Methotrexate loaded in alginate beads for controlled drug release against breast cancer.

Methotrexate (MTX), as a folate antagonist is used for breast cancer chemotherapy, but its application due to the adverse side effects was limited. In this study, MTX were encapsulated in magnetic alginate beads coated with glutaraldehyde to control its release in order to reduce the side effects and improve its stability. The complex was characterized by physicochemical studies. The encapsulation efficiency was 75 % and the complex showed acceptable controlled release behavior. The cell cytotoxicity assessed using methylthiazol tetrazolium (MTT) method showed that magnetic alginate beads-MTX, in lower dosage has higher anticancer effect compared to the free MTX. The real-time polymerase chain reaction (PCR) was used to evaluate apoptotic factors Bcl2 associated X gene (Bax), B-cell lymphoma 2 (Bcl-2), and neuroinflammatory marker tumor necrosis factor-alpha (TNF-α) genes expression level on the treated cells. The findings demonstrated the significant increase of expression of Bax and a significant decrease in the expressions of Bcl-2 and TNF-α in Michigan cancer foundation-7 (MCF-7) cells. These results indicated that the developed drug can overcome the side effects of MTX and offer a controlled drug release for a sustained period with the long-term treatment of breast cancer.

Design of highly sensitive nano-biosensor for diagnosis of hydatid cyst based on gold nanoparticles.

Cystic echinococcosis, a zoonotic parasitic infection, is a major public health and economic concern, with worldwide distribution. The development of sensitive diagnostic methods for hydatid disease is important. We designed a highly sensitive nano-biosensor for the diagnosis of hydatid cyst based on gold nanoparticles (AuNPs). AuNPs were synthesized. Echinococcus granulosus antigen was coated on the ELISA microwells. Then, the E. granulosus IgG antibody was added to the microwells. After incubation and washing, the Ag-Ab complex was incubated with a human IgG HRP​-conjugated antibody. Then, the synthesized AuNPs and tetramethylbenzidine (TMB), as a chromogenic substrate of HRP, were added to the reaction. Finally, the absorption rate was measured by spectrophotometry. The results showed that the enzyme peroxide and TMB change the color of the reaction from red to yellow by oxidizing AuNPs. The sensitivity and specificity of the designed method were investigated. The linear equation and regeneration of nanobiosensor designed for red color Y = 0.0312X + 0.649, R2 9962 and for yellow color Y = 0.013X + 0.398, R2 9851 were determined. The limit of detection of the designed nanobiosensor was 0.001 µg mL-1. The results confirmed that the designed nanobiosensor was completely specific for the detection of E. granulosus antibody.

Enhanced sensitivity and efficiency of detection of Staphylococcus aureus based on modified magnetic nanoparticles by photometric systems.

Staphylococcus aureus is an important infectious factor in the food industry and hospital infections. Many methods are used for detecting bacteria but they are mostly time-consuming, poorly sensitive. In this study, a nano-biosensor based on iron nanoparticles (MNPs) was designed to detect S. aureus. MNPs were synthesized and conjugated to Biosensors. Then S. aureus was lysed and nano-biosensor (MNP-TiO2-AP-SMCC-Biosensors) was added to the lysed bacteria. After bonding the bacterial genome to the nano-biosensor, MNPs were separated by a magnet. Bacterial DNA was released from the surface of nano-biosensor and researched by Nano-drop spectrophotometry. The results of SEM and DLS revealed that the size of MNPs was 20-25 nm which increased to 38-43 nm after modification and addition of biosensors. The designed nano-biosensor was highly sensitive and specific for the detection of S. aureus. The limit of detection (LOD) was determined as 230 CFU mL-1. There was an acceptable linear correlation between bacterial concentration and absorption at 3.7 × 102-3.7× 107 whose linear diagram and regression was Y = 0.242X + 2.08 and R2 = .996. Further, in the presence of other bacteria as a negative control, it was absolutely specific. The sensitivity of the designed nano-biosensor was investigated and compared through PCR.

Functionalization of anti-Brucella antibody based on SNP and MNP nanoparticles for visual and spectrophotometric detection of Brucella.

