Advances in Bioscience and Biotechnology

Published by Scientific Research Publishing
Online ISSN: 2156-8456
The innate immune response is a complex process involving multiple pathogen-recognition receptors, including toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors. Complement is also a critical component of innate immunity. While complement is known to interact with TLR-mediated signals, the interactions between NOD-like receptors and complement are not well understood. Here we report a synergistic interaction between C5a and Nod2 signaling in RAW 264.7 macrophages. Long-term treatment with muramyl dipeptide (MDP), a NOD2 ligand, enhanced C5a-mediated expression of chemokine mRNAs in RAW 264.7 cells. This response was dependent on NOD2 expression and was associated with a decrease in expression of C5L2, a receptor for C5a which acts as a negative modulator of C5a receptor (C5aR) activity. MDP amplified C5a-mediated phosphorylation of p38 MAPK. Treatment of RAW264.7 cells with an inhibitor of p38 attenuated the synergistic effects of C5a on MDP-primed cells on MIP-2, but not MCP-1, mRNA. In contrast, inhibition of AKT prevented C5a stimulation of MCP-1, but not MIP-2, mRNA, in MDP-primed cells. Taken together, these data demonstrated a synergistic interaction between C5a and NOD2 in the regulation of chemokine expression in macrophages, associated with a down-regulation of C5L2, a negative regulator of C5a receptor activity.
Harmful algal blooms (HABs) are a serious environmental problem globally. The ability of cyanobacteria, one of the major causative agents of HABs, to grow in heavy metal polluted areas is proving a challenge to environmental restoration initiatives. Some cyanobacteria secrete toxins, such as microcystin, that are potentially dangerous to animals and humans. In this study, the physiology of a cyanobacterium was assessed to nickel chloride exposure. Cell growths were monitored throughout the study with various nickel chloride concentrations (0, 10, 25 or 50 mg/L). Morphological abnormalities were observed with microscopic image analyses. Inductively coupled plasma mass spectrometry (ICP-MS) was carried out to trace the distribution of nickel during the growth period. This study provides insight on potential nickel response mechanisms in freshwater cyanobacteria, which may lead to effective HAB prevention strategy development.
LOOH-dependent EET formation by CYP isoforms (nmol EETs/nmol P450/min). 
VitC-dependent rat kidney microsomal EET formation. (a) Dose-dependent VitC-activated EET formation at 37˚C in 10 min incubations; (b) Timedependent EET formation. Rat kidney microsomes (1 ml, 0.4 mg protein) and AA (0.1 mM) were incubated at 37˚C, n = 4-6. Production of EETs including DHETs was analyzed by LC/MS/MS analysis after extraction. 
Plasma concentrations of individual cis-and trans- EETs in control and VitC-treated Sprague-Dawley rats. 
VitC levels in mouse brain cortex and kidney. 
Inhibition and stimulation of VitC-dependent microsomal EET formation. VitC (0.1 mM) was applied to all incubations except for the control with rat kidney miosomes (1 ml, 0.5 mg protein) and AA (10 M) for 30 min at 37˚C and EET formation including DHETs was analyzed with LC/MS/MS. * p < 0.05 vs VitC 0.1 mM, n = 3-6. 
The cardiovascular effects of vitamin C (VitC) could be mediated by epoxyeicosatrienoic acids (EETs). We aimed to study the mechanism of VitC-dependent microsomal formation of cis- and trans-EETs and the regulation of EET levels in rat isolated perfused kidneys and in vivo. VitC biphasically stimulated rat kidney microsomal cis- and trans-EET formation in a ratio of 1:2, involving the participation of lipid hydroperoxides (LOOHs), Fe(2+), and cytochrome P450 (CYP). Levels of LOOHs correlated with microsomal EET production. LOOH stimulation of CYP isoforms resulted in preferred trans-over cis-EET formation from arachidonic acid and was associated with the cleavage of LOOHs, which indicated a CYP peroxygenase activity. EETs contributed to VitC-induced vasodilator responses in rat isolated perfused kidneys. VitC (1 mg/ml) given in the drinking water for 9 days doubled rat urinary EET excretion, increased plasma levels of EETs, mostly trans-EETs, by 40%, and reduced plasma levels of 20-hydroxyeicosatetraenoic acid. Depletion of VitC in brain cortex and kidney tissues by more than 20- and 50-fold, respectively, in gulonolactone oxidase-knockout mice was associated with mild increases in tissue EETs. These data suggest that LOOHs are a determinant factor for EET formation in vivo in which VitC exerts a key regulatory effect. VitC-activated CYP peroxygenase activity may represent a CYP interaction with lipoxygenases and cyclooxygenases to mediate the cardiovascular effects of VitC via formation of EETs.
(a) Caspase-1 activity in rat urothelium 2 h after injection of 80 mg/kg CP or saline control. Data points are the mean ± SEM. Asterisks indicate values significantly different from the mean value at 0 h, n = 6, p < 0.05. (b) (upper) diagram of the protocol for treatment with YVAD-AOM, CP and DMSO. (b) (lower) caspase-3/7 activity in the various treatment groups. Data points are mean ± SEM. Asterisks indicate values significantly different from DMSO control; n = 6, p < 0.05. No other differences between groups were significant. (c) (upper) diagram of the protocol for treatment with Anakinra (Ana), CP and saline. (c) (lower) caspase-3/7 activity in the various treatment groups. Data points are mean ± SEM. Asterisks indicate values significantly different from saline control; n = 5-6, p < 0.05. No other differences between groups were significant. 
