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The GH (growth hormone)/IGFs (insulin-like growth factors) system has an important function in the regulation of growth. In this system, IGFBPs play a crucial regulatory role in IGF functions. As a member of the IGFBP family, IGFBP2 can bind to IGF and regulate IGF functions to regulate development and growth. In addition, IGFBP2 shows key regulatory functions in cell proliferation and metabolism. In this study, the igfbp2 gene was cloned from grass carp (Ctenopharyngodon idellus) liver. The ORF of grass carp igfbp2 is 834 bp long and encodes 277 amino acids. The tissue distribution results showed that igfbp2 is expressed in multiple tissues in grass carp and has a high expression level in the liver. In the OGTT, igfbp2 expression was significantly decreased in the liver and brain after 6 h of treatment with glucose. In vitro, igfbp2 expression in grass carp's primary hepatocytes was significantly suppressed by insulin after treatment for 6 and 12 h. Moreover, igfbp2 expression was markedly increased in a dose-dependent manner with glucagon incubation in grass carp's primary hepatocytes. To the best of our knowledge, this is the first report about Igfbp2 in grass carp. These results will provide a basis for the in-depth study of grass carp Igfbp2.

Leaf angle is a key factor in plant architecture and crop yield. Brassinosteroids (BRs) regulate many developmental processes, especially the leaf angle in monocots. However, the BR signalling pathway is complex and includes many unknown members. Here, we propose that Oryza sativa BRASSINOSTEROID-RESPONSIVE LEAF ANGLE REGULATOR 1 (OsBLR1) encodes a bHLH transcription factor, and positively regulates BR signalling to increase the leaf angle and grain length in rice (Oryza sativa L.). Lines overexpressing OsBLR1 (blr1-D and BLR1-OE-1/2/3) had similar traits, with increased leaf angle and grain length. Conversely, OsBLR1-knockout mutants (blr1-1/2/3) had erect leaves and shorter grains. Lamina joint inclination, coleoptile elongation, and root elongation assay results indicated that these overexpression lines were more sensitive to BR, while the knockout mutants were less sensitive. There was no significant difference in the endogenous BR contents of blr1-1/2 and wild-type plants. These results suggest that OsBLR1 is involved in BR signal transduction. The blr1-D mutant, with increased cell growth in the lamina joint and smaller leaf midrib, showed significant changes in gene expression related to the cell wall and leaf development compared with wild-type plants; furthermore, the cellulose and protopectin contents in blr1-D were reduced, which resulted in the increased leaf angle and bent leaves. As the potential downstream target gene of OsBLR1, the REGULATOR OF LEAF INCLINATION1 (OsRLI1) gene expression was up-regulated in OsBLR1-overexpression lines and down-regulated in OsBLR1-knockout mutants. Moreover, we screened OsRACK1A as an interaction protein of OsBLR1 using a yeast two-hybrid assay and glutathione-S-transferase pull-down.

Female moths use sex pheromones to attract males, and corresponding regulatory mechanism underlying sex pheromone biosynthesis is species-dependent. However, the detailed mechanism involved in sex pheromone biosynthesis in Ostrinia furnacalis has not yet been fully addressed. In the present study, transcriptome sequencing of O. furnacalis pheromone glands screened a serials of candidate genes involved in sex pheromone biosynthesis. Our analysis showed that sex pheromone release in O. furnacalis females arrives its peak at the 2nd scotophase, consistent with its mating behavior. Pheromone biosynthesis-activating neuropeptide (PBAN) was confirmed to regulate sex pheromone biosynthesis, and Ca2+ is the secondary messenger of PBAN signaling in O. furnacalis. The functional analysis of candidate genes demonstrated that the decreased mRNA levels or activities of calcineurin (CaN) and acetyl-CoA carboxylase (ACC) led to significant decrease in sex pheromone production and female capability to attract males, as demonstrated by RNAi-mediated knockdown and pharmacological inhibitor assay. Most importantly, the activities of CaN and ACC depend on the activation of PBAN/PBANR/Ca2+. Furthermore, fatty-acyl reductase 14 was involved in PBAN-mediated sex pheromone biosynthesis. Altogether, our results demonstrated that PBAN regulates sex pheromone biosynthesis through PBANR/Ca2+/CaN/ACC pathway to promote sex pheromone biosynthesis in O. furnacalis and provided a reference for non-model organism to study neuropeptide signal transduction.

Pyruvate kinase (PYK) is a speed-limited enzyme of glycolysis that catalyzes the formation of pyruvate, and plays an important role in acetyl-CoA synthesis. The acetyl-CoA is the precursor of sex pheromone biosynthesis in Helicoverpa armigera. However, the role of PYK in sex pheromone biosynthesis remains elusive. Here, PYK in H. armigera (HaPYK) was found to be highly expressed in the pheromone glands (PGs). The developmental expression profile of HaPYK was consistent with the fluctuation of sex pheromone release. Function analysis revealed that the knockdown of HaPYK led to a decrease in the levels of pyruvic acid and acetyl-CoA in PGs, which in turn caused a significant decrease in cis-11-hexadecenal (Z11-16: Ald) production, female capability to attract males, and mating frequency. Further study demonstrated that sugar feeding (5% sugar) increased the transcription and enzyme activity of HaPYK, thereby facilitating sex pheromone biosynthesis. Moreover, pheromone biosynthesis activating neuropeptide (PBAN) upregulated HaPYK activity through protein kinase C (PKC), as shown by PKC-specific inhibitor analysis. Altogether, our results revealed that PBAN activated HaPYK by Ca2+/PKC, thereby regulating the synthesis of pyruvate and subsequent acetyl-CoA, ensuring the supply of sex pheromone precursor, and finally facilitating sex pheromone biosynthesis and mating behavior.

Optimized pore size is essential for maximizing the encapsulation efficiency of molecules in solid supports. In this work, we prepared porous silica nanosheets with mesopore in the size range from ...

Helicoverpa armigera (cotton bollworm) is one of the most destructive pests worldwide. Due to resistance to Bacillus thuringiensis and conventional insecticides, an effective management strategy to control this pest is urgently needed. Spinosad, a natural pesticide, is considered an alternative; however, the mechanism underlying the developmental effects of sublethal spinosad exposure remains elusive. In this study, the mechanism was examined using an insect model of H. armigera. Results confirmed that exposure to sublethal spinosad led to reduced larval wet weight, delayed larval developmental period, caused difficulty in molting, and deformed pupae. Further investigation demonstrated that exposure to sublethal spinosad caused a significant decrease in 20E titer and increase in JH titer, thereby leading to the discordance between 20E and JH titers, and consequently alteration in the expression levels of HR3 and Kr-h1. These results suggested that sublethal spinosad caused hormonal disorders in larvae, which directly affect insect development. Our study serves as a reference and basis for the toxicity evaluation of spinosad on molting and pupation in insect metamorphosis, which may contribute to identifying targets for effective control of cotton bollworm.

ObjectivesTo identify echocardiographic signatures featuring left ventricular longitudinal strain (LS) associated with genetic risk for cardiac amyloidosis (CA) due to theTTRVal142Ile (V142I) variant in African American (AA) and Hispanic/Latinx (H/L) individuals.BackgroundHereditary transthyretin amyloidosis (hATTR) can cause CA in ∼60-70% of older V142I carriers, but amyloid deposition progresses over many years. Disease-modifying therapy for CA is now available and early initiation is a priority for improving outcomes. Genomic screening programs and familial cascade genetic testing uncover pre-symptomatic V142I carriers, yet no guidelines exist for early CA detection.MethodsExome sequencing data linked to electronic health records (EHRs) of BioMebiobank participants were queried for AA or H/LTTR- andTTR+ (V142I) subjects without hATTR diagnoses and with prior echocardiograms suitable for retrospective LS analysis. Systemic “red flag” features of ATTR were extracted from EHRs ofTTR+ subjects. Speckle tracking echocardiography was retrospectively applied to determine global (GLS) and segmental LS. Relative apical sparing (RAS) was calculated.Results57TTR+ and 46TTR-age- and ancestry-matched subjects were included. GLS declined with age in females but not males, and was abnormal (TTR+ and 7 (15.2%)TTR-subjects (p = 0.066). Apical sparing was observed in 13 (22.8%)TTR+ and 11 (23.9%)TTR-subjects (p = 1.0). After adjusting for relevant demographic and echocardiographic covariates, neither GLS nor RAS was associated withTTR+ V142I status. Red flag features were not associated with GLS or RAS inTTR+ subjects.ConclusionsNeither GLS nor RAS were significantly different betweenTTR+ andTTR-subjects. Since >50% ofTTR+ subjects were ≥ 60 years old, penetrance of CA by echocardiography among unselected V142I carriers may be lower than previously estimated. These findings indicate that surveillance for CA in individuals at increased genetic risk due to V142I should not rely solely on echocardiography, even with LS.

