Phenotypes of lung smooth muscle cells in health and disease are poorly characterized. This is due, in part, to a lack of methodologies that allow for the independent and direct isolation of bronchial smooth muscle cells (BSMCs) and vascular smooth muscle cells (VSMCs) from the lung. In this paper, we describe the development of a bi-fluorescent mouse that permits purification of these two cell populations by cell sorting. By subjecting this mouse to an acute allergen based-model of airway inflammation that exhibits many features of asthma, we utilized this tool to characterize the phenotype of so-called asthmatic BSMCs. First, we examined the biophysical properties of single BSMCs from allergen sensitized mice and found increases in basal tone and cell size that were sustained ex vivo. We then generated for the first time, a comprehensive characterization of the global gene expression changes in BSMCs isolated from the bi-fluorescent mice with allergic airway inflammation. Using statistical methods and pathway analysis, we identified a number of differentially expressed mRNAs in BSMCs from allergen sensitized mice that code for key candidate proteins underlying changes in matrix formation, contractility, and immune responses. Ultimately, this tool will provide direction and guidance for the logical development of new markers and approaches for studying human lung smooth muscle.
Despite improvements in treatment strategies for head and neck squamous cell carcinoma (HNSCC), outcomes have not significantly improved; highlighting the importance of identifying novel therapeutic approaches to target this disease. To address this challenge, we proceeded to evaluate the role of iron in HNSCC.
Expression levels of iron-related genes were evaluated in HNSCC cell lines using quantitative RT-PCR. Cellular phenotypic effects were assessed using viability (MTS), clonogenic survival, BrdU, and tumor formation assays. The prognostic significance of iron-related proteins was determined using immunohistochemistry.
In a panel of HNSCC cell lines, hemochromatosis (HFE) was one of the most overexpressed genes involved in iron regulation. In vitro knockdown of HFE in HNSCC cell lines significantly decreased hepcidin (HAMP) expression and intracellular iron level. This in turn, resulted in a significant decrease in HNSCC cell viability, clonogenicity, DNA synthesis, and Wnt signalling. These cellular changes were reversed by re-introducing iron back into HNSCC cells after HFE knockdown, indicating that iron was mediating this phenotype. Concordantly, treating HNSCC cells with an iron chelator, ciclopirox olamine (CPX), significantly reduced viability and clonogenic survival. Finally, patients with high HFE expression experienced a reduced survival compared to patients with low HFE expression.
Our data identify HFE as potentially novel prognostic marker in HNSCC that promotes tumour progression via HAMP and elevated intracellular iron levels, leading to increased cellular proliferation and tumour formation. Hence, these findings suggest that iron chelators might have a therapeutic role in HNSCC management.
Few mitochondrial gene rearrangements are found in vertebrates and large-scale changes in these genomes occur even less frequently. It is difficult, therefore, to propose a mechanism to account for observed changes in mitogenome structure. Mitochondrial gene rearrangements are usually explained by the recombination model or tandem duplication and random loss model.
In this study, the complete mitochondrial genomes of four flatfishes, Crossorhombus azureus (blue flounder), Grammatobothus krempfi, Pleuronichthys cornutus, and Platichthys stellatus were determined. A striking finding is that eight genes in the C. azureus mitogenome are located in a novel position, differing from that of available vertebrate mitogenomes. Specifically, the ND6 and seven tRNA genes (the Q, A, C, Y, S1, E, P genes) encoded by the L-strand have been translocated to a position between tRNA-T and tRNA-F though the original order of the genes is maintained.
These special features are used to suggest a mechanism for C. azureus mitogenome rearrangement. First, a dimeric molecule was formed by two monomers linked head-to-tail, then one of the two sets of promoters lost function and the genes controlled by the disabled promoters became pseudogenes, non-coding sequences, and even were lost from the genome. This study provides a new gene-rearrangement model that accounts for the events of gene-rearrangement in a vertebrate mitogenome.
Overexpression of the sonic hedgehog (SHH) signaling pathway is an essential characteristic of pancreatic cancer stem cells (PCSCs) and arsenic trioxide (ATO) is described as a SHH inhibitor. This study evaluates whether ATO has the potential to inhibit viability of PCSCs via binding to SHH-Gli proteins.
