FunRich: An open access standalone functional enrichment and interaction network analysis tool

As high‐throughput techniques including proteomics become more accessible to individual laboratories, there is an urgent need for a user‐friendly bioinformatics analysis system. Here, we describe FunRich, an open access, standalone functional enrichment and network analysis tool. FunRich is designed to be used by biologists with minimal or no support from computational and database experts. Using FunRich, users can perform functional enrichment analysis on background databases that are integrated from heterogeneous genomic and proteomic resources (>1.5 million annotations). Besides default human specific FunRich database, users can download data from the UniProt database, which currently supports 20 different taxonomies against which enrichment analysis can be performed. Moreover, the users can build their own custom databases and perform the enrichment analysis irrespective of organism. In addition to proteomics datasets, the custom database allows for the tool to be used for genomics, lipidomics and metabolomics datasets. Thus, FunRich allows for complete database customization and thereby permits for the tool to be exploited as a skeleton for enrichment analysis irrespective of the data type or organism used. FunRich ( http://www.funrich.org ) is user‐friendly and provides graphical representation (Venn, pie charts, bar graphs, column, heatmap and doughnuts) of the data with customizable font, scale and color (publication quality).     

Effects of the heavy metal Cu2+ on growth, development, and population dynamics of Spodoptera litura (Lepidoptera: Noctuidae)

Spodoptera litura (F.) larvae were fed with artificial food containing four different concentrations of copper (25, 50, 100, and 200 mg/kg). Copper affected growth, development, and population dynamics of late instar larvae. Our results indicate that the fourth and fifth instar survival rates were significantly reduced at 50-200 mg/kg copper (Cu2+). Moreover, all the Cu2+ treatments significantly reduced pupation and moth emergence rates in a dose-dependent manner. Larvae fed 25 or 50 mg/kg Cu2+ showed significant reductions in development period compared with non-Cu controls, and the pupation duration of animals fed 200 mg/kg was significantly longer than in non-Cu controls. All Cu2+ concentrations significantly reduced individual pupal weights compared with controls. In addition, Cu2+ at some concentrations significantly affected fertility parameters, such as doubling population time (DT), the net reproduction rate (R(o)), the mean generation time (T), the intrinsic rate of increase (r(m)), and the finite increase rate (A). Our study demonstrates that low concentrations of Cu2+ in the diet (25 and 50 mg/kg) shorten the generation time by 4-5 d, whereas higher Cu2+ concentrations (100 and 200 mg/kg) increase DT for 1-2 d.     

Chemical transfection of dye-conjugated microRNA precursors for microRNA functional analysis of M2 macrophages

MicroRNAs (miRNAs) are short noncoding ribonucleic acids known to affect gene expression at the translational level and there is mounting evidence that miRNAs play a role in the function of tumor-associated macrophages (TAMs). To aid the functional analyses of miRNAs in an in-vitro model of TAMs known as M2 macrophages, a transfection method to introduce artificial miRNA constructs or miRNA molecules into primary human monocytes is needed. Unlike differentiated macrophages or dendritic cells, undifferentiated primary human monocytes have been known to show resistance to lentiviral transduction. To circumvent this challenge, other techniques such as electroporation and chemical transfection have been used in other applications to deliver small gene constructs into human monocytes. To date, no studies have compared these two methods objectively to evaluate their suitability in the miRNA functional analysis of M2 macrophages. Of the methods tested, the electroporation of miRNA-construct containing plasmids and the chemical transfection of miRNA precursor molecules are the most efficient approaches. The use of a silencer siRNA labeling kit (Ambion) to conjugate Cy 3 fluorescence dyes to the precursor molecules allowed the isolation of successfully transfected cells with fluorescence-activated cell sorting. The chemical transfection of these dye-conjugated miRNA precursors yield an efficiency of 37.5 ± 0.6% and a cell viability of 74 ± 1%. RNA purified from the isolated cells demonstrated good quality, and was fit for subsequent mRNA expression qPCR analysis. While electroporation of plasmids containing miRNA constructs yield transfection efficiencies comparable to chemical transfection of miRNA precursors, these electroporated primary monocytes seemed to have lost their potential for differentiation. Among the most common methods of transfection, the chemical transfection of dye-conjugated miRNA precursors was determined to be the best-suited approach for the functional analysis of M2 macrophages.     