Since discovery, glucagon biology has remained enigmatic. Studies have shown that glucagon may have physiological effects beyond glucose regulation by binding to its receptor (Gcgr). The potential roles of glucagon and its receptor in the crosstalk between glucose metabolism and immune function of fish remain to be investigated. In the present study, we sequenced and characterized gcgra and gcgrb in grass carp. Phylogenetic analysis showed that they were highly homologous with their counterparts in fish. The gcgra and gcgrb mRNAs were ubiquitously expressed in all the tissues examined. The gcgra mRNA was highly expressed in the liver and gcgrb was mainly expressed in the liver and brain regions of grass carp. Furthermore, the results of oral glucose tolerance test (OGTT) showed that the expression levels of the two receptors were significantly increased at 12 h after treatment. In the hepatocytes, insulin inhibited gcgra and gcgrb mRNA expressions at 3−12 h and 6−12 h, respectively. While glucagon promoted their expressions at 3−12 h and 3 h, respectively. Intraperitoneal injection of lipopolysaccharide (LPS) increased hepatic gcgra mRNA level at 6 h post treatment, and up-regulated gcgrb mRNA abundance in the liver at 24 h. In response to polyinosinic polycytidylic acid [poly (I:C)] stimuli, however, gcgra and gcgrb mRNA levels were decreased. These results suggested that glucagon receptors were affected by bacterial-like and virus-like infection beyond their roles in the glucose metabolism of grass carp.

Glucose transporter 2 (glut2) has been studied in mammals, aves, and several fish, while the comparative studies of glut2 in common carp are still lacking. In this study, glut2 was firstly isolated and characterized from the liver of common carp. The full-length cDNA of glut2 was 2351 bp with an open reading frame (ORF) of 1512 bp, encoding 503 amino acids. Alignment of glut2 amino acid sequences from different species revealed that common carp glut2 showed higher sequence identity with teleosts, and lower homology with mammals and amphibians. Tissue distribution demonstrated that glut2 mRNA level was mainly expressed in liver, foregut, and midgut. To investigate the actions of glut2 on glucose metabolism, the level of glut2 mRNA was detected after intraperitoneal injection of glucose, human insulin and glucagon (100 ng/g), respectively. Following glucose administration, glut2 gene expression was significantly upregulated at 3 h in the foregut. However, no change was found in hepatic glut2 mRNA level, indicating that glut2 may have a role in intestinal glucose uptake rather than in the liver. Following insulin treatment, the expression of glut2 was markedly downregulated at 3 h and 6 h in the liver, and at 3 h in the foregut, respectively. Furthermore, glut2 mRNA expression was unaffected by glucagon injection in the liver and foregut. These results suggested that the expression of glut2 regulated by pancreatic hormones was different. Taken together, our studies firstly revealed the structure of the glut2 gene and its potential functions in glucose metabolism of common carp.

Lepidoptera sex pheromone biosynthesis is regulated by pheromone biosynthesis activating neuropeptide (PBAN). PBAN regulates not only female sex pheromone biosynthesis but also male sex pheromone biosynthesis. Previous research has confirmed that PBAN regulates sex pheromone biosynthesis using Ca2+ as a secondary messenger in all examined species to date. However, the downstream signal of Ca2+ has remained elusive. In the present study, calcineurin A (CNA), a downstream signal of Ca2+ , was discovered in Helicoverpa armigera male hairpencil and named HaCNA. Sequence analysis demonstrated that the open reading frame of HaCNA contains 1488 nucleotides encoding 495 amino acid residues. A homology search revealed that HaCNA shares a high amino acid identity with the CNA of other insects. Developmental and spatial expression analyses revealed that the mRNA levels of HaCNA peaked at 24 h after emergence and that HaCNA expression was ubiquitous in all examined tissues. Activity analysis revealed that PBAN activates HaCNA, and a Ca2+ inhibitor, Lacl3 , attenuated the effect of PBAN by decreasing HaCNA activity. Pharmacological inhibitor and RNA interference-mediated knockdown assays revealed that both activity inhibition and decreased mRNA levels of HaCNA led to a significant decrease in the production of the male sex pheromone components [octadecanol and (Z)-11 hexadecanol)] and in the efficacy of female mating acceptance. Our results demonstrate that HaCNA acts as downstream signal of PBAN/Ca2+ and plays an important role in PBAN-induced male sex pheromone biosynthesis and female mating acceptance.

Colon cancer is the third most common malignancy and one of the leading causes of cancer-associated mortality worldwide. Neuronal pentraxin 1 (NPTX1) is associated with tumor progression in some types of tumors. However, its expression and role in colon cancer has not been yet reported. Here we observed that NPTX1 was down-regulated in colon cancer. Additionally, we explored the functional significance of NPTX1 in colon cancer. We found that over-expression of NPTX1 inhibited colon cancer cell growth by performing MTT, colony formation, Edu corporation assays, and cell cycle analysis. In vivo mouse experiments also confirmed the anti-proliferative role of NPTX1 in colon cancer. Further mechanistic study showed that over-expression of NPTX1 inhibited the expression of cyclin A2 and CDK2 in colon cancer cells, thereby regulating the Rb-E2F signaling. In summary, these findings reveal that NPTX1 suppress the colon cancer cell growth and might serve as a useful potential target for treatment of colon cancer.

Adiponectin plays critical roles in energy metabolism and immunity of mammals by binding to its receptors (AdipoR1 and AdipoR2). However, they have not been fully understood in fish. The present study examined the distribution of adiponectin receptors (AdipoRs) mRNA in grass carp, and the changes of their expressions under glucose load, insulin or glucagon stimulation, lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid [poly (I:C)] challenge. As for glucose load, fish were orally administrated with glucose (1.67 mg/g BW) for 1, 3, 6, 12 h. For insulin or glucagon stimulation, the primary hepatocytes of grass carp were incubated with 0, 10, 100, 1000 nM insulin or glucagon for 1, 3, 6, 12 h. In the immune challenge experiment, grass carp were intraperitoneally injected with LPS or poly (I:C) (5 μg/g BW) for 3, 6, 12, 24, 48 h. Finally, AdipoRs mRNA expression in all experiments was detected by Real-time quantitative PCR. The results showed that AdipoRs mRNA was widely expressed in various tissues of grass carp. The adipoR1a mRNA was highly expressed in the brain regions, and the highest level of adipoR2 mRNA was observed in the adipose tissue. Glucose load up-regulated adipoR1b and adipoR2 mRNA expressions in the liver at 6 h post administration and decreased adipoR1a expression in the muscle at 3 h. Insulin significantly enhanced adipoR1b and adipoR2 mRNA expressions in the hepatocytes at 1–12 h, and promoted adipoR1a mRNA expression at 1, 3 and 12 h, while glucagon had little effect on their expressions. LPS and poly (I:C) stimuli induced AdipoRs expression in the liver at 6 and 12 h, and their expressions in the muscle were decreased slightly or remained unchanged. In addition, LPS promoted AdipoRs expression in the kidney and spleen, whereas poly (I:C) only induced adipoR1a mRNA expression in the kidney, and increased adipoR2 mRNA level in the spleen. These results suggest that AdipoRs may act as one of the targets of insulin and immune response in grass carp, but whether they are direct target remains to be further studied.

The present study aimed to explore the function of miR‑34b promoter methylation in cell proliferation in children's acute leukemia. Quantitative PCR and methylation‑specific PCR were performed to measure the levels of miR‑34b and its promoter methylation in normal cells, eight leukemia cell lines as well as primary leukemic cells isolated from patients newly diagnosed with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) and mixed lymphocytic lymphoma. miR‑34b levels in leukemia cell lines and primary leukemic cells were significantly lower than those in normal cells. The miR‑34b promoter was found to be methylated in all leukemia cell lines, 24 of 31 ALL patients and 8 of 19 AML patients, but not in the 23 normal controls. miR‑34b expression and methylation of its promoter were not associated with most clinical parameters assessed; however, miR‑34b levels in prednisone‑sensitive ALL were significantly different from those in insensitive ALL. A cell counting kit‑8 assay showed that transfection of miR‑34b mimics into K562 cells inhibited their proliferation. Furthermore, treatment with the demethylating agent 5‑aza‑2‑deoxycytidine significantly enhanced miR‑34b expression levels and decreased the methylation status of its promoter in HL‑60 and K562 cells. In conclusion, the results of the present study indicated that in pediatric leukemia cells and leukemia cell lines, the expression of miR‑34b is inhibited by methylation of its promoter, which impairs the restraining effects of miR‑34b on cell proliferation. It was also indicated that the expression of miR‑34b in ALL patients may affect their response to early treatments.

