Highly sensitive and novel dual-emission fluorescence nanosensor utilizing hybrid carbon dots-quantum dots for ratiometric determination of chlorpromazine.

Herein, a ratiometric fluorimetric nanosensor is introduced for the sensitive and selective analysis of chlorpromazine (CPZ) via employing blue-emitting B-doped carbon dots (B-CDs) as the reference fluorophore and green-emitting CdTe capped thioglycolic acid (TGA) quantum dots (TGA-CdTe-QDs) as the specific recognition probe. The sensor exhibits dual emission centered at 440 and 560 nm, under a single excitation wavelength of 340 nm. Upon the addition of ultra-trace amount of CPZ, the fluorescence signal of TGA-CdTe-QDs declines due to electron transfer process from excited TGA-CdTe-QDs to CPZ molecules, whereas the fluorescence peak of B-CDs is unaffected. Therefore, a new fluorimetric platform was prepared for the assay of CPZ in the range of 2.2 × 10-10 to 5.0 × 10-9 M with a detection limit of 1.3 × 10-10 M. Moreover, the practicability of the designed strategy was investigated for the detection of CPZ in biological samples and the results demonstrate that it possesses considerable potential to be utilized in practical applications.

The potential therapeutic effect of melatonin in oxaliplatin combination therapy against chemoresistant colorectal cancer cells.

BACKGROUND: Oxaliplatin is one of the main therapeutics in colorectal cancer (CRC) chemotherapy. However, in light of multidrug resistance (MDR) phenotype development, the efficacy of oxaliplatin has decreased. This study aimed to assess the potential therapeutic effect of melatonin in oxaliplatin combination therapy for drug-resistant colorectal cancer cells. METHODS AND RESULTS: Initially, the oxaliplatin-resistant cell line was created of LS174T (LS174T/DR) by using the oxaliplatin IC50 concentration and resting cycles. MTT assays and flow cytometry were applied for assessing cell viability and apoptotic cells. The mRNA expression level of Bax, Bcl2, MT1, MT2, and ABCB1 as well as protein levels of ABCB1, Bcl2, BAX were measured by the qRT-PCR and western blot techniques respectively. P-gp activity was assessed by Rho123 staining. The IC50 concentration of oxaliplatin in resistant cells was increased from 500.7 ± 0.2 nM to 7119 ± 0.1 nM. Bcl2, MT1, MT2, and ABCB1 mRNA plus protein expression levels of Bcl2 and ABCB1 were significantly reduced in resistant cells, along with a marked increase in Bax mRNA and protein levels compared to parental cells. Rho 123 staining revealed a marked reduction in P-gp activities in the combination-treated group compared to the oxaliplatin-treated group. CONCLUSIONS: The results of cytotoxicity assays, MTT, and flow cytometry revealed that the combination of melatonin and oxaliplatin exerts synergistic effects on induction of oxaliplatin's cytotoxicity in CRC. Our research suggests that combining the treatments of melatonin and oxaliplatin may be considered as a new approach to overcoming oxaliplatin resistance in CRC patients.

Oncostatin M and its receptor in women with polycystic ovary syndrome and association with assisted reproductive technology outcomes.

The role of adipokines in ovarian-related disorders such as polycystic ovary syndrome (PCOS) has been reported. However, the involvement of Oncostatin M (OSM), a recently identified adipokine, in ovarian function is unknown. Therefore, we investigated the association of the OSM signaling pathway with ovarian functions and PCOS pathogenesis. This case-control study enrolled 30 PCOS and 30 healthy women who underwent the intracytoplasmic sperm injection procedure. OSM and OSM receptor (OSMR) levels were evaluated in the follicular fluid (FF). Moreover, the expression of insulin receptor substrates (IRS1 and IRS2), OSM, OSMR, suppressor of cytokine signaling 3 (SOCS3), and androgen receptor (AR) genes were analyzed in the isolated cumulus cells (CCs). For the in-vitro experiment, the effect of recombinant OSM on the expression of related genes in isolated CCs was analyzed. Follicular concentrations of OSM and OSMR were significantly lower in PCOS (123.91±48.58 pg/ml and 0.93±0.35 ng/ml, respectively) compared to control women (283.53 ± 96.62 pg/ml and 1.45 ± 0.18 ng/ml, respectively; p < 0.001) and were positively correlated with the oocyte maturation (r = 0.611 and r = 0.611, respectively) and fertilization (r = 0.592 and r = 0.627, respectively) rates in the PCOS group. Furthermore, the SOCS3 expression was upregulated about eight times in PCOS patients compared to the controls (p < 0.05). The treatment of cells with recombinant OSM significantly increased SOCS3, OSMR, IRS-1, and -2 expression and decreased AR expression. The decreased levels of OSM and its receptor in PCOS patients, possibly mediated by SOCS3, could negatively affect oocyte maturation and fertilization rates.

