Biological assays for the detection and measurement of environmental pollutants

The environmental pollution threatens human health as well as wildlife in the past decades. As a result, it is necessary to develop sensitive, effective, and inexpensive methods which can efficiently monitor and determine the presence and amount of hazards in the environment. In this study, we report two biologically analytical methods to detect and measure the environmental pollutants. Firstly, we describe the construction and characterization of the fluorescent and the luminescent Escherichia coli whole-cell biosensors for the detection of bioavailable toluene and its related compounds. The biosensor strains Escherichia coli DH5α carrying pTOLGFP or pTOLLUX were developed based on the expression of reporter genes: gfp or luxCDABE under the control of the Pu promoter and xylR gene of Pseudomonas putita plasmid pWW0. To assess their applicability for analyzing environmentally relevant samples, the biosensor harboring pTOLLUX was field-tested on water and soil samples collected from toluene contaminated sites. Our results demonstrate that nonpathogenic bacterial biosensors developed in the present study is useful and applicable in determining the bioavailability of toluene and its related compounds with high sensitivity in environmental samples, and they suggest a potential for its inexpensive application in field-ready tests. Secondly, alterations of glutathione levels as well as the mRNA levels of HSP70 in tilapia fish were investigated under arsenite exposure. Tilapia fish were exposed to waterborne arsenite (0, 1, 2, and 4 ppm, respectively) for 1 day and 3 day exposure duration. After the treatment, arsenite concentrations in gill, intestine, liver, and muscle were measured by means of inductively coupled plasma (ICP). Meanwhile, the GSH contents in fish tissues were measured and the mRNA expression of HSP70 was analyzed with semi-quantitative RT-PCR. Our results indicate that HSP70 mRNA expression and GSH levels exhibited a correlation with the arsenite exposure condition as well as the arsenic accumulation, indicating their usefulness as the biomarker of arsenite exposure in tilapia. Furthermore, the bioaccumulations of arsenic differentially distributed in various fish tissues assist in providing information of bioavailability. We conclude that biological assays, such as bacterial biosensors and biomarker presented in this study, for detecting and determining the environmental contaminants can compensate the disadvantages of traditional analytical methods such as not capable to reflect the bioavailability and failing to show the effects of contaminants mixtures on organisms, and thus providing critical data that can be useful in risk assessment.致 謝 I 中文摘要 III ABSTRACT V TABLE OF CONTENTS VII LIST OF TABLES IX LIST OF FIGURES X ABBREVIATIONS XI CHAPTER 1 INTRODUCTION 1 Overview of environmental pollution 1 1.1 Bacterial biosensor of toluene and its related compounds 2 1.1.1 Toluene 2 1.1.2 Regulation of xyl operon 3 1.1.3 Bacterial biosensor 6 1.2 Biomarker of arsenic exposure 9 1.2.1 Arsenic 9 1.2.2 Biomarker 11 1.2.3 Tilapia 12 1.2.4 HSP70 12 1.2.5 Glutathione 13 1.3 Purposes of study 15 1.3.1 Bacterial biosensor of toluene and its related compounds 15 1.3.2 Biomarker of arsenite exposure 16 CHAPTER 2 MATERIALS AND METHODS 17 Chemicals 17 2.1 Bacterial biosensor of toluene 17 2.1.1 Construction of biosensor plasmids 17 2.1.2 Bacteria Cultivation and induction experiments 19 2.1.3 Measurements of GFP fluorescence and luminescence in culture 21 2.1.4 Microscopic detection of fluorescent biosensor 22 2.1.5 Selectivity studies of biosensors 22 2.1.6 Kinetic analysis of GFP fluorescence and luminescence induced by toluene 23 2.1.7 Testing of contaminated soil and water samples with bacteria biosensors 23 2.1.8 Data analysis 24 2.2 Biomarker of arsenic exposure 24 2.2.1 Test organisms 24 2.2.2 Treatment of fish 25 2.2.3 Isolation of total RNA from fish 25 2.2.4 Reverse transcription and polymerase chain reaction (RT-PCR) 26 2.2.5 Chemical analysis of arsenic 28 2.2.6 Intracellular GSH measurement 28 CHAPTER 3 RESULTS 30 3.1 Bacterial biosensor of toluene and its related compounds 30 3.1.1 Construction of the bacterial biosensors 30 3.1.2 Selectivity analysis of biosensor to toluene-related compounds 30 3.1.3 Time-dependent induction of biosensor with effector 33 3.1.4 Dose-dependent induction of biosensor with effector 35 3.1.5 Testing of contaminated water and soil with the biosensor 37 3.2 Biomarker of arsenic exposure 41 3.2.1 Arsenic accumulation in tilapia 41 3.2.2 HSP70 as biomarker of arsenic exposure 41 3.3.3 GSH as biomarker of arsenic exposure 43 CHAPTER 4 DISCUSSION 50 4.1 Bacterial biosensor of toluene and its related compounds 50 4.1.1 Development of bacterial biosensors of toluene 50 4.1.2 Characterization of bacterial biosensors of toluene 50 4.1.3 Comparison of the bacterial biosensor assay with the traditional analysis method 52 4.1.4 Comparison of reporter protein in biosensor 54 4.1.5 Field testing of environmental samples using a biosensor approach 56 4.2 Biomarker of arsenic exposure 57 4.2.1 Arsenic accumulation in tilapia 57 4.2.2 HSP70 mRNA expression as biomarker of arsenic exposure 58 4.2.3 GSH as biomarker of arsenic exposure 59 CHAPTER 5 CONCLUSION 61 5.1 Bacterial biosensor for toluene and its related compounds 61 5.2 Biomarker of the arsenic exposure 61 REFERENCE 62 APPENDIX 7