Cell counting kit-8 and flow cytometry were used for analyzing apoptosis in cells in vitro. The animal model was an athymic nude mouse model bearing subcutaneous xenografts of SW1990 pancreatic cancer cells. The terminal deoxynucleotidyl transferase dUTP nick end labeling assay and immunohistochemistry were used for tumor tissue analysis. The interaction between Gli1 and ATO was examined by a confocal system and an ultraviolet absorption spectrum assay.
ATO induced apoptosis in pancreatic cancer cells, especially CD24+CD44+ cells in vitro. Combination treatment of ATO and low dose gemcitabine inhibited tumor growth by 60.9% (P = 0.004), and decreased the expression of CD24, CD44, and aldehyde dehydrogenase 1 family, member A1 significantly in vivo. ATO changed the structure of the recombinant Gli1 zinc finger peptides in a cell-free condition and the binding action of ATO to recombinant Gli1 was observed in cultured pancreatic cancer cells.
ATO may have the potential to inhibit viability of PCSCs via binding to SHH-Gli proteins in vitro and in vivo.
pancreatic cancer; stem cells; gemcitabine; arsenic trioxide; sonic hedgehog; Gli
Direct reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) provides an invaluable resource for regenerative medicine. Because of some ethical and logistical barriers, human iPSCs cannot be used to generate a chimera, which is one of markers representing pluripotency. As the most attractive model for preclinical studies, pigs offer another path to improve clinical medicine. In this study, porcine adult stem cells (pASCs), including adipose mesenchymal stem cells (AMSCs) and bone marrow mesenchymal stem cells (BMSCs), were collected and cultured under the same conditions in vitro. Real-time PCR, immunocytochemical staining, apoptosis analysis, and induced differentiation and reprogramming techniques were used to investigate the proliferative capacity and pluripotent characteristics of pASCs. Our results showed that both AMSCs and BMSCs displayed a similar immunophenotype, and their proliferative capacity appeared as a downward trend as the cell passage number increased. The cell proliferative capacity of AMSCs was significantly lower than that of BMSCs (p<0.05). Moreover, each type of pASCs went through 20 passages without undergoing alterations in the expression of reprogramming transcriptional factors (Oct4, Sox2, c-Myc, and Nanog). All pASCs had adipogenic and osteogenic differentiation potential. In addition, they also could be reprogrammed to pig induced pluripotent stem cells (piPSCs) with similar time and efficiency. In conclusion, porcine BMSCs had a higher proliferative capacity than AMSCs, and the pluripotency of pASCs was stable in long-term culture.
The outer domain of the HIV-1 gp120 envelope glycoprotein contains the epitope for broadly neutralizing antibodies directed to the CD4-binding site, many of which are able to neutralize over 90% of circulating HIV-1 isolates. While the outer domain is conformationally more stable than other portions of the HIV-1 envelope, efforts to express the outer domain as an immunogen for eliciting broadly neutralizing antibodies have not been successful, potentially because natural outer domain variants do not bind strongly to antibodies such as VRC01. In this study, we optimized the antigenic properties of the HIV-1 Env outer domain to generate OD4.2.2, from the KER2018 strain of clade A HIV-1, enabling it to bind antibodies such as VRC01 with nanomolar affinity. The crystal structure of OD4.2.2 in complex with VRC-PG04 was solved at 3.0-Å resolution and compared to known crystal structures including (i) the structure of core gp120 bound by VRC-PG04 and (ii) a circularly permutated version of the outer domain in complex with antibody PGT128. Much of the VRC-PG04 epitope was preserved in the OD4.2.2 structure, though with altered N and C termini conformations. Overall, roughly one-third of the outer domain structure appeared to be fixed in conformation, independent of alterations in termini, clade, or ligand, while other portions of the outer domain displayed substantial structural malleability. The crystal structure of OD4.2.2 with VRC-PG04 provides atomic-level details for an HIV-1 domain recognized by broadly neutralizing antibodies and insights relevant to the rational design of an immunogen that could elicit such antibodies by vaccination.
