Parallel Affinity-Based Isolation of Leukocyte Subsets Using Microfluidics: Application for Stroke Diagnosis

We report the design and performance of a polymer microfluidic device that can affinity select multiple types of biological cells simultaneously with sufficient recovery and purity to allow for the expression profiling of mRNA isolated from these cells. The microfluidic device consisted of four independent selection beds with curvilinear channels that were 25 μm wide and 80 μm deep and were modified with antibodies targeting antigens specifically expressed by two different cell types. Bifurcated and Z-configured device geometries were evaluated for cell selection. As an example of the performance of these devices, CD4+ T-cells and neutrophils were selected from whole blood as these cells are known to express genes found in stroke-related expression profiles that can be used for the diagnosis of this disease. CD4+ T-cells and neutrophils were simultaneously isolated with purities >90% using affinity-based capture in cyclic olefin copolymer (COC) devices with a processing time of ∼3 min. In addition, sufficient quantities of the cells could be recovered from a 50 μL whole blood input to allow for reverse transcription-polymerase chain reaction (RT-PCR) following cell lysis. The expression of genes from isolated T-cells and neutrophils, such as S100A9, TCRB, and FPR1, was evaluated using RT-PCR. The modification and isolation procedures demonstrated here can also be used to analyze other cell types as well where multiple subsets must be interrogated

Regulatory subunits of PKA define an axis of cellular proliferation/differentiation in ovarian cancer cells

<p>Abstract</p> <p>Background</p> <p>The regulatory subunit of cAMP-dependent protein kinase (PKA) exists in two isoforms, RI and RII, which distinguish the PKA isozymes, type I (PKA-I) and type II (PKA-II). Evidence obtained from a variety of different experimental approaches has shown that the relative levels of type I and type II PKA in cells can play a major role in determining the balance between cell growth and differentiation. In order to characterize the effect of PKA type I and type II regulatory subunits on gene transcription at a global level, the PKA regulatory subunit genes for RIα and RIIβ were stably transfected into cells of the ovarian cancer cell line (OVCAR8).</p> <p>Results</p> <p>RIα transfected cells exhibit hyper-proliferative growth and RIIβ transfected cells revert to a relatively quiescent state. Profiling by microarray revealed equally profound changes in gene expression between RIα, RIIβ, and parental OVCAR cells. Genes specifically up-regulated in RIα cells were highly enriched for pathways involved in cell growth while genes up-regulated in RIIβ cells were enriched for pathways involved in differentiation. A large group of genes (~3600) was regulated along an axis of proliferation/differentiation between RIα, parental, and RIIβ cells. RIα/wt and RIIβ/wt gene regulation was shown by two separate and distinct gene set analytical methods to be strongly cross-correlated with a generic model of cellular differentiation.</p> <p>Conclusion</p> <p>Overexpression of PKA regulatory subunits in an ovarian cancer cell line dramatically influences the cell phenotype. The proliferation phenotype is strongly correlated with recently identified clinical biomarkers predictive of poor prognosis in ovarian cancer suggesting a possible pivotal role for PKA regulation in disease progression.</p

Twenty-three unsolved problems in hydrology (UPH) – a community perspective

This paper is the outcome of a community initiative to identify major unsolved scientific problems in hydrology motivated by a need for stronger harmonisation of research efforts. The procedure involved a public consultation through on-line media, followed by two workshops through which a large number of potential science questions were collated, prioritised, and synthesised. In spite of the diversity of the participants (230 scientists in total), the process revealed much about community priorities and the state of our science: a preference for continuity in research questions rather than radical departures or redirections from past and current work. Questions remain focussed on process-based understanding of hydrological variability and causality at all space and time scales. Increased attention to environmental change drives a new emphasis on understanding how change propagates across interfaces within the hydrological system and across disciplinary boundaries. In particular, the expansion of the human footprint raises a new set of questions related to human interactions with nature and water cycle feedbacks in the context of complex water management problems. We hope that this reflection and synthesis of the 23 unsolved problems in hydrology will help guide research efforts for some years to come

Skewing X chromosome choice by modulating sense transcription across the Xist locus

The X-inactive-specific transcript (Xist) locus is a cis-acting switch that regulates X chromosome inactivation in mammals. Over recent years an important goal has been to understand how Xist is regulated at the initiation of X inactivation. Here we report the analysis of a series of targeted mutations at the 5′ end of the Xist locus. A number of these mutations were found to cause preferential inactivation, to varying degrees, of the X chromosome bearing the targeted allele in XX heterozygotes. This phenotype is similar to that seen with mutations that ablate Tsix, an antisense RNA initiated 3′ of Xist. Interestingly, each of the 5′ mutations causing nonrandom X inactivation was found to exhibit ectopic sense transcription in embryonic stem (ES) cells. The level of ectopic transcription was seen to correlate with the degree of X inactivation skewing. Conversely, targeted mutations which did not affect randomness of X inactivation also did not exhibit ectopic sense transcription. These results indicate that X chromosome choice is determined by the balance of Xist sense and antisense transcription prior to the onset of random X inactivation

