An extensive class of small RNAs in Caenorhabditis elegans.

The lin-4 and let-7 antisense RNAs are temporal regulators that control the timing of developmental events in Caenorhabditis elegans by inhibiting translation of target mRNAs. let-7 RNA is conserved among bilaterian animals, suggesting that this class of small RNAs [microRNAs (miRNAs)] is evolutionarily ancient. Using bioinformatics and cDNA cloning, we found 15 new miRNA genes in C. elegans. Several of these genes express small transcripts that vary in abundance during C. elegans larval development, and three of them have apparent homologs in mammals and/or insects. Small noncoding RNAs of the miRNA class appear to be numerous and diverse.

Frequency dependence of electric field modulation of fibroblast protein synthesis.

The effect of electric current on protein biosynthesis in mammalian fibroblasts was investigated with neonatal bovine fibroblast-populated collagen matrices. The field strength dependence of electric field modulation of proline incorporation into extracellular and intracellular protein was measured over a frequency range from 0.1 to 1000 hertz. A frequency- and amplitude-dependent reduction in the rate of incorporation was observed. In tissues containing cells aligned either parallel or perpendicular to the electric field, this response was dependent on the orientation of the cells relative to the direction of the applied electric field. This study demonstrates that currents of physiological strength can stimulate alterations in biosynthesis and thereby may influence tissue growth, remodeling, and repair.

Applications of artificial intelligence: relationships between mass spectra and pharmacological activity of drugs.

The possibility that the mass spectrum and pharmacological activity of a compound may be directly related has been explored with the help of various computer-based pattern-recognition techniques. The relationship appears to hold at least for tranquilizers and sedatives, and compounds with one or the other of these two pharmacological activities can thus be classified from their mass spectra with a high degree of accuracy.

Drosophila LAR regulates R1-R6 and R7 target specificity in the visual system.

Different classes of photoreceptor neurons (R cells) in the Drosophila compound eye connect to specific targets in the optic lobe. Using a behavioral screen, we identified LAR, a receptor tyrosine phosphatase, as being required for R cell target specificity. In LAR mutant mosaic eyes, R1-R6 cells target to the lamina correctly, but fail to choose the correct pattern of target neurons. Although mutant R7 axons initially project to the correct layer of the medulla, they retract into inappropriate layers. Using single cell mosaics, we demonstrate that LAR controls targeting of R1-R6 and R7 in a cell-autonomous fashion. The phenotypes of LAR mutant R cells are strikingly similar to those seen in N-cadherin mutants.

Structure and function analysis of LIN-14, a temporal regulator of postembryonic developmental events in Caenorhabditis elegans.

During postembryonic development of Caenorhabditis elegans, the heterochronic gene lin-14 controls the timing of developmental events in diverse cell types. Three alternative lin-14 transcripts are predicted to encode isoforms of a novel nuclear protein that differ in their amino-terminal domains. In this paper, we report that the alternative amino-terminal domains of LIN-14 are dispensable and that a carboxy-terminal region within exons 9 to 13 is necessary and sufficient for in vivo LIN-14 function. A transgene capable of expressing only one of the three alternative lin-14 gene products rescues a lin-14 null mutation and is developmentally regulated by lin-4. This shows that the deployment of alternative lin-14 gene products is not critical for the ability of LIN-14 to regulate downstream genes in diverse cell types or for the in vivo regulation of LIN-14 level by lin-4. The carboxy-terminal region of LIN-14 contains an unusual expanded nuclear localization domain which is essential for LIN-14 function. These results support the view that LIN-14 controls developmental timing in C. elegans by regulating gene expression in the nucleus.

Kinetics of the chondrocyte biosynthetic response to compressive load and release.

To gain insight regarding the rate at which cartilage tissue can sense and respond to a dynamic mechanical stimulus, we have examined the time-course of changes in biosynthetic activity following both the application and release of a static compressive stress. Cartilage harvested from the reserve zone of calf epiphyseal plate was subjected to unconfined static compressive stresses of 0, 0.25 and 0.5 MPa. Incorporation of [35S]sulfate and [3H]proline was measured during loading periods of less than 1 to 26 h and after preloading periods of 0.5, 2 or 12 h. During loading, total incorporation decreased to steady levels with time constants estimated to be 0.25-4 h (proline) and 1-5 h (sulfate). Proline incorporation exceeded control levels for 3 h after release of a 2 or 12 h preload. Sulfate incorporation remained depressed for at least 4 h after release of a 12 h preload and remained at control levels following release of 0.5 and 2 h preloads. We conclude that the modulation of proline incorporation by both loading and load release is faster than the modulation of sulfate incorporation. Furthermore, the response to unloading is not just the inverse of the response to loading; this nonlinearity suggests that the response to dynamic loading would not be determined simply by the time average component of the dynamic load.