The down-regulation of microRNA-196b (miR-196b) has been reported, but its contribution to cervical cancer progression remains to be investigated. In this study, we first demonstrated that miR-196b down-regulation was significantly associated with worse disease-free survival (DFS) for cervical cancer patients treated with combined chemo-radiation. Secondly, using a tri-modal approach for target identification, we determined that homeobox-B7 (HOXB7) was a bona fide target for miR-196b, and in turn, vascular endothelial growth factor (VEGF) was a downstream transcript regulated by HOXB7. Reconstitution of miR-196b expression by transient transfection resulted in reduced cell growth, clonogenicity, migration and invasion in vitro, as well as reduced tumor angiogenesis and tumor cell proliferation in vivo. Concordantly, siRNA knockdown of HOXB7 or VEGF phenocopied the biological effects of miR-196b over-expression. Our findings have demonstrated that the miR-196b/HOXB7/VEGF pathway plays an important role in cervical cancer progression; hence targeting this pathway could be a promising therapeutic strategy for the future management of this disease.
Fibrodysplasia ossificans progressiva (FOP) is a rare heritable disease characterized by progressive heterotopic ossification of connective tissues, for which there is presently no definite treatment. A recurrent activating mutation (c.617G→A; R206H) of activin receptor-like kinase 2 (ACVR1/ALK2), a BMP type I receptor, has been shown as the main cause of FOP. This mutation constitutively activates the BMP signaling pathway and initiates the formation of heterotopic bone. In this study, we have designed antisense oligonucleotides (AONs) to knockdown mouse ALK2 expression by means of exon skipping. The ALK2 AON could induce exon skipping in cells, which was accompanied by decreased ALK2 mRNA levels and impaired BMP signaling. In addition, the ALK2 AON potentiated muscle differentiation and repressed BMP6-induced osteoblast differentiation. Our results therefore provide a potential therapeutic approach for the treatment of FOP disease by reducing the excessive ALK2 activity in FOP patients.
The glycoprotein 130 (gp130) dependent family of cytokines comprises interleukin-6 (IL-6), IL-11, leukemia inhibitory factor (LIF), cardiotrophin-like cytokine (CLC), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1) and oncostatin M (OSM). These cytokines share the membrane gp130 as a common signal transducer. Recently, it was demonstrated that IL-6 promotes, whereas LIF inhibits fetal lung branching. Thus, in this study, the effects on fetal lung morphogenesis of the other classical members of the gp130-type cytokines (IL-11, CLC, CNTF, CT-1 and OSM) were investigated. We also provide the first description of these cytokines and their common gp130 receptor protein expression patterns during rat lung development. Fetal rat lung explants were cultured in vitro with increasing concentrations of IL-11, CLC, CNTF, CT-1 and OSM. Treated lung explants were morphometrically analyzed and assessed for MAPK, PI3K/AKT and STAT3 signaling modifications. IL-11, which similarly to IL-6 acts through a gp130 homodimer receptor, significantly stimulated lung growth via p38 phosphorylation. On the other hand, CLC, CNTF, CT-1 and OSM, whose receptors are gp130 heterodimers, inhibited lung growth acting in different signal-transducing pathways. Thus, the present study demonstrated that although cytokines of the gp130 family share a common signal transducer, there are specific biological activities for each cytokine on lung development. Indeed, cytokine signaling through gp130 homodimers stimulate, whereas cytokine signaling through gp130 heterodimers inhibit lung branching.
Poly(lauryl methacrylate) (PLMA) thin film doped with Mn:ZnSe quantum dots (QDs) was spin-deposited on the front surface of Si solar cell for enhancing the solar cell efficiency via photoluminescence (PL) conversion. Significant solar cell efficiency enhancements (approximately 5% to 10%) under all-solar-spectrum (AM0) condition were observed after QD-doped PLMA coatings. Furthermore, the real contribution of the PL conversion was precisely assessed by investigating the photovoltaic responses of the QD-doped PLMA to monochromatic and AM0 light sources as functions of QD concentration, combined with reflectance and external quantum efficiency measurements. At a QD concentration of 1.6 mg/ml for example, among the efficiency enhancement of 5.96%, about 1.04% was due to the PL conversion, and the rest came from antireflection. Our work indicates that for the practical use of PL conversion in solar cell performance improvement, cautions are to be taken, as the achieved efficiency enhancement might not be wholly due to the PL conversion.
