Role of glycogen synthase kinase following myocardial infarction and ischemia-reperfusion.

Glycogen synthase kinase-3 beta (GSK3ß) is principally is a glycogen synthase phosphorylating enzyme that is well known for its role in muscle metabolism. GSK3ß is a serine/threonine protein Kinase, which is responsible for several essential roles in mammalian cells. This enzyme is implicated in the pathophysiology of many conditions involved in homeostasis and cellular immigration. GSK3ß is involved in several pathways leading to neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Increasing evidence has shown the potential importance of GSK3ß in ischemic heart disease and ischemia-reperfusion pathologies. Reperfusion injury may occur in tissues after prolonged ischemia following reperfusion. Reperfusion injury can be life threatening. Reperfusion injury occurs due to a change in ionic homeostasis, excess free radical production, mitochondrial damage and cell death. There are however clear, cardiac-protective signals; although the molecular pathophysiology is not clearly understood. In normal physiology, GSK3ß has a critical role in the cytoprotective pathway. However, it`s controversial role in ischemia and ischemia-reperfusion is a topic of current interest. In this review, we have opted to focus on GSK3ß interactions with mitochondria in ischemic heart disease and expand on the therapeutic interventions.

IL-4 induces the formation of multinucleated giant cells and expression of ß5 integrin in central giant cell lesion.

BACKGROUND: It is now well established that IL-4 has a central role in the development of monocytes to multinucleated giant cells (MGCs) by inducing the expression of integrins on the surface of monocytes. The aim of this study was to investigate the potential role of IL-4 in induction of ß5 integrin expression in the peripheral blood samples of patients with giant cell granuloma. MATERIAL AND METHODS: Monocytes were isolated from peripheral blood samples of patients with central giant cell granuloma (CGCG) and healthy controls using human Monocyte Isolation Kit II. Isolated monocytes were then cultured in the absence or presence of IL-4 (10 and 20 ng/mL), and following RNA extraction and cDNA synthesis, Real-time PCR was performed to determine the level of ß5 integrin expression. The formation of CGCGs and morphological analyses were done under light microscopy. For confirmation of CGCGs, immunocytochemistry technique was also carried out by anti-RANK (receptor-activator of NF-κB ligand) antibody. RESULTS: In both patient and control groups, ß5 levels were significantly enhanced by increasing the IL-4 dose from 10 to 20 ng/mL. In addition, these differences were significant between patient and control groups without IL-4 treatment. On the other hand, the number of cells which expressed RANK and therefore the number of giant cells were significantly higher in the patient group in comparison to controls, as assessed by immunohistochemistry evaluations. CONCLUSIONS: In this study, we showed an elevation in the expression levels of ß5 integrin when stimulated by IL-4. It is strongly indicated that this integrin acts as an important mediator during macrophage to macrophage fusion and development of giant cells.

Silencing of myeloid cell leukemia-1 by small interfering RNA improves chemosensitivity to etoposide in u-937 leukemic cells.

A key issue in the treatment of acute myeloid leukemia (AML) is the development of drug resistance to chemotherapeutic agents. Overexpression of myeloid cell leukemia-1 (Mcl-1), an anti-apoptotic protein, is associated with tumor progression and drug resistance in leukemia and several cancers. The purpose of this study was to investigate the effect of specific Mcl-1 small interference RNA (siRNA) on the proliferation and chemosensitivity of U-937 AML cell to etoposide. The siRNA transfection was conducted using Lipofectamine™ 2000. Quantitative real-time RT-PCR (qRT-PCR) and Western blot analysis were employed to measure the expression levels of mRNA and protein, respectively. To evaluate tumor cell growth after siRNA transfection, Trypan blue exclusion assay was conducted. The cytotoxic effects of siRNA and etoposide were determined using MTT assay on their own and in combination. DNA-histone ELISA and annexin-V/FITC assays were performed to study the apoptosis. Mcl-1 siRNA transfection significantly blocked the expression of Mcl-1 mRNA and protein in a time-dependent manner, leading to a strong growth inhibition and enhanced apoptosis (P less than 0.05). Furthermore, pretreatment with Mcl-1 siRNA, synergistically enhanced the cytotoxic and apoptotic effects of etoposide (P less than 0.05). Our results demonstrated that Mcl-1 plays a fundamental role in the survival and resistance of U-937 cells to etoposide. Therefore, Mcl-1 can be considered an attractive target in gene therapy of AML patients and siRNA-mediated silencing of this gene may be a novel strategy in AML treatment.

