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Respiratory syncytial virus (RSV) causes severe infections in infants, immunocompromised or elderly individuals resulting in annual epidemics of respiratory disease. Currently, limited clinical surveillance and the lack of predictable seasonal dynamics limit the public health response. Wastewater-based epidemiology (WBE) has recently been used globally as a key metric in determining prevalence of severe acute respiratory syndrome coronavirus 2 in the community, but its application to other respiratory viruses is limited. In this study, we present an integrated genomic WBE approach, applying reverse-transcription quantitative polymerase chain reaction and partial G-gene sequencing to track RSV levels and variants in the community. We report increasing detection of RSV in wastewater concomitant with increasing numbers of positive clinical cases. Analysis of wastewater-derived RSV sequences permitted identification of distinct circulating lineages within and between seasons. Altogether, our genomic WBE platform has the potential to complement ongoing global surveillance and aid the management of RSV by informing the timely deployment of pharmaceutical and nonpharmaceutical interventions., Competing Interests: Potential conflicts of interest . All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Many pathological conditions are predominantly associated with oxidative stress, arising from reactive oxygen species (ROS); therefore, the modulation of redox activities has been a key strategy to restore normal tissue functions. Current approaches involve establishing a favorable cellular redox environment through the administration of therapeutic drugs and redox-active nanomaterials (RANs). In particular, RANs not only provide a stable and reliable means of therapeutic delivery but also possess the capacity to finely tune various interconnected components, including radicals, enzymes, proteins, transcription factors, and metabolites. Here, we discuss the roles that engineered RANs play in a spectrum of pathological conditions, such as cancer, neurodegenerative diseases, infections, and inflammation. We visualize the dual functions of RANs as both generator and scavenger of ROS, emphasizing their profound impact on diverse cellular functions. The focus of this review is solely on inorganic redox-active nanomaterials (inorganic RANs). Additionally, we deliberate on the challenges associated with current RANs-based approaches and propose potential research directions for their future clinical translation.

"Flash heating" that transiently generates high temperatures above 1000 °C has great potential in synthesizing new materials with unprecedently properties. Up to now, the realization of "flash heating" still relies on the external power, which requires sophisticated setups for vast energy input. In this study, a mechanochemically triggered, self-powered flash heating approach is proposed by harnessing the enthalpy from chemical reactions themselves. Through a model reaction between Mg 3 N 2 /carbon and P 2 O 5 , it is demonstrated that this self-powered flash heating is controllable and compatible with conventional devices. Benefit from the self-powered flash heating, the resulting product has a nanoporous structure with a uniform distribution of phosphorus (P) nanoparticles in carbon (C) nanobowls with strong P─-C bonds. Consequently, the P/C composite demonstrates remarkable energy storage performance in lithium-ion batteries, including high capacity (1417 mAh g -1 at 0.2 A g -1 ), robust cyclic stability (935 mAh g -1 at 2 A g -1 after 800 cycles, 91.6% retention), high-rate capability (739 mAh g -1 at 20 A g -1 ), high loading performance (3.6 mAh cm -2 after 100 cycles), and full cell cyclic stability (90% retention after 100 cycles). This work broadens the flash heating concept and can potentially find application in various fields., (© 2024 Wiley‐VCH GmbH.)

Emerging regenerative cell therapies for alveolar bone loss have begun to explore the use of cell laden hydrogels for minimally invasive surgery to treat small and spatially complex maxilla-oral defects. However, the oral cavity presents a unique and challenging environment for in vivo bone tissue engineering, exhibiting both hard and soft periodontal tissue as well as acting as key biocenosis for many distinct microbial communities that interact with both the external environment and internal body systems, which will impact on cell fate and subsequent treatment efficacy. Herein, we design and bioprint a facile 3D in vitro model of a human dentine interface to probe the effect of the dentine surface on human mesenchymal stem cells (hMSCs) encapsulated in a microporous hydrogel bioink. We demonstrate that the dentine substrate induces osteogenic differentiation of encapsulated hMSCs, and that both dentine and β-tricalcium phosphate substrates stimulate extracellular matrix production and maturation at the gel-media interface, which is distal to the gel-substrate interface. Our findings demonstrate the potential for long-range effects on stem cells by mineralized surfaces during bone tissue engineering and provide a framework for the rapid development of 3D dentine-bone interface models., (© 2024. The Author(s).)

Nanomaterial composition, morphology, and mechanical performance are critical parameters for tissue engineering. Within this rapidly expanding space, tubular nanomaterials (TNs), including carbon nanotubes (CNTs), titanium oxide nanotubes (TNTs), halloysite nanotubes (HNTs), silica nanotubes (SiNTs), and hydroxyapatite nanotubes (HANTs) have shown significant potential across a broad range of applications due to their high surface area, versatile surface chemistry, well-defined mechanical properties, excellent biocompatibility, and monodispersity. These include drug delivery vectors, imaging contrast agents, and scaffolds for bone tissue engineering. This review is centered on the recent developments in TN-based biomaterials for structural tissue engineering, with a strong focus on bone tissue regeneration. It includes a detailed literature review on TN-based orthopedic coatings for metallic implants and composite scaffolds to enhance in vivo bone regeneration.

