Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts. Traction forces are generated through myosin-actin interactions and play an important role in our physiology. During development, they enable cells to move from one location to the next in order to form the early structures of tissue. Traction forces help in the healing processes. They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body. These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor. The most common method by which to study cells in vitro has been to use a glass or polystyrene dish. However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces. Our lab has developed a technique to overcome these limitations. The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process. The pillars we use are 3 μm in diameter, 10 μm tall, and are configured in a regular array with 9 μm center-to-center spacing. But these physical dimensions can be readily varied to accommodate a variety of studies. We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS. We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections. We call these substrates microfabricated post-array-detectors, or mPADs. Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.     

Changes in surface topologies of chondrocytes subjected to mechanical forces: an AFM analysis

The cartilage is composed of chondrocytes embedded in a matrix of collagen fibrils interspersed within a network of proteoglycans and is constantly exposed to biomechanical forces during normal joint movement. Characterization of the surface morphology, cytoskeletal structure, adherance and elastic properties of these mechanosensitive cells are crucial in understanding the effects of mechanical forces around a cell and how a cell responds to changes in its physical environment. In this work, we employed the atomic force microscope (AFM) to image cultured chondrocytes before and after subjecting them to mechanical forces in the presence or absence of interleukin-1β to mimic inflammatory conditions. Nanoscale imaging and quantitative measurements from AFM data revealed that there are distinct changes in cell-surface topology and cytoskeleton arrangement in the cells following treatment with mechanical forces, IL-1β or both. Our findings for the first time demonstrate that cultured chondrocytes are amenable to high-resolution AFM imaging and dynamic tensile forces may help overcome the effect of inflammatory factors on chondrocyte response.     

New approach to isolate mesenchymal stem cell (MSC) from human umbilical cord blood

HUCB (human umbilical cord blood) has been frequently used in clinical allogeneic HSC (haemopoietic stem cell) transplant. However, HUCB is poorly recognized as a rich source of MSC (mesenchymal stem cell). The aim of this study has been to establish a new method for isolating large number of MSC from HUCB to recognize it as a good source of MSC. HUCB samples were collected from women following their elective caesarean section. The new method (Clot Spot method) was carried out by explanting HUCB samples in mesencult complete medium and maintained in 37°C, in a 5% CO2 and air incubator. MSC presence was established by quantitative and qualitative immunophenotyping of cells and using FITC attached to MSC phenotypic markers (CD29, CD73, CD44 and CD105). Haematopoietic antibodies (CD34 and CD45) were used as negative control. MSC differentiation was examined in neurogenic and adipogenic media. Immunocytochemistry was carried out for the embryonic markers: SOX2 (sex determining region Y-box 2), OLIG-4 (oligodendrocyte-4) and FABP-4 (fatty acid binding protein-4). The new method was compared with the conventional Rosset Sep method. MSC cultures using the Clot Spot method showed 3-fold increase in proliferation rate compared with conventional method. Also, the cells showed high expression of MSC markers CD29, CD73, CD44 and CD105, but lacked the expression of specific HSC markers (CD34 and CD45). The isolated MSC showed some differentiation by expressing the neurogenic (SOX2 and Olig4) and adipogenic (FABP-4) markers respectively. In conclusion, HUCB is a good source of MSC using this new technique.     

Measurement of mechanical forces generated by plant P-protein aggregates (forisomes)

Mechanical forces generated by forisomes were measured using a microfabricated polymer cantilever sensor. The forces were simultaneously measured in both the longitudinal and radial directions. Sensors were fabricated from polystyrene using the sacrificial layer micromolding process. The sensor response was simulated using finite element analysis. Forces in the longitudinal direction ranged from 84 to 136 nN and forces in the radial direction were 22–61 nN. This device offers a new approach to measuring small magnitude biological forces. In addition, the ability to accurately measure forces generated by forisomes is an important step toward their implementation as functional structures in microdevices.     