Autophagy is required for hematopoietic stem cell multilineage differentiation, but the underlying mechanism is unknown. Using a conditional mouse model and human leukemia cells, we uncovered a mechanistic link between autophagy and hematopoietic stem cell differentiation. Loss of autophagy in mouse hematopoietic stem cells diminished the bone marrow generation of functional blood cells, in particular lymphocytes, and resulted in a leukemic phenotype and elevated Notch signaling. Physiological autophagy activity in mice was inversely correlated with Notch signaling during adult hematopoietic stem cell differentiation, while pathologically low autophagy was associated with upregulated Notch signaling in dysfunctional hematopoietic stem cells of acute leukemia patients. Furthermore, we show that autophagy directly degraded intracellular Notch, the active form of Notch receptor cleaved from the full-length Notch molecule by γ-secretase. Finally, we show that hematopoietic multilineage differentiation potential was restored in autophagy defective hematopoietic stem and progenitor cells when their Notch signaling was abrogated either pharmacologically with γ-secretase inhibitor DAPT or genetically with RNA interference of Notch effector RBPJ. Hence, we propose that autophagy sustains hematopoiesis by direct targeting Notch.

Autophagy is integral to hematopoiesis and protects against leukemogenesis. However, the fundamentals of the required molecular machinery have yet to be fully explored. Using conditional mouse models to create strategic defects in the hematopoietic hierarchy, we have shown that recovery capacities in stem cells and somatic cells differ if autophagy is impaired or flawed. An in vivo Atg7 deletion in hematopoietic stem cells completely ablates the autophagic response, leading to irreversible and ultimately lethal hematopoiesis. However, while no adverse phenotype is manifested in vivo by Atg7-deficient myeloid cells, they maintain active autophagy that is sensitive to brefeldin A, an inhibitor targeting Golgi-derived membranes destined for autophagosome formation in alternative autophagy. Removing Rab9, a key regulatory protein, in alternative autophagy, disables autophagy altogether in Atg7-deficient macrophages. Functional analysis indicates that ATG7-dependent canonical autophagy is physiologically active in both hematopoietic stem cells and in terminally differentiated hematopoietic cells; however, only terminally differentiated cells such as macrophages are rescued by alternative autophagy if canonical autophagy is ineffective. Thus, it appears that hematopoietic stem cells rely solely on ATG7-dependent canonical autophagy, whereas terminally differentiated or somatic cells are capable of alternative autophagy in the event that ATG7-mediated autophagy is dysfunctional. These findings offer new insight into the transformational trajectory of hematopoietic stem cells, which in our view renders the autophagic machinery in stem cells more vulnerable to disruption.

IGFBPs play a pivotal role in regulating the physiological function of IGFs (insulin-like growth factors). As an important member of IGFBPs, IGFBP3 is involved in the regulation of physiological functions of IGFs. To investigate the functional role of Igfbp3 in a herbivorous fish species, grass carp igfbp3 (GenBank accession no. MN251843) was isolated from the liver by molecular cloning. The ORF of grass carp igfbp3 was 882 bp, which encoded 293 amino acids, and the first 22 amino acids comprised the signal peptide. The predicted molecular weight of grass carp Igfbp3 is 31.95 kDa, and the theoretical isoelectric point is 8.32. Grass carp Igfbp3 displayed a high identity with its counterparts of common carp and zebrafish. And phylogenetic tree analysis showed that the grass carp Igfbp3 was clustered into the Igfbp3 subgroups of common carp and zebrafish. Tissue distribution study showed that igfbp3 was ubiquitously expressed in all tissues examined in the present study. High expression level of igfbp3 was detected in the heart, brain and fat of grass carp. The OGTT demonstrated that igfbp3 mRNA expression was significantly elevated in the liver of grass carp in response to glucose treatment. In vitro study showed that insulin could markedly stimulated igfbp3 mRNA expression in primary grass carp hepatocytes. Moreover, igfbp3 mRNA levels were also regulated by glucagon in grass carp primary hepatocytes. These results may provide the theoretical foundation for investigating the role of Igfbp3 in fish metabolism.

Pathogenic gene variants in transthyretin ( TTR ) can cause heritable amyloidosis (hATTR) and confer a 60% risk of heart failure. Echocardiography with global longitudinal strain (GLS) discriminates fully manifest cardiac amyloid (CA) but its ability to detect preclinical CA is unknown. We

Generally, fish are thought to have a limited ability to utilize carbohydrate. Postprandial blood glucose is cleared sluggishly in fish, resulting in prolonged hyperglycemia. Facilitative glucose transporters (GLUTs) play an important role in glucose utilization. In the present study, the expression levels of glut2 in different tissues were detected in grass carp. Furthermore, the effects of oral glucose administration on glut2 mRNA expression in the liver, intestine and kidney were investigated, and we also evaluated the response of glut2 mRNA to insulin and glucagon in the primary hepatocytes of grass carp. The expression level of glut2 mRNA was highest in the liver, followed by the intestine and kidney, but lower in other tissues. The result of glucose tolerance test (GTT) showed that serum glucose reached the highest level at 3 h after GTT and recovered to the basic level at 6 h. The glut2 mRNA in the intestine was up-regulated at 1 h after GTT. However, the glut2 mRNA expression in the liver of grass carp was unchanged after GTT for 1, 3, 6 h, and even decreased at 12 h after GTT. In addition, the expression of glut2 mRNA in the primary hepatocytes was enhanced by insulin and glucagon at 3 h post treatment. These results suggested that glut2 expression in the liver of grass carp was sensitive to insulin and glucagon, but not blood glucose. The up-regulation of glut2 by these hormones might be involved in the bi-directional transportation of glucose in the liver.

Background HPV 18 is one of the most prevalent oncogenic types, only second to HPV 16, and included in the licensed vaccines on the market. In this study, we describe the production and characterization of a panel of monoclonal antibodies (mAb) to HPV18. Methods The immunocompetence of 1B1 and 4C2 mAbs for HPV L1 protein was evaluated by SDS-PAGE analysis, neutralization assays, affinity identification, and ELISA. The 1B1 and 4C2 genes were sequenced and analyzed. Finally, the detection kit with the two mAbs was assessed for linearity, repeatability and specificity. Results Both mAbs specifically recognized HPV18 L1 and virus-like particles (VLPs). These mAbs are conformation-neutralizing antibodies that have high affinity and type specificity. Based on these characteristics of these mAbs, we developed an ELISA kit for specifically detecting HPV 18 antigen. We showed that this kit displayed good linearity, repeatability and sensitivity for detecting HPV18 L1 pentamer and HPV18 VLP. Conclusions We characterized two monoclonal neutralizing antibodies for HPV L1 protein, and developed an ELISA kit for specifically detecting HPV 18 antigen. This newly developed kit can be used to monitor the potency of HPV vaccines throughout the entire production process as well as preliminary analysis of HPV18 infections.

To investigate the distribution of mercury (Hg) in Futian mangrove wetland, surface sediments from land to sea were collected, including Kandelia obovata, Avicennia marina, Sonneratia caseolaris, and mud flat. The ecological risks of Hg in sediments were also assessed. The results showed that mangrove forests acidified sediments and promoted the accumulation of salinity and organic matter in sediments. Hg concentrations in both mangrove forests (154.7–218.4 ng g−1) and mud flat sediments (226.3–251.9 ng g−1) surpassed the background level (71.0 ng g−1). Furthermore, Hg concentrations in sediments decreased gradually from sea to land at all depth. From the bottom to the top layer sediment, Hg concentration decreased gradually in the sediments near land, while it kept vertically stable in the coastal area, indicating its pollution may mainly come from the coastal area rather than the land to some extent. Although the mean values of geo-accumulation indexes revealed uncontaminated to moderately contaminated levels, the mean values of potential ecological risk coefficients revealed considerable ecological risk of Hg to the environment, deserving further attention.