A series of metal silicates, NaMSi 10 Ox (M = Cu, Mn and Ni), were prepared by in situ doping of metals into mesoporous SBA-15 under a hydrothermal process, displaying a continuous framework of SiO 4 structure with a narrow pore size distribution. These metal silicate materials were tested for CO 2 adsorption behavior in the absence and presence of water. The results exhibited that the effect of H 2 O on the CO 2 capture capability of metal silicates depends on the types of metal inserted into SBA-15. Compared to the dry condition, H 2 O addition enhances CO 2 uptake dramatically for NaCuSi 10 Ox by 25%, and slightly for NaNiSi 10 Ox (∼10%), whereas little effect is shown on NaMnSi 10 Ox. The metal silicate materials are stable after adsorption of CO 2 under wet conditions, which is benefited from their synthesis method, hydrothermal conditions. The improvement of CO 2 uptake on metal silicates by H 2 O is attributed to the competitive and synergistic adsorption mechanism on the basis of IR investigations, where initially adsorbed H 2 O acts as a promoter for further CO 2 capture through a hydration reaction, i.e. , formation of bicarbonate and carbonates on the surface of the samples. These observations provide new possibilities for the design and synthesis of porous metal silicate materials for CO 2 capture under practical conditions where moisture is present., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

An efficient synthetic protocol involving iron-catalyzed cross-coupling reactions between organolithium compounds and alkenyl iodides as key coupling partners was achieved. More than 30 examples were obtained with moderate to good yields and high stereospecificity. Gram-scale and synthetic applications of this procedure are recorded herein to demonstrate its feasibility and potential utilization.

FUS-DDIT3 belongs to the FET (FUS, EWSR1, and TAF15) family of fusion oncogenes, which collectively are considered to be key players in tumor development. Even though over 90% of all myxoid liposarcomas (MLS) have a FUS-DDIT3 gene fusion, there is limited understanding of the signaling pathways that regulate its expression. In order to study cell proliferation and FUS-DDIT3 regulation at mRNA and protein levels, we first developed a direct cell lysis approach that allows DNA, mRNA, and protein to be analyzed in the same sample using quantitative PCR, reverse transcription quantitative qPCR and proximity ligation assay, respectively. We screened 70 well-characterized kinase inhibitors and determined their effects on cell proliferation and expression of FUS-DDIT3 and FUS at both mRNA and protein levels in the MLS 402-91 cell line, where twelve selected inhibitors were evaluated further in two additional MLS cell lines. Both FUS-DDIT3 and FUS mRNA expression correlated with cell proliferation and both transcripts were co-regulated in most conditions, indicating that the common 5' FUS promotor is important in transcriptional regulation. In contrast, FUS-DDIT3 and FUS protein levels displayed more cell line dependent expression. Furthermore, most JAK inhibitors caused FUS-DDIT3 downregulation at both mRNA and protein levels. In conclusion, defining factors that regulate FUS-DDIT3 expression opens new means to understand MLS development at the molecular level.

Background: The proximity ligation assay (PLA) detects proteins via their interaction with pairs of proximity probes, which are antibodies coupled to noncomplementary DNA oligonucleotides. The binding of both proximity probes to their epitopes on the target protein brings the oligonucleotides together, allowing them to be bridged by a third oligonucleotide with complementarity to the other two. This enables their ligation and the detection of the resulting amplicon by real-time quantitative PCR (qPCR), which acts as a surrogate marker for the protein of interest. Hence PLA has potential as a clinically relevant diagnostic tool for the detection of pathogens where nucleic acid based tests are inconclusive proof of infection., Methods: We prepared monoclonal and polyclonal proximity probes targeting Clostridium difficile toxins A (TcdA) and B (TcdB) and used hydrolysis probe-based qPCR and digital PCR (dPCR) assays to detect antibody/antigen interactions., Results: The performance of the PLA assays was antibody-dependent but both TcdA and TcdB assays were more sensitive than comparable ELISAs in either single- or dualplex formats. Both PLAs could be performed using single monoclonal antibodies coupled to different oligonucleotides. Finally, we used dPCR to demonstrate its potential for accurate and reliable quantification of TcdA., Conclusions: PLA with either qPCR or dPCR readout have potential as new diagnostic applications for the detection of pathogens where nucleic acid based tests do not indicate viability or expression of toxins. Importantly, since it is not always necessary to use two different antibodies, the pool of potential antibodies useful for PLA diagnostic assays is usefully enhanced.

Cell culture is a technique that requires vigilance from the researcher. Common cell culture problems, including contamination with microorganisms or cells from other cultures, can place the reliability and reproducibility of cell culture work at risk. Here we use survey data, contributed by research scientists based in Australia and New Zealand, to assess common cell culture risks and how these risks are managed in practice. Respondents show that sharing of cell lines between laboratories continues to be widespread. Arrangements for mycoplasma and authentication testing are increasingly in place, although scientists are often uncertain how to perform authentication testing. Additional risks are identified for preparation of frozen stocks, storage and shipping., (© 2015 UICC.)

Dielectrophoretic separation of particles finds a variety of applications in the capture of species such as cells, viruses, proteins, DNA from biological systems, as well as other organic and inorganic contaminants from water. The ability to capture particles is constrained by poor volumetric scaling of separation force with respect to particle diameter, as well as the weak penetration of electric fields in the media. In order to improve the separation of sub-micron colloids, we present a scheme based on multiple interdigitated electrode arrays under mixed AC/DC bias. The use of high frequency longitudinal AC bias breaks the shielding effects through electroosmotic micromixing to enhance electric fields through the electrolyte, while a transverse DC bias between the electrode arrays enables penetration of the separation force to capture particles from the bulk of the microchannel. We determine the favorable biasing conditions for field enhancement with the help of analytical models, and experimentally demonstrate the improved capture from sub-micron colloidal suspensions with the mixed AC/DC electrostatic excitation scheme over conventional AC-DEP methods.

