Miniaturised nucleic acid analysis systems

The design and implementation of miniaturised systems for analysis of nucleic acids from various biological samples has undergone extensive development. Several advances have been made particularly with the integration of nucleic acid amplification and detection, where amplification is most often polymerase chain reaction (PCR). Sample preparation remains a major obstacle for achieving a quantitative analysis employing full miniaturised integration. Miniaturised devices for nucleic acid sample preparation, amplification and detection have to be further developed in order to achieve a fully integrated system, which ultimately can perform single cells genomic analysis with sample-in-answer-out ability. In this thesis, three miniaturised systems have been presented, which can be used for purification and preconcentration of DNA, pre-amplification and long-term storage of DNA, and amplification with real-time detection of DNA, respectively. The first miniaturised system applies isotachophoresis for pretreatment of DNA, where the DNA sample can be purified and concentrated using a discontinuous electrolyte system. Both qualitative and quantitative information can be acquired simultaneously. The second miniaturised system employs simple isothermal multiple displacement amplification, (MDA) for whole genome amplification (WGA) of human genomic DNA. The miniaturised WGA process showed a high efficiency of 95.8%, and the fidelity of the amplified products is extremely high as suggested by single-nucleotide polymorphisms analysis. For the last system, we developed a bidirectional shunting PCR microdevice equipped with real-time fluorescence detection, which allows higher flexibility and fast thermocycling by combining both advantages of stationary PCR and continuous-flow PCR. Real-time monitoring of RNase P PCR amplification from lower concentration human genomic DNA down to ~24 copy numbers or 12 cells was achieved. The three systems described in this thesis can be readily adapted to current reported miniaturised platforms. Such a fully integrated device capable of quantitative nucleic acid analysis remains an enigma, and with further development will represent significant importance for the development of point-of-care device.Das Design und die Implementierung miniaturisierter Systeme für die Analyse von Nukleinsäuren aus verschiedenen biologischen Proben haben eine beträchtliche Entwicklung erlebt. Fortschritte wurden insbesondere bei der Integration der Nukleinsäure-Vervielfältigung und -Detektion gemacht, wobei die Vervielfältigung meistens auf der Polymerase-Kettenreaktion (Polymerase Chain Reaction, PCR) beruht. Die Probenvorbereitung bleibt ein Haupthemmnis bei dem Versuch, eine quantitative Analyse mit vollständig miniaturisierten Systemen zu verwirklichen. Miniaturisierte Geräte für die Probenvorbereitung, Vervielfältigung und Detektion von Nukleinsäuren müssen weiter entwickelt werden, um ein vollständig integriertes System zu verwirklichen, das letztendlich in der Lage ist, die Genomanalyse einzelner Zellen mit "sample-in-answer-out"-Fähigkeit durchzuführen. In dieser Arbeit werden drei miniaturisierte Systeme präsentiert, die jeweils für die DNA-Aufreinigung und -Vorkonzentrierung, deren Vor-Vervielfältigung und Langzeit-Speicherung bzw. der Vervielfältigung mit Echtzeitdetektion von DNA verwendet werden können. Das erste miniaturisierte System nutzt die Isotachophorese zur Vorbehandlung der DNA, bei der die DNA-Probe in einem diskontinuierlichen Elektrolytsystem gereinigt und aufkonzentriert werden kann. Dabei können sowohl qualitative als auch quantitative Informationen simultan aufgenommen werden. Das zweite miniaturisierte System verwendet die simple Methode der isothermischen Multiplen Displacement Amplification (MDA) für die Vervielfältigung des gesamten Genoms (Whole Genome Amplification, WGA) der humanen, genomischen DNA. Der miniaturisierte WGA-Prozess zeigte eine hohe Effizienz von 95,8% und die Wiedergabetreue des vervielfachten Produkts ist extrem hoch, was durch die Ergebnisse einer Single Nucleotide Polymorphism (SNP) Analyse angedeutet wurde. Für des letzte System entwickelten wir ein kleines bidirektionales shunting-PCR-Instrument, in dem die injizierte DNA durch eine temperierte Zone mehrfach hin und her verschoben wird. Ausgestattet mit einem Fluoreszens Detektor, erreicht man eine höhere Flexibilität und schelle Temperaturzyklen, und kombiniert so die Vorteile der stationären PCR und der Durchfluss-PCR. Eine Echtzeitdetektion der RNase-P-PCR-Vervielfältigung von niedrig konzentrierter, humaner genomischer DNA mit einem Minimum von ~24 Kopien oder 12 Zellen wurde erreicht. Die drei in dieser Arbeit beschriebenen Systeme können direkt an aktuelle miniaturisierte Aufbauten angepasst werden. Ein solches vollständig integriertes, zur quantitativen Nukleinsäure-Analyse fähiges Gerät bleibt ein Mysterium, und zusammen mit weiteren Verbesserungen wäre es von großer Bedeutung für die Entwicklung von point-of-care Geräten

