FAIRshake: Toolkit to Evaluate the FAIRness of Research Digital Resources.

As more digital resources are produced by the research community, it is becoming increasingly important to harmonize and organize them for synergistic utilization. The findable, accessible, interoperable, and reusable (FAIR) guiding principles have prompted many stakeholders to consider strategies for tackling this challenge. The FAIRshake toolkit was developed to enable the establishment of community-driven FAIR metrics and rubrics paired with manual and automated FAIR assessments. FAIR assessments are visualized as an insignia that can be embedded within digital-resources-hosting websites. Using FAIRshake, a variety of biomedical digital resources were manually and automatically evaluated for their level of FAIRness.

The National Institutes of Health's Big Data to Knowledge (BD2K) initiative: capitalizing on biomedical big data.

Biomedical research has and will continue to generate large amounts of data (termed 'big data') in many formats and at all levels. Consequently, there is an increasing need to better understand and mine the data to further knowledge and foster new discovery. The National Institutes of Health (NIH) has initiated a Big Data to Knowledge (BD2K) initiative to maximize the use of biomedical big data. BD2K seeks to better define how to extract value from the data, both for the individual investigator and the overall research community, create the analytic tools needed to enhance utility of the data, provide the next generation of trained personnel, and develop data science concepts and tools that can be made available to all stakeholders.

NHLBI support of systems biology.

The National Heart, Lung, and Blood Institute (NHLBI) has recognized the importance of the systems biology approach for understanding normal physiology and perturbations associated with heart, lung, blood, and sleep diseases and disorders. In 2006, NHLBI announced the Exploratory Program in Systems Biology program, followed in 2010 by the NHLBI Systems Biology Collaborations program. The goal of these programs is to support collaborative teams of investigators in using experimental and computational strategies to integrate the component parts of biological networks and pathways into computational models that are based firmly on and validated using experimental data. These validated models are then applied to gain insights into the mechanisms of altered system function in disease, to generate novel hypotheses regarding these mechanisms that can be tested experimentally, and to then use the results of experiments to refine the models. This perspective reviews the history of dedicated systems biology programs at NHLBI and reviews some promising directions for future research in this area.

Genetic basis of murine antibacterial defense to streptococcal lung infection.

To evaluate the effect of genetic background on antibacterial defense to streptococcal infection, eight genetically diverse strains of mice (A/J, DBA/2J, CAST/Ei, FVB/NJ, BALB/cJ, C57BL/6J, 129/SvImJ, and C3H/HeJ) and tlr2-deficient mice (C57BL/6(tlr2-/-)) were infected with three doses of Streptococcus zooepidemicus (500, 5,000, or 50,000 colony-forming units) by alveolar challenge. There was a range of susceptibility between the strains at each dose and time point (6, 24, and 96 h). At the lowest dose, the 129/SvImJ and C3H/HeJ strains had significantly higher bacterial counts at all time points after infection, when compared to A/J, DBA/2J, CAST/Ei, FVB/NJ, which were resistant to infection at the low dose of innoculum. At the medium dose, 129/SvImJ and C3H/HeJ had higher bacterial counts, while A/J, DBA/2J, and BALB/cJ showed reduced streptococcal growth. After the highest dose of Streptococcus, there were minimal differences between strains, suggesting the protective impact of modifier genes can be overcome. TLR2-deficient animals contained increased bacterial load with reduced cytokines after 96 h when compared to C57BL/6J controls suggesting a role of innate immunity in late antibacterial defense. Overall, we identify vulnerable (129/SvlmJ and C3H/HeJ) and resistant (A/J, FVB, and DBA) mouse strains to streptococcal lung infection, which demonstrate divergent genetic expression profiles. These results demonstrate that innate differences in pulmonary host defense to S. zooepidemicus are dependent on host genetic factors.

Independence and reproducibility across microarray platforms.

Microarrays have been widely used for the analysis of gene expression, but the issue of reproducibility across platforms has yet to be fully resolved. To address this apparent problem, we compared gene expression between two microarray platforms: the short oligonucleotide Affymetrix Mouse Genome 430 2.0 GeneChip and a spotted cDNA array using a mouse model of angiotensin II-induced hypertension. RNA extracted from treated mice was analyzed using Affymetrix and cDNA platforms and then by quantitative RT-PCR (qRT-PCR) for validation of specific genes. For the 11,710 genes present on both arrays, we assessed the relative impact of experimental treatment and platform on measured expression and found that biological treatment had a far greater impact on measured expression than did platform for more than 90% of genes, a result validated by qRT-PCR. In the small number of cases in which platforms yielded discrepant results, qRT-PCR generally did not confirm either set of data, suggesting that sequence-specific effects may make expression predictions difficult to make using any technique.

