Control of embryonic lung branching morphogenesis by the Rho activator,cytotoxic necrotizing factor 1

BACKGROUND: Lung development is sensitive to physiological stresses, and its development may be impaired by physical distortion, as in patients with congenital diaphragmatic hernia. Yet, little is known about how mechanical forces can influence lung morphogenesis. Studies with cultured cells suggest that cytoskeletal tension may play a key role in growth control. Since the small GTPase Rho plays an important role in the control of cell tension generation, we carried out studies to test the hypothesis that changes in Rho-mediated cell tension may influence branching morphogenesis. METHODS: Embryonic lung buds from timed pregnant Swiss Webster mice were microdissected on Embryonic Day 12 (E12), and whole organs were cultured in serum-free medium in the presence of the Rho activator cytotoxic necrotizing factor 1 (CNF-1) for 48 h. Serial measurements of the degree of epithelial branch formation and tissue maturation were performed using light microscopy and computerized image analysis. RESULTS: At 48 h, embryonic lungs treated with 2 ng/ml CNF-1 increased their terminal bud count by 236 +/- 18% (P = 0.01) compared with 132 +/- 2% for untreated controls. However, dose-response experiments revealed biphasic behavior: at a higher dose of CNF-1 (200 ng/ml), bud number was actually decreased relative to controls (43 +/- 1%, P < 0.001). Histological analysis revealed that individual glands appeared to be more highly developed at low-dose CNF-1, whereas the high dose produced gland contraction. CONCLUSIONS: These data support a potential role for Rho and cytoskeletal tension in control of epithelial pattern formation during lung development.     

Cell tension, matrix mechanics, and cancer development

Oncologists often diagnose cancer based on a change of tissue stiffness sensed by palpation, yet cancer researchers generally focus on biochemical signaling mechanisms. Tumors are more rigid because they have a stiffer extracellular matrix. A new study shows that this alteration of matrix mechanics activates integrins, which not only promotes mitogenic signaling through Erk but also cell contractility through Rho, which can further increase matrix stiffness. This establishes a positive feedback loop that switches on the malignant phenotype in mammary epithelial cells. This mechanical "autocrine loop" brings solid-state mechanotransduction on a par with oncogenic signaling pathways in malignant transformation.     

Thimerosal – Is it really irrelevant?

Recently, several investigators claimed that thimerosal is one of the most irrelevant allergens existing in screening for contact dermatitis. 508 patients who were suspected to have allergic contact dermatitis were patch tested at our clinic. They completed a questionnaire including medical, demographic and occupational details. We used the standard tray of chemotechnique diagnostics (Malmö, Sweden) and additional series, which were case relevant. The relevance of the allergic reaction to thimerosal was scored from 1 to 6. 19 patients (3.7%) had an allergic reaction to thimerosal. 6 (31.5%) had a definite relevance and 8 (42.1%) had a probable relevance. Only 3 patients (15.8%) had an irrelevant reaction. SPIN value (significance–prevalence index number) was 2281. We found a high proportion of mechanics (42.1%) among the patients who had positive reaction to thimerosal (P < 0.0001). Although previous reports found thimerosal highly irrelevant, our daily experience being supported by the above data indicates that positive reactions to thimerosal could be relevant for many patients.     

Tropism of herpes simplex virus type 1 to nonmelanoma skin cancers

Background Current treatments for nonmelanoma skin cancer include surgery, Mohs micrographic surgery, radiation, cryosurgery, photodynamic therapy, local chemotherapy and application of immunomodulators such as imiquimod. However, all have a 5‐year recurrence rate of 1–40%. Gene therapy for the treatment of skin cancers is a promising new approach, as delivery of the vectors to the skin is simple and safety issues can be properly addressed. Objectives To develop an ex‐vivo organ culture system for basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) tumours, and to test the feasibility of applying oncolytic viruses to these tumours. Methods We first optimized conditions for the maintenance of BCC and SCC tissues in organ culture, and demonstrated viability of the tissues ex vivo for 3–7 days. The tropism of two potential oncolytic viral vectors, herpes simplex virus type 1 (HSV‐1) and adenovirus (AD), was next evaluated. Results Immunohistological analysis revealed that HSV‐1 targeted tumour cells that expressed p63 and did not express keratin 15 or keratin 14 markers of keratinocytes. Further examination indicated that uninfected BCC and SCC tissues express two isoforms of p63 mRNA, and HSV‐1 infection specifically enhanced expression of the TAp63 isoform. Furthermore, following infection, both HSV‐1 and AD induced apoptosis in the BCC and SCC cells as indicated by the induction of activated caspase‐3. Conclusions The results indicated a specific pattern of viral tropism to skin cancer cells that are critical for maintenance of the tumour. This new experimental system should aid in the analysis of new therapeutic modalities, such as oncolytic viruses, for future treatment of these skin tumours.     

Angiostatic steroids. Method of discovery and mechanism of action

A new class of steroids has been found that inhibits angiogenesis in the presence of nonanticoagulant heparin. Tetrahydrocortisol is the most potent of the naturally occurring angiostatic steroids. It is a metabolite of cortisone that circulates in the blood, appears in the urine, and was previously believed to be biologically inactive. Both the plasma form and the urinary form are antiangiogenic. The mechanism of action of these steroids depends on their ability to specifically alter basement membrane turnover in growing capillary blood vessels. These steroids represent a prototype of angiogenesis inhibitors that may find potential therapeutic use as adjuncts to anticancer therapy, and in diseases dominated by abnormal neovascularization, i.e., angiogenesis-dependent diseases.     

