The tethering of platelets on the injured vessel surface mediated by glycoprotein Ibα (GPIbα) - Von Willebrand factor (vWF) bonds, as well as the interaction between flowing platelets and adherent platelets, are two key events that take place immediately following blood vessel injury. This early-stage platelet deposition and accumulation triggers the initiation of hemostasis, a self-defensive mechanism to prevent the body from excessive blood loss. To understand and predict this complex process, one must integrate experimentally determined information on the mechanics and biochemical kinetics of participating receptors over very small time frames (1–1000 µs) and length scales (10–100 nm), to collective phenomena occurring over seconds and tens of microns. In the present study, a unique three dimensional multiscale computational model, platelet adhesive dynamics (PAD), was applied to elucidate the unique physics of (i) a non-spherical, disk-shaped platelet interacting and tethering onto the damaged vessel wall followed by (ii) collisional interactions between a flowing platelet with a downstream adherent platelet. By analyzing numerous simulations under different physiological conditions, we conclude that the platelet’s unique spheroid-shape provides heterogeneous, orientation-dependent translocation (rolling) behavior which enhances cell-wall interactions. We also conclude that platelet-platelet near field interactions are critical for cell-cell communication during the initiation of microthrombi. The PAD model described here helps to identify the physical factors that control the initial stages of platelet capture during this process.
Adhesion; Multiscale modeling; Platelet; Receptors; Shear flow; Von Willebrand factor
microRNAs (miRNAs) are a class of small non-coding RNAs that play important roles in carcinogenesis. In the present study, we investigated the effect of miR-212 on pancreatic ductal adenocarcinoma (PDAC) and its target protein.
Quantitative real-time PCR(qRT-PCR) was performed to detect the expression of miR-212 in PDAC tissues and pancreatic cancer cell lines. miR-212 mimic, miR-212 inhibitor and negative control were transfected into pancreatic cancer cells and the effect of miR-212 up-regulation and down-regulation on the proliferation, migration and invasion of cells were investigated. Furthermore, the mRNA and protein levels of Patched-1(PTCH1) were measured. Meanwhile, luciferase assays were performed to validate PTCH1 as miR-212 target in PDAC.
miR-212 was up-regulated in PDAC tissues and cells.Using both gain-of function and loss-of function experiments, a pro-oncogenic function of miR-212 was demonstrated in PDAC. Moreover, up-regulated of PTCH1 could attenuate the effect induced by miR-212.
These data suggest that miR-212 could facilitate PDAC progression and metastasis through targeting PTCH1, implicating a novel mechanism for the progression of PDAC.
miR-212; Patched-1; Pancreatic cancer; Proliferation; Migration; Invasion
Resistance to chemotherapy and the side effects of anticancer drugs are the major obstacles for glioma treatment. The aim of the present study was to develop a novel approach for the treatment of gliomas that improved the therapeutic effect; the anticancer drug, doxorubicin (DOX), was combined with short interfering (si)RNA and monomethoxy polyethylene glycol polyethylenimine superparamagnetic iron oxide nanoparticle (mPEG-PEI-SPION), a magnetic resonance imaging (MRI)-visible nanoparticle. Specific siRNA molecules, delivered by mPEG-PEI-SPION, were employed to knockdown the PIN2-interacting protein 1 (PinX1) gene in C6 glioma cells. PinX1 is a nucleolar protein associated with telomere and telomerase. C6 cells were treated with DOX and/or PinX1-siRNA. The results of the transfection experiments revealed that siRNA/mPEG-PEI-SPION was transfected into C6 cells with high efficiency. PinX1-siRNA was unable to inhibit C6 cells, while in the PinX1-siRNA + DOX group, the same dose of DOX caused an increased loss of cell viability. Therefore, mPEG-PEI-SPION was shown to be viable for siRNA delivery into C6 cells and coadministration of DOX with PinX1-siRNA may be a potential therapeutic method for inhibiting gliomas.
