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Background Inflammation is a major contributor in pulmonary arterial hypertension (PAH) pathogenesis. Non-invasive, focused ultrasound stimulation (FUS) of the spleen activates the neuroimmune inflammatory reflex (IR) and suppresses systemic inflammation [1]. Purpose We aimed to explore whether daily FUS of the spleen improves haemodynamics and biomarkers by modulating the IR, in a rat model of PAH. Methods Sprague-Dawley rats (n=12) were injected s.c. with Sugen5416 (VEGF receptor inhibitor) and then, were placed in a hypoxic chamber (FiO2=10%) for 21 days, followed by 14 days of re-exposure to normoxia (FiO2=10%). At day 21, rats were randomized to either FUS (n=6) or sham-stimulation of the spleen (n=6). Each FUS- or sham-stimulation session consisted of 12 minutes. After 14 days of treatment, in a terminal experiment, right ventricular systolic pressure (RVSP) and arterial pressure were measured invasively, as well as biomarkers, in each animal (Figure 1A). Results FUS significantly reduced RVSP compared with the sham-stimulation (Mean±SEM, 50.83±3.57 mmHg vs 72.50±5.81; p=0.009), resulting in a 30% relative reduction (Figure 1B). Mean systemic arterial pressure was similar in the 2 groups (Mean±SEM, 79.83±3.73 mmHg vs. 87.00±2.95; p=0.137) (Figure 1C), as was the change in heart rate between day 1 and day 14 of treatment period (−19.53±5.36% vs. −16.79±3.1, p=0.61) (Figure 1D). Consistently, plasma brain natriuretic peptide (BNP) levels are reduced in the FUS group (119.45±19.93 ng/μl vs. 319.39±91.85; p=0.019), indicative of reduced myocardial wall stress in the FUS group (Figure 1E). Conclusion Non-invasive FUS of the spleen reduced RVSP by ∼30% and BNP without significantly affecting systemic pressure or heart rate, in rats with severe PAH. Non-invasive FUS, by modulating the IR, may exert an anti-inflammatory effect in PAH. FUS of the spleen is noninvasive, safe, widely available and easy to perform, and should be further explored as a possible therapeutic option in PAH. Funding Acknowledgement Type of funding sources: Private hospital(s). Main funding source(s): Northwell Health

New findings What is the central question of this study? Does peripheral non-invasive focused ultrasound targeted to the celiac plexus improve inflammatory bowel disease? What is the main finding and its importance? Peripheral non-invasive focused ultrasound targeted to the celiac plexus in a rat model of ulcerative colitis improved stool consistency and reduced stool bloodiness, which coincided with a longer and healthier colon than in animals without focused ultrasound treatment. The findings suggest that this novel neuromodulatory technology could serve as a plausible therapeutic approach for improving symptoms of inflammatory bowel disease. Abstract Individuals suffering from inflammatory bowel disease (IBD) experience significantly diminished quality of life. Here, we aim to stimulate the celiac plexus with non-invasive peripheral focused ultrasound (FUS) to modulate the enteric cholinergic anti-inflammatory pathway. This approach may have clinical utility as an efficacious IBD treatment given the non-invasive and targeted nature of this therapy. We employed the dextran sodium sulfate (DSS) model of colitis, administering lower (5%) and higher (7%) doses to rats in drinking water. FUS on the celiac plexus administered twice a day for 12 consecutive days to rats with severe IBD improved stool consistency scores from 2.2 ± 1 to 1.0 ± 0.0 with peak efficacy on day 5 and maximum reduction in gross bleeding scores from 1.8 ± 0.8 to 0.8 ± 0.8 on day 6. Similar improvements were seen in animals in the low dose DSS group, who received FUS only once daily for 12 days. Moreover, animals in the high dose DSS group receiving FUS twice daily maintained colon length (17.7 ± 2.5 cm), while rats drinking DSS without FUS exhibited marked damage and shortening of the colon (13.8 ± 0.6 cm) as expected. Inflammatory cytokines such as interleukin (IL)-1β, IL-6, IL-17, tumour necrosis factor-α and interferon-γ were reduced with DSS but coincided with control levels after FUS, which is plausibly due to a loss of colon crypts in the former and healthier crypts in the latter. Lastly, overall, these results suggest non-invasive FUS of peripheral ganglion can deliver precision therapy to improve IBD symptomology.