Deformation and fracture of crystalline tungsten and fabrication of composite STM probes

Fracturing microscale constrictions in metallic wires, such as tungsten, platinum, or platinum-iridium, is a common fabrication method used to produce atomically sharp tips for scanning tunneling microscopy (STM), field-emission microscopy and field ion microscopy. Typically, a commercial polycrystalline drawn wire is locally thinned and then fractured by means of a dislocation slip inside the constriction. We examine a special case where a dislocation-free microscale constriction is created and fractured in a single crystal tungsten rod with a long side parallel to the [100] direction. In the absence of dislocations, vacancies become the main defects in the constriction which breaks under the tensile stress of approximately 10 GPa, which is close to the theoretical fracture strength for an ideal monocrystalline tungsten. We propose that the vacancies are removed early in the tensile test by means of deformation annealing, creating a defect-free tungsten constriction which cleaves along the W(100) plane. This approach enables fabrication of new composite STM probes which demonstrate excellent stability, atomic resolution and magnetic contrast that cannot be attained using conventional methods

Association of the M1V PRKAR1A Mutation with Primary Pigmented Nodular Adrenocortical Disease in Two Large Families

Background: Carney complex (CNC) is a familial multiple neoplasia syndrome frequently associated with primary pigmented nodular adrenocortical disease (PPNAD), a bilateral form of micronodular adrenal hyperplasia that leads to Cushing’s syndrome (CS). Germline PRKAR1A mutations cause CNC and only rarely isolated PPNAD

Does somatostatin have a role in the regulation of cortisol secretion in primary pigmented nodular adrenocortical disease (PPNAD)? A clinical and in vitro investigation

Context: Somatostatin (SST) receptors (SSTRs) are expressed in a number of tissues, including the adrenal cortex, but their role in cortisol secretion has not been well characterized. Objectives: The objective of the study was to investigate the expression of SSTRs in the adrenal cortex and cultured adrenocortical cells from primary pigmented nodular adrenocortical disease (PPNAD) tissues and to test the effect of a single injection of 100 g of the SST analog octreotide on cortisol secretion in patients with PPNAD. Setting and Design: The study was conducted at an academic research laboratory and clinical research center. Expression of SSTRs was examined in 26 PPNAD tissues and the immortalized PPNAD cell line CAR47. Ten subjects with PPNAD underwent a randomized, single-blind, crossover study of their cortisol secretion every 30 minutes over 12 hours (6:00 PM to 6:00 AM) before and after the midnight administration of octreotide 100 μg sc. Methods: SSTRs expression was investigated by quantitative PCR and immunohistochemistry. The CAR47and primary cell lines were studied in vitro. The data of the 10 patients were analyzed before and after the administration of octreotide. Results: All SSTRs, especially SSTR1-3, were expressed in PPNAD at significantly higher levels than in normal adrenal. SST was found to differentially regulate expression of its own receptors in the CAR47 cell line. However, the administration of octreotide to patients with PPNAD did not significantly affect cortisol secretion. Conclusions: SSTRs are overexpressed in PPNAD tissues in comparison with normal adrenal cortex. Octreotide did not exert any significant effect on cortisol secretion in a short clinical pilot study in a small number of patients with PPNAD, but long-acting SST analogs targeting multiple SSTRs may be worth investigating in this condition

Parallel Affinity-Based Isolation of Leukocyte Subsets Using Microfluidics: Application for Stroke Diagnosis

We report the design and performance of a polymer microfluidic device that can affinity select multiple types of biological cells simultaneously with sufficient recovery and purity to allow for the expression profiling of mRNA isolated from these cells. The microfluidic device consisted of four independent selection beds with curvilinear channels that were 25 μm wide and 80 μm deep and were modified with antibodies targeting antigens specifically expressed by two different cell types. Bifurcated and Z-configured device geometries were evaluated for cell selection. As an example of the performance of these devices, CD4+ T-cells and neutrophils were selected from whole blood as these cells are known to express genes found in stroke-related expression profiles that can be used for the diagnosis of this disease. CD4+ T-cells and neutrophils were simultaneously isolated with purities >90% using affinity-based capture in cyclic olefin copolymer (COC) devices with a processing time of ∼3 min. In addition, sufficient quantities of the cells could be recovered from a 50 μL whole blood input to allow for reverse transcription-polymerase chain reaction (RT-PCR) following cell lysis. The expression of genes from isolated T-cells and neutrophils, such as <i>S100A9</i>, <i>TCRB</i>, and <i>FPR1</i>, was evaluated using RT-PCR. The modification and isolation procedures demonstrated here can also be used to analyze other cell types as well where multiple subsets must be interrogated