Retropubic synthetic midurethral slings: Techniques and outcomes

Retropubic midurethral slings have evolved to become one of the standard therapies for treatment of stress urinary incontinence. The current slings can be placed through a small vaginal incision, with minimal dissection, in the ambulatory setting. With improvements in technique, complications are minimal. Although the tension-free vaginal tape has the most prospective data in the literature, the suprapubic arc sling appears to have equal cure and complication rates in shorterterm studies. Other retropubic slings have a paucity of data, and long-term equivalency studies have yet to be reported. This article describes the techniques of the most commonly performed retropubic synthetic slings and reports on efficacy and outcomes based on recently published studies.     

From the Cover: Field-evolved resistance by western corn rootworm to multiple Bacillus thuringiensis toxins in transgenic maize

The widespread planting of crops genetically engineered to produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) places intense selective pressure on pest populations to evolve resistance. Western corn rootworm is a key pest of maize, and in continuous maize fields it is often managed through planting of Bt maize. During 2009 and 2010, fields were identified in Iowa in which western corn rootworm imposed severe injury to maize producing Bt toxin Cry3Bb1. Subsequent bioassays revealed Cry3Bb1 resistance in these populations. Here, we report that, during 2011, injury to Bt maize in the field expanded to include mCry3A maize in addition to Cry3Bb1 maize and that laboratory analysis of western corn rootworm from these fields found resistance to Cry3Bb1 and mCry3A and cross-resistance between these toxins. Resistance to Bt maize has persisted in Iowa, with both the number of Bt fields identified with severe root injury and the ability western corn rootworm populations to survive on Cry3Bb1 maize increasing between 2009 and 2011. Additionally, Bt maize targeting western corn rootworm does not produce a high dose of Bt toxin, and the magnitude of resistance associated with feeding injury was less than that seen in a high-dose Bt crop. These first cases of resistance by western corn rootworm highlight the vulnerability of Bt maize to further evolution of resistance from this pest and, more broadly, point to the potential of insects to develop resistance rapidly when Bt crops do not achieve a high dose of Bt toxin.The global area devoted to transgenic crops producing insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) has increased rapidly over the past 15 y, with Bt crops covering more than 69 million hectares in 2012 (1). Most of this area was planted in Bt cotton and Bt maize (1). Benefits of Bt crops include effective management of target pests, decreased use of conventional insecticides, and reduced harm to nontarget organisms (2–5). However, the evolution of resistance could diminish these benefits. The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a major pest of maize, with larval feeding on maize roots and associated management costs causing economic losses in excess of $1 billion per year (6). Through 2013, three Bt toxins have been used in transgenic maize for management of western corn rootworm: Cry3Bb1, mCry3A, and Cry34/35Ab1 (7).In the United States and elsewhere, commercial registration of a Bt crop is accompanied by a resistance-management plan to delay the onset of pest resistance. Resistance management for Bt crops has focused on the refuge strategy, in which refuges of non-Bt crops allow the survival of Bt-susceptible insects, which may mate with resistant insects that survive on the Bt crop (8). To the extent that the heterozygous progeny from these matings have lower fitness on a Bt crop than their Bt-resistant parent, delays in resistance may be achieved, and these delays in resistance increase with the quantity of refuge (9). Additionally, refuges are far more effective in delaying resistance when Bt crops achieve a high dose of toxin against a target pest. High-dose Bt crops kill more than 99.99% of susceptible insects and render resistance a functionally recessive trait (9, 10). None of the currently commercialized Bt maize targeting the western corn rootworm is high dose, so the risk of resistance is increased (11, 12).In 2003, Cry3Bb1 maize was registered by the United States Environmental Protection Agency (US EPA) for management of western corn rootworm larvae (7). In 2009, farmers in Iowa observed severe injury to Cry3Bb1 maize by larval western corn rootworm in the field, and subsequent laboratory assays revealed that this injury was associated with Cry3Bb1 resistance (13). More fields with Cry3Bb1 resistance were identified in 2010 (14), and research in fields identified in 2009 as harboring Cry3Bb1-resistant western corn rootworm found no difference in survival for this pest between non-Bt maize and Cry3Bb1 maize (11). Current threats to Bt maize include the spread of Bt-resistant western corn rootworm and the loss of additional Bt toxins through the presence of cross-resistance. In this paper we report that injury to Cry3Bb1 maize in the field has persisted through 2011 and expanded to include mCry3A maize. Analysis of western corn rootworm collected in 2011 revealed that (i) severe injury to Cry3Bb1 maize and mCry3A maize in the field was associated with resistance, and (ii) cross-resistance between Cry3Bb1 and mCry3A was present. These results demonstrate that insects can evolve resistance rapidly to Bt crops that are not high dose and raise concerns about the adequacy of current resistance-management strategies.     

Patterned deposition of cells and proteins onto surfaces by using three-dimensional microfluidic systems

Three-dimensional microfluidic systems were fabricated and used to pattern proteins and mammalian cells on a planar substrate. The three-dimensional topology of the microfluidic network in the stamp makes this technique a versatile one with which to pattern multiple types of proteins and cells in complex, discontinuous structures on a surface. The channel structure, formed by the stamp when it is in contact with the surface of the substrate, limits migration and growth of cells in the channels. With the channel structure in contact with the surface, the cells stop dividing once they form a confluent layer. Removal of the stamp permits the cells to spread and divide.