Glioblastoma as a Novel Drug Repositioning Target: Updated State.

Glioblastoma multiforme (GBM) is an aggressive form of adult brain tumor that can arise from a low-grade astrocytoma. In recent decades, several new conventional therapies have been developed that have significantly improved the prognosis of patients with GBM. Nevertheless, most patients have a limited long-term response to these treatments and survive < 1 year. Therefore, innovative anti-cancer drugs that can be rapidly approved for patient use are urgently needed. One way to achieve accelerated approval is drug repositioning, extending the use of existing drugs for new therapeutic purposes, as it takes less time to validate their biological activity as well as their safety in preclinical models. In this review, a comprehensive analysis of the literature search was performed to list drugs with antiviral, antiparasitic, and antidepressant properties that may be effective in GBM and their putative anti-tumor mechanisms in GBM cells.

Current Status and Challenges of Vaccination Therapy for Glioblastoma.

Glioblastoma (GBM), also known as grade IV astrocytoma, is the most common and deadly type of central nervous system malignancy in adults. Despite significant breakthroughs in current GBM treatments such as surgery, radiotherapy, and chemotherapy, the prognosis for late-stage glioblastoma remains bleak due to tumor recurrence following surgical resection. The poor prognosis highlights the evident and pressing need for more efficient and targeted treatment. Vaccination has successfully treated patients with advanced colorectal and lung cancer. Therefore, the potential value of using tumor vaccines in treating glioblastoma is increasingly discussed as a monotherapy or in combination with other cellular immunotherapies. Cancer vaccination includes both passive administration of monoclonal antibodies and active vaccination procedures to activate, boost, or bias antitumor immunity against cancer cells. This article focuses on active immunotherapy with peptide, genetic (DNA, mRNA), and cell-based vaccines in treating GBM and reviews the various treatment approaches currently being tested. Although the ease of synthesis, relative safety, and ability to elicit tumor-specific immune responses have made these vaccines an invaluable tool for cancer treatment, more extensive cohort studies and better guidelines are needed to improve the efficacy of these vaccines in anti-GBM therapy.

Methodological advancements in organ-specific ectopic lipid quantitative characterization: Effects of high fat diet on muscle and liver intracellular lipids.

OBJECTIVE: Ectopic lipid accumulation is a hallmark of metabolic diseases, linking obesity to non-alcoholic fatty liver disease, insulin resistance and diabetes. The use of zebrafish as a model of obesity and diabetes is raising due to the conserved properties of fat metabolism between humans and zebrafish, the homologous genes regulating lipid uptake and transport, the implementation of the '3R's principle and their cost-effectiveness. To date, a method allowing the conservation of lipid droplets (LDs) and organs in zebrafish larvae to image ectopic lipids is not available. Our objectives were to develop a novel methodology to quantitatively evaluate organ-specific LDs, in skeletal muscle and liver, in response to a nutritional perturbation. METHODS: We developed a novel embedding and cryosectioning protocol allowing the conservation of LDs and organs in zebrafish larvae. To establish the quantitative measures, we used a three-arm parallel nutritional intervention design. Zebrafish larvae were fed a control diet containing 14% of nutritional fat or two high fat diets (HFDs) containing 25 and 36% of dietary fats. In muscle and liver, LDs were characterized using immunofluorescence confocal microscopy. In liver, intrahepatocellular lipids were discriminated from intrasinusoid lipids. To complete liver characteristics, fibrosis was identified with Masson's Trichrome staining. Finally, to confirm the conservation and effect of HFD, molecular players of fat metabolism were evaluated by RT-qPCR. RESULTS: The cryosections obtained after setting up the embedding and cryopreservation method were of high quality, preserving tissue morphology and allowing the visualization of ectopic lipids. Both HFDs were obesogenic, without modifying larvae survival or development. Neutral lipid content increased with time and augmented dietary fat. Intramuscular LD volume density increased and was explained by an increase in LDs size but not in numbers. Intrahepatocellular LD volume density increased and was explained by an increased number of LDs, not by their increased size. Sinusoid area and lipid content were both increased. Hepatic fibrosis appeared with both HFDs. We observed alterations in the expression of genes associated with LD coating proteins, LD dynamics, lipogenesis, lipolysis and fatty acid oxidation. CONCLUSIONS: In this study, we propose a reproducible and fast method to image zebrafish larvae without losing LD quality and organ morphology. We demonstrate the impact of HFD on LD characteristics in liver and skeletal muscle accompanied by alterations of key players of fat metabolism. Our observations confirm the evolutionarily conserved mechanisms in lipid metabolism and reveal organ specific adaptations. The methodological advancements proposed in this work open the doors to study organelle adaptations in obesity and diabetes related research such as lipotoxicity, organelle contacts and specific lipid depositions.