Hemorrhagic cystitis (HC or bladder inflammation) affects a significant number of patients undergoing cyclophosphamide (CP) chemotherapy despite treatment with 2-mercaptoethane sulfonate (Mesna) to inactivate the metabolite acrolein. While the mechanism is unknown, there is clearly acrolein-independent damage to the urothelium. In this study we have explored the induction of apoptosis in the urothelium as a marker of damage and the mechanism underlying the acrolein-independent apoptosis. Apoptosis in urothelium (caspase-3/7 activity and Poly (ADP-ribosyl) polymerase (PARP) cleavage) was measured following CP administration (80 mg/kg). Sodium 2-mercaptoethane sulfonate (Mesna) was used to mask acrolein's effect. An IL-1β receptor antagonist and a cell-permeable caspase-1 inhibitor were used to assess the involvement of IL-1β and caspase-1, respectively. Two waves of apoptosis were detected following CP administration, one peaking at 2 h and a second at 48 h. The first wave was independent of acrolein. Caspase-1 was also active at 2 h and activation of caspase-3/7 was blocked by a caspase-1 inhibitor but not an IL-1β receptor antagonist suggesting the direct activation of caspase-3/7 by caspase-1 without the need for IL-1β as an intermediate. Our results indicate that CP initiates an early, acrolein-independent wave of apoptosis that results from direct cleavage of caspase-3/7 by caspase-1.
Fibrotic lung diseases increase with age. Previously we determined that senescence increases tissue expression of fibronectin EDA (Fn-EDA) and decreases fibroblast expression of Thy-1, and that fibrocytes contribute to fibrosis following bleomycin-induced lung injury in mice. In this study we hypothesized that fibroblasts lacking Thy-1 expression produce an extracellular matrix that promotes fibrocyte retention and myofibroblast transdifferentiation, thereby promoting fibrogenesis. Young and old mice were treated with bleomycin intratracheally; fibrocytes in the bone marrow, blood, and lungs were quantified, and lung fibroblast Thy-1 expression was assessed. Bone marrow derived fibrocytes were cultured on matrices derived from Thy-1(+) or Thy-1(−) fibroblasts ± the pro-fibrotic cytokine TGFβ1. Older mice had more fibrocytes in their bone marrows at baseline and more fibrocytes in their lungs following bleomycin treatment. In parallel, lung fibroblasts in older mice had lower expression of Thy-1 at baseline that increased transiently 7 days after bleomycin treatment but then rapidly waned such that 14 days after bleomycin treatment Thy-1 expression was again markedly lower. Fibrocytes cultured on matrices derived from Thy-1(−) fibroblasts + TGFβ1 had increased gene expression for collagen type 1, fibronectin, Fn-EDA, and α-smooth muscle actin. In parallel, whereas the matrices derived from Thy-1(−) fibroblasts stimulated phosphorylation of Akt in cultured fibrocytes, the matrices derived from Thy-1(+) fibroblasts induced apoptosis. These findings suggest that senescence increases fibrocyte recruitment to the lung following injury and that loss of Thy-1 expression by lung fibroblasts promotes fibrocyte retention and myofibroblast transdifferentiation that renders the “aging lung” susceptible to fibrosis.
Diagrams depicting the structure of (A) the intact Ab molecule (IgG); (B) a Fab fragment; and (C) a Fv fragment. The circles in each panel represent the specific Ag of the depicted Ab molecule.  
Ig unfolding and aggregation pathways. The scheme summarizes the pathways discussed along the text. Reference numbers of relevant articles describing these pathways are included in brackets.  
Diagrams depicting the structure of (A) an scFv fragment (IgG); and the derived (B) diabody; (C) triabody; and (D) tetrabody. The circles in each panel represent the specific Ag of the depicted Fv.  
Monoclonal antibodies (mAbs) have proven to be useful for development of new therapeutic drugs and diagnostic techniques. To overcome the difficulties posed by their complex structure and folding, reduce undesired immunogenicity, and improve pharmacokinetic properties, a plethora of different Ab fragments have been developed. These include recombinant Fab and Fv segments that can display improved properties over those of the original mAbs upon which they are based. Antibody (Ab) fragments such as Fabs, scFvs, diabodies, and nanobodies, all contain the variable Ig domains responsible for binding to specific antigenic epitopes, allowing for specific targeting of pathological cells and/or molecules. These fragments can be easier to produce, purify and refold than a full Ab, and due to their smaller size they can be well absorbed and distributed into target tissues. However, the physicochemical and structural properties of the immunoglobulin (Ig) domain, upon which the folding and conformation of all these Ab fragments is based, can limit the stability of Ab-based drugs. The Ig domain is fairly sensitive to unfolding and aggregation when produced out of the structural context of an intact Ab molecule. When unfolded, Ab fragments may lose their specificity as well as establish non-native interactions leading to protein aggregation. Aggregated antibody fragments display altered pharmacokinetic and immunogenic properties that can augment their toxicity. Therefore, much effort has been placed in understanding the factors impacting the stability of Ig folding at two different levels: 1) intrinsically, by studying the effects of the amino acid sequence on Ig folding; 2) extrinsically, by determining the environmental conditions that may influence the stability of Ig folding. In this review we will describe the structure of the Ig domain, and the factors that impact its stability, to set the context for the different approaches currently used to achieve stable recombinant Ig domains when pursuing the development of Ab fragment-based biotechnologies.
