A 3D In-vitro model of the human dentine interface shows long-range osteoinduction from the dentine surface.

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 ß-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.

Genomic Analysis and Surveillance of Respiratory Syncytial Virus (RSV) Using Wastewater-Based Epidemiology (WBE).

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 limits the public health response. Wastewater-based epidemiology (WBE) has recently been used globally as a key metric in determining prevalence of SARS-CoV-2 in the community but its application to other respiratory viruses is limited. In this study, we present an integrated genomic WBE approach, applying RT-qPCR 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 non-pharmaceutical interventions.

Tubular nanomaterials for bone tissue engineering.

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.

H2O-prompted CO2 capture on metal silicates in situ generated from SBA-15.

A series of metal silicates, NaMSi10Ox (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 SiO4 structure with a narrow pore size distribution. These metal silicate materials were tested for CO2 adsorption behavior in the absence and presence of water. The results exhibited that the effect of H2O on the CO2 capture capability of metal silicates depends on the types of metal inserted into SBA-15. Compared to the dry condition, H2O addition enhances CO2 uptake dramatically for NaCuSi10Ox by 25%, and slightly for NaNiSi10Ox (∼10%), whereas little effect is shown on NaMnSi10Ox. The metal silicate materials are stable after adsorption of CO2 under wet conditions, which is benefited from their synthesis method, hydrothermal conditions. The improvement of CO2 uptake on metal silicates by H2O is attributed to the competitive and synergistic adsorption mechanism on the basis of IR investigations, where initially adsorbed H2O acts as a promoter for further CO2 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 CO2 capture under practical conditions where moisture is present.

Stereospecific Iron-Catalyzed Carbon(sp2)-Carbon(sp3) Cross-Coupling with Alkyllithium and Alkenyl Iodides.

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.

Identification of inhibitors regulating cell proliferation and FUS-DDIT3 expression in myxoid liposarcoma using combined DNA, mRNA, and protein analyses.

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.

Homogeneous and digital proximity ligation assays for the detection of Clostridium difficile toxins A and B.

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.

Is cell culture a risky business? Risk analysis based on scientist survey data.

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.

Enhanced sub-micron colloidal particle separation with interdigitated microelectrode arrays using mixed AC/DC dielectrophoretic scheme.

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.

A regenerable oxide-based H2S adsorbent with nanofibrous morphology.

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.

Nano-fabrication with a flexible array of nano-apertures.

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.

Applications of quantitative polymerase chain reaction protein assays during reprogramming.

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.

Synthesis, characterization, and photoactivity of Ta2O5-grafted SiO2 nanoparticles.

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).

Dynamic response of AFM cantilevers to dissimilar functionalized silica surfaces in aqueous electrolyte solutions.

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.

Expanding applications of protein analysis using proximity ligation and qPCR.

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.

An inorganic-organic proton exchange membrane for fuel cells with a controlled nanoscale pore structure.

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.

Nanoporous silica-water interfaces studied by sum-frequency vibrational spectroscopy.

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.

Partially buried microcolumns for micro gas analyzers.

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.

Multidimensional separation of chiral amino acid mixtures in a multilayered three-dimensional hybrid microfluidic/nanofluidic device.

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.