Combination Therapy of Metadichol Nanogel and Lipocalin-2 Engineered Mesenchymal Stem Cells Improve Wound Healing in Rat Model of Excision Injury.

Purpose: Currently, several disorders including burns, trauma, excisional and diabetic wounds, and bedsores threaten the human health. Application of mesenchymal stem cells (MSCs) is recommended for treatment of skin disorders. However, because of oxidative stress and inflammation after skin injury, survival of transplanted MSCs is low which in turn negatively affects the efficiency of the MSCs-based therapy. In an attempt to address the aforementioned challenge and introducing a novel potential therapeutic strategy, we employed combination therapy by lipocalin 2 (Lcn2)-engineered MSCs and a Metadichol (an inverse agonist of vitamin D receptor (VDR)) nanogel in a rat model of excisional wound. Methods: First, human umbilical cord MSCs (hUC-MSCs) was transfected by a recombinant plasmid encoding Lcn2 gene. Next, a combination of Metadichol nanogel and the engineered MSCs was co-applied on wound in rat model of excision injury. Finally the improvement of wound healing in experimental groups was evaluated by photography and histological assessments (hematoxylin and eosin staining). Results: Our findings revealed that the repair rate was higher in the group received combination therapy comparing to control groups. Notably, Metadichol+Lcn2-MSCs showed significantly higher wound contraction rate compared to control group at all time points (P value < 0.001). Furthermore, wound repair rate was 95% 14 days after surgery, and 100% after 21 days in the treatment groups. Our results also revealed that the combination therapy improved and accelerated the wound healing process. Conclusion: Our findings suggest a novel potential therapeutic strategy i.e. Lcn2-engineered MSCs and Metadichol for wound healing. However, further preclinical and clinical studies are required.

Circulating non-coding RNAs as a diagnostic and management biomarker for breast cancer: current insights.

Cancer biomarkers can be used to determine the molecular status of a tumor or its metastases, which either release them directly into body fluids or indirectly through disruption of tumor/metastatic tissue. New minimally invasive and repeatable sample collection methods, such as liquid biopsy, have been developed in the last decade to apply cancer knowledge and track its progression. Circulating non-coding RNAs, which include microRNAs, long non-coding RNAs, and PIWI-interacting RNAs, are increasingly being recognized as potential cancer biomarkers. The growing understanding of cancer's molecular pathogenesis, combined with the rapid development of new molecular techniques, encourages the study of early molecular alterations associated with cancer development in body fluids. Specific genetic and epigenetic changes in circulating free RNA (cf-RNA) in plasma, serum, and urine could be used as diagnostic biomarkers for a variety of cancers. Only a subset of these cf-RNAs have been studied in breast cancer, with the most extensive research focusing on cf-miRNA in plasma. These findings pave the way for immediate use of selected cf-RNAs as biomarkers in breast cancer liquid biopsy, as well as additional research into other cf-RNAs to advance.

Global Scientific Research on SARS-CoV-2 Vaccines: A Bibliometric Analysis.

OBJECTIVE: We performed this bibliometric analysis to identify global scientific research on the SARS-CoV-2 vaccines. MATERIALS AND METHODS: This bibliometric analysis study inclusive search of English-language publications related to the SARS-CoV-2 vaccines was conducted in the Scopus, PubMed, and Dimensions databases without year limitations. The results of bibliometric analysis comprised a time-dependent citation density trend, the name of the journal, journal impact factor (IF), year of publication, type of article, category, subscription or affiliation, co-authorship, and cooccurrence network. RESULTS: A study of the scientific literature from three databases (Scopus, PubMed, Dimensions) shows that investigators have focused more on studying the structure of the coronavirus at different levels (organismic, cellular, and molecular). In addition, the method of virus penetration into the cell and features of the influence of coronavirus on animals are well-studied. Various methods and strategies are being used to develop the vaccines, including both animal-tested methods and computer models. The Dimensions database is the most representative in terms of coverage of research on development of the SARS-CoV-2 vaccines. CONCLUSION: This research is a scientific investigation based on bibliometric analysis of papers related to the SARS-CoV-2 vaccines. The Dimensions database provides the most representative research coverage on the creation of a vaccine against coronavirus. It is characterized by a large number of formed verbose terms (length of more than four words) related to coronavirus, which makes it possible to track trends in the development of methods for creating a vaccine.