Hydrogen sulphide is found in raw fuels such as natural gas and coal/biomass-derived syngas. It is poisonous to catalysts and corrosive to metals and therefore needs to be removed. This is often achieved using metal oxides as reactive adsorbents, but metal oxides perform poorly when subjected to repeated cycles of sulphidation and re-oxidation as a result of complex structural and chemical changes. Here, we show that Zn-Ti-O-based adsorbents with nanofibrous morphology can sustain their initial reactivity and sulphur removal capacity over multiple regeneration cycles. These nanostructured sorbents offer rapid reaction rates that overcome the gas-transport limitations of conventional pellet-based sorbents and allow all of the material to be used efficiently. Regeneration can be carried out at the same temperature as the sulphidation step because of the higher reactivity, which prevents sorbent deterioration and reduces energy use. The efficient regeneration of the adsorbent is also aided by structural features such as the growth of hierarchical nanostructures and preferential stabilization of a wurtzite phase in the sulphidation product.

We report fabrication and use of a flexible array of nano-apertures for photolithography on curved surfaces. The batch-fabricated apertures are formed of metal-coated silicone tips. The apertures are formed at the end of the silicone tips by either electrochemical etching of the metal or plasma etching of a protective mask followed by wet chemical etching. The apertures are as small as 250 nm on substrates larger than several millimeters. We demonstrate how the nano-aperture array can be used for nano-fabrication on flat and curved substrates, and show the subsequent fabrication steps to form large arrays of sub-micron aluminum dots or vertical silicon wires.

The capability to reprogram human somatic cells to induced pluripotent stem cells (iPSCs) has opened a new area of biology and provides unprecedented access to patient-specific iPSCs for drug screening, disease models, and transplantation therapies. Although the process of obtaining iPSC lines is technically simple, reprogramming is a slow and inefficient process consisting of a largely uncharacterized chain of molecular events. To date, researchers have reported a wide range of reprogramming efficiencies, from <0.01% to >1%, depending on the specific reprogramming factors used, the mode of delivery of the reprogramming factors, properties of the starting cells, and culture conditions. We have applied a quantitative polymerase chain reaction methodology, TaqMan Protein Assays to directly quantify the kinetics, and cellular levels of crucial transcription factors during the reprogramming process. Further, we have used the assays to ascertain the threshold levels of reprogramming protein factors required to generate iPSC colonies, to characterize the protein expression signatures of different iPSC lines, and to rapidly identify iPS versus non-iPSC colonies based on expression of pluripotency markers. These data demonstrate that TaqMan Protein Assays can be used as tools to dissect and gain greater understanding of the mechanisms guiding reprogramming and to further characterize individual established iPSC lines.

Herein, we discuss the synthesis as well as material and photochemical characterization of nanometer-sized Ta(2)O(5) decorated, in a controlled fashion, on top of 20 nm diameter SiO(2) particles to yield a composite oxide with a tunable band-gap width. Particular emphasis is paid to control of particle size, and control of the distribution of the overlying oxide. The nanoscale dimension imparts a high surface area and introduces quantum confinement effects that displace the conduction band more negatively and the valence band more positively on the electrochemical scale of potentials. This band shift results in an increase of the number of possible participants in photocatalytic reactions. The band shift is shown to result in an increase in driving force for thermodynamically feasible reactions. By decorating SiO(2) with smaller-sized Ta(2)O(5), the interplay of the Lewis acidity of SiO(2) and the contact area between Ta(2)O(5) and SiO(2) is utilized to develop a photocatalyst with higher photoactivity than pure Ta(2)O(5)., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The dynamic response of an oscillating microcantilever with a gold-coated tip interacting with dissimilar functionalized silica surfaces was studied in electrolyte solutions with pH ranging from 4 to 9. Silica surfaces were chemically modified, yielding dissimilar surfaces with -Br, -NH(2), and -CH(3) functional group terminations. The relative hydrophobicity of the surfaces was characterized by contact angle measurements. The surface charge of the functionalized surfaces was first probed with commonly used static AFM measurements and serves as a reference to the dynamic response data. The amplitude and phase of the cantilever oscillation were monitored and used to calculate the effective interaction stiffness and damping coefficient, which relate to the electrical double layer interactions and also to distance-dependent hydrodynamic damping at the solid/water interface. The data for the dynamic response of the AFM over silica surfaces as a function of chemical functionalization and electrolyte pH show that the effective stiffness has a distinctive dependence on the surface charge of functionalized silica surfaces. The hydrodynamic damping also correlates strongly with the relative hydrophobicity of the surface. The data reported here indicate that interfacial properties can be strongly affected by changing the chemical composition of surfaces.

The correlation of gene and protein expression changes in biological systems has been hampered by the need for separate sample handling and analysis platforms for nucleic acids and proteins. In contrast to the simple, rapid, and flexible workflow of quantitative PCR (qPCR) methods, which enable characterization of several classes of nucleic acid biomarkers (i.e. DNA, mRNA, and microRNAs), protein analysis methods such as Western blotting are cumbersome, laborious, and much less quantitative. However, TaqMan(R) Protein Assays, which use the proximity ligation assay (PLA) technology, now expand the range of qPCR applications to include the direct detection of proteins through the amplification of a surrogate DNA template after antibody binding. Here we describe an integrated qPCR approach for measuring relative changes in gene and protein expression from the same starting sample and on a single analytical platform that pairs TaqMan Gene Expression (GEx) Assays with TaqMan Protein Assays. We have monitored the changes in mRNA, microRNA, and protein expression of relevant biomarkers in the pluripotent human embryonal carcinoma cell line, NTERA2, upon differentiation to neuronal cells. In addition, TaqMan Protein Assays have been used to monitor protein expression in induced pluripotent stem cells (iPSC) that have been reprogrammed from human somatic cells. The data presented establishes a general paradigm utilizing real-time PCR instruments and reagents for studying the relationship between the stem cell transcriptome and proteome., (Copyright 2010. Published by Elsevier Inc.)

