Cloning and characterisation of Myf5 and MyoD orthologues in Xenopus tropicalis.

The myogenic regulatory genes MyoD and Myf5 are members of the bHLH transcription factor superfamily. These genes are expressed as an early response to mesoderm induction in the frog Xenopus laevis. This paper describes our work to determine the conservation of sequence, expression and function of the early myogenic genes in the closely related diploid species Xenopus tropicalis. To this end we have cloned and sequenced Xenopus tropicalis homologues of Myf5 and MyoD and found a high degree of conservation in the nucleotide and amino acid sequences of both genes. The expression of Myf5 and MyoD in Xenopus tropicalis was assayed by in situ hybridisation and the expression patterns were found to be similar to those described in Xenopus laevis. The bHLH myogenic regulatory factors are known to be able to auto- and cross-activate expression of the myogenic genes. Interestingly, however, we found that while injection of mRNA coding for either Xenopus tropicalis myf5 (Xtmyf5) or Xenopus tropicalis myoD (XtmyoD) was capable of activating expression of the endogenous Xenopus laevis myoD (XmyoD) and cardiac actin genes, neither was capable of inducing expression from the endogenous Xenopus laevis (Xmyf5) gene.

Asymmetric primitive-model electrolytes: Debye-Hückel theory, criticality, and energy bounds.

Debye-Hückel (DH) theory is extended to treat two-component size- and charge-asymmetric primitive models, focusing primarily on the 1:1 additive hard-sphere electrolyte with, say, negative ion diameters a(--) larger than the positive ion diameters a(++). The treatment highlights the crucial importance of the charge-unbalanced "border zones" around each ion into which other ions of only one species may penetrate. Extensions of the DH approach that describe the border zones in a physically reasonable way are exact at high T and low density rho and, furthermore, are also in substantial agreement with recent simulation predictions for trends in the critical parameters, T(c) and rho(c), with increasing size asymmetry. Conversely, the simplest linear asymmetric DH description, which fails to account for physically expected behavior in the border zones at low T, can violate a new lower bound on the energy (which applies generally to models asymmetric in both charge and size). Other theories, including those based on the mean spherical approximation, predict trends in the critical parameters quite opposite to those established by the simulations.

Simple mechanochemistry describes the dynamics of kinesin molecules.

Recently, Block and coworkers [Visscher, K., Schnitzer, M. J., & Block, S. M. (1999) Nature (London) 400, 184--189 and Schnitzer, M. J., Visscher, K. & Block, S. M. (2000) Nat. Cell Biol. 2, 718--723] have reported extensive observations of individual kinesin molecules moving along microtubules in vitro under controlled loads, F = 1 to 8 pN, with [ATP] = 1 microM to 2 mM. Their measurements of velocity, V, randomness, r, stalling force, and mean run length, L, reveal a need for improved theoretical understanding. We show, presenting explicit formulae that provide a quantitative basis for comparing distinct molecular motors, that their data are satisfactorily described by simple, discrete-state, sequential stochastic models. The simplest (N = 2)-state model with fixed load-distribution factors and kinetic rate constants concordant with stopped-flow experiments, accounts for the global (V, F, L, [ATP]) interdependence and, further, matches relative acceleration observed under assisting loads. The randomness, r(F,[ATP]), is accounted for by a waiting-time distribution, psi(1)(+)(t), [for the transition(s) following ATP binding] with a width parameter nu identical with (2)/<(Delta t)(2)> approximately 2.5, indicative of a dispersive stroke of mechanicity approximately 0.6 or of a few ( greater than or similar to nu - 1) further, kinetically coupled states: indeed, N = 4 (but not N = 3) models do well. The analysis reveals: (i) a substep of d(0) = 1.8--2.1 nm on ATP binding (consistent with structurally based suggestions); (ii) comparable load dependence for ATP binding and unbinding; (iii) a strong load dependence for reverse hydrolysis and subsequent reverse rates; and (iv) a large ( greater than or similar to 50-fold) increase in detachment rate, with a marked load dependence, following ATP binding.

Precise simulation of criticality in asymmetric fluids.