Development of a Cell-Based Biosensor for Residual Detergent Detection in Decellularized Scaffolds.

Ex vivo engineering of organs that uses decellularized whole organs as a scaffold with autologous stem cells is a potential alternative to traditional transplantation. However, one of the main challenges in this approach is preparing cytocompatible scaffolds. So far, high-precision and specific evaluation methods have not been developed for this purpose. Cell-based biosensors (CBBs) are promising tools to measure analytes with high sensitivity and specificity in a cost-effective and noninvasive manner. In this paper, using the NF-κB inducible promoter we developed a CBB for residual detergent detection. Proximal and core sections of the inducible promoter, containing NF-κB binding sequence, are designed and cloned upstream of the reporter gene (secreted alkaline phosphatase (SEAP)). After transfection into HEK293 cells, stable and reliable clones were selected. After confirmation of induction of this gene construct by sodium dodecyl sulfate (SDS), the stability and function of cells treated by qPCR and SEAP activity were measured. This biosensor was also used to evaluate the cytocompatibility of decellularized tissue. Results showed that the developed biosensor could detect very small amounts of SDS detergent (3.467 pM). It has the best performance 8 h after exposure to detergent, and its stability in high passage numbers was not significantly reduced. Applying this biosensor on decellularized tissues showed that SEAP activity higher than 4.36 (U/L) would lead to a viability reduction of transplanted cells below 70%. This paper presents a novel method to evaluate the cytocompatibility of decellularized tissues. The developed CBB can detect residual detergents (such as SDS) in tissues with high sensitivity and efficiency.

Runx2 silencing promotes adipogenesis via down-regulation of DLK1 in chondrogenic differentiating MSCs.

BACKGROUND: For cartilage regeneration, stem cells are a promising cell source; however, even the advances made in the differentiation of stem cells into precursor-differentiated cartilage cells have not been successful with respect to reprograming these cells to achieve complete differentiation and fully functioning cells until now. Previous findings suggest that Runx2 plays a major role in chondrocyte differentiation and maturation. Although targeting Runx2 has enhanced some chondrocyte properties, the adipogenic lineage shift has eventually occurred in these cells. The present study mainly aimed to reveal the mechanism of this adipogenesis. METHODS: To create inducible artificial shRNA-miR expressing vectors, the designed short hairpin RNAs (shRNAs) were inserted into the pri-mir-30 backbone, cloned into lentiviral pLVET-Tet-on, and transducted into mesenchymal stem cells (MSCs). Runx2 gene was silenced in MSCs either for 1 week or 4 weeks and cultured in the chondrogenic medium. At days 7, 14 and 28, cells were harvested, and chondrogenesis, adipogenesis and hypertrophic states were examined using histochemical staining and a real-time polymerase chain reaction assay. RESULTS: The results showed that the designed shRNA-miR effectively targeted Runx2 in mRNA and protein levels. Chondrogenic markers were up-regulated in constantly silenced Runx2 group; however, adipogenic markers and fat droplets appeared gradually. DLK1 gene was also significantly down-regulated in this group, and overexpression of DLK1 abrogated adipogenesis in the Runx2 targeted group. CONCLUSIONS: Based on these results, it can be concluded that DLK1 is responsible for the lineage shift in Runx2 targeted chondrogenic differentiating MSCs.

MicroRNA Tough Decoy Knockdowns miR-195 and Represses Hypertrophy in Chondrocytes.

