Early stages of vertebrate embryogenesis are characterized by a remarkable series of shape changes. The resulting morphological complexity is driven by molecular, cellular, and tissue-scale biophysical alterations. Operating at the cellular level, extracellular matrix (ECM) networks facilitate cell motility. At the tissue level, ECM networks provide material properties required to accommodate the large-scale deformations and forces that shape amniote embryos. In other words, the primordial biomaterial from which reptilian, avian, and mammalian embryos are molded is a dynamic composite comprised of cells and ECM. Despite its central importance during early morphogenesis we know little about the intrinsic micrometer-scale surface properties of primordial ECM networks. Here we computed, using avian embryos, five textural properties of fluorescently tagged ECM networks--(a) inertia, (b) correlation, (c) uniformity, (d) homogeneity, and (e) entropy. We analyzed fibronectin and fibrillin-2 as examples of fibrous ECM constituents. Our quantitative data demonstrated differences in the surface texture between the fibronectin and fibrillin-2 network in Day 1 (gastrulating) embryos, with the fibronectin network being relatively coarse compared to the fibrillin-2 network. Stage-specific regional anisotropy in fibronectin texture was also discovered. Relatively smooth fibronectin texture was exhibited in medial regions adjoining the primitive streak (PS) compared with the fibronectin network investing the lateral plate mesoderm (LPM), at embryonic stage 5. However, the texture differences had changed by embryonic stage 6, with the LPM fibronectin network exhibiting a relatively smooth texture compared with the medial PS-oriented network. Our data identify, and partially characterize, stage-specific regional anisotropy of fibronectin texture within tissues of a warm-blooded embryo. The data suggest that changes in ECM textural properties reflect orderly time-dependent rearrangements of a primordial biomaterial. We conclude that the ECM microenvironment changes markedly in time and space during the most important period of amniote morphogenesis--as determined by fluctuating textural properties.

The orphan nuclear receptor COUP-TFII plays an undefined role in breast cancer. Previously we reported lower COUP-TFII expression in tamoxifen/endocrine-resistant versus sensitive breast cancer cell lines. The identification of COUP-TFII-interacting proteins will help to elucidate its mechanism of action as a transcriptional regulator in breast cancer.

Free-ranging animals often cope with fluctuating environmental conditions such as weather, food availability, predation risk, the requirements of breeding, and the influence of anthropogenic factors. Consequently, researchers are increasingly measuring stress markers, especially glucocorticoids, to understand stress, disturbance, and population health. Studying free-ranging animals, however, comes with numerous difficulties posed by environmental conditions and the particular characteristics of study species. Performing measurements under either physical restraint or chemical sedation may affect the physiological variable under investigation and lead to values that may not reflect the standard functional state of the animal. This study measured the stress response resulting from different handling conditions in northern elephant seals and any ensuing influences on carbohydrate metabolism. Endogenous glucose production (EGP) was measured using [6-(3)H]glucose and plasma cortisol concentration was measured from blood samples drawn during three-hour measurement intervals. These measurements were conducted in weanlings and yearlings with and without the use of chemical sedatives--under chemical sedation, physical restraint, or unrestrained. We compared these findings with measurements in adult seals sedated in the field. The method of handling had a significant influence on the stress response and carbohydrate metabolism. Physically restrained weanlings and yearlings transported to the lab had increased concentrations of circulating cortisol (F(11, 46) = 25.2, p<0.01) and epinephrine (F(3, 12) = 5.8, p = 0.01). Physical restraint led to increased EGP (t = 3.1, p = 0.04) and elevated plasma glucose levels (t = 8.2, p<0.01). Animals chemically sedated in the field typically did not exhibit a cortisol stress response. The combination of anesthetic agents (Telazol, ketamine, and diazepam) used in this study appeared to alleviate a cortisol stress response due to handling in the field without altering carbohydrate metabolism. Measures of hormone concentrations and metabolism made under these conditions are more likely to reflect basal values.

Heart and skeletal inflammation (HSMI) of farmed Atlantic salmon (Salmo salar L.) is a disease characterized by a chronic myocarditis involving the epicardium and the compact and spongious part of the heart ventricle. Chronic myositis of the red skeletal muscle is also a typical finding of HSMI. Piscine reovirus (PRV) has been detected by real-time PCR from farmed and wild salmon with and without typical changes of HSMI and thus the causal relationship between presence of virus and the disease has not been fully determined. In this study we show that the Atlantic salmon reovirus (ASRV), identical to PRV, can be passaged in GF-1 cells and experimental challenge of naïve Atlantic salmon with cell culture passaged reovirus results in cardiac and skeletal muscle pathology typical of HSMI with onset of pathology from 6 weeks, peaking by 9 weeks post challenge. ASRV replicates in heart tissue and the peak level of virus replication coincides with peak of heart lesions. We further demonstrate mRNA transcript assessment and in situ characterization that challenged fish develop a CD8+ T cell myocarditis.