Proton exchange membrane fuel cells have the potential for applications in energy conversion and energy storage, but their development has been impeded by problems with the membrane electrode assembly. Here, we demonstrate that a silicon-based inorganic-organic membrane offers a number of advantages over Nafion--the membrane widely used as a proton exchange membrane in hydrogen fuel cells--including higher proton conductivity, a lack of volumetric size change, and membrane electrode assembly construction capabilities. Key to achieving these advantages is fabricating a silicon membrane with pores with diameters of approximately 5-7 nm, adding a self-assembled molecular monolayer on the pore surface, and then capping the pores with a layer of porous silica. The silica layer reduces the diameter of the pores and ensures their hydration, resulting in a proton conductivity that is two to three orders of magnitude higher than that of Nafion at low humidity. A membrane electrode assembly constructed with this proton exchange membrane delivered an order of magnitude higher power density than that achieved previously with a dry hydrogen feed and an air-breathing cathode.

This article demonstrates the feasibility of making a partially buried micro gas chromatography (micro-GC) column with a rounded channel wall profile, which enables coating the stationary phase more uniformly and shows better separation characteristics than a square deep reactive ion etched (DRIE) wall profile. A buried structure fabrication method was adapted to fabricate 34 cm long, 165 microm wide, and 65 microm deep partially buried microcolumns, which had a unique rounded microcolumn wall profile similar to that of a flattened circular tube. The separation characteristics were compared to that of a 34 cm long, 100 microm x 100 microm square DRIE microcolumn, which had a similar hydraulic diameter. Minimum height equivalent to a theoretical plate (HETP) and reduced HETP of 0.39 mm and 6.02, respectively, with a retention factor of 6.3 were obtained on the coated partially buried microcolumn compared to 0.66 mm and 6.73, respectively, on the coated square DRIE microcolumn with a similar retention factor. The partially buried microcolumn was found to perform closer to the theoretical approximation and this could be attributed to the uniform phase deposition in the partially buried microcolumn compared to the square DRIE microcolumn. A 10 component mix was separated on the partially buried microcolumn in 3.8 s with the maximum peak width at half-height equal to 0.2 s, while a similar mix separated at higher pressure and temperature conditions on the square DRIE microcolumn in 4.6 s. The rounded corners allowed depositing thinner stationary phase, which was reflected in the faster elution of n-C(12) on the partially buried microcolumn compared to the square DRIE microcolumn. The better performance of the partially buried microcolumn may be attributed to either the rounded channel wall profile, the clean channel structures produced by the fabrication process, or the double-etched wall profile, which lowers the Taylor-Aris dispersion.

Using sum-frequency vibrational spectroscopy, we found that water structure at nanoporous silica/water interfaces depended on the nanoporous film structure. For a periodic, self-assembled nanoporous film with monosized 2 nm pores occupying 20% of the top surface area, the surface vibrational spectrum was dominated by water in contact with silica, bare or covered by silane, at the top surface. It resembled the spectral characteristic of the hydrophilic water/silica or the hydrophobic water/silane interface. For a fractal nanoporous film with pores ranging from 5 to 50 nm in size occupying 90% of the top surface, the spectrum for a trimethyl silane-coated superhydrophobic porous film resembled largely that of a water/air interface. Only when the silane was completely removed would the spectrum revert to that characteristic of a hydrophilic water/silica interface. The surface charging behaviors of the bare nanoporous films in water with different pH were monitored by spectroscopic measurements and atomic force microscopy force measurements. The point of zero charge for the periodic porous film is around pH 2, similar to that of the flat silica surface. The point of zero charge could only be determined to be pH<6 for the fractal porous film because the thin fractal solid network limited the amount of surface charge and therefore, the accuracy of the measurements.

Microscale total analysis systems (microTAS) allow high-throughput analyses by integrating multiple processes, parallelization, and automation. Here we combine unit operations of microTAS to create a device that can perform multidimensional separations using a three-dimensional hybrid microfluidic/nanofluidic device composed of alternating layers of patterned poly(methyl methacrylate) and nanocapillary array membranes constructed from nuclear track-etched polycarbonate. Two consecutive electrophoretic separations are performed, the first being an achiral separation followed by a chiral separation of a selected analyte band. Separation conditions are optimized for a racemic mixture of fluorescein-isothiocyanate-labeled amino acids, serine and aspartic acid, chosen because there are endogenous D-forms of these amino acids in animals. The chiral separation is implemented using micellar electrokinetic chromatography using beta-cyclodextrin as the chiral selector and sodium taurocholate as the micelle-forming agent. Analyte separation is monitored by dual-beam laser-induced fluorescence detection. After separation in the first electrophoretic channel, the preselected analyte is sampled by the second-stage separation using an automated collection sequence with a zero-crossing algorithm. The controlled fluidic environment inherent to the three-dimensional architecture enables a series of separations in varying fluidic environments and allows sample stacking via different background electrolyte pH conditions. The ability to interface sequential separations, selected analyte capture, and other fluidic manipulations in the third dimension significantly improves the functionality of multilayer microfluidic devices.