Highlight A new vegetation index insensitive to adaxial and abaxial leaf surfaces was proposed in this paper to accurately estimate leaf chlorophyll content., Quick non-destructive assessment of leaf chlorophyll content (LCC) is important for studying phenotypes related to plant growth and stress resistance. This study was undertaken to investigate the quantitative relationship between LCC and different vegetation indices (VIs) on both adaxial and abaxial surfaces of white poplar (Populus alba), which has dense tubular hairs on its abaxial surface, and Chinese elm (Ulmus pumila var. pendula), which does not show obvious superficial differences except for lighter colour on the abaxial surface. Some published and newly developed VIs were tested to relate them to LCC. The results showed that most of the published VIs had strong relationships with LCC on the one-surface dataset, but did not show a clear relationship with LCC when both adaxial and abaxial surface reflectance data were included. Among the reflectance indices tested, the modified Datt index, (R 719−R 726)/(R 719−R 743), performed best and is proposed as a new index for remote estimation of chlorophyll content in plants with varying leaf surface structures. It explained 92% of LCC variation in this research, and the root mean square error of the LCC prediction was 5.23 μg/cm2. This new index is insensitive to the effects of adaxial and abaxial leaf surface structures and is strongly related to the variation in reflectance caused by chlorophyll content.

Autophagy is implicated in hematopoiesis, but its role in the regulation of the hematopoietic stem and progenitor cell (HSPC) cycle remains obscure. Here, we show that autophagy is essential to maintain and regulate the cell cycle of HSPCs in a nutrient-dependent manner. The loss of autophagy via conditional deletion of its essential gene atg7 ablated the cell cycle of HSPCs. Under physiologic or nutrient-rich conditions, the in vivo activation of autophagy promoted the cell cycle entry of hematopoietic stem cells and upregulated cyclin D3 expression in HSPCs. In contrast, under poor nutrient conditions, the ex vivo inhibition of early, but not late, autophagy signaling events enhanced the G1/S transition of HSPCs. Cyclin D3 was downregulated in HSPCs in response to nutrient stress. A knockdown of cyclin D3 blocked the G1/S transition in HSPCs. Surprisingly, the ex vivo inhibition of starvation-induced early, but not late, autophagy signaling inhibited the ubiquitin-mediated degradation of cyclin D3 in HSPCs. Furthermore, a conditional autophagy defect also had this effect, leading to an elevated cyclin D3 level in these cells. This suggested a dependency of the proteasomal degradation of cyclin D3 on early, but not late, events in autophagy signaling under nutrient stress in HSPCs. Our results thus indicate a dual role of autophagy in the nutrient-dependent modulation of the cell cycle entry of hematopoietic stem cells and G1/S transition of HSPCs via the regulation of cyclin D3 to maintain a proper cell cycle in HSPCs and normal hematopoiesis during adult life.

Polo-like kinase 1 (PLK1) is highly expressed in many cancers and therefore a biomarker of transformation and potential target for the development of cancer-specific small molecule drugs. RO3280 was recently identified as a novel PLK1 inhibitor, however its therapeutic effects in leukemia treatment are still unknown. We found that the PLK1 protein was highly expressed in leukemia cell lines as well as 73.3% (11/15) of pediatric acute myeloid leukemia (AML) samples. PLK1 mRNA expression was significantly higher in AML samples compared with control samples (82.95 ± 110.28 vs. 6.36 ± 6.35, p &lt, 0.001). Kaplan-Meier survival analysis revealed that shorter survival time correlated with high tumor PLK1 expression (p = 0.002). The 50% inhibitory concentration (IC50) of RO3280 for acute leukemia cells was between 74 and 797 nM. The IC50 of RO3280 in primary acute lymphocytic leukemia (ALL) and AML cells was between 35.49 and 110.76 nM and 52.80 and 147.50 nM, respectively. RO3280 induced apoptosis and cell cycle disorder in leukemia cells. RO3280 treatment regulated several apoptosis-associated genes. The regulation of DCC, CDKN1A, BTK, and SOCS2 was verified by western blot. These results provide insights into the potential use of RO3280 for AML therapy, however, the underlying mechanisms remain to be determined.

B-cell acute lymphoblastic leukemia is the most common type of pediatric leukemia. Despite improved remission rates, current treatment regimens for pediatric B-cell acute lymphoblastic leukemia are often associated with adverse effects and central nervous system relapse, necessitating more effective and safer agents. Bafilomycin A1 is an inhibitor of vacuolar H(+)-ATPase that is frequently used at high concentration to block late-phase autophagy. Here, we show that bafilomycin A1 at a low concentration (1 nM) effectively and specifically inhibited and killed pediatric B-cell acute lymphoblastic leukemia cells. It targeted both early and late stages of the autophagy pathway by activating mammalian target of rapamycin signaling and by disassociating the Beclin 1-Vps34 complex, as well as by inhibiting the formation of autolysosomes, all of which attenuated functional autophagy. Bafilomycin A1 also targeted mitochondria and induced caspase-independent apoptosis by inducing the translocation of apoptosis-inducing factor from mitochondria to the nucleus. Moreover, bafilomycin A1 induced the binding of Beclin 1 to Bcl-2, which further inhibited autophagy and promoted apoptotic cell death. In primary cells from pediatric patients with B-cell acute lymphoblastic leukemia and a xenograft model, bafilomycin A1 specifically targeted leukemia cells while sparing normal cells. An in vivo mouse toxicity assay confirmed that bafilomycin A1 is safe. Our data thus suggest that bafilomycin A1 is a promising candidate drug for the treatment of pediatric B-cell acute lymphoblastic leukemia.

Rapamycin has been shown to inhibit the growth of leukemic cells via an unknown mechanism. In our current study, we show that rapamycin activates autophagy in pediatric t(1;19) pre-B acute lymphoblastic leukemia (pre-B ALL) cells and thereby inhibits proliferation and induces growth arrest in these cells. Rapamycin was found to downregulate an extensive array of positive cell cycle regulators, reduce the total DNA and RNA levels, and specifically downregulate the gene transcription of DNA pol δ1 and RNA pol II. Furthermore, we show that both rapamycin and starvation caused a downregulation of the DNA pol δ1 and RNA pol II proteins which was reversed by the autophagy inhibitor 3-MA. Consistent with the results of our autophagic flux analysis, confocal microscopy indicated that both rapamycin and starvation cause the colocalization of DNA pol δ1 and RNA pol II with GFP-LC3 at autophagosomes. This colocalization was blocked by the autophagy inhibitor bafilomycin A1 which inhibits the fusion between autophagosomes and lysosomes. These data suggest that rapamycin inhibits the growth of pediatric t(1;19) pre-B ALL cells through both transcriptional inhibition and autophagic degradation of DNA pol δ1 and RNA pol II.

Histone modification enzymes regulate gene expression by altering the accessibility of promoters to transcription factors. We sought to determine whether the genes encoding histone modification enzymes are dysregulated in pediatric acute lymphoblastic leukemia (ALL). A real-time PCR array was designed, tested and used to profile the expression of 85 genes encoding histone modification enzymes in bone marrow mononuclear cells from 30 pediatric ALL patients and 20 normal controls. The expression profile of histone-modifying genes was significantly different between normal karyotype B cell pediatric ALL and normal controls. Eleven genes were upregulated in pediatric ALL, including the histone deacetylases HDAC2 and PAK1, and seven genes were downregulated, including PRMT2 and the putative tumor suppressor EP300. Future studies will seek to determine whether these genes serve as biomarkers of pediatric ALL. Ingenuity Pathway Analysis revealed that Gene Expression and Organ Morphology was the highest rated network, with 13 focus molecules (significance score = 35). Ingenuity Pathway Analysis also indicated that curcumin and miR-34 are upstream regulators of histone-modifying enzymes; future studies will seek to validate these results and examine the role of curcumin and miR-34 in leukemia. This study provides new clues into the molecular mechanisms of pediatric ALL.

