Optogenetic analysis of a nociceptor neuron and network reveals ion channels acting downstream of primary sensors.

Background: Nociception generally evokes rapid withdrawal behavior in order to protect the tissue from harmful insults. Most nociceptive neurons responding to mechanical insults display highly branched dendrites, an anatomy shared by Caenorhabditis elegans FLP and PVD neurons, which mediate harsh touch responses. Although several primary molecular nociceptive sensors have been characterized, less is known about modulation and amplification of noxious signals within nociceptor neurons.

Three-dimensional in vitro tri-culture platform to investigate effects of crosstalk between mesenchymal stem cells, osteoblasts, and adipocytes.

The bone marrow niche for mesenchymal stem cells (MSCs) contains different amounts of bone and fat that vary with age and certain pathologies. How this dynamic niche environment may affect their differentiation potential and/or healing properties for clinical applications remains unknown, largely due to the lack of physiologically relevant in vitro models. We developed an enabling platform to isolate and study effects of signaling interactions between tissue-scale, laminated hydrogel modules of multiple cell types in tandem.

Pattern formation by graded and uniform signals in the early Drosophila embryo.

The early Drosophila embryo is patterned by graded distributions of maternal transcription factors. Recent studies revealed that pattern formation by these graded signals depends on uniformly expressed transcriptional activators, such as Zelda. Removal of Zelda influences both the timing and the spatial expression domains for most of the genes controlled by maternal gradients.

A multispectral optical illumination system with precise spatiotemporal control for the manipulation of optogenetic reagents.

Optogenetics is an excellent tool for noninvasive activation and silencing of neurons and muscles. Although they have been widely adopted, illumination techniques for optogenetic tools remain limited and relatively nonstandardized. We present a protocol for constructing an illumination system capable of dynamic multispectral optical targeting of micrometer-sized structures in both stationary and moving objects.

Bending amplitude - a new quantitative assay of C. elegans locomotion: identification of phenotypes for mutants in genes encoding muscle focal adhesion components.

The nematode Caenorhabditis elegans uses striated muscle in its body wall for locomotion. The myofilament lattice is organized such that all the thin filament attachment structures (dense bodies, analogous to Z-disks) and thick filament organizing centers (M-lines) are attached to the muscle cell membrane. Thus, the force of muscle contraction is transmitted through these structures and allows locomotion of the worm.

A computational statistics approach for estimating the spatial range of morphogen gradients.

A crucial issue in studies of morphogen gradients relates to their range: the distance over which they can act as direct regulators of cell signaling, gene expression and cell differentiation. To address this, we present a straightforward statistical framework that can be used in multiple developmental systems. We illustrate the developed approach by providing a point estimate and confidence interval for the spatial range of the graded distribution of nuclear Dorsal, a transcription factor that controls the dorsoventral pattern of the Drosophila embryo.

Microfluidic chamber arrays for whole-organism behavior-based chemical screening.

The nematode Caenorhabditis elegans is an important model organism in genetic research and drug screening because of its relative simplicity, ease of maintenance, amenability to simple genetic manipulation, and relevance to human biology. However, their small size and mobility make nematodes difficult to physically manipulate, particularly with spatial and temporal precision. We have developed a microfluidic device to overcome these challenges and enable fast behavior-based chemical screening in C.

Imaging single-cell signaling dynamics with a deterministic high-density single-cell trap array.

Stochasticity in gene expression, protein or metabolite levels contributes to cell-cell variations, the analysis of which could lead to a better understanding of cellular processes and drug responses. Current technologies are limited in their throughput, resolution (in space, time, and tracking individual cells instead of population average) and the ability to control cellular environment. A few microfluidic tools have been developed to trap and image cells; however, in most designs available to date, there is a compromise among loading efficiency, speed, the ability to trap single cells, and density or number of trapped cells.

Gene regulation by MAPK substrate competition.

Developing tissues are patterned by coordinated activities of signaling systems, which can be integrated by a regulatory region of a gene that binds multiple transcription factors or by a transcription factor that is modified by multiple enzymes. Based on a combination of genetic and imaging experiments in the early Drosophila embryo, we describe a signal integration mechanism that cannot be reduced to a single gene regulatory element or a single transcription factor. This mechanism relies on an enzymatic network formed by mitogen-activated protein kinase (MAPK) and its substrates.

Nitric oxide production and its functional link with OIPK in tobacco defense response elicited by chitooligosaccharide.

Chitooligosaccharide (COS) or oligochitosan has been shown to induce tobacco defense responses which are connected with nitric oxide (NO) and OIPK (oligochitosan-induced Ser/Thr protein kinase). The aim of this study was to reveal the relationship between NO production and OIPK pathway in the defense response of tobacco elicited by COS. NO generation was investigated by epidermal strip bioassay and fluorophore microscope using fluorophore diaminofluorescein diacetate (DAF-2DA).

PKN-1, a homologue of mammalian PKN, is involved in the regulation of muscle contraction and force transmission in C. elegans.

To examine the in vivo functions of protein kinase N (PKN), one of the effectors of Rho small guanosine triphosphatases (GTPases), we used the nematode Caenorhabditis elegans as a genetic model system. We identified a C. elegans homologue (pkn-1) of mammalian PKN and confirmed direct binding to C.

Real-time multimodal optical control of neurons and muscles in freely behaving Caenorhabditis elegans.

