Nitroprusside, a vasodilatorynitric oxide donor, is clinically used during vascular surgery and to lower blood pressure in acute hypertension. This paper reports a novel application of blood flow (BF) and blood-oxygenation-level-dependent (BOLD) MRI on a 11.7 Tesla scanner to image the rat chorioretinal BF and BOLD changes associated with graded nitroprusside infusion. At low doses (1 or 2 μg/kg/min), nitroprusside increased BF as expected but decreased BOLD signals, showing an intriguing BF-BOLD uncoupling. At high doses (3–5 μg/kg/min) nitroprusside decreased BF and markedly decreased BOLD signals. To our knowledge, this is the first pharmacological MRI application of the retina. This approach has potential to open up new avenues to study the drug-related hemodynamic functions and evaluate the effects of novel therapeutic interventions on BOLD and BF in the normal and diseased retinas.
Pharmacological MRI; retina; blood flow; BOLD; PO2; nitroprusside; nitric oxide
Amphioxus is a best candidate for studying the evolutionary and developmental mechanisms of vertebrates, because of its vertebrate-like but much simpler morphology, embryonic development and genome structure. Producing live amphioxus embryos throughout the year is an ideal for comparative evolution and developmental studies. However, all amphioxus species have distinct breeding seasons in the wild and laboratory. We recently found that Chinese amphioxus B. belcheri could reproduce repeatedly beyond its natural breeding season when reared under proper conditions. In this study, we were able to extend further and produce embryos throughout the year from October 2011 to October 2012. We found all examined animals had spawned repeatedly during the examined period. In addition, both lancelets B. belcheri and B. japonicum could be induced to spawn by heat-shock method, although the induced spawning efficiency was not as high as that observed in the European lancelet. In general, we have succeeded in producing B. belcheri embryos almost daily throughout the year. This advancement will provide essential embryonic material for evolutionary and developmental studies, and have great implications for the cultivation and spawning induction of other amphioxus species.
Shugoshin (SGO) is a critical factor that enforces cohesion from segregation of paired sister chromatids during mitosis and meiosis. It has been studied mainly in invertebrates. Knowledge of SGO(s) in a mammalian system has only been reported in the mouse and Hela cells. In this study, the functions of SGO1 in bovine oocytes during meiotic maturation, early embryonic development and somatic cell mitosis were investigated. The results showed that SGO1 was expressed from germinal vesicle (GV) to the metaphase II stage. SGO1 accumulated on condensed and scattered chromosomes from pre-metaphase I to metaphase II. The over-expression of SGO1 did not interfere with the process of homologous chromosome separation, although once separated they were unable to move to the opposing spindle poles. This often resulted in the formation of oocytes with 60 replicated chromosomes. Depletion of SGO1 in GV oocytes affected chromosomal separation resulting in abnormal chromosome alignment at a significantly higher proportion than in control oocytes. Knockdown of SGO1 expression significantly decreased the embryonic developmental rate and quality. To further confirm the function(s) of SGO1 during mitosis, bovine embryonic fibroblast cells were transfected with SGO1 siRNAs. SGO1 depletion induced the premature dissociation of chromosomal cohesion at the centromere and along the chromosome arm giving rise to abnormal appearing mitotic patterns. The results of this study infer that SGO1 is involved in the centromeric cohesion of sister chromatids and chromosomal movement towards the spindle poles. Depletion of SGO1 causes arrestment of cell division in meiosis and mitosis.
Calcitonin gene-related peptide (CGRP) promotes osteoblast recruitment and osteogenic activity. However, no evidence suggests that CGRP could affect the differentiation of stem cells toward osteoblasts. In this study, we genetically modified adipose-derived stem cells (ADSCs) by introducing the CGRP gene through adenoviral vector transduction and investigated on cellular proliferation and osteoblast differentiation in vitro and osteogenesis in vivo as well. For the in vitro analyses, rat ADSCs were transducted with adenoviral vectors containing the CGRP gene (Ad-CGRP) and were cultured in complete osteoblastic medium. The morphology, proliferative capacity, and formation of localized regions of mineralization in the cells were evaluated. The expression of alkaline phosphatase (ALP) and special markers of osteoblasts, such as Collagen I, Osteocalcin (BPG) and Osteopontin (OPN), were measured by cytochemistry, MMT, RT-PCR, and Western blot. For the in vivo analyses, the Ad-CGRP-ADSCs/Beta-tricalcium phosphate (β-TCP) constructs were implanted in rat radial bone defects for 12 weeks. Radiography and histomorphology evaluations were carried out on 4 weeks and 12 weeks. Our analyses indicated that heterogeneous spindle-shaped cells and localized regions of mineralization were formed in the CGRP-transduced ADSCs (the transduced group). A higher level of cellular proliferation, a high expression level of ALP on days 7 and 14 (p<0.05), and increased expression levels of Collagen I, BPG and OPN presented in transduced group (p<0.05). The efficiency of new bone formation was dramatically enhanced in vivo in Ad-CGRP-ADSCs/β-TCP group but not in β-TCP group and ADSCs/β-TCP group. Our results reveal that ADSCs transduced with an Ad-CGRP vector have stronger potential to differentiate into osteoblasts in vitro and are able to regenerate a promising new tissue engineering bone in vivo. Our findings suggest that CGRP-transduced ADSCs may serve as seed cells for bone tissue engineering and provide a potential way for treating bone defects.