Emergent Roles of Circular RNAs in Metabolism and Metabolic Disorders

Circular RNAs (circRNAs) are an emerging group of long non-coding RNAs (lncRNAs) and have attracted attention again according to the progress in high-throughput sequencing in recent years. circRNAs are genome transcripts produced from pre-messenger (m)RNA regions in a specific process called “back-splicing,” which forms covalently closed continuous loops. Due to their lack of a 5’ cap and 3’ poly-adenylated tails, circRNAs are remarkably more stable than linear RNAs. Functionally, circRNAs can endogenously sponge to microRNAs, interact with RNA-binding proteins (RBPs), or translate themselves. Moreover, circRNAs can be expressed in cell type- or tissue-specific expression patterns. Therefore, they are proposed to play essential roles in fine-tuning our body’s homeostasis by regulating transcription and translation processes. Indeed, there has been accumulating emergent evidence showing that dysregulation of circRNAs can lead to metabolic disorders. This study explored the current knowledge of circRNAs that regulate molecular processes associated with glucose and lipid homeostasis and related pathogeneses of metabolic disorders. We also suggest the potential role of circRNAs as disease biomarkers and therapeutic targets

Emergent Roles of Circular RNAs in Metabolism and Metabolic Disorders

Circular RNAs (circRNAs) are an emerging group of long non-coding RNAs (lncRNAs) and have attracted attention again according to the progress in high-throughput sequencing in recent years. circRNAs are genome transcripts produced from pre-messenger (m)RNA regions in a specific process called “back-splicing,” which forms covalently closed continuous loops. Due to their lack of a 5’ cap and 3’ poly-adenylated tails, circRNAs are remarkably more stable than linear RNAs. Functionally, circRNAs can endogenously sponge to microRNAs, interact with RNA-binding proteins (RBPs), or translate themselves. Moreover, circRNAs can be expressed in cell type- or tissue-specific expression patterns. Therefore, they are proposed to play essential roles in fine-tuning our body’s homeostasis by regulating transcription and translation processes. Indeed, there has been accumulating emergent evidence showing that dysregulation of circRNAs can lead to metabolic disorders. This study explored the current knowledge of circRNAs that regulate molecular processes associated with glucose and lipid homeostasis and related pathogeneses of metabolic disorders. We also suggest the potential role of circRNAs as disease biomarkers and therapeutic targets

A Profile of Novice and Senior Nurses’ Communication Patterns during the Transition to Practice Period: An Application of the Roter Interaction Analysis System

