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Culturing cells in three-dimensional (3D) environment can obtain a better clinical prediction for evaluating chemotherapy protocols and become a standard culture practice in cancer research. However, it involves tedious and time consuming operation. In the current work, a paper/polymer composited microfluidic platform was developed for screening cell viability and protein expression under chemical gradient environment. Cells were cultured in a paper sheet and expressed cell properties in 3D environment. The paper sheet was encapsulated in the microfluidic platform generating chemical gradient. After the culture course, investigations of cell viability and protein expression were respectively achieved by directly adding reagent and conducting on-paper immunoassay. Activation of respective signaling pathway could be identified and responded to different stimulations including nutrient gradient, IL-6 cytokine gradient, and anti-cancer drug gradient. On-paper analysis of protein expression could be completed within 1.5 h. The present technique integrates tedious operations on a single paper substrate. It provides a first-tier screening tool for cellular response under chemical gradient.

Gene co-expression networks can be used to associate genes of unknown function with biological processes, to prioritize candidate disease genes or to discern transcriptional regulatory programmes. With recent advances in transcriptomics and next-generation sequencing, co-expression networks constructed from RNA sequencing data also enable the inference of functions and disease associations for non-coding genes and splice variants. Although gene co-expression networks typically do not provide information about causality, emerging methods for differential co-expression analysis are enabling the identification of regulatory genes underlying various phenotypes. Here, we introduce and guide researchers through a (differential) co-expression analysis. We provide an overview of methods and tools used to create and analyse co-expression networks constructed from gene expression data, and we explain how these can be used to identify genes with a regulatory role in disease. Furthermore, we discuss the integration of other data types with co-expression networks and offer future perspectives of co-expression analysis.

Alterations in the relative abundance of synaptic proteins may contribute to hippocampal synaptic dysfunction in Alzheimer’s disease (AD). The extent to which perturbations in synaptic protein expression occur during the earliest stages of cognitive decline remains unclear. We examined protein levels of presynaptic synaptophysin (SYP) and synaptotagmin (SYT), and postsynaptic drebrin (DRB), a marker for dendritic spine plasticity, in the hippocampus of people with an antemortem clinical diagnosis of no cognitive impairment (NCI), mild cognitive impairment (MCI) or mild/moderate AD. Although normalized SYP and SYT levels were preserved, DRB was reduced by approximately 40% in the hippocampus of MCI and AD compared to NCI subjects. This differential alteration of synaptic markers in MCI suggests a selective impairment in hippocampal postsynaptic dendritic plasticity in prodromal AD that likely heralds the onset of memory impairment in symptomatic disease.

Background: Mutations in LRRK2 are associated with familial and sporadic Parkinson’s disease (PD). Subjects with PD caused by LRRK2 mutations show pleiotropic pathology that can involve inclusions containing α-synuclein, tau or neither protein. The mechanisms by which mutations in LRRK2 lead to this pleiotropic pathology remain unknown. Objectives: To investigate mechanisms by which LRRK2 might cause PD. Methods: We used systems biology to investigate the transcriptomes from human brains, human blood cells and Caenorhabditis elegans expressing wild-type LRRK2. The role of autophagy was tested in lines of C. elegans expressing LRRK2, V337M tau or both proteins. Neuronal function was measured by quantifying thrashing. Results: Genes regulating autophagy were coordinately regulated with LRRK2. C. elegans expressing V337M tau showed reduced thrashing, as has been noted previously. Coexpressing mutant LRRK2 (R1441C or G2019S) with V337M tau increased the motor deficits. Treating the lines of C. elegans with an mTOR inhibitor that enhances autophagic flux, ridaforolimus, increased the thrashing behavior to the same level as nontransgenic nematodes. Conclusion: These data support a role for LRRK2 in autophagy, raise the possibility that deficits in autophagy contribute to the pathophysiology of LRRK2, and point to a potential therapeutic approach addressing the pathophysiology of LRRK2 in PD.

