CRET-based immunoassay on magnetic beads for selective and sensitive detection of Nanog antigen as a key cancer stem cell marker.

A method is presented for chemiluminescence resonance energy transfer (CRET) using APTES-Fe3O4 as a highly efficient energy acceptor with strong magnetic effectiveness over extended distances, while an Au@BSA-luminol composite acts as the donor. In order to boost the chemiluminescence reactions, CuO nanoparticles were successfully employed. The distance between the donor and acceptor is a crucial factor in the occurrence of the CRET phenomenon. A sensitive and high-throughput sandwich chemiluminescence immunosensor has been developed accordingly with a linear range of 1.0 × 10-7 g/L to 6.0 × 10-5 g/L and a limit of detection of 0.8 × 10-7 g/L. The CRET-based sandwich immunosensor has the potential to be implemented to early cancer diagnosis because of its high sensitivity in detecting Nanog, fast analysis (30 min), and simplicity. Furthermore, this approach has the potential to be adapted for the recognition of other antigen-antibody immune complexes by utilizing the corresponding antigens and their selective antibodies.

Molecular profile of non-coding RNA-mediated glycolysis control in human cancers.

The glycolysis is a common characteristic of cancer and it is responsible for providing enough energy to ensure growth. The glycolysis suppression is beneficial in tumor growth reduction. The stimulation/inhibition of glycolysis in cancer is tightly regulated by ncRNAs. The regulation of glycolysis by ncRNAs can influence proliferation and therapy response of tumor. The miRNAs are capable of inactivating enzymes responsible for glycolysis and suppressing signaling networks resulting in glycolysis induction. By regulation of glycolysis, miRNAs can affect therapy response. The lncRNAs and circRNAs follow a same pathway and by targeting glycolysis, they affect progression and therapy response of tumor. Noteworthy, lncRNAs and circRNAs sponge miRNAs in glycolysis mechanism control in tumor cells. Furthermore, ncRNA-mediated regulation of glycolysis mechanism can influence metastasis to organs of body. The ncRNAs regulating glycolysis are reliable biomarkers in cancer patients and more importantly, exosomal ncRNAs due to their presence in body fluids, are minimally-invasive biomarkers.

Hypoxia inducible factor-1α (HIF-1α) in breast cancer: The crosstalk with oncogenic and onco-suppressor factors in regulation of cancer hallmarks.

Low oxygen level at tumor microenvironment leads to a condition, known as hypoxia that is implicated in cancer progression. Upon hypoxia, HIF-1α undergoes activation and due to its oncogenic function and interaction with other molecular pathways, promotes tumor progression. The HIF-1α role in regulating breast cancer progression is described, Overall, HIF-1α has upregulation in breast tumor and due to its tumor-promoting function, its upregulation is in favor of breast tumor progression. HIF-1α overexpression prevents apoptosis in breast tumor and it promotes cell cycle progression. Silencing HIF-1α triggers cycle arrest and decreases growth. Migration of breast tumor enhances by HIF-1α signaling and it mainly induces EMT in providing metastasis. HIF-1α upregulation stimulates drug resistance and radio-resistance in breast tumor. Furthermore, HIF-1α signaling induces immune evasion of breast cancer. Berberine and pharmacological intervention suppress HIF-1α signaling in breast tumor and regulation of HIF-1α by non-coding RNAs occurs. Furthermore, HIF-1α is a biomarker in clinic.

An insight into the potentials of carbon dots for in vitro live-cell imaging: recent progress, challenges, and prospects.

Carbon dots (CDs) are a strong alternative to conventional fluorescent probes for cell imaging due to their brightness, photostability, tunable fluorescence emission, low toxicity, inexpensive preparation, and chemical diversity. Improving the targeting efficiency by modulation of the surface functional groups and understanding the mechanisms of targeted imaging are the most challenging issues in cell imaging by CDs. Firstly, we briefly discuss important features of fluorescent CDs for live-cell imaging application in this review. Then, the newest modulated CDs for targeted live-cell imaging of whole-cell, cell organelles, pH, ions, small molecules, and proteins are elaborately discussed, and their challenges in these fields are explained.

An experimental investigation on the influence of various buffer concentrations, osmolytes and gold nanoparticles on lysozyme: Spectroscopic and calorimetric study.