Elevated lipoprotein(a) [Lp(a)] levels predict cardiovascular events incidence in patients with coronary artery disease (CAD). Genetic variants in the rs3798220, rs10455872 and rs6415084 single-nucleotide polymorphisms (SNPs) in the Lp(a) gene (LPA) correlate with elevated Lp(a) levels, but whether these SNPs have prognostic value for CAD patients is unknown. The present study evaluated the association of LPA SNPs with incidence of subsequent cardiovascular events in CAD patients after percutaneous coronary intervention (PCI).
TaqMan SNP genotyping assays were performed to detect the rs6415084, rs3798220 and rs10455872 genotypes in 517 Chinese Han patients with CAD after PCI. We later assessed whether there was an association of these SNPs with incidence of major adverse cardiovascular events (MACE: cardiac death, nonfatal myocardial infarction, ischemic stroke and coronary revascularization). Serum lipid profiles were also determined using biochemical methods.
Only the rs6415084 variant allele was associated with higher Lp(a) levels [41.3 (20.8, 74.6) vs. 18.6 (10.3, 40.9) mg/dl, p < 0.001]. During a 2-year follow-up period, 102 patients suffered MACE, and Cox regression analysis demonstrated that elevated Lp(a) (≥30 mg/dl) levels correlated with increased MACE (adjusted HR, 1.69; 95% CI 1.13-2.53), but there was no association between LPA genetic variants (rs6415084 and rs3798220) and MACE incidence (p > 0.05).
Our data did not support a relationship between genetic LPA variants (rs6415084 and rs3798220) and subsequent cardiovascular events after PCI in Chinese Han CAD patients.
Coronary artery disease; Lipoprotein(a); Major adverse cardiovascular events; Percutaneous coronary intervention; Single-nucleotide polymorphism
The present study aimed to quantify retinal and choroidal blood flow (BF) during light, dark adaptation and flicker light stimulation using the microsphere technique.
Materials and Methods
Adult male Sprague–Dawley rats were anesthetized with isoflurane. Eyes were dark (Group I, n = 8), light (Group II, n = 8) adapted or stimulated with 10Hz flicker light (Group III, n = 10). Retinal and choroidal BF were measured by a previously established method, using a mixture of 8 μm yellow-green and 10 μm red fluorescent microspheres. The microspheres were counted ex vivo in the dissected retina and choroid and in the reference arterial blood under a fluorescent microscope.
The choroidal BF was 64.8 ± 29 μl/min (mean ± SD) during dark adaptation, not significantly different from that during light adaptation (66.0 ± 17.8 μl/min). The retinal BF was 13.5 ± 3.2 μl/min during 10 Hz flickering light stimulation, significantly higher than that during dark adaptation in the control fellow eyes (9.9 ± 2.9 μl/min). The choroidal BF values were not statistically different between flicker stimulation and dark adaptation. Retinal BF was 11.6 ± 2.9 μl/min during light adaptation. Dark adaptation did not increase retinal BF (Group I, 8.2 ± 2.4 μl/min; Group II, 9.9 ± 2.9 μl/min).
These findings argue against a dark-induced or flicker-induced functional hyperemia in the choroid as a result of the demands of the outer retina. Retinal BF was not higher during dark adaptation. Our data support the conclusion that the inner retina has a higher energy demand in flicker conditions relative to dark.
Blood flow; choroid; dark adaptation; flicker light stimulation; light adaptation; microspheres; rat; retina
Parasite infections often result in a switch of the human body’s predominant immune reaction from T-helper 1 (Th1)-type to Th2-type. Hence, parasite infections are widely expected to accelerate the progression of human immunodeficiency virus (HIV) infections to acquired immunodeficiency syndrome (AIDS). In the People’s Republic of China, both parasitic diseases and AIDS are epidemic in certain rural areas, and co-infections are relatively common. However, no population-based studies have yet investigated the frequency of HIV and parasite co-infections, and its effects on immune responses. We studied (1) the immune status of an HIV-infected population, and (2) the effect of co-infection of HIV and intestinal parasites on selected parameters of the human immune system.
