Flow-through immunomagnetic separation system for waterborne pathogen isolation and detection: application to Giardia and Cryptosporidium cell isolation.

Simultaneous sample washing and concentration of two waterborne pathogen samples were demonstrated using a rotational magnetic system under continuous flow conditions. The rotation of periodically arranged small permanent magnets close to a fluidic channel carrying magnetic particle suspension allows the trapping and release of particles along the fluidic channel in a periodic manner. Each trapping and release event resembles one washing cycle. The performance of the magnetic separation system (MSS) was evaluated in order to test its functionality to isolate magnetic-labelled protozoan cells from filtered, concentrated tap water, secondary effluent water, and purified water. Experimental protocols described in US Environmental Protection Agency method 1623 which rely on the use of a magnetic particle concentrator, were applied to test and compare our continuous flow cell separation system to the standard magnetic bead-based isolation instruments. The recovery efficiencies for Giardia cysts using the magnetic tube holder and our magnetic separation system were 90.5% and 90.1%, respectively, from a tap water matrix and about 31% and 18.5%, respectively, from a spiked secondary effluent matrix. The recovery efficiencies for Cryptosporidium cells using the magnetic tube holder and our magnetic separation system were 90% and 83.3%, respectively, from a tap water matrix and about 38% and 36%, respectively, from a spiked secondary effluent matrix. Recoveries from all matrices with the continuous flow system were typically higher in glass tubing conduits than in molded plastic conduits.

A self-contained fully-enclosed microfluidic cartridge for lab on a chip.

We describe a self-contained fully-enclosed cartridge for lab-on-a-chip applications where sample and reagents can be applied sequentially as is performed in a heterogeneous immunoassay, or nucleic acid extraction. Both the self-contained and fully-enclosed features of the cartridge are sought to ensure its safe use in the field by unskilled staff. Simplicity in cartridge design and operation is obtained via adopting a valveless concept whereby reagents are stored and used in the form of liquid plugs isolated by air spacers around a fluidic loop. Functional components integrated in the loop include a microfluidic chip specific to the target application, a novel peristaltic pump to displace the liquid plugs, and a pair of removable tubing segments where one is used to introduce biological sample and while the other is to collect eluant. The novel pump is fabricated through soft-lithography technique and works by pinching a planar channel under stainless-steel ball bearings that have been magnetically loaded. The utility of the cartridge is demonstrated for automated extraction and purification of nucleic acids (DNA) from a cell lysate on a battery-operated portable system. The cartridge shown here can be further extended to sample-in-answer-out diagnostic tests.

Developmental expression pattern and DNA-binding properties of the zinc finger transcription factor Krox-26.

The development of teeth through epithelial-mesenchymal interactions is mediated on a molecular level through the iterative and reciprocal action of secreted growth factors and responsive transcription factors. Although zinc finger transcription factors constitute by far the largest class of transcriptional regulators in mammals, little is known about their specific role in the regulation of tissue formation. The C2H2 zinc finger transcription factor Krox-26 is transiently expressed during organ formation, most prominently at sites of early tooth development and subsequently in secretory stage ameloblasts. The objective of this study was to determine the developmental expression pattern of Krox-26 during mouse embryogenesis and to determine its preferred DNA-binding site. Krox-26 protein expression could be detected in multiple tissues, most prominently in developing craniofacial structures. A target detection assay using recombinant Krox-26 protein identified the sequence CAATG as the preferred Krox-26 DNA-binding site. These results suggest that Krox-26 may regulate the expression of target genes through CCAAT box-related sequences in multiple tissues.

Krox-26 is a novel C2H2 zinc finger transcription factor expressed in developing dental and osteogenic tissues.

The development of teeth through epithelial-mesenchymal interactions is mediated on a molecular level by a network of secreted growth factors and responsive transcription factors. Although zinc finger transcription factors constitute by far the largest class of transcriptional regulators with an estimated number of approximately 1000 genes present in mammals [14], little is known about their role in the regulation of mineralized tissue formation. A fragment (Y150) of the novel C2H2 zinc finger transcription factor Krox-26 has initially been isolated from highly proliferative dental pulp tissue in rats [19]. The objective of this study was to clone the full-length cDNA sequence of the murine homologue and to determine its mRNA and protein expression pattern during mouse embryonic development. Mouse Krox-26 contains five C2H2 zinc finger repeats. Its expression was found to be most prominent in the developing craniofacial bones and dental organs. These results suggest Krox-26 as a potential regulator of gene transcription during the development of teeth and the craniofacial skeleton.

Temporal and spatial expression of a novel zinc finger transcription factor, AJ18, in developing murine skeletal tissues.

Bone morphogenetic proteins (BMPs) are characterized by their ability to induce osteoblastic differentiation. However, the mechanism of osteo-induction by BMPs has yet to be determined. Using differential display we previously identified AJ18, a zinc finger transcription factor, as an immediate-early response gene to BMP-7. AJ18 was shown to bind to the osteoblast-specific element2 (OSE2) and to modulate transactivation by Runx2, a master gene in osteoblastic differentiation. Here we describe the temporal and spatial expression of AJ18 in developing mouse tissues. AJ18 mRNA expression was observed in most tissues, except liver, and was generally highest early in embryonic development, decreasing markedly after parturition. Consistent with immunohistochemical analysis, AJ18 mRNA expression was highest in the brain, kidney, and bone of 17 dpc (days post coitum) embryos. In endochondral bones of embryonic and 4-week-old mice, immunostaining for AJ18 was strong in the nuclei of proliferating and pre-hypertrophic chondrocytes, and osteoblasts, whereas there was low or no staining in hypertrophic chondrocytes. In teeth of embryonic and 4-week-old mice, nuclear staining was observed in precursor and mature ameloblasts, odontoblasts, and cementoblasts, respectively. In addition, in 4-week-old mice staining of AJ18 was observed within alveolar bone cells and periodontal ligament cells. In general, the spatial expression of AJ18 in skeletal and non-skeletal tissues of mouse embryos showed striking similarity to the expression of BMP-7 mRNA. Therefore, the expression of AJ18 is consistent with its perceived role as a transcriptional factor that regulates developmental processes downstream of BMP-7.