Considering importance and several industrial applications of lysozyme, including natural antibiotic and preservative, identifier for the diagnosis of diseases, and extraction purposes, its reversibility and stability studies can be very important. In this paper, the role that buffer and osmolytes concentrations play on the thermodynamic stability of lysozyme denaturation process, that is a new simple and inexpensive method, was evaluated by Nano-DSC III, far- and near-UV CD and fluorescence techniques. In thermal denaturation study, RI and ΔG of protein increased from 25.62% to 58.82% and 48.87 to 63.63 kJ mol-1 with the increment of buffer and osmolytes concentrations, respectively. These changes showed a significant increase of 129.59% in RI and 28.16% in ΔG. The effect of buffer and osmolytes concentrations on the secondary and tertiary structures of protein was also investigated. The results indicated that increment of buffer and osmolytes concentrations increase rigidity and thermodynamic stability of protein. Also, structure of protein may be changed by its interaction with GNPs. Hence, interaction of lysozyme with GNPs was studied at the buffer and osmolytes concentrations that gives the maximum RI and ΔG, respectively. The results showed that molten globule-like state was formed by lysozyme in the presence of GNPs.

Evaluation of Rationally Designed Label-free Stem-loop DNA Probe Opening in the Presence of miR-21 by Circular Dichroism and Fluorescence Techniques.

The characteristic features of stem-loop structured probes make them robust tools to detect targets with high sensitivity and selectivity. The basis of the hairpin based sensors operation is a conformational change that occurs upon hybridization of target with stem-loop probe. The design of the stem-loop probe has an important role in target recognition. Therefore, we designed a label-free stem loop probe for targeting miR-21 as a cancer biomarker investigated by web-based tools; its thermodynamic parameters obtained by thermal UV spectroscopy. The efficiency of stem-loop structure opening in the presence of target and non-target sequences was evaluated by fluorescence spectroscopy and circular dichroism spectro-polarimetry. The results showed that the target sequence opens the structure of hairpin efficiently in comparison to non-target sequences. To optimize the stem-loop hybridization to its target, the buffer ionic strength was changed by adding different concentrations of NaCl, KCl and MgCl2. It was shown that buffering conditions have a significant role in loop structure opening and its optimization, led to an increase in sensitivity detection and have improved LOD from 60 pM to 45 pM.

Surface Functionalization of Gold Nanorods Improves Nanostructure Assemblies on Amyloid Fibril Scaffolds.

A hybrid scaffold containing gold nanorods and lysozyme amyloid fibrils has been fabricated, and the effect of surface modification on improving nanostructure assembly on the biological template has been investigated. The nanohybrid system was characterized by monitoring surface plasmon resonance bands, dynamic light scattering spectroscopy, Thioflavin-T assay, and transmission electron microscopy. Surface of gold nanorods (GNRs) was modified with polystyrene sulfonate (PSS), and possible difference in assembly of the pristine and modified nanostructures was compared upon interaction with amyloid fibrils. Analysis of transmission electron microscopy showed that changing the surface charge of GNRs with biocompatible polymer improved electrostatic interactions between the nanostructures and amyloid fibril templates. Analysis of cell viability assays also showed that surface functionalization of GNRs remarkably improved biocompatibility of the nanoscaffold. Results of this study encourage utilization of modification strategies to fabricate a new generation of nanoscaffolds with fruitful applications in regenerative medicine.

Electrochemical detection of DNA mismatches using a branch-shaped hierarchical SWNT-DNA nano-hybrid bioelectrode.

Common approaches for DNA mutation detection are high cost and have difficult or complex procedure. We propose a fast quantitative method for recognition of DNA mutation based on SWNT/DNA self-assembled nanostructure. Covalent SWNT/DNA hybrid nanostructures are widely used in the fabrication of electrochemical biosensors. Interfacing carbon nanotubes with DNA in particular, is used as a detection method for the analysis of genetic disorders or the detection of mismatches in DNA hybridisation. We have designed a self-assembled, branch-shaped hybrid nanostructure by hybridisation of two sticky oligos that are attached to the ends of SWNTs via a linker oligo. These hybrid nanostructures showed a good conductivity that was greater than free SWNTs. Impedance spectroscopy studies illustrated that the conductivity of these hybrid nanostructures depended on the conformation and structure of the hybridised DNA. We demonstrated that the strategy of using SWNT/DNA self-assembled hybrid nanostructure fabrication yields sensitive and selective tools to discriminate mismatches in DNA. Cyclic voltammetry (CV) and impedance spectroscopy clearly revealed that the conductivity of the branch-shaped and hierarchical hybridised SWNT/DNA nanostructure is higher when matched, than when mismatched in a 1 and 1' hybridised SWNT/DNA nanostructure. Rapid biosensing of match and mismatch nanostructure based on carbon printed electrode showed similar results which can be used for rapid and fast detection of DNA mismatch.