Background Acute myeloid leukemia (AML) is the second-most common form of leukemia in children. Aberrant DNA methylation patterns are a characteristic feature of AML. GATA4 has been suggested to be a tumor suppressor gene regulated by promoter hypermethylation in various types of human cancers although the expression and promoter methylation of GATA4 in pediatric AML is still unclear. Methods Transcriptional expression levels of GATA4 were evaluated by semi-quantitative and real-time PCR. Methylation status was investigated by methylation-specific PCR (MSP) and bisulfate genomic sequencing (BGS). The prognostic significance of GATA4 expression and promoter methylation was assessed in 105 cases of Chinese pediatric acute myeloid leukemia patients with clinical follow-up records. Results MSP and BGS analysis showed that the GATA4 gene promoter is hypermethylated in AML cells, such as the HL-60 and MV4-11 human myeloid leukemia cell lines. 5-Aza treatment significantly upregulated GATA4 expression in HL-60 and MV4-11 cells. Aberrant methylation of GATA4 was observed in 15.0 % (3/20) of the normal bone marrow control samples compared to 56.2 % (59/105) of the pediatric AML samples. GATA4 transcript levels were significantly decreased in AML patients (33.06 ± 70.94; P = 0.011) compared to normal bone marrow/idiopathic thrombocytopenic purpura controls (116.76 ± 105.39). GATA4 promoter methylation was correlated with patient leukocyte counts (WBC, white blood cells) (P = 0.035) and minimal residual disease MRD (P = 0.031). Kaplan-Meier survival analysis revealed significantly shorter overall survival time in patients with GATA4 promoter methylation (P = 0.014). Conclusions Epigenetic inactivation of GATA4 by promoter hypermethylation was observed in both AML cell lines and pediatric AML samples; our study implicates GATA4 as a putative tumor suppressor gene in pediatric AML. In addition, our findings imply that GATA4 promoter methylation is correlated with WBC and MRD. Kaplan-Meier survival analysis revealed significantly shorter overall survival in pediatric AML with GATA4 promoter methylation but multivariate analysis shows that it is not an independent factor. However, further research focusing on the mechanism of GATA4 in pediatric leukemia is required. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1760-5) contains supplementary material, which is available to authorized users.

The hepatic fibrogenesis and sexual dimorphism of hepatitis B virus-related liver cirrhosis (HBV-LC) are related to estrogen and its receptors. Abnormal expression of estrogen receptor α (ESR1) is implicated in the development of cirrhosis in both animal models and humans. Here, we examine whether the ESR1 polymorphisms are related to HBV-LC risk among chronic HBV carriers, and we investigate the functional significance of positively associated polymorphisms. A total of 2,404 unrelated Chinese HBV carriers were recruited to conduct the two-stage designed case-control study. Two ESR1 haplotype tagging polymorphisms, c.30T>C (rs2077647) and c.453-397T>C (rs2234693), were genotyped in 1,285 patients with HBV-LC and in 1,119 asymptomatic HBV carriers. We observed a significantly increased susceptibility to HBV-LC associated with the c.30C allele (P = 4.2 × 10(-8) ), c.453-397C allele (P = 2.0 × 10(-8) ), and [c.30C; c.453-397C] haplotype (Dominant model, P = 8.85 × 10(-10) , odds ratio = 1.50, 95% CI 1.32∼1.71) compared with the T alleles and (c.30T; c.453-397T) haplotype of c.30T>C and c.453-397T>C polymorphisms, respectively. Functional analyses were conducted to verify the biological functions of the associated genetic variations and showed that the c.453-397T>C polymorphism is a novel c.453-397C allele-specific and c-myb-dependent enhancer-like cis-acting regulatory variation and could be part of the genetic variations underlying the susceptibility of individuals to HBV-LC.

The T-box transcription factor T-bet is a key regulator for the lineage commitment in CD4 Th1 cells and CD8 T cells by activating the hallmark production of interferon-γ, and its expression level is linked to autoimmune diseases. T-1993C and T-1514C polymorphisms in the TBX21 gene (encoding T-bet) promoter can affect transcription activity. We investigated the distributions of these functional polymorphisms in 84 adult patients with type 1 autoimmune hepatitis (AIH-1) and 318 healthy controls. Intracellular T-bet staining of polarized CD4 Th1 cells from healthy controls corresponding to T-1993C genotypes were analyzed by flow cytometry. The -1993C allele frequency was 3.0% in AIH-1 and 11.8% in controls (p = 0.000 25). Individuals carrying the -1993C allele had a decreased risk to AIH-1 compared with those without the -1993C allele (p = 0.0016, odds ratio [OR] = 0.22, 95% confidence interval = 0.09-0.56). No association was found between the T-1514C polymorphism and AIH-1. The onset age of AIH-1 was also accelerated among -1993TT homozygotic individuals (p = 0.013). The fractions of T-bet positive Th1 cells in the -1993TT homozygotes were 2.2-fold higher than those in -1993CC homozygotes (p = 0.002). Our results suggest that the T-1993C polymorphism in the TBX21 promoter influences susceptibility to AIH-1 in a Chinese population.

Objective To investigate the proliferation capabilities, immunophenotype changes, level of secreted cytokines and activities against lymphoma cells under the condition that cytokine-induced killer (CIK) cells co-cultured with dendritic cells (DC) in vitro.

MHC type II (MHC II) expression is tightly regulated in macrophages and potently induced by IFN-γ (type II IFN). In contrast, type I IFNs (IFN-Is), which are far more widely expressed, fail to induce MHC II expression, even though both classes of IFNs direct target gene expression through Stat1. The unexpected finding that IFN-Is effectively induce MHC II expression in Stat2−/− macrophages provided an opportunity to explore this conundrum. The ensuing studies revealed that deletion of Stat2, which uniquely transduces signals for IFN-Is, leads to a loss in the IFN-I-dependent induction of suppressor of cytokine signaling-1. Impairment in the expression of this important negative regulator led to a striking prolongation in IFN-I-dependent Stat1 activation, as well as enhanced expression of the target gene, IFN-regulatory factor-1. The prolonged activity of these two transcription factors synergized to drive the transcription of CIITA, the master regulator of MHC II expression, analogous to the pattern observed in IFN-γ-treated macrophages. Thus, IFN-I-dependent suppressor of cytokine signaling-1 expression plays an important role in distinguishing the biological response between type I and II IFNs in macrophages.

Long non-coding RNA (lncRNA) plays a role in gene transcription, protein expression and epigenetic regulation; and altered expression results in cancer development. Acute myeloid leukemia (AML) is rare in children; and thus, this study profiled lncRNA expression in bone marrow samples from pediatric AML patients. Arraystar Human LncRNA Array V3.0 was used to profile differentially expressed lncRNAs in three bone marrow samples obtained from each pediatric AML patient and normal controls. Quantitative polymerase chain reaction (qRT-PCR) was performed to confirm dysregulated lncRNA expressions in 22 AML bone marrow samples. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to construct the lncRNA-mRNA co-expression network. A total of 372 dysregulated lncRNAs (difference ≥10-fold) were found in pediatric AML patients compared to normal controls. Fifty-one mRNA levels were significantly upregulated, while 85 mRNA levels were significantly downregulated by >10-fold in pediatric AML, compared to normal controls. GO terms and KEGG pathway annotation data revealed that cell cycle pathway-related genes were significantly associated with pediatric AML. As confirmed by qRT-PCR, expression of 24 of 97 lncRNA was altered in pediatric AML compared to normal controls. In pediatric AML, ENST00000435695 was the most upregulated lncRNA, while ENST00000415964 was the most downregulated lncRNA. Data from this study revealed dysregulated lncRNAs and mRNAs in pediatric AML versus normal controls that could form gene pathways to regulate cell cycle progression and immunoresponse. Further studies are required to determine whether these lncRNAs could serve as novel therapeutic targets and bbdiagnostic biomarkers in pediatric AML.

ObjectiveTo evaluate the recanalization efficacy and safety of a novel stent retriever in a swine model of acute arterial occlusion.MethodsThe JRecan thrombectomy device, a stent retriever with a weaving stent design, was evaluated in 18 occluded cervicocerebral vessels of swine. The flow restoration effect immediately upon deployment, the reperfusion rate after retrieval, thromboembolic events, and complications were assessed. The histologic structure of the renal arteries after retriever passage was measured to further assess the safety of JRecan.ResultsImmediate flow restoration was achieved in 66.7% of occlusions (12/18). The reperfusion rate was 94.4% (two Thrombolysis in Cerebral Infarction (TICI) 2b and 15 TICI 3). Distal thromboembolic events did not occur. Microscopic examination of the arteries after retrieval showed mild degrees of endothelial loss in 96.6% (29/30), fibrin or platelet deposition in 53.3% (16/30), and disruption of the internal elastic lamina in 10% (3/30), without severe pathologic lesions.ConclusionsThe JRecan is highly effective at clot removal with a favorable safety profile and merits further development as a stent retriever for the treatment of acute ischemic stroke.

