Evaluation of a gene expression biomarker to identify operationally tolerant liver transplant recipients: the LITMUS trial.

LITMUS was a single-centre, Phase 2a study designed to investigate whether the gene biomarker FGL2/IFNG previously reported for the identification of tolerance in murine models could identify operationally tolerant liver transplant recipients. Multiplex RT-PCR was used to amplify eight immunoregulatory genes in peripheral blood mononuclear cells (PBMC) from 69 adult liver transplant recipients. Patients with PBMC FGL2/IFNG ≥ 1 and a normal liver biopsy underwent immunosuppression (IS) withdrawal. The primary end point was the development of operational tolerance. Secondary end points included correlation of tolerance with allograft gene expression and immune cell markers. Twenty-eight of 69 patients (38%) were positive for the PBMC tolerance biomarker and 23 proceeded to IS withdrawal. Nine of the 23 patients had abnormal baseline liver biopsies and were excluded. Of the 14 patients with normal biopsies, eight (57%) have achieved operational tolerance and are off IS (range 12-57 months). Additional studies revealed that all of the tolerant patients and only one non-tolerant patient had a liver gene ratio of FOXP3/IFNG ≥ 1 prior to IS withdrawal. Increased CD4+ T regulatory T cells were detected both in PBMC and livers of tolerant patients following IS withdrawal. Higher expression of SELE (gene for E-selectin) and lower expression of genes associated with inflammatory responses (GZMB, CIITA, UBD, LSP1, and CXCL9) were observed in the pre-withdrawal liver biopsies of tolerant patients by RNA sequencing. These results suggest that measurement of PBMC FGL2/IFNG may enrich for the identification of operationally tolerant liver transplant patients, especially when combined with intragraft measurement of FOXP3/IFNG. Clinical Trial Registration: ClinicalTrials.gov (LITMUS: NCT02541916).

Multicapillary gel electrophoresis based analysis of genetic variants in the WFS1 gene.

The WFS1 gene is one of the thoroughly investigated targets in diabetes research, variants of the gene were suggested to be the genetic components of the common forms (type 1 and type 2) of diabetes. Our project focused on the analysis of polymorphisms (rs4689388, rs148797429, rs4273545) localized in the WFS1 promoter region. Although submarine gel electrophoresis based approaches were also employed in the genetic tests, it was demonstrated that multicapillary electrophoresis offers a state of the art approach for reliable high-throughput SNP and VNTR analysis. Association studies were carried out in a case-control setup. Luciferase reporter assay was employed to test the effect of the investigated loci on the activity of gene expression in vitro. Significant association could be demonstrated between all three polymorphisms and type 2 diabetes in both allele- and genotype-wise settings even using Bonferroni correction. It is notable; however, that the three loci were in strong linkage disequilibrium, thus the observed associations cannot be considered as separate effects. Molecular analyses showed that the rs4273545 GT SNP played a role in the regulation of transcription in vitro. However, this effect took place only in the presence of the region including the rs148797429 site, although this latter locus did not have its own impact on the regulation of gene expression. The paper provides genotyping protocols readily applicable in any multiplex SNP and VNTR analyses, moreover confirms and extends previous results about the role of WFS1 polymorphisms in the genetic risk of diabetes mellitus.

Vaccine Plasmid Topology Monitoring by Capillary Gel Electrophoresis.

BACKGROUND: Plasmid DNA has been widely used in vaccination as well as in cell and gene therapy. It exists in multiple isoforms, including supercoiled, nicked or open circular and linear forms. Regulatory agencies recommend having more than 80% of the supercoiled isoform for the bulk release of plasmid products; thus, it should be analyzed accordingly. METHODS AND RESULTS: The traditional analysis method for plasmid DNA is agarose gel electrophoresis. However, due to time-consuming manual sample loading, visualization, and data analysis, it has limitations in obtaining consistently quantitative results. In this short communication, we introduced a fast, sensitive, and robust plasmid analysis method using capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF). CGE-LIF analysis of the supercoiled isoform and its open circular counterpart was completed in 20 minutes with excellent sensitivity by using a common fluorescent DNA binding dye. The advantage of the method was demonstrated by the purity analysis of two large plasmids (7 kb and 10 kb). The fully automated sample loading, separation and data analysis featured enhanced assay repeatability and ease of quantitation over agarose gel electrophoresis. CONCLUSION: As a worked example, analysis of plasmid samples treated at elevated temperature during an accelerated stability test also demonstrated the applicability of CGE-LIF to monitor plasmid topology and possible degradation.