The dynamic response of amplitude-modulated atomic force microscopy (AM-AFM) is studied at the solid/water interface with respect to changes in ionic concentration, applied surface potential, and surface protonation. Each affects the electric double layer in the solution, charge on the tip and the sample surface, and thus the forces affecting the dynamic response. A theoretical model is developed to relate the effective stiffness and hydrodynamic damping of the AFM cantilever that is due to the tip/surface interaction with the phase and amplitude signals measured in the AM-AFM experiments. The phase and amplitude of an oscillating cantilever are measured as a function of tip-sample distance in three experiments: mica surface in potassium nitrate solutions with different concentrations, biased gold surface in potassium nitrate solution, and carboxylic acid-terminated self-assembled monolayers (SAMs) on gold in potassium nitrate pH buffers. Results show that, over the range where the higher harmonic modes of the oscillation are negligible, the effective stiffness of the AFM cantilever increases to a maximum as the tip approaches the surface before declining again as a result of the repulsive electrical double layer interaction. For attractive electrical double-layer interactions, the effective stiffness declines monotonically as the tip approaches the surface. Similarly, the hydrodynamic damping of the tip increases and then decreases as the tip approaches the solid/water interface, with the magnitude depending on the species present in the solution.

This article demonstrates how to prepare microfabricated columns (microcolumns) for organophosphonate and organosulfur compound separation that rival the performance of commercial capillary columns. Approximately 16,500 theoretical plates were generated with a 3 m long OV-5-coated microcolumn with a 0.25 microm phase thickness using helium as the carrier gas at 20 cm/s. Key to the advance was the development of deactivation procedures appropriate for silicon microcolumns with Pyrex tops. Active sites in a silicon-Pyrex microcolumn cause peak tailing and unwanted adsorption. Experimentally, we found that organosilicon hydride deactivation lowers adsorption activity in microcolumns more than silazane and silane treatments. But without further treatment, the phosphonate peaks continue to tail after the coating process. We found that heat treatment with pinacolyl methylphosphonic acid (PMP) eliminated the phosphonate peak tailing. In contrast, conventional resilylation employing N, O-bis(trimethylsilyl)acetamide, hexamethyldisilazane, and 1-(trimethylsilyl)imidazole does not eliminate peak tailing. Column activity tests show that the PMP treatment also improves the peaks for 2,6-dimethyl aniline, 1-octanol, and 1-decanol implying a decrease in the column's hydrogen bonding sites with the PMP treatment. FT-IR analysis shows that exposure to PMP forms a bond to the stationary phase that deactivates the active sites responsible for organophosphonate peak tailing.

Due to the numerous toxicological effects of lead, its presence in the environment needs to be effectively monitored. Incorporating a biosensing element within a microfluidic platform enables rapid and reliable determinations of lead at trace levels. A microchip-based lead sensor is described here that employs a lead-specific DNAzyme (also called catalytic DNA or deoxyribozyme) as a recognition element that cleaves its complementary substrate DNA strand only in the presence of cationic lead (Pb(2+)). Fluorescent tags on the DNAzyme translate the cleavage events to measurable, optical signals proportional to Pb(2+) concentration. The DNAzyme responds sensitively and selectively to Pb(2+), and immobilizing DNAzyme in the sensor permits both sensor regeneration and localization of the detection zone. Here, the DNAzyme has been immobilized on a PMMA surface using the highly specific biotin-streptavidin interaction. The strategy includes using streptavidin physisorbed on a PMMA surface to immobilize DNAzyme both on planar PMMA and on the walls of a PMMA microfluidic device. The immobilized DNAzyme retains its Pb(2+) detection activity in the microfluidic device and can be regenerated and reused. The DNAzyme shows no response to other common metal cations and the presence of these contaminants does not interfere with the lead-induced fluorescence signal. While prior work has shown lead-specific catalytic DNA can be used in its solubilized form and while attached to gold substrates to quantitate Pb(2+) in solution, this is the first use of the DNAzyme immobilized within a microfluidic platform for real time Pb(2+) detection.

Details are presented for the formulation, fabrication, and mechanical characterization of mesoscopic freestanding polydimethylsiloxane (PDMS) elastomer membranes, 10.0 microm thick and 5.0 mm in diameter, used to probe the rheology of a living epithelial sheet. In what is described as a composite diaphragm inflation (CDI) experiment, freestanding PDMS membranes are utilized as substrates for the culture of a sheet of epithelial cells. Together, the cell layer and the PDMS elastomer form a composite diaphragm (CD) that is suitable for mechanical testing in an axisymmetric membrane inflation experiment. In order to distinguish the rheological behavior of the epithelial sheet from the mechanical response of the elastomer using inflation test data, freestanding PDMS membranes should exhibit a highly compliant yet mechanically invariant finite load-deformation response when subjected to multiple inflation cycles following intermittent periods of cell culture. Given these considerations, we describe a method for preparing freestanding PDMS elastomer membrane specimens that are optically transparent, tensed, and wrinkle-free. Surface modifications intended to facilitate cell culture, namely water vapor plasma and ultraviolet light treatments, were shown to dramatically stiffen the mechanical response of the membranes, rendering them unusable as CD substrates. In this study, only PDMS membranes with physiosorbed collagen demonstrated the mechanical compliance, fatigue resistance, and environmental stability necessary for reliable use in CDI experiments.

We propose a new type of micro/nano fluidic mixer based on non-equilibrium electrokinetics and demonstrate its mixing performance. We fabricate the device with two-step reactive ion etching, one for nanochannels and one for microchannels. Mixing is achieved by strong vortex structures formed near the micro/nano channel interface. We expect the proposed device to be beneficial in the development of micro total analysis systems, since it is simple in its design with minimal fabrication complications.

One of the most pervasive problems afflicting people throughout the world is inadequate access to clean water and sanitation. Problems with water are expected to grow worse in the coming decades, with water scarcity occurring globally, even in regions currently considered water-rich. Addressing these problems calls out for a tremendous amount of research to be conducted to identify robust new methods of purifying water at lower cost and with less energy, while at the same time minimizing the use of chemicals and impact on the environment. Here we highlight some of the science and technology being developed to improve the disinfection and decontamination of water, as well as efforts to increase water supplies through the safe re-use of wastewater and efficient desalination of sea and brackish water.

