Gold nanostructure-enhanced immunosensing: ultra-sensitive detection of VEGF tumor marker for early disease diagnosis.

We present an advanced electrochemical immunosensor designed to detect the vascular endothelial growth factor (VEGF) precisely. The sensor is constructed on a modified porous gold electrode through a fabrication process involving the deposition of silver and gold on an FTO substrate. Employing thermal annealing and a de-alloying process, the silver is eliminated from the electrode, producing a reproducible porous gold substrate. Utilizing a well-defined protocol, we immobilize the heavy-chain (VHH) antibody against VEGF on the gold substrate, facilitating VEGF detection through various electrochemical methods. Remarkably, this immunosensor performs well, featuring an impressive detection limit of 0.05 pg/mL and an extensive linear range from 0.1 pg/mL to 0.1 µg/mL. This emphasizes it's to measure biomarkers across a wide concentration spectrum precisely. The robust fabrication methodology in this research underscores its potential for widespread application, offering enhanced precision, reproducibility, and remarkable detection capabilities for the developed immunosensor.

Bioluminescence measurement of superoxide anion in infertile men with oxidative stress.

Infertility is such an important issue in society today. In some cases of male infertility, the main cause is oxidative stress and the presence of reactive oxygen species in the environment or in sperm cells. All current techniques that measure oxidative stress, including the nitroblue tetrazolium Test, DNA Fragmentation Index, Malondialdehyde, and Endz Test are qualitative and semi-quantitative. These methods do not have good sensitivity and specificity. Semen samples from 50 infertile patients and 10 normal individuals were collected. The samples were examined for laboratory routine tests according to the WHO 2010 protocol. Oxidative stress tests, including DFI, NBT, and MDA, were performed for these two groups. Bioluminescence inhibition assay was performed for detection of O2.- in semen samples by aequorin. The normal individuals showed significantly better semen parameters than the patient's group. Significantly lower O2.- levels were seen in the patient's group compared to normal individuals. The cut-off value of O2.- levels in normal individuals was determined to be 8 × 105 RLU/s with a sensitivity of 100% and a specificity of 100%. Infertile patients, despite having reduced quality of semen parameters, have high O2.- levels, and this causes the intensity of bioluminescence to be quenched in these people.

Expression of VEGFR2 Ligand Binding Domain in Pichia pink™ 4 Cells and Evaluation of Its Interactions with VEGF-A165 Receptor Binding Domain.

Vascular endothelial growth factor A165 (VEGF-A165) and VEGF receptor 2 (KDR) are important mediators of angiogenesis. We aimed to express the soluble KDR ligand-binding domain (sKDR1-3) and evaluate its interaction with the VEGF-A165 receptor-binding domain (VEGFA165-RBD). sKDR1-3 DNA was designed and subcloned into pPinkα-HC plasmid. The cassette was transfected into the Pichia pink™ 4 genome by homologous recombination. We optimized the expression of sKDR1-3 under the induction of different methanol concentrations. VEGFA165-RBD was expressed in E. coli BL21 harboring pET28a( +)─VEGFA165-RBD vector under induction with IPTG with/without lactose. Interaction and biological activity of sKDR1-3 and VEGFA165-RBD were investigated by ELISA and anti-proliferation tests. sKDR1-3 migrated on SDS-PAGE gel as a 35-180 kDa protein due to glycosylation. The relative expression level of sKDR1-3 under 1% methanol was higher than 0.5% and 4% methanol induction. IPTG and cysteine were suitable for induction and refolding of VEGFA165-RBD. 25 ng sKDR1-3 and 20 ng VEGFA165-RBD showed strong binding. sKDR1-3 bound to VEGFA165-RBD and VEGF-A165 with dissociation constants of 0.148 and 0.2 nM, respectively. 4-10 nM concentrations of sKDR1-3 inhibited the proliferation of HUVE cells induced by 5 nM VEGFA165-RBD. In consideration, sKDR1-3 in the nanomolar concentration range, is a promising anticancer drug to inhibit angiogenesis.

Design, synthesis, spectroscopic characterizations, antidiabetic, in silico and kinetic evaluation of novel curcumin-fused aldohexoses.