We hypothesized that current antimicrobial peptides should be susceptible to proteolytic digestion. The antimicrobial peptides: Griffithinsin, RC-101, LL-37, LSA-5, PSC-RANTES and DJ007 were degraded by commercially available proteases. Two different species of anaerobic vaginal flora, Prevotella bivia and Porphyromonas asaccharolytica also degraded the materials. Griffithsin was resistant to digestion by 8 of the 9 proteases and the bacteria while LL-37 was the most sensitive to protease digestion. These data suggests most of the molecules may not survive for very long in the proteolytic rich environments in which they are intended to function.
Fibroblasts perform critical functions during the normal host response to tissue injury, but the inappropriate accumulation and persistent activation of these cells results in the development of tissue fibrosis. The mechanisms accounting for the aberrant accumulation of fibroblasts during fibrotic repair are poorly understood, although evidence supports a role for fibroblast resistance to apoptosis as a contributing factor. We have shown that TGF-β1 and endothelin-1 (ET-1), soluble mediators implicated in fibrogenesis, promote fibroblast resistance to apoptosis. Moreover, we recently found that ET-1 induced apoptosis resistance in normal lung fibroblasts through the upregulation of survivin, a member of the Inhibitor of Apoptosis (IAP) protein family. In the current study, we sought to determine the role of survivin in the apoptosis resistance of primary fibroblasts isolated from the lungs of patients with Idiopathic Pulmonary Fibrosis (IPF), a fibrotic lung disease of unclear etiology for which there is no definitive therapy. First, we examined survivin expression in lung tissue from patients with IPF and found that there is robust expression in the fibroblasts residing within fibroblastic foci (the "active" lesions in IPF which correlate with mortality). Next, we show that survivin expression is increased in fibroblasts isolated from IPF lung tissue compared to cells from normal lung tissue. Consistent with a role in fibrogenesis, we demonstrate that TGF-β1 increases survivin expression in normal lung fibroblasts. Finally, we show that inhibition of survivin enhances susceptibility of a subset of IPF fibroblasts to apoptosis. Collectively, these findings suggest that increased survivin expression represents one mechanism contributing an apoptosis-resistant phenotype in IPF fibroblasts.
Oxygen uptake data acquired from bacterial cultures and Lake Washington sediment. (a) Example oxygen consumption rates obtained from ~ 1.7 × 10 5 M. extorquens AM1 cells grown on succinate or methanol with an optical density (600 nm) of ~0.10. (b) Summary of respiration rates obtained from 3 replicates for the top aerobic layer of Lake Washington sediment. 
Oxygen consumption is a fundamental component of metabolic networks, mitochondrial function, and global carbon cycling. To date there is no method available that allows for replicate measurements on attached and unattached biological samples without compensation for extraneous oxygen leaking into the system. Here we present the Respiratory Detection System, which is compatible with virtually any biological sample. The RDS can be used to measure oxygen uptake in microliter-scale volumes with a reversibly sealed sample chamber, which contains a porphyrin-based oxygen sensor. With the RDS, one can maintain a diffusional seal for up to three hours, allowing for the direct measurement of respiratory function of samples with fast or slow metabolic rates. The ability to easily measure oxygen uptake in small volumes with small populations or dilute samples has implications in cell biology, environmental biology, and clinical diagnostics.
Breast cancer bone metastases. When active TGF-β is released from the bone matrix upon bone resorption by osteoclasts it acts on breast cancer cells to stimulate the production of osteolytic factors, such as parathyroid hormone-related protein (PTHrP), connective tissue growth factor (CTGF) and interleukin-(IL) 6 and −11. These factors increase the RANKL/OPG expression ratio in osteoblasts, which bind to the RANK receptors expressed on osteoclasts and activate osteoclastogenesis. TGF-β can directly stimulate osteoclast activity and inhibiting osteoblast differentiation thus, TGF-β can stimulate tumor growth.
Breast cancer is the most prevalent cancer among females worldwide leading to approximately 350,000 deaths each year. It has long been known that cancers preferentially metastasize to particular organs, and bone metastases occur in ~70% of patients with advanced breast cancer. Breast cancer bone metastases are predominantly osteolytic and accompanied by increased fracture risk, pain, nerve compression and hypercalcemia, causing severe morbidity. In the bone matrix, transforming growth factor-β (TGF-β) is one of the most abundant growth factors, which is released in active form upon tumor-induced osteoclastic bone resorption. TGF-β, in turn, stimulates bone metastatic tumor cells to secrete factors that further drive osteolytic bone destruction adjacent to the tumor. Thus, TGF-β is a crucial factor responsible for driving the feed-forward vicious cycle of cancer growth in bone. Moreover, TGF-β activates epithelial-to-mesenchymal transition, increases tumor cell invasiveness and angiogenesis and induces immunosuppression. Blocking the TGF-β signaling pathway to interrupt this vicious cycle between breast cancer and bone offers a promising target for therapeutic intervention to decrease skeletal metastasis. This review will describe the role of TGF-β in breast cancer and bone metastasis, and pre-clinical and clinical data will be evaluated for the potential use of TGF-β inhibitors in clinical practice to treat breast cancer bone metastases.