Hybrid microfluidic/nanofluidic devices offer unique capabilities for manipulating and analyzing minute volumes of expensive or hard-to-obtain samples. Here, multilayer poly-(methyl methacrylate) microchips, with multiple spatially isolated microfluidic channels interconnected by nanocapillary array membranes (NCAMs), are fabricated using an adhesive contact printing process. The NCAMs, positioned between the microfluidic channel layers, add functionality to the inter-microchannel fluid transfer unit operation. They do so because the transport of specific analytes through the NCAM can be controlled by adjusting the ionic strength, the polarity of the applied bias, the surface charge density, and the pore size. A simplified, floating injection technique for NCAM-coupled nanofluidic devices is described and compared with conventional biased injection. In the floating injection approach, a voltage is applied across the injection channel and the slight electric field extension at the cross-section is used to transfer analytes through the nanopores to the separation channel. Floating injection excels in plug reproducibility, separation resolution, and operation simplicity, although it decreases assay throughput relative to biased injection. Floating injection can avoid the uneven distribution of analytes in the microfluidic channel that sometimes results from biased injection because of the volume mismatch between NCAM nanopore transport capacity and the supply of fluid. Moreover, the pressure-driven flow caused by the mismatch of the EOFs in the microfluidic channels connected by an NCAM must be considered when using NCAMs with pore diameters below 50 nm.

Details are given for the design, calibration, and operation of an apparatus for measuring the finite load-deformation behavior of a sheet of living epithelial cells cultured on a mesoscopic freestanding elastomer membrane, 10 microm thick and 5 mm in diameter. Although similar in concept to bulge tests used to investigate the mechanical properties of micromachined thin films, cell-elastomer composite diaphragm inflation tests pose a unique set of experimental challenges. Composite diaphragm (CD) specimens are extremely compliant (E<50 kPa), experience large displacements when subject to small inflation pressures (approximately 100 Pa), and must be continuously immersed in a bath of liquid culture medium during the acquisition of load-deformation measurements. Given these considerations, we have constructed an inflation apparatus consisting of an air-piston-cylinder pump integrated with a modular specimen mounting fixture that constitutes a horizontally semi-infinite reservoir of liquid culture medium. In a deformation-controlled inflation test, pressurized air is used to inflate a CD specimen into the liquid reservoir with minimum disturbance of the liquid-air interface. Piston displacements and absolute pump chamber air pressures are utilized as feedback to cycle the displaced (or inflated) CD volume V in a 0.05 Hz triangular or sinusoidal wave form (V(MIN)=0 microl, V(MAX)

An experimental technique is presented to measure reflectance at high sample temperature with respect to room temperature in the infrared using Fourier transform infrared fitted with a reflectometer. Sample temperature artifacts are accounted for by sequential measurements taken with the lamp source on and with the lamp source off. The sequential measurements are shown mathematically to correct for the modulation of sample and detector thermal emissions. Further, the technique is applied to a polyimide (PMDA-ODA) film on a layer of gold deposited on a thermally oxidized Si wafer. It is shown that the optical properties (index of refraction and extinction coefficient) remain relatively constant with temperature (from room temperature to 380 degrees C) in the 4000-6000 cm(-1) spectral region. The significant changes that occur with temperature are the change in thickness of the film and also the spectral properties in the 2000-4000 cm(-1) region. Also, by using a Lorentz oscillator model, it is shown that this method is able to discern that spectral features corresponding to the OH stretching bands at 3630 and 3470 cm(-1) show significant variation with increasing temperature.

We report here a technique to print small volumes of liquid on a hydrophobic substrate. This process is based on the control of the critical parameters that govern a quasi-equilibrium liquid transfer process from one surface to another. We present a qualitative model that describes the physics of a transfer printing process between hydrophobic surfaces, and we use the parameters outlined in this model to manipulate the amount of liquid transferred between surfaces. We demonstrate the printing of discrete, small volumes (approximately 70 fL) of different liquid inks on a polymer substrate starting with volumes that are 8 orders of magnitude larger (a droplet of approximately 10 microL) in a simple two-step procedure.

This paper demonstrates a chemical surface modification method for covalent attachment of various polymers by using silane-based "click" chemistry on silica surfaces and within glass microchannels suitable for CE systems. Modified surfaces are characterized by contact angle measurements, X-ray photoelectron spectroscopy, and Fourier transform infrared-attenuated total reflection spectroscopy. Electroosmotic flow (EOF) measurements in modified and unmodified channels are provided. Spectroscopic and transport data show that various polymers can be covalently attached to glass surfaces with a measurable change in EOF.

Sheets of normal human epidermal keratinocytes (NHEKs) were reconstituted in vitro on tensed but highly compliant, freestanding polydimethylsiloxane (PDMS) membranes, 5.0 mm in diameter and 10 mum thick. NHEK-PDMS composite diaphragm (CD) specimens were then subjected to cyclical axisymmetric inflation tests to investigate epithelial sheet rheology under conditions of physiologically severe deformations (~50% nominal polar biaxial strains). Because the compliance of the specially formulated PDMS membrane was greater than that of the attached cell layer, the finite load-deformation behavior (mechanical response) of the living NHEK sheet was inferred from differences between the mechanical behavior of the CD specimen and the response of the underlying PDMS membrane measured prior to cell culture. In these composite diaphragm inflation (CDI) experiments, interconnected NHEKs exhibited rheological behaviors that were suggestive of a viscoelastic-plastic stress response. Remarkably, specimens returned to quiescent culture following a sequence of inflation tests recovered at least 80% of their original ability to store elastic strain energy, evidence of biological adaptation and recovery or restitutio ad integrum. Unlike methodologies that assay the morphological or biochemical response of cultured cells to an applied mechanostimulus, CDI experiments can be used to probe the load-bearing functions of desmosomes and adherens junctions within a living epithelial sheet, as well as to assess the rheological behaviors of the intermediate filament and microfilament networks that these cell-cell junctions serve to interconnect.

