Evolutionary triage governs fitness in driver and passenger mutations and suggests targeting never mutations.

Genetic and epigenetic changes in cancer cells are typically divided into 'drivers' and 'passengers'. Drug development strategies target driver mutations, but inter- and intratumoral heterogeneity usually results in emergence of resistance. Here we model intratumoral evolution in the context of a fecundity/survivorship trade-off. Simulations demonstrate that the fitness value of any genetic change is not fixed but dependent on evolutionary triage governed by initial cell properties, current selection forces and prior genotypic/phenotypic trajectories. We demonstrate that spatial variations in molecular properties of tumour cells are the result of changes in environmental selection forces such as blood flow. Simulated therapies targeting fitness-increasing (driver) mutations usually decrease the tumour burden but almost inevitably fail due to population heterogeneity. An alternative strategy targets gene mutations that are never observed. Because up or downregulation of these genes unconditionally reduces cellular fitness, they are eliminated by evolutionary triage but can be exploited for targeted therapy.

Expression of OB receptor splice variants during prenatal development of the mouse.

In adult animals, signaling through the leptin receptor (OB-R) has been shown to play a critical role in fat metabolism. However, it is not known when these receptors are first expressed and what their role may be during embryonic development. To date, at least 6 splice variants of the OB-R have been identified. Although the function of each of these individual splice variants are unknown, only one of them, ob-rL ,encodes a receptor with a long intracellular domain that is implicated in OB-R signaling. In this study we have used in situ hybridization to examine the localization of OB-R splice variants during embryonic development of C57B1/6J mice. Using a probe, ob-r, that recognizes all of the splice variants, ob-r mRNA was found to be distributed in developing bone, mesenchyme, notochord and liver. In addition, epithelial structures including leptomeninges, choroid plexi and hair follicles also expressed ob-r. No ob-r mRNA was detected in the CNS. ob-rL, expression was only detected in notochord, bone and mesenchyme. The differential expression of these two mRNA isoforms suggests that the extracellular and intracellular domains of the OB receptor perform different biological functions.

Postnatal neuronal proliferation in mice lacking Ink4d and Kip1 inhibitors of cyclin-dependent kinases.

Development of the central nervous system requires proliferation of neuronal and glial cell precursors followed by their subsequent differentiation in a highly coordinated manner. The timing of neuronal cell cycle exit and differentiation is likely to be regulated in part by inhibitors of cyclin-dependent kinases. Overlapping and sustained patterns of expression of two cyclin-dependent kinases, p19(Ink4d) and p27(Kip1), in postmitotic brain cells suggested that these proteins may be important in actively repressing neuronal proliferation. Animals derived from crosses of Ink4d- null with Kip1-null mice exhibited bradykinesia, proprioceptive abnormalities, and seizures, and died at about 18 days after birth. Metabolic labeling of live animals with bromodeoxyuridine at postnatal days 14 and 18, combined with immunolabeling of neuronal markers, showed that subpopulations of central nervous system neurons were proliferating in all parts of the brain, including normally dormant cells of the hippocampus, cortex, hypothalamus, pons, and brainstem. These cells also expressed phosphorylated histone H3, a marker for late G(2) and M-phase progression, indicating that neurons were dividing after they had migrated to their final positions in the brain. Increased proliferation was balanced by cell death, resulting in no gross changes in the cytoarchitecture of the brains of these mice. Therefore, p19(Ink4d) and p27(Kip1) cooperate to maintain differentiated neurons in a quiescent state that is potentially reversible.

Changes in the numbers of neurons and astrocytes during the postnatal development of the rat inferior olive.

