bub1 by binding to the 3′-untranslated region of Bub1 mRNA. We found that the overexpression of miR-450a-3p in mouse embryonic fibroblast (MEF) cells down-regulated Bub1 protein level, repressed cell proliferation, increased apoptosis and restricted most cells in G1 phase of the cell cycle. Furthermore, when the fertilized eggs were microinjected with miR-450a-3p mimics, the cleavage of zygotes was effectively suppressed. Our results strongly suggest that an abnormally decreased Bub1 level regulated by miRNAs may be implicated in the pathogenesis of spontaneous miscarriage. Therefore, the blockade of miR-450a-3p may be explored as a novel therapeutic strategy for preventing spontaneous miscarriages.
Alzheimer’s disease (AD) is a progressive and irreversible neurodegenerative disorder in which the aggregation and deposition of amyloid-β (Aβ) peptides in the brain are central to its pathogenesis. In healthy brains, Aβ is effectively metabolized with little accumulation. Cellular uptake and subsequent degradation of Aβ is one of the major pathways for its clearance in the brain. Increasing evidence has demonstrated significant roles for the low-density lipoprotein receptor-related protein 1 (LRP1) in the metabolism of Aβ in neurons, glia cells, and along the brain vasculatures. Heparan sulphate proteoglycan (HSPG) has also been implicated in several pathogenic features of AD, including its co-localization with amyloid plaques. Here, we demonstrate that HSPG and LRP1 cooperatively mediate cellular Aβ uptake. Fluorescence-activated cell sorter and confocal microscopy revealed that knockdown of LRP1 suppresses Aβ uptake while over-expression of LRP1 enhances this process in neuronal cells. Heparin, which antagonizes HSPG, significantly inhibited cellular Aβ uptake. Importantly, treatment with heparin or heparinase blocked LRP1-mediated cellular uptake of Aβ. We further showed that HSPG is more important for the binding of Aβ to the cell surface than LRP1. The critical roles of HSPG in cellular Aβ binding and uptake were confirmed in Chinese hamster ovary cells genetically deficient in HSPG. We also showed that heparin and a neutralizing antibody to LRP1 suppressed Aβ uptake in primary neurons. Our findings demonstrate that LRP1 and HSPG function in a cooperative manner to mediate cellular Aβ uptake and define a major pathway through which Aβ gains entry to neuronal cells.
amyloid-β; Alzheimer’s disease; HSPG; LRP1; heparin; endocytosis
amylose extender (ae−) starches characteristically have modified starch granule morphology resulting from amylopectin with reduced branch frequency and longer glucan chains in clusters, caused by the loss of activity of the major starch branching enzyme (SBE), which in maize endosperm is SBEIIb. A recent study with ae− maize lacking the SBEIIb protein (termed ae1.1 herein) showed that novel protein–protein interactions between enzymes of starch biosynthesis in the amyloplast could explain the starch phenotype of the ae1.1 mutant. The present study examined an allelic variant of the ae− mutation, ae1.2, which expresses a catalytically inactive form of SBEIIb. The catalytically inactive SBEIIb in ae1.2 lacks a 28 amino acid peptide (Val272–Pro299) and is unable to bind to amylopectin. Analysis of starch from ae1.2 revealed altered granule morphology and physicochemical characteristics distinct from those of the ae1.1 mutant as well as the wild-type, including altered apparent amylose content and gelatinization properties. Starch from ae1.2 had fewer intermediate length glucan chains (degree of polymerization 16–20) than ae1.1. Biochemical analysis of ae1.2 showed that there were differences in the organization and assembly of protein complexes of starch biosynthetic enzymes in comparison with ae1.1 (and wild-type) amyloplasts, which were also reflected in the composition of starch granule-bound proteins. The formation of stromal protein complexes in the wild-type and ae1.2 was strongly enhanced by ATP, and broken by phosphatase treatment, indicating a role for protein phosphorylation in their assembly. Labelling experiments with [γ-32P]ATP showed that the inactive form of SBEIIb in ae1.2 was phosphorylated, both in the monomeric form and in association with starch synthase isoforms. Although the inactive SBEIIb was unable to bind starch directly, it was strongly associated with the starch granule, reinforcing the conclusion that its presence in the granules is a result of physical association with other enzymes of starch synthesis. In addition, an Mn2+-based affinity ligand, specific for phosphoproteins, was used to show that the granule-bound forms of SBEIIb in the wild-type and ae1.2 were phosphorylated, as was the granule-bound form of SBEI found in ae1.2 starch. The data strongly support the hypothesis that the complement of heteromeric complexes of proteins involved in amylopectin synthesis contributes to the fine structure and architecture of the starch granule.