Ellman’s reaction.
Cholinesterase activity comparison. Cholinesterase activity values determined from HemogloBindTM vs classical blood (RBC + plasma) processing methods (U/mL). n = 45. Slope = 0.80; 95% CI = 0.73, 0.86; p < 0.0001.
Acetylcholine is an essential neurotransmitter found throughout the nervous system. Its action on postsynaptic receptors is regulated through hydrolysis by various carboxylesterases, especially cholinesterases (ChEs). The acute toxicity of organophosphate (OP) compounds is directly linked to their action as inhibitors of ChE. One widely used assay for evaluating ChE activity is a spectrophotometric method developed by Ellman et al. When the enzyme source is from tissues or, in particular, blood, hemoglobin displays a spectrophotometric peak at the same wavelength used to analyze cholinergic activity. This creates a substantial background that interferes with the Ellman's assay and must be overcome in order to accurately monitor cholinesterase activity. Herein, we directly compare blood processing methods: classical method (1.67 ± 0.30 U/mL) and HemogloBind™ treatment (1.51 ± 0.17 U/mL), and clearly demonstrate that pretreatment of blood samples with Hemoglobind™ is both a sufficient and rapid sample preparation method for the assessment of ChE activity using the Ellman's method.
Enhanced florescence in the FITC-channel upon ligand-induced endocytosis of hPTH1R-pHluorin2. HEK293 cells expressing hPTH1R-pHluorin2 were treated with either vehicle (a) or 100 nM PTH(1-34) (b) Representative confocal images using the FITC-channel with identical laser power and gain are shown. 
Green florescent protein (GFP) variants that are sensitive to changes in pH are invaluable reagents for the analysis of protein dynamics associated with both endo- and exocytotic vesicular trafficking. Ratiometric pHluorin is a GFP variant that displays a bimodal excitation spectrum with peaks at 395 and 475 nm and an emission maximum at 509 nm. Upon acidification, pHluorin excitation at 395 nm decreases with a corresponding increase in the excitation at 475 nm. GFP2, a GFP variant that contains mammalianized codons and the folding enhancing mutation F64L, displays ~8-fold higher florescence compared to pHluorin upon excitation at 395 nm. Using GFP2 as a template, an enhanced ratiometric pHluorin (pHluorin2) construct was developed to contain fully mammalianized codons, the F64L mutation and ten of the thirteen pHluorin-specific mutations. As a result, pHluorin2 displays markedly higher florescence when compared to pHluorin while maintaining the ratiometric pH-sensitivity. Unlike native pHluorin, pHluorin2 expressed in the ligand-binding domain of the parathyroid hormone 1 receptor is readily detectable by confocal microscopy and displays a marked increase in florescence upon ligand-induced endocytosis to intracellular vesicles. Thus, pHluorin2's enhanced florescence while sustaining ratiometric pH-sensitivity represents a significant improvement for this methodological approach.
Nested in the environment of the nucleus of the cell, the 23 sets of chromosomes that comprise the human genome function as one integrated whole system, orchestrating the expression of thousands of genes underlying the biological characteristics of the cell, individual and the species. The extraction of meaningful information from this complex data set depends crucially upon the lens through which the data are examined. We present a biophysical perspective on genomic information encoded in single nucleotide polymorphisms (SNPs), and introduce metrics for modeling information encoded in the genome. Information, like energy, is considered to be a conserved physical property of the universe. The information structured in SNPs describes the adaptation of a human population to a given environment. The maintained order measured by the information content is associated with entropies, energies, and other state variables for a dynamic system in homeostasis. "Genodynamics" characterizes the state variables for genomic populations that are stable under stochastic environmental stresses. The determination of allelic energies allows the parameterization of specific environmental influences upon individual alleles across populations. The environment drives population-based genome variation. From this vantage point, the genome is modeled as a complex, dynamic information system defined by patterns of SNP alleles and SNP haplotypes.