High Throughput Multiplex SNP-analysis in Chronic Obstructive Pulmonary Disease and Lung Cancer.

BACKGROUND: A number of human inflammatory diseases and tumors have been shown to cause alterations in the glycosylation pattern of plasma proteins in a specific manner. These highly variable and versatile post-translational modifications finetune protein functions by influencing sorting, folding, enzyme activity and subcellular localization. However, relatively little is known about regulatory factors of this procedure and about the accurate causative connection between glycosylation and disease. OBJECTIVE: The aim of the present study was to investigate whether certain single nucleotide polymorphisms (SNPs) in genes encoding glycosyltransferases and glycosidases could be associated with elevated risk for chronic obstructive pulmonary disease (COPD) and lung adenocarcinoma. METHODS: A total of 32 SNPs localized in genes related to N-glycosylation were selected for the association analysis. Polymorphisms with putative biological functions (missense or regulatory variants) were recruited. SNPs were genotyped by a TaqMan OpenArray platform. A single base extension-based method in combination with capillary gel electrophoresis was used for verification. RESULTS: The TaqMan OpenArray approach provided accurate and reliable genotype data (global call rate: 94.9%, accuracy: 99.6%). No significant discrepancy was detected between the obtained and expected genotype frequency values (Hardy-Weinberg equilibrium) in the healthy control sample group in case of any SNP confirming reliable sampling and genotyping. Allele frequencies of the rs3944508 polymorphism localized in the 3' UTR of the MGAT5 gene significantly differed between the sample groups compared. CONCLUSION: Our results suggest that the rs34944508 SNP might modulate the risk for lung cancer by influencing the expression of MGAT5. This enzyme catalyzes the addition of N-acetylglucosamine (GlcNAc) in beta 1-6 linkage to the alpha-linked mannose of biantennary N-linked oligosaccharides, thus, increasing branching that is the characteristic of invasive malignancies.

Cancer Niche as a Garbage Disposal Machine: Implications of TCM-Mediated Balance of Body-Disease for Treatment of Cancer.

Cancer epidemic led to worldwide to search for a new "game changer" concept to govern cancer research and cancer treatment. Western medicine-based cancer research has been extending the impasse without resolution in sigh for improving survival of patients with solid malignant tumors in the last four decades due to heterogeneity in cancer tissues. Such a deadlock charts a course to learn lessons from the developing countries, directly or indirectly to complement the exhausted Western medicine. We propose a new concept of "Cancer niche as a garbage disposal machine" with implications of traditional Chinese medicine-mediated restoration of normal balance between body and disease to bring the fight against cancer under control.

Breathing Signature as Vitality Score Index Created by Exercises of Qigong: Implications of Artificial Intelligence Tools Used in Traditional Chinese Medicine.

Rising concerns about the short- and long-term detrimental consequences of administration of conventional pharmacopeia are fueling the search for alternative, complementary, personalized, and comprehensive approaches to human healthcare. Qigong, a form of Traditional Chinese Medicine, represents a viable alternative approach. Here, we started with the practical, philosophical, and psychological background of Ki (in Japanese) or Qi (in Chinese) and their relationship to Qigong theory and clinical application. Noting the drawbacks of the current state of Qigong clinic, herein we propose that to manage the unique aspects of the Eastern 'non-linearity' and 'holistic' approach, it needs to be integrated with the Western "linearity" "one-direction" approach. This is done through developing the concepts of "Qigong breathing signatures," which can define our life breathing patterns associated with diseases using machine learning technology. We predict that this can be achieved by establishing an artificial intelligence (AI)-Medicine training camp of databases, which will integrate Qigong-like breathing patterns with different pathologies unique to individuals. Such an integrated connection will allow the AI-Medicine algorithm to identify breathing patterns and guide medical intervention. This unique view of potentially connecting Eastern Medicine and Western Technology can further add a novel insight to our current understanding of both Western and Eastern medicine, thereby establishing a vitality score index (VSI) that can predict the outcomes of lifestyle behaviors and medical conditions.

Tissue Elasticity Bridges Cancer Stem Cells to the Tumor Microenvironment Through microRNAs: Implications for a "Watch-and-Wait" Approach to Cancer.