The potential impact of carboxylic-functionalized multi-walled carbon nanotubes on trypsin: A Comprehensive spectroscopic and molecular dynamics simulation study.

In this study, we report a detailed experimental, binding free energy calculation and molecular dynamics (MD) simulation investigation of the interactions of carboxylic-functionalized multi-walled carbon nanotubes (COOH-f-MWCNTs) with porcine trypsin (pTry). The enzyme exhibits decreased thermostability at 330K in the presence of COOH-f-MWCNTs. Furthermore, the activity of pTry also decreases in the presence of COOH-f-MWCNTs. The restricted diffusion of the substrate to the active site of the enzyme was observed in the experiment. The MD simulation analysis suggested that this could be because of the blocking of the S1 pocket of pTry, which plays a vital role in the substrate selectivity. The intrinsic fluorescence of pTry is quenched with increase in the COOH-f-MWCNTs concentration. Circular dichroism (CD) and UV-visible absorption spectroscopies indicate the ability of COOH-f-MWCNTs to experience conformational change in the native structure of the enzyme. The binding free energy calculations also show that electrostatics, π-cation, and π-π stacking interactions play important roles in the binding of the carboxylated CNTs with pTry. The MD simulation results demonstrated that the carboxylated CNTs adsorb to the enzyme stronger than the CNT without the-COOH groups. Our observations can provide an example of the nanoscale toxicity of COOH-f-MWCNTs for proteins, which is a critical issue for in vivo application of COOH-f-MWCNTs.

Assembly of Gold Nanorods on HSA Amyloid Fibrils to Develop a Conductive Nanoscaffold for Potential Biomedical and Biosensing Applications.

Today, Gold Nanorods have promised variety of applications in conjugation with biomolecules of interest. Discovery of functional amyloids has also been highlighted with possible use in designing high performance materials. To exploit dual properties of both Nano and Bio counterparts in new functional materials, this effort has focused on synthesis of a potential hybrid system of Gold nanorods (GNRs) and HSA amyloid fibrils to develop a conductive nanoscaffold. UV-Vis spectroscopy, Thioflavin T (ThT) assay, Far-UV Circular Dichroism (CD) spectropolarimetry, fluorescence and Transmission Electron microscopy were used to characterize formation of the nanostructures and amyloid fibrils. Surface plasmon resonance of GNRs was also monitored upon interaction with HSA amyloid fibrils, showing that the plasmonic component of the hybrid system has maintained its characteristic rod morphology without any perturbations. Analysis of Nyquist plots for the hybrid nanoscaffold showed that the electronic behavior of the hybrid system has been enhanced due to the presence of the assembled GNRs. Results of this investigation highlight the possibility of fabricating hybrid nano-bioscaffolds as promising candidates in versatile biomedical and biosensing applications.

Rational design toward developing a more efficient laccase: Catalytic efficiency and selectivity.

Laccases are multicopper oxidases that catalyze the oxidation of variety of substrates. The specificity and efficiency of laccases are clearly the important components leading to their remarkable uses. To develop an improved biocatalysts, site directed mutagenesis of laccase from Bacillus HR03 was carried out in the current study. Based on the ABTS-bound crystal structure of CotA from B. subtilis and alignment with closely related enzymes, T415 and T418 at the vicinity of the type 1 copper site were chosen and several mutants (T415I, T418I, T415G, T415G/T418I) were made. Kinetic parameters of the constructs were then determined using ABTS and SGZ as substrates. In comparison with the wild-type, catalytic efficiency toward ABTS was improved by 4 fold in T415I and 1.5 fold in T418I and T415G. T415I and T418I variants were identified to be almost 11 and 27 times more specific for ABTS than for SGZ compared with the wild type. T415I was also found to acquire enhanced thermal stability with the half-life of 60min at 80°C. Secondary and tertiary structure of mutants were analyzed by CD and fluorescence spectroscopy. Our result illustrated that replacement of residues in the substrate-binding pocket would change the specificity and efficiency of variants.