Extensive grand canonical Monte Carlo simulations have been performed for the hard-core square-well fluid with interaction range b=1.5 sigma. The critical exponent for the correlation length has been estimated in an unbiased fashion as nu=0.63+/-0.03 via finite-size extrapolations of the extrema of properties measured along specially constructed, asymptotically critical loci that represent pseudosymmetry axes. The subsequent location of the critical point achieves a precision of five parts in 10(4) for Tc and about 0.3% for the critical density rhoc. The effective exponents gamma+(eff) and beta(eff) indicate Ising-type critical-point values to within 2% and 5.6%, respectively, convincingly distinguishing the universality class from the "nearby" XY and n=0 (self-avoiding walk) classes. Simulations of the heat capacity CV(T,rho) and d2psigma/dT2, where psigma is the vapor pressure below Tc, suggest a negative but small Yang-Yang anomaly, i.e., a specific-heat-like divergence in the corresponding chemical potential derivative (d2 musigma/dT2) that requires a revision of the standard asymptotic scaling description of asymmetric fluids.

Force-velocity relation for growing microtubules.

Forces generated by microtubule polymerization and depolymerization are important for the biological functioning of cells. The mean growth velocity, V, under an opposing force, F, has been measured by; Science 278:856-860) for single microtubules growing in vitro, but their analysis of the data suggested that V decreased more rapidly with F than equilibrium (or "thermodynamic") theory predicted and entailed negative values for the dissociation rate and undefined (or unreasonable) values for the stall force, at which V vanishes. By contrast, considering the mean work done against the external load and allowing for load-distribution factors for the "on" and "off" rates, we find good agreement with a simple theory that yields a plausible stalling force. Although specific numerical results are sensitive to choice of fitting criteria, about 80% of the variation with load is carried by the "off" (or dissociation) rate, but, since that is small (in accordance with independent observations), the dominant force dependence comes from the "on" rate, which is associated with a displacement length, d(1), significantly longer than d(0) approximately 1/13(8.2 nm), the mean length increase per added tubulin dimer. Measuring the dispersion in length of the growing microtubules could provide a check. The theory implies that the stationary stall state (at V = 0) is not one of simple associative thermal equilibrium, as previously supposed; rather, it appears to be dissipative and kinetically controlled.

Transcript quantitation in total yeast cellular RNA using kinetic PCR.

Kinetically monitored, reverse transcriptase-initiated PCR (kinetic RT-PCR, kRT-PCR) is a novel application of kinetic PCR for high throughput transcript quantitation in total cellular RNA. The assay offers the simplicity and flexibility of an enzyme assay with distinct advantages over DNA microarray hybridization and SAGE technologies for certain applications. The reproducibility, sensitivity and accuracy of the kRT-PCR were assessed for yeast transcripts previously quantitated by a variety of methods including SAGE analysis. Changes in transcript levels between different genetic or physiological cell states were reproducibly quantitated with an accuracy of +/-20%. The assay was sufficiently sensitive to quantitate yeast transcripts over a range of more than five orders of magnitude, including low abundance transcripts encoding cell cycle and transcriptional regulators.

The force exerted by a molecular motor.

The stochastic driving force exerted by a single molecular motor (e. g., a kinesin, or myosin) moving on a periodic molecular track (microtubule, actin filament, etc.) is discussed from a general viewpoint open to experimental test. An elementary "barometric" relation for the driving force is introduced that (i) applies to a range of kinetic and stochastic models, (ii) is consistent with more elaborate expressions entailing explicit representations of externally applied loads, and (iii) sufficiently close to thermal equlibrium, satisfies an Einstein-type relation in terms of the velocity and diffusion coefficient of the (load-free) motor. Even in the simplest two-state models, the velocity-vs.-load plots exhibit a variety of contrasting shapes (including nonmonotonic behavior). Previously suggested bounds on the driving force are shown to be inapplicable in general by analyzing discrete jump models with waiting time distributions.

Identity of the universal repulsive-core singularity with Yang-Lee edge criticality.

Lattice and continuum fluid models with repulsive-core interactions typically display a dominant, critical-type singularity on the real, negative activity axis. Lai and Fisher recently suggested, mainly on numerical grounds, that this repulsive-core singularity is universal and in the same class as the Yang-Lee edge singularities, which arise above criticality at complex activities with positive real part. A general analytic demonstration of this identification is presented here using a field-theory approach with separate representations of the repulsive and attractive parts of the pair interactions.