An overview of the history, applications, advantages, disadvantages and prospects of gene therapy.

Gene therapy has become a significant issue in science-related news. The principal concept of gene therapy is an experimental technique that uses genes to treat or prevent disease. Although gene therapy was originally conceived as a way to treat life-threatening disorders (inborn defects, cancers) refractory to conventional treatment, it is now considered for many non–life-threatening conditions, such as those adversely impacting a patient’s quality of life. An extensive range of efficacious vectors, delivery techniques, and approaches for developing gene-based interventions for diseases have evolved in the last decade. The lack of suitable treatment has become a rational basis for extending the scope of gene therapy. The aim of this review is to investigate the general methods by which genes are transferred and to give an overview to clinical applications. Maximizing the potential benefits of gene therapy requires efficient and sustained therapeutic gene expression in target cells, low toxicity, and a high safety profile. Gene therapy has made substantial progress albeit much slower than was initially predicted. This review also describes the basic science associated with many gene therapy vectors and the present progress of gene therapy carried out for various surface disorders and diseases. The conclusion is that, with increased pathobiological understanding and biotechnological improvements, gene therapy will become a standard part of clinical practice.

Urtica dioica dichloromethane extract induce apoptosis from intrinsic pathway on human prostate cancer cells (PC3).

Prostate cancer is considered as the major cause of death among men around the world. There are a number of medicinal plants triggering apoptosis response in cancer cells, thus have a therapeutic potential. Therefore, further studies to characterize beneficial properties of these plants in order to introduce novel anti-cancer drugs are the interest of recent researches on the alternative medicine. On the other hand, due to traditional uses and availability of Urtica dioica extract, we decided to evaluate the efficacy of this medicinal herb on pc3 prostate cancer cell line. In the present study the cytotoxic effects of Urtica dioica extract were assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and trypan blue viability dye. Then, DNA fragmentation and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were exploited to measure cell death and apoptosis stage. The expression levels of caspase 3, caspase 9 and Bcl-2 genes were quantified by Real-Time PCR. Finally, Cell cycle was analyzed by flow cytometry. MTT assay showed that dichloromethanolic extract of Urtica dioica significantly inhibited the cell growth. According to the DNA fragmentation and TUNEL assay results, the herbal extract was able to induce apoptosis in prostate cancer cells. Our findings also demonstrated that the plant extract substantially increases the caspase 3 and 9 mRNA expression, while decreases Bcl-2. Cell cycle arrest was occurred in G2 stage, due to the results of flow cytometry. These results indicate that dichloromethanolic extract of Urtica dioica can successfully induce apoptosis in PC3 cells. Therefore, it could be used as a novel therapeutic candidate for prostate tumor treatment.

Effects of Urtica dioica dichloromethane extract on cell apoptosis and related gene expression in human breast cancer cell line (MDA-MB-468).

Breast cancer is the most common cancer among women in worldwide, especially in developing countries. Therefore, a large number of anticancer agents with herbal origins have been reported against this deadly disease. This study is the first to examine the cytotoxic and apoptotic effects of Urtica dioica in MDA-MB-468, human breast adenocarcinoma cells. The 3-(4,5-dimethylethiazol-2 yl)-2,5- diphenyltetrazolium (MTT) reduction and trypan-blue exclusion assay were performed in MDA-MB-468 cells as well as control cell line L929 to analyze the cytotoxic activity of the dichloromethane extract. In addition, Apoptosis induction of Urtica dioica on the MDA-MB-468 cells was assessed using TUNEL (terminal deoxy transferase (TdT)-mediated dUTP nick- end labeling) assay and DNA fragmentation analysis and real-time polymerase chain reaction (PCR). The results showed that the extract significantly inhibited cell growth and viability without inducing damage to normal control cells. Nuclei Staining in TUNEL and DNA fragments in DNA fragmentation assay and increase in the mRNA expression levels of caspase-3, caspase-9, decrease in the bcl2 and no significant change in the caspase-8 mRNA expression level, showed that the induction of apoptosis was the main mechanism of cell death that induce by Urtica dioica extract. Our results suggest that urtica dioica dichloromethane extract may contain potential bioactive compound(s) for the treatment of breast adenocarcinoma.