Microwave-assisted facile synthesis of N, P co-doped fluorescent carbon dot probe for the determination of nifedipine.

A simple and fast microwave synthesis method was applied for the preparation of several carbon dots (CDs) from various combinations of urea, phosphoric acid, and B-alanine as nitrogen, phosphorus, and carbon precursors. The maximum quantum yield (44%) was obtained for nitrogen and phosphorus co-doped carbon dots (N, P-CDs) prepared from urea, B-alanine, and phosphoric acid. Furthermore, N, P-CDs were exploited to synthesize a simple and sensitive fluorometric probe to determine nifedipine (NFD). We determined that the analytical response of the designed sensor could be affected by the kind of dopant and synthesis precursors. It is worth mentioning that the fluorescence intensity of N, P-CDs was weakened by NFD, and no fluorescence quenching was observed for other prepared CDs. The NFD-developed sensor demonstrated a linear response range of 3.3 × 10-8-3.2 × 10-5 mol/L, with the detection limit of 1.0 × 10-8 mol/L. The sensor was successfully applied to measure NFD in human biological fluids.

Evaluation of the Diagnostic and Predictive Value of Glutamic Acid Decarboxylase, Zinc Transporter 8 Serum Levels in Alzheimer's Disease.

BACKGROUND: Identification of validated peripheral biomarkers for Alzheimer's Disease, leading to an early diagnosis of the disease, would be valuable for predicting progression and targeted therapeutics. In this regard, serum levels of GADA, ZnT8A, Zn, vitamin D, and leukocyte expression of brain-derived neurotrophic factor (BDNF) gene were investigated in Alzheimer's patients and control group. METHODS: Serum levels of GADA, ZnT8A, Zn, and vitamin D and leukocyte expression of the BDNF gene were evaluated in 40 AD patients and 40 control cases. The diagnostic value of investigated factors was examined with the receiver operating characteristic (ROC). RESULTS: The results showed significant differences of p < 0.0001, p < 0.0001, and p = 0.0006 between AD patients and control individuals in GADA, Zn, and ZnT8A serum levels, respectively. No significant difference was observed in the serum concentration of vitamin D between AD patients and control cases (p = 0.2993). The expression level of the BDNF gene in AD patients was different from control cases, but it was not statistically significant (p > 0.05). Moreover, ROC curve analysis disclosed a diagnostic potency for serum levels of GADA, Zn, and ZnT8A for AD with an area under the ROC curve of > 0.7 (p < 0.0001, p < 0.0001, and p = 0.0007, respectively). CONCLUSIONS: The results demonstrated the higher serum levels of GADA and ZnT8A and lower serum concentrations of Zn in the patient group. Therefore, these parameters can be discussed as possibly diagnostic in AD cases.

A turn off-on fluorometric and paper-based colorimetric dual-mode sensor for isoniazid detection.

In the present study, cobalt oxyhydroxide (CoOOH) nanosheets were applied for establishing a dual fluorometric and smartphone-paper-based colorimetric method to detect isoniazid. CoOOH nanosheets quenched the fluorescence emission of sulfur and nitrogen co-doped carbon dots (S,N-CDs) due to inner filter effect (IFE). The quenched fluorescence intensity of S,N-CDs restored in the presence of isoniazid due to destroying CoOOH nanosheets by this drug. Moreover, with adding isoniazid the solution color of CoOOH nanosheets altered from brownish yellow to pale yellow. We exploited these facts to design a turn off-on fluorometric and paper-based colorimetric sensor for isoniazid measurement at the range 0.5-5 and 5-100 µM with detection limits of 0.28 µM and 4.0 µM, respectively. The introduced dual sensor was used for pharmaceutical, environmental and biological analysis of isoniazid with satisfactory results. The paper-based colorimetric sensor can be applied for isoniazid portable monitoring using a smartphone as a detector or even the naked eye.

