Abstract. We have used an isometric force transducer to study contraction of two types of nonmuscle cells in tissue culture . This method permits the quantitative measurement of contractile force generated by cells of defined type under the influence of external agents while allowing detailed morphological observation . Chick embryo fibroblasts (CEF), which form a contractile network inside a collagen matrix, and human umbilical vein endothelial cells (HUVE), which are located in a monolayer on the surface of the collagen matrix, were studied . CEF and HUVE in 10% FCS produce a substantial tension of 4.5 ± 0.2 x 104 dynes/cm2 and 6.1 x 104 dynes/cm2, respectively. Both M MST of what is known about force generation in animal cells has come from studies of muscle. However, the generation of force is essential to such nonmuscle cell functions as wound contraction, gastrulation, and connective tissue morphogenesis . We have developed a simple quantitative technique to study isometric contraction of cells in tissue culture . This technique allows mechanical study of a variety ofnonmuscle and smooth muscle cells undergoing biochemical and genetic manipulations .Nonmuscle cells are believed to generate force through interaction between actin and myosin as in smooth and skeletal muscle (1,20,25,26) . Recently it has been demonstrated that nonmuscle cells express isoforms ofnonsarcomeric myosin distinct from those expressed in smooth muscle (18,19) . The nature of these isoforms has been characterized ; however, their function remains unclear. Thus, a quantitative study of isometric contraction in a tissue culture system may provide insight into the mechanical specialization of nonmuscle myosin isoforms.Unlike skeletal muscle, in which the cytoskeleton is arranged in a well-defined, stable organization, nonmuscle cells rearrange their cytoskeletons in response to physiological stimuli such as growth factors and secretagogues. It is clear that studies correlating cell mechanics with cytoskeletal morphology and biochemistry are needed to understand mechanisms through which nonmuscle cells control their mechanical machinery. In this study, we present the results of initial efforts to assay quantitatively the force produced in cell types contract when stimulated with thrombin, generating a force per cell cross-sectional area of -105 dynes/cm2, a value approximately an order of magnitude less than smooth muscle . The integrity of the actin cytoskeleton is essential for force generation, as disruption of actin microfilaments with cytochalasin D results in a rapid disappearance of force . Intact microtubules appear to reduce isometric force exerted by CEF, as microtubule-disrupting drugs result in increased tension . Contraction by HUVE precedes a dramatic rearrangement of actin microfilaments from a circumferential ring to stress fibers .vitro by nonmuscle cells as they undergo stimulation and morphological change. We present measurements of the isometric force developed by a contractile network ofchick embryo fibroblasts (CEF...

Tissue models reconstituted from cells and extracellular matrix (ECM) simulate natural tissues. Cytoskeletal and matrix proteins govern the force exerted by a tissue and its stiffness. Cells regulate cytoskeletal structure and remodel ECM to produce mechanical changes during tissue development and wound healing. Characterization and control of mechanical properties of reconstituted tissues are essential for tissue engineering applications. We have quantitatively characterized mechanical properties of connective tissue models, fibroblast-populated matrices (FPMs), via uniaxial stretch measurements. FPMs resemble natural tissues in their exponential dependence of stress on strain and linear dependence of stiffness on force at a given strain. Activating cellular contractile forces by calf serum and disrupting F-actin by cytochalasin D yield "active" and "passive" components, which respectively emphasize cellular and matrix mechanical contributions. The strain-dependent stress and elastic modulus of the active component were independent of cell density above a threshold density. The same quantities for the passive component increased with cell number due to compression and reorganization of the matrix by the cells.