In the developing nervous system, cell death is an important component of refining axonal projections. In the developing rat inferior olive, previous studies have demonstrated cell death as temporally incongruent with both initial axon-target interactions and subsequent axon collateral regression. Furthermore, these studies identified a late rise in neuron numbers that is concurrent with climbing fibre regression. As axonal regression has not previously been associated with increasing neuron numbers, and since immature neurons and glia have similar morphological characteristics, it was decided to reassess the timing of cell death within the inferior olive in animals in which neurons and glia had been differentially stained. Glia were identified by the presence of glial cytoskeletal proteins, S100, or glial fibrillary acidic protein, and stereological counts were made of both neurons and glia in the inferior olive from rats of ages 0, 5, 10, 15, and 30 days. The number of inferior olivary neurons was approximately 22,000 between birth and day 10, which decreased to about 17,500 by day 30 (P<0.05). In contrast, the number of glia rose from about 5,000 at birth to approximately 15,000 by day 10 (P<0.001), after which there was no further increase. The changes in neurons and glia caused the neuron-to-glia ratio to fall to approximately 1.5 by the time of functional maturation within the olive. These results confirm that there is neuronal death in the inferior olive but that it is temporally correlated with both climbing fibre regression and functional maturation of the olivocerebellar projection.

Sonographic findings in testicular sarcoidosis simulating malignant nodule.

Testicular involvement by sarcoidosis is rare. We report a case of a patient with known sarcoid who had a unilateral testicular nodule with apparent capsular invasion on sonography. The epididymis was normal on both sides. Despite these atypical features, pathology showed the nodule to be a sarcoid granuloma. In patients with sarcoidosis, the differential diagnosis of an intratesticular mass should include testicular involvement by sarcoid.

Microtubule assembly is regulated by externally applied strain in cultured smooth muscle cells.

Mechanical forces clearly regulate the development and phenotype of a variety of tissues and cultured cells. However, it is not clear how mechanical information is transduced intracellularly to alter cellular function. Thermodynamic modeling predicts that mechanical forces influence microtubule assembly, and hence suggest microtubules as one potential cytoskeletal target for mechanical signals. In this study, the assembly of microtubules was analyzed in rat aortic smooth muscle cells cultured on silicon rubber substrates exposed to step increases in applied strain. Cytoskeletal and total cellular protein fractions were extracted from the cells following application of the external strain, and tubulin levels were quantified biochemically via a competitive ELISA and western blotting using bovine brain tubulin as a standard. In the first set of experiments, smooth muscle cells were subjected to a step-increase in strain and the distribution of tubulin between monomeric, polymeric, and total cellular pools was followed with time. Microtubule mass increased rapidly following application of the strain, with a statistically significant increase (P<0.05) in microtubule mass from 373+/-32 pg/cell (t=0) to 514+/-30 pg/cell (t=15 minutes). In parallel, the amount of soluble tubulin decreased approximately fivefold. The microtubule mass decreased after 1 hour to a value of 437+/-24 pg/cell. In the second set of experiments, smooth muscle cells were subjected to increasing doses of externally applied strain using a custom-built strain device. Monomeric, polymeric, and total tubulin fractions were extracted after 15 minutes of applied strain and quantified as for the earlier experiments. Microtubule mass increased with increasing strain while total cellular tubulin levels remained essentially constant at all strain levels. These findings are consistent with a thermodynamic model which predicts that microtubule assembly is promoted as a cell is stretched and compressional loads on the microtubules are presumably relieved. Furthermore, these data suggest microtubules are a potential target for translating changes in externally applied mechanical stimuli to alterations in cellular phenotype.

The glucose/insulin system and vitamin C: implications in insulin-dependent diabetes mellitus.

The cellular uptake of vitamin C (ascorbic acid, ASC) is promoted by insulin and inhibited by hyperglycemia. If a rise in plasma ASC is uncoupled from insulin replacement in insulin-dependent diabetes mellitus (IDDM) then the degree of hyperglycemia could account for "tissue scurvy" in IDDM. Leukocyte ASC is lower in IDDMs compared with nondiabetics when vitamin C consumption is adequate and our data suggest that this is a variable component of the pathophysiology of IDDM. The complications of diabetes mellitus are believed to result from either the intracellular accumulation of sorbitol or the nonenzymatic glycoxidation of proteins or both. With respect to the abnormal cellular accumulation of sorbitol, vitamin C supplementation has been shown to be effective in several studies of adults with diabetes; the situation regarding the prevention of protein glycoxidations by supplementation is presently unclear. The roles of ASC as an aldose reductase inhibitor and a water soluble antioxidant in body fluids are potentially very important as adjuncts to tight glycemic control in the management of diabetes. Tissue saturation and maximal physiologic function in IDDM may require supplemental vitamin C intake.