Amylopectin; amyloplasts; amylose extender; high-amylose; protein phosphorylation; protein–protein interactions; starch synthase; starch branching enzyme; starch phosphorylase; starch granule-bound proteins; starch synthesis
A long and ever-expanding roster of small (∼20–30 nucleotides) RNAs has emerged during the last decade, and most can be subsumed under the three main headings of microRNAs(miRNAs), Piwi-interacting RNAs(piRNAs), and short interferingRNAs(siRNAs). Among the three categories, miRNAs is the most quickly expanded group. The most recent number of identified miRNAs is 16,772 (Sanger miRbase, April 2011). However, there are insufficient publications on their primary forms, and no tissue-specific small RNAs precursors have been reported in the epididymis. Here, we report the identification in rats of an epididymis-specific, chimeric, noncoding RNA that is spliced from two different chromosomes (chromosomes 5 and 19), which we named HongrES2. HongrES2 is a 1.6 kb mRNA-like precursor that gives rise to a new microRNA-like small RNA (mil-HongrES2) in rat epididymis. The generation of mil-HongrES2 is stimulated during epididymitis. An epididymis-specific carboxylesterase named CES7 had 100% cDNA sequence homology at the 3′end with HongrES2 and its protein product could be downregulated by HongrES2 via mil-HongrES2. This was confirmed in vivo by initiating mil-HongrES2 over-expression in rats and observing an effect on sperm capacitation.
The prognosis of most hepatocellular carcinoma (HCC) patients is poor due to the high metastatic rate of the disease. Understanding the molecular mechanisms underlying HCC metastasis is extremely urgent. The role of CD24 and NDRG2 (N-myc downstream-regulated gene 2), a candidate tumor suppressor gene, has not yet been explored in HCC.
The mRNA and protein expression of CD24 and NDRG2 was analyzed in MHCC97H, Huh7 and L-02 cells. Changes in cell adhesion, migration and invasion were detected by up- or down-regulating NDRG2 by adenovirus or siRNA. The expression pattern of NDRG2 and CD24 in HCC tissues and the relationship between NDRG2 and HCC clinical features was analyzed by immunohistochemical and western blotting analysis.
NDRG2 expression was negatively correlated with malignancy in HCC. NDRG2 exerted anti-tumor activity by regulating CD24, a molecule that mediates cell-cell interaction, tumor proliferation and adhesion. NDRG2 up-regulation decreased CD24 expression and cell adhesion, migration and invasion. By contrast, NDRG2 down-regulation enhanced CD24 expression and cell adhesion, migration and invasion. Immunohistochemical analysis of 50 human HCC clinical specimens showed a strong correlation between NDRG2 down-regulation and CD24 overexpression (P = 0.04). In addition, increased frequency of NDRG2 down-regulation was observed in patients with elevated AFP serum level (P = 0.006), late TNM stage (P = 0.009), poor differentiation grade (P = 0.002), tumor invasion (P = 0.004) and recurrence (P = 0.024).
Our findings indicate that NDRG2 and CD24 regulate HCC adhesion, migration and invasion. The expression level of NDRG2 is closely related to the clinical features of HCC. Thus, NDRG2 plays an important physiological role in HCC metastasis.