Si solar cell; Quantum dots; Photoluminescence conversion; Antireflectin; 78.55.-m; 84.60.Jt
Plant height is an important botanical feature closely related to yield. Two populations consisting of 118 and 262 accessions respectively were used to identify elite alleles for plant height and to validate their allelic effects. Plant height was measured from the early booting to the flowering stages. Simple sequence repeat markers for candidate quantitative trait locus (QTL) regions with large effects identified in a doubled haploid (DH) population (Hanxuan 10 × Lumai 14) were selected for further verification by association analysis. Nine loci significantly (P < 0.001) associated with plant height were detected 13 times in the population with 118 accessions. Three loci (Xgwm11-1B, Xwmc349-4B and Xcfd23-4D) were identified in three, two and two periods of plant height growth, respectively. Markers Xbarc168-2D, Xgwm249-2D, Xwmc349-4B, Xcfd23-4D and Xgwm410-5A located at or near additive QTL regions in the DH population proved to coincide with known Rht loci. The results showed a consistency between linkage analysis and association mapping, and also confirmed the value of fine mapping of QTL through combined linkage and association analyses. For final plant height, the alleles Xgwm11-1B208, Xwmc349-4B103 and Xcfd23-4D202 exhibited negative effects, i.e. reducing plant height; Xwmc349-4B101 and Xcfd23-4D205 showed significant positive effects. A second larger population (262 accessions) was used to validate the effects of these large-effect alleles and the efficacy of pyramiding in eight environments (year × site × water regime combinations). Strong correlations between final plant height and numbers of large-effect alleles indicated that the alleles contributed additively to plant height. The additive effects showed that pyramiding elite alleles for target traits has significant potential for wheat breeding.
Electronic supplementary material
The online version of this article (doi:10.1007/s11032-013-9873-5) contains supplementary material, which is available to authorized users.
Association mapping; Development; Elite allele; Gene pyramiding; Plant height; Triticum aestivum
Epigenetic regulation participates broadly in many fundamentally cellular and physiological processes. In this study, we found that DDB1, a protein originally identified as a factor involved in DNA repairing, plays important roles in regulating organ size, growth habit and photosynthesis in tomato via an epigenetic manner. We generated transgenic tomato plants overexpressing an alternatively spliced DDB1 transcript (DDB1F, prevalently present in tomato tissues) and found the primary transformants displayed small-fruited “cherry tomato” in companion with strikingly enhanced shoot branching and biomass, dark-green leaves with elevated chlorophyll accumulation, and increased soluble solids in fruits. Significantly, these phenotypic alterations did not segregate with the DDB1F transgene in subsequent generations, suggesting that the effect of DDB1F on multiple agronomic traits is implemented via an epigenetic manner and is inheritable over generations. We speculate that DDB1, as a core subunit in the recently identified CUL4-based E3 ligase complex, mediates the 26S proteasome-dependent degradation of a large number of proteins, some of which might be required for perpetuating epigenetic marks on chromatins.
DDB1; epigenetics; organogenesis; fruit size; growth habit; soluble solids; tomato
Accumulating studies have demonstrated that 1,25-Dihydroxyvitamin D(3) (1,25(OH)2D3) reduces proteinuria and protects podocytes from injury. Recently, urokinase receptor (uPAR) and its soluble form have been shown to cause podocyte injury and focal segmental glomerulosclerosis (FSGS). Here, our findings showed that 1,25(OH)2D3 did inhibit podocyte uPAR expression and attenuate proteinuria and podocyte injury.
In this study, the antiproteinuric effect of 1,25(OH)2D3 was examined in the lipopolysaccharide mice model of transient proteinuria (LPS mice) and in the 5/6 nephrectomy rat FSGS model(NTX rats). uPAR protein expression were tested by flow cytometry, immune cytochemistry and western blot analysis, and uPAR mRNA expression by real-time quantitative PCR in cultured podocytes and kidney glomeruli isolated from mice and rats. Podocyte motility was observed by transwell migration assay and wound healing assay. Podocyte foot processes effacement was identified by transmission electron microscopy. We found that 1,25(OH)2D3 inhibited podocyte uPAR mRNA and protein synthesis in LPS-treated podocytes, LPS mice and NTX rats, along with 1,25(OH)2D3 reducing proteinuria in NTX rats and LPS mice.1,25(OH)2D3 reduced glomerulosclerosis in NTX rats and alleviated podocyte foot processes effacement in LPS mice. Transwell migration assay and wound healing assay showed that LPS-induced podocyte motility, irrespective of random or directed motility, were substantially reduced by 1,25(OH)2D3.