Background Peripheral nerve reflexes enable organ systems to maintain long-term physiological homeostasis while responding to rapidly changing environmental conditions. Electrical nerve stimulation is commonly used to activate these reflexes and modulate organ function, giving rise to an emerging class of therapeutics called bioelectronic medicines. Dogma maintains that immune cell migration to and from organs is mediated by inflammatory signals (i.e. cytokines or pathogen associated signaling molecules). However, nerve reflexes that regulate immune function have only recently been elucidated, and stimulation of these reflexes for therapeutic effect has not been fully investigated. Methods We utilized both electrical and ultrasound-based nerve stimulation to activate nerve pathways projecting to specific lymph nodes. Tissue and cell analysis of the stimulated lymph node, distal lymph nodes and immune organs is then utilized to measure the stimulation-induced changes in neurotransmitter/neuropeptide concentrations and immune cellularity in each of these sites. Results and conclusions In this report, we demonstrate that activation of nerves and stimulated release of neurotransmitters within a local lymph node results in transient retention of immune cells (e.g. lymphocytes and neutrophils) at that location. Furthermore, such stimulation results in transient changes in neurotransmitter concentrations at distal organs of the immune system, spleen and liver, and mobilization of immune cells into the circulation. This report will enable future studies in which stimulation of these long-range nerve connections between lymphatic and immune organs can be applied for clinical purpose, including therapeutic modulation of cellularity during vaccination, active allergic response, or active auto-immune disease.

While peripheral glucose sensors are known to relay signals of substrate availability to integrative nuclei in the brain, the importance of these pathways in maintaining energy homeostasis and their contribution to disease remain unknown. Herein, we demonstrate that selective activation of the hepatoportal neural plexus via transient peripheral focused ultrasound (pFUS) induces glucose homeostasis in models of well-established insulin resistant diabetes. pFUS modulates sensory projections to the hindbrain and alters hypothalamic concentrations of neurotransmitters that regulate metabolism, resulting in potentiation of hypothalamic insulin signaling, leptin-independent inhibition of the orexigenic neuropeptide Y system, and therapeutic alteration in autonomic output to peripheral effector organs. Multiomic profiling confirms pFUS-induced modifications of key metabolic functions in liver, pancreas, muscle, adipose, kidney, and intestines. Activation of the hepatic nutrient sensing pathway not only restores nervous system coordination of peripheral metabolism in three different species but does so across these organ systems; several of which are current targets of antidiabetic drug classes. These results demonstrate the potential of hepatic pFUS as a novel/non-pharmacologic therapeutic modality to restore glucose homeostasis in metabolic diseases, including type II diabetes.One Sentence SummaryWe utilize a non-invasive ultrasound technique to activate a liver-brain sensory pathway and demonstrate its potential to induce durable normalization of glucose homeostasis in models of well-established insulin resistant diabetes.

Obesity, a growing health concern, is associated with an increased risk of morbidity and mortality. Chronic low-grade inflammation is implicated in obesity-driven metabolic complications. Peripheral focused ultrasound stimulation (pFUS) is an emerging non-invasive technology that modulates inflammation. Here, we reasoned that focused ultrasound stimulation of the liver may alleviate obesity-related inflammation and other comorbidities. After 8 weeks on a high-fat high-carbohydrate “Western” diet, C57BL/6J mice were subjected to either sham stimulation or focused ultrasound stimulation at the porta hepatis. Daily liver-focused ultrasound stimulation for 8 weeks significantly decreased body weight, circulating lipids and mitigated dysregulation of adipokines. In addition, liver-focused ultrasound stimulation significantly reduced hepatic cytokine levels and leukocyte infiltration. Our findings demonstrate the efficacy of hepatic focused ultrasound for alleviating obesity and obesity-associated complications in mice. These findings suggest a previously unrecognized potential of hepatic focused ultrasound as a possible novel noninvasive approach in the context of obesity.