Micronutrients as nutriceutical interventions in diabetes mellitus.

Hyperglycemia portends chronic complications in insulin-dependent diabetes mellitus (IDDM) and substantial benefits are associated with "tight" glycemic control. Other interventions should either enhance glycemic control per se or add benefit to an established degree of glycemic control. Several micronutrients enhance insulin action and others offer promise in countering the untoward consequences of hyperglycemia. Supplements of micronutrients including the vitamins niacin (as niacinamide), C and E and the minerals zinc, chromium and vanadium have been studied. For the purpose of this review, the term "nutriceutic" refers to supplementation on the order of 2 to 10 times the RDA for which a benefit is linked to a mechanism of action. Benefits associated with "nutriceutic" supplementation are reported in small trials for vitamins C and E and these supplements are safe and affordable from food or tablet sources. A dietary strategy adding 200-600 mg of vitamin C and 100 IU of vitamin E to a healthy dietary pattern is worthy of consideration as an intervention for individuals with IDDM.

Physiologic hypoalbuminemia is well tolerated by severely burned children.

BACKGROUND: Physiologic hypoalbuminemia, defined as a plasma albumin (pl-ALB) of 1.0 to 2.5 g/dL, is a component of the injury response. A consensus on the need for albumin supplementation in this setting is lacking. METHODS: We examined 27 consecutive children (age, 7 +/- 6 years) with > 40% body surface burns (mean, 59 +/- 18%) during their initial 4 weeks of care. Patients were managed with an albumin-supplementation protocol that tolerated profound physiologic hypoalbuminemia. Intravenous albumin was administered by infusion of 1 to 2 g/kg/d when pl-ALB fell below 1.0 g/dL, or below 1.5 g/dL in the presence of enteral feeding intolerance or pulmonary dysfunction. Supplementation was stopped when pl-ALB reached 2.0 g/dL. RESULTS: Mean pl-ALB was 1.7 g/dL overall. Infusion for pl-ALB < 1.0 g/dL was needed for 70% (n = 19) of the patients. Profound physiologic hypoalbuminemia was constant, that is, mean weekly pl-ALB never exceed 2.5 g/dL in any patient. Mean plasma globulin rose during the 4 week period from 2.3 +/- 0.1 at week 1 to 3.1 +/- 0.1 at week 4. Diarrhea was negligible (19 of 756 patient days), nasogastric feedings were well tolerated, PaO2/FiO2 ratios remained well above 150, wounds healed satisfactorily, and all children survived and have been discharged home. CONCLUSIONS: Profound physiologic hypoalbuminemia (pl-ALB of 1.0-2.5 g/dL) does not have adverse effects on pulmonary or gut function, wound healing, or outcome in severely burned children, perhaps because of a compensatory increase in acute-phase proteins reflected in plasma globulin.

Energy and protein provisions for thermally injured children revisited: an outcome-based approach for determining requirements.

Energy and protein provisions for adequate wound healing and weight maintenance were examined among severely burned children. Actual intakes were documented for 27 patients admitted with a more than 40% total body surface area burn. Mean energy intake over the 4-week study period averaged 140% of the predicted basal metabolic rate (PBMR), and mean protein intake was 2.8 +/- 0.2 grams per kilogram daily. Wound healing progressed satisfactorily in all patients; at 4 weeks, the open wound area (% open) was 20% or less in 22 patients. Average weight at discharge was 88% +/- 2.6% of ideal body weight. Discharge weights were significantly higher (p < 0.05) among patients whose energy intake exceeded PBMR x 1.7 for at least 1 of the study weeks. We suggest that energy intakes approximating PBMR x 1.2 with a minimum of 3 grams of protein per kilogram will support adequate wound healing, whereas higher energy provisions (PBMR x 1.7) will enhance weight status.

