Systemic collapse: Medical care in the aftermath of Hurricane Katrina

This article describes and analyzes key aspects of the medical response to Hurricane Katrina in New Orleans. It is based on interviews with individuals involved in the response and on analysis of published reports and news articles. Findings include: (1) federal, state, and local disaster plans did not include provisions for keeping hospitals functioning during a large-scale emergency; (2) the National Disaster Medical System (NDMS) was ill-prepared for providing medical care to patients who needed it; (3) there was no coordinated system for recruiting, deploying, and managing volunteers; and (4) many Gulf Coast residents were separated from their medical records. The article makes recommendations for improvement.     

Positive-pressure ventilation equipment for mass casualty respiratory failure

In the event of an influenza pandemic, patients with severe acute respiratory failure (ARF) due to influenza will require positive-pressure ventilation (PPV) in order to survive. In countries with widely available critical care services, PPV is delivered almost exclusively through use of full-feature mechanical ventilators in intensive care units (ICUs) or specialized hospital wards. But the supply of these ventilators is limited even during the normal course of hospital functioning. Purchasing and maintaining additional full-feature mechanical ventilators to be held in reserve and used only during mass casualty events is too expensive to allow the stockpiling of such equipment. Consequently, planning and preparedness efforts to respond to a severe influenza pandemic have stimulated consideration of limited-feature, less-expensive ventilation devices to augment traditional PPV capacity. This article offers guidance to authorities charged with preparing for mass casualty PPV in deciding which PPV equipment would be adequate for ventilating patients for days, weeks, or even months during a medical catastrophe.     

Biological dosimetry by the triage dicentric chromosome assay: potential implications for treatment of acute radiation syndrome in radiological mass casualties

Biological dosimetry is an essential tool for estimating radiation dose. The dicentric chromosome assay (DCA) is currently the tool of choice. Because the assay is labor-intensive and time-consuming, strategies are needed to increase throughput for use in radiation mass casualty incidents. One such strategy is to truncate metaphase spread analysis for triage dose estimates by scoring 50 or fewer metaphases, compared to a routine analysis of 500 to 1000 metaphases, and to increase throughput using a large group of scorers in a biodosimetry network. Previously, the National Institutes for Allergies and Infectious Diseases (NIAID) and the Armed Forces Radiobiology Research Institute (AFRRI) sponsored a double-blinded interlaboratory comparison among five established international cytogenetic biodosimetry laboratories to determine the variability in calibration curves and in dose measurements in unknown, irradiated samples. In the present study, we further analyzed the published data from this previous study to investigate how the number of metaphase spreads influences dose prediction accuracy and how this information could be of value in the triage and management of people at risk for the acute radiation syndrome (ARS). Although, as expected, accuracy decreased with lower numbers of metaphase spreads analyzed, predicted doses by the laboratories were in good agreement and were judged to be adequate to guide diagnosis and treatment of ARS. These results demonstrate that for rapid triage, a network of cytogenetic biodosimetry laboratories can accurately assess doses even with a lower number of scored metaphases.     

Intercolony variation in growth of black brant goslings on the Yukon-Kuskokwim Delta,Alaska

Recent declines in black brant (Branta bernicla nigricans) are likely the result of low recruitment. In geese, recruitment is strongly affected by habitat conditions experienced by broods because gosling growth rates are indicative of forage conditions during brood rearing and strongly influence future survival and productivity. In 2006–2008, we studied gosling growth at 3 of the 4 major colonies on the Yukon-Kuskokwim Delta, Alaska. Estimates of age-adjusted gosling mass at the 2 southern colonies (approx. 30% of the world population of breeding black brant) was low (gosling mass at 30.5 days ranged 346.7 ± 42.5 g to 627.1 ± 15.9 g) in comparison to a third colony (gosling mass at 30.5 days ranged 640.0 ± 8.3 g to 821.6 ± 13.6 g) and to most previous estimates of age-adjusted mass of brant goslings. Thus, our results are consistent with the hypothesis that poor gosling growth is negatively influencing the brant population. There are 2 non-mutually exclusive explanations for the apparent growth rates we observed. First, the population decline may have been caused by density-independent factors and habitat capacity has declined along with the population as a consequence of the unique foraging feedback between brant and their grazing habitats. Alternatively, a reduction in habitat capacity, as a result of changes to the grazing system, may have negatively influenced gosling growth, which is contributing to the overall long-term population decline. We found support for both explanations. For colonies over habitat capacity we recommend management to enhance foraging habitat, whereas for colonies below habitat capacity we recommend management to increase nesting productivity. © 2010 The Wildlife Society.     

