Study on Simultaneous Colonization of Rhizophagus irregularis and Serendipita indica in Barley under Different P Levels Using Monoclonal Antibody

Introduction Recent studies show that most crops and horticultural plants can form symbiosis with the arbuscular mycorrhizal fungi (AMF) and the endophytic Serendepita indica, simultaneously. The endophytic fungus plays an important role in alleviating environmental stresses in plants. It has also been shown that excessive available phosphorus in soil limits the root colonization by arbuscular mycorrhizal fungi. No information is available on how soil phosphorus affects the establishment of endophytic fungus in root. Barley roots can be colonized by both mycorrhizal fungi and the endophytic fungus Serendipita indica. The objective of this study was to evaluate the effects of single or dual inoculation with Rhizophagus irregularis and Serendipita indica on barley roots under different phosphorus (P) levels. The researchers utilized a monoclonal antibody called MAb32B11 to assess the presence of glomalin, a signature molecule of arbuscular mycorrhizal (AM) fungi, in the roots. The glomalin content was quantified using the enzyme-linked immunosorbent assay (ELISA) method with the MAb32B11 antibody. Materials and Methods In this experiment, barley plants were inoculated with Rhizaphagus irregularis (AMF) and Serendepita indica (endophytic fungus) with three levels of phosphorus from triple super phosphate source. At the end of the vegetative growth period (about three months), the plants were harvested and phosphorus concentration in the plant were measured. A subsample from roots was stored in -20 ºC for determination of glomalin content. The glomalin content in the roots was analyzed using the monoclonal antibody MAb32B11. This antibody was employed to differentiate between the two fungi present in the roots and to quantify the abundance of arbuscular mycorrhizal fungi (AMF) specifically in plants treated with Rhizophagus irregularis. Additionally, the content of glomalin in the soil was determined at the end of the experiment using the same method as described above. The experiment was designed as a factorial completely randomized design (CRD) with three replications. Results and Discussion The results showed that the fresh and dry weights of shoot and root increased significantly in dual inoculation. At zero phosphorus level, shoot and root phosphorus concentrations were significantly higher in treatments with R. irregularis than in those without fungus (control). Under individual inoculation with R. irregularis, or S. indica as well as their dual inoculations, increasing level of phosphorus had no significant effect on shoot and root phosphorus concentration. In dual inoculation, the percentage of total colonization (88%) was significantly higher than that of individual inoculation treatment (68%) but the contribution of each fungus in root colonization under dual inoculation was significantly reduced as estimated by glomalin content of root and determination of total colonization. It was found that with increasing phosphorus level, total colonization percentage significantly decreased and the highest percentage of colonization (61%) was observed at zero level of phosphorus. By increasing phosphorus level, the percentage of root colonization was significantly decreased in individual inoculation by R. irregularis, or S. indica as well as dual inoculation. Results of glomalin assay in soil showed that the glomalin content was high in treatments of R. irregularis but control treatments without fungus and individual inoculation with S. indica had low glomalin. Antibody-reactive root glomalin was less in the dual inoculation treatment (1006.9 µg.g-1) than in the R. irregularis treatment alone (1924.5 µg.g-1) indicating that the presence of S. indica, in root inhibits, root colonization by R. irregularis. Moreover, the increasing of phosphorus level, significantly decreased root glomalin. Conclusion The increase of available phosphorus concentration in the soil caused to limit the expansion of the symbiosis of R. irregularis and S. indica, and this limitation was more for R. irregularis. In the case of dual inoculation with both Rhizophagus irregularis and Serendipita indica, the negative impact of phosphorus on colonization percentage was observed to be less compared to single inoculation. Although the percentage of colonization by each fungus decreased in the dual inoculation treatment compared to their individual inoculation, the overall colonization percentage increased significantly. It appears that in the dual inoculation scenario, while the total root colonization percentage increases, the presence of S. indica leads to a decrease in the colonization percentage specifically with R. irregularis. But in general, growth and nutrient absorption in the case of dual inoculation was better than the inoculation of each of them individually. It was also found that increasing the concentration of phosphorus in the soil caused a decrease in root colonization for both fungi, although the negative effect of phosphorus on the colonization of R. irregularis was more than that of S. indica. The measurement of glomalin in soil and root showed that the inhibitory effect of S. indica fungus on R. irregularis is less in soil than in root

Induction of CD14 Expression and Differentiation to Monocytes or Mature Macrophages in Promyelocytic Cell Lines: New Approach