Identification of an opd (organophosphate degradation) gene in an Agrobacterium isolate

We isolated a bacterial strain, Agrobacterium radiobacter P230, which can hydrolyze a wide range of organophosphate (OP) insecticides. A gene encoding a protein involved in OP hydrolysis was cloned from A. radiobacter P230 and sequenced. This gene (called opdA) had sequence similarity to opd, a gene previously shown to encode an OP-hydrolyzing enzyme in Flavobacterium sp. strain ATCC 27551 and Brevundimonas diminuta MG. Insertional mutation of the opdA gene produced a strain lacking the ability to hydrolyze OPs, suggesting that this is the only gene encoding an OP-hydrolyzing enzyme in A. radiobacter P230. The OPH and OpdA proteins, encoded by opd and opdA, respectively, were overexpressed and purified as maltose-binding proteins, and the maltose-binding protein moiety was cleaved and removed. Neither protein was able to hydrolyze the aliphatic OP malathion. The kinetics of the two proteins for diethyl OPs were comparable. For dimethyl OPs, OpdA had a higher k(cat) than OPH. It was also capable of hydrolyzing the dimethyl OPs phosmet and fenthion, which were not hydrolyzed at detectable levels by OPH.     

Ecological aspects of an isolate of Steinernema diaprepesi (Rhabditida: Steinernematidae) from Argentina

Ecological aspects of Steinernema diaprepesi isolate SRC were studied to evaluate the species potential as biological control agent of insect pests. Under laboratory conditions, the following aspects were determined: the nematode life cycle, pathogenicity to several arthropods, reproductive capacity, tolerance to desiccation, effect of temperature on survival and infectivity of infective juveniles (IJs), and influence of soil texture and soil water potential on the isolate. The parasitic cycle on last-instar larvae of Galleria mellonella at 25°C was completed 8 days after infection. The nematode showed high virulence to lepidopteran larvae, being limited or nil in the remaining orders of arthropods evaluated. An acceptable offspring production of S. diaprepesi was confirmed in the species G. mellonella and S. frugiperda, suggesting that the isolate would have potential for control of lepidopteran larvae. Optimum temperature for reproduction was 20–25°C. IJs survived exposure to a range of temperatures between 10 and 40°C, with a significant reduction in the number of live IJs at 40°C. The nematodes remained infective at 20–40°C. IJ mortality was 100% on day 6 of exposure to 85% RH. The movement of IJs observed in the soil column experiments revealed that the isolate uses a cruiser-type search strategy. Soil texture and water potential significantly influenced IJ movement, search and penetration of G. mellonella larvae. The efficacy of this isolate was found to be favoured in sandy soils, regardless of the soil water potential.     

Division orientation: disentangling shape and mechanical forces

Oriented cell divisions are essential for the generation of cell diversity and for tissue shaping during morphogenesis. Cells in tissues are mechanically linked to their neighbors, upon which they impose, and from which they experience, physical force. Recent work in multiple systems has revealed that tissue-level physical forces can influence the orientation of cell division. A long-standing question is whether forces are communicated to the spindle orienting machinery via cell shape or directly via mechanosensing intracellular machinery. In this article, we review the current evidence from diverse model systems that show spindles are oriented by tissue-level physical forces and evaluate current models and molecular mechanisms proposed to explain how the spindle orientation machinery responds to extrinsic force.     

Balanced mechanical forces and microtubule contribution to fibroblast contraction

Fibroblast locomotion is thought to generate tractional forces which lead to contraction and reorganisation of collagen in tissue development and repair. A culture force monitor device (CFM) was used to measure changes in force in fibroblast populated collagen lattices, which resulted from cytoskeletal reorganisation by cytochalasin B, colchicine, vinblastine, and taxol. Microfilament disruption abolished contraction forces, microtubule disruption elicited a new peak of contraction, while taxol stabilisation of microtubules produced a gradual fall in measured force across the collagen gel. Based on these measurements, it is suggested that the cell can be viewed as an engineering structure in which residual intracellular forces, from contractile microfilaments, exert compressive loading on microtubular elements. This microtubular structure appears to act as a “balanced space frame” (analogous to an aeroplane chassis), maintaining cell shape and consequently storing a residual internal tension (RIT). In dermal fibroblasts this hidden RIT was up to 33% of the measurable force exerted on the collagen gel. Phenotypic differences between space frame organisation and RIT levels could explain site and pathological variations in fibroblast contraction. © 1996 Wiley-Liss, Inc.     