A total of 309 HIV-infected individuals were recruited and compared to an age-matched and sex-matched control group of 315 local HIV-negative individuals. Questionnaires were administered to all participants to obtain information on sociodemographic characteristics, sanitation habits, family income, and recent clinical manifestations. Two consecutive stool samples and 10 ml samples of venous blood were also collected from each individual for the diagnosis of parasite infections and quantitative measurements of selected cytokines and CD4+ T-lymphocytes, respectively.
During the study period, 79 HIV-infected individuals were not under highly active antiretroviral therapy (HAART) and were thus included in our analysis; the prevalence of intestinal helminth infections was 6.3% and that of protozoa was 22.8%. The most common protozoan infections were Blastocystis hominis (B. hominis) (13.9%) and Cryptosporidium spp. (10.1%). The prevalence of Cryptosporidium spp. in HIV-infected individuals was significantly higher than that in HIV negative individuals (P < 0.05). Compared to the non-co-infected population, no significant difference was found for any of the measured immunological indicators (P > 0.05). However, the following trends were observed: IFN-γ levels were lower, but the IL-4 level was higher, in the population co-infected with HIV and helminths. In the population co-infected with HIV and B. hominis, the IL-2 level was higher. The population co-infected with HIV and Cryptosporidium spp. had markedly lower CD4+ T-lymphocyte counts.
According to the immunologic profile, co-infection with helminths is disadvantageous to HIV-infected individuals. It was associated with a shift in the Th1/Th2 balance in the same direction as that caused by the virus itself, which might indicate an acceleration of the progress from an HIV infection to AIDS. Co-infection with Cryptosporidium spp. was not associated with a significant change in immune factors but co-infection with Cryptosporidium spp. was associated with a reduced level of CD4 + T-lymphocytes, confirming the opportunistic nature of such infections. Co-infection with B. hominis, on the other hand, was associated with an antagonistic shift in the immunological profile compared to an HIV infection.
HIV; Intestinal parasite; Co-infection; Immunological profile; People’s Republic of China
Penguins are an important seabird species in Antarctica and are sensitive to climate and environmental changes. Previous studies indicated that penguin populations increased when the climate became warmer and decreased when it became colder in the maritime Antarctic. Here we determined organic markers in a sediment profile collected at Cape Bird, Ross Island, high Antarctic, and reconstructed the history of Adélie penguin colonies at this location over the past 700 years. The region transformed from a seal to a penguin habitat when the Little Ice Age (LIA; 1500–1800 AD) began. Penguins then became the dominant species. Penguin populations were the highest during ca. 1490 to 1670 AD, a cold period, which is contrary to previous results in other regions much farther north. Different responses to climate change may occur at low latitudes and high latitudes in the Antarctic, even if for same species.
Human monocytes/macrophages (M/MФ) of the innate immunity sense and respond to microbial products via specific receptor coupling with stimulatory (such as TLR) and inhibitory (such as Tim-3) receptors. Current models imply that Tim-3 expression on M/MØ can deliver negative signaling to TLR-mediated IL-12 expression through trans association with its ligand Galectin-9 (Gal-9) presented by other cells. However, Gal-9 is also expressed within M/MØ, and the effect of intracellular Gal-9 on Tim-3 activities and inflammatory responses in the same M/MØ remains unknown. In this study, our data suggest that Tim-3 and IL-12/IL-23 gene transcriptions are regulated by enhanced or silenced Gal-9 expression within monocytes through synergizing with TLR signaling. Additionally, TLR activation facilitates Gal-9/Tim-3 cis association within the same M/MØ to differentially regulate IL-12/IL-23 expressions through STAT-3 phosphorylation. These results reveal a ligand (Gal-9) compartment-dependent regulatory effect on receptor (Tim-3) activities and inflammatory responses via TLR pathways—a novel mechanism underlying cellular responses to external or internal cues.
We previously reported that Axin1 (Axin) is down-regulated in many cases of lung cancer, and X-ray irradiation increased Axin expression and inhibited lung cancer cells. The mechanisms, however, were not clear.
Four lung cancer cell lines were used to detect the methylation status of Axin with or without X-ray treatment. Real-time PCR was used to quantify the expression of Axin, and western blot analysis was applied to measure protein levels of Axin, β-catenin, Cyclin D1, MMP-7, DNMTS, MeCP2 and acetylated histones. Flow cytometric analysis, colony formation assay, transwell assay and xenograft growth experiment were used to study the biological behavior of the cells with hypermethylated or unmethylated Axin gene after X-ray treatment.
Hypermethylated Axin gene was detected in 2 of 4 cell lines, and it correlated inversely with Axin expression. X-ray treatment significantly up-regulated Axin expression in H446 and H157 cells, which possess intrinsic hypermethylation of the Axin gene (P<0.01), but did not show up-regulation in LTE and H460 cells, which have unmethylated Axin gene. 2Gy X-ray significantly reduced colony formation (from 71% to 10.5%) in H157 cells, while the reduction was lower in LTE cells (from 71% to 20%). After X-ray irradiation, xenograft growth was significantly decreased in H157 cells (from 1.15 g to 0.28 g) in comparison with LTE cells (from 1.06 g to 0.65 g). Significantly decreased cell invasiveness and increased apoptosis were also observed in H157 cells treated with X-ray irradiation (P<0.01). Down-regulation of DNMTs and MeCP2 and up-regulation of acetylated histones could be detected in lung cancer cells.