Tumor-associated macrophages (TAMs) can influence cancer progression and metastasis, but the mechanism remains unclear. Here, we show that breast TAMs abundantly produce CCL18, and its expression in blood or cancer stroma is associated with metastasis and reduced patient survival. CCL18 released by breast TAMs promotes the invasiveness of cancer cells by triggering integrin clustering and enhancing their adherence to extracellular matrix. Furthermore, we identify PITPNM3 as a functional receptor for CCL18 that mediates CCL18 effect and activates intracellular calcium signaling. CCL18 promotes the invasion and metastasis of breast cancer xenografts, whereas suppressing PITPNM3 abrogates these effects. These findings indicate that CCL18 derived from TAMs plays a critical role in promoting breast cancer metastasis via its receptor, PITPNM3.
AIM: To investigate the effects of interferon-alpha (IFN-α) to restrain the growth and invasive potential of hepatocellular carcinoma (HCC) induced by hepatitis B virus (HBV) X protein.
METHODS: The pcDNA3.1-HBx plasmid was transfected into Chang cells by Lipofectamine in vitro, and Chang/HBx was co-cultured with IFN-α. Cell survival growth curve and clonogenicity assay were used to test the growth potential of Chang/pcDNA3.1, Chang/HBx and IFN-α-Chang/HBx in vitro. Growth assay in nude mice was used to detect the growth potential of Chang/pcDNA3.1, Chang/HBx and IFN-α-Chang/HBx in vivo. Wound healing and transwell migration assays were used to detect the invasive ability of Chang/pcDNA3.1, Chang/HBx and IFN-α-Chang/HBx.
RESULTS: Compared with CCL13 cells transfected with pcDNA3.1, CCL13 with stable expression of hepatitis B virus X protein showed the characteristics of malignant cells with high capability of growth and invasion by detecting their growth curves, colony forming efficiency, wound healing , transwell migration assays and growth assays in nude mice. Its capability of growth and invasion could be controlled by IFN-α.
CONCLUSION: IFN-α can restrain the growth and invasive potential of HCC cells induced by HBx protein, which has provided an experimental basis for IFN-α therapy of HCC.
Hepatitis B virus X protein; Interferon-alpha; Hepatocellular carcinoma; Growth; Invasion
The clathrin adaptor complex AP2 is thought to be an obligate heterotetramer. We identify null mutations in the α subunit of AP2 in the nematode Caenorhabditis elegans. α-adaptin mutants are viable and the remaining μ2/β hemicomplex retains some function. Conversely, in μ2 mutants, the alpha/sigma2 hemicomplex is localized and is partially functional. α-μ2 double mutants disrupt both halves of the complex and are lethal. The lethality can be rescued by expression of AP2 components in the skin, which allowed us to evaluate the requirement for AP2 subunits at synapses. Mutations in either α or μ2 subunits alone reduce the number of synaptic vesicles by about 30%; however, simultaneous loss of both α and μ2 subunits leads to a 70% reduction in synaptic vesicles and the presence of large vacuoles. These data suggest that AP2 may function as two partially independent hemicomplexes.
The cell membrane is a busy place, with cell-surface proteins continually added and removed according to the needs of the cell. Each protein extends a polypeptide tail into the cell cytoplasm. When a protein is to be removed from the cell surface, its tail recruits a protein complex known as the AP2 adaptor to the membrane. AP2 then recruits a coat protein called clathrin, which forms a spherical scaffold around the adaptor, the target protein and the surrounding membrane, enclosing them inside a vesicle that breaks off from the membrane and enters the cell.
Endocytosis is particularly common in neurons, which use it as a means of recycling proteins at synapses—the contact points between nerve cells. However, it is unclear whether synaptic-vesicle recycling also involves clathrin and AP2. To address this question, Gu et al. examined mutant nematode worms (C. elegans) in which the composition of AP2 had been altered.