Energetics of fattening and starvation in the long-distance migratory garden warbler,Sylvia borin,during the migratory phase

Garden warblers (Sylvia borin) were subjected to starvation trials during their autumnal migratory phase in order to simulate a period of non-stop migration. Before, during and after this treatment the energy expenditure, activity, food intake and body mass of the subjects were monitored. Assimilation efficiency was constant throughout the experiments. The catabolized (during starvation) and deposited body tissue (during recovery) consisted of 73% fat. Basal metabolic rate was decreased during the starvation period and tended to a gradual increase during the recovery period. The reduced basal metabolic rate can possibly be attributed to a reduced size/function of the digestive system, which is consistent with the sub-maximal food intake immediately after resuming the supply of food to the experimental birds. The observed reductions in basal metabolic rate during starvation and activity during recovery can be viewed as adaptations contributing to a higher economization of energy supplies. The experimental birds were unable to eat large quantities of food directly after a period of starvation leading to a comparatively low, or no increase in body mass. Such a slow mass increase is in agreement with observations of migratory birds on arrival at stop-over sites.Abbreviations BM body mass - BMR basal metabolic rate - LBM lean body mass - RQ respiratory quotient     

Colobine forestomach anatomy and diet

Colobine monkeys have complex, multichambered, foregut-fermenting stomachs with either three (“tripartite”) or four (“quadripartite,” adding the praesaccus) chambers where a commensal microbiome digests plant cell walls and possibly detoxifies defensive plant chemicals. Although different potential functions for the praesaccus have been suggested, little evidence exists to support any of the proposed functions. To address the issue of the function of the praesaccus, we collated literature data on diet and compared tripartite and quadripartite species. Our results suggest that the praesaccus is an adaptation to a dietary niche with a particularly high reliance on leaves as fallback foods in colobine clades with quadripartite stomachs, and a higher reliance on fruits/seeds as foods at times of high fruit availability in clades with tripartite stomachs. This supports the notion that a large gut capacity is an important characteristic by which folivores survive on a high fiber diet, and that this large gut capacity may not be necessary for some species if there are seasonal peaks in fruit availability.     

Giraffe browsing in response to plant traits

Intake rates by large herbivores are governed by among other things plant traits. We used Masai giraffe (Giraffa camelopardalis tippelskirchi Matschie) as study animals, testing whether they as very large browsers would follow the Jarman–Bell principle and maximize intake rate while tolerating low forage quality. We worked in Arusha National Park, Tanzania. We investigated how intake rate was determined by bite mass and bite rate, and show that bite mass and bite rate were determined by plant characteristics, governed by inherent plant traits, plant traits acquired from previous years’ browsing, and season. We predicted that; (1) bite mass would be larger in trees without spines than with (2) bite mass would be larger in the wet season than in the dry, (3) bite rate would be higher in spinescent trees than in non-spinescent, (4) bite rate and/or bite mass would increase with previous years’ browsing, (5) bite mass, bite rate or browsing time per tree would be highest for high trees with large, although still available canopies. Visual observations were used to collect data on tree attributes, number of bites taken and time of browsing. Sample size was 132 observed giraffe. We found that bite mass was larger in spineless than in spinescent trees and was larger in the wet season than in the dry. Bite rate, but not bite mass, increased with increasing browsing in previous years and was highest on two to three meter high trees and in spinescent trees. Intake rate followed bite mass more than bite rate and was higher in spineless than in spinescent trees, higher in the wet season than in the dry, and tended to increase with tree height. Giraffe did not prioritize the highest intake rate, but browsed much on Acacias giving a high quality diet but a low intake rate.     