The relative thermal stability of tissue macromolecules and cellular structure in burn injury.

When tissue is subjected to higher than physiological temperatures, protein and cell organelle structures can be altered resulting in cell death and subsequent tissue necrosis. A burn injury can be stratified into three main zones, coagulation, stasis and edema, which correlate with the extent of heat exposure and thermal properties of the tissue. While there has been considerable effort to characterize the time-temperature dependence of the injury, relatively little attention has been paid to the other important variable, the thermal susceptibility of the tissue. In the present study, we employ a standard physical chemistry approach to predict the level of denaturation at supraphysiological temperatures of 12 vital proteins as well as RNA, DNA and cell membrane components. Melting temperatures and unfolding enthalpies of the cellular components are used as input experimental parameters. This approach allows us to establish a relation between the level of denaturation of critical cellular components and clinical manifestations of the burn through the characteristic zones of the injury. Specifically, we evaluate the degree of molecular alteration for characteristic temperature profiles at two different depths (Mid-Dermis and Dermis-Fat interface) of 80 degrees C; 20s contact burn. The results of this investigation suggest that the thermal alteration of the plasma membrane is likely the most significant cause of the tissue necrosis. The lipid bilayer and membrane-bound ATPases show a high probability of thermal damage (almost 100% for the former and 85% for the latter) for short heat exposure times. These results suggest that strategies to minimize the damage in a burn injury might focus on the stabilization of the cellular membrane and membrane-bound ATPases. Further work will be required to validate these predictions in an in vivo model.

Phytophthora Leaf Spot and Foliar Blight of Pieris japonica Caused by Phytophthora citricola in California.

During 2004, containerized nursery stock of lily-of-the-valley-bush (Pieris japonica 'Flamingo', family Ericaceae) in Santa Cruz County was affected by a foliar disease. Symptoms consisted of large leaf spots, many developing at the leaf tips that ranged in size from 1 to greater than 4 cm in diameter. Spots were dark brown to almost black, generally oval to round, visible from both sides of the leaf, and did not exhibit signs of any pathogen. Lesions typically expanded and affected the entire leaf, leaf petiole, and stems, resulting in blight-like symptoms. Severely affected leaves abscised from the plant. In advanced stages of the disease, the foliage of the plant was killed. These symptoms resembled those caused by the sudden oak death (SOD) pathogen, Phytophthora ramorum (3). A Phytophthora sp. was isolated consistently from symptomatic leaf tissue. However, the species was identified as P. citricola based on morphological traits that included the following: production of semipapillate, noncaducous sporangia that were irregular in shape and occasionally had more than one apex; presence of oospores with paragynous antheridia in single culture; and radiate to slightly petaloid colony morphology (1). P. ramorum and other fungi were not recovered. Pathogenicity of four representative isolates was confirmed by gently abrading the adaxial surfaces of attached leaves with a sterile wire brush, placing a colonized agar plug (5 mm in diameter) on the surface, misting the leaf with sterile water, and then covering the plug with a plastic cap that was secured with a wire clip. Control leaves were treated in the same manner but received sterile agar plugs. Plants were maintained in a greenhouse at 23 to 25°C. After 2 days, all leaves inoculated with the isolates exhibited dark brown lesions and by day 6, lesions measured 3 cm in diameter. P. citricola was reisolated from symptomatic lesions. Sterile plug control leaves developed no symptoms. The test was repeated and the results were similar. To our knowledge, this is the first report of P. citricola causing a foliar disease of Pieris japonica in California. P. citricola has been reported as a pathogen on Pieris spp. in Ohio (2). Our finding is important because P. ramorum causes very similar symptoms on this same host (3). The occurrence of these two foliar Phytophthora spp. on this ornamental plant may complicate P. ramorum field detection during inspections and laboratory confirmations as established by quarantine regulations. References: (1) D. C. Erwin and O. K. Ribeiro. Morphology and Identification of Phytophthora Species. Pages 96-144 in: Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (2) W. W. P. Gerlach et al. Phytopathology 64:1368, 1974. (3) P. W. Tooley et al. Plant Dis. 88:993, 2004.

