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Introduction: Osteopontin (OPN) is recognized as a potent biomarker of Oral lichen planus (OLP) because of vital role in inflammation and repair process. The aim of present study is assessment of OPN in OLP in comparison with healthy controls (HC). Material and Methods: To explore salivary levels of OPN, a group of 20 subjects with OLP were compared with 20 HC. Salivary OPN levels was measured by enzyme-linked immunosorbent (ELISA) assay. Results: Results indicated elevated OPN levels in lesion size < 1 cm compared with 1 - 3 cm lesion size of OLP (p= 0.02). In contrast, we did not find a significant difference in OPN expression level in saliva from OLP patients and healthy controls (P = 0.96). Discussion: The above results suggest that maybe OPN may serve as a potential biomarker for the monitoring of repair process in OLP.

Despite the crucial role of cell metabolism in biological processes, particularly cell division, metabolic aspects of liver regeneration are not well defined. Better understanding of the metabolic activity governing division of liver cells will provide powerful insights into mechanisms of physiological and pathological liver regeneration. Recent studies have provided evidence that metabolic response to liver failure might be a proximal signal to initiate cell proliferation in liver regeneration. In this review, we highlight how lipids, carbohydrates, and proteins dynamically change and orchestrate liver regeneration. In addition, we discuss translational studies in which metabolic intervention has been used to treat chronic liver diseases (CLDs).

Objective: Hepatocellular carcinoma (HCC) as a chronic liver condition is largely associated with immune responses. Previous studies have revealed that different subsets of lymphocytes play fundamental roles in controlling or improving the development and outcome of solid tumors like HCC. Hence, this study aimed to investigate whether immune system changes were related to disease development in HCC patients. Methods: Peripheral blood mononuclear cells were isolated from 30 HCC patients and 30 healthy volunteers using Ficoll density centrifugation. The isolated cells were stained with different primary antibodies and percentages of different immune cells were determined by flow cytometry. Results: HCC patients indicated significant reductions in the numbers of CD4+ cells, Tbet+IFNγ+cells, and GATA+IL-4+cells in peripheral blood in comparison with healthy individuals ( p < 0.05). There was no significant change in IL-17+RORγt+cells between patient and healthy groups. In contrast, Foxp3+CD127lowcell frequency was significantly higher in patients than healthy subjects ( p < 0.0001). The numbers of Th1, Th2, and Th17 cells were significantly lower in HCC patients than healthy control ( p < 0.0001), although the reduction in Th2 cell numbers was not statistically significant. On the contrary, Treg percentage showed a significant increase in patients compared to healthy subjects ( p < 0.0001). Other data revealed that Th1, Th2, and Th17 cell frequencies were significantly higher in healthy individuals than patients with different TNM stages of HCC, with the exception of Th2 in patients with stage II HCC ( p < 0.01–0.05). Treg percentage was significantly increased in patients with different TNM stages ( p < 0.0001). Among all CD4+ T cells, the frequency of Th2 cell was significantly associated with TNM stages of HCC ( p < 0.05). Conclusion: Our data provide further evidence to show that immune changes may participate in determining HCC progression and disease outcome. However, it should be mentioned that more investigations are needed to clarify our results and explain possible impacts of other immune cells on the pathogenesis of HCC.

The BIV1-CovIran vaccine is highly effective against COVID-19. The neutralizing potency of all SARS-CoV-2 vaccines seems to be decreased against variants of concern. We assessed the sensitivity of the Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2) variants to neutralizing antibodies (NAbs) present in sera from individuals who had received the BIV1-CovIran candidate vaccine compared with an original Wuhan-related strain.The ability of vaccine serum to neutralize the variants was measured using the conventional virus neutralization test. The correlation of spike (S) protein antibody and anti-receptor binding domain with neutralizing activity was investigated.The current study demonstrated that 29 of 32 (90.6%; 95% CI: 75.0-98.0) of the vaccinees developed NAbs against a Wuhan-related strain. It is noteworthy that 28 (87.50%) and 24 of 32 (75%) of the recipients were able to produce NAbs against Alpha, Beta, and Delta variants, respectively. Serum virus-neutralizing titres for different SARS-CoV-2 strains were weakly correlated with anti-receptor binding domain antibodies (Spearman r = 36-42, p 0.05), but not S-binding antibodies (p 0.05).Although there was a reduction in neutralization titres against the Alpha, Beta, and Delta variants compared with the Wuhan strain, BIV1-CovIran still exhibited potent neutralizing activity against the SARS-CoV-2 variants of concern.