Quantum processes have been confirmed for various biological phenomena. Here we model a quantum process in cells based on coherent waves of established ultrafast energy transfers in water. We compute wave speed, ~156 km/s, and wavelength, ~9.3 nm, and determine that the waves retain local coherence. The model is compared with observations and diverse numerical applications lend support to the hypothesis that rapid energy transfers in water are characteristic of living cells. Close agreements are found for the dipole moment of water dimers, microwave radiation on yeast, and the Kleiber law of metabolic rates. We find a sphere with diameter ~20 nm is a lower bound for life in this theory. The quantum properties of the model suggest that cellular chemistry favors reactions that support perpetuation of the energy waves
This study aimed to examine the effect of L-ornithine hydrochloride ingestion on serum growth hormone secretion response after strength training in young men who did not regularly engage in high intensity exercise. Ten healthy young males without workout habits (age: 22.2 +/- 1.0 yr). Subjects performed biceps curl strength training after L-ornithine hydrochloride and placebo ingestions. They participated in both of the above conditions randomly with a week interval in between. Serum growth hormone and or-nithine levels were measured before L-ornithine hy-drochloride or placebo ingestions and at 30 minutes after strength training. Serum growth hormone and ornithine level were measured. A change magnitude of serum growth hormone was significantly larger in the L-ornithine hydrochloride condition than in the placebo condition, and the effect size was also large (t = 1.91, p = .044, ES = .75). A significant interaction (F = 280.98, p = 0.000, ηp2 = 0.96) was found in serum ornithine and a multiple comparison test showed that it was greater in the L-ornithine hydrochloride condition. Serum growth hormone level after strength training increases by L-ornithine hydrochloride in gestion in untrained young males.
Quantitative RT-PCR of RAGE. Total RNA was isolated from both control and LL-37 inflamed bladders. Tissue and RNA isolation was performed 24 hrs after either saline or LL-37 exposure. LL-37 inflamed tissues demonstrated a near 10-fold reduction in RAGE mRNA levels (red bar) when compared to saline controls. 
RAGE Western Blot. (A)-lung protein isolate (positive control), (B)-LL-37 instilled tissue harvested after 24 hours, (C)-saline instilled control tissue harvested after 24 hours. Two common RAGE bands were detected at 48kDa and 55kDa. A moderate reduction in RAGE protein levels were observed within the inflamed LL-37 instilled bladders (B). Normalization carried out via baseline actin content. 
To elucidate pathways in bladder inflammation, we employed our physiologically relevant LL-37 induced cystitis model. Based on inflammatory studies involving other organ systems implicating the receptor for advanced glycation end-products (RAGE), we first hypothesized that RAGE is critically involved in LL-37 induced cystitis. We further hypothesized a common RAGE ligand - high mobility group box 1 (HMGB1) is up-regulated in bladders challenged with LL-37. Finally, we hypothesized NF-κB dependent inflammatory genes are activated in LL-37 induced cystitis. Testing our first hypothesis, C57Bl/6 mice were challenged with either saline (control) or 320 μM of LL-37 intravesically for 1 hr. After 12 or 24 hours, tissues were examined with immunohistochemistry (IHC) for RAGE, and both mRNA and protein isolation for respective qRT-PCR and Western Blot analysis. Our second hypothesis was tested by employing HMGB1 IHC. Testing our final hypothesis, qRT-PCR was performed investigating five genes: TNFα, IL-6, IL-1β, GM-CSF, COX-2. In control and LL-37 challenged tissues, IHC for RAGE revealed similar qualitative expression. Evaluation with qRT-PCR and Western Blot for RAGE revealed diminished expression at the mRNA and protein level within LL-37 challenged bladders. IHC for HMGB1 revealed a moderate qualitative increase within LL-37 challenged tissues. Finally, with the exception of TNF α, all NF- κB dependent inflammatory genes yielded substantial up-regulation. We have employed our LL-37 induced cystitis model to gain insight towards a possible mechanistic pathway involved in bladder inflammation. This work provides data for future studies involving the inflammatory ligand HMGB1, RAGE, and receptor pathways that activate NF-κB.
Quantities of each respective reagent used in the synthesis of PCLF.
FT-IR spectra of DEF, PCL and synthesized PCLF.  
Quantities of each respective reagent used in the synthesis of PCLtF.
FT-IR spectra of PCLtF synthesis reaction 1.  
It is known that the body can efficiently repair hard tissue (bone) micro fractures by suturing the defect through the deposition of minerals resulting in an area that is stronger post-injury. Larger defects, however, generally cause more trouble since the body is incapable of repairing them. Bone defects can occur as a result of congenital abnormalities, trauma, or disease. Traditional methods for addressing these defects have involved the use of acellular cadaverous bone or autologous bone. Both contain innate problems associated with them; the former method can result in disease transmission, as well as very low integration with the host due to the lack of viable cells while the latter is associated with two surgical sites and morbidity at the donor site. Alternative methods have been developed, but no method has yet provided a satisfactory solution. As a result, researchers and the medical community are turning toward the promising fields of biomaterial development and tissue engineering to develop new materials and methods of bone regeneration. In this work, a design of experiments (DOE) approach was performed to render commercially available biodegradable polymers (Poly(caprolactone)-diol/triol) photocrosslinkable and resultantly manufacturable using stereolithography (SL), a rapid prototyping technology. To perform the investigations, a commercial SL system (Viper HA, 3D Systems, Valencia, CA) equipped with a solid state laser system (355 nm wavelength) was used to manufacture synthesized poly(caprolactone) trifumarate (PCLtF) 3D porous constructs. Results of the work conducted produced constructs which provided promising chemical and biological results for the intended application.