DMP1 has been shown to play many roles in osteogenesis. We recently demonstrated that calcium-mediated stress kinase activation by DMP1 leads to osteoblast differentiation. In this study we demonstrate that DMP1 can also activate the extracellular signal-regulated kinase (ERK)-MAPK pathway. This activation was mediated through the RGD integrin-binding domain in DMP1. Further, we demonstrate that Runx2, an essential transcription factor, is stimulated by the ERK-MAPK pathway.

The collagen 2.3 and 3.6 promoters have been used to drive Cre expression for generation of conditional transgenic mutant mice. Within the bone, Col3.6 is expressed by mesenchymal precursor cells and their downstream progeny, while Col2.3 is more osteoblast specific. Our generation of transgenic mice with Col2.3-Cre- and Col3.6-Cre-driven deletion of the long-form leptin receptor (ObRb) necessitated a thorough analysis of the nonspecific expression of these promoters in the central nervous system. Both Col2.3 and Col3.6 were capable of forcing loxP recombination in the brain as demonstrated by EGFP expression in ROSA reporter mice. Expression of Col2.3 was limited to the central base of the brain near the third ventricle. In contrast, robust expression of Col3.6 was noted throughout the brain, centering near the distal third ventricle, third ventricle, and aqueduct. We subsequently analyzed the colocalization of leptin-responsive P-Stat3 neurons with Col3.6-expressing neurons. Approximately 5–10% colocalization was noted in leptin-responsive brain areas such as the arcuate nucleus, dorsal medial hypothalamus, ventral premammillary nucleus, and lateral hypothalamus. Injection of 3.6Cre+F/F ObRb knockout mice with leptin confirmed the presence of an intact P-Stat3 response that was dampened in the lateral hypothalamus (p < 0.050). This test was done to explore the contribution of neural leptin signaling to the bone phenotype of the 3.6Cre+F/F mice. Our analysis indicates that neural ObRb deletion, while present, is likely not the sole driver of femoral changes through traditional sympathetic circuits.

This study was undertaken to determine whether periosteum from different bone sources in a donor results in the same formation of bone and cartilage. In this case, periosteum obtained from the cranium and mandible (examples of tissue supporting intramembranous ossification) and the radius and ilium (examples of tissues supporting endochondral ossification) of individual calves was used to produce tissue-engineered constructs that were implanted in nude mice and then retrieved after 10 and 20 weeks. Specimens were compared in terms of their osteogenic and chondrogenic potential by radiography, histology, and gene expression levels. By 10 weeks of implantation and more so by 20 weeks, constructs with cranial periosteum had developed to the greatest extent, followed in order by ilium, radius, and mandible periosteum. All constructs, particularly with cranial tissue although minimally with mandibular periosteum, had mineralized by 10 weeks on radiography and stained for proteoglycans with safranin-O red (cranial tissue most intensely and mandibular tissue least intensely). Gene expression of type I collagen, type II collagen, runx2, and bone sialoprotein (BSP) was detectable on QRT-PCR for all specimens at 10 and 20 weeks. By 20 weeks, the relative gene levels were: type I collagen, ilium >> radial ≧ cranial ≧ mandibular; type II collagen, radial > ilium > cranial ≧ mandibular; runx2, cranial >>> radial > mandibular ≧ ilium; and BSP, ilium ≧ radial > cranial > mandibular. These data demonstrate that the osteogenic and chondrogenic capacity of the various constructs is not identical and depends on the periosteal source regardless of intramembranous or endochondral ossification. Based on these results, cranial and mandibular periosteal tissues appear to enhance bone formation most and least prominently, respectively. The appropriate periosteal choice for bone and cartilage tissue engineering and regeneration should be a function of its immediate application as well as other factors besides growth rate.