Integrin regulation by vascular endothelial growth factor in human brain microvascular endothelial cells: role of alpha6beta1 integrin in angiogenesis

The precise role of vascular endothelial growth factor (VEGF) in regulating integrins in brain microvascular endothelial cells is unknown. Here, we analyzed VEGF effects on integrin expression and activation in human brain microvascular endothelial cells (HBMECs). Using human cDNA arrays and ribonuclease (RNase) protection assays, we observed that VEGF up-regulated the mRNA expression of alpha(6) integrin in HBMECs. VEGF significantly increased alpha(6)beta(1) integrin expression, but not alpha(6)beta(4) integrin expression in these cells. Specific down-regulation of alpha(6) integrin expression by small interfering RNA (siRNA) oligonucleotides inhibited both the capillary morphogenesis of HBMECs and their adhesion and migration. Additionally, VEGF treatment resulted in activation of alpha(6)beta(1) integrins in HBMECs. Functional blocking of alpha(6) integrin with its specific antibody inhibited the VEGF-induced adhesion and migration as well as in vivo angiogenesis, and markedly suppressed tumor angiogenesis and breast carcinoma growth in vivo. Thus, VEGF can modulate angiogenesis via increased expression and activation of alpha(6)beta(1) integrins, which may promote VEGF-driven tumor angiogenesis in vivo.     

Generation and characterization of B7-H4/B7S1/B7x-deficient mice

Members of the B7 family of cosignaling molecules regulate T-cell proliferation and effector functions by engaging cognate receptors on T cells. In vitro and in vivo blockade experiments indicated that B7-H4 (also known as B7S1 or B7x) inhibits proliferation, cytokine production, and cytotoxicity of T cells. B7-H4 binds to an unknown receptor(s) that is expressed on activated T cells. However, whether B7-H4 plays nonredundant immune regulatory roles in vivo has not been tested. We generated B7-H4-deficient mice to investigate the roles of B7-H4 during various immune reactions. Consistent with its inhibitory function in vitro, B7-H4-deficient mice mounted mildly augmented T-helper 1 (Th1) responses and displayed slightly lowered parasite burdens upon Leishmania major infection compared to the wild-type mice. However, the lack of B7-H4 did not affect hypersensitive inflammatory responses in the airway or skin that are induced by either Th1 or Th2 cells. Likewise, B7-H4-deficient mice developed normal cytotoxic T-lymphocyte reactions against viral infection. Thus, B7-H4 plays a negative regulatory role in vivo but the impact of B7-H4 deficiency is minimal. These results suggest that B7-H4 is one of multiple negative cosignaling molecules that collectively provide a fine-tuning mechanism for T-cell-mediated immune responses.     

Hyaluronidase increases the biodistribution of acid alpha-1,4 glucosidase in the muscle of Pompe disease mice: an approach to enhance the efficacy of enzyme replacement therapy

Pompe disease (glycogen storage disease type II) is a glycogen storage disease caused by a deficiency of the lysosomal enzyme, acid maltase/acid alpha-1,4 glucosidase (GAA). Deficiency of the enzyme leads primarily to intra-lysosomal glycogen accumulation, primarily in cardiac and skeletal muscles, due to the inability of converting glycogen into glucose. Enzyme replacement therapy (ERT) has been applied to replace the deficient enzyme and to restore the lost function. However, enhancing the enzyme activity to the muscle following ERT is relatively insufficient. In order to enhance GAA activity into the muscle in Pompe disease, efficacy of hyaluronidase (hyase) was examined in the heart, quadriceps, diaphragm, kidney, and brain of mouse model of Pompe disease. Administration of hyase 3000 U/mouse (intravenous) i.v. or i.p. (intraperitoneal) and 10 min later recombinant human GAA (rhGAA) 20 mg/kg i.v. showed more GAA activity in hyase i.p. injected mice compared to those mice injected with hyase via i.v. Injection of low dose of hyase (3000 U/mouse) or high dose of hyase (10,000 U/mouse) i.p. and 20 min or 60 min later 20 mg/kg rhGAA i.v. increased GAA activity into the heart, diaphragm, kidney, and quadriceps compared to hyase untreated mice. These studies suggest that hyase enhances penetration of enzyme into the tissues including muscle during ERT and therefore hyase pretreatment may be important in treating Pompe disease.     