Synergistic interventions to control COVID-19: Mass testing and isolation mitigates reliance on distancing

Stay-at-home orders and shutdowns of non-essential businesses are powerful, but socially costly, tools to control the pandemic spread of SARS-CoV-2. Mass testing strategies, which rely on widely administered frequent and rapid diagnostics to identify and isolate infected individuals, could be a potentially less disruptive management strategy, particularly where vaccine access is limited. In this paper, we assess the extent to which mass testing and isolation strategies can reduce reliance on socially costly non-pharmaceutical interventions, such as distancing and shutdowns. We develop a multi-compartmental model of SARS-CoV-2 transmission incorporating both preventative non-pharmaceutical interventions (NPIs) and testing and isolation to evaluate their combined effect on public health outcomes. Our model is designed to be a policy-guiding tool that captures important realities of the testing system, including constraints on test administration and non-random testing allocation. We show how strategic changes in the characteristics of the testing system, including test administration, test delays, and test sensitivity, can reduce reliance on preventative NPIs without compromising public health outcomes in the future. The lowest NPI levels are possible only when many tests are administered and test delays are short, given limited immunity in the population. Reducing reliance on NPIs is highly dependent on the ability of a testing program to identify and isolate unreported, asymptomatic infections. Changes in NPIs, including the intensity of lockdowns and stay at home orders, should be coordinated with increases in testing to ensure epidemic control; otherwise small additional lifting of these NPIs can lead to dramatic increases in infections, hospitalizations and deaths. Importantly, our results can be used to guide ramp-up of testing capacity in outbreak settings, allow for the flexible design of combined interventions based on social context, and inform future cost-benefit analyses to identify efficient pandemic management strategies.     

Optimal progeny body size in a solitary bee,Osmia bicornis (Apoidea: Megachilidae)

1. Females of solitary, nest‐constructing bees determine both sex (haplo‐diploidy) as well as body size (by amount of provision) of each single offspring. 2. According to the Optimal Allocation Theory, females should allocate resources in portions that maximise fitness returns. A key fitness component in bees is body size that is determined solely by the provisions supplied by the mother. 3. The optimal progeny body size relies on different factors in both sexes. In females, provision efficiency is crucial for reproductive success. In males, however, fitness depends primarily on mating success. 4. Provision efficiency of Osmia bicornis L. females depends on the capacity to stow pollen (scopa) and their ability to carry the packed loads. Scopa capacity increases isometrically with body size whereas indices of flight performance (EPI, free lift) decrease. These complementary effects substantially contribute to the adjustment of optimal body size in daughters. 5. The impact of body mass on fitness of males is determined by the mating system. Owing to the opportunistic polygyny in O. bicornis, there was no detectable correlation between body size and male mating success. Consequently, mother bees distribute their provisions to many, but small sons to increase the number of descendant competitors in the race for matings. Optimal body size in sons is a trade‐off between a large male advantage in rare scrambles over receptive females and small‐size‐related disadvantages in viability.     

中国三江源国家公园与韩国智异山国家公园的对比研究

自1967年智异山被认定为韩国首个国家公园以来,韩国以国家公园为主体的自然保护地体系建设已历经50余年。这期间韩国在国家公园的建设中积累了大量经验,十分值得我国借鉴。选取我国首个国家公园体制试点——三江源国家公园和韩国首个国家公园——智异山国家公园为研究对象,对二者的管理现状进行了定性分析,并运用基于最优实践的国家公园管理能力评价方法对二者的管理能力进行了定量评价。研究表明:三江源国家公园的管理能力综合得分低于智异山国家公园,在体制建设、保障机制、资源环境管理、社区管理和科普教育五个方面均与智异山国家公园存在一定差距;智异山国家公园在资源本底调查、法制建设、多方参与、环境教育和游憩管理方面的管理能力十分突出,为三江源国家公园管理能力的提升提供了有益借鉴;三江源国家公园在生态补偿和制度约束方面具有比较优势,但在自然资源权属、资源本底调查、社区组织建设和游憩管理方面则亟待提升。在此基础上,针对三江源国家公园建设提出了强化科研支撑、健全多方参与制度、推进全民福利共享三项建议,针对我国国家公园体制建设提出了制定《国家公园法》、设置自然保护地顶层规划两项建议,以期促进三江源国家公园管理能力提升、推动我国国家公园体制建设。     

Familial mitochondrial encephalomyopathy (MERRF): genetic, pathophysiological, and biochemical characterization of a mitochondrial DNA disease

A large MERRF pedigree permitted the direct testing of the predictions for a mitochondrial DNA (mtDNA) mutation. A mtDNA mutation was demonstrated by proving maternal inheritance and by identifying specific deficiencies in muscle energetics and mitochondrial respiratory complexes I and IV. mtDNA heteroplasmy (a mixture of mutant and wild-type mtDNAs) was demonstrated by showing variation in the mitochondrial energetic capacity between family members. The phenotypic consequences of differential tissue-specific reliance on mitochondrial ATP was shown by correlating individual respiratory deficiency with the nature and severity of patients' clinical manifestations. The observed spectrum of clinical manifestations resulting from this heteroplasmic mtDNA mutation implies that mtDNA disease may be much more prevalent than previously anticipated.     