Empiric broad‐spectrum antimicrobial treatments of urinary tract infections (UTIs) have contributed to widespread antimicrobial resistance. Clinical adoption of evidence‐based treatments necessitates rapid diagnostic methods for pathogen identification (ID) and antimicrobial susceptibility testing (AST) with minimal sample preparation. In response, a microfluidic droplet‐based platform is developed for achieving both ID and AST from urine samples within 30 min. In this platform, fluorogenic hybridization probes are utilized to detect 16S rRNA from single bacterial cells encapsulated in picoliter droplets, enabling molecular identification of uropathogenic bacteria directly from urine in as little as 16 min. Moreover, in‐droplet single‐bacterial measurements of 16S rRNA provide a surrogate for AST, shortening the exposure time to 10 min for gentamicin and ciprofloxacin. A fully integrated device and screening workflow were developed to test urine specimens for one of seven unique diagnostic outcomes including the presence/absence of Gram‐negative bacteria, molecular ID of the bacteriaas Escherichia coli, an Enterobacterales, or other organism, and assessment of bacterial susceptibility to ciprofloxacin. In a 50‐specimen clinical comparison study, the platform demonstrates excellent performance compared to clinical standard methods (areas‐under‐curves, AUCs >0.95), within a small fraction of the turnaround time, highlighting its clinical utility., Single‐cell measurements of bacterial 16S rRNA are leveraged in picoliter droplets to achieve simultaneous molecular detection of bacteria and phenotypic assessment of antimicrobial susceptibility, unlocking a 30 min sample‐to‐answer diagnostic platform for urinary tract infections.

Hyperinflammation and uncontrolled cytokine release, which can be seen in severe cases of COVID-19, require therapy to reduce the innate immune response without hindering necessary adaptive immune mechanisms. Here, we show results from the first in-human trials using non-invasive ultrasound stimulation of the spleen to reduce cytokine release in the context of both an acute response in healthy subjects and a chronic inflammatory condition in rheumatoid arthritis patients. Splenic ultrasound results in a reduction in TNF serum levels, as well as IL-1β and IL-8 transcript levels in monocytes. There is also a down regulation of pathways involved in TNF and IL-6 production, and IFNγ- and NFκB-regulated genes. Many of these cytokines or pathways are upregulated in COVID-19 patients. There is also a reduction in chemokine transcript levels and other components of the chemotactic response, suggesting that reduction of cellular migration may contribute to the therapeutic effects of ultrasound. There is no inhibition of the adaptive immune response with ultrasound treatment relating to antibody production. This is consistent with a pre-clinical animal model where enhanced antibody production was achieved with splenic ultrasound. Therefore, this new splenic ultrasound approach has the potential to treat acute and chronic hyper-inflammatory diseases, as it lowers cytokine levels without disrupting the normal adaptive immune response. Portable ultrasound technologies are currently being developed and translated to the clinic to treat various inflammatory disorders, with more recent efforts directed towards combatting the hyperinflammation or ‘cytokine storm’ in COVID-19 patients.

Test disk electrodes were fabricated from carbon nanotubes (CNT) using the Carbon Nanotube Templated Microfabrication (CNT-M) technique. The CNT-M process uses patterned growth of carbon nanotube forests from surfaces to form complex patterns, enabling electrode sizing and shaping. The additional carbon infiltration process stabilizes these structures for further processing and handling. At a macroscopic scale, the electrochemical, electrical and magnetic properties, and magnetic resonance imaging (MRI) characteristics of the disk electrodes were investigated; their microstructure was also assessed. CNT disk electrodes showed electrical resistivity around 1 Ω·cm, charge storage capacity between 3.4 and 38.4 mC/cm2, low electrochemical impedance and magnetic susceptibility of −5.9 to −8.1 ppm, closely matched to that of tissue (∼−9 ppm). Phantom MR imaging experiments showed almost no distortion caused by these electrodes compared with Cu and Pt-Ir reference electrodes, indicating the potential for significant improvement in accurate tip visualization.

We present methods to fabricate high-capacity redox electrodes using thick membrane or fiber casting of conjugated polymer solutions. Unlike common solution casting or printing methods used in current organic electronics, the presented techniques enable production of PEDOT:PSS electrodes with high charge capacity and the capability to operate under applied voltages greater than 100 V without electrochemical overoxidation. The electrodes are shown integrated into several electrokinetic components commonly used in automated bioprocess or bioassay workflows, including electrophoretic DNA separation and extraction, cellular electroporation/lysis, and electroosmotic pumping. Unlike current metal electrodes used in these applications, the high-capacity polymer electrodes are shown to function without electrolysis of solvent (i.e., without production of excess H+, OH-, and H2O2 by-products). In addition, each component fabricated using the electrodes is shown to have superior capabilities compared with those fabricated with common metal electrodes. These innovations in electrokinetics include a low-voltage/high-pressure electroosmotic pump, and a "flow battery" (in which electrochemical discharge is used to generate electroosmotic flow in the absence of an applied potential). The novel electrodes (and electrokinetic demonstrations) enable new applications of organic electronics within the biology, health care, and pharmaceutical fields.