In recent years, adenoviruses that selectively replicate in tumor cells have been developed. However, there is a tremendous need to improve their anticancer efficacy. We wish to investigate whether a strategy that combines the oncolytic effects of an adenoviral vector with simultaneous expression of soluble form of transforming growth factor-β type II receptor (sTGFβRII) offers a therapeutic advantage. We chose to target TGF-βs because they play a pivotal role in late-stage tumorigenesis by enhancing tumor invasion and metastasis. A sTGFβRII cDNA was cloned in conditionally replicating adenoviral vector rAd-sTRII and in a replication-deficient adenovirus Ad-sTRII. Infection of MDA-MB-231 breast cancer cells with rAd-sTRII or Ad-sTRII followed by Western blot analysis indicated the expression of diffused glycosylated forms of sTGFβRII that were also secreted into the extracellular medium. The secreted proteins were shown to bind with TGF-β and antagonize TGF-β–induced p38 mitogen-activated protein kinase activity. However, marked differences in the replication potential of rAd-sTRII and Ad-sTRII were observed in breast tumor cells. Infection of MDA-MB-231 cells with rAd-sTRII resulted in cytotoxicity and significant increase in the adenoviral titers that were comparable with a wild-type adenovirus dl309. However, Ad-sTRII was much less toxic to the tumor cells, and the viral titers of Ad-sTRII remained relatively unchanged. These results suggest that the infection of breast tumor cells with conditionally replicating adenoviral vector rAd-sTRII produced sTGFβRII that can abrogate TGF-β signaling while maintaining the replication potential of the virus, indicating that rAd-sTRII could be a potential anticancer agent. [Mol Cancer Ther 2006;5(2):367–73]

As most solid tumors are hypoxic, dendritic cells (DC) in solid tumors are also exposed to hypoxia. While many adaptation responses of tumor cells to hypoxia are known, it is yet to be determined how hypoxia affects the functions of DC. To explore the effects of hypoxia on the functions of DC, we compared the expression of surface markers, cytokines, chemokine receptors and matrix metalloproteinases (MMP) of human monocyte-derived DC (hmDC) differentiated under hypoxia to those differentiated under normoxia. Both groups of hmDC expressed similar levels of surface markers and cytokines. However, expression of MMP-9 and membrane type-1-MMP, as well as migrating activity, was significantly suppressed in hmDC differentiated under hypoxia compared with their normoxia counterparts. We also demonstrated that trichostatin A restored the production of MMP-9 in hmDC, under hypoxia. Collectively, our findings show that a hypoxic microenvironment suppresses the production of MMP in hmDC, most probably through the deacetylation of promoter regions of MMP, thus suppressing the migrating activity of hmDC. Our results suggest that the hypoxic microenvironment in solid tumor tissues may suppress the function of DC.

We have recently demonstrated that granulocyte colony-stimulating factor (G-CSF) stimulated the production of epithelial-cell-derived-neutrophil attractant-78 (ENA-78) by neutrophils and that ENA-78 might promote the accumulation of neutrophils that had migrated from the intravascular space into inflammatory tissues. In this study, we examined whether other chemokines could be secreted by neutrophils that had accumulated after migrating from the intravascular space into the inflammatory tissues. We demonstrated that adhesion to laminin contained in the basement membrane and Matrigel, which is an artificial basement membrane model, induced macrophage inflammatory protein-1 beta (MIP-1 beta) in neutrophils and that MIP-1 beta secreted by neutrophils induced the chemotaxis of dendritic cells. These findings suggest that neutrophils transmigrating through the basement membrane are stimulated to secrete MIP-1 beta by the basement membrane, inducing the transmigration of dendritic cells from the intravascular space into inflammatory tissues. We propose that neutrophils intervene between innate immune response and specific immune response by secreting MIP-1 beta during the transmigration through the basement membrane.

During hematopoiesis, megakaryopoiesis, megakaryocyte differentiation, and thrombopoiesis are regulated at multiple stages, which involve successive lineage commitment steps and proceed with polyploidization, maturation, and organized fragmentation of the cytoplasm, leading to the release of platelets in circulation. However, the cellular mechanisms by which megakaryocytes derive from their progenitors and differentiate into platelets have not fully been understood. Using an Atg7 hematopoietic conditional knockout mouse model, we found that loss of autophagy, a metabolic process essential in homeostasis and cellular remodeling, caused mitochondrial and cell cycle dysfunction, impeding megakaryopoiesis and megakaryocyte differentiation, as well as thrombopoiesis and subsequently produced abnormal platelets, larger in size and fewer in number, ultimately leading to severely impaired platelet production and failed hemostasis.

Acute myeloid leukemia (AML) is the second-most common form of leukemia in children. Aberrant DNA methylation patterns are characteristic of AML. Zinc finger protein 382 (ZNF382) has been suggested to be a tumor suppressor gene possibly regulated by promoter hypermethylation in various types of human cancer. However, ZNF382 expression and methylation status in pediatric AML is unknown. In the present study, ZNF382 transcription levels were evaluated by quantitative reverse-transcription PCR. Methylation status was investigated by methylation-specific (MSP) PCR and bisulfate genomic sequencing (BGS). The prognostic significance of ZNF382 expression and promoter methylation was assessed in 105 cases of pediatric AML. The array data suggested that the ZNF382 promoter was hypermethylated in the AML cases examined. MSP PCR and BGS analysis revealed that ZNF382 was hypermethylated in leukemia cell lines. Furthermore, treatment with 5-aza-2'-deoxycytidine (5-Aza) upregulated ZNF382 expression in the selected leukemia cell lines. The aberrant methylation of ZNF382 was observed in 10% (2/20) of the control samples compared with 26.7% (28/105) of the AML samples. ZNF382 expression was significantly decreased in the 105 AML patients compared with the controls. Patients with ZNF382 methylation showed lower ZNF382 transcript levels compared with patients exhibiting no methylation. There were no significant differences in clinical characteristics or cytogenetic analysis between the patients with or without ZNF382 methylation. ZNF382 methylation correlated with minimal residual disease (MRD). Kaplan-Meier survival analysis revealed similar survival times in the samples with ZNF382 methylation, and multivariate analysis revealed that ZNF382 methylation was not an independent prognostic factor in pediatric AML. The epigenetic inactivation of ZNF382 by promoter hypermethylation can be observed in AML cell lines and pediatric AML samples. Therefore, our study suggests that ZNF382 may be considered a putative tumor suppressor gene in pediatric AML. However, further studies focusing on the mechanisms responsible for ZNF382 downregulation in pediatric leukemia are required.

Background: Acute myeloid leukemia (AML) is the second most common form of leukemia in children. Aberrant DNA methylation patterns are a characteristic feature in various tumors, including AML. Metallothionein III (MT3) is a tumor suppresser reported to show promoter hypermethylated in various cancers. However, the expression and molecular function of MT3 in pediatric AML is unclear. Methods: Eleven human leukemia cell lines and 41 pediatric AML samples and 20 NBM/ITP (Norma bone marrow/ Idiopathic thrombocytopenic purpura) control samples were analyzed. Transcription levels of MT3 were evaluated by semi-quantitative and real-time PCR. MT3 methylation status was determined by methylation specific PCR (MSP) and bisulfite genomic sequencing (BSG). The molecular mechanism of MT3 was investigated by apoptosis assays and PCR array analysis. Results: The MT3 promoter was hypermethylated in leukemia cell lines. More CpG’s methylated of MT3 was observed 39.0% pediatric AML samples compared to 10.0% NBM controls. Transcription of MT3 was also significantly decreased in AML samples compared to NBM/ITP controls (P< 0.001); patients with methylated MT3 exhibited lower levels of MT3 expression compared to those with unmethylated MT3 (P = 0.049). After transfection with MT3 lentivirus, proliferation was significantly inhibited in AML cells in a dose-dependent manner (P< 0.05). Annexin V assay showed that apoptosis was significantly upregulated MT3-overexpressing AML cells compared to controls. Real-time PCR array analysis revealed 34 dysregulated genes that may be implicated in MT3 overexpression and apoptosis in AML, including FOXO1. Conclusion: MT3 may be a putative tumor suppressor gene in pediatric AML. Epigenetic inactivation of MT3 via promoter hypermethylation was observed in both AML cell lines and pediatric AML samples. Overexpression of MT3 may inhibit proliferation and induce apoptosis in AML cells. FOXO1 was dysregulated in MT3-overexpressing cells, offering an insight into the mechanism of MT3-induced apoptosis. However, further research is required to determine the underlying molecular details.

