BackgroundThe Critical Assessment of Functional Annotation (CAFA) is an ongoing, global, community-driven effort to evaluate and improve the computational annotation of protein function.ResultsHere, we report on the results of the third CAFA challenge, CAFA3, that featured an expanded analysis over the previous CAFA rounds, both in terms of volume of data analyzed and the types of analysis performed. In a novel and major new development, computational predictions and assessment goals drove some of the experimental assays, resulting in new functional annotations for more than 1000 genes. Specifically, we performed experimental whole-genome mutation screening in Candida albicans and Pseudomonas aureginosa genomes, which provided us with genome-wide experimental data for genes associated with biofilm formation and motility. We further performed targeted assays on selected genes in Drosophila melanogaster, which we suspected of being involved in long-term memory.ConclusionWe conclude that while predictions of the molecular function and biological process annotations have slightly improved over time, those of the cellular component have not. Term-centric prediction of experimental annotations remains equally challenging; although the performance of the top methods is significantly better than the expectations set by baseline methods in C. albicans and D. melanogaster, it leaves considerable room and need for improvement. Finally, we report that the CAFA community now involves a broad range of participants with expertise in bioinformatics, biological experimentation, biocuration, and bio-ontologies, working together to improve functional annotation, computational function prediction, and our ability to manage big data in the era of large experimental screens.

Hosts have numerous defenses against parasites, of which behavioral immune responses are an important but under-appreciated component. Here we describe a behavioral immune response Drosophila melanogaster utilizes against endoparasitoid wasps. We found that when flies see wasps they switch to laying eggs in alcohol-laden food sources that protect hatched larvae from infection. This oviposition behavior change, mediated by neuropeptide F, is retained long after wasps are removed. Flies respond to diverse female larval endoparasitoids but not to pupal endoparasitoids or males, showing they maintain specific wasp search images. Furthermore, the response evolved multiple times across the genus Drosophila. Our data reveal a behavioral immune response based on anticipatory medication of offspring, and outline a non-associative memory paradigm based on innate parasite recognition by the host.

Hosts may defend themselves against parasitism through a wide variety of defence mechanisms, but due to finite resources, investment in one defence mechanism may trade-off with investment in another mechanism. We studied resistance strategies against the parasitoid wasp Leptopilina boulardi in two Drosophila species. We found that D. melanogaster had significantly lower physiological resistance against L. boulardi than D. simulans, and hypothesized that D. melanogaster might instead invest more heavily in other forms of defence, such as behavioural defence. We found that when given a choice between clean oviposition sites and sites infested with wasps, both D. melanogaster and D. simulans detected and avoided infested sites, which presumably limits later exposure of their offspring to infection. Unlike D. simulans, however, D. melanogaster laid significantly fewer eggs than controls in the forced presence of wasps. Our findings suggest that D. melanogaster relies more heavily on behavioural avoidance as defence against wasp parasitism than D. simulans, and that this may compensate for a lack of physiological defence.

The present experiments tested the involvement of central catecholaminergic systems in the suckling-induced release of oxytocin (OT) during lactation in the rat. In the first experiment, female rats in midlactation were separated from their offspring for 4 h and then allowed to suckle their litters for 30 or 60 min or to remain nonsuckled. The turnover rates of norepinephrine (NE) and dopamine (DA) were calculated from the rate of decline after synthesis inhibition. Suckling decreased the turnover rate of DA in the median eminence and in the neurointermediate lobe of the pituitary gland. Suckling increased the turnover rate of NE in the rostral paraventricular and supraoptic nuclei, areas that contain most of the OT cells that project to the neural lobe of the pituitary, and in the interstitial nucleus of the stria terminalis, but not in the arcuate or caudal paraventricular nuclei, median eminence, or neurointermediate lobe. In a second experiment, midlactating females received intracerebral microinjections of the catecholamine neurotoxin 6-hydroxydopamine or of vehicle into the vicinity of the paraventricular and supraoptic nuclei 1 week before a suckling test. The release of OT was completely prevented in 6-hydroxydopamine-treated animals, and NE was significantly decreased in the paraventricular, supraoptic, and arcuate nuclei. In a third study, the increase in plasma OT in response to suckling was prevented by stimulation of DA receptors with bromocriptine, while blockade of DA receptors with domperidone significantly increased plasma OT levels in nonsuckled lactating rats. These results suggest that suckling stimulation activates the noradrenergic innervation to the rostral paraventricular nucleus and to the supraoptic nucleus, which exerts an excitatory influence on the release of OT and decreases activity of the tuberohypophyseal DA system, which provides a tonic inhibitory influence over the secretion of OT.