X-ray-induced inhibition of lung cancer cells may be mediated by enhanced expression of Axin via genomic DNA demethylation and histone acetylation. Lung cancer cells with a different methylation status of the Axin gene showed different radiosensitivity, suggesting that the methylation status of the Axin gene may be one important factor to predict radiosensitivity of the tumor.
Axin; Methylation; Proliferation; Invasiveness; Radiosensitivity
The Royal College of Surgeons (RCS) rat is an established animal model of retinitis pigmentosa, a family of inherited retinal diseases which starts with loss of peripheral vision and progresses to eventual blindness. Blood flow (BF), an important physiological parameter, is intricately coupled to metabolic function under normal physiological conditions and is perturbed in many neurological and retinal diseases. This study reports non-invasive high-resolution MRI (44 × 44 × 600 μm) to image quantitative retinal and choroidal BF and layer-specific retinal thicknesses in RCS rat retinas at different stages of retinal degeneration compared with age-matched controls. The unique ability to separate retinal and choroidal BF was made possible by the depth-resolved MRI technique. RBF decreased with progressive retinal degeneration, but ChBF did not change in RCS rats up to post-natal day 90. We concluded that choroidal and retinal circulations have different susceptibility to progressive retinal degeneration in RCS rats. Layer-specific retinal thickness became progressively thinner and was corroborated by histological analysis in the same animals. MRI can detect progressive anatomical and BF changes during retinal degeneration with laminar resolution.
retinal degeneration; retinitis pigmentosa; arterial spin labeling; blood flow; retina; choroid
To investigate the relationship between health-related quality of life (HRQOL) and survival in Chinese patients with chronic liver disease (CLD).
HRQOL was measured with the Chinese version of Short Form 36 (SF-36). SF-36 scores, demographic and clinical data were collected at baseline and after 18 months follow-up. Kaplan-Meier and Cox Proportional Hazard Regression survival analyses were used for interpretation of data. Surviving patients were censored in the analyses.
A total of 415 Chinese patients with CLD and 86 healthy controls were enrolled. During the follow-up period 50 patients died. SF-36 scores in healthy controls and surviving patients were higher compared with those in deceased patients. Scores of physical component summary (PCS) in healthy controls, surviving and deceased patients were 54.1 ± 5.2, 48.9 ± 7.7 and 33.5 ± 8.2 respectively (p < 0.001). Scores of mental component summary (MCS) in healthy controls, surviving and deceased patients were 56.6 ± 8.2, 53.0 ± 5.6 and 37.1 ± 12.1 (p < 0.001) respectively. Survival was significantly associated with PCS and MCS scores, and the presence of ascites.
HRQOL was associated with survival in patients with CLD. PCS and MCS scores were predictors of survival.
Health-related quality of life; Chronic liver disease; SF-36; Survival
AIM: To further analyse cancer involvement of basic transcription factor 3 (BTF3) after detection of its upregulation in gastric tumor samples.
METHODS: BTF3 transcription rates in human gastric tumor tissue samples (n = 20) and adjacent normal tissue (n = 18) specimens as well as in the gastric cancer cell lines AGS, SGC-7901, MKN-28, MKN-45 and MGC803 were analyzed via quantitative real-time polymerase chain reaction. The effect of stable BTF3 silencing via infection with a small interfering RNA (siRNA)-BTF3 expressing lentivirus on SGC-7901 cells was measured via Western blotting analysis, proliferation assays, cell cycle and apoptosis profiling by flow cytometry as well as colony forming assays with a Cellomic Assay System.
RESULTS: A significant higher expression of BTF3 mRNA was detected in tumors compared to normal gastric tissues (P < 0.01), especially in section tissues from female patients compared to male patients, and all tested gastric cancer cell lines expressed high levels of BTF3. From days 1 to 5, the relative proliferation rates of stable BTF3-siRNA transfected SGC7901 cells were 82%, 70%, 57%, 49% and 44% compared to the control, while the percentage of cells arrested in the G1 phase was significantly decreased (P = 0.000) and the percentages of cells in the S (P = 0.031) and G2/M (P = 0.027) phases were significantly increased. In addition, the colony forming tendency was significantly decreased (P = 0.014) and the apoptosis rate increased from 5.73% to 8.59% (P = 0.014) after BTF3 was silenced in SGC7901 cells.
CONCLUSION: BTF3 expression is associated with enhanced cell proliferation, reduced cell cycle regulation and apoptosis and its silencing decreased colony forming and proliferation of gastric cancer cells.