Activation of hepatic stellates (HSCs) is known as the major cause of initiation and progression of liver fibrosis. A wide array of events occurs during HSC activation including induction of hedgehog (Hh) signaling and endoplasmic reticulum (ER) stress. Targeting HSC activation may provide promising insights into liver fibrosis treatment. In this regard, establishing in vitro models which can mimic the molecular pathways of interest is very important. We aimed to activate HSC in which Hh signaling and ER stress are stimulated simultaneously. We used 5 ng/ml TGFβ to activate LX-2 cells, HSC cell line. Gene expression analysis using qRT-PCR, immunostaining and immunoblotting were performed to show HSC activation associated markers. Furthermore, the migration capacity of the TGFβ treated cells is evaluated. The results demonstrated that major fibrogenic markers including collagen1a, lysyl oxidase, and tissue inhibitor of matrix metalloproteinase 1 genes are up-regulated significantly. In addition, our immunofluorescence and immunoblotting results showed that protein levels of GLI-2 and XBP1, were enhanced. Moreover, we found that TGFβ treatment reduced the migration of LX-2 cells. Our results are compatible with high throughput data analysis with respect to differentially expressed genes of activated HSC compared to the quiescent ones. Moreover, our findings suggest that quercetin can reduce fibrogenic markers of activated HSCs as well as osteopontin expression, a target gene of hedgehog signaling.

Cytotoxic small-molecule drugs have a major influence on the fate of antibody-drug conjugates (ADCs). An ideal cytotoxic agent should be highly potent, remain stable while linked to ADCs, kill the targeted tumor cell upon internalization and release from the ADCs, and maintain its activity in multidrug-resistant tumor cells. Lessons learned from successful and failed experiences in ADC development resulted in remarkable progress in the discovery and development of novel highly potent small molecules. A better understanding of such small-molecule drugs is important for development of effective ADCs. The present review discusses requirements making a payload appropriate for antitumor ADCs and focuses on the main characteristics of commonly-used cytotoxic payloads that showed acceptable results in clinical trials. In addition, the present study represents emerging trends and recent advances of payloads used in ADCs currently under clinical trials.

Impaired wound healing is a common complication of diabetes mellitus (DM) and the underlying mechanism of this impairment is still unclear. Fibroblast, as the main reconstructing cell, secretes some critical growth factors and cytokine contributing to wound healing. It is well known that DM alters the behavior of these cells and photobiomodulation therapy (PBMT) compensates some impairments in diabetic fibroblasts. Therefore, the aim of the present study was to demonstrate the impact of diabetes and the role of PBMT through low level laser irradiation on secretory profile of human diabetic fibroblasts. Primary human dermal fibroblasts from normal (HDFs) and diabetic (DHDFs) donors were harvested. For PBMT, the DHDFs were irradiated with a Helium-Neon laser at 632.8 nm wavelength and energy density of 0.5 J/cm2, as laser treated group (LT-DHDFs). Next, some cellular behaviors and secretory profiling array for 60 growth factors/cytokines were investigated in LT-DHDFs and then compared with those of controls. The data showed that the PBMT could compensate such impairments occurred in DHDFs in terms of viability, proliferation, and migration. Furthermore, considering our novel findings, out of those 20 growth factors/cytokines involved in cell proliferation, immune system regulation, and cell-cell communication pathways, which significantly decreased in DHDF as compared with HDFs, the PBMT could compensate seven in LT-DHDFs as compared with DHDFs. The seven growth factor/cytokines, which are mainly involved in cell-cell communication, positive regulation of cell proliferation, and chemokine mediated pathway included BDNF, Eotaxin-3, FGF6, FGF7, Fractalkine, fit-3ligand, and GCP-2. Therefore, it is suggested that scrutinizing these differentially secreted molecules and the impaired pathways in DHDFs, in combination with those compensated in LT-DHDFs, could raise our knowledge to manage diabetic ulcer through a feasible and cost effective intervention, specifically PBMT.