Renin inhibition by soyasaponin I: a potent native anti-hypertensive compound.

One way to control hypertension is inactivation of the Renin- Angiotensin- Aldosterone System (RAAS). Inhibition of renin as a rate-limiting step of this system is an effective way to stop up RAAS. It has been proved that soyasaponin I, an herbal compound obtained from soybeans, has anti-hypertensive effect via renin inhibition, so it has the potential of being an anti-hypertensive drug. Herein, some theoretical approaches such as Docking Simulation, Molecular Dynamics (MD) Simulation and MMPBSA analysis have been used to study how soyasaponin I inhibits renin at the structural level. The results of docking simulation and hydrogen bond pattern show that this ligand is able to bind to the active site of renin and a region near the active site. Results of MD simulation for renin - soyasaponin I complexes confirm that soyasaponin I binds to the active site of renin and has inhibition effect on it via competing with the substrate. Besides, according to MMPBSA analysis, the binding free energy for renin - soyasaponin I complex is -42.61 kcal/mol when it binds to the active site. Comparing to the peptide obtained from angiotensinogen, ΔG = -74.96 kcal/mol, it may inferred that although binding of soyasaponin I to the active site of renin does not have a complete competition with the substrate, it might attenuate the formation of renin - angiotensinogen complex and have partial non-competitive effect. The results of this survey might be helpful to design partial non - competitive renin inhibitors with pharmaceutical capability.

Improving the stability of chondroitinase ABC I via interaction with gold nanorods.

Chondroitinase ABC I (cABC I) cleaves glycosaminoglycan chains which are responsible for most of the inhibition of axon regrowth in spinal cord injury. The application of chondroitinase ABC I (cABC I) in damaged nervous tissue is found to prune glycosaminoglycan chains of proteoglycans and facilitate axon regeneration. However, a limiting factor for such application is the enzyme's instability. In this study, the structure and activity of cABC I have been investigated upon interaction with various concentrations of Gold nanorods. The enzyme preserved its major activity with increase in substrate affinity in the presence of the nanostructures. Analysis of circular dichroism spectropolarimetry data showed that secondary structural content of the enzyme slightly increased. The complex form of the enzyme also showed higher storage stability. Fluorescence studies indicated that enzyme obtained more rigidity in its structure. Taking higher stability of enzyme upon interaction, result of this investigation interaction paves the way for utilizing tiny plasmonic nanostructures for fruitful applications in biomedicine.

Evolutionary conservation of EF-hand ΙΙ loop in aequorin: Priority of intensity to decay rate in bioluminescence emission.

As a Ca2+-regulated photoprotein, aequorin (Aeq) contains four EF-hand motifs, the second one lacks the standard sequence for Ca2+ coordination and doesn't bind to Ca2+. Here, we replaced this loop with a functional loop. According to structural studies, although the global stability of modified aequorin (4EFAeq) is higher than that of Aeq; increasing the local flexibility accompanied by internal structural rearrangements in 4EFAeq result in its penetrability to urea and acrylamide. A fast decay rate was observed for 4EFAeq. Assuming the presence of intermediate states in the luminescent reaction, this observation indicate that the loop replacement leads to the lowering of the half-life of intermediate states which results in increasing the rate of conformational switching of 4EFAeq to light emitting form. However, considerable reduction in initial luminescence intensity of 4EFAeq suggests that the number of functional complexes is reduced. Our findings demonstrate that the conformational effects of the second loop in Aeq elicit a delicate balance between local flexibility and global stability which may be considered as an important functional parameter in photoproteins. It was also concluded that evolutionary conservation of EF-hand ΙΙ in the current form is a consequence of priority of intensity to decay rate in bioluminescent organisms.

Structural and functional study of a simple, rapid, and label-free DNAzyme-based DNA biosensor for optimization activity.