An Immuno-Nano-Biosensor with high sensitivity was designed based on iron and silica nanoparticles to detect B. abortus. Briefly explain, primary polyclonal antibody (IgG1) was conjugated on surface magnetic nanoparticles (MNPs) to form MNP-IgG1. Secondary polyclonal antibody (IgG2) and Horseradish Peroxidase enzyme were conjugated on silica nanoparticles (SNPs) to form HRP-SNP-IgG2. HRP-SNP-IgG2. MNP-IgG1 and HRP-SNP-IgG2 were added to B. abortus. The MNP-IgG1-B.abortus-IgG2-SNP-HRP complex was isolated from the reaction mixture using a magnet. After that, tetramethylbenzidine was added to the complex. The reaction was stopped with HCl and investigated using UV-Vis spectrophotometry. The nanoparticles' structure and size were investigated using SEM and DLS. Immuno-Nano-Biosensor sensitivity and specificity were determined. The SEM and DLS results indicated that the SNPs, MNPs, HRP-SNP-IgG2 and MNP-IgG1 size and structure were 35, 44, 60 and 56 nm, respectively. In addition, a good linear correlation was observed at 102-107 CFU mL-1 concentrations, which their linear equation and regression were Y = 0.3× + 0.18 and R2 0.982, respectively. The limitation of detecting B. abortus was 160 CFU mL-1. Finally, the results demonstrated that those designed Immuno-Nano-Biosensor could be specifically detected B. abortus and B. melitensis in real samples.

A fluorescence Nano-biosensors immobilization on Iron (MNPs) and gold (AuNPs) nanoparticles for detection of Shigella spp.

The highly sensitive and specificity detection are very important in diagnosis of foodborne pathogens and prevention of spread diseases. Therefore, in the present study, a highly sensitive fluorescence Nano-biosensors was designed for detection of Shigella species. For achieved this purpose, DNA probes and gold nanoparticles (AuNPs) were designed and synthesized, respectively. Then, two DNA probes as signal reporter were immobilized on surface of AuNPs. On the other hand, Iron nanoparticles (MNPs) were synthesized and modified with SMCC (Sulfosuccinimidyl 4-Nmaleimidomethyl cyclohexane-1- carboxylate). The 3th DNA probe was immobilized on surface of MNPs for separation of target DNA. The MNP-DNA probe and DNA probe-AuNP-fluorescence DNA probe were added to target DNA. The MNP- DNA probe-target DNA-DNA probe-AuNP-fluorescence DNA probe complex was isolated by a magnet. The fluorescence DNA probe was released on surface of AuNPs and the fluorescence intensity was read by fluorescence spectrophotometry. Sensitivity and specificity of designed Nano-biosensor was determined. The results showed that the fluorescence intensity was increased with increasing of target DNA concentration. Linear related between target DNA and fluorescence intensity was observed in 2.3 × 102 up to 2.3 × 107 CFU mL-1. The linear equation and regression were Y = 1.8 X + 23.4 and R2 0.9953. Limit of detection (LOD) were determined 90 CFUmL-1. The specificity of Nano-biosensor in present of other bacteria was confirmed.

Folic acid functionalized nanoparticles as pharmaceutical carriers in drug delivery systems.

Conventional chemotherapeutic approaches in cancer therapy such as surgery, chemotherapy, and radiotherapy have several disadvantages due to their nontargeted distributions in the whole body. On the other hand, nanoparticles (NPs) based therapies are remarkably progressing to solve several limitations of conventional drug delivery systems (DDSs) including nonspecific biodistribution and targeting, poor water solubility, weak bioavailability and biodegradability, low pharmacokinetic properties, and so forth. The enhanced permeability and retention effect escape from P-glycoprotein trap in cancer cells as a passive targeting mechanism, and active targeting strategies are also other most important advantages of NPs in cancer diagnosis and therapy. Folic acid (FA) is one of the biologic molecules which has been targeted overexpressed-folic acid receptor (FR) on the surface of cancer cells. Therefore, conjugation of FA to NPs most easily enhances the FR-mediated targeting delivery of therapeutic agents. Here, the recent works in FA which have been decorated NPs-based DDSs are discussed and cancer therapy potency of these NPs in clinical trials are presented.

Bio-barcode technology for detection of Staphylococcus aureus protein A based on gold and iron nanoparticles.