Basic transcription factor 3; Gastric cancer; Small interfering RNA; Proliferation; Apoptosis; Cell cycle
Sika deer (Cervus nippon) have different dietary preferences to other ruminants and are tolerant to tannin-rich plants. Because the rumen bacteria in domestic Sika deer have not been comprehensively studied, it is important to investigate its rumen bacterial population in order to understand its gut health and to improve the productivity of domestic Sika deer.
The rumen bacterial diversity in domestic Sika deer (Cervus nippon) fed oak leaves- (OL group) and corn stalks-based diets (CS group) were elucidated using 16S rRNA gene libraries and denaturing gradient gel electrophoresis (DGGE). Overall, 239 sequences were examined from the two groups, 139 clones from the OL group were assigned to 57 operational taxonomic units (OTUs) and 100 sequences from the CS group were divided into 50 OTUs. Prevotella-like sequences belonging to the phylum Bacteroidetes were the dominant bacteria in both groups (97.2% OL and 77% CS), and sequences related to Prevotella brevis were present in both groups. However, Prevotella shahii-like, Prevotella veroralis-like, Prevotella albensis-like, and Prevotella salivae-like sequences were abundant in the OL group compared to those in the CS group, while Succinivibrio dextrinosolvens-like and Prevotella ruminicola-like sequences were prevalent in the CS group. PCR-DGGE showed that bacterial communities clustered with respect to diets and the genus Prevotella was the dominant bacteria in the rumen of domestic Sika deer. However, the distribution of genus Prevotella from two groups was apparent. In addition, other fibrolytic bacteria, such as Clostridium populeti and Eubacterium cellulosolvens were found in the rumen of domestic Sika deer.
The rumen of domestic Sika deer harbored unique bacteria which may represent novel species. The bacterial composition appeared to be affected by diet, and sequences related to Prevotella spp. may represent new species that may be related to the degradation of fiber biomass or tannins. Moreover, the mechanism and biological functions of Prevotella spp. in the rumen ecosystem, and synergistic interactions with other microorganisms should be noticed.
Ecology; Prevotella; Fiber; Tannin
AIM: To explore the effect of lysine acetylation in related proteins on regulation of the proliferation of gastric cancer cells, and determine the lysine-acetylated proteins and the acetylated modified sites in AGS gastric cancer cells.
METHODS: The CCK-8 experiment and flow cytometry were used to observe the changes in proliferation and cycle of AGS cells treated with trichostatin A (TSA). Real time polymerase chain reaction and Western blotting were used to observe expression changes in p21, p53, Bax, Bcl-2, CDK2, and CyclinD1 in gastric cancer cells exposed to TSA. Cytoplasmic proteins in gastric cancer cells before and after TSA treatment were immunoprecipitated with anti-acetylated lysine antibodies, separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and silver-stained to detect the proteins by mass spectrometry after removal of the gel. The acetylated proteins in AGS cells were enriched with lysine-acetylated antibodies, and a high-resolution mass spectrometer was used to detect the acetylated proteins and modified sites.
RESULTS: TSA significantly inhibited AGS cell proliferation, and promoted cell apoptosis, leading to AGS cell cycle arrest in G0/G1 and G2/M phases, especially G0/G1 phase. p21, p53 and Bax gene expression levels in AGS cells were increased with TSA treatment duration; Bcl-2, CDK2, and CyclinD1 gene expression levels were decreased with TSA treatment duration. Two unknown protein bands, 72 kDa (before exposure to TSA) and 28 kDa (after exposure to TSA), were identified by silver-staining after immunoprecipitation of AGS cells with the lysine-acetylated monoclonal antibodies. Mass spectrometry showed that the 72 kDa protein band may be PKM2 and the 28 kDa protein band may be ATP5O. The acetylated proteins and modified sites in AGS cells were determined.
CONCLUSION: TSA can inhibit gastric cancer cell proliferation, which possibly activated signaling pathways in a variety of tumor-associated factors. ATP5O was obviously acetylated in AGS cells following TSA treatment.
Trichostatin A; Acetylation modification; Gastric cancer; Mass spectrometry; ATP5O; PKM2
To investigate the therapeutic effect of different doses of low energy shock wave therapy (LESWT) on the erectile dysfunction (ED) in streptozotocin (STZ) induced diabetic rats. SD rats (n = 75) were randomly divided into 5 groups (normal control, diabetic control, 3 different dose LESWT treated diabetic groups). Diabetic rats were induced by intra-peritoneal injection of STZ (60 mg/kg) and rats with fasting blood glucose ≥ 300 mg/dL were selected as diabetic models. Twelve weeks later, different doses of LESWT (100, 200 and 300 shocks each time) treatment on penises were used to treat ED (7.33 MPa, 2 shocks/s) three times a week for two weeks. The erectile function was evaluated by intracavernous pressure (ICP) after 1 week washout period. Then the penises were harvested for histological study. The results showed LESWT could significantly improve the erectile function of diabetic rats, increase smooth muscle and endothelial contents, up-regulate the expression of α-SMA, vWF, nNOS and VEGF, and down- regulate the expression of RAGE in corpus cavernosum. The therapeutic effect might relate to treatment dose positively, and the maximal therapeutic effect was noted in the LESWT300 group. Consequently, 300 shocks each time might be the ideal LESWT dose for diabetic ED treatment.
