Smart phone, smart science: how the use of smartphones can revolutionize research in cognitive science

Investigating human cognitive faculties such as language, attention, and memory most often relies on testing small and homogeneous groups of volunteers coming to research facilities where they are asked to participate in behavioral experiments. We show that this limitation and sampling bias can be overcome by using smartphone technology to collect data in cognitive science experiments from thousands of subjects from all over the world. This mass coordinated use of smartphones creates a novel and powerful scientific "instrument" that yields the data necessary to test universal theories of cognition. This increase in power represents a potential revolution in cognitive science.     

The role of trimerization in the osmoregulated betaine transporter BetP

The osmoregulated betaine transporter BetP is a stable trimer. Structural studies have shown that individual protomers can adopt distinct transport conformations, implying a functional role for the trimeric state in transport, although the role of trimerization in regulation is not yet understood. We designed putative monomeric mutants by molecular-dynamics simulations and in silico alanine-scanning mutagenesis. Several mutants including BetP-W101A/T351A were monomeric in detergent as well as in the membrane, as shown by blue native gel electrophoresis, crosslinking and electron microscopy. This monomeric form retains the ability to accumulate betaine, but is no longer regulated by hyperosmotic shock.     

The endocrinology of puberty and reproductive functioning in female cotton-top tamarins (Saguinus oedipus) under varying social conditions

Sexual maturation and fertility were assessed in fourteen cotton-top tamarin (Saguinus oedipus) females under various social conditions. Six tamarin females (20-28 mo of age) showed a suppression of fertility while living with their families. Hormonal profiles demonstrated low, acyclic levels of urinary luteinizing hormone (LH) and estrone-conjugates (E1C). A rapid onset of ovarian and pituitary cyclicity occurred when four of the six females were removed from their families and paired with an unrelated male. In one female, an ovulatory LH peak occurred as early as eight days after pairing and resulted in conception and full-term pregnancy. Two of the six females were housed in total isolation for 30 days following their removal from the family and prior to pairing. Gradual increases in hormone concentrations occurred during isolation; however, there was no ovarian cyclicity until each female was paired with an unrelated male. In all six females, conception occurred before or as a result of the third ovulatory cycle. Partial isolation of a 36-mo-old female resulted in elevated LH and E1C levels, but cyclicity was not observed until the female was paired with an unrelated male. These findings indicate that removal of a female from the family alone does not initiate ovarian cycling. Sexual maturation, or puberty, occurs in female tamarins living with their families between 15 and 17 mo of age when mean LH and E1C levels began to increase. However, when a female is removed and paired at 9 mo of age with an unrelated male, elevated levels of LH and E1C may be seen by 10 and 11 mo of age. Our findings indicate that a suppression of fertility occurs in cotton-top tamarins living with their families, but that reproductive suppression does not affect the process of sexual maturation. Both removal from the family environment and stimulation by an unrelated male tamarin were necessary to induce normal reproductive activity. An acceleration of puberty occurred when a female tamarin was removed from her family early in development and paired with a male.     

CYP719B1 Is Salutaridine Synthase, the C-C Phenol-coupling Enzyme of Morphine Biosynthesis in Opium Poppy

