A Unifying Framework for Understanding Biological Structures and Functions Across Levels of Biological Organization.

The relationship between structure and function is a major constituent of the rules of life. Structures and functions occur across all levels of biological organization. Current efforts to integrate conceptual frameworks and approaches to address new and old questions promise to allow a more holistic and robust understanding of how different biological functions are achieved across levels of biological organization. Here, we provide unifying and generalizable definitions of both structure and function that can be applied across all levels of biological organization. However, we find differences in the nature of structures at the organismal level and below as compared to above the level of the organism. We term these intrinsic and emergent structures, respectively. Intrinsic structures are directly under selection, contributing to the overall performance (fitness) of the individual organism. Emergent structures involve interactions among aggregations of organisms and are not directly under selection. Given this distinction, we argue that while the functions of many intrinsic structures remain unknown, functions of emergent structures are the result of the aggregate of processes of individual organisms. We then provide a detailed and unified framework of the structure-function relationship for intrinsic structures to explore how their unknown functions can be defined. We provide examples of how these scalable definitions applied to intrinsic structures provide a framework to address questions on structure-function relationships that can be approached simultaneously from all subdisciplines of biology. We propose that this will produce a more holistic and robust understanding of how different biological functions are achieved across levels of biological organization.

Habitat, latitude and body mass influence the temperature dependence of metabolic rate.

The sensitivity of metabolic rate to temperature constrains the climate in which ectotherms can function, yet the temperature dependence of metabolic rate may evolve in response to biotic and abiotic factors. We compiled a dataset on the temperature dependence of metabolic rate for heterotrophic ectotherms from studies that show a peak in metabolic rate at an optimal temperature (i.e. that describe the thermal performance curve for metabolic rate). We found that peak metabolic rates were lower in aquatic than terrestrial habitats and increased with body mass, latitude and the optimal temperature. In addition, the optimal temperature decreased with latitude. These results support competing hypotheses about metabolic rate adaptation, with hotter being better in the tropics but colder being better towards the poles. Moreover, our results suggest that the temperature dependence of metabolic rate is more complex than previously suggested.

Blocking the VISTA pathway enhances disease progression in (NZB × NZW) F1 female mice.

V-domain Ig suppressor of T-cell activation (VISTA) is a critical negative checkpoint molecule involved in regulating the immune response. Targeting the pathway with an antagonist anti-VISTA antibody designated 13F3 has been shown to enhance disease severity in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. To determine if VISTA plays a role in murine lupus, New Zealand Black × New Zealand White (BWF1) mice were treated with 13F3 or control hamster Ig and disease monitored. Onset of proteinuria was earlier and renal damage more profound in mice treated with 13F3. Cell subset analysis showed an increase of activated splenic T cells and inflammatory splenic myeloid cells, but no effect on B cells, in mice receiving 13F3. Examination of the kidney showed an increase in inflammatory myeloid cell infiltration with 13F3 treatment. This study along with previous EAE data, suggests that interventions that enhance VISTA regulatory activity may be effective for the treatment of autoimmune disease.

The combined effects of reactant kinetics and enzyme stability explain the temperature dependence of metabolic rates.

A mechanistic understanding of the response of metabolic rate to temperature is essential for understanding thermal ecology and metabolic adaptation. Although the Arrhenius equation has been used to describe the effects of temperature on reaction rates and metabolic traits, it does not adequately describe two aspects of the thermal performance curve (TPC) for metabolic rate-that metabolic rate is a unimodal function of temperature often with maximal values in the biologically relevant temperature range and that activation energies are temperature dependent. We show that the temperature dependence of metabolic rate in ectotherms is well described by an enzyme-assisted Arrhenius (EAAR) model that accounts for the temperature-dependent contribution of enzymes to decreasing the activation energy required for reactions to occur. The model is mechanistically derived using the thermodynamic rules that govern protein stability. We contrast our model with other unimodal functions that also can be used to describe the temperature dependence of metabolic rate to show how the EAAR model provides an important advance over previous work. We fit the EAAR model to metabolic rate data for a variety of taxa to demonstrate the model's utility in describing metabolic rate TPCs while revealing significant differences in thermodynamic properties across species and acclimation temperatures. Our model advances our ability to understand the metabolic and ecological consequences of increases in the mean and variance of temperature associated with global climate change. In addition, the model suggests avenues by which organisms can acclimate and adapt to changing thermal environments. Furthermore, the parameters in the EAAR model generate links between organismal level performance and underlying molecular processes that can be tested for in future work.