Recently advances in miniaturization and automation have been utilized to rapidly decrease the time to result for microbiology testing in the clinic. These advances have been made due to the limitations of conventional culture-based microbiology methods, including agar plate and microbroth dilution, which have long turnaround times and require physicians to treat patients empirically with antibiotics before test results are available. Currently, there exist similar limitations in pharmaceutical sterility and bioburden testing, where the long turnaround times associated with standard microbiology testing drive costly inefficiencies in workflows. These include the time lag associated with sterility screening within drug production lines and the warehousing cost and time delays within supply chains during product testing. Herein, we demonstrate a proof-of-concept combination of a rapid microfluidic assay and an efficient cell filtration process that enables a path toward integrating rapid tests directly into pharmaceutical microbiological screening workflows. We demonstrate separation and detection of Escherichia coli directly captured and analyzed from a mammalian (i.e., CHO) cell culture with a 3.0 h incubation. The demonstration is performed using a membrane filtration module that is compatible with sampling from bioreactors, enabling in-line sampling and process monitoring.

This review describes work from several research groups in which ultrasound is being used to target the peripheral nervous system and perform neuromodulation noninvasively. Although these techniques are in their infancy compared to implant-based and electrical nerve stimulation, if successful this new noninvasive method for neuromodulation could solve many of the challenges facing the field of bioelectronic medicine. The work outlined herein shows results in which two different (potentially therapeutic) targets are stimulated, a neuroimmune pathway within the spleen and a nutrient/sensory pathway within the liver. Both data and discussion are provided that compare this new noninvasive technique to implant-based nerve stimulation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Tools for noninvasively modulating neural signaling in peripheral organs will advance the study of nerves and their effect on homeostasis and disease. Herein, we demonstrate a noninvasive method to modulate specific signaling pathways within organs using ultrasound (U/S). U/S is first applied to spleen to modulate the cholinergic anti-inflammatory pathway (CAP), and US stimulation is shown to reduce cytokine response to endotoxin to the same levels as implant-based vagus nerve stimulation (VNS). Next, hepatic U/S stimulation is shown to modulate pathways that regulate blood glucose and is as effective as VNS in suppressing the hyperglycemic effect of endotoxin exposure. This response to hepatic U/S is only found when targeting specific sub-organ locations known to contain glucose sensory neurons, and both molecular (i.e. neurotransmitter concentration and cFOS expression) and neuroimaging results indicate US induced signaling to metabolism-related hypothalamic sub-nuclei. These data demonstrate that U/S stimulation within organs provides a new method for site-selective neuromodulation to regulate specific physiological functions., Stimulation of peripheral nerve activity may be used to treat metabolic and inflammatory disorders, but current approaches need implanted devices. Here, the authors present a non-invasive approach, and show that ultrasound-mediated stimulation can be targeted to specific sub-organ locations in preclinical models and alter the response of metabolic and inflammatory neural pathways.

We demonstrate the efficient separation of blood cells from millilitre volumes of whole blood in minutes using a simple gravity sedimentation device. Blood cell and plasma separation is often the initial step in clinical diagnostics, and reliable separation techniques have remained a major obstacle for the success of point-of-care or remote diagnostics. Unlike plasma collection devices that rely solely on microchannels that restrict sample volume and throughput, we demonstrate the use of a hybrid micro/mesoscale sedimentation chamber to enable >99% capture of cells from millilitre blood samples in less than two minutes.

We demonstrate single biomolecule detection and quantification within sub-nanolitre droplets through application of Cylindrical Illumination Confocal Spectrosocpy (CICS) and droplet confinement within a retractable microfluidic constriction.