Low ceruloplasmin levels during recovery from major burn injury: influence of open wound size and copper supplementation.

Copper (Cu) status is often judged by the plasma level of its chief transport protein, ceruloplasmin (Cp). Only copper deficiency and heredity are known to decrease circulating Cp. Cp is an acute-phase responsive protein in trauma and it is also induced by Cu supplementation. Despite this, plasma concentrations of Cp remain low during the acute recovery from major burn injury. The high provision of vitamin C typically used in burn patients may influence these observations when an indirect oxidase activity assay is used. We employed a radial immunodiffusion (RID) assay specific for the Cp protein as well as an indirect oxidase assay for Cp in a series of 11 burned children who were supplemented with both Cu and vitamin C, either enterally or parenterally. Our findings confirm that low Cp is a characteristic of the acute recovery from major burns. The oxidase assay is shown to be valid for very low Cp levels even during high vitamin C provision. When these data are combined with our previously reported series, a strong relationship between the size of the open wound area and the amount of circulating Cp is demonstrated. Copper supplementation by either the enteral or parenteral routes is only marginally successful in restoring Cp toward normal levels.

Body composition and nutrition support in pediatrics: what to defend and how soon to begin.

Specialized nutrition support is initiated during hospital care on the assumption that sparing the mobilization of body energy reserves is advantageous to recovery. The combined effects of disease and undernutrition on body cell mass, organ function, immune responsiveness, and wound healing are well documented in adults. Children cannot survive a fast as long as adults because of their lesser stores of energy substrates relative to the rate of energy expenditure. The present contribution attempts to estimate the rates of compositional losses for infants and prepubertal children on the basis of available data and reasonable metabolic assumptions. The lesson that emerges from this exercise is one of a very critical need for the early initiation of nutrition support for infants and children. The analysis suggests that an acute risk of protein depletion exists for children of all ages. Especially for infants, the empirical wisdom that "the absence of evidence is not evidence of absence" should be invoked to support early nutrition intervention. This work is submitted for critical review and revision to establish a consensus on the timeline of pediatric morbidity or mortality from semistarvation or starvation.

Hyperzincuria in individuals with insulin-dependent diabetes mellitus: concurrent zinc status and the effect of high-dose zinc supplementation.

The urinary excretion of zinc in individuals with insulin-dependent diabetes mellitus (IDDM) is approximately doubled. In the absence of a compensatory mechanism, this hyperzincuria should induce a deficient or marginal Zn status. We examined parameters of Zn status in plasma and in blood cells with respect to urinary Zn losses and Zn supplementation. We measured Zn levels in the urine, plasma, and erythrocytes of 14 IDDM subjects and 15 nondiabetics who kept dietary records for 3 consecutive days. Subsequently, six IDDM subjects and seven nondiabetics were supplemented with 50 mg Zn daily for 28 days. We measured the above parameters, as well as mononuclear leukocyte Zn (MNL-Zn) and the plasma subfraction of albumin-bound Zn (alb-Zn). The total plasma Zn-binding capacity was also assessed. Plasma copper and erythrocyte Cu were monitored as indicators of potential Zn toxicity. Individuals with IDDM displayed the expected hyperzincuria, but had normal blood Zn parameters. Zincuria increased by a similar amount in both groups during supplementation, as did the MNL-Zn content. However, erythrocyte Zn (e-Zn) was refractory, so a trend toward lower e-Zn among IDDM subjects persisted during Zn supplementation. Hemoglobin A1c (HbA1c) increased markedly in the Zn-supplemented IDDM group. Despite their chronic hyperzincuria, individuals with IDDM appear not to be Zn-deficient. Large-dose Zn supplementation increases MNL-Zn and induces an undesirable elevation of HbA1c in all individuals. This is especially disconcerting for those with IDDM, and may reflect an exacerbation of a chronic "Zn diabetes." These data suggest a potential for toxicity from large-dose Zn supplementation.

