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In many biological applications, for example high-dimensional metabolic data, the measurements consist of several continuous measurements of subjects or tissues over multiple attributes or metabolites. Measurement values are put in a matrix with subjects in rows and attributes in columns. The analysis of such data requires grouping subjects and attributes to provide a primitive guide toward data modeling. A common approach is to group subjects and attributes separately, and construct a two-dimensional dendrogram tree, once on rows and then on columns. This simple approach provides a grouping visualization through two separate trees, which is difficult to interpret jointly. When a joint grouping of rows and columns is of interest, it is more natural to partition the data matrix directly. Our suggestion is to build a dendrogram on the matrix directly, thus generalizing the two-dimensional dendrogram tree to a three-dimensional forest. The contribution of this research to the statistical analysis of metabolic data is threefold. First, a novel spike-and-slab model in various hierarchies is proposed to identify discriminant rows and columns. Second, an agglomerative approach is suggested to organize joint clusters. Third, a new visualization tool is invented to demonstrate the collection of joint clusters. The new method is motivated over gas chromatography mass spectrometry (GCMS) metabolic data, but can be applied to other continuous measurements with spike at zero property.

Coronary artery disease is a leading cause of mortality in the U.S. and percutaneous coronary intervention with stent placement is a common treatment. Stents often fail through coronary in-stent restenosis. Periodically monitoring restenosis could help alert the physician of any problems earlier, reduce myocardial reinfarctions, and improve outcomes. Currently, there are no inexpensive and noninvasive techniques to monitor stent patency. We describe an X-ray-addressable blood pressure (X-BP) microsensor. The X-BP has a column of radio-opaque liquid that changes its length with blood pressure. The X-BP allows for the noninvasive evaluation of the pressure drop across a stent and the fractional flow reserve (FFR) on radiographs. A FFR threshold of 0.75-0.8 is clinically established as the cutoff for the identification of hemodynamically significant stenosis that requires intervention. The X-BP membrane was modeled and the X-ray signal-to-noise ratio of different sensor dimensions was experimentally determined. Based on this data, optimal design parameters were selected. The sensor was prototyped and tested under microscope with radiographs and video fluoroscopy. The sensor has a potential dynamic range of 0-200 mmHg, and can reliably resolve the clinically important pressure drop of 20%-25% across the dynamic range for an FFR value of 0.8-0.75 or less. The X-BP also has a time constant

Identification of optimal transcription factor expression patterns to direct cellular differentiation along a desired pathway presents significant challenges. We demonstrate massively combinatorial screening of temporally-varying mRNA transcription factors to direct differentiation of neural progenitor cells using a dynamically-reconfigurable magnetically-guided spotting technology for localizing mRNA, enabling experiments on millimetre size spots. In addition, we present a time-interleaved delivery method that dramatically reduces fluctuations in the delivered transcription factor copy numbers per cell. We screened combinatorial and temporal delivery of a pool of midbrain-specific transcription factors to augment the generation of dopaminergic neurons. We show that the combinatorial delivery of LMX1A, FOXA2 and PITX3 is highly effective in generating dopaminergic neurons from midbrain progenitors. We show that LMX1A significantly increases TH-expression levels when delivered to neural progenitor cells either during proliferation or after induction of neural differentiation, while FOXA2 and PITX3 increase expression only when delivered prior to induction, demonstrating temporal dependence of factor addition., eLife, 7, ISSN:2050-084X

Neurological drugs are often associated with serious side effects, yet drug screens typically focus only on efficacy. We demonstrate a novel paradigm utilizing high-throughput in vivo electrophysiology and brain activity patterns (BAPs). A platform with high sensitivity records local field potentials (LFPs) simultaneously from many zebrafish larvae over extended periods. We show that BAPs from larvae experiencing epileptic seizures or drug-induced side effects have substantially reduced complexity (entropy), similar to reduced LFP complexity observed in Parkinson’s disease. To determine whether drugs that enhance BAP complexity produces positive outcomes, we used light pulses to trigger seizures in a model of Dravet syndrome, an intractable genetic epilepsy. The highest-ranked compounds identified by BAP analysis exhibit far greater anti-seizure efficacy and fewer side effects during subsequent in-depth behavioral assessment. This high correlation with behavioral outcomes illustrates the power of brain activity pattern-based screens and identifies novel therapeutic candidates with minimal side effects., One challenge in drug screening for neurological disorders is how to accurately capture disease pathology and side effects. Here, the authors developed a multi-channel recording platform based on a zebrafish genetic model of epilepsy to screen for antiepileptic drugs.