Grass-specific CD4(+) T-cells exhibit varying degrees of cross-reactivity, implications for allergen-specific immunotherapy.

BACKGROUND: Conceptually, allergic responses may involve cross-reactivity by antibodies or T-cells. While IgE cross-reactivity among grass-pollen allergens has been observed, cross-reactivity at the allergen-specific T-cell level has been less documented. Identification of the patterns of cross-reactivity may improve our understanding, allowing optimization of better immunotherapy strategies. OBJECTIVES: We use Phleum pratense as model for the studying of cross-reactivity at the allergen-specific CD4(+) T cell level among DR04:01 restricted Pooideae grass-pollen T-cell epitopes. METHODS: After in vitro culture of blood mono-nucleated cells from grass-pollen-allergic subjects with specific Pooideae antigenic epitopes, dual tetramer staining with APC-labelled DR04:01/Phleum pratense tetramers and PE-labelled DR04:01/Pooideae grass homolog tetramers was assessed to identify cross-reactivity among allergen-specific DR04:01-restricted T-cells in six subjects. Direct ex vivo staining enabled the comparison of frequency and phenotype of different Pooideae grass-pollen reactive T-cells. Intracellular cytokine staining (ICS) assays were also used to examine phenotypes of these T-cells. RESULTS: T-cells with various degrees of cross-reactive profiles could be detected. Poa p 1 97-116 , Lol p 1 221-240 , Lol p 5a 199-218 , and Poa p 5a 199-218 were identified as minimally cross-reactive T-cell epitopes that do not show cross-reactivity to Phl p 1 and Phl p 5a epitopes. Ex vivo tetramer staining assays demonstrated T-cells that recognized these minimally cross-reactive T-cell epitopes are present in Grass-pollen-allergic subjects. CONCLUSIONS: Our results suggest that not all Pooideae grass epitopes with sequence homology are cross-reactive. Non-cross-reactive T-cells with comparable frequency, phenotype and functionality to Phl p-specific T-cells suggest that a multiple allergen system should be considered for immunotherapy instead of a mono-allergen system.

Bending the health cost curve: the promise and peril of the Independent Payment Advisory Board.

Underlying today's and the future's health-care reform debate is a consensus that America's health-care financing system is in a slow-moving but deep crisis: care appears substandard in comparison with other advanced industrial countries, and relative costs are exploding beyond all reasonable measures. The Obama Administration's Patient Protection and Affordable Care Act (ACA) attempts to grapple with both of these problems. One of ACA's key instrumentalities is the Independent Payment Advisory Board-the IPAB, designed to discover and authorize ways to reduce the rate of growth of Medicare and other categories of health spending. The IPAB is a peril. Expert boards to perform regulatory tasks in the interest of efficiency and social goals always run a high risk of being captured by the industry they are supposed to regulate. Even should it succeed at its task of reducing the rate of growth of Medicare spending, who is to say that the reductions will not come at a heavy cost in reduced quantity and effectiveness of medical care? But the IPAB also has promise. The need for a better process than our current specialist-driven one to assign value to the medical services provided by Medicare is great. The bellwether status of Medicare payment systems means that commercial insurance consumers and payors would also benefit mightily from bringing more coherent, technocratic, and cost-effectiveness oriented logic to this process. And the current system of relative Medicare reimbursement rates is, in the judgment of many, currently well out of whack. We quail when we consider the magnitude of the tasks the IPAB faces--even its initial task. Nevertheless, we remain optimistic that this administrative agency will manage to bend the long-run healthcare cost curve and moderate future price increases.

The effects of the novel benzodiazepine receptor inverse agonist Ru 34000 on ethanol-maintained behaviors.

