COVID-19 electrochemical immunosensor with Ag-MOF: Rapid and high-selectivity nasal swab testing for effective detection.

Early detection of the coronavirus is acknowledged as a crucial measure to mitigate the spread of the pandemic, facilitating timely isolation of infected individuals, and disrupting the transmission chain. In this study, we leveraged the properties of synthesized Ag-MOF, including high porosity and increased flow intensity. Electrochemical techniques such as cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were employed to develop an economical and portable sensor with exceptional selectivity for COVID-19 detection. The methodology involves the deposition of Ag-MOF onto the surface of a Glassy Carbon Electrode (GCE), which resulted in a progressive augmentation of electric current. Subsequently, the targeted antibodies were applied, and relevant tests were conducted. The sensor demonstrated the capacity to detect the virus within a linear range of 100 fM to 10 nM, boasting a noteworthy Limit of Detection (LOD) of 60 fM. The entire detection process could be completed in a brief duration of 20 min, exhibiting high levels of accuracy and precision, outperforming comparable techniques in terms of speed and efficacy.

Cell Surface Vimentin Detection in Cancer Cells by Peptide-Based Monoclonal Antibody.

Background: Vimentin is a prominent Intermediate Filaments (IFs) protein expressed in different mesenchymal origin cell types. Besides a wide range of cellular function roles associated with vimentin expression, its dysregulation and cell surface expression in the induction of malignancy properties have been reported extensively, making it a promising cancer-specific target. Therefore, this study aimed to generate and characterize anti-vimentin monoclonal antibodies. Methods: A 14-mer synthetic peptide from vimentin was conjugated to Keyhole Limpet Hemocyanin (KLH) and used for immunization of Blab/C mice and monoclonal production by conventional hybridoma technology. The monoclonal antibody was purified using affinity chromatography of supernatants from the selected hybridoma cells. ELISA, Immunoprecipitation-Western blotting (IP-WB), Immunocytochemistry (ICC), and flow cytometry were employed to characterize the produced monoclonal antibody in terms of interaction with vimentin immunizing peptide as well as vimentin protein. Results: Amid the several obtained producing anti-vimentin antibody hybridomas, the 7C11-D9 clone (IgG1 isotype with kappa light chain) showed higher reactivity with the immunizing peptide, and led to its selection for purification and characterization. The purified antibody could detect vimentin protein in IP-WB, ICC and flow cytometry of the normal and cancerous cells with different origin. No vimentin expression was found in normal healthy Peripheral Blood Mononuclear Cell (PBMC). Conclusion: Taken together, 7C11-D9 anti-vimentin monoclonal antibody might be used as immune diagnostic or immune therapeutic tool where detection or targeting of vimentin in a wide range of organisms is required.

Investigating the inhibitory and penetrating properties of three novel anticancer and antimicrobial scorpion peptides via molecular docking and molecular dynamic simulation.

The two types of bladder cancer, muscle invasive and non-muscle invasive (NMIBC), are among the most prevalent cancers worldwide. Despite this, even though muscle-invasive bladder cancer is more deadly, NMIBC requires more therapy due to a greater recurrence rate and more extended and expensive care. Immunotherapy, intravesical chemotherapy, cystoscopy, and transurethral resection (TUR) are among the treatments available. Crude scorpion venomand purified proteins and peptides, can suppress cancer metastasis in an in vitro or in vivo context, suppress cancer growth, halt the cell cycle, and cause cell apoptosis, according to an increasing number of experimental and preclinical studies. In this research, three novels discovered peptides (P2, P3 and P4. ProteomeXchange: PXD036231) from Buthotus saulcyi and, Odontobuthus doriae scorpions were used along with a peptide called pantinin (as a control). The phylogenetic tree showed that the peptides belong to Chaperonin HSP60, Chrysophsin2 and Pheromone-binding protein2, respectively. These peptides were docked with four known antigens, BAGE, BLCAP, PRAME and ROR1 related to bladder cancer and three bacterial antigens FliC, FliD and FimH to investigate their antimicrobial and anticancer properties. The results showed that peptides 2 and 3 have the best binding rate. The MD simulation results also confirmed the binding of peptides 2 and 3 to antigens. The penetration power of peptides 2 and 3 in the membrane of cancer cells and bacterial cells was also simulated, and the results of RMSD and PD confirmed it. QSAR suggests that peptides 2 and 3 can act as anti-cancer and anti-microbial peptides.Communicated by Ramaswamy H. Sarma.