erectile dysfunction; diabetes mellitus; low energy shock wave therapy; corpus cavernosum
Water-related parasitic diseases are directly dependent on water bodies for their spread or as a habitat for indispensable intermediate or final hosts. Along with socioeconomic development and improvement of sanitation, overall prevalence is declining in the China. However, the heterogeneity in economic development and the inequity of access to public services result in considerable burden due to parasitic diseases in certain areas and populations across the country. In this review, we demonstrated three aspects of ten major water-related parasitic diseases, i.e., the biology and pathogenicity, epidemiology and recent advances in research in China. General measures for diseases control and special control strategies are summarized.
parasitic disease; water; protozoa; helminths; epidemiology; China
Although embryonic stem (ES) cell-derived hepatocytes have the capacity for liver engraftment and repopulation, their in vivo hepatic function has not been analyzed yet. We aimed to determine the metabolic function and therapeutic action of ES cell-derived hepatocytes after serial liver repopulations in fumaryl acetoacetate hydrolase knockout (Fah−/−) mice. Albumin expressing (Alb+) cells were obtained by hepatic differentiation of ES cells using two frequently reported methods. After transplantation, variable levels of liver repopulation were found in Fah−/− mice recipients. FAH expressing (FAH+) hepatocytes were found either as single cells or as nodules with multiple hepatocytes. After serial transplantation, the proportion of the liver that was repopulated by the re-transplanted FAH+ hepatocytes increased significantly. ES cell-derived FAH+ hepatocytes were found in homogenous nodules and corrected the liver metabolic disorder of Fah−/− recipients and rescued them from death. ES cell-derived hepatocytes had normal karyotype, hepatocytic morphology and metabolic function both in vitro and in vivo. In conclusion, ES cell-derived hepatocytes were capable of liver repopulation and correction of metabolic defects after serial transplantation. Our results are an important piece of evidence to support future clinical applications of ES cell-derived hepatocytes in treating liver diseases.
Embryonic stem cell; Hepatocyte; Metabolic function; Liver repopulation; Cell transplantation
Protein phosphatase magnesium-dependent 1δ (PPM1D) is an oncogene, overexpressed in many solid tumors, including ovarian cancer and breast cancer. The current study examined the expression and the prognostic value of PPM1D mRNA in human hepatocellular carcinoma (HCC).
Total RNA was extracted from 86 HCC and paired non-cancerous liver tissues. PPM1D mRNA expression was determined by real-time quantitative reverse transcriptase-polymerase chain reaction (qPCR). Immunohistochemistry assay was used to verify the expression of ppm1d protein in the HCC and non-cancerous liver tissues. HCC patients were grouped according to PPM1D mRNA expression with the average PPM1D mRNA level in non-cancerous liver tissue samples as the cut-off. Correlations between clinicopathologic variables, overall survival and PPM1D mRNA expression were analyzed.
PPM1D mRNA was significantly higher in HCC than in the paired non-cancerous tissue (p<0.01). This was confirmed by ppm1d staining. 56 patients were classified as high expression group and the other 30 patients were categorized as low expression group. There were significant differences between the two groups in term of alpha-fetoprotein (α-FP) level (p<0.01), tumor size (p<0.01), TNM stage (p<0.01), recurrence incidence (p<0.01) and family history of liver cancer (p<0.01). The current study failed to find significant differences between the two groups in the following clinical characteristics: age, gender, portal vein invasion, lymphnode metastasis, hepatitis B virus (HBV) infection and alcohol intake. Survival time of high expression group was significantly shorter than that of low expression group (median survival, 13 months and 32 months, respectively, p<0.01).
Up-regulation of PPM1D mRNA was associated with progressive pathological feature and poor prognosis in HCC patients. PPM1D mRNA may serve as a prognostic marker in HCC.