Cartilage hypertrophy is a condition in which the cells are completely differentiated, and new morphological changes and mineralization prevent proper cellular functions. The occurrence of hypertrophy during differentiation fails current regenerative strategies for treatment. Strategies to minimize hypertrophy in chondrocytes are categorized into two levels of protein and gene. Among these strategies, one way to affect multiple pathways involved in the development of hypertrophy is to manage microRNA activity in cells. Recent miRNA profiling studies have shown that miR-195-5p upregulates through the transition from chondrogenic to hypertrophic state. Bioinformatics assessment of microRNA targets also indicates that several genes repressed by miR-195-5p play important roles in processes related to hypertrophy. The aim of this study was to develop a microRNA Tough Decoy to suppress miR-195-5p and investigate whether it can prevent a hypertrophic state in chondrocytes. The Tough Decoy (TUD) was designed and evaluated bioinformatically and then cloned into the pLVX-Puro plasmid. The TUD function was validated by Dual-Luciferase assay and qRT-PCR. After delivering TUD to C28/I2 chondrocytes cultured in a hypertrophic medium, hypertrophic differentiation was assessed by histochemical staining, quantitative RT-PCR of hypertrophy marker genes, and alkaline phosphatase activity. Results showed that the TUD could inhibit miRNA efficiently and downregulate hypertrophic markers such as RUNX2, alkaline phosphatase, and collagen 10 significantly compared with the control group. Alcian blue and alizarin red staining also demonstrated the optimal effect of gene constructs on tissue properties and mineralization of the TUD group. Delivering the miR-195-5p Tough Decoy to the cartilage cells can prevent the occurrence of hypertrophy in chondrocytes and could be considered as a candidate for the treatment of other diseases such as osteoarthritis.

CRISPR/Cas9-mediated knockout of Lcn2 effectively enhanced CDDP-induced apoptosis and reduced cell migration capacity of PC3 cells.

AIMS: Lipocalin 2 (Lcn2/NGAL) belongs to lipocalin superfamily with diverse functions. The precise function of Lcn2, particularly in cancer development, remains to be elucidated yet. In an attempt to knockout of Lcn2 expression by CRISPR/Cas 9 technology in a highly aggressive and invasive prostate cancer cell line and to evaluate the combination therapy with cisplatin (CDDP), this study was conducted. MAIN METHODS: Control CRISPR/Cas9 plasmid and homology-directed repair plasmid or validated human Lcn2 CRISPR/Cas9 KO plasmids were co-transfected into PC3 cells using fugene HD transfection reagent. The stable cells were selected in the presence of puromycin. Correspondingly, knock out of Lcn2 was evaluated by RT-PCR, ELISA, and immunocytochemistry. PC3-Scr (control) and Lcn2-KO (PC3 cells in which lcn2 has been knocked out) were treated with or without cisplatin (CDDP). Cell proliferative ability was measured by WST-1 and colony-formation assays. Apoptosis was evaluated by DAPI staining, in situ cell death detection (TUNEL) assay, and cell death detection ELISA plus methods. The migration capabilities were studied by wound healing/scratch and transwell assays. KEY FINDINGS: Lcn2 knock out in a highly aggressive and invasive cancer cell like PC3 decreased cell proliferation and increased the sensitivity of CDDP. Conspicuously, loss of Lcn2 expression effectively enhanced CDDP-induced apoptosis in PC3 cells. Lcn2 knock out by CRISPR/Cas9 technology decreased the cell migration capacity of PC3 cells as well. SIGNIFICANCE: Lcn2 not only is a valuable and useful biomarker for diagnosis and prognosis of prostate cancer but also and more importantly is a potential novel emerging therapeutic target.

Design, development and evaluation of microRNA-199a-5p detecting electrochemical nanobiosensor with diagnostic application in Triple Negative Breast Cancer.

The new sensitive diagnostic method, electrochemical nanobiosensor, is applied to cancer detection by using specific biomarkers such as miRNA. Studies have shown that for the diagnosis of Triple Negative Breast Cancer (TNBC) patients, in the early stages of the disease, miR-199a-5p as circulating miRNA can be a suitable candidate. In this study, a new electrochemical nanobiosensor for serum miR-199a-5p detection was designed by using modified glassy carbon electrode (GCE) with graphene oxide (GO) and gold nanoroad (GNR) on which a thiolated probe was immobilized. The electrochemical impedance method (EIS) was used to examine the electrochemical characteristics and behavior of nanobiosensor and for verifying the nanomaterial synthesis the scanning electron microscope (SEM) were applied, field emission scanning electron microscope (FE-SEM), UV-Vis spectrophotometry and energy dispersive spectroscopy (EDS). Optimum conditions were investigated for designed nanobiosensor and in optimal conditions, following results were obtained; the linear range of the calibration curve from 15 fM to 148 pM, the detection limit of 4.5 fM, and the standard deviation of 2.9%. The research data showed that our designed electrochemical nanobiosensor has a sensitivity and desirable precision for evaluation of miR-199a-5p that can be used to measure low concentrations of miR-199a-5p in blood serum sample.

