Application of a novel rectangular filtering microfluidic device for microfilarial detection.

The rectangular filtering microfluidic chip was invented using microfluidics device fabrication technology and can separate living microfilariae from blood samples without a syringe pump. The diagnostic results are highly effective. The device is based on the principle of separating millions of blood cells from microfilariae using a rectangular filter structure. It disperses fluid evenly into the flow-passage channel, and its rectangular filter structure is the key to success in reducing the pressure and separating blood cells from microfilariae effectively. The flow rate and blood cell concentration were optimized in our study. The chip is intended to be a point-of-care device that can reduce the use of superfluous instrumentation in the field. The technology is designed to be rapid, accurate, and easy-to-use for all users, especially those in remote areas.

Immunohistochemical and molecular profiling of CD 117, Oct-4, and Sox-2 in canine cutaneous mast cell tumor of the crossbred dogs in Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.

BACKGROUND AND AIM: CD 117 (c-KIT) internal tandem duplication (ITD), octamer-binding transcription factor 4 (Oct-4), and sex-determining region Y-box 2 (Sox-2) may govern the oncogenicity and aggressiveness of canine cutaneous mast cell tumor (MCT) in the crossbred dogs. Thus, a comprehension of this matter may help us establishing a novel platform to treat the disease in those dogs. However, evidence has lacked so far. Thus, this study aimed to survey CD 117 ITD, Oct-4, and Sox-2 expressions and their relations to the 2-tier grading in a group of Thai crossbreed dogs. The study was done using polymerase chain reaction (PCR), Reverse transcription PCR (RT-PCR), and immunohistochemistry. MATERIALS AND METHODS: Thirty-three MCT specimens graded by the 2-tier histopathology grading were collected from the crossbred and purebred dogs. CD 117 ITD was detected by conventional PCR and immunohistochemistry. While, Oct-4 and Sox-2 expression levels were determined at the protein and mRNA levels by immunohistochemistry and RT-PCR, respectively. The expression magnitude of each parameter was then related to the grades and breeds. RESULTS: About 60.61% of specimens were low grade, while 39.39% were high grade. CD 117 ITD was not detected in all specimens. A significant increase of Oct-4 expression was found in the high-grade, crossbred dogs. Meanwhile, Sox-2 expressions were increased both in the purebred and crossbred dogs with high-grade MCT. CONCLUSION: The study finding has indicated that the level of Sox-2 expression may be a useful tumorigenic and prognostic biomarker because it correlates to the 2-tier grades but not dog breeds.

A Potential Application of Triangular Microwells to Entrap Single Cancer Cells: A Canine Cutaneous Mast Cell Tumor Model.

Cellular heterogeneity is a major hindrance, leading to the misunderstanding of dynamic cell biology. However, single cell analysis (SCA) has been used as a practical means to overcome this drawback. Many contemporary methodologies are available for single cell analysis; among these, microfluidics is the most attractive and effective technology, due to its advantages of low-volume specimen consumption, label-free evaluation, and real-time monitoring, among others. In this paper, a conceptual application for microfluidic single cell analysis for veterinary research is presented. A microfluidic device is fabricated with an elastomer substrate, polydimethylsiloxane (PDMS), under standard soft lithography. The performance of the microdevice is high-throughput, sensitive, and user-friendly. A total of 53.1% of the triangular microwells were able to trap single canine cutaneous mast cell tumor (MCT) cells. Of these, 38.82% were single cell entrapments, while 14.34% were multiple cell entrapments. The ratio of single-to-multiple cell trapping was high, at 2.7:1. In addition, 80.5% of the trapped cells were viable, indicating that the system was non-lethal. OCT4A-immunofluorescence combined with the proposed system can assess OCT4A expression in trapped single cells more precisely than OCT4A-immunohistochemistry. Therefore, the results suggest that microfluidic single cell analysis could potentially reduce the impact of cellular heterogeneity.

Investigation of Leukocyte Viability and Damage in Spiral Microchannel and Contraction-Expansion Array.