monomethoxy polyethylene glycol polyethyleneimine superparamagnetic iron oxide nanoparticle; doxorubicin; PIN2-interacting protein 1; short interfering RNA; glioma
Phytogenic compounds with anti-oxidant and anti-inflammatory properties, such as ginsenoside metabolite compound K (CK) or berberine (BBR), are currently discussed as promising complementary agents in the prevention and treatment of cancer and inflammation. The latest study showed that ginsenoside Rb1 and its metabolites could inhibit TNBS-induced colitis injury. However, the functional mechanisms of anti-inflammation effects of ginsenoside, particularly its metabolite CK are still not clear. Here, using dextran sulfate sodium (DSS)-induced colitis in mice, clinical parameters, intestinal integrity, pro-inflammatory cytokines production, and signaling pathways in colonic tissues were determined. In mild and sever colitis mice, CK and BBR (as a positive agent) alleviated colitis histopathology injury, ameliorated myeloperoxidase (MPO) activity, reduced pro-inflammatory cytokines production, such as, IL-6, IL-1β, TNF-α, and increased anti-inflammatory cytokine IL-10 production in both mice colon tissues and blood. Nevertheless, the results revealed that CK and BBR inhibited NF-κB p65 nuclear translocation, downregulated p-IκBα and upregulated IκBα, indicating that CK, as well as BBR, suppressed the activation of the NF-κB pathway in the progression of colitis with immunofluorescence, immunohistochemical and western blotting analysis. Furthermore, CK inhibited pro-inflammatory cytokines production in LPS-activated macrophages via down-regulation of NF-κB signaling pathway. Taken together, our results not only reveal that CK promotes the recovery of the progression of colitis and inhibits the inflammatory responses by suppressing NF-κB activation, but also suggest that CK downregulates intestinal inflammation through regulating the activation of macrophages and pro-inflammatory cytokines production.
Management of solid wastes with high nicotine content, such as those accumulated during tobacco manufacturing, poses a major challenge, which can be addressed by using bacteria such as Pseudomonas and Arthrobacter. In this study, a new species of Pseudomonas geniculata, namely strain N1, which is capable of efficiently degrading nicotine, was isolated and identified. The optimal growth conditions for strain N1 are a temperature of 30°C, and a pH 6.5, at a rotation rate of 120 rpm min−1 with 1 g l−1 nicotine as the sole source of carbon and nitrogen. Myosmine, cotinine, 6-hydroxynicotine, 6-hydroxy-N-methylmyosmine, and 6-hydroxy-pseudooxynicotine were detected as the five intermediates through gas chromatography-mass and liquid chromatography-mass analyses. The identified metabolites were different from those generated by Pseudomonas putida strains. The analysis also highlighted the bacterial metabolic diversity in relation to nicotine degradation by different Pseudomonas strains.
Brain retraction causes great distortion that limits the accuracy of an image-guided neurosurgery system that uses preoperative images. Therefore, brain retraction correction is an important intraoperative clinical application.
We used a linear elastic biomechanical model, which deforms based on the eXtended Finite Element Method (XFEM) within a framework for brain retraction correction. In particular, a laser range scanner was introduced to obtain a surface point cloud of the exposed surgical field including retractors inserted into the brain. A brain retraction surface tracking algorithm converted these point clouds into boundary conditions applied to XFEM modeling that drive brain deformation. To test the framework, we performed a brain phantom experiment involving the retraction of tissue. Pairs of the modified Hausdorff distance between Canny edges extracted from model-updated images, pre-retraction, and post-retraction CT images were compared to evaluate the morphological alignment of our framework. Furthermore, the measured displacements of beads embedded in the brain phantom and the predicted ones were compared to evaluate numerical performance.
The modified Hausdorff distance of 19 pairs of images decreased from 1.10 to 0.76 mm. The forecast error of 23 stainless steel beads in the phantom was between 0 and 1.73 mm (mean 1.19 mm). The correction accuracy varied between 52.8 and 100 % (mean 81.4 %).
The results demonstrate that the brain retraction compensation can be incorporated intraoperatively into the model-updating process in image-guided neurosurgery systems.