BACKGROUND: Targeting the tumor microenvironment (TME) through which cancer stem cells (CSCs) crosstalk for cancer initiation and progression, may open new treatments different from those centered on the original hallmarks of cancer genetics thereby implying a new approach for suppression of TME driven activation of CSCs. Cancer is dynamic, heterogeneous, evolving with the TME and can be influenced by tissue-specific elasticity. One of the mediators and modulators of the crosstalk between CSCs and mechanical forces is miRNA, which can be developmentally regulated, in a tissue- and cellspecific manner. OBJECTIVE: Here, based on our previous data, we provide a framework through which such gene expression changes in response to external mechanical forces can be understood during cancer progression. Recognizing the ways mechanical forces regulate and affect intracellular signals with applications in cancer stem cell biology. Such TME-targeted pathways shed new light on strategies for attacking cancer stem cells with fewer side effects than traditional gene-based treatments for cancer, requiring a "watchand- wait" approach. We attempt to address both normal brain microenvironment and tumor microenvironment as both works together, intertwining in pathology and physiology - a balance that needs to be maintained for the "watch-and-wait" approach to cancer. CONCLUSION: This review connected the subjects of tissue elasticity, tumor microenvironment, epigenetic of miRNAs, and stem-cell biology that are very relevant in cancer research and therapy. It attempts to unify apparently separate entities in a complex biological web, network, and system in a realistic and practical manner, i.e., to bridge basic research with clinical application.

Control dominating subclones for managing cancer progression and posttreatment recurrence by subclonal switchboard signal: implication for new therapies.

In contrast to hematological malignancies, meaningful improvements in survival statistics for patients with malignant brain tumors have not been realized in >40 years of clinical research. Clearly, a new medical approach to brain cancers is needed. Recent research has led to a new concept that needs to destroy all cancer subclones to control the cancer progression. However, this new concept fails to distinguish the difference between dominating subclones and dormant subclones. Here, we address the issue of clonal switch and emphasize that there may be one or more than one dominant clones within the tumor mass at any time. Destructing one dominant clone triggers activating other dormant subclones to become dominating subclones, causing cancer progress and post-treatment cancer recurrence. We postulate the concept of subclonal switchboard signaling and the pathway that involved in this process. In the context of stem cell and development, there is a parallel with the concept of quiescent/dormant cancer stem cells (CSC) and their progeny, the differentiated cancer cells; these 2 populations communicate and co-exist. The mechanism with which determines to extend self-renewal and expansion of CSC is needed to elucidate. We suggest eliminating the "dominating subclonal switchboard signals" that shift the dormant subclones to dominating subclones as a new strategy.

Mechanisms for progenitor cell-mediated repair for ischemic heart injury.

Recent studies have shown that treatments involving injection of stem cells into animals with damaged cardiac tissue result in improved cardiac functionality. Clinical trials have reported conflicting results concerning the recellularization of post-infarct collagen scars. No clear mechanism has so far emerged to fully explain how injected stem cells, specifically the commonly used mesenchymal stem cells (MSC) and endothelial precursor cells (EPC), help heal a damaged heart. Clearly, these injected stem cells must survive and thrive in the hypoxic environment that results after injury for any significant repair to occur. Here we discuss how ischemic preconditioning may lead to increased tolerance of stem cells to these harsh conditions and increase their survival and clinical potential after injection. As injected cells must reach the site in numbers large enough for repair to be functionally significant, homing mechanisms involved in stem cell migration are also discussed. We review the mechanisms of action stem cells may employ once they arrive at their target destination. These possible mechanisms include that the injected stem cells (1) secrete growth factors, (2) differentiate into cardiomyocytes to recellularize damaged tissue and strengthen the post-infarct scar, (3) transdifferentiate the host cells into cardiomyocytes, and (4) induce neovascularization. Finally, we discuss that tissue engineering may provide a standardized platform technology to produce clinically applicable stem cell products with these desired mechanistic capacities.

Convergence of normal stem cell and cancer stem cell developmental stage: Implication for differential therapies.