Our results demonstrated that 1,25(OH)2D3 inhibited podocyte uPAR expression in vitro and in vivo, which may be an unanticipated off target effect of 1,25(OH)2D3 and explain its antiproteinuric effect in the 5/6 nephrectomy rat FSGS model and the LPS mouse model of transient proteinuria.
Cases of ectopic pregnancy (EP) following levonorgestrel (LNG) emergency contraception (EC) failure were reported, however, the effects of LNG on tubal microenvironment or chorionic villi in EP have not yet been documented.
Fifty-five women with tubal pregnancy were divided into two groups according to whether LNG-EC was administrated during the cycle of conception. The serum concentrations of beta-hCG, E2 and P were measured. The mRNA and protein expressions of estrogen and progesterone receptors, leukemia inhibitory factor, vascular endothelial growth factor, inducible nitric oxide synthase, and endocannabinoid receptor - CB1 in the ectopic implantation site and chorionic villi were examined.
Compared to those unexposed to LNG-EC, women with tubal pregnancy exposed to LNG-EC during the cycle of conception had no statistically significances in the serum concentrations of beta-hCG, E2 P, nor in the pathological types of tubal pregnancy or the expressions of ER-alpha, PR, LIF, VEGF, iNOS and CB1.
The expressions of candidate molecules in the fallopian tube and chorionic villi were not altered by exposure to LNG-EC. A routine therapy with no additional intervention might thus be applied to tubal pregnancy exposed to LNG-EC.
Tubal pregnancy; Levonorgestrel; Emergency contraception; Fallopian tube; Chorionic villi
As a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors, the glucocorticoid receptor (GR) is essential for normal embryonic development. To date, the role of mesenchymal glucocorticoid signaling during development has not been fully elucidated. In the present study, we investigated the role of the GR during embryogenesis specifically in mesenchymal tissues. To this aim, we crossed GRflox mice with Dermo1-Cre mice to generate GRDermo1 mice, where the GR gene was deleted within mesenchymal cells. Compared to their wild type littermates, GRDermo1 mice displayed severe pulmonary atelectasis, defects in abdominal wall formation resulting in intestinal herniation, abnormal extracellular matrix synthesis in connective tissues and high postnatal lethality. Lungs of GRDermo1 mice failed to progress from the canalicular to saccular stage, as evidenced by the presence of immature air sacs, thickened interstitial mesenchyme and an underdeveloped vascular network between E17.5 and E18.5. Furthermore, myofibroblasts and vascular smooth muscle cells, although present in normal numbers in GRDermo1 animals, were characterized by significantly reduced elastin synthesis, whilst epithelial lining cells of the immature saccules were poorly differentiated. A marked reduction in normal elastin and collagen deposits were also observed in connective tissues adjacent to the umbilical hernia. This study demonstrates that eliminating the GR in cells of the mesenchymal lineage results in marked effects on interstitial fibroblast function, including a significant decrease in elastin synthesis. This results in lung atelectasis and postnatal lethality, as well as additional and hitherto unrecognized developmental defects in abdominal wall formation. In addition, altered glucocorticoid signaling in the mesenchyme attenuates normal lung epithelial differentiation.
The effects of large-dose oral arginine administration on the secretion of insulin by islet β-cells in healthy adults were determined. Eight non-obese healthy volunteers with normal glucose tolerance participated randomly in tests with four stages (with an interval of at least 3 days): the 300 ml purified water stage (PWS), the 75 g glucose stage (GSS), the 30 g arginine stage (ARS) and the 75 g glucose with 30 g arginine stage (GAS). Venous blood samples were collected to detect the concentrations of glucose and insulin at baseline (0) and at 15, 30, 45, 60 and 120 min after drug administration. The glucose and insulin levels were steady in the PWS. The remaining three stages had similarly shaped insulin concentration-time curves, which differed from that of the PWS. The peak concentration of blood insulin and the net incremental area under the curve of blood insulin in the GSS, ARS and GAS were significantly higher compared with those in the PWS (P<0.05). In the ARS, the glucose levels remained stable; however, the net incremental area under the curve for blood insulin in the ARS was much lower compared with that in the GSS or GAS (P<0.05). Large-dose oral arginine administration may slightly stimulate insulin secretion by islet β-cells in healthy adults with normal glucose tolerance in a manner that is independent of glucose concentration.