Curcumin (bis-α,ß-unsaturated ß-diketone) plays an important role in the prevention of numerous diseases, including diabetes. Curcumin, as an enzyme inhibitor, has ideal structural properties including hydrophobic nature, flexible backbone, and several available hydrogen bond (H-bond) donors and acceptors. In this study, curcumin-fused aldohexose derivatives 3(a-c) were synthesized and used as influential agents in the treatment of diabetes with inhibitory properties against two carbohydrate-hydrolyzing enzymes α-glucosidase (α-Gls) and α-amylase (α-Amy) which are known to be significant therapeutic targets for the reduction of postprandial hyperglycemia. These compounds were isolated, purified, and then spectrally characterized via FT-IR, Mass, 1H, and 13C NMR, which strongly confirmed the targeted product's formation. Also, their inhibitory properties against α-Gls and α-Amy were evaluated spectroscopically. The Results indicated that all compounds strongly inhibited α-Amy and α-Gls by mixed and competitive mechanisms, respectively. The intrinsic fluorescence of α-Amy was quenched by the interaction with compounds 1 and 3b through a dynamic quenching mechanism, and the 1 and 3b/α-Amy complexes were spontaneously formed, mainly driven by the hydrophobic interaction and hydrogen bonding. Fourier transform infrared spectra (FT-IR) comprehensively verified that the binding of compounds 1 and 3b to α-Amy would change the conformation and microenvironment of α-Amy, thereby inhibiting the enzyme activity. Docking and molecular dynamics (MD) simulations showed that all compounds interacted with amino acid residues located in the active pocket site of the proteins. In vivo studies confirmed the plasma glucose diminution after the administration of compound 3b to Wistar rats. Accordingly, the results of the current work may prompt the scientific communities to investigate the possibility of compound 3b application in the clinic.

Synthesis and Potential Antidiabetic Properties of Curcumin-Based Derivatives: An In Vitro and In Silico Study of α-Glucosidase and α-Amylase Inhibition.

BACKGROUND: Over the past twenty years, the prevalence of diabetes as one of the most common metabolic diseases has become a public health problem worldwide. Blood glucose control is important in delaying the onset and progression of diabetes-related complications. α-Glycosidase (α- Glu) and α-amylase (α-Amy) are important enzymes in glucose metabolism. Diabetic control through the inhibition of carbohydrate hydrolyzing enzymes is established as an effective strategy. METHODS: In this study, curcumin-based benzaldehyde derivatives with high stability, bioavailability, and favorable efficiency were synthesized. RESULTS: The results showed that L13, L8, and L11 derivatives have the highest inhibitory effect on α-Glu with IC50 values of 18.65, 20.6, and 31.7 µM and, also L11, L13, and L8 derivatives have the highest inhibitory effect on α-Amy with IC50 value of 14.8, 21.8, and 44.9 µM respectively. Furthermore, enzyme inhibitory kinetic characterization was also performed to understand the mechanism of enzyme inhibition. CONCLUSION: L13, compared to the other compounds, exhibited acceptable inhibitory activity against both enzymes. The L13 derivative could be an appropriate candidate for further study through the rational drug design to the exploration of a new class of powerful anti-diabetic drugs considering the antioxidant properties of the synthesized compounds. The derivative helps reduce the glycemic index and limits the activity of the major reactive oxygen species (ROS) producing pathways.

A mutation in Arabidopsis SAL1 alters its in vitro activity against IP3 and delays developmental leaf senescence in association with lower ROS levels.

KEY MESSAGE: Our manuscript is the first to find a link between activity of SAL1/OLD101 against IP3 and plant leaf senescence regulation and ROS levels assigning a potential biological role for IP3. Leaf senescence is a genetically programmed process that limits the longevity of a leaf. We identified and analyzed the recessive Arabidopsis stay-green mutation onset of leaf death 101 (old101). Developmental leaf longevity is extended in old101 plants, which coincided with higher peroxidase activity and decreased H2O2 levels in young 10-day-old, but not 25-day-old plants. The old101 phenotype is caused by a point mutation in SAL1, which encodes a bifunctional enzyme with inositol polyphosphate-1-phosphatase and 3' (2'), 5'-bisphosphate nucleotidase activity. SAL1 activity is highly specific for its substrates 3-polyadenosine 5-phosphate (PAP) and inositol 1, 4, 5-trisphosphate (IP3), where it removes the 1-phosphate group from the IP3 second messenger. The in vitro activity of recombinant old101 protein against its substrate IP3 was 2.5-fold lower than that of wild type SAL1 protein. However, the in vitro activity of recombinant old101 mutant protein against PAP remained the same as that of the wild type SAL1 protein. The results open the possibility that the activity of SAL1 against IP3 may affect the redox balance of young seedlings and that this delays the onset of leaf senescence.