School nurses as volunteers in a bioterrorism event

Most public health departments do not have enough public health nurses to adequately respond to a bioterrorist attack or other large-scale health emergency and would need to look to volunteers to help with staffing needs in an emergency. The National Association of School Nurses supports the use of school nurses, a skilled workforce, as responders in a mass casualty event, whether it is naturally occurring or as a result of bioterrorism. This article explores the use of school nurses to supplement public health staff during an emergency. A Likert-scale survey was used to assess the preparedness and willingness of school nurses to respond to a health threat. Although school nurses are willing to respond, the findings highlight the importance of continued education on risk and personal protection.     

The increase in human plasma antioxidant capacity after apple consumption is due to the metabolic effect of fructose on urate, not apple-derived antioxidant flavonoids

Regular fruit consumption lowers the risk of cardiovascular diseases and certain cancers, which has been attributed in part to fruit-derived antioxidant flavonoids. However, flavonoids are poorly absorbed by humans, and the increase in plasma antioxidant capacity observed after consumption of flavonoid-rich foods often greatly exceeds the increase in plasma flavonoids. In the present study, six healthy subjects consumed five Red Delicious apples (1037 +/- 38 g), plain bagels (263.1 +/- 0.9 g) and water matching the carbohydrate content and mass of the apples, and fructose (63.9 +/- 2.9 g) in water matching the fructose content and mass of the apples. The antioxidant capacity of plasma was measured before and up to 6 h after food consumption as ferric reducing antioxidant potential (FRAP), without or with ascorbate oxidase treatment (FRAPAO) to estimate the contribution of ascorbate. Baseline plasma FRAP and FRAPAO were 445 +/- 35 and 363 +/- 35 microM trolox equivalents, respectively. Apple consumption caused an acute, transient increase in both plasma FRAP and FRAPAO, with increases after 1 h of 54.6 +/- 8.7 and 61.3 = 17.2 microM trolox equivalents, respectively. This increase in plasma antioxidant capacity was paralleled by a large increase in plasma urate, a metabolic antioxidant, from 271 +/- 39 microM at baseline to 367 +/- 43 microM after 1 h. In contrast, FRAP and FRAPAO time-dependently decreased after bagel consumption, together with urate. Consumption of fructose mimicked the effects of apples with respect to increased FRAP, FRAPAO, and urate, but not ascorbate. Taken together, our data show that the increase in plasma antioxidant capacity in humans after apple consumption is due mainly to the well-known metabolic effect of fructose on urate, not apple-derived antioxidant flavonoids.     

三江源国家公园游憩承载力模拟仿真研究

游憩承载力评估与预测是国家公园游憩规划与动态管理的重要工具。采用系统动力学模拟仿真软件Vensim＿PLE,构建三江源国家公园游憩承载力系统动力学模型,包括资源空间、生态环境、社会经济、社会心理4个子系统。以2015—2035年为系统仿真区间,模拟了原始情景、社会驱动模式、生态保护优先模式、协调发展模式4种情景下各子系统的发展演化趋势,为三江源国家公园游憩规划与管理提供参考。结果表明：(1)原始情景模式下,三江源国家公园游憩承载力呈先增后降的趋势,在2031年面临游憩承载力超载风险,在加大游憩设施投入的社会驱动模式下,游憩承载力将有所提升,但2031年后提升效果微弱。(2)生态保护优先模式通过提高污染治理能力和适度控制游客规模,游憩承载力有所提升,并将游憩承载力超载风险由2031年延迟至2033年。(3)协调发展模式下,生态环境承载力和资源空间承载力缓慢上升,在4种模式中均为最高值,游憩承载力也达到最大值,游憩承载力超载的风险由2031年延长至2035年以后。对国家公园游憩承载力的模拟预测,可为合理及时调控提供科学参考,有利于三江源国家公园生态保护与游憩质量提升。     