Recent advances in sequencing technology allow genome-scale approaches to cancer mutation discovery. Such data-intensive methods have been applied to cutaneous squamous cell carcinomas (SCCs) and melanomas but have not, to our knowledge, been applied to basal cell carcinomas (BCCs). We used whole-exome sequencing to characterize the mutational landscape of sporadic BCCs. We show that BCCs are the most mutated type of human cancer. Tumors from anatomical regions with chronic UV exposure were associated with higher mutation rates than those with intermittent exposure. The majority of all mutations (75.7%) were UV signature. Using a conventional binomial probability model, several genes were found mutated significantly. However, this model assumes a uniform distribution of mutations throughout the genome. We also used a more stringent approach called InVEx that uses a permutation-based framework to pick drivers from passengers. After correction for multiple hypothesis testing, InVEx identified only PTCH1 (Patched 1) as having a significant functional mutation burden. We also found three genes, STAT5B, CRNKL1, and NEBL, with mutational hot spots at a single base in 3 of 12 tumors sequenced. Our findings support the central role of PTCH1 mutations in BCC genesis. Moreover, our discovery of the uniquely high number of mutations in this tumor may lend insight into its biological behavior.

Microtubules have been proposed to function as rigid struts which oppose cellular contraction. Consistent with this hypothesis, microtubule disruption strengthens the contractile force exerted by many cell types. We have investigated an alternative explanation for the mechanical effects of microtubule disruption: that microtubules modulate the mechanochemical activity of myosin by influencing phosphorylation of the myosin regulatory light chain (LC2o). We measured the force produced by a population of fibroblasts within a collagen lattice attached to an isometric force transducer. Treatment of cells with nocodazole, an inhibitor of microtubule polymerization, stimulated an isometric contraction that reached its peak level within 30 min and was typically 30-45% of the force increase following maximal stimulation with 30%o fetal bovine serum. The contraction following nocodazole treatment was associated with a 2-to 4-fold increase in LC2. phosphorylation. The increases in both force and LC20 phosphorylation, after addition of nocodazole, could be blocked or reversed by stabilizing the microtubules with paclitaxel (former generic name, taxol). Increasing force and LC20 phosphorylation by pretreatment with fetal bovine serum decreased the subsequent additional contraction upon microtubule disruption, a finding that appears inconsistent with a load-shifting mechanism. Our results suggest that phosphorylation of LC20 is a common mechanism for the contractions stimulated both by microtubule poisons and receptor-mediated agonists. The modulation of myosin activity by alterations in microtubule assembly may coordinate the physiological functions of these cytoskeletal components.

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The phosphatidylcholine formula popularly used in subcutaneous injections for fat dissolution works primarily as a detergent causing nonspecific lysis of cell membranes. Our findings suggest that sodium deoxycholate is the major active component responsible for cell lysis. Detergent substances may have a role in eliminating unwanted adipose tissue. It is advised that physicians use caution until adequate safety data are available.

BACKGROUND Mesotherapy was originally conceived in Europe as a method of utilizing cutaneous injections containing a mixture of compounds for the treatment of local medical and cosmetic conditions. Although mesotherapy was traditionally employed for pain relief, its cosmetic applications, particularly fat and cellulite removal, have recently received attention in the United States. Another treatment for localized fat reduction, which was popularized in Brazil and uses injections of phosphatidylcholine, has been erroneously considered synonymous with mesotherapy. Despite their attraction as purported ''fat-dissolving'' injections, the safety and efficacy of these novel cosmetic treatments remain ambiguous to most patients and physicians.

Detection and sequencing of mutations from clinical specimens is often complicated by the presence of an excess of nonmutated cells. To facilitate the detection and sequencing of minority mutations from clinical specimens, we developed wild-type blocking polymerase chain reaction (WTB-PCR). This technique allows sensitive detection of minority mutations in a tissue sample containing excess wild-type DNA. In WTB-PCR, a nonextendable locked nucleic acid (LNA) oligonucleotide binds tightly to a region of wild-type DNA known to develop point mutations. This LNA sequence blocks amplification of wild-type DNA during PCR while permitting amplification of mutant exon 15. Our results show that the LNA blocking oligonucleotide inhibits amplification of wildtype DNA in a dose-dependent manner. WTB-PCR was able to detect mutant DNA in clinical samples of melanoma tissue containing an excess of nonmelanoma cells. This method was also able to detect small amounts of point mutated or tandem mutated DNA diluted with a much larger concentration of wild-type DNA. This rapid and simple assay overcomes the limitations of current methods to detect minority mutations. The potential applications of WTB-PCR include early diagnosis and prognosis of various cancers.