Spatial and Temporal Control of 3D Hydrogel Viscoelasticity through Phototuning.

The mechanical properties of the extracellular environment can regulate a variety of cellular functions, such as spreading, migration, proliferation, and even differentiation and phenotypic determination. Much effort has been directed at understanding the effects of the extracellular matrix (ECM) elastic modulus and, more recently, stress relaxation on cellular processes. In physiological contexts such as development, wound healing, and fibrotic disease progression, ECM mechanical properties change substantially over time or space.

The matricellular protein Drosophila Cellular Communication Network Factor is required for synaptic transmission and female fertility.

Within the extracellular matrix, matricellular proteins are dynamically expressed nonstructural proteins that interact with cell surface receptors, growth factors, and proteases, as well as with structural matrix proteins. The cellular communication network factors family of matricellular proteins serve regulatory roles to regulate cell function and are defined by their conserved multimodular organization. Here, we characterize the expression and neuronal requirement for the Drosophila cellular communication network factor family member.

Intact central nervous system cellular quantitation reveals sexual dimorphism.

Establishing with precision the quantity and identity of the cell types of the brain is a prerequisite for a detailed compendium of gene and protein expression in the central nervous system (CNS). Currently, however, strict quantitation of cell numbers has been achieved only for the nervous system of . Here, we describe the development of a synergistic pipeline of molecular genetic, imaging, and computational technologies designed to allow high-throughput, precise quantitation with cellular resolution of reporters of gene expression in intact whole tissues with complex cellular constitutions such as the brain.

A conditional GABAergic synaptic vesicle marker for Drosophila.

Background: Throughout the animal kingdom, GABA is the principal inhibitory neurotransmitter of the nervous system. It is essential for maintaining the homeostatic balance between excitation and inhibition required for the brain to operate normally. Identification of GABAergic neurons and their GABA release sites are thus essential for understanding how the brain regulates the excitability of neurons and the activity of neural circuits responsible for numerous aspects of brain function including information processing, locomotion, learning, memory, and synaptic plasticity, among others.

A conditional glutamatergic synaptic vesicle marker for Drosophila.

Glutamate is a principal neurotransmitter used extensively by the nervous systems of all vertebrate and invertebrate animals. It is primarily an excitatory neurotransmitter that has been implicated in nervous system development, as well as a myriad of brain functions from the simple transmission of information between neurons to more complex aspects of nervous system function including synaptic plasticity, learning, and memory. Identification of glutamatergic neurons and their sites of glutamate release are thus essential for understanding the mechanisms of neural circuit function and how information is processed to generate behavior.

Miniature neurotransmission is required to maintain Drosophila synaptic structures during ageing.

The decline of neuronal synapses is an established feature of ageing accompanied by the diminishment of neuronal function, and in the motor system at least, a reduction of behavioural capacity. Here, we have investigated Drosophila motor neuron synaptic terminals during ageing. We observed cumulative fragmentation of presynaptic structures accompanied by diminishment of both evoked and miniature neurotransmission occurring in tandem with reduced motor ability.

Advances in Extracellular Matrix-Mimetic Hydrogels to Guide Stem Cell Fate.

In the fields of regenerative medicine and tissue engineering, stem cells offer vast potential for treating or replacing diseased and damaged tissue. Much progress has been made in understanding stem cell biology, yielding protocols for directing stem cell differentiation toward the cell type of interest for a specific application. One particularly interesting and powerful signaling cue is the extracellular matrix (ECM) surrounding stem cells, a network of biopolymers that, along with cells, makes up what we define as a tissue.

Engineering hydrogels for personalized disease modeling and regenerative medicine.

Technological innovations and advances in scientific understanding have created an environment where data can be collected, analyzed, and interpreted at scale, ushering in the era of personalized medicine. The ability to isolate cells from individual patients offers tremendous promise if those cells can be used to generate functional tissue replacements or used in disease modeling to determine optimal treatment strategies. Here, we review recent progress in the use of hydrogels to create artificial cellular microenvironments for personalized tissue engineering and regenerative medicine applications, as well as to develop personalized disease models.

Octopamine neuron dependent aggression requires dVGLUT from dual-transmitting neurons.

Neuromodulators such as monoamines are often expressed in neurons that also release at least one fast-acting neurotransmitter. The release of a combination of transmitters provides both "classical" and "modulatory" signals that could produce diverse and/or complementary effects in associated circuits. Here, we establish that the majority of Drosophila octopamine (OA) neurons are also glutamatergic and identify the individual contributions of each neurotransmitter on sex-specific behaviors.

Characterization of Drosophila octopamine receptor neuronal expression using MiMIC-converted Gal4 lines.

Octopamine, the invertebrate analog of norepinephrine, is known to modulate a large variety of behaviors in Drosophila including feeding initiation, locomotion, aggression, and courtship, among many others. Significantly less is known about the identity of the neurons that receive octopamine input and how they mediate octopamine-regulated behaviors. Here, we characterize adult neuronal expression of MiMIC-converted Trojan-Gal4 lines for each of the five Drosophila octopamine receptors.

Demonstration of a Simple Epitope Tag Multimerization Strategy for Enhancing the Sensitivity of Protein Detection Using vAChT.

The expression and distribution of a protein can provide critical information about its function in a cell. For some neuronal proteins this information may include neurotransmitter (NT) usage and sites of NT release. However, visualizing the expression of a protein within a given neuron is often challenging because most neurons are intricately intermingled with numerous other neurons, making individual neuronal expression difficult to discern, especially since many neuronal genes are expressed at low levels.

Identification of cell context-dependent YAP-associated proteins reveals β and β integrin mediate YAP translocation independently of cell spreading.