Brain retraction; Extended finite element method; Laser range scanner; Image-guided neurosurgery system
Microorganisms such as Pseudomonas putida play important roles in the mineralization of organic wastes and toxic compounds. To comprehensively and accurately elucidate key processes of nicotine degradation in Pseudomonas putida, we measured differential protein abundance levels with MS-based spectral counting in P. putida S16 grown on nicotine or glycerol, a non-repressive carbon source. In silico analyses highlighted significant clustering of proteins involved in a functional pathway in nicotine degradation. The transcriptional regulation of differentially expressed genes was analyzed by using quantitative reverse transcription-PCR. We observed the following key results: (i) The proteomes, containing 1,292 observed proteins, provide a detailed view of enzymes involved in nicotine metabolism. These proteins could be assigned to the functional groups of transport, detoxification, and amino acid metabolism. There were significant differences in the cytosolic protein patterns of cells growing in a nicotine medium and those in a glycerol medium. (ii) The key step in the conversion of 3-succinoylpyridine to 6-hydroxy-3-succinoylpyridine was catalyzed by a multi-enzyme reaction consisting of a molybdopeterin binding oxidase (spmA), molybdopterin dehydrogenase (spmB), and a (2Fe-2S)-binding ferredoxin (spmC) with molybdenum molybdopterin cytosine dinucleotide as a cofactor. (iii) The gene of a novel nicotine oxidoreductase (nicA2) was cloned, and the recombinant protein was characterized. The proteins and functional pathway identified in the current study represent attractive targets for degradation of environmental toxic compounds.
Pseudomonas putida strains are among the microorganisms that have acquired the capability to use toxic and xenobiotic compounds, such as nicotine, for growth. Although nicotine degradation by Pseudomonas was first discovered more than 50 years ago, the underlying molecular mechanisms remain unclear. In the last few years, we have made significant efforts to identify the key genes for the hydroxylation of 3-succinoylpyridine (SP) through genomic library screening and purification of wild-type enzymes. However, these efforts did not result in identifying any genes related to SP hydroxylation. In this study, by using comparative genetic analysis, we report the identification of 3 key genes, spmA, spmB and spmC from P. putida S16. The heterotrimeric enzyme encoded by these genes requires molybdopterin-cytosine dinucleotide as a cofactor. The proteomes of strain S16 grown on nicotine or glycerol contain 1,292 observed proteins, and provide a detailed view of enzymes involved in nicotine degradation. Our comparative analysis of the proteomic profiles of nicotine grown versus glycerol grown bacterial cells reveals a wide range of cellular processes and functions related to nicotine catabolism.
Tumorigenesis is a complex dynamic biological process that includes multiple steps of genetic and epigenetic alterations, aberrant expression of noncoding RNA, and changes in the expression profiles of coding genes. We call the collection of those perturbations in genome space the “cancer initiatome.” Long noncoding RNAs (lncRNAs) are pervasively transcribed in the genome and they have key regulatory functions in chromatin remodeling and gene expression. Spatiotemporal variation in the expression of lncRNAs has been observed in development and disease states, including cancer. A few dysregulated lncRNAs have been studied in cancers, but the role of lncRNAs in the cancer initiatome remains largely unknown, especially in esophageal squamous cell carcinoma (ESCC). We conducted a genome-wide screen of the expression of lncRNAs and coding RNAs from ESCC and matched adjacent nonneoplastic normal tissues. We identified differentially expressed lncRNAs and coding RNAs in ESCC relative to their matched normal tissue counterparts and validated the result using polymerase chain reaction analysis. Furthermore, we identified differentially expressed lncRNAs that are co-located and co-expressed with differentially expressed coding RNAs in ESCC and the results point to a potential interaction between lncRNAs and neighboring coding genes that affect ether lipid metabolism, and the interaction may contribute to the development of ESCC. These data provide compelling evidence for a potential novel genomic biomarker of esophageal squamous cell cancer.
Cylindrical blood vessels, ellipsoid platelets and biconcave-shaped deformable erythrocytes (RBCs) are important participants in hemostasis and thrombosis. However, due to the challenge of combining these components in simulation tools, few simulation studies have included all of them in realistic three-dimensional models. In the present study, we apply a recently developed simulation model to incorporate these components and analyze the flow in a thrombotic tubular arteriole, particularly the detailed hydrodynamic interactions between the thrombus shape, RBCs and platelets. It was found that at certain azimuth positions, the velocity drops in the proximity of both the upstream and downstream edge of the thrombus, which is accompanied by a rapid velocity increase in the narrowed region. The RBCs alter the flow profiles significantly from the typical low Reynolds (Re) number flow, and also enhance the deposition of free flowing platelets onto the thrombus. By evaluating the platelet-thrombus interaction and platelet-RBC interaction together, several mechanisms of platelet deposition augmentation are identified. With in vivo data comparison, our model illustrates the potential of future thrombosis studies that incorporate detailed receptor-ligand adhesion modules.