siRNA-mediated inhibition of survivin gene enhances the anti-cancer effect of etoposide in U-937 acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is one of the most frequent types of leukemia which mostly affects adult people. Resistance to therapeutic drugs is considered as a major clinical concern resulting in a weaker response to chemotherapy, disease relapse and decreased survival rate. Survivin, a member of Inhibitor of Apoptosis Proteins (IAPs), is associated with drug resistance and inhibition of apoptotic mechanisms in numerous hematological malignancies. In the present study, we examined the combined effect of etoposide and siRNA-mediated silencing of survivin on U-937 acute myeloid leukemia cells. The AML cells were transfected with survivin specific siRNA and gene knockdown was confirmed by quantitative real time PCR and western blotting. Subsequently, U-937 cells were assessed for response to etoposide treatment and apoptosis rate was measured with flowcytometery. The cytotoxic effects in siRNA-etoposide group were measured and compared to etoposide single therapy group. Survivin siRNA effectively knocked down the mRNA and protein levels of survivin, which led to lower cell proliferation and enhanced apoptosis. Furthermore, combined treatment of etoposide and survivin siRNA synergistically increased the cell toxic effects of etoposide and its ability to induce apoptosis.

Extracts of Scrophularia frigida Boiss display potent antitumor effects in human breast cancer cells by inducing apoptosis and inhibition of expression of the human epidermal growth factor receptor 2.

Some species of the Scrophularia genus have been extensively used as a natural remedy for treatment of various medical conditions. The objective of this study was to evaluate the growth inhibitory activity of Scrophularia frigida Boiss extracts as well as to study the effect of the potent extracts on the induction of apoptosis and cell cycle arrest on human breast cancer cells. S. frigida Boiss extracts exhibited obvious inhibitory effects on the growth of cancer cells and induced apoptosis. It is suggested that the extracts exert their anti-proliferative effect through multiple implications such as suppressing growth, arresting the cell cycle, increased DNA fragmentation, downregulation of the expression of human epidermal growth factor receptor 2 and myeloid cell Leukemia-1, and upregulation of pro-apoptotic messenger RNAs like caspase-3 and caspase-9. Taken together, the results obtained indicate that S. frigida Boiss extracts may contain effective compounds that can be used as a therapeutic anticancer agent.

SiRNA/DOX lodeded chitosan based nanoparticles: Development, Characterization and in vitro evaluation on A549 lung cancer cell line.

High-mobility group AT-hook2 (HMGA2), involved in epithelial mesenchymal transition (EMT) process, has a pivotal role in lung cancer metastasis. Lung cancer therapy with HMGA2 suppressing small interfering RNA (siRNA) has been introduced recently while doxorubicin (DOX) has been used as a frequent cancer chemotherapy agent. Both reagents have been faced with obstacles in clinic which make them ineffective. NanoParticles (NPs) provided a platform for efficient co delivery of the anticancer drugs. The aim of this study was production and in vitro characterization of different pharmacological groups (siRNA, DOX or siRNA-DOX) of carboxymethyl dextran thrimethyl chitosan nanoparticles (CMDTMChiNPs) on cytotoxicity, gene expression, apoptosis and migration of metastatic lung cancer cell line (A-549). CMDTMChiNPs were synthesized and encapsulated with siRNA, DOX or siRNA-DOX. Then the effects of HMGA2 siRNA and DOX co delivery was assessed in A549 viability and target genes (HMGA2, Ecadherin, vimentin and MMP9) by MTT and real time PCR, respectively. In addition capability of apoptosis induction and anti-migratory features of formulated NPs were analyzed by flowcytometry and wound healing assays. SiRNA-DOX-CMDTM ChiNPs approximate size were 207±5 with poly dispersity index (PDI) and zeta potential of 0.4 and 16.3±0.3, respectively. NPs loaded with DOX and siRNA were the most efficient drug formulations in A549 cell cytotoxicity, altering of EMT markers, apoptosis induction and migration inhibition. Generally our results showed that co delivery of HMGA2 siRNA and DOX by novel designed CMDTMChiNPs is a new therapeutic approach with great potential efficiency for lung cancer treatment.