Microfluidic DNA amplification - a review

The application of microfluidic devices for DNA amplification has recently been extensively studied. Here, we review the important development of microfluidic polymerase chain reaction (PCR) devices and discuss the underlying physical principles for the optimal design and operation of the device. In particular, we focus on continuous-flow microfluidic PCR on-chip, which can be readily implemented as an integrated function of a micro-total-analysis system. To overcome sample carryover contamination and surface adsorption associated with microfluidic PCR, microdroplet technology has recently been utilized to perform PCR in droplets, which can eliminate the synthesis of short chimeric products, shorten thermal-cycling time, and offers great potential for single DNA molecule and single-cell amplification. The work on chip-based PCR in droplets is highlighted

Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the most common type of leukemia. The Cancer Genome Atlas Research Network has demonstrated the increasing genomic complexity of acute myeloid leukemia (AML). In addition, the network has facilitated our understanding of the molecular events leading to this deadly form of malignancy for which the prognosis has not improved over past decades. AML is a highly heterogeneous disease, and cytogenetics and molecular analysis of the various chromosome aberrations including deletions, duplications, aneuploidy, balanced reciprocal translocations and fusion of transcription factor genes and tyrosine kinases has led to better understanding and identification of subgroups of AML with different prognoses. Furthermore, molecular classification based on mRNA expression profiling has facilitated identification of novel subclasses and defined high-, poor-risk AML based on specific molecular signatures. However, despite increased understanding of AML genetics, the outcome for AML patients whose number is likely to rise as the population ages, has not changed significantly. Until it does, further investigation of the genomic complexity of the disease and advances in drug development are needed. In this review, leading AML clinicians and research investigators provide an up-to-date understanding of the molecular biology of the disease addressing advances in diagnosis, classification, prognostication and therapeutic strategies that may have significant promise and impact on overall patient survival

Lab-on-PCB Devices

Lab-on-PCB devices can be considered an emerging technology. In fact, most of the contributions have been published during the last 5 years. It is mainly focussed on both biomedical and electronic applications. The book includes an interesting guide for using the different layers of the Printed Circuit Boards for developing new devices; guidelines for fabricating PCB-based electrochemical biosensors, and an overview of fluid manipulation devices fabricated using Printed Circuit Boards. In addition, current PCB-based devices are reported, and studies for several aspects of research and development of lab-on-PCB devices are described

Il-15/il-15rα signalling and synaptic transmission: a crosstalk between the immune and the nervous system?

Immune and nervous system have been traditionally considered separately, but from ‘90s many studies had unraveled the deep interconnection and interdependence between these two systems, enough to coin the term “neuroimmune system” to define this relationship. While it was well known that central nervous system (CNS) actively communicates with the immune system to control immune responses both centrally and peripherally, the opposite action was just recently discovered. Related to the role of immune system in defending and react, the interactions between immune system and CNS have been classically studied in contexts of neuroinflammation such as trauma, injury and disease [1] [2]. Recent evidences about the neuroinflammatory process in non-pathological conditions and the discovery of the important involvement of adaptive immune system in healthy brain development and activity [3], have opened many questions about physiological neuroimmune cross-talk. In this view, the cytokine network, well known to operate in a bidirectional way affecting both immune and nervous system, has a pivotal role in neuroimmune cross-talk [4]. Traditionally seen as immunomodulators, in the last years has been evident that cytokines are also potent neuromodulators [5]. In the complex cytokine system, interleukin 15 (IL-15) is considered a bridge between adaptive and innate immune system and it is one of the first upregulated cytokines in neuroinflammation [6]. It has many bioregulatory roles which range from those of modulator of selected adaptive immune responses [7] [8] and central player in the development and homeostasis of several immunocyte populations [9] to those of a potent, general inhibitor of apoptosis in multiple systems [9]. Interestingly, has been shown that IL-15 and IL-15Rα deletions affect memory and neurotransmitters concentration suggesting a major role of this signalling in cerebral functions which cannot be compensated during the development [10] [11] [12]. IL-15Rα KO mice, in particular, show decreased retention of spatial memory and contextual fear, both related to hippocampus-dependent memory, and alteration in GABA concentration. Their hippocampal ultrastructure is, however, well preserved, suggesting that the modulatory changes may involve neural plasticity even if the exact role of IL15 in modulating neurotransmission has not been investigated so far. The understandings about the mechanism by which IL-15/IL-15Rα system affect the synaptic transmission may be useful to get insight into the mechanisms of cross talk between the immune and the nervous system and eventually to develop strategies to treat pathologies whose symptoms are memory impairments and neuroinflammation