Purpose: CD14, one of the main differentiation markers on the surface of myeloid lineage cells, acts as a key role in activation of LPS-induced monocytes. LPS (lipopolysaccharide) binds to LPS-binding protein in plasma and are delivered to the cell surface receptor CD14. In this study, Various stimuli [Dimethyl Sulfoxide (DMSO), active 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and LPS], either alone or in combination, have been recognized that have an effect on the level of CD14 expression in the human HL-60 and U937 promonocytic cell lines and therefore induce their terminal differentiation into monocytes or mature macrophages. Methods: U937 and HL-60 cells were cultured in RPMI 1640 supplemented with 10% FBS. For each cell line, 1×106 cells were seeded for 72 hours with DMSO, 14 days with LPS and 18 days with 1, 25-D3 in each well plate; then ELISA method was used to study their responses to the factors by means of anti-CD14. Results: ELISA assay demonstrated that U937 and HL-60 cells were induced by both [1,25(OH)2D3] and DMSO to obtain characteristics of adherent cells and express CD14 protein; moreover, LPS at a low dose increased CD14 expression on surface of this cells. Conclusion: According to the our results, it is speculated that CD14 gene expression may be induced in human U937 and HL-60 cell lines by different factors including 1,25-D3, DMSO and LPS

The current landscape of CAR T-cell therapy for solid tumors: Mechanisms, research progress, challenges, and counterstrategies

The successful outcomes of chimeric antigen receptor (CAR) T-cell therapy in treating hematologic cancers have increased the previously unprecedented excitement to use this innovative approach in treating various forms of human cancers. Although researchers have put a lot of work into maximizing the effectiveness of these cells in the context of solid tumors, few studies have discussed challenges and potential strategies to overcome them. Restricted trafficking and infiltration into the tumor site, hypoxic and immunosuppressive tumor microenvironment (TME), antigen escape and heterogeneity, CAR T-cell exhaustion, and severe life-threatening toxicities are a few of the major obstacles facing CAR T-cells. CAR designs will need to go beyond the traditional architectures in order to get over these limitations and broaden their applicability to a larger range of malignancies. To enhance the safety, effectiveness, and applicability of this treatment modality, researchers are addressing the present challenges with a wide variety of engineering strategies as well as integrating several therapeutic tactics. In this study, we reviewed the antigens that CAR T-cells have been clinically trained to recognize, as well as counterstrategies to overcome the limitations of CAR T-cell therapy, such as recent advances in CAR T-cell engineering and the use of several therapies in combination to optimize their clinical efficacy in solid tumors

Epigenetic Modifications and Therapy in Chronic Obstructive Pulmonary Disease (COPD): An Update Review.

Chronic obstructive pulmonary disease (COPD) that is one of the most prevalent chronic adult diseases and the third leading cause of fatality until 2020. Elastase/anti-elastase hypothesis, chronic inflammation, apoptosis, oxidant-antioxidant balance and infective repair cause pathogenesis of COPD are among the factors at play. Epigenetic changes are post-translational modifications in histone proteins and DNA such as methylation and acetylation as well as dysregulation of miRNAs expression. In this update review, we have examined recent studies on the upregulation or downregulation of methylation in different genes associated with COPD. Dysregulation of HDAC activity which is caused by some factors and miRNAs plays a key role in the suppression and reduction of COPD development. Also, some therapeutic approaches are proposed against COPD by targeting HDAC2 and miRNAs, which have therapeutic effects

Recent Advances in Immunotherapeutic Approaches for Recurrent Reproductive Failure

Human reproduction is an insufficient process, disturbed by various factors, such as immunologic aberrations of mother. Immunologic abnormalities, including cellular and humoral immunity imbalance, cause dysregulated immune responses against embryo, fetus, and associated components and lack of maternal immunotolerance, which compromise the maintenance of pregnancy. Therefore, evaluation of immunologic parameters, including cellular and humoral immunity assessment (T and B lymphocyte, T helper subtypes, NK cells, cytokines, and autoantibodies), especially in women with a history of pregnancy loss or implantation failure, would help clinicians to manage the disorder and prevent next unfavorable pregnancy outcomes. Moreover, several immunomodulatory approaches have been introduced to modulate the abnormal immunologic responses in patients who experience reproduction failure, especially those diagnosed with immunologic basis. Anticoagulants, corticosteroids, intravenous immunoglobulin, immunosuppressive medications used in inhibition of graft rejection, such as calcineurin inhibitors, recombinant cytokines, and cell therapy approaches, are among these modalities. Here, we discuss the proposed mechanisms of immunologic abnormalities involved in the etiopathogenesis of reproduction disorders, besides the suggested immunologic tests and immunotherapeutic approaches which may be helpful in management of these disorders