Although effluent treatment systems within pulp and paper mills remove many toxicants and improve wastewater quality, there is a need to understand and quantify the effectiveness of the treatment process. At a combined news and kraft pulp and paper mill in northwestern Ontario, Canada, fathead minnow (FHM) reproduction and physiology were examined before, during, and after a short-term (6-d) exposure to 10% (v/v) untreated kraft mill effluent (UTK), 25% (v/v) secondary treated kraft mill effluent (TK), and 100% (v/v) combined mill outfall (CMO). Although UTK exposure significantly decreased egg production, neither TK nor CMO caused any reproductive changes. The expression of six genes responsive to endocrine-disrupting compounds, stress, or metals was then examined in livers of these fish using real-time polymerase chain reaction. In female FHMs, none of the three effluents induced significant expression changes in any genes investigated. By contrast, in males there were significant increases in the mRNA levels of androgen receptor, estrogen receptor (ER) β, and cytochrome P4501A (CYP1A) upon UTK and TK exposure but no changes in ERα or vitellogenin (VTG) gene expression, whereas CMO exposure significantly increased the mRNA levels of ERα, VTG, and CYP1A. Together, these results suggest that kraft effluent before and after biological treatment contained compounds able to induce androgenic effects in FHMs, and that combination of kraft and newsmill effluents eliminated the androgenic compounds while inducing distinct and significant patterns of gene expression changes that were likely due to estrogenic compounds produced by the newsmill. Environ. Toxicol. Chem. 2010;29:430–439. © 2009 SETAC

The differentiation of osteoblasts from mesenchymal precursors requires a series of cell fate decisions controlled by a hierarchy of transcription factors. These include RUNX2, Osterix (OSX), ATF4 and a large number of nuclear coregulators. During bone development, initial RUNX2 expression coincides with the formation of mesenchymal condensations and precedes the branching of chondrogenic and osteogenic lineages. Given its central role in bone development, it is not surprising that RUNX2 is subject to a variety of controls. These include posttranslational modification, especially phosphorylation, and interactions with accessory nuclear factors. Specific examples of RUNX2 regulation to be reviewed include phosphorylation by the ERK/MAP kinase pathway and interactions with DLX5. RUNX2 is regulated via phosphorylation of critical serine residues in the proline/serine/threonine domain. In vivo, the transgenic expression of constitutively active MAP kinase in osteoblasts accelerated skeletal development, while a dominant-negative MAPK retarded development in a RUNX2-dependent manner. DLX5-RUNX2 complexes can be detected in osteoblasts and this interaction plays a critical role in maintaining osteoblast-specific expression of the bone sialoprotein gene. These studies allow us to begin understanding the complex mechanisms necessary to fine-tune bone formation as mesenchymal progenitors progress down the osteoblast lineage.

Culturing aortic valvular interstitial cells in an environment that models the aortic valve is an essential step towards understanding the progression of calcific aortic valve disease. Here the adaption of a three-dimensional (3-D) stacked paper-based culture system is presented for analyzing valve cells in a thick collagen gel matrix. Filter paper layers, modeled after a 96-well plate design, were printed with a wax well-plate template and then seeded with valve cell and collagen mixtures that quickly gelled into 3-D cultures. Stacking these layers permitted extensive customization of culture thickness and cell density profiles to model the full thickness of native valve tissue. Aortic valvular interstitial cells seeded into the paper-based constructs consistently demonstrated high survival up to 14 days of culture with significant increases in cell number through the first 3 days of culture. After 4 days following seeding, valve cells in single layer cultures showed reduced smooth muscle α-actin expression with a stabilized cell density, suggesting a transition from an activated phenotype to a more quiescent state. Valve cells in multilayer cultures demonstrated the ability to migrate from layer to layer and had the highest smooth muscle α-actin expression in areas with predicted low oxygen tensions. These results establish the filter-paper-based method as a viable culture system for analyzing valve cells in an in vitro 3-D model of the aortic valve.

Juvenile whitefish (Coregonus lavaretus L. s.l.) were exposed by caging in the field to diluted effluents from three operating pulp, paper, and paperboard mills in Southern Lake Saimaa, Finland. The expression of the vitellogenin gene, used as a biomarker of estrogenic contamination of effluents, was measured using a Northern blotting method. Increased mRNA levels, the most specific and reliable evidence for estrogen receptor-mediated actions in vivo, were found in fish caged in the vicinity of one of three mills studied. This mill was found to discharge wood-derived compounds, such as sterols and resin acids, into Lake Saimaa in amounts considerably exceeding those from the other two mills. The increased vitellogenin gene expression suggests that the effluent is a source of estrogenic contaminants.