Light pulses do not induce c-fos or per1 in the SCN of hamsters that fail to reentrain to the photocycle.

Circadian activity rhythms of most Siberian hamsters (Phodopus sungorus sungorus) fail to reentrain to a 5-h phase shift of the light-dark (LD) cycle. Instead, their rhythms free-run at periods close to 25 h despite the continued presence of the LD cycle. This lack of behavioral reentrainment necessarily means that molecular oscillators in the master circadian pacemaker, the SCN, were unable to reentrain as well. The authors tested the hypothesis that a phase shift of the LD cycle rendered the SCN incapable of responding to photic input. Animals were exposed to a 5-h phase delay of the photocycle, and activity rhythms were monitored until a lack of reentrainment was confirmed. Hamsters were then housed in constant darkness for 24 h and administered a 30-min light pulse 2 circadian hours after activity onset. Brains were then removed, and tissue sections containing the SCN were processed for in situ hybridization. Sections were probed with Siberian hamster c-fos and per1 mRNA probes because light rapidly induces these 2 genes in the SCN during subjective night but not at other circadian phases. Light pulses induced robust expression of both genes in all animals that reentrained to the LD cycle, but no expression was observed in any animal that failed to reentrain. None of the animals exhibited an intermediate response. This finding is the first report of acute shift in a photocycle eliminating photosensitivity in the SCN and suggests that a specific pattern of light exposure may desensitize the SCN to subsequent photic input.

Cardiac transcriptional response to acute and chronic angiotensin II treatments.

Exposure of experimental animals to increased angiotensin II (ANG II) induces hypertension associated with cardiac hypertrophy, inflammation, and myocardial necrosis and fibrosis. Some of the most effective antihypertensive treatments are those that antagonize ANG II. We investigated cardiac gene expression in response to acute (24 h) and chronic (14 day) infusion of ANG II in mice; 24-h treatment induces hypertension, and 14-day treatment induces hypertension and extensive cardiac hypertrophy and necrosis. For genes differentially expressed in response to ANG II treatment, we tested for significant regulation of pathways, based on Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Microarray Pathway Profiler (GenMAPP) databases, as well as functional classes based on Gene Ontology (GO) terms. Both acute and chronic ANG II treatments resulted in decreased expression of mitochondrial metabolic genes, notably those for the electron transport chain and Krebs-TCA cycle; chronic ANG II treatment also resulted in decreased expression of genes involved in fatty acid metabolism. In contrast, genes involved in protein translation and ribosomal activity increased expression following both acute and chronic ANG II treatments. Some classes of genes showed differential response between acute and chronic ANG II treatments. Acute treatment increased expression of genes involved in oxidative stress and amino acid metabolism, whereas chronic treatments increased cytoskeletal and extracellular matrix genes, second messenger cascades responsive to ANG II, and amyloidosis genes. Although a functional linkage between Alzheimer disease, hypertension, and high cholesterol has been previously documented in studies of brain tissue, this is the first demonstration of induction of Alzheimer disease pathways by hypertension in heart tissue. This study provides the most comprehensive available survey of gene expression changes in response to acute and chronic ANG II treatment, verifying results from disparate studies, and suggests mechanisms that provide novel insight into the etiology of hypertensive heart disease and possible therapeutic interventions that may help to mitigate its effects.

Homeostatic regulation of sleep in arrhythmic Siberian hamsters.

Sleep is regulated by independent yet interacting circadian and homeostatic processes. The present study used a novel approach to study sleep homeostasis in the absence of circadian influences by exposing Siberian hamsters to a simple phase delay of the photocycle to make them arrhythmic. Because these hamsters lacked any circadian organization, their sleep homeostasis could be studied in the absence of circadian interactions. Control animals retained circadian rhythmicity after the phase shift and re-entrained to the phase-shifted photocycle. These animals displayed robust daily sleep-wake rhythms with consolidated sleep during the light phase beginning about 1 h after light onset. This marked sleep-wake pattern was circadian in that it persisted in constant darkness. The distribution of sleep in the arrhythmic hamsters over 24 h was similar to that in the light phase of rhythmic animals. Therefore, daily sleep amounts were higher in arrhythmic animals compared with rhythmic ones. During 2- and 6-h sleep deprivations (SD), it was more difficult to keep arrhythmic hamsters awake than it was for rhythmic hamsters. Because the arrhythmic animals obtained more non-rapid eye movement sleep (NREMS) during the SD, they showed a diminished compensatory response in NREMS EEG slow-wave activity during recovery sleep. When amounts of sleep during the SD were taken into account, there were no differences in sleep homeostasis between experimental and control hamsters. Thus loss of circadian control did not alter the homeostatic response to SD. This supports the view that circadian and homeostatic influences on sleep regulation are independent processes.

Melatonin production accompanies arousal from daily torpor in Siberian hamsters.