Background Reduction/alkylation is one of the leading strategies for the development of antibody drug conjugates (ADCs). Precise control of the reduction process would not only yield a defined number of free thiols per antibody but also result in development of more homogenous conjugates. Methods In the present study, we investigated the effect of various dithiothreitol (DTT) concentrations, temperature conditions, and DTT exposure times on antibody reduction. After antibody reduction, the Ellman's test and SDS-PAGE analysis were used to evaluate free thiols produced and confirm the reduction process, respectively. Results DTT concentration seems to be a potential factor in the reduction process. Concentrations of 0.1, 1, 5, 10, 20, 50, and 100 mM DTT at 37°C for 30 minutes resulted in approximately 0.4, 1.2, 5.4, 7, 8, 8, and 8 thiols per antibody, respectively. Conclusion Optimized site-specific conjugation can provide better process control and reproducibility for the development of disulfide-based ADCs.

Background: Mesenchymal stem cells (MSCs) are important candidates for MSC-based cellular therapy. Current paradigm states that MSCs support local progenitor cells in damaged tissue through paracrine signaling. Therefore, the study of paracrine effects and secretome of MSCs could lead to the appreciation of mechanisms and molecules associated with the therapeutic effects of these cells. This study analyzed anti-inflammatory and immune-modulatory effects of MSC secretomes derived from embryonic stem cells (ESCs) and bone marrow cells after hypoxia and normoxia preconditioning. Methods: ESCs differentiated into MSCs and characterized by flow cytometry as well as by differentiation into adipocytes and osteoblasts. The experimental groups were consisted of individual groups of ESC-MSCs and BM-MSCs (bone marrow-derived mesenchymal stromal cells), which were preconditioned with either hypoxia or normoxia for 24, 48 and 72 h. After collecting the cell-free medium from each treatment, secretomes were concentrated by centrifugal filters. Using a peripheral blood mononuclear cell (PBMC) assay and ELISA, IL-10 concentration in PBMCs was evaluated after their incubation with different secretomes from preconditioned and non-preconditioned MSCs. Results: A significant difference was observed between ESC-MSC normoxia and ESC-MSC hypoxia in IL-10 concentration, and normoxia secretomes increased IL-10 secretion from PBMCs. Moreover, the strongest IL-10 secretion from PBMCs could be detected after the stimulation by ESC-MSC conditioned secretomes, but not BM-MSC conditioned medium. Conclusions: Human hypoxia preconditioned ESC-MSC secretome indicated stronger immune-modulatory effects compared to BM-MSC conditioned medium. It could be suggested that induced MSCs confer less immune-modulatory effects but produce more inflammatory molecules such as tumor necrosis factor α, which needs further investigation.

Adult tissue-derived mesenchymal stem cells (MSCs) show tremendous promise for a wide array of therapeutic applications predominantly through paracrine activity. Recent reports showed that human embryonic stem cell (ESC)-derived MSCs are an alternative for regenerative cellular therapy due to manufacturing large quantities of MSCs from a single donor. However, no study has been reported to uncover the secretome of human ESC-MSCs as treatment of an acute liver failure (ALF) mouse model. We demonstrated that human ESC-MSCs showed similar morphology and cell surface markers compared with bone marrow-derived MSCs. ESC-MSCs exhibited a higher growth rate during early in vitro expansion, along with adipogenic and osteogenic differentiation potential. Treatment with ESC-MSC-conditioned medium (CM) led to statistically significant enhancement of primary hepatocyte viability and increased immunomodulatory interleukin-10 secretion from lipopolysaccharide-induced human blood mononuclear cells. Analysis of the MSCs secretome by a protein array screen showed an association between higher frequencies of secretory proteins such as vascular endothelial growth factor (VEGF) and regulation of cell proliferation, cell migration, the development process, immune system process, and apoptosis. In this thioacetamide-induced mouse model of acute liver injury, we observed that systemic infusion of VEGF led to significant survival. These data have provided the first experimental evidence of the therapeutic potential of human ESC-MSC-derived molecules. These molecules show trophic support to hepatocytes, which potentially creates new avenues for the treatment of ALF, as an inflammatory condition.