Vitamin C: an aldose reductase inhibitor that normalizes erythrocyte sorbitol in insulin-dependent diabetes mellitus.

OBJECTIVE: Diabetic hyperglycemia promotes sorbitol production from glucose via aldose reductase. Since the intracellular accumulation of sorbitol, or its sequelae, are postulated to contribute to the progression of chronic diabetic complications, aldose reductase inhibitors (ARI) offer therapeutic promise. Others have shown that vitamin C at pharmacologic doses decreases erythrocyte (RBC) sorbitol. We examined whether smaller, physiologic doses of vitamin C were also effective in individuals with insulin-dependent diabetes mellitus (IDDM) and whether vitamin C was an ARI in vitro. METHODS: Vitamin C supplements (100 or 600 mg) were taken daily for 58 days by young adults with IDDM and nondiabetic adults in an otherwise free-living design. Diabetic control was monitored by fasting plasma glucose, glycosylated hemoglobin, and glycosuria and was moderate to poor throughout the study. RBC sorbitol was measured at baseline and again at 30 and 58 days. Three-day dietary records and 24-hour urine collections were performed for each sampling day. RESULTS: RBC sorbitol levels were significantly elevated in IDDMs, on average doubled, despite their more than adequate dietary intakes of vitamin C and normal plasma concentrations. Vitamin C supplementation at either dose normalized the RBC sorbitol in IDDMs within 30 days. This correction of sorbitol accumulation was independent of changes in diabetic control. Furthermore, our in vitro studies show that ascorbic acid inhibited aldose reductase activity. CONCLUSIONS: Vitamin C supplementation is effective in reducing sorbitol accumulation in the erythrocytes of diabetics. Given its tissue distribution and low toxicity, we suggest a superiority for vitamin C over pharmaceutic ARIs.

Effects of chromium picolinate supplementation on body composition, strength, and urinary chromium loss in football players.

The effects of 9 weeks of daily chromium supplementation (200 microgram Cr as picolinate) were investigated in a double-blind design in football players during spring training. Testing was done pre-, mid-, and postsupplementation on the following criterion measures: urinary chromium excretion, girth and skinfold measures, percent body fat and lean body mass, and isometric and dynamic strength. With the exception of 2 variables (of 65 variables analyzed), no significant group by trials interactions were found (based on a repeated measures ANOVA). The two exceptions were unrelated and inconsequential. For 27 of the 38 subjects, average urinary chromium loss at pre was 0.36 microgram/24 hr, whereas it was undetectable (<0.1 microgram/24 hr) for 10 subjects and excessive in 1 subject (2.4 micrograms/24 hr). Subjects receiving chromium supplements demonstrated urinary chromium losses five times greater than those in the placebo group at mid and post. Chromium picolinate supplementation was ineffective in bringing about changes in body composition or strength during a program of intensive weight-lifting training.

Burn severity, copper dose, and plasma ceruloplasmin in burned children during total parenteral nutrition.

Copper (Cu) is an essential nutrient with known metabolic roles in wound healing. Ceruloplasmin (CP), the primary Cu-transport protein, responds as an acute-phase reactive protein after trauma. However, for severe burn trauma, this response is absent in the early catabolic phase despite Cu provision. We report data for 14 severely burned children receiving Cu in total parenteral nutrition (TPN-Cu) ranging from 7 to 26 micrograms Cu.kg-1 x day-1. All patients manifested low plasma levels of CP. The reduction in CP reflected burn severity but also appeared to be dependent on the Cu dose. Increasing Cu supplementation to improve CP raises a concern for hepatotoxicity, which is accompanied by an elevation in the plasma nonceruloplasmin Cu (nonCP-Cu). The calculated plasma nonCP-Cu in our series is consistent with a lack of increased risk and suggests that TPN-Cu at the general pediatric guideline of 20 micrograms.kg-1 x day-1 is safe and reasonable for severely burned children. Cu supplementation > 20 micrograms/kg may be beneficial; however, monitoring of both CP and total Cu should continue as standard practice in the management of these patients.