arginine; insulin; glucose tolerance
Injured endothelium is an important target for drug and/or gene therapy because brain microvascular endothelial cells (BMECs) play critical roles in various pathophysiological conditions. RNA-mediated gene silencing presents a new therapeutic approach for treating such diseases, but major challenge is to ensure minimal toxicity and target delivery of siRNA to injured BMECs. Injured BMECs overexpress tissue factor (TF), which the fusion protein EGFP-EGF1 could be targeted to. In this study, TNF alpha (TNF-α) was chosen as a stimulus for primary BMECs to produce injured endothelium in vitro. The EGFP-EGF1-PLGA nanoparticles (ENPs) with loaded TF-siRNA were used as a new carrier for targeted delivery to the injured BMECs. The nanoparticles then produced intracellular RNA interference against TF. We compared ENP-based transfections with NP-mediated transfections, and our studies show that the ENP-based transfections result in a more efficient downregulation of TF. Our findings also show that the TF siRNA-loaded ENPs had minimal toxicity, with almost 96% of the cells viable 24 h after transfection while Lipofectamine-based transfections resulted in only 75% of the cells. Therefore, ENP-based transfection could be used for efficient siRNA transfection to injured BMECs and for efficient RNA interference (RNAi). This transfection could serve as a potential treatment for diseases, such as stroke, atherosclerosis and cancer.
It is well-established that lipid disorder is an important cardiovascular risk factor, and failure to reach optimal lipid levels significantly contributes to the residual cardiovascular risks. However, limited information is available on the management and the attainment of recommended cholesterol targets in real-world practice in China.
Methods and Results
A nationally representative sample of 12,040 patients with dyslipidemia from 19 provinces and 84 hospitals across China were consecutively enrolled in this survey. Risk stratification and individual cholesterol target was established for all participants. This survey identified a high-risk cohort, with over 50% of patients had hypertension, 37.5% had coronary artery disease, and more than 30% had peripheral artery disease. Thirty-nine percent of all participants received lipid lowering medications. And the majority of them (94.5%) had statins (42.5% with atorvastatin, 29.0% with simvastatin, and 15.2% with rosuvastatin). However, the overall attainment for low-density lipoprotein cholesterol (LDL-C) target is low (25.8%), especially, in female (22.2%), and in patients with increased body mass index (BMI) (38.3% for BMI<18.5, 28.1% for BMI 18.5–24.9, 26.0% for BMI 25.0–29.9, and 17.4% for BMI≥30, P<0.0001). Subgroup analysis also showed the attainment is significantly lower in patients who were stratified into high (19.9%) and very high (21.1%) risk category. In logistic regression analysis, eight factors (BMI, gender, coronary artery disease, systolic and diastolic blood pressure, hypertension, family history of premature coronary artery disease and current smoking) were identified as independent predictors of LDL-C attainment.
Despite the proven benefits of lipid-lowering therapies, current management of dyslipidemia continues to be unsatisfied. A considerable proportion of patients failed to achieve guideline-recommended targets in China, and this apparent treatment gap was more pronounced among patients with increased BMI, higher risk stratification and women.
Read alignment is an ongoing challenge for the analysis of data from sequencing technologies. This article proposes an elegantly simple multi-seed strategy, called seed-and-vote, for mapping reads to a reference genome. The new strategy chooses the mapped genomic location for the read directly from the seeds. It uses a relatively large number of short seeds (called subreads) extracted from each read and allows all the seeds to vote on the optimal location. When the read length is <160 bp, overlapping subreads are used. More conventional alignment algorithms are then used to fill in detailed mismatch and indel information between the subreads that make up the winning voting block. The strategy is fast because the overall genomic location has already been chosen before the detailed alignment is done. It is sensitive because no individual subread is required to map exactly, nor are individual subreads constrained to map close by other subreads. It is accurate because the final location must be supported by several different subreads. The strategy extends easily to find exon junctions, by locating reads that contain sets of subreads mapping to different exons of the same gene. It scales up efficiently for longer reads.
A physical conceptual model for water retention in fractured rocks is derived while taking into account the effect of pore size distribution and tortuosity of capillaries. The formula of calculating relative hydraulic conductivity of fractured rock is given based on fractal theory. It is an issue to choose an appropriate capillary pressure-saturation curve in the research of unsaturated fractured mass. The geometric pattern of the fracture bulk is described based on the fractal distribution of tortuosity. The resulting water content expression is then used to estimate the unsaturated hydraulic conductivity of the fractured medium based on the well-known model of Burdine. It is found that for large enough ranges of fracture apertures the new constitutive model converges to the empirical Brooks-Corey model.