The success of therapeutic strategies within the fields of regenerative medicine, including tissue engineering, biomaterials engineering, and cell and tissue transplantation science, relies on researchers' understanding of the complex cellular microenvironments that occur within functional tissue. Microfabricated biomedical platforms provide tools for researchers to study cellular response to various stimuli with micro- and nanoscale spatial control. Initial studies utilizing relatively passive means of microenvironmental control have provided the fundamental knowledge required to begin to design microculture platforms that closely mimic these biological systems. In this review, we discuss second-generation cell and tissue culture platforms that utilize active components, borrowed from work in the development of microelectromechanical systems (MEMS). These microsystems offer the unprecedented opportunity to fabricate culture platforms designed to match tissue-specific growth parameters. In addition, the adoption of MEMS components opens up the door for future integration with the burgeoning field of microanalytical systems, providing analytical platforms that retain the sensitivity and resolution required within low-volume, microfluidic culture technologies.

In vitro models of the alveolo-pulmonary barrier consist of microvascular endothelial cells and alveolar epithelial cells cultured on opposing sides of synthetic porous membranes. However, these simple models do not reflect the physiological microenvironment of pulmonary cells, wherein cells are exposed to a complex milieu of mechanical and soluble stimuli. In this report, we studied alveolar epithelial (A549) and microvascular endothelial (HMEC-1) cells within varying microfluidic environments as a first step towards building a microfluidic analog of the gas-exchange interface. We fabricated polydimethylsiloxane (PDMS) microdevices for parallel studies of cell growth under multiple flow rates. Cells adhered and proliferated in the microculture chambers for shear stresses up to approximately 2 x 10(-3) dynes/cm(2), corresponding to media turnover rates of approximately 53 seconds. Proliferation of these cells into confluent monolayers and expression of cell-specific markers (SP-A and CD-31) demonstrated successful pulmonary cell culture in microscale devices, a first for alveolar epithelial cells. These results represent the initial steps towards the development of microfluidic analogs of the alveolo-pulmonary barrier and tissue engineering of the lung.

Evaluation of: Neely LA, Patel S, Garver J et al.: A single-molecule method for the quantitation of microRNA gene expression. Nat. Methods 3(1), 41–46 (2006) [1] . This study extended single-molecule detection (SMD) techniques to accurately quantitate microRNA (miRNA) gene expression in human tissue. In all, the expression of 45 different miRNAs was quantified from 16 different tissues and several mature miRNAs were found to be expressed at low levels in tissues where they were undetectable previously. This SMD assay showed the capability of quantitating miRNA expression from as little as 50 ng of total RNA. Furthermore, by incorporating locked nucleic acid (LNA)–DNA oligonucleotide probes, this method was shown to be highly specific and capable of discrimination between miRNA targets that differed by as little as a single nucleotide. Future extensions of SMD miRNA assays and integration with current microfluidic and nanotechnologies may prove essential in pushing miRNA profiling to the fundamental limits for discerning variation in the spatiotemporal regulation of miRNA expression across various tissue and cell types, as well as disease states, and establishing the clinical utility of miRNA expression in cancer diagnosis.

Structured Abstract Authors – Elisseeff J, Puleo C, Yang F, Sharma B. Objectives – Tissue engineering has the potential to make a significant impact on improving tissue repair in the craniofacial system. The general strategy for tissue engineering includes seeding cells on a biomaterial scaffold. The number of scaffold and cell choices for tissue engineering systems is continually increasing and will be reviewed. Design – Multilayered hydrogel systems were developed to coculture different cell types and develop osteochondral tissues for applications including the temporomandibular joint. Experimental variable – Hydrogels are one form of scaffold that can be applied to cartilage and bone repair using fully differentiated cells, adult and embryonic stem cells. Outcome measure – Case studies represent an overview of our laboratory's investigations. Results – Bilayered scaffolds to promote tissue development and the formation of more complex osteochondral tissues were developed and proved to be effective. Conclusion – Tissue engineering provides a venue to investigate tissue development of mutant or diseased cells and potential therapeutics.

The unique photophysical properties of semiconductor quantum dots (QDs) have made them ideal for use as spectral labels and luminescent probes. In this review, applications are presented in which QDs function as active participants in nanoscale biosensor assemblies, where replacing traditional molecular fluorophores results in improved assay performance. Specific focus is on disease detection with applications including multiplexed target detection, mutation detection by coincidence analysis and QD-based FRET reporters for miRNA detection and DNA methylation analysis.