Ru 34000 [5-ethyl-7-methoxy-imidazo (1,2-a) pyrimidin-2-yl cyclopropyl methanone] is a novel imidazopyrimidine benzodiazepine inverse agonist that exhibits low affinity for central benzodiazepine receptors (Ki approximately 0.98 microM). The present study examined the in vivo actions of Ru 34000 (0.5-5 mg/kg) following intraperitoneal (i.p.), subcutaneous (s.c), oral (p.o.), and intraventral tegmental administration in alcohol-preferring (P) rats trained under a concurrent operant schedule (FR4-FR4) for ethanol (10% v/v) and a palatable saccharin (0.025% or 0.75% w/v) reinforcer. Ru 34000 (i.p., s.c., p.o.) markedly reduced ethanol responding by 28-96% of control levels without affecting saccharin responding, except for the highest dose level. Clear dose-dependent suppressant effects were observed with all routes of administration on ethanol responding. Flumazenil [ethyl-8-fluro-5, 6-dihydro-5-methyl-6-4H-imidazo [1,5-a]-[1,4]-benzodiazepine-3-carboxylate] (6 mg/kg; i.p.), a benzodiazepine receptor antagonist reversed the Ru 34000-reduction of ethanol responding, suggesting that the effects were mediated at the benzodiazepine receptor. Bilateral microinjections of Ru 34000 (50, 100, 200 ng) into the ventral tegmental area dose-dependently reduced ethanol responding by as much as 97% of control levels. The results suggest that the in vivo actions of Ru 34000 are determined not only by its binding affinity, but also by its bioavailability at active benzodiazepine sites and route of drug administration. Low affinity imidazopyrimidines may be useful pharmacological probes to further understand the role of the GABA(A)-benzodiazepine receptor complex in ethanol motivated behaviors.

High-affinity benzodiazepine antagonists reduce responding maintained by ethanol presentation in ethanol-preferring rats.

In the present study, we examined two high-affinity and selective benzodiazepine (BDZ) receptor antagonists (ZK 93426, CGS 8216) in ethanol (EtOH)-preferring rats whose responding (i.e., lever pressing) was maintained by the presentation of EtOH. The in vivo actions of CGS 8216 (1-30 mg/kg) and ZK 93426 (5-75 mg/kg) were examined after intraperitoneal or oral administration. Flumazenil (10-40 mg/kg) was used as a reference BDZ antagonist. EtOH (10% v/v) and saccharin (0.05 g/v) solutions were concurrently available for 30 min each day under a two-lever fixed-ratio schedule in which four responses on one lever produced the EtOH solution and four responses on the other lever produced the saccharin solution. A 40 mg/kg intraperitoneal injection of flumazenil given on the first injection day (day 1) nonsignificantly reduced control levels of responding maintained by EtOH by 36%. No effects were observed 24 hr after drug administration (day 2). Oral administration of flumazenil was without effect. On day 1, intraperitoneal administration of CGS 8216 (1-20 mg/kg) and ZK 93426 (1-50 mg/kg) reduced control levels of responding maintained by EtOH by 44% to 73%; on day 2, EtOH maintained responding continued to be suppressed with the highest doses (> or = 20 mg/kg) suppressing control levels of responding by as much as 62%. Oral administration of higher doses of CGS 8216 (5-30 mg/kg) and ZK 93426 (10-75 mg/kg) reduced responding maintained by EtOH by as much as 54% to 84% of controls; however, no effects were seen on day 2. Only the highest intraperitoneal dose of ZK 93426 (50 mg/kg) suppressed responding maintained by saccharin. These findings demonstrate that some BDZ antagonists decrease responding maintained by EtOH. The findings suggest that certain BDZ antagonists may have potential as pharmacotherapies to prevent or decrease EtOH abuse in humans.

A congestive heart failure project with measured improvements in care.

This project was designed to improve the in-hospital management of Medicare beneficiaries with congestive heart failure (CHF). Eleven hospitals were studied using two indicators: (a) assessment of left ventricular (LV) function, and (b) use of angiotensin converting enzyme (ACE) inhibitors in patients with systolic dysfunction. Baseline performance rates were obtained for 990 cases with the Diagnosis Related Group (DRG) 127 for CHF discharged January 1994 to December 1994. Baseline data feedback presentations in 1995 spurred quality improvement plans with interventions such as physician education, critical care maps, and standing orders. Follow-up abstractions were performed on 612 discharges October 1995 through April 1997. The study demonstrated 12% improvement (53% to 65%, p < .01) in assessing LV function and 20% improvement (54% to 74%, p < .01) in appropriate ACE inhibitor use. Projects emphasizing Health Care Quality Improvement Program (HCQIP) principles can successfully affect health care management for the Medicare population.