A novel co-culture assay to evaluate the effects of sympathetic innervation on vascular smooth muscle differentiation.

Dedifferentiation of vascular smooth muscle cells (VSMCs) from a functional phenotype to an inverse synthetic phenotype is a symptom of cardiovascular disorders, such as atherosclerosis and hypertension. The sympathetic nervous system (SNS) is an essential regulator of the differentiation of vascular smooth muscle cells (VSMCs). In addition, numerous studies suggest that SNS also stimulates VSMCs to retain their contractile phenotype. However, the molecular mechanisms for this stimulation have not been thoroughly studied. In this study, we used a novel in vitro co-culture method to evaluate the effective cellular interactions and stimulatory effects of sympathetic neurons on the differentiation of VSMCs. We co-cultured rat neural-like pheochromocytoma cells (PC12) and rat aortic VSMCs with this method. Expression of VSMCs contractile genes, including smooth muscle actin (acta2), myosin heavy chain (myh11), elastin (eln), and smoothelin (smtn), were determined by quantitative real-time-PCR analysis as an indicator of VSMCs differentiation. Fold changes for specific contractile genes in VSMCs grown in vitro for seven days in the presence (innervated) and absence (non-innervated) of sympathetic neurons were 3.5 for acta2, 6.5 for myh11, 4.19 for eln, and 4 for smtn (normalized to Tata Binding Protein (TBP)). As a result, these data suggest that sympathetic innervation promotes VSMCs' contractile gene expression and also maintains VSMCs' functional phenotype.

The effect of Nano-liposomal sodium nitrite on smooth muscle cell growth in a tissue-engineered small-diameter vascular graft.

BACKGROUND: Nitric oxide is a chemical agent produced by endothelial cells in a healthy blood vessel, inhibiting the overgrowth of vascular smooth muscle cells and regulating vessel tone. Liposomes are biocompatible and biodegradable drug carriers with a similar structure to cell bilayer phospholipid membrane that can be used as useful nitric oxide carriers in vascular grafts. METHOD: Using a custom-designed apparatus, the sheep carotid arteries were decellularized while still maintaining important components of the vascular extracellular matrix (ECM), allowing them to be used as small-diameter vascular grafts. A chemical signal of sodium nitrite was applied to control smooth muscle cells' behavior under static and dynamic cell culture conditions. The thin film hydration approach was used to create nano-liposomes, which were then used as sodium nitrite carriers to control the drug release rate and enhance the amount of drug loaded into the liposomes. RESULTS: The ratio of 80:20:2 for DPPC: Cholesterol: PEG was determined as the optimum formulation of the liposome structure with high drug encapsulation efficiency (98%) and optimum drug release rate (the drug release rate was 40%, 65%, and 83% after 24, 48, and 72 h, respectively). MTT assay results showed an improvement in endothelial cell proliferation in the presence of nano-liposomal sodium nitrite (LNS) at the concentration of 0.5 µg/mL. Using a suitable concentration of liposomal sodium nitrite (0.5 µg/mL) put onto the constructed scaffold resulted in the controllable development of smooth muscle cells in the experiment. The culture of smooth muscle cells in a pulsatile perfusion bioreactor indicated that in the presence of synthesized liposomal sodium nitrite, the overgrowth of smooth muscle cells was inhibited in dynamic cell culture conditions. The mechanical properties of ECM graft were measured, and a multi-scale model with an accuracy of 83% was proposed to predict mechanical properties successfully. CONCLUSION: The liposomal drug-loaded small-diameter vascular graft can prevent the overgrowth of SMCs and the formation of intimal hyperplasia in the graft. Aside from that, the effect of LNS on endothelial has the potential to stimulate endothelial cell proliferation and re-endothelialization.