A better understanding of cancer stem cells (CSCs) may facilitate the prevention and treatment of cancers. Epithelial-mesenchymal transition (EMT) is a process activated during invasion and metastasis of tumors. EMT induction in normal and tumor cells makes them more resistant to chemotherapy. E-cadherin is a membrane protein and plays a role in tumor invasion, metastasis, and prognosis. Downregulation of E-cadherin is a hallmark of EMT. Here, we created a model of cancer stem-like cells enrichment via EMT induction using E-cadherin downregulation in HT29 cell line using a lentiviral vector carrying shRNA. We aimed to evaluate cancer and anti-CSC chemotherapeutics screening. The markers of EMT and CSCs were assessed and compared with control cells using flow cytometry, real-time PCR, immunocytochemistry, western blot, migration assay, invasion assay, and colony formation assay. The transduced cells showed a mesenchymal morphology. High levels of EMT-related proteins were also expressed. These results confirmed that the transduced cells underwent EMT. In addition, we observed an increased population of E-cadherin-downregulated HT29 cell line among the cells expressing colon CSC markers (CD133+ and CD44+ ) after EMT induction. E-cadherin-downregulated cells were morphologically like mesenchymal cells, and the number of CD133+ - and CD44+ -cells (CSC-like cells) increased. These cells can be used as stable models to study cancer cells and screening of antitumor therapeutics.

Polymerase chain reaction followed by high resolution melting (PCR-HRM) analysis is a simple, rapid and accurate method for molecular detection of various nematode species. The objective of the present study was, for the first time, to develop a PCR-HRM assay for the detection of various animal Trichostrongylus spp. A pair of primers targeting the ITS-2 rDNA region of the Trichostrongylus spp. was designed for the development of the HRM assay. DNA samples were extracted from 30 adult worms of Trichostrongylus spp., the ITS-2-rDNA region was amplified using PCR, and the resultant products were sequenced and characterized. Afterwards, the PCR-HRM analysis was conducted to detect and discriminate Trichostrongylus spp. Molecular sequence analysis revealed that 24, 4, and 1 of the samples were T. colubriformis, T. vitrinus and T. capricola, respectively. Results from PCR-HRM indicated that complete agreement was relatively found between speciation by HRM analysis and DNA sequencing for the detection of Trichostrongylus species. The PCR-HRM analysis method developed in the present study is fast and low-cost; the method can be comparable with other molecular detection techniques, representing a reliable tool for the identification of various species within the Trichostrongylus genus.

Present strategy introduces a novel method established for the synthesis of spherical shape ErVO4/MnWO4 heterostructures by a sonochemical method. This heterostructures with optima morphology can be synthesized by changing power and time ultrasound irradiation without any capping agent. BET analysis revealed that ErVO4/MnWO4 prepared in the presence of ultrasonic procedure has 75 times specific surface area as much as that of those was produced in the absence of ultrasonic rays. A variety of analyses (i.e., BET, XRD, TEM, EDS, FT-IR, and SEM) were applied for characterization of the ErVO4/MnWO4. Next, a selective and sensitive nanostructured sensor based on ErVO4/MnWO4 nanocomposite modified carbon paste electrode (ErVO4/MnWO4/CPE) was constructed for electrochemical detection of tyrosine (Tyr). The electrochemical characterizations were performed using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV). Compared with the unmodified CPE, the oxidation peak current was significantly enhanced for Tyr. The impact of effective parameters on voltammetric response of Tyr was analyzed with design of experiments (DOE) and response surface methodology (RSM). Under the optimized conditions, the oxidation peak current of Tyr was linear over a range of 0.08–400.0 μM with a detection limit of 7.7 nM. Finally, the usage of the proposed method was confirmed by the recovery tests of Tyr in biological samples.