AP2 has four subunits, called α, β2, μ2 and σ2, and Gu et al. produced worms that lack either the α- or μ2-subunit, or both. Few worms that lacked both subunits survived. Surprisingly, however, worms that lacked just one subunit were viable, despite previous evidence that AP2 requires all four subunits to be functional. Nevertheless, these single mutants produced 30% fewer synaptic vesicles compared to wild-type worms. To examine the consequences of both subunits being absent, Gu et al. rescued the double mutants by selectively expressing AP2 in their skin. These animals—which still lack AP2 in their nervous systems—produced 70% fewer synaptic vesicles than their wild-type counterparts.
The results show that AP2 does not need all four of its subunits and that it can exist as two semi-independent hemicomplexes. Moreover, Gu et al. show that C. elegans uses at least two endocytotic mechanisms (AP2-dependent and independent) to recycle vesicles and so maintain synaptic function.
apa-2; apm-2; synaptic vesicle endocytosis; AP2; C. elegans
Recent reports have demonstrated that somatic cells can be directly converted to other differentiated cell types through ectopic expression of sets of transcription factors, directly avoiding the transition through a pluripotent state. Our previous experiments generated induced neural progenitor-like cells (iNPCs) by a novel combination of five transcription factors (Sox2, Brn2, TLX, Bmi1 and c-Myc). Here we demonstrated that the iNPCs not only possess NPC-specific marker genes, but also have qualities of primary brain-derived NPCs (WT-NPCs), including tripotent differentiation potential, mature neuron differentiation capability and synapse formation. Importantly, the mature neurons derived from iNPCs exhibit significant physiological properties, such as potassium channel activity and generation of action potential-like spikes. These results suggest that directly reprogrammed iNPCs closely resemble WT-NPCs, which may suggest an alternative strategy to overcome the restricted proliferative and lineage potential of induced neurons (iNCs) and broaden applications of cell therapy in the treatment of neurodegenerative disorders.
Cigarette smoking is associated with a decreased incidence of Parkinson disease (PD) through unknown mechanisms. Interestingly, a decrease in the numbers of α4β2 nicotinic acetylcholine receptors (α4β2-nAChRs) in PD patients suggests an α4β2-nAChR-mediated cholinergic deficit in PD. Although oligomeric forms of α-synuclein have been recognized to be toxic and involved in the pathogenesis of PD, their direct effects on nAChR-mediated cholinergic signaling remains undefined. Here, we report for the first time that oligomeric α-synuclein selectively inhibits human α4β2-nAChR-mediated currents in a dose-dependent, non-competitive and use-independent manner. We show that pre-loading cells with guanyl-5′-yl thiophosphate fails to prevent this inhibition, suggesting that the α-synuclein-induced inhibition of α4β2-nAChR function is not mediated by nAChR internalization. By using a pharmacological approach and cultures expressing transfected human nAChRs, we have shown a clear effect of oligomeric α-synuclein on α4β2-nAChRs, but not on α4β4- or α7-nAChRs, suggesting nAChR subunit selectivity of oligomeric α-synuclein-induced inhibition. In addition, by combining the size exclusion chromatography and atomic force microscopy (AFM) analyses, we find that only large (>4 nm) oligomeric α-synuclein aggregates (but not monomeric, small oligomeric or fibrillar α-synuclein aggregates) exhibit the inhibitory effect on human α4β2-nAChRs. Collectively, we have provided direct evidence that α4β2-nAChR is a sensitive target to mediate oligomeric α-synuclein-induced modulation of cholinergic signaling, and our data imply that therapeutic strategies targeted toward α4β2-nAChRs may have potential for developing new treatments for PD.
U1 snRNP (U1), in addition to its splicing role, protects pre-mRNAs from drastic premature termination by cleavage and polyadenylation (PCPA) at cryptic polyadenylation signals (PASs) in introns. Here, a high throughput sequencing strategy of differentially expressed transcripts (HIDE-seq), mapped PCPA sites genome-wide in divergent organisms. Surprisingly, while U1 depletion terminated most nascent gene transcripts within ~1 kb, moderate functional U1 level decreases, insufficient to inhibit splicing, dose-dependently shifted PCPA downstream, eliciting mRNA 3′ UTR shortening and proximal 3′ exon switching characteristic of activated immune and neuronal cells, stem cells and cancer. Activated neurons’ signature mRNA shortening could be recapitulated by U1 decrease and antagonized by U1 over-expression. Importantly, we show that rapid and transient transcriptional up-regulation inherent to neuronal activation physiology creates U1 shortage relative to pre-mRNAs. Additional experiments suggest co-transcriptional PCPA counteracted by U1 association with nascent-transcripts, a process we term telescripting, ensuring transcriptome integrity and regulating mRNA length.