An Anti-TAZ Monoclonal Antibody Recognizing Cell Surface Expressed TAZ Protein in Human Tumor Cells.

Background: WWTR1 or TAZ is a transcriptional co-activator protein expressed in cytoplasm which functions as the main downstream effector of the Hippo signaling pathway. This pathway is an evolutionally conserved signal cascade, which plays a pivotal role in organ size control and tumorigenesis. Ectopic expression of TAZ has already been observed in many malignancies, while the ectopic localization of TAZ is reported for the first time. The aim of this study was to produce a specific monoclonal antibody (mAb) against a synthetic peptide derived from WWTR1 protein to be used as a research tool in human carcinomas. Methods: A 21-mer synthetic peptide (derived from human TAZ protein) was used for immunization of BALB/c mice after conjugation with Keyhole Limpet Haemocyanin (KLH) using hybridoma technology. The generated mAb reacted with the immunizing peptide employing ELISA assay. The reactivity of the antibody with native TAZ protein was assessed through Western blot, immunocytochemistry, and flow cytometry using different cancer cell lines. Results: The produced mAb could recognize the immunizing peptide in ELISA and Kaff was 0.6×10-9 M. The produced anti-TAZ mAb unlike available commercial anti-TAZ antibody, was capable of specifically recognizing cell surface TAZ in human carcinoma cell lines including MCF-7, Raji, and A431 in Western blot, immunocytochemistry, and flow cytometry assays. As expected, no reactivity was observed using normal Peripheral Blood Mononuclear Cell (PBMC) from healthy donors. Conclusion: Based on the results, TAZ is ectopically expressed on the surface of tumor cell lines which is not the case in normal cells. The generated mAb has a potential to be used as a research tool in studying the expression of TAZ in human carcinomas in different applications.

Investigation of electrical stimulation on phenotypic vascular smooth muscle cells differentiation in tissue-engineered small-diameter vascular graft.

In the development of vascular tissue engineering, particularly in the case of small diameter vessels, one of the key obstacles is the blockage of these veins once they enter the in vivo environment. One of the contributing factors to this problem is the aberrant proliferation and migration of vascular smooth muscle cells (VSMCs) from the media layer of the artery to the interior of the channel. Two distinct phenotypes have been identified for smooth muscle cells, namely synthetic and contractile. Since the synthetic phenotype plays an essential role in the unusual growth and migration, the aim of this study was to convert the synthetic phenotype into the contractile one, which is a solution to prevent the abnormal growth of VSMCs. To achieve this goal, these cells were subjected to electrical signals, using a 1000 µA sinusoidal stimulation at 10 Hz for four days, with 20 min duration per 24 h. The morphological transformations and changes in the expression of vimentin, nestin, and ß-actin proteins were then studied using ICC and flow cytometry assays. Also, the expression of VSMC specific markers such as smooth muscle myosin heavy chain (SMMHC) and smooth muscle alpha-actin (α-SMA) were evaluated using RT-PCR test. In the final phase of this study, the sheep decellularized vessel was employed as a scaffold for seeding these cells. Based on the results, electrical stimulation resulted in some morphological alterations in VSMCs. Furthermore, the observed reductions in the expression levels of vimentin, nestin and ß-actin proteins and increase in the expression of SMMHC and α-SMA markers showed that it is possible to convert the synthetic phenotype to the contractile one using the studied regime of electrical stimulation. Finally, it can be concluded that electrical stimulation can significantly affect the phenotype of VSMCs, as demonstrated in this study.