Adeno-associated virus (AAV)-derived vectors are currently the most common type of viral vectors used in gene therapy clinical trials. The presence of neutralizing antibodies (NAbs) against wild-type AAVs in the host body is one of the limitations for the successful use of AAV vectors. AAV capsid manipulation, by which recombinant vectors lose their ability to interact with NAbs, can help overcome this obstacle. Various methods can be used for this purpose, including directed evolution as well as conjugation of certain chemical groups to AAV epitopes. The present review concisely explains the use of AAV vectors in the clinic for gene therapy of some diseases, their limitations, and solutions to these limitations.

Targeted delivery of therapeutic molecules into cancer cells is considered as a promising strategy to tackle cancer. Antibody-drug conjugates (ADCs), in which a monoclonal antibody (mAb) is conjugated to biologically active drugs through chemical linkers, have emerged as a promising class of anticancer treatment agents, being one of the fastest growing fields in cancer therapy. The failure of early ADCs led researchers to explore strategies to develop more effective and improved ADCs with lower levels of unconjugated mAbs and more-stable linkers between the drug and the antibody, which show improved pharmacokinetic properties, therapeutic indexes, and safety profiles. Such improvements resulted in the US Food and Drug Administration approvals of brentuximab vedotin, trastuzumab emtansine, and, more recently, inotuzumab ozogamicin. In addition, recent clinical outcomes have sparked additional interest, which leads to the dramatically increased number of ADCs in clinical development. The present review explores ADCs, their main characteristics, and new research developments, as well as discusses strategies for the selection of the most appropriate target antigens, mAbs, cytotoxic drugs, linkers, and conjugation chemistries.

Targeted therapy using specific monoclonal antibodies (mAbs) conjugated to chemotherapeutic agents or toxins has become one of the top priorities in cancer therapy. Antibody-drug conjugates (ADCs) are emerging as a promising strategy for cancer-targeted therapy. In this study, trastuzumab, a humanized monoclonal anti-HER2 antibody, was reduced by dithiothreitol and conjugated to the microtubule-disrupting agent monomethyl auristatin E (MMAE) through a valine-citrulline peptide linker (trastuzumab-MC-Val-Cit-PABC-MMAE [trastuzumab-vcMMAE]). After conjugation, ADCs were characterized by using UV-vis, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and flow cytometry. The antitumor activity of the ADC was evaluated in breast cancer cells in vitro. In addition, ADCs were further characterized using purification by the protein A chromatography, followed by assessment using apoptosis and MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assays. Hydrophobic interaction chromatography was used to determine drug-to-antibody ratio species of ADCs produced. Our finding showed that approximately 5.12 drug molecules were conjugated to each mAb. H2L2, H2L, HL, H2, H, and L forms of ADCs were detected in nonreducing SDS-PAGE. The binding of trastuzumab-vcMMAE to HER2-positive cells was comparable with that of the parental mAb. The MTT assay showed that our ADCs induced significant cell death in HER2-positive cells, but not in HER2-negative cells. The ADCs produced was a mixture of species, unconjugated trastuzumab (14.147%), as well as trastuzumab conjugated with two (44.868%), four (16.886%), six (13.238%), and eight (10.861%) molecules of MMAE. These results indicated that MMAE-conjugated trastuzumab significantly increases the cytotoxic activity of trastuzumab, demonstrating high affinity, specificity, and antitumor activity in vitro. Trastuzumab-vcMMAE is an effective and selective agent for the treatment of HER2-positive breast tumors.