Drug repositioning based on gene expression data for human HER2-positive breast cancer.

Human epidermal growth factor receptor 2 (HER2)-positive breast cancer represents approximately 15-30% of all invasive breast cancers. Despite the recent advances in therapeutic practices of HER2 subtype, drug resistance and tumor recurrence still have remained as major problems. Drug discovery is a long and difficult process, so the aim of this study is to find potential new application for existing therapeutic agents. Gene expression data for breast invasive carcinoma were retrieved from The Cancer Genome Atlas (TCGA) database. The normal and tumor samples were analyzed using Linear Models for Microarray Data (LIMMA) R package in order to find the differentially expressed genes (DEGs). These genes were used as entry for the library of integrated network-based cellular signatures (LINCS) L1000CDS2 software and suggested 24 repurposed drugs. According to the obtained results, some of these drugs including vorinostat, mocetinostat, alvocidib, CGP-60474, BMS-387032, AT-7519, and curcumin have significant functional similarity and structural correlation with FDA-approved breast cancer drugs. Based on the drug-target network, which consisted of the repurposed drugs and their target genes, the aforementioned drugs had the highest degrees. Moreover, the experimental approach verified curcumin as an effective therapeutic agent for HER2 positive breast cancer. Hence, our work suggested that some repurposed drugs based on gene expression data can be noticed as potential drugs for the treatment of HER2-positive breast cancer.

The application of the QDs/H2O2 chemiluminescence system in HRP assay and HRP-based immunoassay.

Horseradish peroxidase (HRP) is usually used as a label enzyme in immunoassay so the method used for HRP detection in enzyme immunoassay (EIA) plays a key role in sensitivity and precision. The catalytic activity of HRP does not strictly follow classic Michaelis-Menten kinetics, probably due to the inactivation of the enzyme at high concentrations of H2O2. In this paper, a highly sensitive alternative procedure for the HRP assay using H2O2-sensitive CdTe quantum dots as a chemiluminescence (CL) system is reported. This method can measure a much more accurate and reliable value of Km (187 mM H2O2) in comparison with the standard detection method. This system also was applied to thyroid hormone (T4) detection using HRP-based immunoassay. The QD/H2O2 system exhibits a higher linear range of 0.2-16 µg/dL with the improved LOD value of 0.06 µg/dL and selective response to T4, which was better than the commercial colorimetric immunoassay. Meanwhile, the proposed method has been successfully applied to the clinical determination of T4 in the serum samples, and the results confirmed an excellent correlation with the conventional ELISA method (R2 = 0.9832), indicating the potential applications of the method for clinical diagnosis as well.

Thermophilic iron containing type superoxide dismutase from Cohnella sp. A01.

Superoxide dismutases (SODs) (EC 1.15.1.1) are well known antioxidant enzymes that play critical roles in cellular defenses of living organisms against harmful superoxide radicals during oxidative stress. This study details on cloning, biochemical and functional characterization of an iron containing type superoxide dismutase (SOD) from a novel thermophilic bacteria Cohnella sp. A01 (CaSOD). The secondary and three dimensional structure of the protein were predicted. CaSOD gene was subsequently cloned into pET-26b(+) expression vector and expression of the recombinant protein (rCaSOD) was optimized in E. coli BL21 (DE3) and the purified recombinant SOD showed a single band with an apparent molecular weight of 26 kDa by SDS-PAGE. The half-life and thermodynamic parameters including ΔH⁎, ΔS⁎, and ΔG⁎ were 187 min at 60 °C, 7.3 kJ.mol-1, -76.8 kJ.mol-1.°K-1, and 84.1 kJ.mol-1, respectively. The rCaSOD exhibited catalytic activity in a very broad range of pH (6.0-10.0) and temperatures (35-75 °C), as well as stability in a broad pH range, from 3.0 to 11.0, and wide range of temperature, different concentrations of detergent agents, metal ions, organic solvents and other chemicals. The results suggest that this novel enzyme could be used for various industrial applications in cosmetic, food, and pharmaceutical industries.