Resuscitation with fresh frozen plasma (FFP) is associated with improved outcomes after hemorrhagic shock (HS). Many trauma centers are using thawed plasma which has been stored for up to 5 days at 4°C (refrigeration), yet the effect of refrigeration on FFP is relatively unknown. Previously our group showed that refrigeration of FFP changed its coagulation factors and diminished its beneficial effects on endothelial cell (EC) function and resuscitation in an animal model of HS.We hypothesize that growth factor composition of FFP is altered during refrigeration, leading to a diminished beneficial effect on EC. Transforming growth factor (TGF)-β is a potent inhibitor of EC migration and is released during refrigeration of platelets. We found increased TGF-β1 protein levels and greater activation of downstream mediators Smad2/3 during refrigeration of FFP. Both Day 0 FFP (used on the same day after being thawed) and Day 5 FFP (used after being thawed and refrigerated for 5 days) stimulated EC migration in vitro; however, the EC migration in Day 5 FFP was significantly reduced. Inhibition of TGF-β type I receptor blocked FFP-induced Smad3 signaling in EC cells and restored the effectiveness of Day 5 FFP on EC migration to a comparable level seen in Day 0 FFP. These data suggest that the increased TGF-β levels during FFP refrigeration contribute to the deterioration of refrigerated FFP's effects on EC migration. This study identifies a novel molecular mechanism contributing to the reduced efficacy of refrigerated FFP.
Hemorrhagic shock; resuscitation; Smad; TGF-β type I receptor
Endogenous mechanisms exist to limit inflammation. One such molecule is netrin. This study examined the impact of ischemia-reperfusion (I/R) on netrin expression and the role of netrin in preventing renal inflammation and injury. All three isoforms of netrin (1, 3, and 4) are expressed in normal kidney. I/R significantly downregulated netrin-1 and -4 mRNA expression, whereas expression of netrin-3 was moderately upregulated at 24 h of reperfusion. The netrin receptor UNC5B mRNA increased at 3 h and but decreased at later time points. Expression of a second netrin receptor, DCC, was not altered significantly. I/R was associated with dramatic changes in netrin-1 protein abundance and localization. Netrin-1 protein levels increased between 3 and 24 h after reperfusion. Immunolocalization showed an interstitial distribution of netrin-1 in sham-operated kidneys which colocalized with Von Willebrand Factor suggesting the presence of netrin-1 in peritubular capillaries. After I/R, interstitial netrin-1 expression decreased and netrin-1 appeared in tubular epithelial cells. By 72 h after reperfusion, netrin-1 reappeared in the interstitium while tubular epithelial staining decreased significantly. Downregulation of netrin-1 in the interstitium corresponded with increased MCP-1 and IL-6 expression and infiltration of leukocytes into the reperfused kidney. Administration of recombinant netrin-1 significantly improved kidney function (blood urea nitrogen: 161 ± 7 vs. 104 ± 24 mg/dl, creatinine: 1.3 ± 0.07 vs. 0.75 ± 0.16 mg/dl, P < 0.05 at 24 h) and reduced tubular damage and leukocyte infiltration in the outer medulla. These results suggest that downregulation of netrin-1 in vascular endothelial cells may promote endothelial cell activation and infiltration of leukocytes into the kidney thereby enhancing tubular injury.
netrin-4; inflammation; endothelial cell; chemokine
Long-term follow-up studies in critical care have described survivors’ outcomes, but provided less insight into the patient/disease characteristics and intensive care therapies (“exposures”) associated with these outcomes. Such insights are essential for improving patients’ long-term outcomes. This report describes the development of a strategy for comprehensively measuring relevant exposures for long-term outcomes research, and presents empirical results from its implementation.
Materials and Methods
A multi-step, iterative process was used to develop the exposures strategy. First, a comprehensive list of potential exposures was generated and subsequently reduced based on feasibility, redundancy, and relevance criteria. Next, data abstraction methods were designed and tested. Finally, the strategy was implemented in 150 acute lung injury patients with iterative refinement.