Postmortem detection of acute septicemia in broilers.

Septicemia is an unwholesome condition diagnosed during postmortem inspection in poultry slaughter establishments on the basis of macroscopic lesions. Early identification of septicemia has important public health implications. In this study, Pasteurella multocida, Escherichia coli, and Staphylococcus aureus septicemia were induced in broilers in order to determine if lesions of acute septicemia can be grossly detected in the visceral organs of broiler carcasses prior to the development of changes in the skeletal muscle. Increased spleen and liver weights were observed during the acute phase of septicemia. Airsacculitis, pericarditis, and perihepatitis were observed during the acute phase of P. multocida- and E. coli-induced septicemia; and arthritis was the earliest indicator of S. aureus-induced septicemia. These macroscopic lesions were sufficient to identify unwholesome septicemic broiler carcasses prior to the development of changes in the skeletal muscle of the carcass.

Hair growth cycle affects hair follicle destruction by ruby laser pulses.

It has been shown that normal mode ruby laser pulses (694 nm) are effective in selectively destroying brown or black pigmented hair follicles in adult Caucasians. This study investigated how the various stages of the hair follicle growth cycle influence follicle destruction by ruby laser treatment, using a model of predictable synchronous hair growth cycles in the infantile and adolescent mice. A range of ruby laser pulse fluences was delivered during different stages of the hair growth cycle, followed by histologic and gross observations of the injury and regrowth of hair. Actively growing and pigmented anagen stage hair follicles were sensitive to hair removal by normal mode ruby laser exposure, whereas catagen and telogen stage hair follicles were resistant to laser irradiation. Selective thermal injury to follicles was observed histologically, and hair regrowth was fluence dependent. In animals exposed during anagen, intermediate fluences induced nonscarring alopecia, whereas high fluences induced scarring alopecia. The findings of this study suggest treatment strategies for optimal laser hair removal.

Flow cytometric technique for quantitating cytotoxic response to photodynamic therapy.

A simple flow cytometric technique for rapid measurement of multilog cytotoxic responses to photosensitization of cellular systems is described. This technique is particularly useful for cell lines with a low colony-forming efficiency, for which a nonclonogenic assay is required. The assay separates cell-sized objects from cellular debris by gating on forward scatter versus side scatter, identifies viable cells by positive calcein AM and negative ethidium homodimer-1 staining and measures cell concentration relative to an internal standard of polystyrene beads. Large numbers of cells can be analyzed rapidly. Two patient-derived small cell lung cancer cell lines, NCI-H209 and SV-E, were used to test the technique. Photordiation survival curves of the response of these cell lines to 5-aminolevulinic acid-induced protoprophyrin IX photosensitization correlated with the extent of photosensitizer accumulation. There was good agreement between the results obtained using the tritiated thymidine incorporation assay and the flow cytometric cytotoxicity assay. The technique can be used to measure cytotoxic responses to photosensitization of cell lines regardless of their plating efficiencies.

Optimal control of tumor size used to maximize survival time when cells are resistant to chemotherapy.

The high failure rates encountered in the chemotherapy of some cancers suggest that drug resistance is a common phenomenon. In the current study, the tumor burden during therapy is used to slow the growth of the drug-resistant cells, thereby maximizing the survival time of the host. Three types of tumor growth model are investigated--Gompertz, logistic, and exponential. For each model, feedback controls are constructed that specify the optimal tumor mass as a function of the size of the resistant subpopulation. For exponential and logistic tumor growth, the tumor burden during therapy is shown to have little impact upon survival time. When the tumor is in Gompertz growth, therapies maintaining a large tumor burden double and sometimes triple the survival time under aggressive therapies. Aggressive therapies aim for a rapid reduction in the sensitive cell subpopulation. These conclusions are not dependent upon the values of the model constants that determine the mass of resistant cells. Since treatments maintaining a high tumor burden are optimal for Gompertz tumor growth and close to optimal for exponential and logistic tumor growth, it may no longer be necessary to know the growth characteristics of a tumor to schedule anticancer drugs.

Low-intensity combination chemotherapy maximizes host survival time for tumors containing drug-resistant cells.