Here, we describe an automated platform suitable for large-scale deep-phenotyping of zebrafish mutant lines, which uses optical projection tomography to rapidly image brain-specific gene expression patterns in 3D at cellular resolution. Registration algorithms and correlation analysis are then used to compare 3D expression patterns, to automatically detect all statistically significant alterations in mutants, and to map them onto a brain atlas. Automated deep-phenotyping of a mutation in the master transcriptional regulator fezf2 not only detects all known phenotypes but also uncovers important novel neural deficits that were overlooked in previous studies. In the telencephalon, we show for the first time that fezf2 mutant zebrafish have significant patterning deficits, particularly in glutamatergic populations. Our findings reveal unexpected parallels between fezf2 function in zebrafish and mice, where mutations cause deficits in glutamatergic neurons of the telencephalon-derived neocortex., National Institutes of Health (U.S.) (Director’s Pioneer Award DP1-NS082101), David & Lucile Packard Foundation. Award in Science and Engineering, Broad Institute of MIT and Harvard (SPARC Award), Epilepsy Foundation of America (Postdoctoral Fellowship)

We report the development of an add-on, chip-based, optical module—termed the Microfluidic-based Oil-immersion Lenses (μOIL) chip—which transforms any stereo microscope into a high-resolution, large field of view imaging platform. The μOIL chip consists of an array of ball mini-lenses that are assembled onto a microfluidic silicon chip. The mini-lenses are made out of high refractive index material (sapphire) and they are half immersed in oil. Those two key features enable submicron resolution and a maximum numerical aperture of ~1.2. The μOIL chip is reusable and easy to operate as it can be placed directly on top of any biological sample. It improves the resolution of a stereo microscope by an order of magnitude without compromising the field of view; therefore, we believe it could become a versatile tool for use in various research studies and clinical applications.

We present a wireless and power-free, optomechanical, implantable microsensor that can potentially be used to accurately monitor intracranial pressure (ICP) over long periods of time. The developed microsensor vertically integrates a glass mini-lens with a two-wavelength quantum dot (QD) micropillar that is photolithographically patterned on an ICP-exposed silicon nitride membrane. The operation principle is based on a novel optomechanical transduction scheme that converts ICP changes into changes in the intensity ratio of the two-wavelength, near-infrared fluorescent light emitted from the QDs. The microsensor is microfabricated using silicon bulk micromachining, and it operates at an ICP clinically relevant pressure dynamic range (0-40 mmHg). The microsensor has a maximum error of less than 15% throughout its dynamic range, and it is extremely photostable. We believe that the proposed microsensor will open up a new direction not only in ICP monitoring but in other pressure-related biomedical applications.

This paper proposes a new approach based on missing value pattern discovery for classifying incomplete data. This approach is particularly designed for classification of datasets with a small number of samples and a high percentage of missing values where available missing value treatment approaches do not usually work well. Based on the pattern of the missing values, the proposed approach finds subsets of samples for which most of the features are available and trains a classifier for each subset. Then, it combines the outputs of the classifiers. Subset selection is translated into a clustering problem, allowing derivation of a mathematical framework for it. A trade off is established between the computational complexity (number of subsets) and the accuracy of the overall classifier. To deal with this trade off, a numerical criterion is proposed for the prediction of the overall performance. The proposed method is applied to seven datasets from the popular University of California, Irvine data mining archive and an epilepsy dataset from Henry Ford Hospital, Detroit, Michigan (total of eight datasets). Experimental results show that classification accuracy of the proposed method is superior to those of the widely used multiple imputations method and four other methods. They also show that the level of superiority depends on the pattern and percentage of missing values.

Circulating tumor cells (CTCs) are believed to play an important role in metastasis, a process responsible for the majority of cancer-related deaths. But their rarity in the bloodstream makes microfluidic isolation complex and time-consuming. Additionally the low processing speeds can be a hindrance to obtaining higher yields of CTCs, limiting their potential use as biomarkers for early diagnosis. Here, a high throughput microfluidic technology, the OncoBean Chip, is reported. It employs radial flow that introduces a varying shear profile across the device, enabling efficient cell capture by affinity at high flow rates. The recovery from whole blood is validated with cancer cell lines H1650 and MCF7, achieving a mean efficiency >80% at a throughput of 10 mL h(-1) in contrast to a flow rate of 1 mL h(-1) standardly reported with other microfluidic devices. Cells are recovered with a viability rate of 93% at these high speeds, increasing the ability to use captured CTCs for downstream analysis. Broad clinical application is demonstrated using comparable flow rates from blood specimens obtained from breast, pancreatic, and lung cancer patients. Comparable CTC numbers are recovered in all the samples at the two flow rates, demonstrating the ability of the technology to perform at high throughputs.