The strategy resulted in development of >60 unique exposures requiring <45 minutes per patient-day for data collection. The vast majority of exposures had minimal missing data and adequate reliability. These data revealed that evidence-based practices including lower tidal volume ventilation, spontaneous breathing trials, sedation interruption, adequate nutrition, and blood glucose <6.1 mmol/L (110 mg/dl) occurred in only 23%-50% of assessments.
Using a multi-step, iterative process, a comprehensive and feasible exposure measurement strategy for long-term outcomes research was successfully developed and implemented.
Apoptosis is an essential cellular process in multiple diseases and a major pathway for neuronal death in neurodegeneration. The detailed signaling events/pathways leading to apoptosis, especially in neurons, require further elucidation. Here we identify a β-amyloid precursor protein (APP)-interacting protein, designated as appoptosin, whose levels are upregulated in brain samples from Alzheimer’s disease and infarct patients, and in rodent stroke models, as well as in neurons treated with β-amyloid (Aβ) and glutamate. We further demonstrate that appoptosin induces reactive oxygen species release and intrinsic caspase-dependent apoptosis. The physiological function of appoptosin is to transport/exchange glycine/5-amino-levulinic acid across the mitochondrial membrane for heme synthesis. Downregulation of appoptosin prevents cell death and caspase activation caused by glutamate or Aβ insults. APP modulates appoptosin-mediated apoptosis through interaction with appoptosin. Our study identifies appoptosin as a crucial player in apoptosis and a novel proapoptotic protein involved in neuronal cell death, providing a possible new therapeutic target for neurodegenerative disorders and cancers.
The rpoS mRNA, which encodes the master regulator σS of general stress response, requires Hfq-facilitated base pairing with DsrA small RNA for efficient translation at low temperatures. It has recently been proposed that one mechanism underlying Hfq action is to bridge a transient ternary complex by simultaneously binding to rpoS and DsrA. However, no structural evidence of Hfq simultaneously bound to different RNAs has been reported. We detected simultaneous binding of Hfq to rpoS and DsrA fragments. Crystal structures of AU6A•Hfq•A7 and Hfq•A7 complexes were resolved using 1.8- and 1.9-Å resolution, respectively. Ternary complex has been further verified in solution by NMR. In vivo, activation of rpoS translation requires intact Hfq, which is capable of bridging rpoS and DsrA simultaneously into ternary complex. This ternary complex possibly corresponds to a meta-stable transition state in Hfq-facilitated small RNA–mRNA annealing process.
We conducted an unbiased metagenomics survey using plasma from patients with chronic hepatitis B, chronic hepatitis C, autoimmune hepatitis (AIH), non-alcoholic steatohepatitis (NASH), and patients without liver disease (control). RNA and DNA libraries were sequenced from plasma filtrates enriched in viral particles to catalog virus populations. Hepatitis viruses were readily detected at high coverage in patients with chronic viral hepatitis B and C, but only a limited number of sequences resembling other viruses were found. The exception was a library from a patient diagnosed with hepatitis C virus (HCV) infection that contained multiple sequences matching GB virus C (GBV-C). Abundant GBV-C reads were also found in plasma from patients with AIH, whereas Torque teno virus (TTV) was found at high frequency in samples from patients with AIH and NASH. After taxonomic classification of sequences by BLASTn, a substantial fraction in each library, ranging from 35% to 76%, remained unclassified. These unknown sequences were assembled into scaffolds along with virus, phage and endogenous retrovirus sequences and then analyzed by BLASTx against the non-redundant protein database. Nearly the full genome of a heretofore-unknown circovirus was assembled and many scaffolds that encoded proteins with similarity to plant, insect and mammalian viruses. The presence of this novel circovirus was confirmed by PCR. BLASTx also identified many polypeptides resembling nucleo-cytoplasmic large DNA viruses (NCLDV) proteins. We re-evaluated these alignments with a profile hidden Markov method, HHblits, and observed inconsistencies in the target proteins reported by the different algorithms. This suggests that sequence alignments are insufficient to identify NCLDV proteins, especially when these alignments are only to small portions of the target protein. Nevertheless, we have now established a reliable protocol for the identification of viruses in plasma that can also be adapted to other patient samples such as urine, bile, saliva and other body fluids.