Morphine is a powerful analgesic natural product produced by the opium poppy Papaver somniferum. Although formal syntheses of this alkaloid have been reported, the morphine molecule contains five stereocenters and a C-C phenol linkage that to date render a total synthesis of morphine commercially unfeasible. The C-C phenol-coupling reaction along the biosynthetic pathway to morphine in opium poppy is catalyzed by the cytochrome P450-dependent oxygenase salutaridine synthase. We report herein on the identification of salutaridine synthase as a member of the CYP719 family of cytochromes P450 during a screen of recombinant cytochromes P450 of opium poppy functionally expressed in Spodoptera frugiperda Sf9 cells. Recombinant CYP719B1 is a highly stereo- and regioselective enzyme; of forty-one compounds tested as potential substrates, only (R)-reticuline and (R)-norreticuline resulted in formation of a product (salutaridine and norsalutaridine, respectively). To date, CYP719s have been characterized catalyzing only the formation of a methylenedioxy bridge in berberine biosynthesis (canadine synthase, CYP719A1) and in benzo[c]phenanthridine biosynthesis (stylopine synthase, CYP719A14). Previously identified phenol-coupling enzymes of plant alkaloid biosynthesis belong only to the CYP80 family of cytochromes. CYP719B1 therefore is the prototype for a new family of plant cytochromes P450 that catalyze formation of a phenol-couple.The C-O or C-C phenol-couple is widely present in the plant kingdom in natural product biosynthetic processes such as alkaloid (1), lignan (2), lignin (3), and gallotannin (4) formation. Phenol-coupling reactions in nature were thought to be catalyzed by a variety of oxidative enzymes with broad substrate specificity such as peroxidases, polyphenol oxidases, and laccases. More recently, several enzymes discovered to be responsible for the formation of intermolecular C-O phenol and intramolecular C-C phenol-couples were found to be highly regio- and/or stereoselective catalysts. The first intermolecular C-O phenol-coupling enzyme identified was the cytochrome P450-dependent oxidase berbamunine synthase (CYP80A1) of bisbenzylisoquinoline alkaloid biosynthesis in Berberis cell cultures (5, 6) (Fig. 1). This enzyme is regiospecific, but will accept either (R)- and (S)-N-methylcoclaurine to form R-R and R-S phenol-coupled products. Absolute regio- and stereospecificity is demonstrated in the formation of the lignan (+)-pinoresinol from two molecules of coniferyl alcohol, a reaction guided by dirigent proteins that can be catalyzed by a range of oxidases or oxidants (7). The aporphine alkaloid intramolecular C-C phenol-couple is catalyzed in Coptis japonica cell cultures by the cytochrome P450-dependent oxidase CYP80G2; this enzyme accepts six tetrahydrobenzylisoquinoline alkaloids as substrate (8).Open in a separate windowFIGURE 1.Selected phenol-coupling reactions of alkaloid biosynthesis. Berbamunine synthase (CYP80A1) catalyzes the C-O intermolecular phenol-coupling reaction of bisbenzyisoquinoline alkaloid biosynthesis. (S)-Corytuberine synthase (CYP80G2) catalyzes formation of the intramolecular C-C phenol-couple in magnoflorine biosynthesis. Salutaridine synthase forms the C-C intramolecular phenol-couple of salutaridine in morphine biosynthesis.Morphine has often been described as the king of alkaloids. Although formal syntheses of this powerful analgesic have been reported, yields are low (Ref. 9 and references therein); attempts in organic chemistry to mimic the biosynthetic formation of the C-C phenol-couple of salutaridine (Fig. 1) have been either unsuccessful, yielding rather isoboldine or pallidine (10), or have resulted in very low yield of salutaridine (11) or in a mixture of isoboldine and salutaridine, with the reaction favoring formation of isoboldine by a factor of ∼5 (12). Along with the five stereocenters present in this molecule, the C-C phenol-couple renders a chemical synthesis of morphine commercially unfeasible. The enzyme catalyzing this reaction in planta was sought unsuccessfully for many years and was discovered finally in the opium poppy Papaver somniferum to be a cytochrome P450-dependent oxidase that stereo- and regiospecifically produces salutaridine by C-C phenol-coupling of (R)-reticuline (Fig. 1) (1, 13). The native enzyme salutaridine synthase was unstable, which precluded protein purification for further characterization.Herein, we describe the identification and functional expression of opium poppy salutaridine synthase, a member of the cytochrome P450 family, in Spodoptera frugiperda Sf9 cells. The recombinant enzyme was sufficiently stable in insect cell culture to be characterized with respect to substrate specificity and steady state kinetic values. Recombinant salutaridine synthase converted (R)-reticuline exclusively to salutaridine and (R)-norreticuline exclusively to norsalutaridine (N-demethylsalutaridine).     

Formation of lipid reserves in fat body and eggs of the yellow fever mosquito, Aedes aegypti

We examined the accumulation of lipids in adult females of the mosquito, Aedes aegypti. Females emerged with about 100 μg lipid in the fat body. With access to sugar water lipids increased over seven days to 300 μg. After a blood meal on day five, sugar-fed females accumulated 120-140 μg of lipids in their ovaries within 2 days. At the same time the lipid content of the fat body decreased by 100 μg, indicating transfer of lipids from fat body to oocytes. Experiments in which fat body lipids were prelabelled support this conclusion. Label was transferred to oocytes: in mature oocytes the specific radioactivity of lipids was 80% of the specific radioactivity of prelabeled fat body lipids. Components of blood meals are also used to synthesize oocyte lipids. Fat bodies of females starved for four days had only 27 μg of lipids left. When these females were given a blood meal, they matured oocytes, although the number of ooyctes was reduced and ovaries contained only half the amount of lipids found in ovaries of females which had first fed on sugar water. Fat body lipids of these females had only slightly increased to 36 μg. This demonstrates that female Ae. aegypti use sugar to synthesize lipids, but they can also use components of blood for this purpose.     