Aim: The high mortality rate of melanoma is due to partially the lack of good diagnostic markers and treatment strategies. Over the past several years, several microRNA (miRNA) profiling studies have been performed on melanoma tissues, but with extremely inconsistency, the diagnostic value of miRNA candidates in melanoma remains under debate. Thus, this study aims to systematically evaluate the consistency of miRNAs tissue in multiple independent studies in melanoma. Method: Eligible studies were screened and selected from the PubMed, EMBASE, and Web of Science. A systematic analysis of published miRNA expression studies that compared the miRNA expression profiles between melanoma tissues and normal skin tissue was conducted. A vote-counting strategy was followed with the collection of information. Real time PCRs were employed to validate miRNA candidates with high consistency. Targets of consistent miRNAs were predicted by online programs (like miRTarBase, and TargetScanHuman 6.2). Enrichment analyses for gene ontology (GO) terms and Kyoto encyclopedia of genes and genomes (KEGG) pathways were carried out with Database for Annotation, Visualization, and Integrated Discovery (DAVID). Results: A total of 303 differentially expressed miRNAs were reported in the 10 miRNA-profiling studies during comparison of melanoma tissues with normal tissues; 132 were up-regulated in melanoma, and 171 were down-regulated. However, in the group of consistently reported miRNAs (cutoff > 3 times), only moderate numbers of consistent and differentially expressed miRNAs were selected. miRNA-21 was found increased in 5 different studies, miRNA-146b, miRNA-17 and miRNA- 18a were reported up-regulated in 4 profiling studies. Meanwhile, miRNA-204 and miRNA- 125b were found down-regulated in 5 studies, miRNA-141, miRNA-149, miRNA-224, miRNA-200b, miRNA-200c were consistently decreased in just 4 out of 10 profiling studies in total. The directions of differential expression of these miRNA candidates were confirmed by real time PCRs. Enrichment analyses demonstrated that programmed cell death and transcription regulation played very important roles in the involvement of miRNAs in tumorigenesis of melanoma. Conclusion: This systematic study of melanoma miRNA profiling studies would provide rich information on miRNAs with potential role as the biomarkers and therapeutic agents with high consistency in melanoma.
In this work, sequential optimization strategy, based on statistical designs, was employed to enhance the production of α-amylase by Aspergillus niger ATCC 16404. This statistical study consists of optimizing the factors that influence the production of α-amylase of A. niger ATCC 16404. Indeed, another statistical study has allowed the selection of 5 factors (pH, starch, yeast extract, “corn steep liquor”, CaCl2 and salts) affecting both the development of mould (biomass) and that of the enzyme production. The central composite design allows the determination of the optimum of these selected factors and a quadratic model explains the factor reaction. Thus, the “ridge analysis” method, has led to maximizing the experimental reaction. The results indicate that the production rate of α-amylase is maximized in the presence of starch at 8.97 g/l, yeast extract at 2.86 g/l, CaCl2 at 1.224 g/l, salts (composed of 25% FeSO4, 7H2O, 25% MnSO4 and 50% MgCl2, 6H2O): FeSO4, 7H2O, MnSO4 0.1518 g/l and MgCl2, 6H2O at 0.3036 g/l. As for the pH, it is maintained at the rate of 5.68.
Effect of temperature on chitinase activity. 
The chitin is extracted from the cuttlebone of S. inermis and the mineral contents are predictable. The structure and degree of deacetylation of extracted cuttlebone chitin is dogged through Fourier Transform Infrared (FT-IR) spectroscopy. The extracted cuttlebone chitin is used as the substrate for the production of chitinase from Vibrio sp. The extra cellular proteins are concentrated by ammonium sulphate precipitation, dialysed and then purified by using gel (sephadex G-100) chromatography. Among the 40 fractions, only two fractions (active fractions) showed maximum absorbance at 280 nm, which are pooled, dialysed and free-dried. The enzyme activity (0.104 μmoles/ml) and molecular weight (50 - 60 kDa) of purified chitinase is also determined through SDS- PAGE. The optimal condition for chitinase activity is pH between 6.0 - 6.5 and 45℃.
The combined effect of SULT1A1 G638A polymor- phism and cigarette smoking on BC risk.
Cigarette smoking is a major risk factor for bladder cancer (BC). Sulfotransferase 1A1 (SULT1A1), a phase II enzyme, plays an important role in the metabolism of several carcinogens contained in cigarettes. The aim of this study was to investigate the relationship between SULT1A1 G638A polymorphism, cigarette smoking and bladder cancer risk in Taiwan. A total of 150 BC patients and 150 cancer-free controls were recruited from February 2002 to February 2009. Genotyping of the SULT1A1 G638A polymerphism was determined using the polymerase chain reac-tion-restricted fragment length polymorphism (PCR-RFLP) method. The odds ratio (OR) and 95% confi-dence interval (CI) were calculated as a measure of the combined effect of cigarette smoking and the SULT1A1 G638A polymorphism on BC risk. In the present study, we found that study subjects with the G/G genotype of the SULT1A1 gene had a signifi-cantly higher BC risk of 1.7 (95% CI = 1.3 -3.2) com-pared with those carrying the combination of G/A and A/A genotypes. Moreover, ever smokers who car-ried the G/G genotype of the SULT1A1 gene had a significantly increased UC risk of 3.5 (95% CI = 2.5 -10.2) compared with never smokers who carried the G/A and A/A genotypes as the reference group. In conclusion, our findings suggest that SULT1A1 G638A polymorphism is associated with the development of BC, especially among cigarette smokers.