Nanoparticle-assisted micropatterning of active proteins on solid substrate

Micropatterning of proteins on silica substrate was achieved using a new method. Proteins were first immobilized onto silica nanoparticles which were then dispensed into arrayed microwells on silicon. Atomic force microscopy (AFM), fluorescence microscopy and Fourier transform infrared (FTIR) spectroscopy were used to characterize the samples. The results showed that, compared to a planar surface, curved surfaces of nanoparticles provide more space for attaching proteins and thus increases the intensity of fluorescence signal. Furthermore, after attaching to silica nanoparticles, bovine serum albumin (BSA) maintains its major structure and the cytokine IFN-gamma maintains its ability to bind to its antibody. Use of this method can be extended to micropatterning of other biomolecules, such as DNA and enzymes.     

The possibility of useful usage of biodegradation products of sunflower plants

Heads of sunflower (Helianthus annuus L.) were first biodegraded at 55 degrees C by their microflora. Biodegradation products of sunflower heads (BPSH) were analyzed. They were found to contain 9.77% dry matter, 3.98% total ash, 3.18% crude protein and 5.75% total organic matter. BOD load was determined as 4.80 g/100 g. Subsequently, the effects of BPSH at various concentrations (1.0%, 2.5%, 10% and 100%) on the seed germination and some growth parameters of Phaseolus vulgaris L., Cicer arietinum L. and Triticum vulgare L. were investigated. The percentages of seed germination and germination index of seeds were similar between the control and 1.0% and 2.5% BPSH groups, but these values decreased at higher concentrations. On the other hand, growth of the seedlings gradually increased up to a concentration of 10% BPSH and decreased at 100% concentration. As a result, at concentrations up to 10% the product was found to be beneficial for growth of plants.     

Physical and functional interactions between human mitochondrial single-stranded DNA-binding protein and tumour suppressor p53

Single-stranded DNA-binding proteins (SSB) form a class of proteins that bind preferentially single-stranded DNA with high affinity. They are involved in DNA metabolism in all organisms and serve a vital role in replication, recombination and repair of DNA. In this report, we identify human mitochondrial SSB (HmtSSB) as a novel protein-binding partner of tumour suppressor p53, in mitochondria. It binds to the transactivation domain (residues 1–61) of p53 via an extended binding interface, with dissociation constant of 12.7 (± 0.7) μM. Unlike most binding partners reported to date, HmtSSB interacts with both TAD1 (residues 1–40) and TAD2 (residues 41–61) subdomains of p53. HmtSSB enhances intrinsic 3′-5′ exonuclease activity of p53, particularly in hydrolysing 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) present at 3′-end of DNA. Taken together, our data suggest that p53 is involved in DNA repair within mitochondria during oxidative stress. In addition, we characterize HmtSSB binding to ssDNA and p53 N-terminal domain using various biophysical measurements and we propose binding models for both.     

Quantitation of green fluorescent protein using a gel-based imaging method

Green fluorescent protein (GFP) is a versatile reporter protein and has been widely used in biological research. However, its quantitation requires expensive equipment such as a spectrofluorometer. In the current study, a gel documentation imaging system using a native polyacrylamide gel for the quantitation of GFP was developed. The assay was evaluated for its precision, linearity, reproducibility, and sensitivity in the presence of Escherichia coli cells and was compared with the spectrofluorometric method. Using this newly established, gel-based imaging technique; the amount of GFP can be quantified accurately.