Novice nurses’ successful transition to practice is impacted by their interactions with senior nurses. Ensuring that novice nurses are adequately supported during their transition to practice has wide-ranging and significant implications. The aim of this study is to explore the communication patterns between novice and senior nurses by applying an interaction analysis technique. Trimonthly onboarding evaluations between novice and senior nurses were recorded. The Roter Interaction Analysis System was adapted and deployed to identify communication patterns. In total, twenty-two interactions were analyzed. Senior nurses spoke more (64.5%). Task-focused exchange was predominant amongst senior (79.7%) and novice (59.5%) nurses. Senior nurses’ talk was concentrated in clusters of information-giving (45%) and advice or instructions (17.2%), while emotional expression (1.4%) and social talk (0.4%) were rare. Novice nurses’ talk was concentrated in clusters-information giving (57%) and positive talk (39.5%). The communication patterns between senior and novice nurses during the onboarding period indicate aspects of novice nurse transition that could be addressed, such as encouraging novice nurses to use these interactions to communicate more, or emphasizing the importance of social talk. These insights can be used to inform mentorship and preceptorship training to ensure that senior nurses are able to adequately support novice nurses through all parts of the transition to practice period

Salvia miltiorrhiza Extract and Individual Synthesized Component Derivatives Induce Activating-Transcription-Factor-3-Mediated Anti-Obesity Effects and Attenuate Obesity-Induced Metabolic Disorder by Suppressing C/EBPα in High-Fat-Induced Obese Mice

Pharmacological studies indicate that Salvia miltiorrhiza extract (SME) can improve cardiac and blood vessel function. However, there is limited knowledge regarding the effects (exerted through epigenetic regulation) of SME and newly derived single compounds, with the exception of tanshinone IIA and IB, on obesity-induced metabolic disorders. In this study, we administered SME or dimethyl sulfoxide (DMSO) as controls to male C57BL/J6 mice after they were fed a high-fat diet (HFD) for 4 weeks. SME treatment significantly reduced body weight, fasting plasma glucose, triglyceride levels, insulin resistance, and adipogenesis/lipogenesis gene expression in treated mice compared with controls. Transcriptome array analysis revealed that the expression of numerous transcriptional factors, including activating transcription factor 3 (ATF3) and C/EBPα homologous protein (CHOP), was significantly higher in the SME group. ST32db, a novel synthetic derivative similar in structure to compounds from S. miltiorrhiza extract, ameliorates obesity and obesity-induced metabolic syndrome in HFD-fed wild-type mice but not ATF3−/− mice. ST32db treatment of 3T3-L1 adipocytes suppresses lipogenesis/adipogenesis through the ATF3 pathway to directly inhibit C/EBPα expression and indirectly inhibit the CHOP pathway. Overall, ST32db, a single compound modified from S. miltiorrhiza extract, has anti-obesity effects through ATF3-mediated C/EBPα downregulation and the CHOP pathway. Thus, SME and ST32db may reduce obesity and diabetes in mice, indicating the potential of both SME and ST32db as therapeutic drugs for the treatment of obesity-induced metabolic syndrome

Highly Absorbent Antibacterial Hemostatic Dressing for Healing Severe Hemorrhagic Wounds

To accelerate healing of severe hemorrhagic wounds, a novel highly absorbent hemostatic dressing composed of a Tencel®/absorbent-cotton/polylactic acid nonwoven base and chitosan/nanosilver antibacterial agent was fabricated by using a nonwoven processing technique and a freeze-drying technique. This study is the first to investigate the wicking and water-absorbing properties of a nonwoven base by measuring the vertical wicking height and water absorption ratio. Moreover, blood agglutination and hemostatic second tests were conducted to evaluate the hemostatic performance of the resultant wound dressing. The blending ratio of fibers, areal weight, punching density, and fiber orientation, all significantly influenced the vertical moisture wicking property. However, only the first two parameters markedly affected the water absorption ratio. After the nonwoven base absorbed blood, scanning electron microscope (SEM) observation showed that erythrocytes were trapped between the fibrin/clot network and nonwoven fibers when coagulation pathways were activated. Prothrombin time (PT) and activated partial thromboplastin time (APTT) blood agglutination of the resultant dressing decreased to 14.34 and 50.94 s, respectively. In the femoral artery of the rate bleeding model, hemostatic time was saved by 87.2% compared with that of cotton cloth. Therefore, the resultant antibacterial wound dressing demonstrated greater water and blood absorption, as well as hemostatic performance, than the commercially available cotton cloth, especially for healing severe hemorrhagic wounds