The present experiments investigated the time course of maternal modulation of GH secretion and examined the possible role of milk in the regulation of GH secretion in neonatal rats. Serum GH concentrations in neonatal rats were high at birth and declined over time postpartum. Separation of rat pups from their mothers decreased, while a subsequent period of suckling increased, serum GH levels in rat pups on postpartum days 1-14, but not on day 20. The water-soluble fraction (infranatant) of rat milk contained immunoreactive (ir) rat GH-releasing hormone (rGHRH)-like material (725.06 +/- 81.29 pg/ml), ir-somatostatin-like activity (1.64 +/- 0.2 ng/ml), and irGH (4.79 +/- 0.73 ng/ml). The concentrations of these hormones tended to decrease with time postpartum and were positively correlated with each other (r = 0.70; P less than or equal to 0.0001). IrPRL was also present in the infranatant (148.44 +/- 14.55 ng/ml), but levels were not correlated with the other hormones detected. Milk infranatant stimulated GH secretion from perifused neonatal rat pituitary glands in vitro. Milk infranatant also stimulated GH secretion in vivo when administered sc or intragastrically to 2- or 8-day-old rat pups. The GH-releasing effect was not due to gastric distension or nonspecific nutritive components, as neither 0.9% saline nor nutrients (5% glucose and 10% BSA) increased serum GH levels. The presence of high concentrations of irGHRH in rat milk infranatant and the strong correlation between the irGHRH concentrations of milk samples and the in vitro GH response (r = 0.71; P less than or equal to 0.005) suggested that this peptide is a major candidate for producing the in vitro and in vivo GH-stimulating activity in rat milk. However, the minimally effective concentration of synthetic rGHRH required to stimulate GH release in the superfusion system was between 1-10 nM, which exceeds milk irGHRH levels by 100- to 1000-fold. Moreover, in vivo administration of synthetic rGHRH (sc or intragastrically) was unable to increase serum GH concentrations in 2-day-old pups, although a large dose (100 ng/g) of human GHRH sc was effective. These findings indicate that rat milk may be an important maternal factor that modulates GH secretion and, consequently, growth during the neonatal period. Rat milk has GH-releasing activity both in vivo and in vitro in neonatal rats, but the GH-releasing activities of milk are probably only minimally due to its rGHRH content.

Prolactin (PRL)-like bioactivity (in Nb2 lymphoma assay) and immunoreactivity (in radioimmunoassay (RIA)) in rat milk, maternal and neonatal sera and in neonatal rat pituitary cultures were investigated. The PRL-like bioactivity in the water-soluble fraction of rat milk was high and exceeded its immunoreactivity 5.8-, 4.0- and 2.1-fold, on days 2, 12 and 22 of lactation respectively. The elevated bioactivity to immunoreactivity (B/I) ratio of PRL in milk was not due to the presence of interleukin-2 (IL-2) in milk, since the proliferation of the CTLL-2 murine T cells, which are not sensitive to PRL, was promoted by IL-2 but not by milk. Serum levels of immunoreactive PRL were low in sera of non-weaned rat pups on days 2, 12 and 22 postpartum. Similar to milk, the B/I ratio of PRL in sera of rat pups was high and decreased with time postpartum. Pituitary glands of pups obtained on days 2, 12 and 22 secreted progressively increasing amounts of PRL in vitro; the B/I ratio ranged between 1.2 and 2.1 without a significant change. The relative concentrations of size variants in milk were not proportional to those in serum of lactating rats on day 2 postpartum as assessed by Sepharcryl S-100 HR gel permeation chromatography and Nb2 bioassay or RIA. Size variants of biologically active PRL were abundant in early milk and gradually diminished as lactation progressed: a partially resolved peak representing monomeric to dimeric PRL variants (relative molecular weights ranging between 18 k and 42 k) became progressively narrower between days 2 and 22. Biologically active and immunoreactive PRLs displayed disparate elution profiles. The elution profile of PRL in sera of neonatal rats on day 2 post-partum was different from that of maternal serum or milk. The major immunological (and possibly biological) PRL-like activity eluted as two adjacent peaks at 2.2 k and 1.5 k, raising the possibility that fragments of milk-borne PRL were absorbed from the gut after partial proteolytic degradation. In contrast with PRL, GH (which is present in rat milk only in minute concentrations) did not show heterogeneity in sera of 2-day-old rat pups in gel permeation chromatography. The present results demonstrate that the concentrations of PRL-like activity in rat milk and newborn rat serum have been grossly underestimated because levels have been measured by RIA. The high B/I ratio of PRL in milk and neonatal sera is due to the presence of PRL-related compounds.(ABSTRACT TRUNCATED AT 250 WORDS)