Peroxidase-mimicking DNAzyme has a potential to self-assemble into a G-quadruplex and shows peroxidase activity. In comparison to proteins, peroxidase-mimicking DNAzyme is less expensive and more stable. Herein, it is used in fabricating non-labeling biosensors. This paper investigates the structural and functional properties of a DNA biosensor based on split DNAzyme with a detection limit in nM range (9.48 nM). Two halves of DNAzyme were linked by a complementary sequence of DNA target. Hybridization of the DNA target pulled two DNAzyme halves apart and peroxidase activity decreased. This study can be divided into 3 stages. First, the characteristics of DNAzyme were studied by Circular Dichroism technique and UV-Vis spectroscopy to find out DNAzyme's optimum activity. It is worth to note that some divalent cations were used to form G-quadruplex, in addition to common monovalent cations. Furthermore, the hemin incubation was also optimized. Secondly, the structural and functional properties of two types of split DNAzyme were compared with DNAzyme. Thirdly, the hybridization of DNA target was monitored. The results revealed that peroxidase activities of split types decreased by half without any specific conformational changes. Interestingly, the catalytic activities of split DNAzymes could be promoted by adding Mg2+ . Besides, it was demonstrated that the structure, peroxidation reaction, and DNA target hybridization of 2:2 and 3:1 split modes were almost alike. It was also illustrated that magnesium promoted the possibility of hybridization.

Ca2+ Binding and Conformational Switch of the Photoprotein Mnemiopsin.

BACKGROUND: Bioluminescence in Ca2+-binding photoproteins is an intramolecular reaction triggered by the addition of Ca2+. A comparative study has been done on Ca2+-depleted and Ca2+-loaded apo-mnemiopsin to understand the structural transition of the photoprotein by Ca2+ binding. Ca2+ is removed by TCA (trichloroacetic acid) precipitation to obtain Ca2+-depleted apomnemiopsin. METHOD: UV-visible, CD and fluorescence spectroscopic studies demonstrate that the addition of Ca2+ is brought about by the overall structure of apo-mnemiopsin becomes more open in a concentration- dependent manner without significantly influencing the secondary structure and indicate that the Ca2+-depleted form of apo-mnemiopsin, in contrast to most other EF-hand calcium binding proteins, adopt a closed conformation when compared to the Ca2+-loaded form. On the other hand, dynamic quenching and limited proteolysis analysis revealed that Ca2+-loaded apo-mnemiopsin became much more flexible than Ca2+ free apo-mnemiopsin. RESULTS: It seems that increased flexibility of the protein, which occurs due to calcium binding, is a critical factor in oxidative decarboxylation reaction on coelenterazine and consequently light emission.

Ultra-sensitive, rapid gold nanoparticle-quantum dot plexcitonic self-assembled aptamer-based nanobiosensor for the detection of human cardiac troponin I.

Acute myocardial infarction (AMI) is one of the leading causes of death throughout the world. Usual methods for detecting AMI are expensive, time-consuming and using blood samples as biological samples. Therefore, creating an ultra-fast, sensitive and non-invasive diagnostic test is necessary. Herein, a novel ultra-sensitive, fluorescent, plasmon-exciton coupling hybrid of a gold nanoparticle-quantum dot (PQ)-based aptamer nanobiosensor is presented for the detection of human cardiac troponin I (cTnI), the golden biomarker of AMI, and a preclinical test is performed within saliva. The binding of the cTnI protein to aptamer leads to a fluorescence enhancement of the plexcitonic hybrid system. The response range of this nanobiosensor is 0.4-2500 fM and the limit of detection is 0.3 fM. It seems that this novel design of nanobiosensor in the form of the PQ plexcitonic hybrid system can presents new opportunities for nanobiosensor progress.

MicroRNA and Transcription Factor Gene Regulatory Network Analysis Reveals Key Regulatory Elements Associated with Prostate Cancer Progression.