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3(rd) instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

We present a novel implantable X-ray-addressable MEMS Blood Pressure sensor, the X-BP, for the noninvasive and cost-effective surveillance of coronary in-stent restenosis. We successfully fabricated and tested the X-BP sensor and its pressure response curve. We placed the X-BP sensor in a coronary stent and prove adequate visibility in a clinically realistic scenario.

We developed a compact, single-cell imaging system for measuring red blood cell (RBC) concentration from diluted, whole blood samples. Key element of the design is the integration of a microfabricated lens array with a commercial haemocytometer. To image large areas and therefore a larger number of cells, the field of view (FOV) can be adjusted by simply increasing the thickness of the haemocytometer coverslip. We used a FOV = 200 μm, corresponding to a resolution of 0.53 μm, to demonstrate RBC counting: counts obtained from our system were in excellent agreement with counts obtained from standard microscopy and flow cytometry over a range of dilution factors.

Frequent and accurate measurement of intraocular pressure (IOP) is required in monitoring patients who are at high risk of developing glaucoma. We present a novel electronic-free, wireless, optomechanical implantable IOP microsensor. The microsensor has a total volume of less than 1mm3 and is integrated into a keratoprosthesis (KPro). We performed ex-vivo experiments by implanting the microsensor/KPro assembly into human eyebank globes for two weeks. Our microsensor had 2 mmHg accuracy and less than 1 mmHg zero-drift.

In clinical problems, numerous factors are usually involved in a medical syndrome. New advances in medicine provide a broad range of diagnosis methods to cover all aspects of a disease. However, huge amounts of raw information may confuse clinicians and decrease decision accuracy. Computerized knowledge extraction is an active area of research in medical informatics. This paper suggests a new medical data mining approach using an advanced swarm intelligence data mining algorithm. Considering medical knowledge discovery difficulties, this approach addresses common issues such as missing value management and interactive rule extraction. Here, surgery candidate selection in temporal lobe epilepsy is the main target application. However, the general idea can be applied to other medical knowledge discovery problems. Experimental results show noticeable performance improvement in the final rule-set quality while the method is flexible and fast.

Circulating tumor cells (CTCs) are believed to play an important role in metastasis, a process accounting for the majority of cancer-related deaths. They offer a non-invasive biopsy technique to study tumors and are shown to be useful prognostic indicators. Because of their rarity in the bloodstream, microfluidic isolation techniques are complex and time-consuming, and provide yields of CTCs insufficient or non-viable for studies other than enumeration. Additionally, due to the low processing speeds, the volumes of blood processed remain limited and can be a hindrance to obtaining higher yields of CTCs and their potential use as biomarkers of early diagnosis. Here we report a novel high throughput microfluidic technology, the OncoBean Chip, employing radial flow that introduces a varying shear profile across the device and enables efficient cell capture by affinity at high flow rates. The cell recovery from whole blood was validated with cancer cell lines H1650 and MCF7, and the OncoBean Chip achieved an efficiency >80% at a throughput of 10 mL/hr, a flow rate yet achieved only in physical size based separation techniques. A cell viability of 92.91% shows that the cells recovered at high throughput could still be used for downstream analysis. Clinical competence was demonstrated in blood specimens from breast, pancreatic and lung cancer patients. The OncoBean Chip thus holds potential applications in the diagnosis of early stage cancers, where the low numbers of CTCs could be enriched using this novel device. Citation Format: Vasudha Murlidhar, Rishindra M. Reddy, Mina Zeinali, Svetlana Grabauskiene, Mostafa Ghannad-Rezaie, Max S. Wicha, Diane M. Simeone, Nithya Ramnath, Sunitha Nagrath. Radial flow microfluidic device for high-throughput affinity-based isolation of circulating tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3069. doi:10.1158/1538-7445.AM2014-3069

With powerful genetics and a translucent cuticle, the Drosophila larva is an ideal model system for live imaging studies of neuronal cell biology and function. Here, we present an easy-to-use approach for high resolution live imaging in Drosophila using microfluidic chips. Two different designs allow for non-invasive and chemical-free immobilization of 3(rd) instar larvae over short (up to 1 hour) and long (up to 10 hours) time periods. We utilized these 'larva chips' to characterize several sub-cellular responses to axotomy which occur over a range of time scales in intact, unanaesthetized animals. These include waves of calcium which are induced within seconds of axotomy, and the intracellular transport of vesicles whose rate and flux within axons changes dramatically within 3 hours of axotomy. Axonal transport halts throughout the entire distal stump, but increases in the proximal stump. These responses precede the degeneration of the distal stump and regenerative sprouting of the proximal stump, which is initiated after a 7 hour period of dormancy and is associated with a dramatic increase in F-actin dynamics. In addition to allowing for the study of axonal regeneration in vivo, the larva chips can be utilized for a wide variety of in vivo imaging applications in Drosophila.