Ontogenetic shift to dependence on salmon-derived nutrients in Dolly Varden char from the Iliamna River,Alaska

The upstream migration, spawning, and death of anadromous, semelparous Pacific salmon brings nutrients to terrestrial and aquatic communities around the Pacific Rim. Many fishes use these resources but the relationship between fish body size and the reliance on salmon-derived nutrients might follow one of several patterns related to the onset of egg consumption with body size as fish grow, and possible shifts to alternative prey such as fishes as they grow larger still. In this study, these size-dependent hypotheses of marine subsidy use by resident Dolly Varden, Salvelinus malma, were tested using diet and stable isotope analyses. S. malma did not shift abruptly to a reliance on salmon eggs after they became large enough to eat eggs (i.e., no gape limitation). Rather, fish large enough to eat eggs but < 150 mm showed diets that blended salmon nutrients with aquatic insects, likely because they were spatially segregated from the highest concentration of spawning sockeye salmon, Oncorhynchus nerka. From intermediate through the largest sizes observed (150 to > 600 mm long) S. malma received ca. 80 % of their nutrients from salmon (eggs, flesh, and maggots that had scavenged dead salmon) based on diet analysis and stable isotope ratios despite being large enough to consume fish, as many similarly-sized salmonids do in other ecosystems. The few fish sampled in June, prior to the availability of salmon subsidies, had stable isotope signatures that also reflected heavy (ca. 90 %) reliance on marine sources, likely because they had eaten little since the end of the salmon run the previous fall. This apparent avoidance of piscivory in favor a rich yet pulsed marine subsidy highlights the importance of healthy salmon runs for the sake of not only the salmon but resident fishes that consume them.     

Regulation of lean mass, bone mass, and exercise tolerance by the central melanocortin system

Signaling via the type 4-melanocortin receptor (MC4R) is an important determinant of body weight in mice and humans, where loss of function mutations lead to significant obesity. Humans with mutations in the MC4R experience an increase in lean mass. However, the simultaneous accrual of fat mass in such individuals may contribute to this effect via mechanical loading. We therefore examined the relationship of fat mass and lean mass in mice lacking the type-4 melanocortin receptor (MC4RKO). We demonstrate that MC4RKO mice display increased lean body mass. Further, this is not dependent on changes in adipose mass, as MC4RKO mice possess more lean body mass than diet-induced obese (DIO) wild type mice with equivalent fat mass. To examine potential sources of the increased lean mass in MC4RKO mice, bone mass and strength were examined in MC4RKO mice. Both parameters increase with age in MC4RKO mice, which likely contributes to increases in lean body mass. We functionally characterized the increased lean mass in MC4RKO mice by examining their capacity for treadmill running. MC4R deficiency results in a decrease in exercise performance. No changes in the ratio of oxidative to glycolytic fibers were seen, however MC4RKO mice demonstrate a significantly reduced heart rate, which may underlie their impaired exercise performance. The reduced exercise capacity we report in the MC4RKO mouse has potential clinical ramifications, as efforts to control body weight in humans with melanocortin deficiency may be ineffective due to poor tolerance for physical activity.     

Concordance between Patient Self-Reports and Claims Data on Clinical Diagnoses,Medication Use,and Health System Utilization in Taiwan

Purpose

The aim of this study was to evaluate the concordance between claims records in the National Health Insurance Research Database and patient self-reports on clinical diagnoses, medication use, and health system utilization.

Methods

In this study, we used the data of 15,574 participants collected from the 2005 Taiwan National Health Interview Survey. We assessed positive agreement, negative agreement, and Cohen''s kappa statistics to examine the concordance between claims records and patient self-reports.

Results

Kappa values were 0.43, 0.64, and 0.61 for clinical diagnoses, medication use, and health system utilization, respectively. Using a strict algorithm to identify the clinical diagnoses recorded in claims records could improve the negative agreement; however, the effect on positive agreement and kappa was diverse across various conditions.

Conclusion

We found that the overall concordance between claims records in the National Health Insurance Research Database and patient self-reports in the Taiwan National Health Interview Survey was moderate for clinical diagnosis and substantial for both medication use and health system utilization.     