In an effort to improve the physiologic relevance of existing in vitro models for alveolar cells, we present a microfluidic platform which provides an air-interface in a dynamic system combining microfluidic and suspended membrane culture systems. Such a system provides the ability to manipulate multiple parameters on a single platform along with ease in cell seeding and manipulation. The current study presents a comparison of the efficacy of the hybrid system with conventional platforms using assays analyzing the maintenance of function and integrity of A549 alveolar epithelial cell monolayer cultures. The hybrid system incorporates bio-mimetic nourishment on the basal side of the epithelial cells along with an open system on the apical side of the cells exposed to air allowing for easy access for assays.

We performed on-chip DNA methylation analysis using methylation-specific PCR (MSP) within a high throughput microfluidic droplet array. The device uses of the oil phase as a companion fluid for both sample actuation and compartmentalization. These technical advantages allow for infusion of minute amounts of sample for arrayed MSP analysis, without the added complexities inherent in microfluidic droplet-based studies. Ease of use of this micro device is exemplified by analysis of two tumor suppressor promoters, p15 and TMS1 using an on-chip methylation assay. These results were consistent with standard MSP protocols, yet the simplicity of the droplet-in-oil microfluidic PCR platform provides an easy and efficient tool for DNA methylation analysis in a large-scale arrayed manner.

The development of single-molecule detection (SMD) technologies that allow measurements of intra and intermolecular interactions at the most fundamental level has greatly advanced basic research in biophysics and biochemistry over the past two decades. Incorporated with the use of a variety of new molecular probes, probe strategies and functional nanomaterials, SMD has recently been demonstrated as a powerful tool in medical applications. The use of SMD in quantitative study of both specific DNA sequences and mutations has resulted in extraordinary performance characteristics (sensitivity, specificity and accuracy) unmatched by conventional ensemble detection methods. The rapid advances in microfluidics and MEMS technologies have further made possible on-chip SMD analysis in a high-throughput and cost-effective manner. An integrated molecular analysis platform based on a combination of SMD, nanotechnology and microfluidics promises to tackle the current technological challenges in genomic analysis such as amplification-free detection of genomic DNA and accurate quantification of minute gene expression changes.

Fluorescence correlation spectroscopy (FCS) can resolve the intrinsic fast-blinking kinetics (FBKs) of fluorescent molecules that occur on the order of microseconds. These FBKs can be heavily influenced by the microenvironments in which the fluorescent molecules are contained. In this work, FCS is used to monitor the dynamics of fluorescence emission from Cy5 labeled on DNA probes. We found that the FBKs of Cy5 can be tuned by having more or less unpaired guanines (upG) and thymines (upT) around the Cy5 dye. The observed FBKs of Cy5 are found to predominantly originate from the isomerization and back-isomerization processes of Cy5, and Cy5-nucleobase interactions are shown to slow down these processes. These findings lead to a more precise quantification of DNA hybridization using FCS analysis, in which the FBKs play a major role rather than the diffusion kinetics. We further show that the alterations of the FBKs of Cy5 on probe hybridization can be used to differentiate DNA targets with single-nucleotide differences. This discrimination relies on the design of a probe-target-probe DNA three-way-junction, whose basepairing configuration can be altered as a consequence of a single-nucleotide substitution on the target. Reconfiguration of the three-way-junction alters the Cy5-upG or Cy5-upT interactions, therefore resulting in a measurable change in Cy5 FBKs. Detection of single-nucleotide variations within a sequence selected from the Kras gene is carried out to validate the concept of this new method.

The demand for safer and more efficient non-viral gene vectors has increased with the recent progress of genetic medicine. Appropriate nanocomplex assembly of DNA and gene carriers is critical for successful cellular entry and transfection. However, there is a lack of knowledge on this self-assembly process, let alone the controllability of monodisperse nanocomplexes. This paper describes a novel platform integrating nanobiophotonics (quantum dots-mediated FRET) and microfluidic technology to determine binding kinetics that govern the structural and chemical properties of DNA nanocomplexes. We anticipate that this method will elucidate mechanistic and kinetic insights into the self-assembly process of nanocomplexes which may facilitate the rational design of more efficient gene carriers. In addition, a microfluidic platform offers many advantages, including small volume, fast response to external stimulations, continuous monitoring and real-time control of reaction environments, which may be potentially used to generate more monodisperse complexes.