Background: After age, the second largest risk factor for Alzheimer’s disease (AD) is apolipoprotein E (APOE) genotype, where APOE4 is associated with lower apoE protein levels, more severer brain pathology, enhanced inflammation and disease. Small peptides corresponding to the receptor-binding region of apoE mimic the anti-inflammatory activity of the apoE holoprotein. These apoE mimetics greatly improve behavioral outcomes and neuronal survival in head trauma models that display AD pathology and neuronal loss. Objective: To determine whether apoE mimetics change behavior, inflammation and pathology in CVND-AD (SwDI-APP/NOS2–/–) transgenic mice. Methods: Starting at 9 months, apoE peptides were subcutaneously administered 3 times per week for 3 months followed by behavioral, histochemical and biochemical testing. Results: Treatment with apoE mimetics significantly improved behavior while decreasing the inflammatory cytokine IL-6, neurofibrillary tangle-like and amyloid plaque-like structures. Biochemical measures matched the visible pathological results. Conclusions: Treatment with apoE mimetics significantly improved behavior, reduced inflammation and reduced pathology in CVND-AD mice. These improvements are associated with apoE-mimetic-mediated increases in protein phosphatase 2A activity. Testing in additional AD models showed similar benefits, reinforcing this novel mechanism of action of apoE mimetics. These data suggest that the combination of anti-inflammatory and neuroprotective activities of apoE mimetics represents a new generation of potential therapeutics for AD.

Field experiments were conducted in early summer 2004 by caging brown trout (Salmo trutta m. lacustris) in Southern Lake Saimaa (SE Finland) for 10 days. Cages were positioned into two depths at four distances (5 to 17 km) from the pulp and paper mill; one set with direct contact to the sediment and other locating at the three meters' depth from the water surface. Resin acids were measured from sediment, water, and fish bile. Possible toxicities were investigated by measuring liver EROD-activity, HSP70-protein, and gene expression profiles by microarray. The concentration of resin acids in the sediment showed clear gradient along the distance from the mill, and their concentrations in water ranged approximately from one to 30 microg L(-1). In the trout bile, concentrations were higher near the surface than on the bottom, indicating the role of current releases as sources of resin acids. When compared to trout held upstream from the effluent source, no differences were found in liver EROD nor HSP70 in bottom cages. However, near surface, a slight but statistically significant increase in EROD and HSP70 was found. Altogether 145 genes were found to be differentially regulated at downstream sites when compared 10 km upstream from the mill. The numbers of genes at each site varied from 5 to 45 without consistent differences between bottom and surface cages. Overall, neither expression profiles nor composition of the transcription signatures made possible to conclude an overt toxicity in exposed brown trout.

Fundamental investigations of human biology, and the development of therapeutics, commonly rely on 2D cell-culture systems that do not accurately recapitulate the structure, function, or physiology of living tissues. Systems for 3D cultures exist but do not replicate the spatial distributions of oxygen, metabolites, and signaling molecules found in tissues. Microfabrication can create architecturally complex scaffolds for 3D cell cultures that circumvent some of these limitations; unfortunately, these approaches require instrumentation not commonly available in biology laboratories. Here we report that stacking and destacking layers of paper impregnated with suspensions of cells in extracellular matrix hydrogel makes it possible to control oxygen and nutrient gradients in 3D and to analyze molecular and genetic responses. Stacking assembles the “tissue”, whereas destacking disassembles it, and allows its analysis. Breast cancer cells cultured within stacks of layered paper recapitulate behaviors observed both in 3D tumor spheroids in vitro and in tumors in vivo: Proliferating cells in the stacks localize in an outer layer a few hundreds of microns thick, and growth-arrested, apoptotic, and necrotic cells concentrate in the hypoxic core where hypoxia-sensitive genes are overexpressed. Altering gas permeability at the ends of stacks controlled the gradient in the concentration of the O 2 and was sufficient by itself to determine the distribution of viable cells in 3D. Cell cultures in stacked, paper-supported gels offer a uniquely flexible approach to study cell responses to 3D molecular gradients and to mimic tissue- and organ-level functions.