S. aureus is one of important causes of disease, food poisoning in humans and animals. The generally methods for detection of S. aureus is time consuming. Therefore, a new method is necessary for rapid, sensitive and specific diagnosis of S. aureus. In the present study, two probes and a Bio-barcode DNA were designed for detection of S. aureus (Protein A). Firstly, magnetic nanoparticle (MNPs) and gold nanoparticle (AuNPs) were synthesized at 80 °C and 100 °C, respectively. The AuNPs and the MNPs were functionalized with probe1, Bio-barcode DNA and probe2, respectively. Target DNA was added into the nanomaterial's system containing bio-barcode DNA-AuNPs-probe1 and probe2-MNPs to formed bio-barcode DNA-AuNPs-probe1-target DNA-probe2-MNPs complex. The bio-barcode DNA-AuNPs-probe1-target DNA-probe2-MNPs complex was separated with magnetic field. Finally, the bio-barcode DNA was released from surface of complex using DTT (0.8 M) and there was isolated of nanoparticles by magnetic field and centrifuge. The fluorescence intensity of bio-barcode DNA was measured in different concentrations of S. aureus (101 to 108 CFU mL-1) by fluorescence spectrophotometry. The results showed that standard curve was linearly from 102 to 107 CFU mL-1. Limit of detection of bio-barcode assay for both PBS and real samples was 86 CFU mL-1.

Highly selective and sensitive detection of Staphylococcus aureus with gold nanoparticle-based core-shell nano biosensor.

Staphylococcus aureus is a gram-positive and opportunistic pathogen that is one of the most common causes of nosocomial infections; therefore, its rapid diagnosis is important and valuable. Today, the use of nanoparticles is expanding due to their unique properties. The purpose of the present study is the determination of S. aureus by a colorimetric method based on gold nanoparticles (AuNPs). Firstly, S. aureus was cultured on both LB media (broth and agar) and their chromosomal DNA was extracted. Afterwards, primers and biosensor were designed based on Protein A sequence data in the gene bank. PCR assay was performed under optimal conditions and the PCR product was electrophoresed on 2-percent agarose gel. The synthesized biosensors were conjugated with AuNPs and, eventually, a single-stranded genome was added to the conjugated AuNPs and hybridization was performed. The results were evaluated based on color change detected by the naked eye, optical spectrophotometry, and transient electron microscopy. Finally, the sensitivity and specificity of the AuNP-biosensor were determined. The results of the present study showed a 390 bp band on the agarose electrophoresis gel, which confirmed the presence of Protein A genes on the chromosome of the bacteria. The PCR and colorimetric methods were compared with each other. The sensitivity of the PCR and colorimetric methods were 30 ng µL-1 and 10 ng µL-1, respectively. The limit of detection (LOD) equaling 8.73 ng µL-1 was determined and the specificity of the method was confirmed by the DNA of other bacteria. According to the results, the present method is rapid and sensitive in detecting S. aureus.

Spectrophotometric, colorimetric and visually detection of Pseudomonas aeruginosa ETA gene based gold nanoparticles DNA probe and endonuclease enzyme.

Colorimetric DNA detection is preferred over other methods for clinical molecular diagnosis because it does not require expensive equipment. In the present study, the colorimetric method based on gold nanoparticles (GNPs) and endonuclease enzyme was used for the detection of P. aeruginosa ETA gene. Firstly, the primers and probe for P. aeruginosa exotoxin A (ETA) gene were designed and checked for specificity by the PCR method. Then, GNPs were synthesized using the citrate reduction method and conjugated with the prepared probe to develop the new nano-biosensor. Next, the extracted target DNA of the bacteria was added to GNP-probe complex to check its efficacy for P. aeruginosa ETA gene diagnosis. A decrease in absorbance was seen when GNP-probe-target DNA cleaved into the small fragments of BamHI endonuclease due to the weakened electrostatic interaction between GNPs and the shortened DNA. The right shift of the absorbance peak from 530 to 562nm occurred after adding the endonuclease. It was measured using a UV-VIS absorption spectroscopy that indicates the existence of the P. aeruginosa ETA gene. Sensitivity was determined in the presence of different concentrations of target DNA of P. aeruginosa. The results obtained from the optimized conditions showed that the absorbance value has linear correlation with concentration of target DNA (R: 0.9850) in the range of 10-50ngmL-1 with the limit detection of 9.899ngmL-1. Thus, the specificity of the new method for detection of P. aeruginosa was established in comparison with other bacteria. Additionally, the designed assay was quantitatively applied to detect the P. aeruginosa ETA gene from 103 to 108CFUmL-1 in real samples with a detection limit of 320CFUmL-1.