Endometrial stromal sarcoma mimicking submucosal myoma protruding to the vagina: MRI findings.

A 46-year-old woman complained of persistent abnormal vaginal bleeding over ten days. Her intrauterine device had been removed two years before. Soon after, she suffered from menorrhagia and metrorrhagia. An incidental finding of severe anemia was also noted. In this admission, our initial T2-weighted magnetic resonance imaging (MRI) revealed a well-demarcated mass predominantly in the uterine cavity. The mass was depicted by an isointense signal relative to the myometrium on T1-weighted images, high signal intensity on T2-weighted images, and slightly heterogeneous enhancement on post-contrast images. The patient refused surgery. After two years, follow-up MRI showed a pedunculated mass protruding into the upper third of the vagina with a stalk connecting to the posterior wall of the uterine cavity, simulating submucosal myoma. Histological diagnosis was compatible with low-grade endometrial stromal sarcoma.

Total-body skeletal muscle mass: development and cross-validation of anthropometric prediction models.

BACKGROUND: Skeletal muscle (SM) is a large body compartment of biological importance, but it remains difficult to quantify SM with affordable and practical methods that can be applied in clinical and field settings. OBJECTIVE: The objective of this study was to develop and cross-validate anthropometric SM mass prediction models in healthy adults. DESIGN: SM mass, measured by using whole-body multislice magnetic resonance imaging, was set as the dependent variable in prediction models. Independent variables were organized into 2 separate formulas. One formula included mainly limb circumferences and skinfold thicknesses [model 1: height (in m) and skinfold-corrected upperarm, thigh, and calf girths (CAG, CTG, and CCG, respectively; in cm)]. The other formula included mainly body weight (in kg) and height (model 2). The models were developed and cross-validated in nonobese adults [body mass index (in kg/m(2)) < 30]. RESULTS: Two SM (in kg) models for nonobese subjects (n = 244) were developed as follows: SM = Ht x (0.00744 x CAG(2) + 0.00088 x CTG(2) + 0.00441 x CCG(2)) + 2.4 x sex - 0.048 x age + race + 7.8, where R:(2) = 0.91, P: < 0.0001, and SEE = 2.2 kg; sex = 0 for female and 1 for male, race = -2.0 for Asian, 1.1 for African American, and 0 for white and Hispanic, and SM = 0.244 x BW + 7.80 x Ht + 6.6 x sex - 0.098 x age + race - 3.3, where R:(2) = 0.86, P: < 0.0001, and SEE = 2.8 kg; sex = 0 for female and 1 for male, race = -1.2 for Asian, 1.4 for African American, and 0 for white and Hispanic. CONCLUSION: These 2 anthropometric prediction models, the first developed in vivo by using state-of-the-art body-composition methods, are likely to prove useful in clinical evaluations and field studies of SM mass in nonobese adults.

An independent, inverse association of high-density-lipoprotein-cholesterol concentration with nonadipose body mass.

BACKGROUND: Increasing body mass index (BMI) is associated with progressively lower serum HDL-cholesterol concentrations, although the underlying body-composition compartment accounting for this unfavorable lipid change remains uncertain. OBJECTIVE: Because growing evidence favors a role of lean tissue in HDL homeostasis, the hypothesis was tested that non-adipose tissue components of body mass explain the inverse association of HDL cholesterol and BMI. DESIGN: Fasting serum lipid concentrations and body composition [total, subcutaneous, and visceral adipose tissue; adipose tissue-free mass (ATFM); and skeletal muscle by whole-body magnetic resonance imaging and body cell mass by 40K counting) were evaluated in healthy adults. Body-composition compartments were expressed as height2-normalized indexes. RESULTS: An inverse correlation was observed between serum HDL cholesterol and BMI in women (n = 68; R2 = 0.08, P = 0.023) and men (n = 61; R2 = 0.07, P = 0.046). Significant inverse correlations (P = 0.005-0.02) were also observed between HDL cholesterol and nonadipose components (ie, ATFM, skeletal muscle, and body cell mass) but not between HDL cholesterol and any adipose tissue component. The association between HDL cholesterol and ATFM remained significant after serum triacylglycerol was controlled for. When BMI was entered into the HDL cholesterol-ATFM regression model, BMI was not a significant independent variable. The strongest correlate of serum triacylglycerol was visceral adipose tissue (P = 0.002 for both women and men). CONCLUSIONS: Lean tissues and body cell mass appear to account in part for the long-observed inverse association of HDL cholesterol and BMI. These observations suggest a link between nonadipose tissue compartments and the greater cardiovascular risk associated with high BMI.