Arylimidamides (AIAs) have shown outstanding in vitro potency against intracellular kinetoplastid parasites, and the AIA 2,5-bis[2-(2-propoxy)-4-(2-pyridylimino)aminophenyl]furan dihydrochloride (DB766) displayed good in vivo efficacy in rodent models of visceral leishmaniasis (VL) and Chagas' disease. In an attempt to further increase the solubility and in vivo antikinetoplastid potential of DB766, the mesylate salt of this compound and that of the closely related AIA 2,5-bis[2-(2-cyclopentyloxy)-4-(2-pyridylimino)aminophenyl]furan hydrochloride (DB1852) were prepared. These two mesylate salts, designated DB1960 and DB1955, respectively, exhibited dose-dependent activity in the murine model of VL, with DB1960 inhibiting liver parasitemia by 51% at an oral dose of 100 mg/kg/day × 5 and DB1955 reducing liver parasitemia by 57% when given by the same dosing regimen. In a murine Trypanosoma cruzi infection model, DB1960 decreased the peak parasitemia levels that occurred at 8 days postinfection by 46% when given orally at 100 mg/kg/day × 5, while DB1955 had no effect on peak parasitemia levels when administered by the same dosing regimen. Distribution studies revealed that these compounds accumulated to micromolar levels in the liver, spleen, and kidneys but to a lesser extent in the heart, brain, and plasma. A 5-day repeat-dose toxicology study with DB1960 and DB1955 was also conducted with female BALB/c mice, with the compounds administered orally at 100, 200, and 500 mg/kg/day. In the high-dose groups, DB1960 caused changes in serum chemistry, with statistically significant increases in serum blood urea nitrogen, lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase levels, and a 21% decrease in body weight was observed in this group. These changes were consistent with microscopic findings in the livers and kidneys of the treated animals. The incidences of observed clinical signs (hunched posture, tachypnea, tremors, and ruffled fur) were more frequent in DB1960-treated groups than in those treated with DB1955. However, histopathological examination of tissue samples indicated that both compounds had adverse effects at all dose levels.
β-amyloid (Aβ) accumulation is described as a hallmark of Alzheimer’s disease (AD). Aβ perturbs a number of synaptic components including nicotinic acetylcholine receptors containing α7 subunits (α7-nAChRs), which are abundantly expressed in the hippocampus and found on GABAergic interneurons. We have previously demonstrated the existence of a novel, heteromeric α7β2-nAChR in basal forebrain cholinergic neurons that exhibits high sensitivity to acute Aβ exposure. To extend our previous work, we evaluated the expression and pharmacology of α7β2-nAChRs in hippocampal interneurons and their sensitivity to Aβ.
GABAergic interneurons in the CA1 subregion of the hippocampus expressed functional α7β2-nAChRs, which were characterized by relatively slow whole-cell current kinetics, pharmacological sensitivity to dihydro-β-erythroidine (DHβE), a nAChR β2* subunit selective blocker, and α7 and β2 subunit interaction using immunoprecipitation assay. In addition, α7β2-nAChRs were sensitive to 1 nM oligomeric Aβ. Similar effects were observed in identified hippocampal interneurons prepared from GFP-GAD mice.
These findings suggest that Aβ modulation of cholinergic signaling in hippocampal GABAergic interneurons via α7β2-nAChRs could be an early and critical event in Aβ-induced functional abnormalities of hippocampal function, which may be relevant to learning and memory deficits in AD.