An enzyme-mediated controlled release system for curcumin based on cyclodextrin/cyclodextrin degrading enzyme.

In this study, an enzyme-triggered system based on ß-cyclodextrin (ß-CD) has been developed to achieve controlled release of hydrophobic drugs in the presence of maltogenic amylase (MAase). The inclusion complex formation of curcumin (CUR), as a model anticancer compound, with ß-CD was characterized by fluorescence and Fourier transform infrared (FTIR) spectroscopy. CUR was loaded into ß-CD with an encapsulation efficiency of approximately 30 %. The in vitro profiles of CUR release from ß-CD showed that 100 % of the drug was released after one hour incubation in the presence of MAase with cyclodextrin degrading activity. Fluorescence microscopy images indicate a significantly greater cellular uptake of CUR using ß-CD-CUR/MAase system compared to ß-CD-CUR inclusion complex without MAase. The ß-CD-CUR/MAase system exhibited lower IC50 values and greater anti-proliferative effects in comparison with free CUR and ß-CD-CUR in MCF-7 and Huh-7 cancer cells. The results from fluorescence microscopy and flow cytometric assay using the acridine orange/ethidium bromide and Annexin V-PE/7-AAD staining suggest that the ß-CD-CUR/MAase system exhibited higher cytotoxic and apoptotic effects on cancer cells compared to other formulations. This triggered release of CUR in the presence of MAase is owing to the ß-CD degradation by MAase resulting ring opening and chain scission in ß-CD. We demonstrate that this enzyme-mediated controlled release system has a potential application for controlled release of poorly water-soluble drugs or hydrophobic compounds such as CUR.

Stress-dependent conformational changes of artemin: Effects of heat and oxidant.

Artemin is an abundant thermostable protein in Artemia embryos and it is considered as a highly efficient molecular chaperone against extreme environmental stress conditions. The conformational dynamics of artemin have been suggested to play a critical role in its biological functions. In this study, we have investigated the conformational and functional changes of artemin under heat and oxidative stresses to identify the relationship between its structure and function. The tertiary and quaternary structures of artemin were evaluated by fluorescence measurements, protein cross-linking analysis, and dynamic light scattering. Based on the structural analysis, artemin showed irreversible substantial conformational lability in responses to heat and oxidant, which was mainly mediated through the hydrophobic interactions and dimerization of the chaperone. In addition, the chaperone-like activity of heated and oxidized artemin was examined using lysozyme refolding assay and the results showed that although both factors, i.e. heat and oxidant, at specific levels improved artemin potency, simultaneous incubation with both stressors significantly triggered the chaperone activation. Moreover, the heat-induced dimerization of artemin was found to be the most critical factor for its activation. It was suggested that oxidation presumably acts through stabilizing the dimer structures of artemin through formation of disulfide bridges between the subunits and strengthens its chaperoning efficacy. Accordingly, it is proposed that artemin probably exists in a monomer-oligomer equilibrium in Artemia cysts and environmental stresses and intracellular portion of protein substrates may shift the equilibrium towards the active dimer forms of the chaperone.

RepCOOL: computational drug repositioning via integrating heterogeneous biological networks.