Background The sexual dimorphism of hepatitis B virus (HBV) -related liver diseases is related with estrogen and its receptors. Recent reports indicate that abnormal expression of estrogen receptor alpha (ESR1) may be a hallmark for the progression of liver disease and HBV carriers presenting variant ESR1 have an extremely aggressive clinical course. Here we examine whether the ESR1 polymorphisms or its haplotypes are related to HBV-related acute liver failure (ALF) risk among chronic HBV carriers in a Chinese population. Methods A total of 1216 unrelated Han Chinese HBV carriers were recruited in this hospital-based case–control study, including 359 HBV surface antigen (HBsAg) carriers affected with ALF and 857 asymptomatic HBsAg carriers. Two ESR1 haplotype tagging polymorphisms, c.30 T > C (rs2077647) and c.453-397 T > C (rs2234693), were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. Results We observed a significantly increased susceptibility to HBV-ALF associated with the c.30 C allele (P = 8.65 × 10-4), c.453-397 C allele (5.37 × 10-4) and [c.30 C; c.453-397 C] haplotype (Dominant model, P =0.0004, odds ratio = 1.53, 95% CI 1.23 ~ 1.96) compared with the T alleles and [c.30 T; c.453-397 T] haplotype of c.30 T > C and c.453-397 T > C polymorphisms, respectively. Conclusions Our study suggests that [c.30 C; c.453-397 C] hapotype may be a risk factor for genetic susceptibility to HBV-related ALF in the Chinese population. It also emphasizes the importance of ESR1 in the pathophysiology of HBV-related ALF on the population level.

In this report, a Ti:Sapphire oscillator was utilized to realize synchronization-free stimulated emission depletion (STED) microscopy. With pump power of 4.6 W and sample irradiance of 310 mW, we achieved super-resolution as high as 71 nm. With synchronization-free STED, we imaged 200 nm nanospheres as well as all three cytoskeletal elements (microtubules, intermediate filaments, and actin filaments), clearly demonstrating the resolving power of synchronization-free STED over conventional diffraction limited imaging. It also allowed us to discover that, Dylight 650, exhibits improved performance over ATTO647N, a fluorophore frequently used in STED. Furthermore, we applied synchronization-free STED to image fluorescently-labeled intracellular viral RNA granules, which otherwise cannot be differentiated by confocal microscopy. Thanks to the widely available Ti:Sapphire oscillators in multiphoton imaging system, this work suggests easier access to setup super-resolution microscope via the synchronization-free STED. © 2012 Liu et al.

Cell transplantation has been well explored for cartilage regeneration. We recently showed that the entire articular surface of a synovial joint can regenerate by endogenous cell homing and without cell transplantation. However, the sources of endogenous cells that regenerate articular cartilage remain elusive. Here, we studied whether cytokines not only chemotactically recruit adipose stem cells (ASCs), mesenchymal stem cells (MSCs), and synovium stem cells (SSCs) but also induce chondrogenesis of the recruited cells. Recombinant human transforming growth factor-β3 (TGF-β3; 100 ng) and/or recombinant human stromal derived factor-1β (SDF-1β; 100 ng) was control released into an acellular collagen sponge cube with underlying ASCs, MSCs, or SSCs in monolayer culture. Although all cell types randomly migrated into the acellular collagen sponge cube, TGF-β3 and/or SDF-1β recruited significantly more cells than the cytokine-free control group. In 6 wk, TGF-β3 alone recruited substantial numbers of ASCs (558±65) and MSCs (302±52), whereas codelivery of TGF-β3 and SDF-1β was particularly chemotactic to SSCs (400±120). Proliferation of the recruited cells accounted for some, but far from all, of the observed cellularity. TGF-β3 and SDF-1β codelivery induced significantly higher aggrecan gene expression than the cytokine-free group for ASCs, MSCs, and SSCs. Type II collagen gene expression was also significantly higher for ASCs and SSCs by SDF-1 and TGF-β3 codelivery. Remarkably, the expression of aggrecan and type II collagen was detected among all cell types. Thus, homing of multiple stem/progenitor cell populations may potentially serve as an alternative or adjunctive approach to cell transplantation for cartilage regeneration.—Mendelson, A., Frank, E., Allred, C., Jones, E., Chen, M., Zhao, W., Mao, J. J. Chondrogenesis by chemotactic homing of synovium, bone marrow, and adipose stem cells in vitro.

Aim: Transcription factor T-bet is responsible for the differentiation of naive T lymphocytes, and its expression level is linked with different responses to some viral infections, including hepatitis B virus (HBV) infection. In this report we examine whether promoter polymorphisms of the TBX21 gene (encoding T-bet) are associated with susceptibility to HBV persistence or disease progression in chronic HBV carriers. Methods: Three previously reported promoter polymorphisms, T-1993C, T-1514C and G-1499A, were analyzed by polymerase chain reaction restriction fragment length polymorphism analysis. Two common polymorphisms, T-1993C and T-1514C, were selected for genotyping in 1074 chronic HBV carriers, 310 spontaneously recovered controls and 374 HBV naive controls. Of 1074 HBV carriers, 234 were considered to be asymptomatic carriers and 840 were found to have chronic progressive liver disease including cirrhosis and hepatocellular carcinoma. Haplotypes were constructed for each subject and associations with the susceptibility to persistent HBV infection were estimated by logistic regression. Results: The –1993C allele in the TBX21 promoter was significantly more common among chronic HBV carriers compared with recovered controls (χ2 = 6.65, P = 0.01). In contrast, the frequency of TT haplotype at positions –1993/–1514, was significantly higher in recovered controls than chronic HBV carriers (P = 0.0027, odds ratio = 1.57; 95% confidence interval, 1.16–2.12). In HBV carriers, the TBX21 promoter polymorphisms were not linked to disease progression. Conclusion: The TBX21 promoter polymorphisms do not appear to be determinant of disease progression in Chinese HBV carriers. The T-1993C polymorphism in the TBX21 promoter influences susceptibility to persistent HBV infection.

Summary. Recent reports indicated that high levels of interleukin 10 (IL-10) contribute to the monocytes paralysis and poor clinical outcome in acute liver failure (ALF). Polymorphisms in the promoter region of IL-10 affect IL-10 production and confer susceptibility to inflammatory diseases. The aim of this study was to determine the possible association of the three polymorphisms (A-1082G, T-819C, A-592C) in the IL-10 gene promoter with the susceptibility to hepatitis B virus (HBV)-related ALF in a Chinese population. The IL-10 gene promoter polymorphisms were genotyped in 414 unrelated healthy blood donors, 367 asymptomatic HBV carriers and 345 HBV-related ALF patients. Functional analyses were conducted to verify the biological significances of the associated genetic variations. The allele frequencies of IL-10−592C and −819C were significantly higher in HBV-related ALF patients than in blood donors and asymptomatic HBV carriers. Logistic regression analysis and stratification analysis with adjustment for age and sex indicated that the polymorphisms of A-592C and T-819C were associated with susceptibility to HBV-related ALF (P = 6.9 × 10−7), and the -1082A-819C-592C haplotype in the IL-10 gene promoter were associated with an increased susceptibility to ALF in HBV carriers (dominant model, P = 0.0002, odds ratio = 1.60, 95% CI 1.25–2.07). Functional analyses showed that the A-592C polymorphism is a nuclear proteins binding site, and the disease susceptible −592C allele had a higher transcription activity compared with −592A allele. This study emphasizes the importance of IL-10 in the pathophysiology of HBV-related ALF on the population level.

Mammalian type I IFNs (IFN-Is) mediate their potent biological activities through an evolutionarily conserved IFN-α receptor (IFNAR), consisting of IFNAR1 and IFNAR2. These two chains direct the rapid activation of two founding members of the STAT family of transcription factors, STAT1 and STAT2. To understand how IFN-Is direct the recruitment and activation of STATs, a series of mutant murine IFNAR1 and IFNAR2 receptors were generated and evaluated in IFNAR1 and IFNAR2 knockout cells. These studies reveal that a single conserved IFNAR2 tyrosine, Y510, plays a critical role in directing the IFN-I-dependent activation of STAT1 and STAT2, both in murine fibroblasts and macrophages. A second IFNAR2 tyrosine, Y335, plays a more minor role. Likewise, Y510 > Y335 play a critical role in the induction of genes and antiviral activity traditionally associated with IFN-Is.