Primary causes of medical instability of Medicare patients at discharge 1990-1991.

BACKGROUND: HCFA-sponsored Peer Review Organizations nationwide have been criticized by organized medicine, Medicare beneficiaries and interested Congressional parties for being punitive without exploiting educational opportunities garnered through their massive data collection capabilities. Until now this data has been poorly analyzed and has not been adequately utilized as a positive motivational tool. METHODS: HCFA has developed, with the cooperation of the medical community, six generic screens for Peer Review Organizations to utilize when evaluating quality care provided Medicare beneficiaries in an acute care setting. Through the sheer volume of cases seen, significant inpatient information can be obtained that cannot be duplicated in any other epidemiological endeavor. Line item discrepancies can be further studied through small-area-analysis to determine "who" as well as "what" may be the problem. RESULTS: By compiling and reviewing data in a systematic manner over two years (1990-1991), the Alabama Quality Assurance Foundation (AQAF) has singled out problems with patient medical stability at discharge as the predominant concern for care providers and reviewers alike. CONCLUSIONS: Having determined the primary problem in rendering quality care in a Prospective Payment System to Medicare beneficiaries lies in premature discharges, subsequent small-area-analysis further narrows the focus of concerned individuals. Hopefully, practitioners and providers alike will act on their own initiative in correcting this problem. Follow-up review will be utilized to assure this self-corrective action.

Apical Na+ permeability of frog skin during serosal Cl- replacement.

Gluconate substitution for serosal Cl- reduces the transepithelial short-circuit current (Isc) and depolarizes short-circuited frog skins. These effects could result either from inhibition of basolateral K+ conductance, or from two actions to inhibit both apical Na+ permeability (PapNa) and basolateral pump activity. We have addressed this question by studying whole-and split-thickness frog skins. Intracellular Na+ concentration (CcNa) and PapNa have been monitored by measuring the current-voltage relationship for apical Na+ entry. This analysis was conducted by applying trains of voltage pulses, with pulse durations of 16 to 32 msec. Estimates of PapNa and CcNa were not detectably dependent on pulse duration over the range 16 to 80 msec. Serosal Cl- replacement uniformly depolarized short-circuited tissues. The depolarization was associated with inhibition of Isc across each split skin, but only occasionally across the whole-thickness preparations. This difference may reflect the better ionic exchange between the bulk medium and the extracellular fluid in contact with the basolateral membranes, following removal of the underlying dermis in the split-skin preparations. PapNa was either unchanged or increased, and CcNa either unchanged or reduced after the anionic replacement. These data are incompatible with the concept that serosal Cl- replacement inhibits PapNa and Na,K-pump activity. Gluconate substitution likely reduces cell volume, triggering inhibition of the basolateral K+ channels, consistent with the data and conclusions of S.A. Lewis, A.G. Butt, M.J. Bowler, J.P. Leader and A.D.C. Macknight (J. Membrane Biol. 83:119-137, 1985) for toad bladder. The resulting depolarization reduces the electrical force favoring apical Na+ entry. The volume-conductance coupling serves to conserve volume by reducing K+ solute loss. Its molecular basis remains to be identified.

Apical sodium entry in split frog skin: current-voltage relationship.

Apical Na+ entry into frog skin epithelium is widely presumed to be electrodiffusive in nature, as for other tight epithelia. However, in contrast to rabbit descending colon and Necturus urinary bladder, the constant field equation has been reported to fit the apical sodium current (INa)-membrane potential (psi mc) relationship over only a narrow range of apical membrane potentials or to be inapplicable altogether. We have re-examined this issue by impaling split frog skins across the basolateral membrane and examining the current-voltage relationships at extremely early endpoints in time after initiating pulses of constant transepithelial voltage. In this study, the rapid transient responses in psi mc were completed within 0.5 to 3.5 msec. Using endpoints to 1 to 25 msec, the Goldman equation provided excellent fits of the data over large ranges in apical potential of 300 to 420 mV, from approximately -200 to about +145 mV (cell relative to mucosa). Split skins were also studied when superfused with high serosal K+ in order to determine whether the INapsi mc relationship could be generated purely by transepithelial measurements. Under these conditions, the basolateral membrane potential was found to be -10 +/- 3 mV (cell relative to serosa, mean +/- SE), the basolateral fractional resistance was greater than zero, and the transepithelial current was markedly and reversibly reduced. For these reasons, use of high serosal K+ is considered inadvisable for determining the INa-psi mc relationship, at least in those tissues (such as frog skin) where more direct measurements are technically feasible. Analysis of the INa-psi mc relationships under baseline conditions provided estimates of intracellular Na+ concentration and of apical Na+ permeability of 9 to 14 mM and of approximately 3 X 10(-7) cm X sec-1, respectively, in reasonable agreement with estimates obtained by different techniques.