Nicotinic acetylcholine receptor; Amyloid; Hippocampal interneuron; Patch-clamp; Acutely dissociated neuron
MicroRNA-27a (miR-27a) is thought to be an onco-microRNA that promotes tumor growth and metastasis by downregulating ZBTB10. The potential predictive value of miR-27a was studied in breast cancer patients.
The expression of miR-27a and ZBTB10 was examined in 102 breast cancer cases using in situ hybridization (ISH) and immunohistochemistry techniques and were evaluated semi-quantitatively by examining the staining index. The Correlation of miR-27a and ZBTB10 expression was analyed by Spearman Rank Correlation. The association of miR-27a and ZBTB10 expression with clinicopathological characteristics was analyzed using the χ2 test, and their effects on patient survival were analyzed by a log-rank test and the Kaplan-Meier method. Univariate and multivariate Cox regression analyses were used to evaluate the prognostic values of miR-27a and ZBTB10.
miR-27a was markedly up-regulated in invasive breast cancers that expressed low levels of ZBTB10 (P<0.001). A reverse correlation between miR-27a and ZBTB10 was also observed in breast cancer tissue samples (rs = −0.478, P<0.001). Furthermore, the expression of miR-27a and ZBTB10 was significantly correlated with clinicopathological parameters, including tumor size, lymph node metastasis and distant metastasis (P<0.05), but not with receptor status. Patients with high miR-27a or low ZBTB10 expression tended to have significantly shorter disease-free survival times (57 months and 53 months, respectively, P <0.001) and overall survival times (58 months and 55 months, respectively, P <0.001). Univariate and multivariate analysis showed that both miR-27a and ZBTB10 were independent prognostic factors of disease-free survival in breast cancer patients (P <0.001), while only miR-27a was an independent predictor of overall survival (P <0.001).
High miR-27a expression is associated with poor overall survival in patients with breast cancer, which suggests that miR-27a could be a valuable marker of breast cancer progression.
The increasing prevalence of human immunodeficiency virus type 1 (HIV-1) subtype C infection worldwide calls for efforts to develop a relevant animal model for evaluating AIDS candidate vaccines. In China, the prevalent HIV strains comprise a circulating recombinant form, BC (CRF07_BC), in which the envelope belongs to subtype C. To evaluate potential AIDS vaccines targeting Chinese viral strains in nonhuman primate models, we constructed a simian-human immunodeficiency virus (SHIV) carrying most of the envelope sequence of a primary HIV-1 clade C strain isolated from an HIV-positive intravenous drug user from YunNan province in China. Infection of six Chinese rhesus macaques with SHIV-CN97001 resulted in a low level of viremia and no significant alteration in CD4+ T-lymphocyte counts. To determine whether in vivo adaptation would enhance the infectivity of SHIV-CN97001, the parental infectious strain was serially passaged through eight Chinese rhesus macaques. The hallmarks of SHIV infectivity developed gradually, as shown by the increasingly elevated peak viremia with each passage. These findings establish that the R5-tropic SHIV-CN97001/Chinese rhesus macaque model should be very useful for evaluation of HIV-1 subtype C vaccines in China.
Clade C; Passage; Rhesus macaques; SHIV
The vaccinia virus Guang9 strain (VG9), derived from the vaccinia virus Tian Tan strain (VTT) has been found to be less virulent than VTT.
To investigate whether VG9 could be a potential replicating virus vector, the TK genes in VG9 and VTT were replaced with the HIV-1 envelope gene via homologous recombination, resulting in the recombinant viruses, VG9-E and VTT-E. The biology, virulence, humoral and cellular immunological responses of VG9-E and VTT-E were evaluated. Our results indicated no obvious difference in range of host cells and diffusion between two recombinant viruses. Neurovirulence for VG9-E in weanling and suckling mice, and skin virulence in rabbits, were lower than that of VTT-E. The humoral immune responses, including binding antibody and neutralizing antibody responses, induced by VG9-E were not significantly different from those for VTT-E whilst IFN-γ response which represented cellular immune response induced by VG9-E was significantly higher than that did by VTT-E.