B-cell acute lymphoblastic leukemia (B-ALL) accounts for the most cancer incidences in children. We present here that autophagy is downregulated in pediatric B-ALL, suggesting a possible link between autophagy failure and pediatric B-ALL leukemogenesis. With a pediatric t(1;19) B-ALL xenograft mouse model, we show here that activation of autophagy by preventive administration of rapamycin improved the survival of leukemia animals by partial restoration of hematopoietic stem/progenitor cells, whereas treatment of the animals with rapamycin caused leukemia bone marrow cell-cycle arrest. Activation of autophagy in vitro or in vivo by rapamycin or starvation downregulated oncogenic fusion protein E2A/Pbx1. Furthermore, E2A/Pbx1 was found to be colocalized with autophagy marker LC3 in autolysosomes and with ubiquitin in response to autophagy stimuli, whereas autophagy or ubiquitination inhibitor blocked these colocalizations. Together, our data suggest a collaborative action between autophagy and ubiquitination in the degradation of E2A/Pbx1, thereby revealing a novel strategy for targeted preventive or treatment therapy on the pediatric ALL.

Background: B cell acute lymphoblastic leukemia (B-ALL) accounts for the most cancer incidences in children. The t(1;19) translocation leukemia accounts for a quarter of pre-B ALL and up to 5% of all ALL patients, in which the transcriptional activator E2A and homeobox pre-B-cell leukemia transcription factor 1 (PBX1) fuses, resulting in expression of the chimeric transcription factor E2A/PBX1. E2A/PBX1 has been proved to be an oncogene and could induce malignant transformation. Methods: (1) Childhood B-ALL patients were collected and the stem/progenitor cells (CD34+CD38-) and leukemia cells (CD19+) were sorted with BD FACS Aria III. The autophagy level in these cells was measured by real-time Q-PCR, including gene expression of Beclin1, Atg7, Atg5, LC3 and p62. Normal bone marrow cells from healthy donors were used as control. (2) E2A/PBX1 fusion gene positive pre-B ALL 697 cells were used to establish leukemia mouse model and the autophagy activity in the mice was enhanced by administration of rapamycin. Mice were sacrificed three weeks post treatment, leukemia phenotype was then identified and E2A/PBX1 oncoprotein of liver was detected by western blotting. (3) Autophagy and ubiquitination were manipulated with inhibitors or starvation in 697 cells and the degradation mechanism of E2A/PBX1 was explored. Co-localization of E2A/PBX1-LC3 and E2A/PBX1-Ub was observed by confocal microscopy and quantified by Amnis image flow cytometry. Results: (1) B-ALL primary cells from childhood patients show down-regulated level of autophagy. (2) The NOD-SCID mouse model study shows that activating autophagy of mice by rapamycin improved the survival of leukemia animals, prevented leukemiagenesis by inhibition on the transplanted leukemia cells (examined by blood cell counting, liver HE staining and expression of CD 45, 10, 19 from transplanted human 697 cells by flow cytometry), promoted the degradation of oncoprotein E2A/PBX1 (by Western blotting)), and more importantly, restored hematopoietic stem cells (LSKCD34- cell number detected by flow cytometry). (3) The ALL 697 cell line study shows that activation of autophagy by rapamycin and starvation could down-regulate E2A/PBX1 expression detected by flow cytometry and western blotting. The confocal microscopic results show co-localization of E2A/PBX1 with autophagy marker GFP-LC3 in both rapamycin and starvation treatment groups. To confirm the degradation mechanism, autophagy inhibitor (3-MA or Baf-A1) and ubiquitin-proteasome inhibitor (MG132) were used to treat 697 cells. The results show that inhibition of autophagy in the early stage by 3-MA fails to degrade E2A/PBX1 in 697 cells, but ubiquitination also contributes to the degradation of E2A/PBX1. Quantitative analysis shows increased co-localization percentage of E2A/PBX1-LC3 in rapamycin and starvation treatment groups and increased co-localization of E2A/PBX1 with Ubiquitin in starvation group; but MG132 treatment inhibited the co-localization of E2A/PBX1-Ub induced by starvation, indicating a collaborative role between autophagy and ubiquitination in the degradation of E2A/PBX1. Conclusions: B-ALL primary cells from patients show low autophagy activity; Autophagy activation fights against B-ALL by inhibition on transplanted leukemia cells, degradation of oncoprotein E2A/Pbx1 and restoration of hematopoietic stem cells in the NOD-SCID B-ALL mouse model; autophagy collaborates with ubiquitination in the degradation of E2A/PBX1 in the 697 cells, thereby proposing a novel strategy for targeted therapy on childhood B-ALL. Disclosures No relevant conflicts of interest to declare.

Transforming growth factor (TGF) betas are multifunctional polypeptides that regulate several cellular functions, including cell growth and differentiation, extra cellular matrix production, motility and immunosuppression. The growth-inhibiting properties of TGFbeta have gained much attention into its role as a tumor suppressor. There is, however, now increasing evidence that TGFbeta switches roles, from tumor suppressor to tumor promoter, as the tumor progresses. Given the integral role of TGFbeta in the tumor progression, it follows that TGFbeta signaling offers an attractive target for cancer therapy. Several strategies including the use of antisense oligonucleotides for TGFbeta, TGFbeta antibodies, dominant negative TGFbeta receptor II, and small drug-molecules to inhibit TGFbeta receptor I kinase have shown great promise in the preclinical studies. These new findings, coupled with progressing clinical trials indicate that inhibition of TGFbeta signaling may, indeed, be a viable option to cancer therapy. This review summarizes the TGFbeta signaling, the dual role of TGFbeta--as a tumor suppressor and tumor promoter, and various strategies targeted against TGFbeta signaling for cancer therapy. The next few years promise to better our understanding of approaching cancer therapy with an eye to the inhibition of TGFbeta signaling.

We have previously reported that transforming growth factor beta (TGF-beta) produced by rat hepatoma cell line KDH-8 cells suppressed the interleukin-2 (IL-2) production of T cells and the tumoricidal activity of macrophages in KDH-8 tumor-bearing rats and that the inhibition of TGF-beta production by low-dose bleomycin restored these activities significantly. In this study, we established three transfectant clones with stable expression of soluble TGF-beta receptor type II (sTRII), namely KT1, KT2 and KT3, and one with an empty vector used as control vector (KV), and then investigated the effects of sTRII on the tumorigenicity of KDH-8 cells and immune responses in syngeneic Wistar King Aptekman/Hok (WKAH) rats. We found that sTRII expressed in sTRII transfectants could abolish growth inhibition of Mv1Lu cells by TGF-beta1 produced by the cells themselves, and that tumor growth of KT2 and KT3 clones in vivo was suppressed significantly compared with that of parent, KV and KT1 clones. Furthermore, we demonstrated that IL-2 production of splenocytes and IL12p40 mRNA expression in tumor tissues were restored in rats inoculated with KT2 and KT3 clones, whereas such restoration was not observed in rats inoculated with parent, KV and KT1 clones. Combined with a low expression of sTRII in KT1 tumor tissues, these results suggest that sTRII may to some extent be able to abolish the tumor-promoting activity of TGF-beta, and imply that sTRII might have a therapeutic effect on TGF-beta-producing tumors.

Leukemia has long been considered unpreventable, and how leukemogenesis is initially triggered remains largely unknown. We observed that autophagy is frequently downregulated in the hematopoietic stem cells of various types of leukemia patients. Using a conditional Becn1 knockout mouse model, we found that impaired autophagy results in rapid accumulation of reactive oxygen species (ROS) that causes DNA damages, genome instabilities and chromosomal translocations of hematopoietic stem cells, leading to their transformation to leukemia stem cells, ultimately attributed to various leukemogenenesis. Restoration of autophagy in the hematopoietic stem cells of the autophagy defective mice by constitutive expression of Becn1 could clean up ROS, inhibit DNA damages and chromosomal translocations, and block the transformation of hematopoietic stem cells to leukemia stem cells, ultimately inhibiting leukemogenesis. Furthermore, enhancement of autophagy by rapamycin could effectively remove the ROS experimentally induced in hematopoietic stem cells and eliminate the leukemia stem cells either transplanted to wild-type recipient mice or transformed from normal hematopoietic stem cells due to pharmacological inhibition of autophagy. Our data thus suggest that autophagy prevent leukemogenesis by protecting normal hematopoietic stem cells from transforming to leukemia stem cells and that leukemia prevention is possible by securing autophagy machinery in hematopoietic stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-40. doi:10.1158/1538-7445.AM2011-LB-40