Rituximab is a chimeric monoclonal antibody directed against B-lymphocyte specific antigen CD20, which is used for the treatment of B-cell malignancies. However, the effectiveness of rituximab is limited partly due to treatment resistance. The aim of this study was to develop rituximab-based antibody drug conjugate (ADC) to enhance rituximab activity. In this study, monomethyl auristatin E (MMAE) was covalently conjugated to dithiothreitol -reduced rituximab via a valine-citrulline peptide linker (rituximab-vcMMAE). The conjugates were then characterized by using nonreducing sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) and cell-based enzyme-linked immunosorbent assay (ELISA). The cytotoxic activity of the ADC was evaluated against Raji (human B-cell lymphoma; CD20-positive) and MOLT-4 (T lymphoblast; acute lymphoblastic leukemia; CD20-negative) cell lines. In addition, the colony formation assay was used to identify the propagation ability of ADC-treated cells in vitro. Results from nonreducing SDS-PAGE revealed various species of rituximab-MC-Val-Cit-PABC-MMAE (rituximab-vcMMAE), as compared with unconjugated rituximab. The binding capacity of rituximab-vcMMAE to the CD20-positive cell was similar to that of the parental rituximab. Most importantly, our results revealed that rituximab-vcMMAE was highly potent against the CD20-positive cell line, but not against the CD20-negative cell. At the same time, rituximab-vcMMAE was able to inhibit colony formation in CD20-positive cells. These data indicate that rituximab-vcMMAE may be a highly effective and selective therapy for the treatment of B-cell lymphoma.

Background Myc (c-Myc) alone activates the embryonic stem cell-like transcriptional module in both normal and transformed cells. Its dysregulation might lead to increased cancer stem cells (CSCs) population in some tumor cells. Objective In order to investigate the potential of Myc decoy oligodeoxynucleotides for differentiation therapy, mouse embryonic stem cells (mESCs) were used in this study as a model of CSCs. To our best of knowledge this is the first report outlining the application of Myc decoy in transcription factor decoy "TFD" strategy for inducing differentiation in mESCs. Methods A 20-mer double-stranded Myc transcription factor decoy and scrambled oligodeoxynucleotides (ODNs) were designed, analyzed by electrophoretic mobility shift (EMSA) assay and transfected into the mESCs under 2 inhibitors (2i) condition. Further investigations were carried out using fluorescence and confocal microscopy, cell proliferation and apoptosis analysis, alkaline phosphatase and embryoid body formation assay, real-time PCR and western blotting. Results EMSA data showed that Myc decoy ODNs bound specifically to c-Myc protein. They were found to be localized in both cytoplasm and nucleus of mESCs. Our results revealed the potential capability of Myc decoy ODNs to decrease cell viability by (16.1±2%), to increase the number of cells arrested in G0/G1 phases and apoptosis by (14.2±3.1%) and (12.1±3.2%), respectively regarding the controls. Myc decoy could also modulate differentiation in mESCs despite the presence of 2i/LIF in our medium the presence of 2i/LIF in our medium. Conclusion The optimized Myc decoy ODNs approach might be considered as a promising alternative strategy for differentiation therapy investigations.

Background: Bone marrow mesenchymal stem cells (BM-MSCs) have emerged as a potential therapy for various inflammatory diseases. Because of some limitations, several recent studies have suggested the use of embryonic stem cell-derived MSCs (ESC-MSCs) as an alternative for BM-MSCs. Some of the therapeutic effects of the ESC-MSCs are related to the secretion of a broad array of cytokines and growth factors, known as secretome. Harnessing this secretome for therapeutic applications requires the optimization of production of secretary molecules. It has been shown that aggregation of MSCs into 3D spheroids, as a preconditioning strategy, can enhance immunomodulatory potential of such cells. In this study, we investigated the effect of secretome derived from human ESC-MSCs (hESC-MSCs) spheroids on secretion of IL-1β, IL-10, and tumor necrosis factor α (TNF-α) from lipopolysaccharide (LPS)-induced peripheral blood mononuclear cells (PBMCs). Methods: In the present study, after immunophenotyping and considering mesodermal differentiation of hESC-MSCs, the cells were non-adherently grown to prepare 3D aggregates, and then conditioned medium or secretome was extracted from the cultures. Afterwards, the anti-inflammatory effects of the secretome were assessed in an in vitro model of inflammation. Results: Results from this study showed that aggregate-prepared secretome from hESC-MSCs was able to significantly decrease the secretion of TNF-α (301.7 ± 5.906, p < 0.0001) and IL-1β (485.2 ± 48.38, p < 0.001) from LPS-induced PBMCs as the indicators of inflammation, in comparison with adherent culture-prepared secretome (TNF-α: 166.6 ± 8.04, IL-1β: 125.2 ± 2.73). Conclusion: Our study indicated that cell aggregation can be an appropriate strategy to increase immunomodulatory characteristics of hESC-MSCs.