Technological and methodological advances in multi-omics data generation and integration approaches help elucidate genetic features of complex biological traits and diseases such as prostate cancer. Due to its heterogeneity, the identification of key functional components involved in the regulation and progression of prostate cancer is a methodological challenge. In this study, we identified key regulatory interactions responsible for primary to metastasis transitions in prostate cancer using network inference approaches by integrating patient derived transcriptomic and miRomics data into gene/miRNA/transcription factor regulatory networks. One such network was derived for each of the clinical states of prostate cancer based on differentially expressed and significantly correlated gene, miRNA and TF pairs from the patient data. We identified key elements of each network using a network analysis approach and validated our results using patient survival analysis. We observed that HOXD10, BCL2 and PGR are the most important factors affected in primary prostate samples, whereas, in the metastatic state, STAT3, JUN and JUNB are playing a central role. Benefiting integrative networks our analysis suggests that some of these molecules were targeted by several overexpressed miRNAs which may have a major effect on the dysregulation of these molecules. For example, in the metastatic tumors five miRNAs (miR-671-5p, miR-665, miR-663, miR-512-3p and miR-371-5p) are mainly responsible for the dysregulation of STAT3 and hence can provide an opportunity for early detection of metastasis and development of alternative therapeutic approaches. Our findings deliver new details on key functional components in prostate cancer progression and provide opportunities for the development of alternative therapeutic approaches.

Unfolding of chondroitinase ABC Ι is dependent on thermodynamic driving force by kinetically rate constant-amplitude compensation: A stopped-flow fluorescence study.

We had previously investigated the role of a loop on the activity and conformational stability of chondroitinase ABC Ι (cABC Ι) by constructing some representative mutants in which a network interaction around Asp689 was manipulated. Here we extended our study by measuring the proteolytic resistance, long term and thermal stability as well as unfolding kinetics of these variants. Long term stability data at 4 and 25°C for 3 weeks indicates that all mutants remain considerably active at 4°C. Thermoinactivation rates for all variants shows that the wild type (WT) enzyme retained 50% of its activity after 2min keeping at 40°C, while L701T, H700N and H700N/L701T as conformationally stabilized variants, have slower inactivation rate. It was also found that compact and thermodynamically stabilized variants are more resistant to tryptolytic digestion. Also, kinetic curves of chemical unfolding of the enzyme variants from stopped-flow fluorescence measurements were best fitted into a three-exponential function with three rate constants and corresponding amplitudes. We found that the energy barrier of the fast unfolding phase is lower in stabilized variants; while the amplitude of this phase to the whole amplitude of the unfolding reaction is lower than that of destabilized variants, indicating more population of stabilized mutants unfold via slower unfolding phase. We concluded that the rate of local conformational change alone is not the same that is expected from global thermodynamic stability; however the corresponding amplitude can compensate the rate constant toward thermodynamic stability.

Applying Central Composite Design and Response Surface Methodology to Optimize Growth and Biomass Production of Haemophilus influenzae Type b.

BACKGROUND: Haemophilus influenzae type b (Hib) is the leading cause of bacterial meningitis, otitis media, pneumonia, cellulitis, bacteremia, and septic arthritis in infants and young children. The Hib capsule contains the major virulence factor, and is composed of polyribosyl ribitol phosphate (PRP) that can induce immune system response. Vaccines consisting of Hib capsular polysaccharide (PRP) conjugated to a carrier protein are effective in the prevention of the infections. However, due to costly processes in PRP production, these vaccines are too expensive. OBJECTIVES: To enhance biomass, in this research we focused on optimizing Hib growth with respect to physical factors such as pH, temperature, and agitation by using a response surface methodology (RSM). MATERIALS AND METHODS: We employed a central composite design (CCD) and a response surface methodology to determine the optimum cultivation conditions for growth and biomass production of H. influenzae type b. The treatment factors investigated were initial pH, agitation, and temperature, using shaking flasks. After Hib cultivation and determination of dry biomass, analysis of experimental data was performed by the RSM-CCD. RESULTS: The model showed that temperature and pH had an interactive effect on Hib biomass production. The dry biomass produced in shaking flasks was about 5470 mg/L, which was under an initial pH of 8.5, at 250 rpm and 35° C. CONCLUSIONS: We found CCD and RSM very effective in optimizing Hib culture conditions, and Hib biomass production was greatly influenced by pH and incubation temperature. Therefore, optimization of the growth factors to maximize Hib production can lead to 1) an increase in bacterial biomass and PRP productions, 2) lower vaccine prices, 3) vaccination of more susceptible populations, and 4) lower risk of Hib infections.