Chlorpyrifos is of great environmental concern due to its widespread use in the past several decades and its potential toxic effects on human health. Thus, the degradation study of chlorpyrifos has become increasing important in recent years. A fungus capable of using chlorpyrifos as the sole carbon source was isolated from organophosphate-contaminated soil and characterized as Cladosporium cladosporioides Hu-01 (collection number: CCTCC M 20711). A novel chlorpyrifos hydrolase from cell extract was purified 35.6-fold to apparent homogeneity with 38.5% overall recovery by ammoniumsulfate precipitation, gel filtration chromatography and anion-exchange chromatography. It is a monomeric structure with a molecular mass of 38.3 kDa. The pI value was estimated to be 5.2. The optimal pH and temperature of the purified enzyme were 6.5 and 40°C, respectively. No cofactors were required for the chlorpyrifos-hydrolysis activity. The enzyme was strongly inhibited by Hg²⁺, Fe³⁺, DTT, β-mercaptoethanol and SDS, whereas slight inhibitory effects (5-10% inhibition) were observed in the presence of Mn²⁺, Zn²⁺, Cu²⁺, Mg²⁺, and EDTA. The purified enzyme hydrolyzed various organophosphorus insecticides with P-O and P-S bond. Chlorpyrifos was the preferred substrate. The Km and Vmax values of the enzyme for chlorpyrifos were 6.7974 μM and 2.6473 μmol·min⁻¹, respectively. Both NH2-terminal sequencing and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometer (MALDI-TOF-MS) identified an amino acid sequence MEPDGELSALTQGANS, which shared no similarity with any reported organophosphate-hydrolyzing enzymes. These results suggested that the purified enzyme was a novel hydrolase and might conceivably be developed to fulfill the practical requirements to enable its use in situ for detoxification of chlorpyrifos. Finally, this is the first described chlorpyrifos hydrolase from fungus.

The peptide drug candidate NAP (davunetide) has demonstrated protective effects in various in vivo and in vitro models of neurodegeneration. NAP was shown to reduce tau hyperphosphorylation as well as to prevent caspase-3 activation and cytochrome-3 release from mitochondria, both characteristic of apoptotic cell death. Recent studies suggest that caspases may play a role in tau pathology. The purpose of this study was to evaluate the effect of NAP on tau hyperphosphorylation and caspase activity in the same biological system. Our experimental setup used primary neuronal cultures subjected to oxygen-glucose deprivation (OGD), with and without NAP or caspase inhibitor. Cell viability was assessed by measuring mitochondrial activity (MTS assay), and immunoblots were used for analyzing protein level. It was shown that apoptosis was responsible for all cell death occurring following ischemia, and NAP treatment showed a concentration-dependent protection from cell death. Ischemia caused an increase in the levels of active caspase-3 and hyperphosphorylated tau, both of which were prevented by either NAP or caspase-inhibitor treatment. Our data suggest that, in this model system, caspase activation may be an upstream event to tau hyperphosphorylation, although additional studies will be required to fully elucidate the cascade of events.

Airway mucin secretion and MC (mast cell) degranulation must be tightly controlled for homoeostasis of the lungs and immune system respectively. We found the exocytic protein Munc18b to be highly expressed in mouse airway epithelial cells and MCs, and localized to the apical pole of airway secretory cells. To address its functions, we created a mouse with a severely hypomorphic Munc18b allele such that protein expression in heterozygotes was reduced by ~50%. Homozygous mutant mice were not viable, but heterozygotes showed a ~50% reduction in stimulated release of mucin from epithelial cells and granule contents from MCs. The defect in MCs affected only regulated secretion and not constitutive or transporter-mediated secretion. The severity of passive cutaneous anaphylaxis was also reduced by ~50%, showing that reduction of Munc18b expression results in an attenuation of physiological responses dependent on MC degranulation. The Munc18b promoter is controlled by INR (initiator), Sp1 (specificity protein 1), Ets, CRE (cAMP-response element), GRE (glucocorticoid-response element), GATA and E-box elements in airway epithelial cells; however, protein levels did not change during mucous metaplasia induced by allergic inflammation. Taken together, the results of the present study identify Munc18b as an essential gene that is a limiting component of the exocytic machinery of epithelial cells and MCs.