Protein is quantitatively the most expensive nutrient in swine diets. Hence it is imperative to understand the physiological roles played by amino acids in growth, development, lactation, reproduction, and health of pigs to improve their protein nutrition and reduce the costs of pork production. Due to incomplete knowledge of amino acid biochemistry and nutrition, it was traditionally assumed that neonatal, post-weaning, growing-finishing, and gestating pigs could synthesize sufficient amounts of all "nutritionally nonessential amino acids" (NEAA) to support maximum production performance. Therefore, over the past 50 years, much emphasis has been placed on dietary requirements of nutritionally essential amino acids as building blocks for tissue proteins. However, a large body of literature shows that NEAA, particularly glutamine, glutamate, arginine and proline regulate physiological functions via cell signaling pathways, such as mammalian target of rapamycin, AMP-activated protein kinase, extracellular signal-related kinase, Jun kinase, mitogen-activated protein kinase, and NEAA-derived gaseous molecules (e.g., nitric oxide, carbon monoxide, and hydrogen sulfide). Available evidence shows that under current feeding programs, only 70% and 55% of dietary amino acids are deposited as tissue proteins in 14-day-old sow-reared piglets and in 30-day-old pigs weaned at 21 days of age, respectively. Therefore, there is an urgent need to understand the roles and dietary requirements of NEAA in swine nutrition. This review highlights the basic biochemistry and physiology of absorption and utilization of amino acids in young pigs to enhance the efficacy of utilization of dietary protein and to minimize excretion of nitrogenous wastes from the body.
Amino acids; Metabolism; Nutrition; Pigs
Quantitative real-time polymerase chain reaction (qPCR) has been previously applied to estimate transgene copy number in transgenic plants. However, the results can be erroneous owing to inaccurate estimation of PCR efficiency. Here, a novel qPCR approach, named standard addition qPCR (SAQPCR), was devised to accurately determine transgene copy number without the necessity of obtaining PCR efficiency data. The procedures and the mathematical basis for the approach are described. A recombinant plasmid harboring both the internal reference gene and the integrated target gene was constructed to serve as the standard DNA. It was found that addition of suitable amounts of standard DNA to test samples did not affect PCR efficiency, and the guidance for selection of suitable cycle numbers for analysis was established. Samples from six individual T0 tomato (Solanum lycopersicum) plants were analyzed by SAQPCR, and the results confirmed by Southern blot analysis. The approach produced accurate results and required only small amounts of plant tissue. It can be generally applied to analysis of different plants and transgenes. In addition, it can also be applied to zygosity analysis.
Recent data has associated improved survival after hemorrhagic shock with the early use of plasma based resuscitation. Our lab has shown that FFP5 has decreased hemostatic potential compared to freshly thawed plasma (FFP0). We hypothesized that FFP5 would increase bleeding and mortality compared to FFP0 in a rodent bioassay model of uncontrolled liver hemorrhage.
Hemostatic potential of plasma was assessed with the Calibrated Automated Thrombogram (CAT) assay. Rats underwent isovolemic hemodilution by 15% of blood volume with the two human plasma groups (FFP0 and FFP5) and two controls (sham and lactated Ringers). A liver injury was created by excising a portion of liver resulting in uncontrolled hemorrhage. Rats that lived for 30 minutes after liver injury were resuscitated to their baseline blood pressure and followed for 6 hours. Hemostasis was assessed by thromboelastography.
Hemostatic potential of FFP5 decreased significantly in all areas measured in the CAT assay as compared to FFP0 (p<0.01). In the FFP5 group overall survival was 54%, compared to 100% in the FFP0 and sham group (p=0.03). For animals that survived 30 minutes and were resuscitated, there was no difference in bleeding and/or coagulopathy between groups. Irrespective of treatment, animals that died following resuscitation demonstrated increased intraperitoneal fluid volume (14.85 ± 1.9 mL vs. 7.02 ± 0.3 mL, p<0.001).
In this model of mild pre-injury hemodilution with plasma, rats that received FFP5 had decreased survival after uncontrolled hemorrhage from hepatic injury. There were no differences in coagulation function or intraperitoneal fluid volume between the two plasma groups.
thawed plasma; resuscitation; blood storage; endothelial permeability
Growing evidence supports BH3-interacting domain death agonist (Bid) playing a dual role in DNA damage response. However, the effects of Bid on hepatocellular carcinoma (HCC) cell proliferation in response to etoposide-induced DNA damage have not been sufficiently investigated.