The design and fabrication of a multilayered polymer micro-nanofluidic chip is described that consists of poly(methylmethacrylate) (PMMA) layers that contain microfluidic channels separated in the vertical direction by polycarbonate (PC) membranes that incorporate an array of nanometre diameter cylindrical pores. The materials are optically transparent to allow inspection of the fluids within the channels in the near UV and visible spectrum. The design architecture enables nanofluidic interconnections to be placed in the vertical direction between microfluidic channels. Such an architecture allows microchannel separations within the chip, as well as allowing unique operations that utilize nanocapillary interconnects: the separation of analytes based on molecular size, channel isolation, enhanced mixing, and sample concentration. Device fabrication is made possible by a transfer process of labile membranes and the development of a contact printing method for a thermally curable epoxy based adhesive. This adhesive is shown to have bond strengths that prevent leakage and delamination and channel rupture tests exceed 6 atm (0.6 MPa) under applied pressure. Channels 100 microm in width and 20 microm in depth are contact printed without the adhesive entering the microchannel. The chip is characterized in terms of resistivity measurements along the microfluidic channels, electroosmotic flow (EOF) measurements at different pH values and laser-induced-fluorescence (LIF) detection of green-fluorescent protein (GFP) plugs injected across the nanocapillary membrane and into a microfluidic channel. The results indicate that the mixed polymer micro-nanofluidic multilayer chip has electrical characteristics needed for use in microanalytical systems.

Separation rates and resolutions within capillary electrophoretic (CE) systems can be enhanced when surface zeta potentials are uniform with minimum deviations from ideal pluglike flow. Microfluidic CE devices based on poly(methyl methacrylate) (PMMA) are being developed due to the optical clarity, availability, stability, and reproducible electroosmotic flow (EOF) rates displayed by this polymer. Control of EOF in polymer-based CE systems can be achieved by surface zeta potential alteration through chemical modification. Herein, a method will be presented for the surface functionalization of PMMA with chemistry analogous to formation of trichlorosilane self-assembled monolayers on SiO2. The current method involves two separate steps. First, surface activation with water-vapor plasma introduces surface hydroxylation. Second, treatment of the plasma-treated PMMA with a substituted trichlorosilane solution forms the functional surface layer. The modified surfaces were characterized using several analytical techniques, including water contact angle, X-ray photoelectron spectroscopy, Fourier transform infrared-attenuated total reflection, secondary ion mass spectroscopy, and measurement of EOF velocities within PMMA microchannels.

The mammalian Nell1 gene encodes a protein kinase C-beta1 (PKC-beta1) binding protein that belongs to a new class of cell-signaling molecules controlling cell growth and differentiation. Over-expression of Nell1 in the developing cranial sutures in both human and mouse induces craniosynostosis, the premature fusion of the growing cranial bone fronts. Here, we report the generation, positional cloning and characterization of Nell1(6R), a recessive, neonatal-lethal point mutation in the mouse Nell1 gene, induced by N-ethyl-N-nitrosourea. Nell1(6R) has a T-->A base change that converts a codon for cysteine into a premature stop codon [Cys(502)Ter], resulting in severe truncation of the predicted protein product and marked reduction in steady-state levels of the transcript. In addition to the expected alteration of cranial morphology, Nell1(6R) mutants manifest skeletal defects in the vertebral column and ribcage, revealing a hitherto undefined role for Nell1 in signal transduction in endochondral ossification. Real-time quantitative reverse transcription-PCR assays of 219 genes showed an association between the loss of Nell1 function and reduced expression of genes for extracellular matrix (ECM) proteins critical for chondrogenesis and osteogenesis. Several affected genes are involved in the human cartilage disorder Ehlers-Danlos Syndrome and other disorders associated with spinal curvature anomalies. Nell1(6R) mutant mice are a new tool for elucidating basic mechanisms in osteoblast and chrondrocyte differentiation in the developing skull and vertebral column and understanding how perturbations in the production of ECM proteins can lead to anomalies in these structures.

A highly automated RT-PCR-based approach has been established to validate novel human gene predictions with no prior experimental evidence of mRNA splicing (ab initio predictions). Ab initio gene predictions were selected for high-throughput validation using predicted protein classification, sequence similarity to other genomes, colocalization with an MPSS tag, or microarray expression. Initial microarray prioritization followed by RT-PCR validation was the most efficient combination, resulting in approximately 35% of the ab initio predictions being validated by RT-PCR. Of the 7252 novel genes that were prioritized and processed, 796 constituted real transcripts. In addition, high-throughput RACE successfully extended the 5' and/or 3' ends of >60% of RT-PCR-validated genes. Reevaluation of these transcripts produced 574 novel transcripts using RefSeq as a reference. RT-PCR sequencing in combination with RACE on ab initio gene predictions could be used to define the transcriptome across all species.