Dibutyl phthalate (DBP) is a commonly used plasticizer and additive to adhesives, printing inks and nail polishes. Because it has been found to be a powerful reproductive and developmental toxicant, a sensor to monitor DBP in some working spaces and the environment is required. In this work polyaniline nanofibers were deposited on the electrode of a quartz crystal oscillator to form a Quartz Crystal Microbalance gas sensor. The coated quartz crystal and a non-coated quartz crystal were mounted in a sealed chamber, and their frequency difference was monitored. When DBP vapor was injected into the chamber, gas adsorption decreased the frequency of the coated quartz crystal oscillator and thereby caused an increase in the frequency difference between the two crystals. The change of the frequency difference was recorded as the sensor response. The sensor was extremely sensitive to DBP and could be easily recovered by N2 purging. A low measurement limit of 20 ppb was achieved. The morphologies of the polyaniline films prepared by different approaches have been studied by SEM and BET. How the nanofiber-structure can improve the sensitivity and stability is discussed, while its selectivity and long-term stability were investigated.
QCM; gas sensor; dibutyl phthalate; nanofiber; polyaniline; PACS 07.07.Df (Gas sensor); 77.65.Fs (Quartz resonator); 82.35.Np (Polymers nanoparticles in); 81.07.-b (Nanoscale materials fabrication and characterization)
In the study of circadian rhythms, it has been a puzzle how a limited number of circadian clock genes can control diverse aspects of physiology. Here we investigate circadian gene expression genome-wide using larval zebrafish as a model system. We made use of a spatial gene expression atlas to investigate the expression of circadian genes in various tissues and cell types. Comparison of genome-wide circadian gene expression data between zebrafish and mouse revealed a nearly anti-phase relationship and allowed us to detect novel evolutionarily conserved circadian genes in vertebrates. We identified three groups of zebrafish genes with distinct responses to light entrainment: fast light-induced genes, slow light-induced genes, and dark-induced genes. Our computational analysis of the circadian gene regulatory network revealed several transcription factors (TFs) involved in diverse aspects of circadian physiology through transcriptional cascade. Of these, microphthalmia-associated transcription factor a (mitfa), a dark-induced TF, mediates a circadian rhythm of melanin synthesis, which may be involved in zebrafish's adaptation to daily light cycling. Our study describes a systematic method to discover previously unidentified TFs involved in circadian physiology in complex organisms.
For most animals whose lives are dependent on the sun, circadian clocks govern their daily behaviors and physiology. In different animals, novel functions under the circadian clock's control can evolve as adaptations to their specific environment. A zebrafish demonstrates a remarkably high level of interplay between external light and its internal circadian clock due to its transparent nature. In a genome-wide study, we identified a large number of circadian oscillating genes as well as genes whose expression is highly sensitive to the light or dark in zebrafish. Our computational analysis of gene regulatory networks revealed a number of transcription factors (TFs) that mediate novel circadian functions. We investigated one example in depth, a key TF that relays the control of the circadian clock to the enzymes synthesizing melanin in a dark-induced pathway thus causing the daily change of pigmentation in zebrafish. This dark-induced circadian melanogenesis can lead to an anticipatory change in zebrafish skin color allowing zebrafish to adapt to its environment. This mechanism allows zebrafish to better evade predators and effectively adjust its daily light-sensitivity in the pigment cells. Our study provides an excellent example of how the circadian clock is adapted in a specific organism to control its behavior, thus enabling evolutionary adaptation to the organism's ecological niche.
Diabetic microvascular changes are considered to be influenced by angiogenic factors. As a compensatory mechanism, the expression of some angiogenic factors are elevated in ischemic myocardium. The aim of this study was to investigate the changes of serum angiogenic factors, and the association among these angiogenic factors, the severity of coronary artery stenosis and collateral vessels form in patients with diabetes and unstable angina pectoris (UAP).
42 patients with diabetes (diabetes group), 57 patients with UAP (UAP group), and 36 age-matched healthy people (control group) were selected. Serum concentrations of angiogenic factors were measured using cytokine array technology. The severity of coronary artery stenosis was scored using the angiographic Gensini score. Coronary collateral vessels were scored according to Rentrop's classification.
No significant differences in the serum concentrations of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), angiogenin, angiostatin, basic fibroblast growth factor (bFGF) and platelet-derived growth factor-BB (PDGF-BB) were detected between control group and diabetes group. But in patients with diabetes complicated with UAP and in patients with UAP without diabetes, serum concentrations of VEGF and Ang-2 were elevated (p < 0.01, p < 0.01). Only serum Ang-2 concentrations were significantly correlated with Gensini score (r=0.585, p < 0.001), left ventricular end diastolic diameter (r=0.501, p < 0.001), left ventricular end systolic diameter (r=0.563, p < 0.001) and left ventricular ejection fraction (r=−0.523, p < 0.001).
Serum concentrations of VEGF and Ang-2 were increased, and diabetes didn’t affect this increases in patients with UAP. Serum Ang-2 concentrations were correlated with the severity of coronary artery stenosis.
Diabetes; Unstable angina pectoris; Angiogenic factors; Gensini score; Collateral vessels
Increasing evidences have suggested that autoantibodies against muscarinic-2 acetylcholine receptor (anti-M2-R) may play an important role in the development of atrial fibrillation (AF). Predictive value of pre-procedural anti-M2-R for the recurrence of AF after radiofrequency catheter ablation is still unclear.