Physical and chemical quality, biodiversity, and thermodynamic prediction of adhesion of bacterial isolates from a water purification system: a case study

The objective of this study was to evaluate the quality of water purification system and identify the bacteria this system, predict bacterial adherence according to the hydrophobicity of these microorganisms and of the polypropylene distribution loop for purified water. The assessment of drinking water that supplies the purification system allowed good-quality physical, chemical, and microbiological specifications. The physicochemical specifications of the distributed purified water were approved, but the heterotrophic bacteria count was higher than allowed (>;2 log CFU mL-1).The sanitation of the storage tank with chlorine decreased the number of bacteria adhered to the surface (4.34 cycles log). By sequencing of the 16SrDNA genes, six species of bacteria were identified. The contact angle was determined and polypropylene surface and all bacteria were considered to be hydrophilic, and adhesion was thermodynamically unfavorable. This case study showed the importance of monitoring the water quality in the purified water systems and the importance of sanitization with chemical agents. The count of heterotrophic bacteria on the polypropylene surface was consistent with the predicted thermodynamics results because the number of adhered cells reached approximate values of 5 log CFU cm-2

Population Structure and Genetic Diversity of the Boll Weevil (Coleoptera: Curculionidae) on Gossypium in North America

Although the boll weevil, Anthonomus grandis grandis Boheman (Coleoptera: Curculionidae), is a devastating pest in the United States and Mexico, its population structure and genetic diversity in Mexico on wild and cultivated cotton hosts (genus Gossypium) is poorly understood. Past studies using morphology, host use, and distribution records suggest that A.grandis grandis comprises three forms with host-associated characteristics: the southeastern form (from domesticated Gossypium hirsutum L., southeastern United States and northeastern Mexico), the thurberia form (from Gossypium thurberi Todaro, Arizona and northwestern Mexico), and the Mexican form (from multiple Gossypium species and other malvaceous plant genera in the remainder of Mexico and Central America). However, the phylogenetic relationships, host preferences, and distributions of these forms are not completely understood. An alternative hypothesis of an eastern and western form of the boll weevil is suggested by the suspected phylogeographic range expansion from an ancestral distribution in the tropics northward along both Mexican coasts, culminating in the maximally contrasting phenotypes observed in the northeastern and northwestern arms of the current distribution. In this study, we sequenced one mitochondrial and four nuclear genes to gain insight into the evolutionary relationships among the putative forms and their distributions on wild and domesticated cotton hosts. Using models of evolution, we compared the three-form to the two-form classification and to two alternative classifications that incorporate geography and host use traits. The genetic data at most loci provide stronger support for the two-form than the three-form hypothesis, with an eastern and western group separated by the Sierra Madre Occidental mountain range. They do not support separate taxonomic status for boll weevils developing onG. thurberi