Yes-associated protein (YAP) is a transcriptional regulator and mechanotransducer, relaying extracellular matrix (ECM) stiffness into proliferative gene expression in 2D culture. Previous studies show that YAP activation is dependent on F-actin stress fiber mediated nuclear pore opening, however the protein mediators of YAP translocation remain unclear. Here, we show that YAP co-localizes with F-actin during activating conditions, such as sparse plating and culturing on stiff 2D substrates.

Matrix stiffness induces a tumorigenic phenotype in mammary epithelium through changes in chromatin accessibility.

In breast cancer, the increased stiffness of the extracellular matrix is a key driver of malignancy. Yet little is known about the epigenomic changes that underlie the tumorigenic impact of extracellular matrix mechanics. Here, we show in a three-dimensional culture model of breast cancer that stiff extracellular matrix induces a tumorigenic phenotype through changes in chromatin state.

Varying PEG density to control stress relaxation in alginate-PEG hydrogels for 3D cell culture studies.

Hydrogels are commonly used as artificial extracellular matrices for 3D cell culture and for tissue engineering. Viscoelastic hydrogels with tunable stress relaxation have recently been developed, and stress relaxation in the hydrogels has been found to play a key role in regulating cell behaviors such as differentiation, spreading, and proliferation. Here we report a simple but precise materials approach to tuning stress relaxation of alginate hydrogels with polyethylene glycol (PEG) covalently grafted onto the alginate.

Conditional Synaptic Vesicle Markers for .

The release of neurotransmitters from synaptic vesicles (SVs) at pre-synaptic release sites is the principle means by which information transfer between neurons occurs. Knowledge of the location of SVs within a neuron can thus provide valuable clues about the location of neurotransmitter release within a neuron and the downstream neurons to which a given neuron is connected, important information for understanding how neural circuits generate behavior. Here the development and characterization of four conditional tagged SV markers for is presented.

t-GRASP, a targeted GRASP for assessing neuronal connectivity.

Background: Understanding how behaviors are generated by neural circuits requires knowledge of the synaptic connections between the composite neurons. Methods for mapping synaptic connections, such as electron microscopy and paired recordings, are labor intensive and alternative methods are thus desirable.

New Method: Development of a targeted GFP Reconstitution Across Synaptic Partners(GRASP) method, t-GRASP, for assessing neural connectivity is described.

Analysis of the Postoperative Periarticular Environment and Influence on Sustained Drug Delivery From a Gel Formulation.

Regional intraarticular delivery of local anesthetics is effective in treating postoperative pain following total knee or hip replacement. Recent research efforts have been only partially successful in achieving sustained release of the analgesic agent, in part due to limited understanding of the biological environment into which these formulations are administered. This study aimed to detail the composition and properties of postoperative periarticular fluid (PO-PAF).

Stress relaxing hyaluronic acid-collagen hydrogels promote cell spreading, fiber remodeling, and focal adhesion formation in 3D cell culture.

The physical and architectural cues of the extracellular matrix (ECM) play a critical role in regulating important cellular functions such as spreading, migration, proliferation, and differentiation. Natural ECM is a complex viscoelastic scaffold composed of various distinct components that are often organized into a fibrillar microstructure. Hydrogels are frequently used as synthetic ECMs for 3D cell culture, but are typically elastic, due to covalent crosslinking, and non-fibrillar.

Maintenance of neural progenitor cell stemness in 3D hydrogels requires matrix remodelling.

Neural progenitor cell (NPC) culture within three-dimensional (3D) hydrogels is an attractive strategy for expanding a therapeutically relevant number of stem cells. However, relatively little is known about how 3D material properties such as stiffness and degradability affect the maintenance of NPC stemness in the absence of differentiation factors. Over a physiologically relevant range of stiffness from ∼0.

Extracellular Matrix Stiffening Induces a Malignant Phenotypic Transition in Breast Epithelial Cells.

Tumors are much stiffer than healthy tissue, and progressively stiffen as the cancer develops. Tumor stiffening is largely the result of extracellular matrix (ECM) remodeling, for example, deposition and crosslinking of collagen I. Well established models have demonstrated the influence of the microenvironment in regulating tissue homeostasis, with matrix stiffness being a particularly influential mediator.

Fibrin-based stem cell containing scaffold improves the dynamics of burn wound healing.

For severe burn injuries, successful medical intervention is accomplished by rapidly and safely providing physical barriers that can cover damaged skin tissues, thereby preventing critical danger of extensive bleeding and infection. Despite availability of a large assortment of wound coverage options, the etiology of wound healing is rather complex leading to significant defects in skin repair. The use of cell-mediated treatment approaches in combination with bioengineered wound coverage constructs may provide the missing tool to improve wound healing outcomes.

Injectable thermosensitive gelling delivery system for the sustained release of lidocaine.

Background: Patients undergoing arthroplasty require appropriate postsurgical pain relief. Analgesia is typically achieved through bolus doses of short-acting local anesthetics and with oral analgesics such as opiates, which are associated with systemic side effects. By formulating an injectable thermosensitive gelling system containing lidocaine, sustained and local delivery can be achieved following a single administration.

Dynamic phototuning of 3D hydrogel stiffness.

Hydrogels are widely used as in vitro culture models to mimic 3D cellular microenvironments. The stiffness of the extracellular matrix is known to influence cell phenotype, inspiring work toward unraveling the role of stiffness on cell behavior using hydrogels. However, in many biological processes such as embryonic development, wound healing, and tumorigenesis, the microenvironment is highly dynamic, leading to changes in matrix stiffness over a broad range of timescales.