New insights into the mechanisms of multidrug resistance in cancers.

Drug resistance is one of the major obstacles in the treatment of various cancers. Since chemotherapy serves as a most beneficial method for the repression of tumor progression and due to its desirable cell death potency in tumors which reducing metastasis, failure of such a pivotal treatment lead to tumor recurrence and consequent mortality. Multidrug resistance, the principal mechanism by which many cancers develop resistance to chemotherapy drugs, is a major factor in the failure of many forms of chemotherapy. MDR1 overexpression is one form of the multidrug resistance (MDR) phenotype, which can be acquired by patients initially responsive to chemotherapy. In this review, we briefly look inside the recent mechanisms of chemotherapeutic resistance, the MDR1 gene expression in tumors and some novel inhibition-based approaches.

Micro-RNAs: The new potential biomarkers in cancer diagnosis, prognosis and cancer therapy.

MicroRNAs (miRNAs) are a large class of small noncoding RNAs approximately 22 nucleotides in length. They are the main regulators of gene expression, regulating specific oncogenes, tumor suppressors, cancer stem cells and metastasis. MicroRNAs have become valuable to cancer research in recent years. They appear as a significant biomarker in tumorigenesis. Briefly, the capacities of miRNA to identify between tumor and normal tissue, to distinguish between various subgroups of tumors and to foretell results or responses to therapy have attracted scientist's attention to these small RNAs. MicroRNAs' remarkable stability in both the tissue and bloodstream of cancer patients has elevated the possibility that miRNAs may prove to be a novel diagnostic biomarker. This review focuses on the utility of miRNAs as key biomarkers in cancer diagnosis, cancer prognosis and cancer therapy.

Differential effects of peroxisome proliferator-activated receptor agonists on doxorubicin-resistant human myelogenous leukemia (K562/DOX) cells.

P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) in tumor cells is still a main obstacle for the chemotherapeutic treatment of cancers. Therefore, identification of safe and effective MDR reversing compounds with minimal adverse side effects is an important approach in the cancer treatment. Studies show that peroxisome proliferator-activated receptor (PPARs) ligands can inhibit cell growth in many cancers. Here, we investigated the effect of different PPAR agonists include fenofibrate, troglitazone and aleglitazar on doxorubicin-resistant human myelogenous leukemia (K562/DOX) cells. The effects of doxorubicin (DOX) following treatment with PPAR agonists on cell viability were evaluated using MTT assay and the reversal fold (RF) values. Rhodamine123 (Rh123) assays were used to determine P-gp functioning. P-gp mRNA/protein expression was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analysis after incubation with troglitazone and aleglitazar. Our results showed that troglitazone and aleglitazar significantly enhanced the cytotoxicity of DOX and decreased the RF values in K562/DOX cells, however, no such results were found for fenofibrate. Troglitazone and aleglitazar significantly down regulated P-gp expression in K562/DOX cells; in addition, the present study revealed that aleglitazar elevated intracellular accumulation of Rh123in K562/DOX cells as short-term effects, which also contribute to the reversal of MDR. These findings show that troglitazone and especially aleglitazar exhibited potent effects in the reversal of P-gp-mediated MDR, suggesting that these compounds may be effective for combination therapy strategies and circumventing MDR in K562/DOX cells to other conventional chemotherapeutic drugs.

siRNA-mediated silencing of MDR1 reverses the resistance to oxaliplatin in SW480/OxR colon cancer cells.