Assessment of E. coli Expression System for Overexpression of Active Recombinant Ocriplasmin.

Purpose: Ocriplasmin (Jetrea TM) is a FDA approved recombinant enzyme utilized in the treatment of vitreomacular adhesion (VMA). This is a recombinant C-terminal fragment of human plasmin produced using yeast Pichia pastoris. Since ocriplasmin does not contain any Oor N-glycosylation or some other post-translational modifications, bacterial expression systems such as Escherichia coli could be considered as an economical host for recombinant expression. In the present study, we aimed to evaluate the efficiency of E. coli expression system for highlevel expression of recombinant ocriplasmin. Methods: The gene coding for ocriplasmin was cloned and expressed in E. coli BL21. The bacterial cells were cultured on large scale and the expressed recombinant protein was purified using Ni-NTA chromatography. Refolding of denatured ocriplasmin to active enzyme was carried out by the stepwise removal of denaturant. The identity of recombinant ocriplasmin was confirmed using western blotting and ELISA assays. The presence of the active ocriplasmin was monitored by the hydrolytic activity assay against the chromogenic substrate S-2403. Results: The final yield of E. coli BL21-produced ocriplasmin was approximately 1 mg/mL which was greater than that of P. pastoris. Using western blotting and ELISA assay, the identity of recombinant ocriplasmin was confirmed. The hydrolysis of chromogenic substrate S-2403 verified the functional activity of E. coli produced ocriplasmin. Conclusion: The results of this study indicated that E. coli could be used for high level expression of ocriplasmin. Although the recombinant protein was expressed as inclusion body, the stepwise refolding leads to the biologically active proteins.

Role of adipokines in the ovarian function: Oogenesis and steroidogenesis.

Adipokines are mainly produced by adipose tissue; however, their expression has been reported in other organs including female reproductive tissues. Therefore, adipokines have opened new avenues of research in female fertility. In this regard, studies reported different roles for certain adipokines in ovarian function, although the role of other recently identified adipokines is still controversial. It seems that adipokines are essential for normal ovarian function and their abnormal levels could be associated with ovarian-related disorders. The objective of this study is to review the available information regarding the role of adipokines in ovarian functions including follicular development, oogenesis and steroidogenesis and also their involvement in ovary-related disorders.

Phosphorylation Modulates Survivin Function in Behcet's Disease.

Purpose: Survivin is critical for proliferation, maturation, homeostasis and differentiation of effector and memory lymphocytes. In this study the baculoviral inhibitors of apoptosis proteins (IAPs) repeat containing 5 (BIRC5) mRNA, survivin, and phosphorylated survivin expression were evaluated in peripheral blood mononuclear cells (PBMCs), and plasma of patients with Behcet's disease (BD). Methods: In this study, 26 Iranian Azari patients diagnosed with BD and 30 healthy controls were recruited. Total RNA was extracted from PBMCs. The expression level of survivin was measured by quantitative real-time polymerase chain reaction (PCR). Survivin plasma levels were measured using survivin Enzyme-linked immunosorbent assays. Also, western blotting analysis was performed to measure phosphorylated-survivin and survivin levels in PBMCs and plasma of patients with BD. Results: In a pilot study, we showed that BIRC5 gene expression increased in BD patients compared with healthy controls (P<0.05). Western blotting analysis indicated that there was an increase in phosphorylated survivin expression in PBMCs of BD patients. Our data from western blot analysis showed survivin level in plasma samples of BD patients was similar to healthy controls. No significant differences were observed between plasma survivin levels in the BD patients compared with control group (P>0.05). The expression of phosphorylated survivin at Thr34 in PBMCs of BD patients with active disease was increased. Plasma phosphorylated survivin levels in having BD patients were also downregulated compared to healthy individuals. Conclusion: Analysis of PBMCs indicated increasing expression level of phosphorylated survivin in PBMCs of BD patients. There was also a downregulation in phosphorylated survivin levels in plasma of BD patients.