Inertial separation techniques in a microfluidic system have been widely employed in the field of medical diagnosis for a long time. Despite no requirement of external forces, it requires strong hydrodynamic forces that could potentially cause cell damage or loss during the separation process. This might lead to the wrong interpretation of laboratory results since the change of structures and functional characteristics of cells due to the hydrodynamic forces that occur are not taken into account. Therefore, it is important to investigate the cell viability and damage along with the separation efficacy of the device in the design process. In this study, two inertial separation techniques-spiral microchannel and contraction-expansion array (CEA)-were examined to evaluate cell viability, morphology and intracellular structures using a trypan blue assay (TB), Scanning Electron Microscopy (SEM) and Wright-Giemsa stain. We discovered that cell loss was not significantly found in a feeding system, i.e., syringe, needle and tube, but mostly occurred in the inertial separation devices while the change of cell morphology and intracellular structures were found in the feeding system and inertial separation devices. Furthermore, percentage of cell loss was not significant in both devices (7-10%). However, the change of cell morphology was considerably increased (30%) in spiral microchannel (shear stress dominated) rather than in CEA (12%). In contrast, the disruption of intracellular structures was increased (14%) in CEA (extensional and shear stress dominated equally) rather than spiral microchannel (2%). In these experiments, leukocytes of canine were used as samples because their sizes are varied in a range between 7-12 µm, and they are commonly used as a biomarker in many clinical and medical applications.

The Viability of Single Cancer Cells after Exposure to Hydrodynamic Shear Stresses in a Spiral Microchannel: A Canine Cutaneous Mast Cell Tumor Model.

Our laboratory has the fundamental responsibility to study cancer stem cells (CSC) in various models of human and animal neoplasms. However, the major impediments that spike our accomplishment are the lack of universal biomarkers and cellular heterogeneity. To cope with these restrictions, we have tried to apply the concept of single cell analysis, which has hitherto been recommended throughout the world as an imperative solution pack for resolving such dilemmas. Accordingly, our first step was to utilize a predesigned spiral microchannel fabricated by our laboratory to perform size-based single cell separation using mast cell tumor (MCT) cells as a model. However, the impact of hydrodynamic shear stresses (HSS) on mechanical cell injury and viability in a spiral microchannel has not been fully investigated so far. Intuitively, our computational fluid dynamics (CFD) simulation has strongly revealed the formations of fluid shear stress (FSS) and extensional fluid stress (EFS) in the sorting system. The panel of biomedical assays has also disclosed cell degeneration and necrosis in the model. Therefore, we have herein reported the combinatorically detrimental effect of FSS and EFS on the viability of MCT cells after sorting in our spiral microchannel, with discussion on the possibly pathogenic mechanisms of HSS-induced cell injury in the study model.

A novel immune-type receptor of Japanese flounder (Paralichthys olivaceus) is expressed in both T and B lymphocytes.

A cDNA encoding a novel immune-type receptor (NITR) of Japanese flounder, Paralichthys olivaceus, was isolated from a kidney cDNA library. The cDNA encoded 357 amino acid residues. The amino acid sequence identities between Japanese flounder NITR1 (poNITR1) and previously reported fish NITRs were approximately 30%-40%. The poNITR1 consisted of two extracellular immunoglobulin (Ig) domains (V and V/C2), a transmembrane domain, an immunoreceptor tyrosine-based inhibitor motif (ITIM) and an ITIM-like motif (itim) in the cytoplasmic region. Five potential N-link glycosylation sites (N-X-S/T) are present in the extracellular Ig domains. Seven cysteine (Cys) residues, which are conserved in previously reported NITRs were observed in the extracellular domain of poNITR1. The poNITR1 gene is composed of five exons and four introns spanning approximately 3.4kb. The poNITR1 transcripts were mainly detected in gill, head kidney, trunk kidney, intestine, while it was weakly detected in heart, liver, muscle, peripheral blood leucocytes, skin, spleen and stomach. However, poNITR1 gene expression was not detected in muscle or ovary. NITR gene expression was not induced by LPS or poly I:C. In situ hybridization revealed that Japanese flounder NITR is expressed in both TCR-alpha- and IgM-presenting cells.