Bisphenol A Exposure, Adipogenic Mechanism and Effect on Childhood Adiposity

Bisphenol A (BPA) is a common component in plastic consumer products and epoxy resin linings. Initially developed in the 1930s-40s as a synthetic hormone treatment, it is now widely considered an endocrine disrupting chemical (EDC). A growing body of epidemiological literature suggests that ubiquitous exposures to BPA may be contributing to the global epidemic of obesity, with children a particularly vulnerable population. Obesity in children, defined by a body mass index (BMI) greater than or equal to the 95th percentile for age and sex, is an epidemic of great concern in the United States. As with other chemicals, the prenatal and early life period are critical windows of exposure to BPA; however, the mechanism by which BPA may influence the development of body size in children remains unclear. Experimental studies have found that BPA influences adipogenesis in both murine and adult human preadipocyte cell lines and BPA is hypothesized to play a role in enhancing adipogenic regulation by nuclear receptors such as peroxisome proliferator-activated receptor gamma (PPARγ). While the timeline of the processes involved in adipogenesis in humans is not universally agreed upon, it is accepted that PPARγ is highly expressed in adipose tissue and considered to be the master regulator of adipogenesis. To answer the question of both timing and developmental origin of BPA effects on adipogensis, we employed both an epidemiological approach, and experimental methodologies using primordial cell lines, mesenchymal stem cells (MSCs). Our study characterizes early life exposures to BPA, explores the adipogenic mechanism of BPA in human MSCs via cellular morphometrics and PPARγ gene expression, and identifies associations between early life exposure to BPA and childhood obesity and adiposity. For our epidemiological assessments, we studied a birth cohort of African American and Dominican mother and child dyads in New York City. BPA was measured in spot urine samples collected during pregnancy and at child ages 3, 5, and 7 years, from mothers and children (n=568 dyads) in the Columbia Center for Children’s Environmental Health (CCCEH). We compared BPA concentrations across paired samples. We explored relationships between BPA and the class of phthalate chemicals, another common plasticizer. BPA was detected in nearly all urine samples from prenatal third trimester and childhood ages 3 years, 5 years and 7 years. Prenatal urinary BPA concentrations were significantly lower than postnatal urinary BPA concentrations (p<0.001). BPA and phthalate metabolites were correlated prenatally and at 3, 5, and 7 years (all p-values < 0.02). BPA concentrations were correlated with phthalate metabolite concentrations prenatally, and at 3, 5 and 7 years(all p-values < 0.05). Geometric means of BPA were higher among African Americans than among Dominicans in prenatal (p<0.01), 5 year (p<0.001) and 7 year (p=0.02) samples. Postnatal BPA concentrations were significantly higher among children with mothers who had never marrried marital status and were significantly higher in summer than in all other seasons (all p-values < 0.05). These findings reveal widespread BPA exposure in an inner-city minority population. Our in vitro experiment was a feasibility study which sought to determine whether exposure to BPA by human umbilical cord mesenchymal stem cells (HUMSC) induces morphological changes and PPARγ gene expression during adipogenesis. An anonymous sample of n=18 umbilical cords was collected at delivery from mothers registered at New York-Presbyterian Sloane Hospital for Women and New York-Presbyterian Allen Hospital in New York City. HUMSCs were harvested from umbilical cords using an adhesion technique. HUMSCs were then induced in culture to differentiate into adipocytes using: a standard differentiation induction mix medium, a negative vehicle control medium, a positive control medium and experimental control media. Differences in cell surface area and cell count in all cultures were assessed using ImageJ software (version 1.49n, 2014). Gene expression of PPARγ in all cultures was evaluated by RT-PCR. Cell morphometric results were based on 11,676 cells from 3 umbilical cord samples. PPARγ1 and PPARγ2 gene expression was assessed during differntiation phase and early terminal phase adipogenesis (0 to 72 hours). Cell morphometrics were assessed during middle to late terminal phase adipogenesis (days 14 and 21). No differences in cell count were observed for experimental conditions compared to standard induction medium. A significant decrease in surface area was seen in cells exposed to 100 μM concentration of BPA as compared to exposure to standard induction medium at day 14 (t=-37.02 p=0.001). Differences in cell surface area were not observed at day 21. A twofold increased expression of PPARγ1 was observed in cells exposed to 10 μM concentration of BPA by 72 hours of adipogenic induction which was higher than the increase in expression observed for cells exposed to the positive control induction medium containing 10 μM concentration of rosiglitazone. All induction media conditions had negligible effects on PPARγ2 expression. As BPA increases expression of PPARγ1 in HUMSCs during the transition into the early terminal differentiation phase of adipogenesis, HUMSCs may be an approximate target tissue for evaluating BPA effects in adipogenesis. Finally, using a longitudinal research design, we analyzed the possible effect of prenatal and postnatal BPA exposures, measured in urine, on childhood anthropometric outcome measures. Participants in the CCCEH have been followed since the third trimester of pregnancy, providing us with anthropometric data on children from birth through the age of seven years. Available anthropometric outcome measures include body mass index z-scores (BMIZ) at 5 and 7 years, as well as fat mass index (FMI), percent body fat (%BF), and waist circumference (WC) at 7 years. Prenatal urinary BPA concentrations were positively associated with child age 7 FMI (beta=0.31 kg/m2, p-value=0.04, [95%CI 0.01, 0.60]), %BF (beta=0.79, p-value=0.04, [95%CI 0.03, 1.55]), and WC (beta=1.29 cm, p-value=0.01, [95%CI 0.29, 2.30]). Child urinary BPA concentrations were not associated with childhood BMI or other anthropometric outcomes. As the prenatal exposures were associated with childhood measures of adiposity, prenatal BPA exposure may have an effect on adiposity as children age that cannot be determined by the use of BMI alone. Our results suggest BPA may contribute to the developmental origins of obesity and adiposity