Novel nano-vehicle for delivery and efficiency of anticancer auraptene against colon cancer cells.

The aim of this study is to devise, prepare and characterize nano encapsulated auraptene (AUR) and evaluate cytotoxic and apoptotic effects on HT-29 colon cancer cells. Herein, AUR nano formulations were prepared by triblock (PCL-PEG-PCL) and pentablock (PLA-PCL-PEG-PCL-PLA) biodegradable copolymers in order to increase AUR bioavailability as an anticancer agent. The preparation of nano particles (NPs) was done with rotor stator homogenization (RSH) and Ultrasonic homogenization (USH) methods. The physicochemical characteristics of prepared nanoparticles (NPs) were studied using HNMR, FTIR, GPC, DLS and SEM techniques. The smaller hydrodynamic size (110 nm) and polydispersity index (PDI: 0.288) as well as higher cellular uptake (89%) were observed in PB NPs rather than TB NPs. The highest cytotoxic and apoptotic effects were observed in AUR loaded PB NPs compared to AUR loaded TB NPs and free AUR obtained by MTT assay, cell cycle arrest, Annexin V-FITC, DAPI staining and RT-PCR techniques. Real time PCR results indicated that Bax /Bcl2 expression ratio as an apoptosis predicting criterion, in free AUR, AUR loaded TB and AUR loaded PB have increased 6, 9 and 13 times, respectively (p value < 0.05). In conclusion, using biodegradable nano-vehicles for sustained delivery of natural anti-cancer compounds may open new perspectives for treatment of cancer patients.

Synthesized Nano particle derivation of poly (Styrene - co- Maleic Anhydride) and sour cherry Rock for removing nickel (II) ion from aqueous solutions.

In this study, Prunus Cerasus Rock (PCR) and Poly (Styrene - co- Maleic Anhydride) modified with Melamine-Oxalic acid (SMA-MO) were used to prepare a cheap adsorbent through chemical modification. The maximum removal was observed at pH = 6.0 and adsorbent dose 1.5 g/L for initial Nickel -ions concentration 30 mg/L. Study of temperature effect proved that the process is endothermic. Langmuir and Freundlich isotherm models were used for equilibrium adsorption data. Langmuir isotherm proved to be a better fit. Pseudo first order and pseudo second order kinetic models were applied to analyze the kinetic mechanism of adsorption.

Selection of a fully human single domain antibody specific to Helicobacter pylori urease.

Helicobacter pylori bacteria are involved in gastroduodenal disorders, including gastric adenocarcinoma. Since the current therapies encounter with some significant shortcomings, much attention has been paid to the development of new alternative diagnostic and treatment modalities such as immunomedicines to target H. pylori. Having used phage display technology, we isolated fully humane small antibody (Ab) fragment (VL) against the Flap region of urease enzyme of H. pylori to suppress its enzymatic activity. Solution biopanning (SPB) and screening process against a customized biotinylated peptide corresponding to the enzyme Flap region resulted in the selection of VL single domain Abs confirmed by the enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blotting. The selected Ab fragments showed a high affinity with a KD value of 97.8 × 10-9 and specificity to the enzyme with high inhibitory impact. For the first time, a VL single domain Ab was isolated by SPB process against a critical segment of H. pylori urease using a diverse semi-synthetic library. Based on our findings, the selected VL Ab fragments can be used for the diagnosis, imaging, targeting, and/or immunotherapy of H. pylori. Further, Flap region shows great potential for vaccine therapy.

New Developments in Pichia pastoris Expression System, Review and Update.

BACKGROUND: Although Pichia pastoris is an outstanding host among conventional expression systems for production of recombinant proteins, a new interest has been emerged to this system due to the inherent advantages and new developments in this expression host. The potential for secretory and soluble expression of heterologous glycoproteins in P. pastoris proposed this system as a candidate for the production of complex eukaryotic proteins. METHODS: Several new developments have occurred in different areas related to P. pastoris expression system including hosts, vectors, glycosylation pattern and fermentation technology. Strain engineering using Crispr/Cas9 technology to produce human-like glycoproteins and protease deficient strains are two new areas of development with high importance. RESULTS: This review is dedicated to discuss the most important characteristics of P. pastoris with emphasis on new developments, especially in the field of glycoengineering, efficient expression vectors and promoters. CONCLUSION: New developments that occurred in the P. pastoris expression system converted this system to a versatile host for the production of complex proteins. This progress paved the way for several proteins to enter the clinical trials or industrial processes with this valuable expression host.