Our results indicated that VG9-E was less virulent, yet induced higher cellular immune response than VTT-E. Therefore, it could be an ideal replicating vaccinia vector for HIV vaccine research and development.
Deregulated apoptosis is a hallmark of cancer, and the B-cell lymphoma-2 (Bcl-2) family of proteins is pivotal to mediating the intrinsic pathway of this process. Recent advances have yielded both pan-Bcl-2 small molecule inhibitors (SMIs) that inhibit both the Bcl-2 and the Mcl-1 arm of the Bcl-2 family anti-apoptotic proteins, as well as selective SMIs to differentially target the two arms. Of these SMIs, ABT-263 (navitoclax), AT-101 [(-)-gossypol], and obatoclax (GX15-070) are currently in clinical trials for multiple cancers. While pan-Bcl-2 inhibitors such as AT-101 and obatoclax can be more toxic for inhibiting all members of the anti-apoptotic Bcl-2 family of proteins, resistance can quickly develop for ABT-263, a selective Bcl-2 inhibitor. In this article, we discuss the current status of Bcl-2 family SMIs in preclinical and clinical development. As Mcl-1 upregulation is a major mechanism of ABT-263 resistance, Mcl-1-specific inhibitors are expected to be efficacious both in combination/sequential treatments and as a single agent against cancers resistant to ABT-263.
Drug discovery; Bcl-2; Mcl-1; apoptosis; cancer
Thioesterases remove the fatty acyl moiety from the fatty acyl-acyl carrier proteins (ACPs), releasing them as free fatty acids (FFAs), which can be further used to produce a variety of fatty acid-based biofuels, such as biodiesel, fatty alcohols and alkanes. Thioesterases play a key role in the regulation of the fatty acid synthesis in Escherichia coli. Therefore, exploring more promising thioesterases will contribute to the development of industrial microbial lipids production.
We cloned and expressed a cytosolic Acinetobacter baylyi thioesterase (‘AcTesA) in E. coli by deleting its leader sequence. Protein sequence alignment, structure modeling and site-directed mutagenesis demonstrated that Ser10, Gly48, Asn77, Asp158 and His161 residues composed the active centre of ‘AcTesA. The engineered strain that overexpressed ‘AcTesA achieved a FFAs titer of up to 501.2 mg/L in shake flask, in contrast to only 20.5 mg/L obtained in wild-type E. coli, demonstrating that the expression of ‘AcTesA indeed boosted the synthesis of FFAs. The ‘AcTesA exhibited a substrate preference towards the C8-C16 acyl groups, with C14:0, C16:1, C12:0 and C8:0 FFAs being the top four components. Optimization of expression level of ‘AcTesA made the FFAs production increase to 551.3 mg/L. The FFAs production further increased to 716.1 mg/L by optimization of the culture medium. Fed-batch fermentation was also carried out to evaluate the FFAs production in a scaleable process. Finally, 3.6 g/L FFAs were accumulated within 48 h, and a maximal FFAs yield of 6.1% was achieved in 12–16 h post induction.
For the first time, an A. baylyi thioesterase was cloned and solubly expressed in the cytosol of E. coli. This leaderless thioesterase (‘AcTesA) was found to be capable of enhancing the FFAs production of E. coli. Without detailed optimization of the strain and fermentation, the finally achieved 3.6 g/L FFAs is encouraging. In addition, ‘AcTesA exhibited different substrate specificity from other thioesterases previously reported, and can be used to supply the fatty acid-based biofuels with high quality of FFAs. Altogether, this study provides a promising thioesterase for FFAs production, and is of great importance in enriching the library of useful thioesterases.