Increased evidence shows that normal stem cells may contribute to cancer development and progression by acting as cancer-initiating cells through their interactions with abnormal environmental elements. We postulate that normal stem cells and cancer stem cells (CSC) possess similar mechanisms of self-renewal and differentiation. CSC can be the key to the elaboration of anti-cancer-based therapy. In this article, we focus on a controversial new theme relating to CSC. Tumorigenesis may have a critical stage characterized as a "therapeutic window", which can be identified by association of molecular, biochemical and biological events. Identifying such a stage can allow the production of more effective therapies (e.g. manipulated stem cells) to treat several cancers. More importantly, confirming the existence of a similar therapeutic window during the conversion of normal stem cells to malignant CSC may lead to targeted therapy specifically against CSC. This conversion information may be derived from investigating the biological behaviour of both normal stem cells and cancerous stem cells. Currently, there is little knowledge about the cellular and molecular mechanisms that govern the initiation and maintenance of CSC. Studies on co-evolution and interdependence of cancer with normal tissues may lead to a useful treatment paradigm of cancer. The crosstalk between normal stem cells and cancer formation may converge developmental stages of different types of stem cells (e.g. normal stem cells, CSC and embryonic stem cells). The differential studies of the convergence may result in novel therapies for treating cancers.

A biological global positioning system: considerations for tracking stem cell behaviors in the whole body.

Many recent research studies have proposed stem cell therapy as a treatment for cancer, spinal cord injuries, brain damage, cardiovascular disease, and other conditions. Some of these experimental therapies have been tested in small animals and, in rare cases, in humans. Medical researchers anticipate extensive clinical applications of stem cell therapy in the future. The lack of basic knowledge concerning basic stem cell biology-survival, migration, differentiation, integration in a real time manner when transplanted into damaged CNS remains an absolute bottleneck for attempt to design stem cell therapies for CNS diseases. A major challenge to the development of clinical applied stem cell therapy in medical practice remains the lack of efficient stem cell tracking methods. As a result, the fate of the vast majority of stem cells transplanted in the human central nervous system (CNS), particularly in the detrimental effects, remains unknown. The paucity of knowledge concerning basic stem cell biology--survival, migration, differentiation, integration in real-time when transplanted into damaged CNS remains a bottleneck in the attempt to design stem cell therapies for CNS diseases. Even though excellent histological techniques remain as the gold standard, no good in vivo techniques are currently available to assess the transplanted graft for migration, differentiation, or survival. To address these issues, herein we propose strategies to investigate the lineage fate determination of derived human embryonic stem cells (hESC) transplanted in vivo into the CNS. Here, we describe a comprehensive biological Global Positioning System (bGPS) to track transplanted stem cells. But, first, we review, four currently used standard methods for tracking stem cells in vivo: magnetic resonance imaging (MRI), bioluminescence imaging (BLI), positron emission tomography (PET) imaging and fluorescence imaging (FLI) with quantum dots. We summarize these modalities and propose criteria that can be employed to rank the practical usefulness for specific applications. Based on the results of this review, we argue that additional qualities are still needed to advance these modalities toward clinical applications. We then discuss an ideal procedure for labeling and tracking stem cells in vivo, finally, we present a novel imaging system based on our experiments.

Identification and validation of genes affecting aortic lesions in mice.

Atherosclerosis represents the most significant risk factor for coronary artery disease (CAD), the leading cause of death in developed countries. To better understand the pathogenesis of atherosclerosis, we applied a likeli-hood-based model selection method to infer gene-disease causality relationships for the aortic lesion trait in a segregating mouse population demonstrating a spectrum of susceptibility to developing atherosclerotic lesions. We identified 292 genes that tested causal for aortic lesions from liver and adipose tissues of these mice, and we experimentally validated one of these candidate causal genes, complement component 3a receptor 1 (C3ar1), using a knockout mouse model. We also found that genes identified by this method overlapped with genes progressively regulated in the aortic arches of 2 mouse models of atherosclerosis during atherosclerotic lesion development. By comparing our gene set with findings from public human genome-wide association studies (GWAS) of CAD and related traits, we found that 5 genes identified by our study overlapped with published studies in humans in which they were identified as risk factors for multiple atherosclerosis-related pathologies, including myocardial infarction, serum uric acid levels, mean platelet volume, aortic root size, and heart failure. Candidate causal genes were also found to be enriched with CAD risk polymorphisms identified by the Wellcome Trust Case Control Consortium (WTCCC). Our findings therefore validate the ability of causality testing procedures to provide insights into the mechanisms underlying atherosclerosis development.