Phage display-derived antibody fragments against conserved regions of VacA toxin of Helicobacter pylori.

Infection with Helicobacter pylori may result in the emergence of gastric adenocarcinoma. Among various toxins assisting pathogenesis of H. pylori, the vacuolating cytotoxin A (VacA) is one of the most potent toxins known as the major cause of the peptic ulcer and gastric adenocarcinoma. To isolate single-chain variable fragments (scFvs) against two conserved regions of VacA, we capitalized on the phage display technology and a solution-phase biopanning (SPB). Characterization of scFvs was carried out by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and surface plasmon resonance (SPR). Bioinformatics analyses were also performed in order to characterize the structural and functional properties of the isolated scFvs and the interaction(s) between the isolated antibodies (Ab)-antigen (Ag). After four rounds of biopanning, the positive colonies detected by scFv ELISA were harvested to extract the plasmids and perform sequencing. Of several colonies, three colonies showed high affinity to the VacA1 and two colonies for the VacA2. Further complementary examinations (e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western blot, SPR, and flow cytometry) displayed the high affinity and specificity of the isolated scFvs to the VacA. Docking results revealed the interaction of the complementarity-determining regions (CDRs) with the VacA peptide. In conclusion, for the first time, we report on the isolation of several scFvs against conserved residues of VacA toxin with high affinity and specificity, which may be used as novel diagnostic/therapeutic tool in the H. pylori infection.

Hydrogen bonding recognition and colorimetric detection of isoprenaline using 2-amino-5-mercapto-1,3,4-thiadiazol functionalized gold nanoparticles.

In this paper, we describe a rapid, low-cost and highly sensitive colorimetric method for the detection of isoprenaline, based on 2-amino-5-mercapto-1,3,4-thiadiazol (AMTD) functionalized gold nanoparticles (AMTD-AuNPs) as a sensing element. Hydrogen bonding interaction between isoprenaline and AMTD resulted in the aggregation of AuNPs and a consequent color change of AuNPs from red to blue. The concentration of isoprenaline could be detected with the naked eye or a UV-visible spectrometer. Results showed that the absorbance ratio (A650/A524) was linear with isoprenaline concentrations in the range of 0.2 to 2.6µM (R=0.997). The detection limit of this method was 0.08µM. The proposed method is simple, without using complicated instruments and adding salts for enhancing sensitivity. This probe could be successfully applied to the determination of isoprenaline in human serum samples and urine samples after deproteinization.

Ionic Liquid-Bonded Fused Silica as a New Solid-Phase Microextraction Fiber for the Liquid Chromatographic Determination of Bisphenol A as an Endocrine Disruptor.

Linear ionic liquid bonded to fused silica and its application as a solid-phase microextraction fiber for the extraction of bisphenol A (BPA) from water samples were studied. After optimization of microextraction conditions (15 mL sample volume, extraction time of 40 min, extraction temperature of 30 ± 1°C, 300 µL acetonitrile as the desorption solvent, and desorption time of 7 min), the fiber was used to extract BPA from packed mineral water, followed by HPLC-UV on an XDB-C18 column (150 × 4.6 mm id, 3.5 µm particle) with a mobile phase of acetonitrile-water (45 + 55%, v/v) and flow rate of 1 mL . min-1). A low LOD (0.20 µg . L-1) and good linearity (0.9977) in the calibration graph indicated that the proposed method was suitable for the determination of BPA.

Pleiotropic cytotoxicity of VacA toxin in host cells and its impact on immunotherapy.