Detection of COVID-19: A Smartphone-Based Machine-Learning-Assisted ECL Immunoassay Approach with the Ability of RT-PCR CT Value Prediction.

The unstoppable spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has severely threatened public health over the past 2 years. The current ubiquitously accepted method for its diagnosis provides sensitive detection of the virus; however, it is relatively time-consuming and costly, not to mention the need for highly skilled personnel. There is a clear need to develop novel computer-based diagnostic tools to provide rapid, cost-efficient, and time-saving detection in places where massive traditional testing is not practical. Here, we develop an electrochemiluminescence (ECL)-based detection system whose results are quantified as reverse transcriptase polymerase chain reaction (RT-PCR) cyclic threshold (CT) values. A concentration-dependent signal is generated upon the introduction of the virus to the electrode and is recorded with a smartphone camera. The ECL images are used to train machine learning algorithms, and a model using artificial neural networks (ANNs) for 45 samples was developed. The model demonstrated more than 90% accuracy in the diagnosis of 50 unknown real samples, detecting up to a CT value of 32 and a limit of detection (LOD) of 10-12 g mL-1 in the testing of artificial samples.

A novel ADC targeting cell surface fibromodulin in a mouse model of triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancers (TNBCs) are highly aggressive and metastatic. To date, finding efficacious targeted therapy molecules might be the only window of hope to cure cancer. Fibromodulin (FMOD), is ectopically highly expressed on the surface of Chronic Lymphocytic Leukemia (CLL) and bladder carcinoma cells; thus, it could be a promising molecule for targeted therapy of cancer. The objective of this study was to evaluate cell surface expression of FMOD in two TNBC cell lines and develop an antibody-drug conjugate (ADC) to target FMOD positive TNBC in vitro and in vivo. MATERIALS AND METHODS: Two TNBC-derived cell lines 4T1 and MDA-MB-231 were used in this study. The specific binding of anti-FMOD monoclonal antibody (mAb) was evaluated by flow cytometry and its internalization was verified using phAb amine reactive dye. A microtubulin inhibitor Mertansine (DM1) was used for conjugation to anti-FMOD mAb. The binding efficacy of FMOD-ADC was assessed by immunocytochemistry technique. The anti-FMOD mAb and FMOD-ADC apoptosis induction were measured using Annexin V-FITC and flow cytometry. Tumor growth inhibition of anti-FMOD mAb and FMOD-ADC was evaluated using BALB/c mice injected with 4T1 cells. RESULTS: Our results indicate that both anti-FMOD mAb and FMOD-ADC recognize cell surface FMOD molecules. FMOD-ADC could induce apoptosis in 4T1 and MDA-MB-231 cells in vitro. In vivo tumor growth inhibition was observed using FMOD-ADC in 4T1 inoculated BALB/c mice. CONCLUSION: Our results suggests high cell surface FMOD expression could be a novel bio-marker TNBCs. Furthermore, FMOD-ADC could be a promising candidate for targeting TNBCs.

Development of sandwich electrochemiluminescence immunosensor for COVID-19 diagnosis by SARS-CoV-2 spike protein detection based on Au@BSA-luminol nanocomposites.

Coronavirus disease (COVID-19) is a new and highly contagious disease posing a threat to global public health and wreaking havoc around the world. It's caused by the Coronavirus that causes severe acute respiratory syndrome (SARS-CoV-2). In the current pandemic situation, rapid and accurate SARS-CoV-2 diagnosis on a large scale is critical for early-stage diagnosis. Early detection and monitoring of viral infections can aid in controlling and preventing infection in large groups of people. Accordingly, we developed a sensitive and high-throughput sandwich electrochemiluminescence immunosensor based on antigen detection for COVID-19 diagnosis (the spike protein of SARS-CoV-2). For the spike protein of SARS-CoV-2, the ECL biosensor had a linear range of 10 ng mL-1 to 10 µg mL-1 with a limit of detection of 1.93 ng mL-1. The sandwich ECL immunosensor could be used in early clinical diagnosis due to its excellent recovery in detecting SARS-CoV-2, rapid analysis (90 min), and ease of use.