The ability to optically excite or silence specific cells using optogenetics has become a powerful tool to interrogate the nervous system. Optogenetic experiments in small organisms have mostly been performed using whole-field illumination and genetic targeting, but these strategies do not always provide adequate cellular specificity. Targeted illumination can be a valuable alternative but it has only been shown in motionless animals without the ability to observe behavior output.

Predicting cytotoxic T-cell age from multivariate analysis of static and dynamic biomarkers.

Adoptive T-cell transfer therapy relies upon in vitro expansion of autologous cytotoxic T cells that are capable of tumor recognition. The success of this cell-based therapy depends on the specificity and responsiveness of the T cell clones before transfer. During ex vivo expansion, CD8+ T cells present signs of replicative senescence and loss of function.

A microfluidic array for large-scale ordering and orientation of embryos.

Quantitative studies of embryogenesis require the ability to monitor pattern formation and morphogenesis in large numbers of embryos, at multiple time points and in diverse genetic backgrounds. We describe a simple approach that greatly facilitates these tasks for Drosophila melanogaster embryos, one of the most advanced models of developmental genetics. Based on passive hydrodynamics, we developed a microfluidic embryo-trap array that can be used to rapidly order and vertically orient hundreds of embryos.

[Protective effect of Salvia on ischemia-reperfusion hepatic injury in rats].

Objective: To detect the expressions of nuclear factor-Kappa B (NF-κB) and intercellular adhesion molecule-1 (ICAM-1) after hepatic ischemia-reperfusion injury so as to study the protective effect of Salvia in rats.

Methods: The hepatic ischemia-reperfusion rat model was established by the method of Nauta. The rats were randomly divided into 3 groups: sham operation (S), ischemia-reperfusion (I/R) and Salvia preconditioning (P).

High-throughput study of synaptic transmission at the neuromuscular junction enabled by optogenetics and microfluidics.

Over the past several years, optogenetic techniques have become widely used to help elucidate a variety of neuroscience problems. The unique optical control of neurons within a variety of organisms provided by optogenetics allows researchers to probe neural circuits and investigate neuronal function in a highly specific and controllable fashion. Recently, optogenetic techniques have been introduced to investigate synaptic transmission in the nematode Caenorhabditis elegans.

Long-term high-resolution imaging and culture of C. elegans in chip-gel hybrid microfluidic device for developmental studies.

Developmental studies in multicellular model organisms such as Caernohabditis elegans rely extensively on the ability to cultivate and image animals repeatedly at the cell or subcellular level. However, standard high-resolution imaging techniques require the use of anaesthetics for immobilization, and may have undesirable side effects on development. Thus such techniques are not ideal in allowing the same animals to grow and be imaged throughout development to observe specific developmental processes.

Long-term spatially defined coculture within three-dimensional photopatterned hydrogels.

Spatially controlled coculture in three-dimensional environments that appropriately mimic in vivo tissue architecture is a highly desirable goal in basic scientific studies of stem cell physiological processes (e.g., proliferation, matrix production, and tissue repair) and in enhancing the development of novel stem-cell-based clinical therapies for a variety of ailments.

Microfluidics-enabled phenotyping, imaging, and screening of multicellular organisms.

This paper reviews the technologies that have been invented in the last few years on high-throughput phenotyping, imaging, screening, and related techniques using microfluidics. The review focuses on the technical challenges and how microfluidics can help to solve these existing problems, specifically discussing the applications of microfluidics to multicellular model organisms. The challenges facing this field include handling multicellular organisms in an efficient manner, controlling the microenvironment and precise manipulation of the local conditions to allow the phenotyping, screening, and imaging of the small animals.

Elevated homocysteine and hypertension in Xinjiang Province, China.

Background: Few data are available comparing homocysteine (Hcy) levels in patients with hypertension in different ethnic groups.

Objectives: This study sought to determine whether there are associations of blood pressure and Hcy levels in patients with hypertension from Xinjiang Province, China.

Methods: We examined the serum total Hcy levels in a total of 451 Chinese of various ethnic groups (Han n=234 [51.

Continuously perfused, non-cross-contaminating microfluidic chamber array for studying cellular responses to orthogonal combinations of matrix and soluble signals.

We present a microfluidic cell culture array with unique versatility and parallelization for experimental trials requiring perfusion cultures. Specifically, we realize a rectangular chamber array in a PDMS device with three attributes: (i) continuous perfusion; (ii) flow paths that forbid cross-chamber contamination; and (iii) chamber shielding from direct perfusion to minimize shear-induced cell behaviour. These attributes are made possible by a bridge-and-underpass architecture, where flow streams travel vertically to pass over (or under) channels and on-chip valves.

Pachymic acid, a novel compound for anti-rejection: effect in rats following cardiac allograft transplantation.

Background: Pachymic acid (PA), a natural triterpenoid, is known to significantly reduce cell proliferation and induce apoptosis in vitro through initiation of mitochondria dysfunction. However, its effect on immune cells and anti-rejection following organ transplantation remains unknown.

Methods: In this study, we investigated PA as a treatment to control acute rejection occurred in rats which had accepted cardiac transplantation.

Automated high-throughput cell microsurgery on-chip.

Laser cell kill is an established tool for studying cells' roles during development and behavior, but its use has been limited due to the manual and low-throughput nature. We demonstrate here a technique combining multiplexing microfluidic manipulation of Caenorhabditis elegans and software for image processing and automation, allowing for high-throughput cell ablations.