Variations in humanized and defined culture conditions supporting derivation of new human embryonic stem cell lines

The evolution of "humanized" (i.e., free of animal sourced reagents) and ultimately chemically defined culture systems for human embryo stem cell (hESC) isolation and culture is of importance to improving their efficacy and safety in research and therapeutic applications. This can be achieved by integration of a multitude of individual approaches to replace or eliminate specific animal sourced reagents into a single comprehensive protocol. In the present study our objective was to integrate strategies obviating reliance on some of the most poorly defined and path-critical factors associated with hESC derivation, namely the use of animal immune compliment to isolate embryo inner cell mass, and animal sourced serum products and feeder cells to sustain hESC growth and attachment. As a result we report the derivation of six new hESC lines isolated by outgrowth from whole blastocysts on an extracellular matrix substrate of purified human laminin (Ln) with transitional reliance on mitotically inactivated human fibroblast (HDF) feeder cells. With this integrated system hESC lines were isolated using either HDF conditioned medium supplemented with a bovine-sourced serum replacement (bSRM), or a defined serum-free medium (SFM) containing only human sourced and recombinant protein. Further, outgrowth of embryonic cells from whole blastocysts in both media could be achieved for up to 1 week without reliance on feeder cells. All variant conditions sustained undifferentiated cell status, a stable karyotype and the potential to form cells representative of all three germinal lineages in vitro and in vivo, when transitioned off of feeders onto Laminin or Matrigel. Our study thus demonstrates the capacity to integrate derivation strategies eliminating a requirement for animal immune compliment and serum products, with a transitional requirement for human feeder cells. This represents another sequential step in the generation of therapeutic grade stem cells with reduced risk of zoonotic pathogen transmission.     

Hydrophobic interaction chromatography of proteins. II. Binding capacity,recovery and mass transfer properties

Hydrophobic interaction chromatography media suited for large scale separations were compared regarding dynamic binding capacity, recovery and mass transfer properties. In all cases, pore diffusion was the rate limiting step. Reduced heights equivalent to a theoretical plate for bovine serum albumin derived from breakthrough curves at reduced velocities between 60 and 1500 ranged from 10 to 700. Pore diffusion coefficients were derived from pulse response experiments for the model proteins alpha-lactalbumin, lysozyme, beta-lactoglobulin, bovine serum albumin and immunoglobulin G. Diffusivity of lysozyme did not follow the trend of decreasing diffusivity with increasing molecular mass, as observed for the rest of the proteins. In general, mass transfer coefficients were smaller compared to ion-exchange chromatography. Dynamic binding capacities for the model protein bovine serum albumin varied within a broad range. However, sorbents based on polymethacrylate showed a lower dynamic capacity than media based on Sepharose. Some sorbents could be clustered regarding binding capacity affected by salt. These sorbents exhibited a disproportional increase of binding capacity with increasing ammonium sulfate concentration. Recovery of proteins above 75% could be observed for all sorbents. Several sorbents showed a recovery close to 100%.     

Altered TNF-alpha, glucose, insulin, and amino acids in islets of Langerhans cultured in a microgravity model system

The present studies were designed to determine effects of a microgravity model system upon lipopolysaccharide (LPS)-stimulated tumor necrosis factor-alpha (TNF-alpha) activity and indexes of insulin and fuel homeostasis of pancreatic islets of Langerhans. Islets (1,726 +/- 117, 150 islet equivalent units) from Wistar-Furth rats were treated as 1) high aspect ratio vessel (HARV) cell culture, 2) HARV plus LPS, 3) static culture, and 4) static culture plus LPS. TNF-alpha (L929 cytotoxicity assay) was significantly increased in LPS-induced HARV and static cultures; yet the increase was more pronounced in the static culture group (P < 0.05). A decrease in insulin concentration was demonstrated in the LPS-stimulated HARV culture (P < 0.05). We observed a greater glucose concentration and increased disappearance of arginine in islets cultured in HARVs. Although nitrogenous compound analysis indicated a ubiquitous reliance on glutamine in all experimental groups, arginine was converted to ornithine at a twofold greater rate in the islets cultured in the HARV microgravity model system (P < 0.05). These studies demonstrate alterations in LPS-induced TNF-alpha production of pancreatic islets of Langerhans, favoring a lesser TNF activity in the HARV. These alterations in fuel homeostasis may be promulgated by gravity-averaged cell culture methods or by three-dimensional cell assembly.