An investigation of a possible role for mitochondrial nuclease in apoptosis

Since mitochondrial factors have been implicated in apoptosis, experiments were designed to assess whether or not the potent mitochondrial nuclease could be one of these factors. Nuclei isolated by two different methods were found to contain mitochondrial nuclease in masked form. This nuclease was released by treatment with the non-ionic detergent NP-40 and rendered trypsin-sensitive. It was not removed appreciably from the nuclei by washing and sedimentation of the nuclei through a sucrose cushion. Levels of the mitochondrial nuclease were followed during drug-induced apoptosis. Time courses of apoptosis in cultures of HL-60 cells were monitored by flow cytometry of propidium iodide-stained cells and by agarose gel electrophoresis of extracted DNA. Changes in the inner mitochondrial transmembrane potential were monitored by flow cytometry of chloromethyl-X-Rosamine-stained cells. Apoptosis was induced by treatment with either the chemotherapeutic agent etoposide (VP-16 at 10 M) over an 8 h period or with the anti-rheumatic agent hydroxychloroquine (HCQ at 0.28 mM) over a 24 h period. These two drugs likely act in different pathways of apoptosis. VP-16 caused loss of the mitochondrial transmembrane potential 1.0–1.5 h before apoptosis was detected. On the other hand, treatment with HCQ caused these processes to occur in parallel possibly indicating that the mitochondrial changes are secondary events. No losses of masked mitochondrial nuclease were detected with either drug treatment during the course of apoptosis. HL-60 mitochondrial DNA was also not degraded during apoptosis induced by either agent. These observations likely explain why the mitochondrial DNA is not degraded and make it unlikely that mitochondrial nuclease plays any role in vivo in chromatin DNA fragmentation.     

Dibutyrylic cyclic AMP and theophylline inhibit proliferation of accessory cells in primary cultures of adrenomedullary cells

Summary Studies on isolated adrenal chromaffin cells in primary cultures may be seriously hampered by the presence of non-chromaffin, mainly fibroblast-like cells, which always occur in dissociates of adrenal medullary tissue and often outnumber the chromaffin cells by the end of the first week of culture, when no measures are taken to control their proliferation. The present study offers a new means to inhibit effectively the proliferation of these accessory cells by treating the cultures with dibutyrylic cyclic AMP (dbcAMP, 0.1 or 0.01 mM) and equimolar amounts of the phosphodiesterase inhibitor theophylline. With this treatment cultures of young rat adrenal chromaffin cells remain virtually free of accessory cells for two weeks of culture. Cultures of bovine adrenomedullary cells retain their initial amounts of non-chromaffin cells, which largely depends upon whether the primary cell suspensions have undergone differential plating prior to seeding. Suppression of accessory cell proliferation with dbcAMP and theophylline is partly due to maintaining differentiation of cortical cells, which otherwise dedifferentiate into rapidly dividing fibroblast-like elements. However, a more direct action of dbcAMP on accessory cells in terms of growth control is also conceivable. DbcAMP and theophylline in the doses applied do not impair the viability, ultrastructure and catecholamine-storing capacity of cultured chromaffin cells.     

The JAK1/2 inhibitor ruxolitinib delays premature aging phenotypes

Hutchinson–Gilford progeria syndrome (HGPS) is caused by an LMNA mutation that results in the production of the abnormal progerin protein. Children with HGPS display phenotypes of premature aging and have an average lifespan of 13 years. We found earlier that the targeting of the transmembrane protein PLA2R1 overcomes senescence and improves phenotypes in a mouse model of progeria. PLA2R1 is regulating the JAK/STAT signaling, but we do not yet know whether targeting this pathway directly would influence cellular and in vivo progeria phenotypes. Here, we show that JAK1/2 inhibition with ruxolitinib rescues progerin‐induced cell cycle arrest, cellular senescence, and misshapen nuclei in human normal fibroblasts expressing progerin. Moreover, ruxolitinib administration reduces several premature aging phenotypes: bone fractures, bone mineral content, grip strength, and a trend to increase survival in a mouse model of progeria. Thus, we propose that ruxolitinib, an FDA‐approved drug, should be further evaluated as a drug candidate in HGPS therapy.     

Exposure to infant scent lowers serum testosterone in father common marmosets (Callithrix jacchus)

Common marmoset (Callithrix jacchus) males are bi-parental non-human primates that show extensive paternal behaviour. Fathers are in direct sensory contact with their infants during the natal period. We found that fathers exposed to isolated scents of their infant displayed a significant drop in serum testosterone levels within 20min after exposure, whereas parentally naive males did not. These data suggest that infant's scent may have a causal role in regulating paternal testosterone in their fathers. This is the first study to demonstrate that olfactory cues have an acute effect on paternal care.     

Protein-catalyzed phospholipid exchange in bilayer vesicles determined by flow cytometry and electron microscopy

The protein-induced lipid transfer between phosphatidylcholine vesicles was investigated. Measurements of the degree of polarization at single vesicles were made by flow cytometry using diphenylhexatriene as the optical probe. Vesicles differing in phase transition temperature could be distinguished by their degree of polarization at a temperature where one population was in the fluid ($\text{T > T}{}_{\mathrm{<mtext>t</mtext>}}$) and the other one in the quasi-crystalline ($\text{T < T}{}_{\mathrm{<mtext>t</mtext>}}$) state. Besides vesicles containing exchanged lipids we also observed fractions of unaffected vesicles. The lipid exchange was visualized directly by freeze-fracture electron microscopy. The characteristic ‘ripple’ structure of phosphatidylcholine vesicles disappeared upon exchange with lipid in the fluid state.