Monoclonal Antibody Against Sortilin Induces Apoptosis in Human Breast Cancer Cells.

Background: Sortilin has an important role in various malignances and can be used as a promising target to eradicate cancer cells. Methods: In this study, the expression of sortilin in 4T1 and MDA-MB231 cell lines was evaluated by flow cytometry and immunocytochemistry. Apoptosis assay was also applied to evaluate apoptosis induction in 4T1 and MDA-MB231 cell lines. Results: Based on cell surface flow cytometry results, anti-sortilin (2D8-E3) mAb could recognize sortilin molecules in 79.2% and 90.3% of 4T1 and MDA-MB231 cell-lines, respectively. The immunocytochemistry staining results confirmed sortilin surface expression. Apoptosis assay indicated that anti-sortilin mAb could induce apoptosis in 4T1 and MDA-MB231 cell lines. Conclusion: Our study revealed the important role of surface sortilin in breast carcinoma cell survival and its possible application as a therapeutic agent in cancer targeted therapies.

Diagnostic and Therapeutic Implications of Sortilin Expressed on the Surface of Bladder Carcinoma Cells.

Background & Objective: Cell surface expression of sortilin in different types of cancer signifies it as a therapeutic target for cancer therapy. The aim of this study was to detect sortilin expression in bladder cancer cells using an anti-sortilin monoclonal antibody (mAb) to evaluate sortilin as a target for developing diagnostic and therapeutic agents against bladder carcinoma. Methods: The protein expression of sortilin in bladder cancer tissues and cell lines (5637 and EJ138) was investigated by immunohistochemistry (IHC), immune-cytochemistry (ICC), and flow cytometry. Furthermore, the capability of anti-sortilin mAb in apoptosis induction in bladder cancer cells was evaluated. Results: A high expression level was observed in bladder carcinoma tissues (P≤0.001) and cell lines, using IHC and ICC, respectively. Flow cytometry results showed cell surface expression of 27.5±3% (P≤0.01), 74.4±7.8% (P≤0.001), and 4.2±0.4% of sortilin in EJ138, 5637, and HFFF cells, respectively. In EJ138 anti-sortilin mAb induced apoptosis in 25.2±11.5% (P≤0.05) (early) and 4.5±1.1% (P>0.05) (late) after 6 h incubation, while for 12 h, the values of 11.6±3.8% (P>0.05) and 20.7±4.4% (P≤0.05) were achieved. In 5637 cells, 6 h incubation resulted in 10.2±0.3% (P>0.05) and 6.6±1.4% (P>0.05) apoptosis induction, while these values were 12.1±0.8% (P>0.05) and 27.4±4.5% (P≤0.01) after 12 h. The HFFF cells did not show significant apoptosis. Conclusion: The overexpression of sortilin in bladder tumor cells and its potential in inducing apoptosis via directed targeting with the specific monoclonal antibody may represent this protein as a potential candidate of targeted therapy in bladder carcinoma.

Developing of specific monoclonal recombinant antibody fused to alkaline phosphatase (AP) for one-step detection of fig mosaic virus.