Crigler-Najjar Syndrome: Current Perspectives and the Application of Clinical Genetics.

BACKGROUND: Crigler-Najjar syndrome (CNS, OMIM: 218800) is the paradigm of an inborn error of metabolism and a rare genetic disease with an estimated incidence of 0.6-1.0 per million live births. Discrimination between CNS subtypes is usually done on the basis of the clinical criteria, such as response to phenobarbital treatment and other molecular and functional characteristics. METHODS: The identification of four novel pathogenic mutations and the analysis of residual activity of missense in UGT1A1 gene are useful for clinical diagnosis, and may reveal a new insight in enzyme activity, whereas the identification of pathogenic mutations will accelerate genetic counseling for newly identified CNS patients. RESULTS: Phototherapy, orthotropic liver transplantation, liver cell transplantation and gene therapy are treatment choices and candidates to fight back this syndrome. Due to the promising reports of gene therapy in small animal models, gene therapy approaches are expected to continue in preclinical research for developing safe and effective treatment of CNS. Gene transfer vectors using recombinant viruses, such as Adenovirus have been applied successfully in transferring UGT1A1 gene to the liver of Gunn rat model of CNS. CONCLUSION: In spite of remaining safety and efficiency issues, gene therapy promises to be a realistic treatment modality for CNS during the future decade.

MicroRNA-Based Linkage between Aging and Cancer: from Epigenetics View Point.

Ageing is a complex process and a broad spectrum of physical, psychological, and social changes over time. Accompanying diseases and disabilities, which can interfere with cancer treatment and recovery, occur in old ages. MicroRNAs (miRNAs) are a set of small non-coding RNAs, which have considerable roles in post-transcriptional regulation at gene expression level. In this review, we attempted to summarize the current knowledge of miRNAs functions in ageing, with mainly focuses on malignancies and all underlying genetic, molecular and epigenetics mechanisms. The evidences indicated the complex and dynamic nature of miRNA-based linkage of ageing and cancer at genomics and epigenomics levels which might be generally crucial for understanding the mechanisms of age-related cancer and ageing. Recently in the field of cancer and ageing, scientists claimed that uric acid can be used to regulate reactive oxygen species (ROS), leading to cancer and ageing prevention; these findings highlight the role of miRNA-based inhibition of the SLC2A9 antioxidant pathway in cancer, as a novel way to kill malignant cells, while a patient is fighting with cancer.

Inducible expression of indoleamine 2,3-dioxygenase attenuates acute rejection of tissue-engineered lung allografts in rats.

Lung disease remains one of the principal causes of death worldwide and the incidence of pulmonary diseases is increasing. Complexity in treatments and shortage of donors leads us to develop new ways for lung disease treatment. One promising strategy is preparing engineered lung for transplantation. In this context, employing new immunosuppression strategies which suppresses immune system locally rather than systemic improves transplant survival. This tends to reduce the difficulties in transplant rejection and the systemic impact of the use of immunosuppressive drugs which causes side effects such as serious infections and malignancies. In our study examining the immunosuppressive effects of IDO expression, we produced rat lung tissues with the help of decellularized tissue, differentiating medium and rat mesenchymal stem cells. Transduction of these cells by IDO expressing lentiviruses provided inducible and local expression of this gene. To examine immunosuppressive properties of IDO expression by these tissues, we transplanted these allografts into rats and, subsequently, evaluated cytokine expression and histopathological properties. Expression of inflammatory cytokines IFNγ and TNFα were significantly downregulated in IDO expressing allograft. Moreover, acute rejection score of this experimental group was also lower comparing other two groups and mRNA levels of FOXP3, a regulatory T cell marker, upregulated in IDO expressing group. However, infiltrating lymphocyte counting did not show significant difference between groups. This study demonstrates that IDO gene transfer into engineered lung allograft tissues significantly attenuates acute allograft damage suggesting local therapy with IDO as a strategy to reduce the need for systemic immunosuppression and, thereby, its side effects.