Using a stable Bid-overexpression HCC cell line, Bid/PLC/PRF/5, overexpression of Bid promoted loss of viability in response to etoposide-induced DNA damage. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]- and BrdU (5′-bromo-2′-deoxyuridine)-labeling assays revealed that etoposide-inhibited HCC cells grew in concentration-and time-dependent manners. The phosphorylations of Akt and mitogen-activated protein kinases (MAPKs) in response to etoposide-induced DNA damage were analyzed by Western blotting.
The survival rates of 100 μM etoposide on the cells with control vector and Bid/PLC/PRF/5 at 48 hours amounted to 71% ± 0.75% and 59% ± 0.60% with MTT assay, and similar results of 85% ± 0.08% and 63% ± 0.14% with BrdU-labeling assay respectively. Moreover, overexpression of Bid sensitized the cells to apoptosis at a high dose of etoposide (causing irreparable damage). However, it had little effect on the proliferation at a low dose of etoposide (repairable damage). Furthermore, the phosphorylation status of Akt and MAPKs were investigated. Overexpression of Bid suppressed the activation of Akt with respect to etoposide-induced DNA damage. Similar to Akt, the levels of phosphorylated p38 and phosphorylated c-Jun were attenuated by Bid-overexpression. On the contrary, the level of phosphorylated ERK1/2 was sustained at a high level, especially in Bid/PLC/PRF/5 cells.
Taken together, these results suggest that overexpression of Bid suppressed the activation of Akt, p38, and c-Jun, and promoted the activation of ERK1/2 induced by etoposide, suggesting that the promotion of ERK1/2 activation may have a negative effect on Bid-mediated HCC DNA damage induced by etoposide.
HCC; mitogen-activated protein kinases; BH3-interacting domain death agonist
The mammalian circadian system controls various physiology processes and behavior responses by regulating thousands of circadian genes with rhythmic expressions. In this study, we redefined circadian-regulated genes based on published results in the mouse liver and compared them with other gene groups defined relative to circadian regulations, especially the non-circadian-regulated genes expressed in liver at multiple molecular levels from gene position to protein expression based on integrative analyses of different datasets from the literature. Based on the intra-tissue analysis, the liver circadian genes or LCGs show unique features when compared to other gene groups. First, LCGs in general have less neighboring genes and larger in both genomic and 3′-UTR lengths but shorter in CDS (coding sequence) lengths. Second, LCGs have higher mRNA and protein abundance, higher temporal expression variations, and shorter mRNA half-life. Third, more than 60% of LCGs form major co-expression clusters centered in four temporal windows: dawn, day, dusk, and night. In addition, larger and smaller LCGs are found mainly expressed in the day and night temporal windows, respectively, and we believe that LCGs are well-partitioned into the gene expression regulatory network that takes advantage of gene size, expression constraint, and chromosomal architecture. Based on inter-tissue analysis, more than half of LCGs are ubiquitously expressed in multiple tissues but only show rhythmical expression in one or limited number of tissues. LCGs show at least three-fold lower expression variations across the temporal windows than those among different tissues, and this observation suggests that temporal expression variations regulated by the circadian system is relatively subtle as compared with the tissue expression variations formed during development. Taken together, we suggest that the circadian system selects gene parameters in a cost effective way to improve tissue-specific functions by adapting temporal variations from the environment over evolutionary time scales.
Acute lung injury (ALI) is a condition characterized by acute onset of severe hypoxemia and bilateral pulmonary infiltrates. ALI patients typically require mechanical ventilation in an intensive care unit. Low tidal volume ventilation (LTVV), a time-varying dynamic treatment regime, has been recommended as an effective ventilation strategy. This recommendation was based on the results of the ARMA study, a randomized clinical trial designed to compare low vs. high tidal volume strategies (The Acute Respiratory Distress Syndrome Network, 2000) . After publication of the trial, some critics focused on the high non-adherence rates in the LTVV arm suggesting that non-adherence occurred because treating physicians felt that deviating from the prescribed regime would improve patient outcomes. In this paper, we seek to address this controversy by estimating the survival distribution in the counterfactual setting where all patients assigned to LTVV followed the regime. Inference is based on a fully Bayesian implementation of Robins’ (1986) G-computation formula. In addition to re-analyzing data from the ARMA trial, we also apply our methodology to data from a subsequent trial (ALVEOLI), which implemented the LTVV regime in both of its study arms and also suffered from non-adherence.