BACKGROUND: It often takes more than 10 years and costs more than 1 billion dollars to develop a new drug for a particular disease and bring it to the market. Drug repositioning can significantly reduce costs and time in drug development. Recently, computational drug repositioning attracted a considerable amount of attention among researchers, and a plethora of computational drug repositioning methods have been proposed. This methodology has widely been used in order to address various medical challenges, including cancer treatment. The most common cancers are lung and breast cancers. Thus, suggesting FDA-approved drugs via drug repositioning for breast cancer would help us to circumvent the approval process and subsequently save money as well as time. METHODS: In this study, we propose a novel network-based method, named RepCOOL, for drug repositioning. RepCOOL integrates various heterogeneous biological networks to suggest new drug candidates for a given disease. RESULTS: The proposed method showed a promising performance on benchmark datasets via rigorous cross-validation. The final drug repositioning model has been built based on a random forest classifier after examining various machine learning algorithms. Finally, in a case study, four FDA approved drugs were suggested for breast cancer stage II. CONCLUSION: Results show the potency of the proposed method in detecting true drug-disease relationships. RepCOOL suggested four new drugs for breast cancer stage II namely Doxorubicin, Paclitaxel, Trastuzumab, and Tamoxifen.

Mutual effects of protein corona formation on CdTe quantum dots.

Future biomedical applications of nanoparticles will encounter these particles with patients' serum which might affect the properties and stability of quantum dots and serum proteins at the desired site of action. Therefore, it is essential to clarify the patient-specific serum components, serve as major interaction partners, the spatial distribution of these, and consequently the time-dependent effects of nanoparticle-protein interaction. Here, a biochemical and structural study was performed on the protein corona formation and the corresponding interaction of different sizes of CdTe QDs with human serum proteins to determine if the mutual effects on optical properties by using electrophoresis, chemiluminescence, and fluorescence spectroscopy. The results revealed that interaction with human serum significantly enhanced the stability and photoluminescence of quantum dots. Structural studies of HSA-coated CdTe QDs also showed that corona formation has no adverse effects on protein structure, and the reduction in fluorescence emissions of HSA is due to the direct quenching of aromatics residues by the quantum dot. Improving nanoparticle properties, as well as the lack of structural changes in HSA, can be very useful in biomedical applications and in vivo studies where stability is important.

Development of a phage display-mediated immunoassay for the detection of vascular endothelial growth factor.

Because of the critical role of vascular endothelial growth factor (VEGF) in angiogenesis and its significantly increased serum levels in early stages of cancer, VEGF is considered an important prognostic biomarker in different cancers. Herein, the amplification power of PCR combined with phage displaying anti-VEGF VHH, a sensitive real-time immunoassay, was precisely designed based on phage display-mediated immuno-PCR (PD-IPCR) for the detection of VEGF. This system benefits from strong and specific binding of antigen and antibody in a sandwich immunosorbent assay platform using avastin (anti-VEGF monoclonal antibody) as the capture antibody. The anti-VEGF phage particles were used as both anti-VEGF agent and DNA template in the PD-IPCR. Anti-VEGF phage ELISA showed a linear range of 3-250 ng/ml and a limit of detection (LOD) of 1.1 ng/ml. Using the PD-IPCR method, the linear range of VEGF detection was found to be 0.06-700 ng/ml, with a detection limit of 3 pg/ml. The recovery rate in serum ranged from 83% to 99%, with a relative standard deviation of 1.2-4.9%. These values indicate that the method has good sensitivity for use in clinical analysis. The proposed method was successfully applied to the clinical determination of VEGF in human serum samples, and the results showed excellent correlation with conventional ELISA (R2 = 0.995). The novel immunoassay provides a specific and sensitive immunoassay protocol for VEGF detection at very low levels. Graphical abstract.

Crosstalk between melatonin and Ca2+/CaM evokes systemic salt tolerance in Dracocephalum kotschyi.