The somatotropic axis, and particularly growth hormonereleasing hormone (GHRH), is implicated in the regulation of sleep-wake activity. To evaluate sleep in chronic somatotropic deficiency, sleep-wake activity was studied in dwarf (dw/dw) rats that are known to have a defective GHRH signaling mechanism in the pituitary and in normal Lewis rats, the parental strain of the dw/dw rats. In addition, expression of GHRH receptor (GHRH-R) mRNA in the hypothalamus/preoptic region and in the pituitary was also determined by means of reverse transcription-PCR, and GHRH content of the hypothalamus was measured. Hypothalamic/preoptic and pituitary GHRH-R mRNA levels were decreased in the dw/dw rats, indicating deficits in the central GHRHergic transmission. Hypothalamic GHRH content in dw/dw rats was also less than that found in Lewis rats. The dw/dw rats had less spontaneous nonrapid eye movement sleep (NREMS) (light and dark period) and rapid eye movement sleep (REMS) (light period) than did the control Lewis rats. After 4 hr of sleep deprivation, rebound increases in NREMS and REMS were normal in the dw/dw rat. As determined by fast Fourier analysis of the electroencephalogram (EEG), the sleep deprivation-induced enhancements in EEG slow-wave activity in the dw/dw rats were only one-half of the response in the Lewis rats. The results are compared with sleep findings previously obtained in GHRH-deficient transgenic mice. The alterations in NREMS are attributed to the defect in GHRH signaling, whereas the decreases in REMS might result from the growth hormone deficiency in the dw/dw rat.

The presence of prolactin (PRL) mRNA in the mammary gland, placenta, and pituitary gland of lactating and pregnant rats was investigated by polymerase chain reaction (PCR). Polyadenylated RNA was prepared from total RNA samples by oligo(dT)-cellulose chromatography, and complementary cDNAs were synthesized. A standardized amount of cDNA from each sample was used as the template in a Taq PCR under high-stringency conditions. PCR amplified a signal with the predicted size of approximately 375 bp in mammary and pituitary glands of lactating and pregnant rats, and in placentae of pregnant rats. This band specifically hybridized with a probe overlapping the entire sequence of the mature rat (r) PRL mRNA in Southern blot analysis. When the rPRL-specific primers were used, PCR revealed no signal in the liver or in lactating mammary gland explants cultured in vitro for 48 h, while the same cDNA preparations gave strong signals for beta-actin. The viability of the mammary gland explants was also suggested by their ability to secrete immunoreactive casein in vitro. PRL mRNA was localized in the epithelium of alveoli and ducts of the lactating mammary gland by in situ hybridization. These data provide evidence that the PRL gene is expressed in the mammary gland of pregnant and lactating rats, and suggest that the mammary gland might contribute to PRL in milk by de novo synthesis. Thus, while the placenta is an exogenous source of PRL-like activities for the fetus in utero, the mammary gland might take over this function after birth.