Microelectrode study of K+ accumulation by tight epithelia: I. Baseline values of split frog skin and toad urinary bladder.

Toad bladder and split frog skin were impaled with fine-tipped single- and double-barrelled K+-selective microelectrodes. In order to circumvent membrane damage induced by impaling toad bladder, a null point method was developed, involving elevations of mucosal potassium concentration. The results suggest that intracellular potassium activity of short-circuited toad bladder is approximately 82 mM, twice as large as earlier estimates. Far more stable and rigorously defined intracellular measurements were recorded from short-circuited split frog skins. The intracellular positions of the micropipette and microelectrode tips were verified by transient hyperpolarizations of the membrane potential with mucosal amiloride or by transient depolarizations with serosal barium or strophanthidin. Simultaneous impalement of distant cells with separate micropipettes demonstrated that both the baseline membrane potentials and the responses to depolarizing agents were similar, further documenting that frog skin is a functional syncytium. Measurements with double-barrelled microelectrodes and simultaneous single-barrelled microelectrodes and reference micropipettes suggest that the intracellular potassium activity is about 104 mM, lower than previously reported. Taken together with measurements of intracellular potassium concentration, this datum suggests that potassium is uniformly distributed within the epithelial cells.

Microelectrode study of K+ accumulation by tight epithelia: II. Effect of inhibiting transepithelial Na+ transport on reaccumulation following depletion.

The effects of restoring serosal potassium to potassium-depleted toad urinary bladders have been re-examined using double-barrelled microelectrodes. The data confirm the existence of a time-lag phenomenon, a dissociation between potassium reaccumulation and restoration of short-circuit current. Returning serosal potassium stimulates an increase in intracellular potassium activity 21-26 min before any increase can be detected in short-circuit current. The reaccumulation of potassium has been further studied using split frog skin, a far more suitable preparation for electrophysiologic study than toad bladder. Under baseline short-circuited conditions, potassium is accumulated against an electrochemical gradient of 22 +/- 4 mV. Reaccumulation of potassium by potassium-depleted tissues can be blocked by inhibiting the Na,K-exchange pump with high concentrations of ouabain. On the other hand, blocking apical sodium entry by the addition of 10(-4) M amiloride to the outer bathing medium does not interfere with reaccumulation of potassium. The data support the concept that the time-lag phenomenon of toad bladder reflects stimulation of potassium reaccumulation by the sodium pump in exchange for the extrusion of excess cell sodium collected during the period of potassium depletion. This reaccumulation of potassium can proceed before the entry of significant added amounts of sodium across the apical plasma membrane.

Subdural empyema associated with an apparent regional hyperperfusion (luxury perfusion).

A patient with a subdural empyema demonstrated a late arterial "blush" during a 99mTc-pertechnetate cerebral dynamic flow study which "washed out" in the delayed images. Cerebral arteriography and subsequent autopsy documented inflammatory tissue surrounding the empyema, resulting in this scintigraphic pattern.

A pressure- and flow-insensitive reference electrode liquid junction.

The design and construction of a pressure- and flow-insensitive reference liquid junction for use in ion concentration electrode measuring systems is described. The junction is inexpensive, is very easily and rapidly constructed, is rugged, and is adaptable to various applications. When used in a pH-measuring system, drift, pressure artifacts, and flow artifacts are negligible. The response time of the system appears to be less than 10 ms. Using the pH electrode device as described, the dissociation reaction rate constant of H2CO3 at 24 degrees C was determined to be 22 s-1.