In this study, the role of calcium/calmodulin (Ca2+/CaM) and melatonin (Mel) as two signal molecules in inducing systemic salt tolerance of Dracocephalum kotschyi Boiss. was investigated. Salinity stress (100 mM NaCl) reduced plant growth and induced ionic, osmotic, and oxidative damages in D. kotschyi leaves. Detection of cytosolic free Ca2+ ([Ca2+]cyt) by the Fura-2 method and the measurement of endogenous Mel by GC-MS demonstrated that salinity induced Ca2+ burst and increased endogenous Mel content in D. kotschyi leaves. Root pretreatment with 5 mM Ca2+ or 100 µM Mel recovered plant growth, reduced leaf electrolytic leakage, H2O2, and MDA contents and improved membrane integrity not only at the application site (roots), but also at the untreated distal parts (leaves) under salt stress. Rhizospheric treatment with Mel and Ca2+ triggered systemic tolerance in D. kotschyi, as judged from improving RWC, increasing proline content, modulating Na+, K+, and Ca2+ homeostasis, and enhancing the activities of SOD, CAT, APX, and POD in the leaves of salt-stressed plants. Mel augmented [Ca2+]cyt, but the rhizospheric application of Ca2+ antagonists impaired the latter responses. Furthermore, root pretreatment with Ca2+ increased Mel content, but the application of p-chlorophenylalanine (as an inhibitor of Mel biosynthesis) decreased the above attributes in the leaves of Ca2+-treated plants, leading to an arrest in the Ca2+-induced systemic salt tolerance. These novel results suggest that interaction of Ca2+/CaM and Mel is involved in overcoming salt-induced ionic, osmotic, and oxidative damages and Ca2+ and Mel may act as long-distance signals for inducing systemic salt tolerance in D. kotschyi.

Anti-amyloidogenic effect of artemin on α-synuclein.

α-Synuclein fibrillation is now regarded as a major pathogenic process in Parkinson's disease and its proteinaceous deposits are also detected in other neurological disorders including Alzheimer's disease. Therefore anti-amyloidegenic compounds may delay or prevent the progression of synucleinopathies disease. Molecular chaperones are group of proteins which mediate correct folding of proteins by preventing unsuitable interactions which may lead to aggregation. The objective of this study was to investigate the anti-amyloidogenic effect of molecular chaperone artemin on α-synuclein. As the concentration of artemin was increased up to 4 µg/ml, a decrease in fibril formation of α-synuclein was observed using thioflavin T (ThT) fluorescence and congo red (CR) assay. Transmission electron microscopy (TEM) images also demonstrated a reduction in fibrils in the presence of artemin. The secondary structure of α-synuclein was similar to its native form prior to fibrillation when incubated with artemin. A cell-based assay has shown that artemin inhibits α-synuclein aggregation and reduce cytotoxicity, apoptosis and reactive oxygen species (ROS) production. Our results revealed that artemin has efficient chaperon activity for preventing α-synuclein fibril formation and toxicity.

Enhanced sensitivity of VEGF detection using catalase-mediated chemiluminescence immunoassay based on CdTe QD/H2O2 system.

BACKGROUND: Since vascular endothelial growth factor (VEGF) is a significant regulator of cancer angiogenesis, it is essential to develop a technology for its sensitive detection. Herein, we sensitized a chemiluminescence (CL) immunoassay through the combination of H2O2-sensitive TGA-CdTe quantum dot (QD) as signal transduction, dextran as a cross-linker to prepare enzyme-labeled antigen and the ultrahigh bioactivity of catalase (CAT) as reporter enzyme. RESULTS: Under the optimized experimental conditions, the chemiluminescence enzyme-linked immunosorbent assay (CL-ELISA) method can detect VEGF in the excellent linear range of 2-35,000 pg mL-1, with a detection limit (S/N = 3) of 0.5 pg mL-1 which was approximately ten times lower than the commercial colorimetric immunoassay. This proposed method has been successfully applied to the clinical determination of VEGF in the human serum samples, and the results illustrated an excellent correlation with the conventional ELISA method (R2 = 0.997). The suitable recovery rate of the method in the serum ranged from 97 to 107%, with a relative standard deviation of 1.2% to 13.4%. CONCLUSIONS: The novel immunoassay proposes a highly sensitive, specific, and stable method for very low levels detection of VEGF that can be used in the primary diagnosis of tumors. With the well-designed sensing platform, this approach has a broad potential to be applied for quantitative analysis of numerous disease-related protein biomarkers for which antibodies are available.

Targeted anticancer prodrug therapy using dextran mediated enzyme-antibody conjugate and ß-cyclodextrin-curcumin inclusion complex.