Present study was performed to develop a fusion recombinant monoclonal antibody for one-step and accurate detection of FMV with a specific single-chain variable fragment (scFv) fused to alkaline phosphatase (AP) named as scFv(FMV-NP)-AP. The gene encoding-specific scFv recombinant antibody binding to nucleocapsid protein of Fig Mosaic Virus (FMV-NP) was fused to upstream of AP gene and integrated in pET26b bacterial expression vector. As vector contain pelB signal peptide, the expressed protein is secreted into periplasmic compartment. Recombinant fusion protein was produced in transformed E. coli following induction by IPTG. Extraction and purification of fusion protein was performed under denatured condition. The results of SDS-PAGE and western blot analysis indicated high integrity and purity with a single band protein with expected size of 72 kDa. The total yield of purified scFv(FMV-NP)-AP fusion protein estimated around 0.5-1 mg/l cultured medium. Subsequent colorimetric analysis confirmed presence of alkaline phosphatase activity in prepared scFv-AP fusion protein. Specificity of generated recombinant fusion antibody against cognate antigen and the native virus presented in infected plant extracts was assessed by ELISA, western blot and dot blot assays. Results revealed that scFv(FMV-NP)-AP is able to detect the presence of FMV in infected fig plants. The novel approach, implementing specific recombinant fusion antibody developed in this research, leads to one-step detection of FMV in plants by avoiding the use of chemical enzyme-labeled secondary antibodies.

The Association of Cell Surface Fibromodulin Expression and Bladder Carcinoma.

BACKGROUND: Fibromodulin (FMOD) is a secretory protein which is considered a major component of extracellular matrix. Its dysregulation in different types of cancer implies it as a promising target for cancer therapy. Within the scope of its rather wide expression in different tumors, we studied expression of FMOD and effect of anti-FMOD antibody in bladder cancer cells in order to identify new target for diagnostic and therapeutic interventions. We report here for the first time the expression of FMOD in bladder cancer cell lines in comparison to the normal cell line and tissues. METHODS: A peptide-based produced anti-FMOD murine monoclonal antibody (mAb) (clone 2C2-A1) was applied for evaluation of FMOD expression in bladder cancer and normal tissues by immunohistochemistry (IHC) staining. Furthermore, the expression of FMOD was examined in human bladder cell lines, 5637 and EJ138, as well as a non-cancerous human cell line, human fetal foreskin fibroblast (HFFF), by immunocytochemistry (ICC) and flow cytometry. The apoptosis induction of anti-FMOD mAb was also evaluated in bladder cancer cells. RESULTS: IHC and ICC analyses revealed that the qualitative expression of FMOD in bladder cancer tissues and cell lines is higher than in normal tissues and cell lines. Flow cytometry analyses revealed that 2C2-A1 mAb could recognize FMOD expression in 84.05 ± 1.85%, 46.1 ± .4% , and 2.56 ± 1.26% of 5637, EJ138, and HFFF cells, respectively. An effective apoptosis induction was detected in 5637 and EJ138 cells with no significant effect on HFFF cell. CONCLUSION: To our knowledge, this is for the first time reporting surface expression of FMOD in bladder cancer. This significant surface expression of FMOD in bladder cancer with no expression in normal bladder tissues and the capacity of inducing apoptosis through directed targeting of FMOD with specific monoclonal antibody might candidates FMOD as a diagnostic marker as well as a potential immunotargeting with monoclonal antibody.

Monoclonal Antibody Against ROR1 Induces Apoptosis in Human Bladder Carcinoma Cells.

BACKGROUND: Receptor tyrosine kinase-like Orphan Receptor 1 (ROR1) is one of the promising cell surface antigens for targeting cancer cells. The aim of this study was to evaluate ROR1 cell surface expression in bladder cancer cells using a murine anti-ROR1 monoclonal antibody (mAb) called 5F1-B10 as well as investigate its potential in apoptosis induction. METHODS: Expression of ROR1 in two human bladder cell lines, 5637 and EJ138, as well as a non-cancerous human cell line, Human Fetal Foreskin Fibroblast (HFFF), was examined by flow cytometry and immunocytochemistry. Immunohistochemical staining of cancer and normal bladder tissues was also performed. RESULTS: The flow cytometry results showed that 5F1-B10 mAb could recognize ROR1 molecules in 86.1% and 45.6% of 5637 and EJ138 cells, respectively. The expression level of ROR1 was 5.49% in HFFF cells. The immunocytochemistry and immunohistochemistry staining results also confirmed the presence of ROR1 on the surface of both bladder cancer cells and tissues, respectively. The obtained data from apoptosis assay demonstrated that 5F1-B10 mAb could induce apoptosis in both 5637 and EJ138 cell lines. CONCLUSION: Taken together, our finding indicates the role of ROR1 in bladder cancer cell survival and suggests this receptor might be a promising target for developing novel therapeutic agents against bladder carcinoma.