Surfactant sealing of membranes permeabilized by ionizing radiation.

Acute tissue injury and subsequent inflammation, including tissue edema and erythema, can be caused by sufficiently high levels of exposure to gamma radiation. The mechanism of this tissue injury is related to the generation of reactive oxygen intermediates (ROI) which chemically alter biological molecules and cell physiology. Cell membrane lipids are vulnerable to ROI-mediated lipid peroxidation that then leads to many of the acute tissue effects. We hypothesize that increased cell membrane permeability leading to osmotic swelling and vascular transudation is one of these effects. Thus we used adult postmitotic rhabdomyocytes in culture and microscopic fluorescence techniques to quantify radiation-induced changes in cell membrane permeability. Based on time-resolved dye flux measurements, a characteristic lag time of 34 +/- 3 min was determined between exposure to 160 Gy of gamma radiation and the decrease in membrane permeability. Administration of 0.1 mM nonionic surfactant Poloxamer 188 added to the cell medium after irradiation completely inhibited the dye loss over the time course of 2 h. Thus a reproducible model was developed for studying the mechanism of acute radiation injury and the efficacy of membrane-sealing agents. As only supportive measures now exist for treating the acute, nonlethal injuries from high-dose radiation exposure, agents that can restore cell membrane function after radiation damage may offer an important tool for therapy.

The future of biothermal engineering.

This is a report on a three-day workshop held at the Allerton House of the University of Illinois. The first day consisted of invited tutorials on topics related to biothermal engineering: biological structures, analysis of microvascular heat transfer, temperature measurement, cryobiology and cryosurgery, burns, and industrial and consumer applications. The rest of the workshop consisted of discussions in small groups and in plenary sessions dealing with relevant topics. Although the discussions endeavored to be as comprehensive as possible, the specific topics were selected by the participants based on their expertise and interests. The main areas examined were: Instrumentation, Priority applications, Mathematical modeling, Thermal injury. The reliable measurement of the temperature distribution inside the living tissue is still the premier problem of instrumentation although the measurement of other parameters, such as properties, blood perfusion or heat flux, is also of great importance. The most important applications are medical, industrial, consumer, agricultural, space, and military. The degree of sophistication needed in the analysis of specific problems varies a great deal from relatively simple heat conduction models to complicated ones including blood perfusion, anisotropy, and the influence of large blood vessels. For many applications new experimental data are still needed. There have been significant advances in the modeling of living tissue with increasing understanding of its thermal behavior. The consensus was, however, that the models will always have to be tissue or organ specific and some new models are still to be developed.

Non-linear microscale alterations in membrane transport by electropermeabilization.

The purpose of this study was to quantify the changes in cell membrane conductance in response to electropermeabilization, which may elucidate the mechanisms of tissue injury resulting from high-voltage electrical shock. A high-speed, space-clamp and voltage-clamp experimental configuration was used. The pulse parameters of an imposed transmembrane potential that are instrumental in membrane properties alteration were precisely controlled. The dynamics of the non-linear electroporation response was characterized.

Dynamics of membrane sealing in transient electropermeabilization of skeletal muscle membranes.

Large supraphysiologic transmembrane electrical potentials are known to alter the molecular organization of the bilayer lipid component of cell membranes, leading to ionic permeabilization or "electroporation". Typically, membrane electroporation is followed by several orders of magnitude increases in electrical conductance and diffusive permeability to low-molecular-weight solutes. Electroporation may be transient or stable depending on whether the membrane eventually seals or remains permeabilized. Factors that control sealing have not been well characterized. This paper describes the kinetics of membrane sealing following electroporation by pulses over a range of supraphysiologic potentials. The increase in membrane conductance is highly nonlinear during a -440-mV, 4-ms pulse and reaches two orders of magnitude greater than baseline. Electroporation and relaxation sealing kinetics are quite different, reflecting a significant hysteresis effect. Thus, it appears that the magnitude and duration of the field pulse are important factors in sealing.