Stabilizing to disruptive transition of focal adhesion response to mechanical forces

Strong mechanical forces can, obviously, disrupt cell–cell and cell–matrix adhesions, e.g., cyclic uniaxial stretch induces instability of cell adhesion, which then causes the reorientation of cells away from the stretching direction. However, recent experiments also demonstrated the existence of force dependent adhesion growth (rather than dissociation). To provide a quantitative explanation for the two seemingly contradictory phenomena, a microscopic model that includes both integrin–integrin interaction and integrin–ligand interaction is developed at molecular level by treating the focal adhesion as an adhesion cluster. The integrin clustering dynamics and integrin–ligand binding dynamics are then simulated within one unified theoretical frame with Monte Carlo simulation. We find that the focal adhesion will grow when the traction force is higher than a relative small threshold value, and the growth is dominated by the reduction of local chemical potential energy by the traction force. In contrast, the focal adhesion will rupture when the traction force exceeds a second threshold value, and the rupture is dominated by the breaking of integrin–ligand bonds. Consistent with the experiments, these results suggest a force map for various responses of cell adhesion to different scales of mechanical force.     

In situ seed baiting to isolate germination-enhancing fungi for an epiphytic orchid,Dendrobium aphyllum (Orchidaceae)

Orchid conservation efforts, using seeds and species-specific fungi that support seed germination, require the isolation, identification, and germination enhancement testing of symbiotic fungi. However, few studies have focused on developing such techniques for the epiphytes that constitute the majority of orchids. In this study, conducted in Xishuangbanna Tropical Botanical Garden, Yunnan, China, we used seeds of Dendrobium aphyllum, a locally endangered and medicinally valuable epiphytic orchid, to attract germination promoting fungi. Of the two fungi isolated from seed baiting, Tulasnella spp. and Trichoderma spp., Tulasnella, enhanced seed germination by 13.6 %, protocorm formation by 85.7 %, and seedling development by 45.2 % (all P?Epulorhiza, another seed germination promoting fungi isolated from Cymbidium mannii, also enhanced seed germination (6.5 %; P?P?Trichoderma suppressed seed germination by 26.4 % (P?Tulasnella was the only treatment that produced seedlings. Light increased seed imbibition, protocorm formation, and two-leaved seed development of Tulasnella inoculated seeds (P?Tulasnella be introduced for facilitating D. aphyllum seed germination at the protocorm formation stage and that light be provided for increasing germination as well as further seedling development. Our findings suggest that in situ seed baiting can be used to isolate seed germination-enhancing fungi for the development of seedling production for conservation and reintroduction efforts of epiphytic orchids such as D. aphyllum.     

Taphonomic and paleoecologic implications of flow-induced forces on concavo-convex articulate brachiopods: an experimental approach

The Orientation of benthic marine organisms may be disturbed by flow-induced forces (i.e. drag and lift) caused by wave and current activity. Drag and lift are partly a function of organism size and shape. Consequently, morphology may affect stability (defined as resistance to reorientation, flipping, or entrainment) both during the life of an organism and after its death. An understanding of drag-and-lift effects is therefore essential to the interpretation of paleoecology and biostratinomic processes. An experimental method for quantifying the relative effects of flow-induced forces is described. These forces are measured during flume experiments using transducers and plaster replicas of fossils. As an illustration of the method's potential for taphonomic research, results from experiments investigating the effects of concavo-convex morphologies of articulate brachiopods are presented. Concave-up and convex-up orientations are commonly used to infer paleohydraulic conditions. Two geniculate brachiopods (Rafinesquina alternata and Leptaena richrnondensis) and three flattened forms (a second morphotype of Rafinesquina altemata, Strophodonta demissa , and Tropidoleptus carinatus) were tested in convex-up and concave-up postures and in three azimuthal orientations (hingeline oriented upstream, hingeline downstream, and hingeline parallel to flow). Concave-up orientations consistently exhibit higher drag than convex-up orientations, and this supports the common observation that valved fossils are typically found convex up in paleoenvironments dominated by traction transport. The presence of geniculation significantly increases drag. Lift is relatively insignificant for all models in most orientations. □ Taphonomy, paleoecology, brachiopods, flow-induced forces, transport.     

5-HT(3)R binding of lerisetron: an interdisciplinary approach to drug-Receptor interactions

The design, synthesis, and use of lerisetron-based molecular probes to investigate the 5-HT(3)R binding site are described. A SAR study, which involved distance and electronic parameter modifications of lerisetron's N-benzyl group, resulted in the discovery of a partial agonist.     