Fluorescence bio-barcode DNA assay based on gold and magnetic nanoparticles for detection of Exotoxin A gene sequence.

Bio-barcode DNA based on gold nanoparticle (bDNA-GNPs) as a new generation of biosensor based detection tools, holds promise for biological science studies. They are of enormous importance in the emergence of rapid and sensitive procedures for detecting toxins of microorganisms. Exotoxin A (ETA) is the most toxic virulence factor of Pseudomonas aeruginosa. ETA has ADP-ribosylation activity and decisively affects the protein synthesis of the host cells. In the present study, we developed a fluorescence bio-barcode technology to trace P. aeruginosa ETA. The GNPs were coated with the first target-specific DNA probe 1 (1pDNA) and bio-barcode DNA, which acted as a signal reporter. The magnetic nanoparticles (MNPs) were coated with the second target-specific DNA probe 2 (2pDNA) that was able to recognize the other end of the target DNA. After binding the nanoparticles with the target DNA, the following sandwich structure was formed: MNP 2pDNA/tDNA/1pDNA-GNP-bDNA. After isolating the sandwiches by a magnetic field, the DNAs of the probes which have been hybridized to their complementary DNA, GNPs and MNPs, via the hydrogen, electrostatic and covalently bonds, were released from the sandwiches after dissolving in dithiothreitol solution (DTT 0.8M). This bio-barcode DNA with known DNA sequence was then detected by fluorescence spectrophotometry. The findings showed that the new method has the advantages of fast, high sensitivity (the detection limit was 1.2ng/ml), good selectivity, and wide linear range of 5-200ng/ml. The regression analysis also showed that there was a good linear relationship (∆F=0.57 [target DNA]+21.31, R2=0.9984) between the fluorescent intensity and the target DNA concentration in the samples.

Determination of sensitivity of male Wistar rats to an equal dose of ketamine/xylazine injection at anesthetic dose in a chronic model of hypernatremia in comparison with control group.

OBJECTIVE: To determine the sensitivity to an equal dose of ketamine/xylazine injection at anesthetic dose in a chronic model of hypernatremia. METHODS: This study was conducted at the Department of Physiology, Zanjan University of Medical Science, Zanjan, Iran in 2004. Sixty male Wistar rats, weighing 250 +/- 20 g were randomly allocated to 3 groups. The control group was provided with tap water, and first and second test groups consumed 1% and 2% salt concentrations for 144 hours. One hundred mg/kg ketamine and 10 mg/kg xylazine were used as an anesthetic agent. The measured anesthetic parameters comprises of righting reflex latency, required time for establishment of animal's immobility, immobility period, required time for appearance of animal's mobility and complete re-establishment of the righting reflex. RESULTS: The required time for inhibition of the righting reflex and animal's mobility in the second group was significantly shorter than the first and control groups. Immobility period, required time for appearance of animal's mobility and complete re-establishment of the righting reflex in the second group were significantly longer than the first and control groups. CONCLUSION: Hypernatremia increases the speed of transition from different steps of ketamine/xylazine anesthesia with significant delay in immobility period and recovery from anesthesia in rats, hence, anesthetic dose reduction in hypernatremia is necessary.

A human protein atlas for normal and cancer tissues based on antibody proteomics.

Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, approximately 400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.

Carbohydrate-active enzymes involved in the secondary cell wall biogenesis in hybrid aspen.