High-performance sono/nano-catalytic system: Fe3O4@Pd/CaCO3-DTT core/shell nanostructures, a suitable alternative for traditional reducing agents for antibodies.

Herein, a novel heterogeneous nanoscale reducing agent for antibody cleavage, made of iron oxide nanoparticles, silica network, palladium on calcium carbonate (10%), and dithiothreitol (Fe3O4@Pd/CaCO3-DTT), is presented as a substantial alternative for traditional homogeneous analogues. Conventionally, antibody fragmentation is accomplished using reducing agents and proteases that digest or cleave certain portions of the immunoglobulin protein structure to provide active thiol sites for drug tagging aims. Then, dialysis process is needed to separate excess chemical structures and purify the reduced antibody. In this work, we have made an effort to design a suitable heterogeneous tool for protein cleavage and skip the dialysis process for purification of the reduced antibody. In this regard, firstly, various preparation methods including microwave irradiation, reflux and ultrasonication have been precisely compared, and it has been proven that the best result is obtained through 10 min ultrasound (US) irradiation using an US bath with 50 KHz frequency and 200 W L-1 power density. Then, all the necessary structural analyses have been done and thoroughly interpreted for the final product. Afterward, the catalytic performance of Fe3O4@Pd/CaCO3-DTT nanoscale system in the presence of US waves (50 KHz, 200 W) has been monitored using some disulphide derivatives. The NPs could be conveniently separated from the mixture through their substantial paramagnetic property. Thus, dialysis process in which various types of membranes are used is practically jumped after the reduction process. In this work, this is clearly demonstrated that there is a constructive synergistic effect between US waves and prepared Fe3O4@Pd/CaCO3-DTT nanoscale reducing agent. Ultimately, trastuzumab (anti HER-2) antibody has been used to test the performance of the prepared Fe3O4@Pd/CaCO3-DTT NPs in a real protein reduction reaction.

Monoclonal and Polyclonal Antibodies Specific to Human Fibromodulin.

BACKGROUND: The unique expression of fibromodulin (FMOD) in patients with chronic lymphocytic leukemia (CLL) has been previously reported. Detecting FMOD in CLL patients using specific anti-FMOD mAbs might provide a promising method in detection, monitoring, and prognosis of CLL. OBJECTIVES: In this study, we aimed for producing specific antibodies against FMOD to facilitate further cohort study of CLL, thus addressing FMOD as a potential target of detection. MATERIALS AND METHODS: Human FMOD gene (1087 bp) was extracted from genome of the CLL patients, and was cloned into the expression vector of pET-22b (+). The recombinant FMOD protein (rFMOD) was expressed in Escherichia coli. The purified rFMOD protein was used as an immunogen in rabbit and mice. Hybridoma technology was used to develop the monoclonal antibodies (mAbs). Polyclonal antibody (pAb) was purified from the rabbit sera using affinity column. The reactivity of anti-FMOD antibodies was assessed in ELISA, immunocytochemistry (ICC) and Western blot. RESULTS: ICC results showed that the anti-FMOD antibodies specifically detected FMOD in CLL PBMCs and cell lines. The developed anti-FMOD pAb detected FMOD in CLL lysates, compared to healthy PBMCs, in Western blot and ELISA. CONCLUSIONS: The developed anti-FMOD mAbs, and pAb specifically detect FMOD in CLL samples and might be used as research tools for further investigations in CLL.

Anchored Fibromodulin as a Novel Target in Chronic Lymphocytic Leukemia: Diagnostic and Therapeutic Implications.