A targeted and controlled drug delivery system based on ß-cyclodextrin (ß-CD) for encapsulation and controlled release of hydrophobic drugs in the presence of maltogenic amylase (MAase), as a cyclodextrin-hydrolyzing enzyme, and trastuzumab antibody has been developed. In this study, the inclusion complex of curcumin (CUR), as a model anticancer compound, with ß-CD was prepared and we constructed an antibody-enzyme bioconjugate (dextran mediated MAase-Trastuzumab bioconjugate) for controlled and targeted release of CUR at HER2 positive cancer cells (including SKBR3 and BT474). Immunocytochemistry analysis indicated that the MAase-Trastuzumab bioconjugate had significant binding affinities to HER2 positive cancer cells and demonstrated high enzyme activity to degrade ß-CD in order to rapid release of CUR on targeted cell surface. Fluorescence microscopy images and cytotoxicity studies represent significantly greater cellular uptake and anti-proliferative effects of CUR by ß-CD-CUR/MAase-Trastuzumab bioconjugate compared to free CUR and ß-CD-CUR in presence and absence of MAase in HER2 positive cells. The results from flow cytometric assay suggest that the ß-CD-CUR/MAase-Trastuzumab conjugate exhibited higher cytotoxic and apoptotic effects on cancer cells compared to other formulation. We demonstrate that this formulation has a potential application for targeted and controlled release of drugs in cancer therapy with increased therapeutic efficiency.

Autolysis, plasmolysis and enzymatic hydrolysis of baker's yeast (Saccharomyces cerevisiae): a comparative study.

Saccharomyces cerevisiae is being used for long as a rich source of proteins, sugars, nucleotides, vitamins and minerals. Autolyzed and hydrolyzed yeast biomass has found numerous applications in the health food industry as well as livestock feeds. Here, we have compared three lysis methods for production of yeast lysates using autolysis, plasmolysis (ethyl acetate 1.5%), and enzymatic hydrolysis (Alcalase 0.2%). The efficiency of each process was compared according to soluble solid and protein contents, cell lysis monitoring, and release of intracellular materials, cell viability and microscopic analysis. Results showed that plasmolysis by ethyl acetate was found to be more efficient compared to autolysis, with a higher recovery of yeast extract (YE) content. In comparison, the content of released solids and proteins were higher during the enzymatic hydrolysis using Alcalase compared to autolysis and plasmolysis treatments. The highest decrease in optical density of 600 nm was monitored for the hydrolyzed cells. Besides, we defined "Degree of Leakage (DL)" as a new index of the lysis process, referring to the percentage of total released proteins from the cells and it was estimated to about 65.8%, which represents an appropriate indicator of the cell lysis. The biochemical and biophysical properties of the hydrolyzed yeast product as well as its biological activity (free radical scavenging activity and bacterial binding capacity) suggest that Alcalase could be used to accelerate the lysis of yeast cells and release the valuable intracellular components used for foodstuffs, feed and fermentation media applications. Production of baker's yeast lysates using autolysis, plasmolysis, and enzymatic hydrolysis methods.

Rapid and simple screening of the apoptotic compounds based on Hsp70 inhibition using luciferase as an intracellular reporter.

HSP70 is a powerful antiapoptotic protein that can block the extrinsic and intrinsic pathways of apoptosis. The present study describes a rapid, sensitive, and inexpensive system using luciferase as a reporter for the functional analysis of apoptotic compounds. For this approach, the co-transformation of Escherichia coli cells was performed with two expression vectors containing Hsp70 and firefly luciferase. It was found that the luciferase inactivated by heat treatment (40-46 °C for 10 min) was approximately reactivated at room temperature and regained 70% of its initial activity before heat inactivation after 60 min. The results show that the reactivation of thermally inactivated luciferase was inhibited in living cells by treatment with VER-155008 and pifitrin-µ as Hsp70 inhibitors, with half-maximal inhibitory concentration of 124 and 384 µM, respectively. The sensitivity of this method for detecting VER-155008 and pifitrin-µ was about 8 and 25 µM, respectively. Also, this reporter system showed no response to doxorubicin and dactinomycin, which bind to DNA, and we used these anticancer compounds as control compounds. Therefore, for the first time, a rapid and simple real-time system using luciferase as a reporter is introduced for the screening of apoptosis-inducing compounds based on suppression of Hsp70 in E. coli cells.