Introduction: In the recent decades, a number of studies have highlighted the importance of Helicobacter pylori in the initiation and development of peptic ulcer and gastric cancer. Some potential virulence factors (e.g., urease, CagA, VacA, BabA) are exploited by this microorganism, facilitating its persistence through evading human defense mechanisms. Among these toxins and enzymes, vacuolating toxin A (VacA) is of a great importance in the pathogenesis of H. pylori. VacA toxin shows different pattern of cytotoxicity through binding to different cell surface receptors in various cells. Methods: To highlight attempts in treatment for H. pylori infection, here, we discussed the VacA potential as a candidate for development of vaccine and targeted immunotherapy. Furthermore, we reviewed the related literature to provide key insights on association of the genetic variants of VacA with the toxicity of the toxin in cells. Results: A number of investigations on the receptor(s) binding of VacA toxin confirmed the pleiotropic nature of VacA that uses a unique mechanism for internalization through some membrane components such as lipid rafts and glycophosphatidylinositol (GPI)-anchored proteins (GPI-AP). Considering the high potency of VacA toxin in the clinical presentations in infection and assisting persistence and colonization of H. pylori, it is considered as one of the pivotal components in production vaccines and monoclonal antibodies (mAbs). Conclusion: It is possible to generate mAbs with a considerable potential to convert into secretory immunoglobulins that could penetrate into the niche of H. pylori and inhibit its normal functionalities. Further, conjugation of H. pylori targeting Ab fragments with the toxic agents or drug delivery systems (DDSs) offers new generation of H. pylori treatments.

Fluorescent Carbon Dot as Nanosensor for Sensitive and Selective Detection of Cefixime Based on Inner Filter Effect.

Here a simple and sensitive fluorescent assay for detecting Cefixime based on inner filter effect (IFE) has been proven, which is conceptually different from the previously reported CEF fluorescent assays. In this sensing platform, fluorescent carbon dots (CDs) were prepared by one-pot synthesis and was directly used as fluorophore in IFE. The method is based on the complexation reaction between cefixime and palladium ion in the presence of acidic buffer solution (pH 4). The Pd(II)-CEFcomplex was capable of functioning as a powerful absorber in IFE to influence the excitation of fluorophore (CDs). Production Pd(II)-CEFcomplex induced the absorption band transition from 310 to 400 nm, which resulted in the complementary overlap with the excitation spectra of CDs. Due to the competitive absorption, the excitation of CDs was significantly weakened, resulting in the quenching of CDs. The present IFE-based sensing strategy showed a good linear relationship from 0.2 × 10-6 M to 8 × 10-6 M (R2 = 0.987) and provided an exciting detection limit of 0.5 × 10-7 (3δ/slope). The proposed method has been successfully applied for the determination of cefixime in raw milk and human urine samples.

Impact of C-rel inhibition of cord blood-derived B-, T-, and NK cells.

The c-Rel transcription factor is a unique member of the nuclear factor (NF)-κB family that has a role in curtailing the proliferation, differentiation, cytokine production, and overall activity of B- and T-cells. In addition, c-Rel is a key regulator of apoptosis in that it influences the expression of anti-apoptotic genes such as Bcl-2 and Bcl-xL; conversely, inhibition of c-Rel increases cell apoptosis. To better understand the relationship between c-Rel expression and effects on B- and T-cell expansion, the current study evaluated c-Rel expression in cord blood mononuclear cells. This particular source was selected as cord blood is an important source of cells used for transplantation and immunotherapy, primarily in treating leukemias. As stem cell factor (SCF) and FLT3 are important agents for hematopoietic stem cell expansion, and cytokines like interleukin (IL)-2, -7, and -15 are essential for T- and B- (and also NK) cell development and proliferation, the current study evaluated c-Rel expression in cord blood mononuclear cells and CD34+ cells, as well as effects on B-, T-, and NK cells associated with alterations in c-Rel expression, using flow cytometry and PCR. The results showed c-Rel expression increased among cells cultured in the presence of SCF and FLT3 but was reduced when IL-2, IL-7, and IL-15 were used all together. Further, inhibition of c-Rel expression by siRNA reduced cord blood-derived B-, T-, and NK cell differentiation and expansion. These results indicated that with cells isolated from cord blood, c-Rel has an important role in B-, T-, and NK cell differentiation and, further, that agents (select cytokines/growth factors) that could impact on its expression might not only affect immune cell profiles in a host but could potentially also limit apoptotic activities in (non-)immune cells in that host. In the context of cancer (immuno)therapy, in particular, when cord blood is used an important source in stem cell transplantation in leukemia patients, such down-regulating changes in c-Rel levels could be counter-productive.