A study on drug delivery tracing with radiolabeled mesoporous hydroxyapatite nanoparticles conjugated with 2DG/DOX for breast tumor cells

Background: Mesoporous nanoparticles have a great potential in targeted therapy approaches due to their ideal properties for encapsulation of various drugs, proteins and also biologically active molecules. Material and methods: We used mesoporous hydroxyapatite (HA) nanoparticles as a drug carrier and developed radiolabeled mesoporous HA containing of 2-deoxy-D-glucose (2DG) and Doxorubicin (DOX) with technetium-99m (99mTc) for imaging in in vitro and in vivo studies. Results: 2DG and DOX in presence of mesoporous HA nanoparticles more reduced the fraction of viable cells in the MDA-MB-231, MCF-7 human and MC4-L2 Balb/c mice breast cancer cells. The radiochemical purity of the nano-2DG-DOX complex with 99mTc was calculated to 96.8%. The results of cellular uptake showed a 44.77% increase in uptake of the [99mTc]-nano-2DG-DOX compared to the complex without nanoparticles (p < 0.001). Conclusion: Radioisotopic imaging demonstrated a high biochemical stability for [99mTc]-nano-2DG-DOX complex. The results demonstrated that [99mTc]-nano-2DG-DOX, may be used as an attractive candidate in cancer imaging and treatment managing.BACKGROUND: Mesoporous nanoparticles have a great potential in targeted therapy approaches due to their ideal properties for encapsulation of various drugs, proteins and also biologically active molecules. MATERIAL AND METHODS: We used mesoporous hydroxyapatite (HA) nanoparticles as a drug carrier and developed ra­diolabeled mesoporous HA containing of 2-deoxy-D-glucose (2DG) and Doxorubicin (DOX) with technetium-99m (99mTc) for imaging in in vitro and in vivo studies. RESULTS: 2DG and DOX in presence of mesoporous HA nanoparticles more reduced the fraction of viable cells in the MDA-MB-231, MCF-7 human and MC4-L2 Balb/c mice breast cancer cells. The radiochemical purity of the nano-2DG-DOX complex with 99mTc was calculated to 96.8%. The results of cellular uptake showed a 44.77% increase in uptake of the [99mTc]- nano-2DG-DOX compared to the complex without nanoparticles (p &lt; 0.001). CONCLUSIONS: Radioisotopic imaging demonstrated a high biochemical stability for [99mTc]-nano-2DG-DOX complex. The results demonstrated that [99mTc]-nano-2DG-DOX, may be used as an attractive candidate in cancer imaging and treatment managing.

Immuno-biosensor for Detection of CD20-Positive Cells Using Surface Plasmon Resonance

Purpose: Surface plasmon resonance (SPR) sensing confers a real-time assessment of molecular interactions between biomolecules and their ligands. This approach is highly sensitive and reproducible and could be employed to confirm the successful binding of drugs to cell surface targets. The specific affinity of monoclonal antibodies (MAb) for their target antigens is being utilized for development of immuno-sensors and therapeutic agents. CD20 is a surface protein of B lymphocytes which has been widely employed for immuno-targeting of B-cell related disorders. In the present study, binding ability of an anti-CD20 MAb to surface antigens of intact target cells was investigated by SPR technique. Methods: Two distinct strategies were used for immobilization of the anti-CD20 MAb onto gold (Au) chips. MUA (11-mercaptoundecanoic acid) and Staphylococcus aureus protein A (SpA) were the two systems used for this purpose. A suspension of CD20-positive Raji cells was injected in the analyte phase and the resulting interactions were analyzed and compared to those of MOLT-4 cell line as CD20-negative control. Results: Efficient binding of anti-CD20 MAb to the surface antigens of Raji cell line was confirmed by both immobilizing methods, whereas this MAb had not a noticeable affinity to the MOLT-4 cells. Conclusion: According to the outcomes, the investigated MAb had acceptable affinity and specificity to the target antigens on the cell surface and could be utilized for immuno-detection of CD20-positive intact cells by SPR method