Mesenchymal stem cells (MSCs) are potentially useful cells for musculoskeletal tissue engineering. However, controlling MSC differentiation and tissue formation in vivo remains a challenge. There is a significant need for well-defined and efficient protocols for directing MSC behaviors in vivo. We hypothesize that morphogenetic signals from chondrocytes may regulate MSC differentiation. In micromass culture of MSCs, incubation with chondrocyte-conditioned medium (CCM) significantly enhanced the production of cartilage specific matrix including type II collagen. In addition, incubation of MSCs with conditioned medium supplemented with osteogenic factors induced more osteogenesis and accumulation of calcium and increased ALP activity. These findings reveal that chondrocyte-secreted factors promote chondrogenesis as well as osteogenesis of MSCs during in vitro micromass culture. Moreover, when MSCs expanded with chondrocyte-conditioned medium were encapsulated in hydrogels and subsequently implanted into athymic mice, basophilic extracellular matrix deposition characteristic of neocartilage was evident. These results indicate that articular chondrocytes produce suitable morphogenetic factors that induce the differentiation program of MSCs in vitro and in vivo.

In this report, we review several single-molecule detection (SMD) methods and newly developed nanocrystal-mediated single-fluorophore strategies for ultrasensitive and specific analysis of genomic sequences. These include techniques, such as quantum dot (QD)-mediated fluorescence resonance energy transfer (FRET) technology and dual-color fluorescence coincidence and colocalization analysis, which allow separation-free detection of low-abundance DNA sequences and mutational analysis of oncogenes. Microfluidic approaches developed for use with single-molecule detection to achieve rapid, low-volume, and quantitative analysis of nucleic acids, such as electrokinetic manipulation of single molecules and confinement of sub-nanoliter samples using microfluidic networks integrated with valves, are also discussed.Copyright © 2007 by ASME

The transcription factor (TF) Sp1 is a well-known RNA polymerase II transcription activator that binds to GC-rich recognition sites in a number of essential cellular and viral promoters. In addition, direct interference of Sp1 binding to DNA cognate sites using DNA-interacting compounds may provide promising therapies for suppression of cancer progression and viral replication. In this study, we present a rapid, sensitive and cost-effective evaluation of a GC intercalative drug, doxorubicin (DOX), in dissociating the Sp1-DNA complex using fluorescence correlation spectroscopy (FCS) in a microfluidic system. FCS allows assay miniaturization without compromising sensitivity, making it an ideal analytical method for integration of binding assays into high-throughput, microfluidic platforms. A polydimethylsiloxane (PDMS)-based microfluidic chip with a mixing network is used to achieve specific drug concentrations for drug titration experiments. Using FCS measurements, the IC50 of DOX on the dissociation of Sp1-DNA complex is estimated to be 0.55 microM, which is comparable to that measured by the electrophoretic mobility shift assay (EMSA). However, completion of one drug titration experiment on the proposed microfluidic-FCS platform is accomplished using only picograms of protein and DNA samples and less than 1 h total assay time, demonstrating vast improvements over traditional ensemble techniques.

This paper describes the fabrication and application of microfluidic devices containing collagen vitrigel (CV) used as both a functional and sacrificial cell growth substrate for the development of corneal microtissue patches. Within the device, vacuum fixation of the CV in a dehydrated state enables quick integration with standard multilayer soft lithographic techniques, while on-chip rehydration results in a gel-like collagen substrate for microfluidic cell culture. Fluidic connectivity to both the apical and basal side of the CV permits bilayered culture of epithelium and supporting stromal cell layers. In addition, microfluidic introduction of a collagenase etching media enables sacrificial degradation of the supporting CV membrane for development of barrier tissue constructs containing minimal synthetic substrate. The utility of this platform was evaluated by miniaturizing the standard transepithelial permeability (TEP) assay in order to measure the integrity of an array of corneal tissue micropatches.

The recent proliferation of platforms designed to handle arrays of nano- and picolitre volumes is in response to the need to perform biological assays on discrete entities, such as single cells. However, a critical challenge associated with this trend for in vitro compartmentalization is the need for highly sensitive, yet low-volume detection platforms. In this paper, we coupled confocal fluorescence detection with recirculating microfluidic control to perform single particle DNA assays within five nL chambers. The performance of this low-volume assay was shown to match that of traditional single molecule detection platforms. However, volume requirements per measurement were nearly 3 orders of magnitude less than conventional systems, enabling future integration with lab-on-a-chip systems that require discrete or digitalized sample processing.