Influenza A virus exerts a large health burden during both yearly epidemics and global pandemics. However, designing effective vaccine and treatment options has proven difficult since the virus evolves rapidly. Therefore, it may be beneficial to identify host proteins associated with viral infection and replication to establish potential new antiviral targets. We have previously measured host protein responses in continuously cultured A549 cells infected with mouse-adapted virus strain A/PR/8/34(H1N1; PR8). We here identify and measure host proteins differentially regulated in more relevant primary human bronchial airway epithelial (HBAE) cells. A total of 3740 cytosolic HBAE proteins were identified by 2D LC-MS/MS, of which 52 were up-regulated ≥2-fold and 41 were down-regulated ≥2-fold after PR8 infection. Up-regulated HBAE proteins clustered primarily into interferon signaling, other host defense processes, and molecular transport, whereas down-regulated proteins were associated with cell death signaling pathways, cell adhesion and motility, and lipid metabolism. Comparison to influenza-infected A549 cells indicated some common influenza-induced host cell alterations, including defense response, molecular transport proteins, and cell adhesion. However, HBAE-specific alterations consisted of interferon and cell death signaling. These data point to important differences between influenza replication in continuous and primary cell lines and/or alveolar and bronchial epithelial cells.

Many plant genes have been identified through whole genome and deep transcriptome sequencing and other methods; yet our knowledge on the function of many of these genes remains limited. The integration and analysis of large gene-expression datasets gives researchers the ability to formalize hypotheses concerning the functionality and interaction between different groups of correlated genes.

During neocortical development, multiple voltage- and ligand-gated ion channels are differentially expressed in neurons thereby shaping their intrinsic electrical properties. One of these voltage-gated ion channels, the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel and its current I(h), is an important regulator of neuronal excitability. Thus far, studies on an early I(h) appearance in rodent neocortex are missing or conflicting. Therefore, we focused our study on perinatal neocortical I(h) and its properties.

Feedback loops, both positive and negative are embedded in the Mitogen Activated Protein Kinase (MAPK) cascade. In the three layer MAPK cascade, both feedback loops originate from the terminal layer and their sites of action are either of the two upstream layers. Recent studies have shown that the cascade uses coupled positive and negative feedback loops in generating oscillations. Two plausible designs of coupled positive and negative feedback loops can be elucidated from the literature; in one design the positive feedback precedes the negative feedback in the direction of signal flow and vice-versa in another. But it remains unexplored how the two designs contribute towards triggering oscillations in MAPK cascade. Thus it is also not known how amplitude, frequency, robustness or nature (analogous/digital) of the oscillations would be shaped by these two designs.

Although mutations and deletions in the p53 tumor suppressor gene lead to resistance to low linear energy transfer (LET) radiation, high-LET radiation efficiently induces cell lethality and apoptosis regardless of the p53 gene status in cancer cells. Recently, it has been suggested that the induction of p53-independent apoptosis takes place through the activation of Caspase-9 which results in the cleavage of Caspase-3 and poly (ADP-ribose) polymerase (PARP). This study was designed to examine if high-LET radiation depresses serine/threonine protein kinase B (PKB, also known as Akt) and Akt-related proteins. Human gingival cancer cells (Ca9-22 cells) harboring a mutated p53 (mp53) gene were irradiated with 2 Gy of X-rays or Fe-ion beams. The cellular contents of Akt-related proteins participating in cell survival signaling were analyzed with Western Blotting 1, 2, 3 and 6h after irradiation. Cell cycle distributions after irradiation were assayed with flow cytometric analysis. Akt-related protein levels decreased when cells were irradiated with high-LET radiation. High-LET radiation increased G(2)/M phase arrests and suppressed the progression of the cell cycle much more efficiently when compared to low-LET radiation. These results suggest that high-LET radiation enhances apoptosis through the activation of Caspase-3 and Caspase-9, and suppresses cell growth by suppressing Akt-related signaling, even in mp53 bearing cancer cells.

Goose circovirus (GCV) presents an immunosuppressive problem in production of geese. The infection's clinical symptoms include growth retardation or feathering disorders but the infection process may remain non-symptomatic what makes the infected birds more susceptible for secondary viral, bacterial and fungal infections. Diagnosis of GCV infection is made by histopathological examination, dot blot hybridization, polymerase chain reaction (PCR) and real-time PCR. However these techniques require application of thermocyclers and qualified staff which may be cost-consuming for some diagnostic units. The aim of this study was to develop loop-mediated isothermal amplification assay (LAMP) as a simple method of GCV detection.

Hypermethylation of promotor CpG islands is a common mechanism that inactivates tumor suppressor genes in cancer. Genes belonging to the RASSF gene family have frequently been reported as epigenetically silenced by promotor methylation in human cancers. Two members of this gene family, RASSF1A and RASSF5A have been reported as methylated in neuroblastoma. Data from our previously performed genome-wide DNA methylation array analysis indicated that other members of the RASSF gene family are targeted by DNA methylation in neuroblastoma.