Sensory innervation of the viscera serves a number of important functions, including regulation of visceral motility and secretory activity, and transmission of visceral sensations, including pain. There are many ways in which the sensitivity of visceral sensory neurones might be modulated, and these are discussed. Altered sensory neurone responsiveness may contribute to pathophysiological states such as irritable bowel syndrome, and the mechanisms leading to sensory neurone sensitisation offer novel targets for the treatment of such disorders.

Xylan is the main carbohydrate found in the hemicellulosic fraction of plant tissues and accounts for one third of all renewable organic carbon available on earth. Xylanase, the major component of an enzymatic consortium, acts in nature by depolymerizing xylan molecules into monomeric pentosan units that are used by bacterial and fungal populations as a primary carbon source. Xylanase producers have been isolated from all ecological niches where plant material is deposited, and microorganisms often contain multiple loci encoding overlapping xylanolytic functions. The numerical excess of genes and the extensive sharing of structural features within beta-glycanase families suggests that extensive gene duplication and conversion events have occurred during xylanase evolution. Hydrolysis of beta-glycosidic linkages is sponsored by a general acid catalytic reaction common to all glycanases, whereas substrate recognition is specified by subsites that interact with adjacent glycosyl units. Under natural conditions xylanases are inducible by the products of their own action and subject to carbon catabolite repression. Bleaching paper pulps with xylanases is the first successful commercial application for these enzymes. The recovery of cellulosic textile fibers is the next logical application and bioconversion of biomass into fuels and chemicals, remains the ultimate target. Recent developments have shown that metabolic pathways can be transferred from one organism to another and proteins can be modified to gain conformational stability, suggesting that naturally occurring systems can be custom engineered to the situation in the fermentation tank. Thus, biotechnologies developed to transform biomass into marketable products that gradually substitute materials derived from non-renewable resources are becoming commercially worthwhile.

Yeast ENO1 promoter was prepared by a chemical synthetic method. Two variant promoters containing a pyrimidine-rich region (CT block), located between the TATA box and the transcription start site, either 32 or 34 base pairs (bp) longer than the native ENO1 promoter were isolated during the chemical synthesis. Gene expression of variant promoters was compared with that of the native promoter by measuring the amount of mRNA and the activity of beta-galactosidase by constructing ENO1-lacZ gene fusions. No significant differences were observed between the native and variant promoters in transcription levels. The start site of transcription was mapped on CAAG, a consensus sequence of transcription start site of yeast glycolytic genes. The results suggest a longer CT block in ENO1 promoter may not affect the expression of the yeast ENO1 gene. In addition, the level of ENO1 gene expression was found to be higher in stationary phase cells than in log phase cells.

Rearrangement and in most cases expression of the T cell rearranging genes gamma (TRG gamma) and T cell antigen receptor beta chain (TCR beta) genes were studied in 19 cases of T cell acute malignancies where the surface phenotype is representative of the different stages of thymic maturation. TCR alpha gene transcription was also studied. TRG gamma and TCR beta genes were found to be rearranged in all but one case. The TRG gamma rearrangement pattern seen in most cases is compatible with biallelic rearrangement by loop excision involving the J gamma 2 regions. The sizes of all but two rearranged bands were identical to those of the rearranged bands seen in polyclonal T lymphocytes also studied in this work. One identical-sized band was found in 11 of the 18 rearranged cases. The expression of TRG gamma mRNA (transcripts of 1.6 kilobases [kb]) was highly variable from case to case and did not correlate with the stage of differentiation of the malignant cells, the expression of the molecules CD4 and CD8, the expression and size of the transcripts of the TCR beta genes, and the transcription of TCR alpha genes. In one CD3 + case, strong expression of the TRG gamma transcripts coexisted with the exclusive presence of TCR beta mRNA of 1.0 kb. The cells from this case did not react with anti-Ti antibody and exhibited no natural killer activity. These findings are suggestive of a malignancy that may express the recently isolated CD3-TRG gamma complex.