The use of collagen-GAG membranes in reconstructive surgery.

Porous collagen-glycosaminoglycan (PCG) membranes with a porous silicone elastomer coating have been useful as a scaffold for dermal replacement in burn victims. Critical physicochemical parameters of these membranes include pore size, cross-link density, the percentage of glycosaminoglycan, and the degree of banding of the collagen. These factors govern the immunobiological response. Optimizing these parameters can reduce inflammation, scarring, and contraction of wounds grafted with PCG membranes. PCG membranes are currently commercially manufactured (Integra, Integra Life Sciences, New Jersey) and available for clinical use. Because clinical outcomes have improved using these membranes for burn wound coverage, other skin reconstruction problems including scar resurfacing, keloids, treatment of donor sites, and treatment of chronic wounds can be considered as potential applications. This manuscript illustrates our early experience using Integra as a CG membrane for dermal replacement in reconstructive surgery. Our results indicate that CG membranes can lead to improved compliance and appearance compared to a meshed graft and may be sequentially placed in multiple layers to correct contour deformities. Also, in one case, we observed that, if placed on a wound bed with embedded skin epithelial cells, the PCG promotes epithelialization through the PCG matrix. The use of this material results in a supple integument with many similarities to normal skin.

Pharmaceutical therapies for sealing of permeabilized cell membranes in electrical injuries.

Several years ago, we proposed that loss of cell membrane structural integrity by electroporation is a substantial cause of tissue necrosis in victims of electrical trauma. Specifically, this involves the permeabilization of the lipid bilayer by thermal and electrical forces. We further suggested that certain mild surfactants in low concentration could induce sealing of permeabilized lipid bilayers and salvage of cells that had not been extensively heat-damaged. Successful restoration of membrane transport properties using the surfactant poloxamer 188 was reported in 1992. The purpose of this study is to further examine the response of electroporated rat skeletal muscle membranes to poloxamer 188 (P188) therapy by direct assay of membrane transport properties. Experimental evidence accumulated to date suggests that P188 is effective in sealing permeabilized cell membranes both in vitro and in vivo.

Management and coordination of postacute medical care for electrical trauma survivors.

The clinical spectrum of electrical injury ranges from the absence of any external physical signs to severe multiple trauma. Reported neuropsychiatric sequelae can vary from vague complaints, which may seem unrelated to the injury in their occurrence over time or by their apparent severity, to sequelae consistent with brain injury accompanying an electrical trauma. In this report, a case study and discussion are presented on the management and coordination of post-acute care of an electrical trauma survivor. Expertise and a multidisciplinary team are essential to cohesive patient care. Patient monitoring for progressive changes and prompt intervention are needed to address the potential difficulties experienced by trauma survivors as they rehabilitate to return to their work and their activities of daily living.

Life after electrical injury. Risk factors for psychiatric sequelae.

Long-term cognitive and emotional deficits have been commonly reported in electrical injury (EI) survivors. However, it remains undetermined what factors may lead to the development of such effects in some patients and not in others. In this study, we hypothesized that certain elements of subjective EI experience may predict specific psychiatric sequelae. A group of 73 post-acute EI patients were included in this retrospective study. Statistical associations were examined between major psychiatric diagnoses (posttraumatic stress disorder and major depression) and such EI descriptors as having experienced "no-let-go" or having been knocked away on contact, as well as loss of consciousness or altered states of consciousness at the scene of the accident (including amnesia for the event). The study results will help physicians determine which patients may be at increased risk of developing psychiatric symptoms and address these issues as part of their total rehabilitation plan.

Oxidative cell membrane alteration. Evidence for surfactant-mediated sealing.

Exposure to very intense ionizing irradiation produces acute tissue sequelae including inflammation, pain, and swelling that often results in tissue fibrosis and/or necrosis. Acute tissue necrosis occurs in hours when sufficiently rapid damage to membrane lipids and proteins leads to altered membrane structure, disrupting the vital electrochemical diffusion barrier necessary for cell survival. This damage mechanism is thought to underlie the interphase death of lethally irradiated postmitotic cells such as neurons, but it has also been implicated in the rapid cell death of lymphocytes and acute vascular changes due to capillary epithelium dysfunction. It is not known whether sealing of radiation-permeabilized cell membranes will prolong survival of lethally irradiated cells or perhaps lead to repair of damaged nucleic acids. The purpose of this study is to begin to address the first question.