Removal efficiency of phenol from aqueous solutions was determined using Pseudomonas putida (NCIM 2102). Experiments were made as a function of pH (4 -9), temperature (28˚C -36˚C), and agitation speed (100 -200 rpm). Optimization of these three process parameters for phenol degradation was studied. Sta-tistically designed experiments using response sur-face methodology was used to get more information about the significant effects and the interactions be-tween these three parameters. A 2 3 full-factorial cen-tral composite design was employed for experimental design and for analysis of the results. A second order polynomial regression model, has been developed using the experimental data. It was found that the degrading potential of P. putida (NCIM 2102) was strongly affected by the variations in pH, tempera-ture and agitation speed. The experimental values were in good agreement with the predicted values and the correlation coefficient was found to be 0.9871. The optimum process conditions for maximizing phenol degradation were recognized as follows: pH (7.49), temperature (29.99˚C), and agitation speed (138.89) rpm.
Phylogenetic tree of the family Allocreadiidae, obtained using maximum parsimony algorithm. Nodal numerals are the bootstrap statistics values (%) for MP/ML/BI algorithms, respectively. Roman numerals correspond to number of cluster, discussed in the text. 
Transition/transversion ratio bias (R * ), obtained by pairwise comparision of 28S rDNA sequences of different Cre- pidostomum species. 
Comparison by KH and SH test of log-likelihood scores among unconstrained and constrained monophyletic trees of the genus Crepidostomum. * P < 0.05. Both tests showed significant differences between unconstrained tree, showed a paraphyly of Cre- pidostomum and phylogenetic tree with constraint monophyly of Crepidostomum for three phylogenetic algorithms used. 
Genetic divergence and phylogenetic relationships of four species of the genus Crepidostomum Braun, 1900 sensu Caira, Bogĕa (2005) were revealed using partial sequences of 28S ribosomal RNA gene. Genetic divergence between C. cf. farionis (Muller, 1784) and C. nemachilus Krotov, 1959 was 3.1%, which corresponds to the mean value of interspecific divergence between Crepidostomum species. These two species, therefore, can be recognized as bonafide species. However, we found no genetic differences between 28S rRNA gene sequences of C. nemachilus and C. cf. metoecus Braun, 1900 in spite of considerable morphological and ecological differences. Maximal values of genetic p-distances were revealed between C. cf. auriculatum Wedl, 1857 and C. cf. farionis. Phylogenetic relationships of Crepidostomum spp. for which sequence data are available, along with species in other related genera (Bunodera Railliet, 1896 and Allocreadium Loss, 1900) showed a paraphyly of the genus Crepidostomum. Considerable differentiation of C. cf. auriculatum from other Crepidostomum species was revealed, which may reflect the original description of this species in a separate genus Arcolichanus Ward, 1917. Our results are consistent with the conventional systematics that places the four genera (Crepidostomum, Bunodera, Megalogonia and Allocreadium) within the same family.
SDS-PAGE analysis of Daspartate oxidase from T. harzianum. lane 1: purified enzyme; lane 2: protein markers. 
Inhibition of D-aspartate oxidase from T. harzianum by carboxylic acids and metals.
Trichoderma harzianum Rifai SKW-36 produced two kinds of D-amino acid oxidizing enzymes. One enzyme was D-aspartate oxidase acting on acidic D-amino acids such as D-aspartate and D-glutamate and another one was D-amino acid oxidase acting on neutral D-amino acid such as D-phenylalanine and D-methionine. These enzymes in the cell-free extract were separated by DEAE-Toyopearl ion-exchange column chromatography. Casamino acids, peptone, and yeast extract as carbon and nitrogen sources were effective for the production of the enzymes. No D-amino acid tested induced the production of the enzymes. Casamino acid (0.33%) as carbon and nitrogen source gave a highest specific activity of D-aspartate oxidase among media tested. D-Aspartate oxidase, which was purified by four-step column chromatography in addition to ammonium sulfate precipitation, exhibited a subunit molecular mass of 40 kDa by SDS-PAGE analysis. D-Aspartate, D-glutamate and N-methyl-D- aspartate were oxidized as substrates with the specific activities of 7.80 U/mg, 4.90 U/mg, and 4.22 U/mg, respectively. D-Asparagine, D-glutamine, D-alanine, and D-valine were slightly oxidized. No other D- amino acids tested were inert. The enzyme exhibited relatively wide substrate specificity compared to D-aspartate oxidases reported so far. The pH and temperature optima were 7.5 - 8.0 and 35°C, respectively. The enzyme was stable at pH 6.0 - 9.0. About 75% of the enzyme activity was retained even after treating the enzyme at 50°C for 10 min. The enzyme activity was inhibited not by benzoate and tartrate, but 60% and 24% by fumarate and malonate, respectively.
Effect of CuCl2 on the signal intensity of the direct ELISA. Direct ELISA using HRP-conjugated antibody 37-11 (a) or 77-3 (b) was performed in the absence (open squares) and the presence of 50 μM CuCl2 (closed circles).  