Systemic administration of nicotine increases dopaminergic (DA) neuron firing in the ventral tegmental area (VTA), which is thought to underlie nicotine reward. Here, we report that the medial prefrontal cortex (mPFC) plays a critical role in nicotine-induced excitation of VTA DA neurons. In chloral hydrate-anesthetized rats, extracellular single-unit recordings showed that VTA DA neurons exhibited two types of firing responses to systemic nicotine. After nicotine injection, the neurons with type-I response showed a biphasic early inhibition and later excitation, whereas the neurons with type-II response showed a monophasic excitation. The neurons with type-I, but not type-II, response exhibited pronounced slow oscillations (SO) in firing. Pharmacological or structural mPFC inactivation abolished SO and prevented systemic nicotine-induced excitation in the neurons with type-I, but not type-II, response, suggesting that these VTA DA neurons are functionally coupled to the mPFC and nicotine increases firing rate in these neurons in part through the mPFC. Systemic nicotine also increased the firing rate and SO in mPFC pyramidal neurons. mPFC infusion of a non-α7 nAChR antagonist mecamylamine blocked the excitatory effect of systemic nicotine on the VTA DA neurons with type-I response, but mPFC infusion of nicotine failed to excite these neurons. These results suggest that nAChR activation in the mPFC is necessary, but not sufficient, for systemic nicotine-induced excitation of VTA neurons. Finally, systemic injection of bicuculline prevented nicotine-induced firing alterations in the neurons with type-I response. We propose that the mPFC plays a critical role in systemic nicotine-induced excitation of VTA DA neurons.
nicotine; prefrontal cortex; ventral tegmental area; dopamine neuron; in vivo recording; slow oscillation
Objective: Side population (SP) cells may play a crucial role in tumorigenesis and the recurrence of cancer. Many kinds of cell lines and tissues have demonstrated the presence of SP cells, including several gastric cancer cell lines. This study is aimed to identify the cancer stem-like cells in the SP of gastric cancer cell line MKN-45. Methods: We used fluorescence activated cell sorting (FACS) to sort SP cells in the human gastric carcinoma cell line MKN-45 (cells labeled with Hoechst 33342) and then characterized the cancer stem-like properties of SP cells. Results: This study found that the SP cells had higher clone formation efficiency than major population (MP) cells. Five stemness-related gene expression profiles, including OCT-4, SOX-2, NANOG, CD44, and adenosine triphosphate (ATP)-binding cassette transporters gene ABCG2, were tested in SP and MP cells using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Western blot was used to show the difference of protein expression between SP and MP cells. Both results show that there was significantly higher protein expression in SP cells than in MP cells. When inoculated into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, SP cells show higher tumorigenesis tendency than MP cells. Conclusions: These results indicate that SP cells possess cancer stem cell properties and prove that SP cells from MKN-45 are gastric cancer stem-like cells.
ATP-binding cassette transporters; Side population cells; Stem cells; Benzimidazole (Hoechst 33342); Stomach neoplasm
Tandem repeats (TRs) in the mitochondrial (mt) genome control region have been documented in a wide variety of vertebrate species. The mechanism by which repeated tracts originate and undergo duplication and deletion, however, remains unclear.
We analyzed DNA sequences of mt genome TRs (mtTRs) in the ridged-eye flounder (Pleuronichthys cornutus), and characterized DNA sequences of mtTRs from other vertebrates using the data available in GenBank. Tandem repeats are concentrated in the control regions; however, we found approximately 16.6% of the TRs elsewhere in the mt genome. The flounder mtTRs possess three motif types with hypervariable characteristics at the 3′ end of the control region (CR).
Based on our analysis of this larger dataset of mtTR sequences, we propose a novel model of Pause Melting Misalignment (PMM) to describe the birth and motif indel of tandem repeats. PMM is activated during a pause event in mitochondrial replication in which a dynamic competition between the nascent (N) heavy strand and the displaced (D) heavy strand may lead to the melting of the N-strand from the template (T) light strand. When mispairing occurs during rebinding of the N-strand, one or several motifs can be inserted or deleted in both strands during the next round of mt-replication or repair. This model can explain the characteristics of TRs in available vertebrate mt genomes.