One of the most challenging aspects of colon cancer therapy is rapid acquisition of multidrug resistant phenotype. The multidrug resistance gene 1 (MDR1) product, p—glycoprotein (P—gp), pump out a variety of anticancer agents from the cell, giving rise to a general drug resistance against chemotherapeutic agents. The aim of this study was to investigate the effect of a specific MDR1 small interference RNA (siRNA) on sensitivity of oxaliplatin—resistant SW480 human colon cancer cell line (SW480/OxR) to the chemotherapeutic drug oxaliplatin. SW480 cells were made resistant by continuous incubation with stepwise serially increased concentrations of oxaliplatin over a 6—months period. Resistance cell were subsequently transfected with specific MDR1 siRNA. Relative MDR1 mRNA expression was measured by Quantitative real—time PCR. Western blot analysis was performed to determine the protein levels of P—gp. The cytotoxic effects of oxaliplatin and MDR1 siRNA, alone and in combination were assessed using MTT and the number of apoptotic cells was determined with the TUNEL assay. MDR1 siRNA effectively reduced MDR1 expression in both mRNA and protein levels. MDR1 down—regulation synergistically increased the cytotoxic effects of oxaliplatin and spontaneous apoptosis SW480/OxR. Our data demonstrates that RNA interference could down regulate MDR1 gene expression and reduce the P—gp level, and partially reverse the drug resistance in SW480/OxR cells in vitro. Therefore, the results could suggest that MDR1 silencing may be a potent adjuvant in human colon chemotherapy.

Restoration of miR-451a-5p/miR-34a-5p could suppress the proliferation and migration of human breast cancer cells through Wnt/ß- catenin and ERK/P-ERK signaling pathways.

MicroRNAs (miRNAs) are a class of small non-coding RNAs with a length of 21-25 nucleotides and play an essential role in the regulation of cancer initiation, development and progression. Breast cancer (BC) is the most commonly detected malignancy in women and one of the leading causes of death worldwide. In this study, the effects of transfection of microRNA-451a-5p and miR-34a-5p (tumor suppressors), individually and in combination on apoptosis, proliferation and migration of breast cancer cells in vitro were investigated. For this study, malignant breast cancer cells (MDA-MB-231) were transfected with the miR-451a-5p and miR-34a-5p mimics. Subsequently cytotoxicity, apoptosis, proliferation, migration protein and gene expression of caspase-3, caspase-8, MMP9, ROCK, vimentin and c-Myc of the cancer cells were analyzed by MTT, flow cytometry, q-RT-PCR (expression level of caspase-3, caspase-8, MMP9, ROCK, vimentin and c-Myc genes), wound healing and Western blot assays. The results showed that miR-34a-5p and miR-451a-5p could additionally induce apoptosis and cell cycle arrest in the sub-G1phase, suppress proliferation and migration in breast cancer cells, and also decrease the expression of ß- catenin and ERK/P-ERK proteins . The present data document that restoration of the tumor suppressor miR-451/miR-34 strongly induces programmed cell death in vitro and apparently inhibits cell proliferation and migration in human breast cancer cells. In summary, miR-451a and miR-34a play an important role in breast cancer cell proliferation and migration via the Wnt/ß-catenin and ERK/P-ERK signaling pathways. Therefore, the simultaneous restoration of the presented tumor suppressor miRNAs can be proposed as a valuable and potential therapeutic strategy in the treatment of breast cancer. However, further studies should be useful.

Autophagy-regulating microRNAs: two-sided coin in the therapies of breast cancer.

Despite recent advances in the treatment of breast cancer (BC), it still remains as a prevalent and deadly cancer in the world. Given that BC is a heterogeneous disease, it is necessary to clarify molecular mechanisms in tumor cells to improve various therapy outcomes and overcome therapy resistance. Autophagy represents one of the most important intracellular degradation pathways involved in diverse biological processes and plays an important bi-directional role in tumor formation and progression. Among the several mechanisms that affect autophagy, microRNAs (miRNAs) play a crucial role as gene regulators. Several in vivo and in vitro studies have reported multiple miRNAs regulating autophagy in BC that affect tumor initiation, progression, and response to various therapies. In the present review, we highlighted the mechanisms through which miRNAs regulate autophagy in BC and their potential use as therapeutic targets.

Evaluation of gamma radiation-induced cytotoxicity of breast cancer cells: Is there a time-dependent dose with high efficiency?