AFM images of soluble aggregates. Soluble aggregates in the absence (a) and presence (b) of 50 μM CuCl2 are shown.  
Amyloid-β42 (Aβ42) accumulates within senileplaque, a pathological hall mark of Alzheimer’s disease (AD). Our previous reports showed that the monoclonal antibodies 37-11 and 77-3 react with conformational epitopes on the surface of the soluble aggregates of Aβ42 and that sandwich ELISA using these two monoclonal antibodies yields high reactivity to detect soluble aggregates of Aβ42. Here, the reactivity of the sandwich ELISA was shown to increase in the presence of 50 μM Cu2+. However, the addition of Cu2+ had only a small effect on the reactivity of a direct ELISA using antibody 37-11 or 77-3, suggesting that Cu2+ has a small effect on the number of epitopes on the surface of the aggregates. Atomic force microscopy images showed that larger aggregates were formed in the presence of Cu2+, as shown in the other reports. Cu2+ may gather the aggregates with distinct epitopes recognized by antibodies 37-11 and 77-3, leading to increased signal intensity of the sandwich ELISA.
Agarose gel electrophoresis of NS1 PCR product. Lane M: Marker Lane 1: NS1 with the EcoRI and XbaI restriction site Lane 2: NS1 with the EcoRI and SalI restriction site.
Avian influenza is a viral contagious disease that af-fects poultry industry and human health. Vaccination has been considered as a preventive tool in the eradi-cation of AI, but it causes some limitations including trade embargoes and interfering with serologic sur-veillance in differentiation between infected and vac-cinated animals (DIVA strategy). Several distinct DIVA strategies have been presented to conquer these limi-tations. In this study, the open reading frame of NS1 gene of a H9N2 subtype of AI virus was amplified by polymerase chain reaction. After extraction and puri-fication of NS1 gene from agarose gel, it was inserted into two different pGEX-4T-1 and pMAL-c2X plas-mids and transferred in DH5α strain of Escherichia coli by using electroporation procedure. The E. coli colonies possessing recombinant NS1 gene were screen-ed using PCR, restriction mapping and sequencing analysis. The expressed rNS1 protein was purified using affinity chromatography based on MBP (pMAL-c2X) and GST (pGEX-4T-1). The MBP-NS1 and GST-NS1 proteins on SDS-PAGE had bands with molecu-lar weight of 68 and 52 kDa respectively. Western blotting with MBP-NS1 protein showed positive reac-tion using antisera obtained from chickens challenged with a H9N2 subtype strain. But, the most sera pre-pared from H9N2 vaccinated chickens were negative in WB. These findings indicated that the MBP-rNS1 protein of 26 kDa expressed by pMAL-c2X plasmid can be used in a DIVA for differentiation of AI in-fected and vaccinated chickens.
Effect of some tropical rhizobia on the nodulation, nitrogen fixation and growth of cowpea. 
This study focused on the development of thermo-philic strain/s of a cowpea (Vigna unguiculata) com-patible nitrogen fixing bacterium. A preliminary plant screening was carried out using some strains of tropical rhizobia and cowpea. Rhizobium species CWP G34A that formed Fix + nodules repeatedly was selected for further studies. First, it was tested for growth at high temperatures of 40˚C to 55˚C at 5˚C interval with 28˚C as the control temperature. Muta-genesis was conducted on the bacterium with ethyl-methane sulphonate (EMS). The wild-type and mu-tants generated were tested for high temperature tolerance by growing them individually in nutrient broth at 60˚C for 24 hours. Optical density (670 nm) was read before and after incubation. The mutants were grouped into classes based on percentage dif-ference in OD values obtained before and after ex-posure to 60˚C. Rhizobium species CWP G34A pro-duced functional pink nodules on the cowpea consis-tently in three different plant tests. There was no growth at all the temperatures tested except at 28˚C and 40˚C after 24 hours of incubation. It grew better at former (51 × 10 10 Cfu/ml) than latter (11 Cfu/ml) temperature. Like the parental strain, all the mutants but one, did not grow after exposure to 60˚C. Sixty degree centigrade caused various reductions in opti-cal density (OD) values of the variants. Eleven classes of the mutants were formed with membership per-centage ranging from 1 to 22%. Class 1 contains only one member while class 11 has the highest mutant population of 22% with OD difference of 0 to 10% and −90 to −100% respectively. The high percentage reduction in the OD of variants in class 11 is similar to that of the unmutated cells (−94.56%). The only mutant that survived the 60 o C and grew was MU70. An increase of 1.67% in OD was obtained for MU70. Mutant MU70 therefore appeared a promising strain that can be further tested to inoculate cowpea in the dry and warm season for increased nitrogen fixation. This will provide encouraging information for farm-ers to grow the cowpea throughout the year particu-larly under high temperatures in summer in order to boost the yield of the legume.
Top-cited authors
Hossain U Shekhar
  • University of Dhaka
Ismail Hosen
  • University of Dhaka
Towhidul Islam
  • Daffodil International University
Taibur Rahman
  • University of Dhaka
Kayode Adekunle
  • Högskolan i Borås