BACKGROUND: We have previously reported the aberrant expression of Fibromodulin (FMOD) in patients with chronic lymphocytic leukemia (CLL). Although FMOD has been considered as a cytoplasmic or secretory protein, we discovered the cell surface expression of FMOD in leukemic B cells via anchoring with glycosylphosphatidylinositol (GPI). OBJECTIVE: To evaluate FMOD as a new biomarker in CLL patients in comparison with healthy individuals. METHODS: A monoclonal antibody was generated against human FMOD. The cell surface expression of FMOD in 52 CLL patients and 45 healthy individuals were compared by flow cytometry. A bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) was used to determine the cell surface localization of FMOD using ELISA and flow cytometry techniques. Annexin V-FITC and propidium iodide (PI) was used to detect apoptosis induction in CLL PBMCs following in vitro incubation with anti-FMOD mAb. RESULTS: The results demonstrated the widespread cell surface expression of GPI-anchored FMOD in CLL patients (median: 79.9 %), although healthy individuals had low FMOD expression (median: 6.2 %) (p≤0.0001). The cut-off value of FMOD expression was estimated with high sensitivity and specificity at 17.9 %. Furthermore, in vitro apoptosis induction of leukemic cells following incubation with anti-FMOD mAb showed a direct apoptosis of CLL cells (27.9%) with very low effect on healthy PBMCs (6%). CONCLUSION: The membrane-anchoring of FMOD by means of a GPI moiety in leukemic cells supports FMOD as a highly potential diagnostic and therapeutic target in CLL patients.

Carboxymethyl kappa carrageenan-modified decellularized small-diameter vascular grafts improving thromboresistance properties.

The development of decellularized small-diameter vascular grafts is a potential solution for patients requiring vascular reconstructive procedures. However, there is a limitation for acellular scaffolds due to incomplete recellularization and exposure of extracellular matrix components to whole blood resulting in platelet adhesion. To address this issue, a perfusion decellularization method was developed using a custom-designed set up which completely removed cell nuclei and preserved three-dimensional structure and mechanical properties of native tissue (sheep carotid arteries). Afterwards, carboxymethyl kappa carrageenan (CKC) was introduced as a novel anticoagulant in vascular tissue engineering which can inhibit thrombosis formation. The method enabled uniform immobilization of CKC on decellularized arteries as a result of interaction between amine functional groups of decellularized arteries and carboxyl groups of CKC. The CKC modified graft significantly reduced platelet adhesion from 44.53 ± 2.05% (control) to 19.57 ± 1.37% (modified) and supported endothelial cells viability, proliferation, and nitric oxide production. Overall, the novel CKC modified scaffold provides a promising solution for thrombosis formation of small-diameter vessels and could be a potent graft for future in vivo applications in vascular bypass procedures. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1690-1701, 2019.

Production of a polyclonal antiserum against recombinant nucleocapsid protein and its application for the detection of fig mosaic virus.

Mosaic disease (MD), caused by Fig mosaic emaravirus (FMV), is the most important and devastating virus disease of fig trees worldwide. The detection of FMV in infected plants is possible only through the use of molecular techniques, i.e. RT-PCR and LAMP, which both offer high sensitivity of detection, but are also considered laborious when dealing with a large number of samples. To cope with this restriction, a polyclonal antiserum through the immunization of a rabbit by injecting the recombinant nucleocapsid protein (NP) of FMV was raised and evaluated for its efficacy in Western Blot, Dot immuno-binding and DAS-ELISA. The results obtained showed that the raised antiserum was able to identify the nucleocapsid protein of FMV (p3) which was found to have an estimated molecular weight of ca. 35 KDa. In addition, the antiserum, when used in the three serological assays, was able to detect the p3 of FMV in protein extracts of infected plants with different levels of efficacy. Dot immuno-binding, using denatured plant protein extract, proved to be the most efficient serological assay for detecting FMV in samples collected from different fig orchards. This is the first report on an antiserum raised against FMV that could be used for immunological detection of the virus.