CONTEXT: Radiotherapy is one of the important treatment modalities in the management of breast cancer. AIMS: The aim of this study is to study the efficient treatment of breast cancer as related to the dose delivery. MATERIALS AND METHODS: The human breast cancer cell lines (MCF-7) cells were cultured and exposed by 1, 2, 4, 6, 8, 10, and 20 Gy of γ-rays. Radiation-induced cell death was detected and evaluated, using three assay methods: Cell viability, clonogenic cell survival assay and induction of apoptosis. The cell viability was determined using trypan blue staining, 24 and 72 h post-irradiation. The survival fraction (SF) was determined by colony counting, 14 days after exposure and the apoptotic cell death was determined using the TUNEL assay. STATISTICAL ANALYSIS USED: One- or two-way analysis of variance was deemed as appropriate, followed by relevant post t-test to determine P values. RESULTS: The difference of MCF-7 cell death through increasing post-radiation time from 24 to 72 h following the dose of 1, 6 and 10 Gy was found to be 2%, 9.6% and 7.14%, respectively. D0of MCF-7 was 220 cGy and the SF in the cells irradiated by 1 Gy and 10 Gy doses were 0.8 and 0.0001, respectively. The estimated variances were 2%, 11.1% and 8.4%, between 24 h and 72 h post-radiation apoptosis death for 1, 6, and 10 Gy, respectively. CONCLUSIONS: The dose and time dependence inducing apoptotic death was significant (P = 0.001). The delayed mortality and apoptosis was observed in MCF-7 cell, but the variance of total cell death and apoptosis in 24 and 72 h post-radiation with 6 Gy was obviously more than that with other doses.

Tumor suppressor microRNAs: Targeted molecules and signaling pathways in breast cancer.

Breast cancer is the most common type of cancer in women whose prevalence is increasing every year. Common strategies for diagnosis, prognosis and specific treatment of breast cancer need improvements to increase patients' survival. For this reason, there is growing number of efforts world-wide with molecular approaches. With the advent of microRNAs (miRNAs), they have been interested for almost all aspects of tumorgenesis and correlation of breast cancer and microRNAs was discovered for the first time in 2005. MiRNAs form a group of small noncoding RNAs which participate in regulation of gene expression and subsequently several biological processes and pathogenesis of various diseases. As other cancers, miRNAs involved in breast cancer are classified in two groups: the first group is tumor inducing miRNAs (also called oncomirs) that can induce tumor initiation and progression, and their expression is increased in cancerous cells. The second group is tumor suppressor miRNAs. In normal situation, tumor suppressor miRNAs prevent beginning and progression of breast cancer through suppressing the expression of various oncogenes. In this review we will give a general overview about miRNAs and breast cancer, and in the following, more discussion about tumor suppressor miRNAs, with focus on the best known of them and their targeted oncogenes and signaling pathways. Finally, we will point to application of this group of miRNAs in diagnosis, prognosis and treatment of patients.

Production and characterization of monoclonal antibodies against human IgG in Balb/c mouse.

Immunoglobulin G (IgG) is the main immunoglobulin in natural human serum. It constitutes 70 to 75 percent of all immunoglobulins. Monoclonal antibodies have many applications in diagnosis, treatment and purification. The conjugated monoclonal antibodies against human IgG are used in most diagnostic kits. For production of monoclonal antibodies against human IgG, spleen cells of the most immune mouse were fused with SP2/0 (myeloma cells) using Poly Ethylene Glycol (PEG). Supernatant of hybridoma cells was screened for detection of antibody by ELISA. The suitable clones were selected for limiting dilution (L.D). Then, the supernatant of suitable monoclones were assessed for cross reactivity with IgM & IgA by ELISA and confirmed by immunobloting. The subclasses of the selected monoclonal antibodies were determined and the clones were frozen and kept in liquid nitrogen. Finally, suitable monoclone was injected into the mouse, intraperitoneally, that has been primed with Pristane. In this study, 127 clones were obtained of which 15 clones had absorbance more than 1 which two of them with absorbance about 1.5 were selected for limiting dilution. The yield of limiting dilution was 6 clones with absorbance about 1.8 which did not show cross reactivity with IgM & IgA. Among these clones, G2 monoclone with IgG1 subclass was selected as suitable one and it was reproduced in FCS free RPMI 1640. For large scale production in invivo, the suitable clone was implanted in the peritoneum of the Balb/c mouse and its titer was measured, which showed 1/100,000 dilution for ascitic fluid, having no cross reactivity with IgM & IgA. Monoclonal antibody was purified by chromatography, confirmed by SDS- PAGE, conjugated with enzyme and applied for diagnostic kits.