Totally 76 AF patients with preserved left ventricular systolic function were prospectively enrolled and subjected to ablation after the detection of serum anti-M2-R by enzyme linked immunosorbent assay. These patients were given follow-up examination for one year after ablation. Risk estimation for the recurrence of AF was performed using the univariate and multivariate logistic regression.
In AF group, serum anti-M2-R was significantly higher than that in the control group in terms of frequency (40.8% versus 11.7%; p < 0.001) and titer (1:116 versus 1:29; p < 0.001). Compared with paroxysmal AF patients, persistent AF patients had higher frequency (57.6% versus 27.9%; p = 0.009) and titer (1:132 versus 1:94; p = 0.012) for autoantibodies. During one-year follow-up examination after ablation, the recurrence of AF was observed in 25 (32.9%) patients. Multivariate analysis showed that pre-procedural serum anti-M2-R was an independent predictor for the recurrence of AF at the time point of 12 months after ablation (odds ratio: 4.701; 95% confidence interval: 1.590-13.894; p = 0.005).
In AF patients, the frequency and titer of serum anti-M2-R were significantly higher than those in the control group with sinus rhythm. Pre-procedural serum anti-M2-R was an independent predictor for the recurrence of AF one year after radiofrequency catheter ablation.
Atrial fibrillation; Radiofrequency catheter ablation; Muscarinic-2 acetylcholine receptor; Autoantibody; Recurrence
In this study, quasi-three-dimensional (3D) microwell patterns were fabricated with poly (l-lactic acid) for the development of cell-based assays, targeting voltage-gated calcium channels (VGCCs).
Methods and materials
SH-SY5Y human neuroblastoma cells were interfaced with the microwell patterns and found to grow as two dimensional (2D), 3D, and near two dimensional (N2D), categorized on the basis of the cells’ location in the pattern. The capability of the microwell patterns to support 3D cell growth was evaluated in terms of the percentage of the cells in each growth category. Cell spreading was analyzed in terms of projection areas under light microscopy. SH-SY5Y cells’ VGCC responsiveness was evaluated with confocal microscopy and a calcium fluorescent indicator, Calcium Green™-1. The expression of L-type calcium channels was evaluated using immunofluorescence staining with DM-BODIPY.
It was found that cells within the microwells, either N2D or 3D, showed more rounded shapes and less projection areas than 2D cells on flat poly (l-lactic acid) substrates. Also, cells in microwells showed a significantly lower VGCC responsiveness than cells on flat substrates, in terms of both response magnitudes and percentages of responsive cells, upon depolarization with 50 mM K+. This lower VGCC responsiveness could not be explained by the difference in L-type calcium channel expression. For the two patterns addressed in this study, N2D cells consistently exhibited an intermediate value of either projection areas or VGCC responsiveness between those for 2D and 3D cells, suggesting a correlative relation between cell morphology and VGCC responsiveness.
These results suggest that the pattern structure and therefore the cell growth characteristics were critical factors in determining cell VGCC responsiveness and thus provide an approach for engineering cell functionality in cell-based assay systems and tissue engineering scaffolds.
replica molding; cell spreading; confocal microscopy; microwell patterns
To employ functional manganese-enhanced MRI (MEMRI) to image layer-specific changes in calcium-dependent activities in the rat retina during light versus dark adaptation.
Functional MEMRI at 20 × 20 × 700 μm was used to study light and dark adaptation in the same animals (N = 10) in which one eye was covered and the fellow eye was not. The activity encoding of the light and dark adaptation was achieved in awake conditions and imaged under anesthesia. T1-weighted MRI at 11.7 tesla (T) was performed using two identical radiofrequency transceiver coils to allow interleaved MRI acquisitions of the two eyes. An intravascular contrast agent was also used to verify layer assignments.
MEMRI detected contrasts among the inner retina, outer retina, and choroid. Independent confirmation of the vascular layers and boundaries between layers was documented with an intravascular contrast agent. The retinal layer thicknesses agreed with published data. The outer retina had lower MEMRI activity in light compared with dark adaption (P < 0.001), consistent with the increased metabolic demand associated with the “dark current.” The inner retina had higher MEMRI activity in light compared with dark adaption (P < 0.05). The choroid MEMRI activity was not statistically different between light and dark adaptation (P > 0.05).
This study demonstrated a high-resolution MEMRI protocol to image functional activities among different layers of the retinas in awake animals during light and dark adaptation. This approach could have potential applications in animal models of retinal dysfunction.
This study employed functional manganese-enhanced MRI (MEMRI) to image layer-specific changes in calcium-dependent activities in the rat retina during light versus dark adaptation. This approach could have potential applications to probe retinal dysfunction in diseased states.