Arousal from deep hibernation is accompanied by a transient rise of melatonin (Mel) in circulation; there are no comparable analyses of Mel concentrations in species that undergo much shallower, shorter duration episodes of daily torpor. Serum Mel concentrations were determined during arousal from both natural daily torpor and torpor induced by 2-deoxy-D-glucose (2-DG) treatment (2,500 mg/kg, intraperitoneal [IP]); blood samples were drawn from the retro-orbital sinus of anesthetized Siberian hamsters. For animals kept in darkness during torpor, Mel concentrations were highest during early arousal when thermogenesis is maximal, and they decreased as body temperature increased during arousal and returned to baseline once euthermia was reestablished. In hamsters kept in the light during the torpor bout, Mel concentrations were elevated above basal values during arousal, but the response was significantly blunted in comparison with values recorded in darkness. Increased Mel concentrations were detected in hamsters only during arousal from torpor (either natural or 2-DG induced) and were not simply a result of the drug treatment; hamsters that remained euthermic or manifested mild hypothermia after drug treatment maintained basal Mel concentrations. We propose that increased Mel production may reflect enhanced sympathetic activation associated with intense thermogenesis during arousal from torpor rather than an adjustment of the circadian rhythm of Mel secretion.

Juvenile Siberian hamsters display torpor and modified locomotor activity and body temperature rhythms in response to reduced food availability.

Siberian hamsters as young as 16 and 28 d displayed torpor in response to treatment with 2,500 mg/kg 2-deoxy-D-glucose and reduced food availability, respectively. In addition, most food-restricted hamsters displayed increased locomotor activity and elevated body temperatures in the 3 h immediately preceding daily food delivery. This anticipatory activity disappeared within a few days of reimposition of ad lib. feeding. Torpor first appeared spontaneously at approximately 13 wk of age in hamsters fed ad lib. and maintained in short day lengths. The onset of this "spontaneous" torpor was unaffected by the hamsters' history of food restriction before age 2 mo. Siberian hamsters born late in the breeding season can conserve energy by undergoing torpor immediately after weaning when they contend with food shortages and concurrent energetic challenges imposed by growth requirements and low ambient temperatures.

Loss of circadian organization of sleep and wakefulness during hibernation.

We investigated circadian and homeostatic regulation of nonrapid eye movement (NREM) sleep in golden-mantled ground squirrels during euthermic intervals between torpor bouts. Slow-wave activity (SWA; 1-4 Hz) and sigma activity (10-15 Hz) represent the two dominant electroencephalographic (EEG) frequency components of NREM sleep. EEG sigma activity has a strong circadian component in addition to a sleep homeostatic component, whereas SWA mainly reflects sleep homeostasis [Dijk DJ and Czeisler CA. J Neurosci 15: 3526-3538, 1995; Dijk DJ, Shanahan TL, Duffy JF, Ronda JM, and Czeisler CA. J Physiol (Lond) 505: 851-858, 1997]. Animals maintained under constant conditions continued to display circadian rhythms in both sigma activity and brain temperature throughout euthermic intervals, whereas sleep and wakefulness showed no circadian organization. Instead, sleep and wakefulness were distributed according to a 6-h ultradian rhythm. SWA, NREM sleep bout length, and sigma activity responded homeostatically to the ultradian sleep-wake pattern. We suggest that the loss of sleep-wake consolidation in ground squirrels during the hibernation season may be related to the greatly decreased locomotor activity during the hibernation season and may be necessary for maintenance of multiday torpor bouts characteristic of hibernating species.

Temperature dependence of gonadal regression in Syrian hamsters exposed to short day lengths.

We sought to determine whether ambient temperature (T(a)) affects gonadal function by altering the rate at which circadian rhythms entrain to short day lengths. Syrian hamsters were housed in cages where they received 14 h of light per day ("long days," 14L) at 22 degrees C. Hamsters were then transferred to cages to receive 10 h of light per day ("short days," 10L) and kept at 5, 22, or 28 degrees C or were maintained in 14L at 22 degrees C. Body mass and estimated testis volume as well as duration of nocturnal locomotor activity (alpha), previously established as a reliable indicator of the duration of nocturnal melatonin secretion, were determined over the course of 24 wk. Testicular regression in short days was accelerated by 4 wk at 5 degrees C and delayed by 3 wk at 28 degrees C relative to 22 degrees C. The interval between alpha-expansion and initiation of testicular regression was markedly affected by T(a) with delays of 0, 3, and 6 wk at 5, 22, and 28 degrees C, respectively. All hamsters held at 5 and 22 degrees C underwent testicular regression, but 25% of those maintained at 28 degrees C failed to do so. We suggest that T(a) modulates testicular regression primarily by affecting responsiveness of neuroendocrine target tissues to long melatonin signals.