Background: A central component of the complex human biological stress response is the modulation of the neuro-endocrine-immune system with its intricate feedback loops that support homeostatic regulation. Well-documented marked gene expression variability among human and animal subjects coupled with sample collection timing and delayed effects, as well as a host of molecular detection challenges renders the quest for deciphering the human biological stress response challenging from many perspectives., Material/methods: A novel Recreational Music-Making (RMM) program was used in combination with a new strategy for peripheral blood gene expression analysis to assess individualized genomic stress induction signatures. The expression of 45 immune response-related genes was determined using a multiplex preamplification step prior to conventional quantitative Real Time Polymerase Chain Reaction (qRT-PCR) mRNA analysis to characterize the multidimensional biological impact of a 2-phase controlled stress induction/amelioration experimental protocol in 32 randomly assigned individuals., Results: In subjects performing the RMM activity following a 1-hour stress induction protocol, 19 out of 45 markers demonstrated reversal with significant (P = 0.05) Pearson correlations in contrast to 6 out of 45 markers in the resting control group and 0 out of 45 in the ongoing stressor group., Conclusions: The resultant amelioration of stress-induced genomic expression supports the underlying premise that RMM warrants additional consideration as a rational choice within our armamentarium of stress reduction strategies. Modulation of individualized genomic stress induction signatures in peripheral blood presents a new opportunity for elucidating the dynamics of the human stress response.

Integrating multiple analytical processes into microfluidic devices is an important research area required for a variety of microchip-based analyses. A microfluidic system is described that achieves preparative separations by intelligent fraction collection of attomole quantities of sample. The device consists of a main microfluidic channel used to perform electrophoresis, which is interconnected at 90 degrees to two vertically displaced channels via a nanocapillary array membrane. The membrane interconnect contains nanometer-diameter pores that provide fluidic communication between the channels. Sample injection and analyte collection are controlled by application of an electrical bias between the microfluidic channels across the nanocapillary array. After the separation, the automated transfer of the FITC-labeled Arg, Gln, and Gly bands occurs; a fluorescence detector located at the separation/collection channel interconnect is used to generate a triggering signal that initiates suitable voltages to allow near-quantitative transfer of analyte from the separation channel to the second fluidic layer. The ability to achieve such sample manipulations from mass-limited samples enables a variety of postseparation processing events.

Bone morphogenetic proteins (BMPs) regulate multiple cellular processes, including cell differentiation and migration. Their signals are transduced by the kinase receptors BMPR-I and BMPR-II, leading to Smad transcription factor activation via BMPR-I. LIM kinase (LIMK) 1 is a key regulator of actin dynamics as it phosphorylates and inactivates cofilin, an actin depolymerizing factor. During a search for LIMK1-interacting proteins, we isolated clones encompassing the tail region of BMPR-II. Although the BMPR-II tail is not involved in BMP signaling via Smad proteins, mutations truncating this domain are present in patients with primary pulmonary hypertension (PPH). Further analysis revealed that the interaction between LIMK1 and BMPR-II inhibited LIMK1's ability to phosphorylate cofilin, which could then be alleviated by addition of BMP4. A BMPR-II mutant containing the smallest COOH-terminal truncation described in PPH failed to bind or inhibit LIMK1. This study identifies the first function of the BMPR-II tail domain and suggests that the deregulation of actin dynamics may contribute to the etiology of PPH.

Mammalian genomes carry hundreds of Krüppel-type zinc finger (ZNF) genes, most of which reside in familial clusters. ZNF genes encoding Krüppel-associated box (KRAB) motifs are especially prone to this type of tandem organization. Despite their prevalence, little is known about the functions or evolutionary histories of these clustered gene families. Here we describe a homologous pair of human and mouse KRAB-ZNF gene clusters containing 21 human and 10 mouse genes, respectively. Evolutionary analysis uncovered only three pairs of putative orthologs and two cases where a single gene in one species is related to multiple genes in the other; several human genes have no obvious homolog in mouse. We deduce that duplication and loss of ancestral cluster members occurred independently in the primate and rodent lineages after divergence, yielding substantially different ZNF gene repertoires in humans and mice. Differences in expression patterns and sequence divergence within the DNA binding regions of predicted proteins suggest that the duplicated genes have acquired novel functions over evolutionary time. Since KRAB-ZNF proteins are predicted to function as transcriptional regulators, the elaboration of new lineage-specific genes in this and other clustered ZNF families is likely to have had a significant impact on species-specific aspects of biology.

The extension of microfluidic devices to include three-dimensional fluidic networks allows complex fluidic and chemical manipulations but requires innovative methods to interface fluidic layers. Externally controllable interconnects, employing nuclear track-etched polycarbonate membranes containing nanometer-diameter capillaries, are described that produce hybrid three-dimensional fluidic architectures. Controllable nanofluidic transfer is achieved by controlling applied bias, polarity, and density of the immobile nanopore surface charge and the impedance of the nanocapillary array relative to the microfluidic channels. Analyte transport between vertically separated microchannels has three stable transfer levels, corresponding to zero, reverse, and forward bias. The transfer can even depend on the properties of the analyte being transferred such as the molecular size, illustrating the flexible character of the analyte transfer. In a specific analysis implementation, nanochannel array gating is applied to capillary electrophoresis separations, allowing selected separated components to be isolated for further manipulation, thereby opening the way for preparative separations at attomole analyte mass levels.

Monte Carlo methods are often applied to the calculation of the apparent emissivities of blackbody cavities. However, for cavities with complex as well as some commonly encountered geometries, the emission Monte Carlo method experiences problems of convergence. The emission and absorption Monte Carlo methods are compared on the basis of ease of implementation and convergence speed when applied to blackbody sources. A new method to determine solution convergence compatible with both methods is developed, and the convergence speeds of the two methods are compared through the application of both methods to a right-circular cylinder cavity. It is shown that the absorption method converges faster and is easier to implement than the emission method when applied to most blackbody and lower emissivity cavities.