Thioesterase; Acinetobacter baylyi; Escherichia coli; Free fatty acid; Substrate specificity; Active-site residues
Influenza A virus NS1 protein has multiple functions in the infected cell during the virus life cycle. Identification of novel cellular factors that interact with NS1 and understanding their functions in virus infection are of great interest. Recombinant viruses carrying a tagged NS1 are valuable for investigation of interactions between NS1 and cellular factors in the context of virus infection. Here, we report the generation of replication-competent recombinant influenza A viruses bearing a Strep tag in the NS1 protein. Purification of a protein complex associated with Strep-tagged NS1 from virus-infected cells followed by mass spectrometry revealed a number of attractive host factors. Among them, we focused our study on RNA helicase A (RHA) in this report. Through biomedical and functional analyses, we demonstrated that RHA interacts with NS1 in an RNA-dependent manner. Knockdown of RHA resulted in a significant reduction on virus yield and polymerase activity in a minigenome assay. Our cell-free viral genome replication assay showed that viral RNA replication and transcription can be enhanced by addition of RHA, and the enhanced effect of RHA required its ATP-dependent helicase activity. In summary, we established a system to identify cellular factors that interact with NS1 protein during virus infection and furthermore demonstrated that RHA interacts with NS1 and enhances viral replication and transcription.
The presence of various levels of Adenovirus serotype 5 neutralizing antibodies (Ad5NAb) is thought to contribute to the inconsistent clinical results obtained from vaccination and gene therapy studies. Currently, two platforms based on high-throughput technology are available for Ad5NAb quantification, chemiluminescence- and fluorescence-based assays. The aim of this study was to compare the results of two assays in the seroepidemiology of Ad5NAb in a local population of donors.
The fluorescence-based neutralizing antibody detection test (FRNT) using recombinant Ad5-EGFP virus and the chemiluminescence-based neutralizing antibody test (CLNT) using Ad5-Fluc were developed and standardized for detecting the presence of Ad5NAb in serum samples from the population of donors in Beijing and Anhui provinces, China. First, the overall percentage of people positive for Ad5NAb performed by CLNT was higher than that obtained by FRNT (85.4 vs 69.9%, p<0.001). There was an 84.5% concordance between the two assays for the 206 samples tested (144 positive in both assays and 30 negative in both assays). All 32 discordant sera were CLNT-positive/FRNT-negative and were confirmed positive by western blot. Secondly, for all 144 sera positive by both assays, the two assays showed high correlation (r = 0.94, p<0.001) and close agreement (mean difference: 0.395 log10, 95% CI: −0.054 log10 to 0.845 log10). Finally, it was found by both assays that there was no significant difference observed for titer or prevalence by gender (p = 0.503 vs 0.818, for two assays); however, age range (p = 0.049 vs 0.010) and geographic origin (p = 0.007 vs 0.011) were correlated with Ad5NAb prevalence in northern regions of China.
The CLNT assay was relatively more simple and had higher sensitivity than the FRNT assay for determining Ad5NAb titers. It is strongly suggested that the CLNT assay be used for future epidemiological studies of Ad5NAb in other localities.
Liver donor shortages stimulate the development of strategies that incorporate damaged organs into the donor pool. Herein we present a simplified machine perfusion system without the need for oxygen carriers or temperature control, which we validated in a model of orthotopic liver transplantation.
Rat livers were procured and subnormothermically perfused with supplemented Williams E medium for 3 hours, then transplanted into healthy recipients (Fresh-SNMP group). Outcome was compared with static cold stored organs (UW-Control group). In addition, a rat liver model of donation after cardiac death was adapted using a 60-minute warm ischemic period, after which the grafts were either transplanted directly (WI group) or subnormothermically perfused and transplanted (WI-SNMP group).
One-month survival was 100% in the Fresh-SNMP and UW-Control groups, 83.3% in the WI-SNMP group and 0% in the WI group. Clinical parameters, postoperative blood work and histology did not differ significantly between survivors.
This work demonstrates for the first time in an orthotopic transplantation model that ischemically damaged livers can be regenerated effectively using practical subnormothermic machine perfusion without oxygen carriers.
Liver; Transplantation; Rat; Machine; Perfusion; Preservation; Ischemia; Donor; Shortage