MicroRNAs in lung diseases: Recent findings and their pathophysiological implications.

Lung diseases are one of the leading causes of mortality and morbidity worldwide and effective therapies are imperfect. Nonetheless, recently some novel strategies have been developed to treat and curtail their debilitating impact. Some of the treatments include the role of MicroRNAs (miRNAs) in stemming the spread of lung morbidities. Micro RNAs are small non-coding RNAs which are known as important players in the posttranscriptional regulation of gene expression in mammalian cells by regulating translation. MiRNAs are involved in basic regulatory mechanisms of cells including influencing inflammation. MiRNA dysregulation, resulting in aberrant expression of a gene, is suggested to play a key role in susceptibility of diseases. MiRNAs are involved in the pathogenesis of lung diseases such as cystic fibrosis, lung cancer, asthma, chronic obstructive pulmonary disease, and Idiopathic pulmonary fibrosis. A better understanding of the involvement of miRNAs in pathogenesis of these diseases could result in the development of new therapeutic and diagnostic tools. In this review, we provide an overview of the current understanding of miRNA biogenesis and role as well as recent insights into role of some miRNAs in different pulmonary diseases.

Recent immunomodulatory strategies in transplantation.

Despite preservation methods, surgical procedures, current immunosuppressive therapy regimens advances, organ transplantation is accompanied with a poor long-term survival and significant mortality. This has led to an increased interest to optimize outcomes while minimizing associated toxicity by using alternative methods for maintenance immunosuppression, organ rejection treatment, and monitoring of immunosuppression. Advance in long-standing allograft outcomes may depend on new drugs with novel mechanisms of action with minimal toxicity. Newer treatment techniques have been developed, including using novel stem cell-based therapies such as mesenchymal stem cells, phagosomes and exosomes. Immunoisolation techniques and salvage therapies, including photopheresis and total lymphoid irradiation have emerged as alternate therapeutic choices. The present review evaluates the recent clinical advances in immunosuppressive therapies for organ transplantation.

Gene therapy modalities in lung transplantation.

Organ transplantation is the ultimate treatment for end-stage organ failure patients. However, the success of lung transplantation is hindered by multiple factors, the most important of which being the adverse effects of systemic immunosuppressive therapy, chronic transplant dysfunction and a severe shortage of donor lungs. The genetic modification of organs or cells is an attractive approach to protect allogeneic transplants from acute rejection and other complications. Many different therapeutic concepts and vector systems have been investigated in the pre-clinical area with a view to prolonging lung allograft survival. This review explores both the candidate genes and ways in which they have been arranged to overcome both immune and non-immune barriers to transplantation success in experimental models.

Immunosuppressive therapy in allograft transplantation: from novel insights and strategies to tolerance and challenges.

Immunosuppression therapy is the key to successful post-transplantation outcomes. The need for ideal immunosuppression became durable maintenance of long-term graft survival. In spite of current immunosuppressive therapy regimens advances, surgical procedures, and preservation methods, organ transplantation is associated with a long-term poor survival and significant mortality. This has led to an increased interest to optimize outcomes while minimizing associated toxicity by using alternative methods for maintenance immunosuppression, organ rejection treatment, and monitoring of immunosuppression. T regulatory (Treg) cells, which have immunosuppressive functions and cytokine profiles, have been studied during the last decades. Treg cells are able to inhibit the development of allergen-specific cell responses and consequently play a key role in a healthy immune response to allergens. Mature dendritic cells (DCs) play a crucial role in the differentiation of Tregs, which are known to regulate allergic inflammatory responses. Advance in long-standing allograft outcomes may depend on new drugs with novel mechanisms of action with minimal toxicity. Newer treatment techniques have been developed, including using novel stem cell-based therapies such as mesenchymal stem cells, phagosomes and exosomes. Immunoisolation techniques and salvage therapies, including photopheresis and total lymphoid irradiation have emerged as alternative therapeutic choices. The present review evaluates the recent clinical advances in immunosuppressive therapies for organ transplantation.