Nutrition in terrestrial isopods (Isopoda: Oniscidea): an evolutionary-ecological approach

The nutritional morphology, physiology and ecology of terrestrial isopods (Isopoda: Oniscidea) is significant in two respects. (1) Most oniscid isopods are truly terrestrial in terms of being totally independent of the aquatic environment. Thus, they have evolved adaptations to terrestrial food sources. (2) In many terrestrial ecosystems, isopods play an important role in decomposition processes through mechanical and chemical breakdown of plant litter and by enhancing microbial activity. While the latter aspect of nutrition is discussed only briefly in this review, I focus on the evolutionary ecology of feeding in terrestrial isopods. Due to their possessing chewing mouthparts, leaf litter is comminuted prior to being ingested, facilitating both enzymatic degradation during gut passage and microbial colonization of egested faeces. Digestion of food through endogenous enzymes produced in the caeca of the midgut glands (hepatopancreas) and through microbial enzymes, either ingested along with microbially colonized food or secreted by microbial endosymbionts, mainly takes place in the anterior part of the hindgut. Digestive processes include the activity of carbohydrases, proteases, dehydrogenases, esterases, lipases, arylamidases and oxidases, as well as the nutritional utilization of microbial cells. Absorption of nutrients is brought about by the hepatopancreas and/or the hindgut epithelium, the latter being also involved in osmoregulation and water balance. Minerals and metal cations are effectively extracted from the food, while overall assimilation efficiencies may be low. Heavy metals are stored in special organelles of the hepatopancreatic tissue. Nitrogenous waste products are excreted via ammonia in its gaseous form, with only little egested along with the faeces. Nonetheless, faeces are characterized by high nitrogen content and provide a favourable substrate for microbial colonization and growth. The presence of a dense microbial population on faecal material is one reason for the coprophagous behaviour of terrestrial isopods. For the same reason, terrestrial isopods prefer feeding on decaying rather than fresh leaf litter, the former also being more palatable and easier to digest. Acceptable food sources are detected through distance and contact chemoreceptors. The 'quality' of the food source determines individual growth, fecundity and mortality, and thus maintenance at the population level. Due to their physiological adaptations to feeding on and digesting leaf litter, terrestrial isopods contribute strongly to nutrient recycling during decomposition processes. Yet, many of these adaptations are still not well understood.     

The ptotic (witch's) chin deformity: an excisional approach.

Ptosis of the chin pad is common and can be seen in patients of all ages. It may be associated with too little or (at times) too much anterior chin projection. Often there is an associated deep submental skin crease present. Frequently, the primary concern of the patient is the appearance or exaggeration of chin ptosis in smiling ("dynamic" ptosis). This report describes a flexible approach to the correction of developmental (and some iatrogenic) ptotic chin deformities. The key element in the approach is the direct excision of sagging or excess chin fat, muscle, and skin. No attempt is made to reposition or lift ptosis-prone soft tissues. If a deep submental skin crease is present, it too is excised. If the chin needs added anterior projection, it is accomplished with a stable alloplastic chin implant. The approach is uniquely suited to correct anterior overprojection caused by an excess of soft tissue at the front of the chin and has been successful in correcting the "dynamic" ptosis that appears with smiling.     

Chemical synthesis of the hexanucleotide d(A-C-C-A-G-C) required to isolate fibroin mRNA on an affinity column.

The synthesis of the hexanucleotide d)A-C-C-A-G-C), complementary to the 2 major triplets of fibroin mRNA, using the phosphotriester methodology is described. The protected dinucleotides ((MeO)2Tr)dbzA.anC, ((MeO)2Tr)danC.bzA and ((meO)2Tr)dacG.anC were synthesized; the latter two were detritylated and joined in stepwize fashion to the 1st to form the protected hexanucleotide ((MeO)2Tr)dbzA.anC.anC.bzA.acG.anC. The latter was deblocked with NH3 and acid to form the hexanucleotide d(A-C-C-A-G-C). In view of the ability of a prototype affinity column, oligo dC-cellulose, to isolate fibroin mRNA, prospects appear excellent for the d(A-C-C-A-G-C)-cellulose affinity column isolation of fibroin mRNA.