Embryonic stem cells (ESCs) are derived from inner cell mass (ICM) and have the potency to differentiate into three germ layers (ectoderm, endoderm, and mesoderm). This potency of ESCs, called pluripotency, is critical for maintaining stemness. Transcriptional regulatory circuitry preserving stemness consists of transcription factors (TFs), epigenetic mechanisms, microRNAs (miRNAs or miRs), and long non-coding RNAs (lncRNAs). In this circuitry, components assist each other to activate essential genes for maintaining pluripotency and suppressing lineage-specific genes. TFs act directly by binding to their binding sites in the genome or indirectly by activating another gene (such as a miR), epigenetic mechanisms play their role by providing an activatory or inhibitory context for transcription, miRNAs regulate gene expression at the post-transcriptional level, and lncRNAs act as a scaffold function for epigenetic elements, regulating gene expression in ESCs. All these factors create a crossroad and collaborate to sustain stemness in the ESCs. Herein, we explain the role of each member in this circuitry and demonstrate the significance of the crossroad for keeping stemness.

Transcription factor decoys (TFDs) are exogenous oligonucleotides which can compete by cis-elements in promoters or enhancers for binding to TFs and downregulating gene expression in a specific manner. It is believed that tumor mass originates from cancer stem cells (CSCs) which the same with embryonic stem cells (ESCs) have the properties of both pluripotency and self-renewal (stemness). Many transcription factors such as Nanog, Oct-4, Sox2, Klf4, and Sall4 act as master regulators in the maintenance of stemness in both cell types. Differentiation therapy is based on this theory that by differentiation of CSCs, tumor mass can be eliminated with common cancer therapy methods. To our knowledge, the present study is the first report of a TFD approach against master regulator of stemness, Nanog, Oct-4, and Klf4, for downregulation purposes in P19 embryonic carcinoma stem cell. Different simple and complex decoys against Nanog, OCT-4, Sox2, and Klf4 were designed and used for this purpose. The results showed that the applied decoys especially Nanog-specific decoy decreased the expression of downstream genes.

Introduction: Auristatin and its derivatives (synthetic analogues of dolastatin 10, an antineoplastic natural product), are highly potent antimitotic agents which have attracted considerable attention because of their cytotoxic activity when targeting tumor cells in the form of antibody-drug conjugates (ADCs). Some sophisticated and expensive equipment such as HPLC are needed for drug/linker isolation. The aim of this study was to provide a simple aqueous work-up procedure for the isolation of such drug/linker constructs. The anti-tumor activity of the extracted drug/linker was also investigated against SKBR3 and HEK293 cancer cell lines, and cell viability was assessed. Methods: In the present study, monomethyl auristatin E (MMAE), a derivative of the cytotoxic tubulin modifier auristatin E, was covalently coupled to maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-vc-PAB), a cathepsin-B-cleavable linker, to obtain MC-vc-PAB-MMAE (vc-MMAE). Afterwards, a non-chromatographic isolation procedure was developed to isolate the drug/linker (vc-MMAE) construct. Silica gel thin-layer chromatography and electrospray ionization mass spectrometry were used to monitor the isolation procedure and to confirm the coupling of the linker to the drug, respectively. Further, the anti-tumor activity of the extracted drug/linker was investigated against SKBR3 and HEK293 cancer cell lines, and cell viability was assessed. Results: After coupling, the isolation process was confirmed as a single spot on the TLC plate. The isolation yield was calculated to be 65%. [M + H]+, [M + 2Na]+ and [M + ACN + 2H]+ species were observed in the mass spectra, showing that the coupling of MMAE to the linker is not adversely affected by the workup method. Our data revealed that the isolated vc-MMAE was highly potent against tumor cell lines, exhibiting that the workup procedure did not affect MMAE-mediated cytotoxicity. Conclusion: The isolation method described in this study can be applied for the development of a wide variety of ADCs.