Wood formation is a fundamental biological process with significant economic interest. While lignin biosynthesis is currently relatively well understood, the pathways leading to the synthesis of the key structural carbohydrates in wood fibers remain obscure. We have used a functional genomics approach to identify enzymes involved in carbohydrate biosynthesis and remodeling during xylem development in the hybrid aspen Populus tremula x tremuloides. Microarrays containing cDNA clones from different tissue-specific libraries were hybridized with probes obtained from narrow tissue sections prepared by cryosectioning of the developing xylem. Bioinformatic analyses using the sensitive tools developed for carbohydrate-active enzymes allowed the identification of 25 xylem-specific glycosyltransferases belonging to the Carbohydrate-Active EnZYme families GT2, GT8, GT14, GT31, GT43, GT47, and GT61 and nine glycosidases (or transglycosidases) belonging to the Carbohydrate-Active EnZYme families GH9, GH10, GH16, GH17, GH19, GH28, GH35, and GH51. While no genes encoding either polysaccharide lyases or carbohydrate esterases were found among the secondary wall-specific genes, one putative O-acetyltransferase was identified. These wood-specific enzyme genes constitute a valuable resource for future development of engineered fibers with improved performance in different applications.

Multiple group-specific sequencing primers for reliable and rapid DNA sequencing.

Pyrosequencing technology is a bioluminometric DNA sequencing method that employs a cascade of four enzymes to deliver sequence signals. To date this technology has been limited to the sequencing of short stretches of DNA. As an improvement to this technique, we have introduced a bacterial group-specific, multiple sequencing primer approach that circumvents sequencing of less informative semi-conservative regions of the 16S rRNA gene. This new approach is suitable for challenging templates, improving sequence data quality, avoiding sequencing of non-specific amplification products, lessening sequencing time, and moreover, this strategy should open the way for many new applications in the future. The group-specific, multiple sequencing primers can be applied in the Sanger dideoxy sequencing method as well. In addition, we have improved the chemistry of the Pyrosequencing system enabling sequencing of longer stretches of DNA, which allows numerous new applications.

Multiple-primer DNA sequencing method.

A multiple-primer DNA sequencing approach suitable for genotyping, detection and identification of microorganisms and viruses has been developed. In this new method two or more sequencing primers, combined in a pool, are added to a DNA sample of interest. The oligonucleotide that hybridizes to the DNA sample will function as a primer during the subsequent DNA sequencing procedure. This strategy is suited for selective detection and genotyping of relevant microorganisms and samples harboring different DNA targets such as multiple variant/infected samples as well as unspecific amplification products. This method is used here in a model system for detection and typing of high-risk oncogenic human papilloma viruses (HPVs) in samples containing multiple infections/variants or unspecific amplification products. Type-specific sequencing primers were designed for four of the most oncogenic (high-risk) HPV types (HPV-16, HPV-18, HPV-33, and HPV-45). The primers were combined and added to a sample containing a mixture of one high-risk (16, 18, 33, or 45) and one or two low-risk types. The DNA samples were sequenced by the Pyrosequencing technology and the Sanger dideoxy sequencing method. Correct genotyping was achieved in all tested combinations. This multiple-sequencing primer approach also improved the sequence data quality for samples containing unspecific amplification products. The new strategy is highly suitable for diagnostic typing of relevant species/genotypes of microorganisms.

Molecular cloning and characterization of a cDNA encoding poplar UDP-glucose dehydrogenase, a key gene of hemicellulose/pectin formation.

Plant UDP-glucose dehydrogenase (UGDH) is an important enzyme in the formation of hemicellulose and pectin, the components of newly formed cell walls. A cDNA clone (Ugdh) corresponding to UGDH was isolated from a cDNA library prepared from cambial zone of poplar (Populus tremula x tremuloides). Within the 1824-nucleotide (nt)-long clone, an open reading frame encoded a protein of 481 amino acids (aa), with a calculated molecular weight of 53.1 kDa. The derived aa sequence showed 90% and 63% identity with UGDHs from soybean and bovine liver, respectively, and had highly conserved aa motifs believed to be of importance for nt binding and catalytic efficiency. In poplar, the Ugdh corresponds to one or two genes, as found by genomic Southern analysis. The gene was expressed predominantly in differentiating xylem and young leaves, with little expression in the phloem zone of the stem. The expression pattern matched that of UGDH protein, as found by immunoblotting. In leaves, the Ugdh expression was upregulated by a short-term feeding with sucrose, sorbitol and polyethylene glycol, and this effect was to some extent mimicked by light exposure. The data suggest that Ugdh is regulated via an osmoticum-dependent pathway, possibly related to the availability of osmotically active carbohydrate precursors to UDP-glucose, a substrate of UGDH.