Recent clinical trials have shown that for some cancers, high-intensity alternating chemotherapy does not significantly improve either survival times or response rates compared with nonalternating therapy. The current study uses optimal control to determine the best way to treat a tumor that contains drug-resistant cells that cannot be destroyed. The delivery of two non-cross-resistant chemotherapeutic agents is limited by bounds on the drug concentration and the dose intensity. This ensures that the drug toxicity stays within a tolerable range. The aim of the therapy is to maximize the host survival time, defined as the time over which the tumor burden can be kept below a fixed bound. The model is posed as a free terminal time, optimal parameter selection problem in which the constraints are continuously parametrized by time and the number of courses of therapy is free to vary. New theory is developed so that the optimal parameter selection problem can be solved as a sequence of fixed terminal time problems using existing optimal control software. Numerical simulations of Gompertz tumor growth showed that a treatment maintaining a high tumor burden doubled and sometimes tripled with survival time under aggressive therapy. When these simulations were repeated using exponential and logistic tumor growth models, the tumor burden during treatment had little influence upon survival time. In all simulations, survival time was not extended by delivering the anticancer drugs concurrently instead of staggering the treatment arms.

Impact of Medicare payment policy on home health resources utilization.

In this study, the association between Medicare regulations and the provision of public home health care is examined. Medicare clients were compared with non-Medicare groups of those 65 years of age or over and those under 65. Results suggested that both age- and payer-related factors contribute to utilization of services. Older patients showed greater need for chronic illness care relative to younger patients; however, Medicare patients used fewer resources and had poorer outcomes relative to older non-Medicare patients.

A semiclosed-loop algorithm for the control of blood glucose levels in diabetics.

In this paper, a theoretical analysis of the control of plasma glucose levels in diabetic individuals is undertaken using a simple mathematical model of the dynamics of glucose and insulin interaction in the blood system. Mathematical optimization techniques are applied to the mathematical model to derive insulin infusion programs for the control of blood levels in diabetic individuals. Based on the results of the mathematical optimization, a semiclosed-loop algorithm is proposed for continuous insulin delivery to diabetic patients. The algorithm is based on three hourly plasma glucose samples. A theoretical evaluation of the effectiveness of this algorithm shows that it is superior to two existing algorithms in controlling hyperglycemia. A glucose infusion term representing the effect of glucose intake resulting from a meal is then introduced into the model equations. Various insulin infusion programs for the control of plasma glucose levels following a meal are then assessed. The theoretical results suggest that the most effective short-term control is achieved by an insulin infusion program which incorporates an injection to coincide with the meal.

A study of patients receiving no billed nursing care.

Although home health care traditionally is conceptualized as nursing care, in today's environment care at home may be delivered by a myriad of professional and nonprofessional practitioners. In fact, many patients who receive home care do not receive billed nursing visits. We studied a group of patients (n = 200) who received no billed nursing care, but rather received billed care from therapists, social workers and home health aides. This cohort of patients differed from patients who received billed nursing care in several ways: they were more frequently referred to home care from the community, their prognoses on admission to home care were significantly better, and their outcomes from home care services were considerably more favorable. Clearly patients not receiving billed nursing care have service and resource needs different from those of the population receiving billed nursing care. Therefore, new perspectives must be used when considering how to assess, deliver and perhaps pay for the requirements of these patients.

Nonreimbursed home health care: beyond the bills.

The unprecedented growth in the home care industry over the past decade has produced a highly competitive environment for care delivery. To survive, most agencies are implementing strategies to maximize reimbursable care. This focus, while likely to improve an agency's financial situation, is fraught with potential problems. Reimbursed care represents only a small portion of the care that is required and delivered. Still, aside from anecdotes, little reporting has been done to document such additional service, much less to consider the consequences of reducing or deleting it. We selected a sample of 350 public health home care episodes throughout Virginia, and reviewed billing records to determine the number of billed home visits attached to them. We then reviewed home health records for the matching episodes and recorded all visits and other care-related activities (e.g., telephone calls, conferences, laboratory trips). Total home visits increased, with an average of 1.5 unbilled home visits per episode. In addition, 10 additional care-related activities take place per patient, none of which is billed. These findings represent one of the first attempts to quantify the amounts and types of nonbilled care delivered by nurses. This care consumes significant nursing resources and, as a result, may be destined to be reduced or totally eliminated.