Ebola viruses (EBOVs) cause severe hemorrhagic fever with a high mortality rate. At present, there are no licensed vaccines or efficient therapies to combat EBOV infection. Previous studies have shown that both humoral and cellular immune responses are crucial for controlling Ebola infection. CD8+ T cells play an important role in mediating vaccine-induced protective immunity. The objective of this study was to identify H-2d-specific T cell epitopes in EBOV glycoproteins (GPs).

Potential antioxidant properties of an aqueous extract of the edible red seaweed Gracilaria tenuistipitata (AEGT) against oxidative DNA damage were evaluated. The AEGT revealed several antioxidant molecules, including phenolics, flavonoids and ascorbic acid. In a cell-free assay, the extract exhibited 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity that significantly reduced H₂O₂-induced plasmid DNA breaks in a dose-response manner (P < 0.001). The AEGT also suppressed H₂O₂-induced oxidative DNA damage in H1299 cells by reducing the percentage of damaged DNA in a dose-response manner (P < 0.001) as measured by a modified alkaline comet-nuclear extract (comet-NE) assay. The MTT assay results showed that AEGT confers significant protection against H₂O₂-induced cytotoxicity and that AEGT itself is not cytotoxic (P < 0.001). Moreover, H₂O₂-induced cell cycle G2/M arrest was significantly released when cells were co-treated with different concentrations of AEGT (P < 0.001). Taken together, these findings suggest that edible red algae Gracilaria water extract can prevent H₂O₂-induced oxidative DNA damage and its related cellular responses.

One of the most important biological characteristics of Glioblastoma multiforme (GBM) is high vascular density. Vadimezan (ASA404, DMXAA) belongs to the class of small molecule vascular disrupting agents (VDA) that cause disruption of established tumor vessels and subsequent tumor hemorrhagic necrosis. Its selective antivascular effect is mediated by intratumoral induction of several cytokines including tumor necrosis factor-α (TNF-α), granulocyte-colony-stimulating factor (G-CSF), interleukin 6 (IL-6) and macrophage inflammatory protein 1α (MIP-1α). Preclinical studies have demonstrated that ASA404 acts synergistically with taxanes. In this study, we investigated if treatment of mice bearing U251 human glioblastoma xenografts with ASA404 and taxol may be synergistic. Therapy response was evaluated by measuring changes in tumor size and metabolic activity using 18F-FDG PET (Fluorodeoxyglucose - positron emision tomography) imaging.

The purpose of this study was to test the stereology method using several grid sizes for measuring liver volume and to find which grid provides an accurate estimate of liver volume.

The generation of myelinating cells from multipotential neural stem cells in the CNS requires the initiation of specific gene expression programs in oligodendrocytes (OLs). We reasoned that microRNAs (miRNAs) could play an important role in this process by regulating genes crucial for OL development. Here we identified miR-7a as one of the highly enriched miRNAs in oligodendrocyte precursor cells (OPCs), overexpression of which in either neural progenitor cells (NPCs) or embryonic mouse cortex promoted the generation of OL lineage cells. Blocking the function of miR-7a in differentiating NPCs led to a reduction in OL number and an expansion of neuronal populations simultaneously. We also found that overexpression of this miRNA in purified OPC cultures promoted cell proliferation and inhibited further maturation. In addition, miR-7a might exert the effects just mentioned partially by directly repressing proneuronal differentiation factors including Pax6 and NeuroD4, or proOL genes involved in oligodendrocyte maturation. These results suggest that miRNA pathway is essential in determining cell fate commitment for OLs and thus providing a new strategy for modulating this process in OL loss diseases.

The kinase complex mechanistic target of rapamycin 1 (mTORC1) plays an important role in controlling growth and metabolism. We report here that the stepwise formation of phosphatidylinositol 3-phosphate (PI(3)P) and phosphatidylinositol 3,5-bisphosphate (PI(3,5)P(2)) regulates the cell type-specific activation and localization of mTORC1. PI(3)P formation depends on the class II phosphatidylinositol 3-kinase (PI3K) PI3K-C2α, as well as the class III PI3K Vps34, while PI(3,5)P(2) requires the phosphatidylinositol-3-phosphate-5-kinase PIKFYVE. In this paper, we show that PIKFYVE and PI3K-C2α are necessary for activation of mTORC1 and its translocation to the plasma membrane in 3T3-L1 adipocytes. Furthermore, the mTORC1 component Raptor directly interacts with PI(3,5)P(2). Together these results suggest that PI(3,5)P(2) is an essential mTORC1 regulator that defines the localization of the complex.