The Study of HLA-G Gene and Protein Expression in Patients withRecurrent Miscarriage

Purpose: Although it has been frequently confirmed that HLA-G plays an important role in thereproduction and pregnancy, the pattern of HLA-G gene and its protein expression are rarelyaddressed in studies. Therefore we conducted this study in regard to evaluate the HLA-G geneand its protein expression in the women’s placenta with recurrent miscarriage.Methods: Placental samples were obtained from the women who were admitted for deliveryor abortion in Al Zahra and Taleghani hospitals, Tabriz, Iran. HLA-G gene expression wasdetermined by real-time polymerase chain reaction (PCR) and HLA-G protein expression wasassessed by western blotting and immunofluorescence staining in the tissue samples.Results: The results showed a significant decrease in the expression of gene and proteins ofHLA-G in the women with recurrent miscarriage compared to the control placental tissues.Conclusion: According to the obtained results, it was concluded that the decrement of HLA-Ggene and protein expressions are associated with recurrent miscarriage. Since there areconflicting results from other studies, it is suggested to conduct a more comprehensive similarstudy with greater sample size

Targeted Co-Delivery of Docetaxel and cMET siRNA for Treatment of Mucin1 Overexpressing Breast Cancer Cells

Purpose: Targeted treatment of breast cancer through combination of chemotherapeutic agents and siRNA had been drawing much attention in recent researches. This study was carried out to evaluate mucin1 aptamer-conjugated chitosan nanoparticles containing docetaxel and cMET siRNA on SKBR3 cells. Methods: Nano-drugs were characterized by transmission electron microscope, Zetasizer and loading efficiency calculation. siRNA entrapment onto nanoparticles, stability of siRNA-loaded nanoparticles and conjugation of mucin1 aptamer to nanoparticles were evaluated via separate electrophoresis. Cellular uptake of the targeted nanoparticles was evaluated through GFP-plasmid expression in mucin1+ SKBR3 vs. mucin1- CHO cells. Protein expression, cell viability and gene expression were assessed by Western Blotting, MTT assay, and Quantitative Real Time-PCR, respectively. Results: Characterization of nano-drugs represented the ideal size (110.5± 3.9 nm), zeta potential (11.6± 0.8 mV), and loading efficiency of 90.7% and 88.3% for siRNA and docetaxel, respectively. Different gel electrophoresis affirmed the conjugation of aptamers to nanoparticles and entrapment of siRNA onto nanoparticles. Increased cellular uptake of aptamer-conjugated nanoparticles was confirmed by GFP expression. cMET gene silencing was confirmed by Western Blotting. The significant (p ≤0.0001) impact of combination targeted therapy vs. control on cell viability was shown. Results of Quantitative Real Time-PCR represented a remarkably decreased (p ≤0.0001) expression of the studied genes involving in tumorigenicity, metastasis, invasion, and angiogenesis (STAT3, IL8, MMP2, MMP9, and VEGF) by targeted combination treatment vs. control. Conclusion: The mucin1 aptamer-conjugated chitosan nanoparticles, containing docetaxel and cMET siRNA, is suggested for treatment of mucin1+ metastatic breast cancer cells. However, further studies should be conducted on animal models

Large Scale Generation and Characterization of Anti-Human CD34 Monoclonal Antibody in Ascetic Fluid of Balb/c Mice

Purpose: Monoclonal antibodies or specific antibodies are now an essential tool of biomedical research and are of great commercial and medical value. The purpose of this study was to produce large scale of monoclonal antibody against CD34 in order to diagnostic application in leukemia and purification of human hematopoietic stem/progenitor cells. Methods: For large scale production of monoclonal antibody, hybridoma cells that produce monoclonal antibody against human CD34 were injected into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. 5 ml ascitic fluid was harvested from each mouse in two times. Evaluation of mAb titration was assessed by ELISA method. The ascitic fluid was examined for class and subclasses by ELISA mouse mAb isotyping Kit. mAb was purified from ascitic fluid by affinity chromatography on Protein A-Sepharose. Purity of monoclonal antibody was monitored by SDS -PAGE and the purified monoclonal antibody was conjugated with FITC. Results: Monoclonal antibodies with high specificity and sensitivity against human CD34 by hybridoma technology were prepared. The subclass of antibody was IgG1 and its light chain was kappa. Conclusion: The conjugated monoclonal antibody could be a useful tool for isolation, purification and characterization of human hematopoietic stem cells