Bayesian inference; Causal inference; Dynamic treatment regime; G-computation formula
In high-dimensional model selection problems, penalized least-square approaches have been extensively used. This paper addresses the question of both robustness and efficiency of penalized model selection methods, and proposes a data-driven weighted linear combination of convex loss functions, together with weighted L1-penalty. It is completely data-adaptive and does not require prior knowledge of the error distribution. The weighted L1-penalty is used both to ensure the convexity of the penalty term and to ameliorate the bias caused by the L1-penalty. In the setting with dimensionality much larger than the sample size, we establish a strong oracle property of the proposed method that possesses both the model selection consistency and estimation efficiency for the true non-zero coefficients. As specific examples, we introduce a robust method of composite L1-L2, and optimal composite quantile method and evaluate their performance in both simulated and real data examples.
Composite QMLE; LASSO; Model Selection; NP Dimensionality; Oracle Property; Robust statistics; SCAD
The role of naturally occurring regulatory T cells (Treg) in the control of the development of systemic lupus erythematosus (SLE) has not been well defined. Therefore, we dissect the phenotypically heterogeneous CD4+FoxP3+ T cells into subpopulations during the dynamic SLE development.
To evaluate the proliferative and suppressive capacities of different CD4+ T cell subgroups between active SLE patients and healthy donors, we employed CD45RA and CD25 as surface markers and carboxyfluorescein diacetatesuccinimidyl ester (CFSE) dilution assay. In addition, multiplex cytokines expression in active SLE patients was assessed using Luminex assay. Here, we showed a significant increase in the frequency of CD45RA+FoxP3low naive Treg cells (nTreg cells) and CD45RA−FoxP3low (non-Treg) cells in patients with active SLE. In active SLE patients, the increased proportions of CD45RA+FoxP3low nTreg cells were positively correlated with the disease based on SLE disease activity index (SLEDAI) and the status of serum anti-dsDNA antibodies. We found that the surface marker combination of CD25+CD45RA+ can be used to defined CD45RA+FoxP3low nTreg cells for functional assays, wherein nTreg cells from active SLE patients demonstrated defective suppression function. A significant correlation was observed between inflammatory cytokines, such as IL-6, IL-12 and TNFα, and the frequency of nTreg cells. Furthermore, the CD45RA+FoxP3low nTreg cell subset increased when cultured with SLE serum compared to healthy donor serum, suggesting that the elevated inflammatory cytokines of SLE serum may promote nTreg cell proliferation/expansion.
Our results indicate that impaired numbers of functional CD45RA+FoxP3low naive Treg cell and CD45RA−FoxP3low non-suppressive T cell subsets in inflammatory conditions may contribute to SLE development. Therefore, analysis of subsets of FoxP3+ T cells, using a combination of FoxP3, CD25 and CD45RA, rather than whole FoxP3+ T cells, will help us to better understand the pathogenesis of SLE and may lead to the development of new therapeutic strategies.
Gut microbiota has become an integral component of the host, and received increasing attention. However, for many domestic animals, information on the microbiota is insufficient and more effort should be exerted to manage the gastrointestinal bacterial community. Understanding the factors that influence the composition of microbial community in the host alimentary canal is essential to manage or improve the microbial community composition. In the present study, 16S rRNA gene sequence-based comparisons of the bacterial communities in the grass carp (Ctenopharyngodon idellus) intestinal contents and fish culture-associated environments are performed. The results show that the fish intestinal microbiota harbors many cellulose-decomposing bacteria, including sequences related to Anoxybacillus, Leuconostoc, Clostridium, Actinomyces, and Citrobacter. The most abundant bacterial operational taxonomic units (OTUs) in the grass carp intestinal content are those related to feed digestion. In addition, the potential pathogens and probiotics are important members of the intestinal microbiota. Further analyses show that grass carp intestine holds a core microbiota composed of Proteobacteria, Firmicutes, and Actinobacteria. The comparison analyses reveal that the bacterial community in the intestinal contents is most similar to those from the culture water and sediment. However, feed also plays significant influence on the composition of gut microbiota.