Two at once: simultaneous increased production of astaxanthin and mycosporines in a single batch culture using a Phaffia rhodozyma mutant strain.

Phaffia rhodozyma is a basidiomycetous yeast characterized by its production of the carotenoid pigment astaxanthin, which holds high commercial value for its significance in aquaculture, cosmetics and as nutraceutics, and the UV-B-absorbing compound mycosporine-glutaminol-glucoside (MGG), which is of great biotechnological relevance for its incorporation into natural sunscreens. However, the industrial exploitation has been limited to the production of astaxanthin in small quantities. On the other hand, the accumulation of MGG in P. rhodozyma was recently reported and could add value to the simultaneous production of both metabolites. In this work, we obtain a mutant strain that overproduces both compounds, furthermore we determined how the accumulation of each is affected by the carbon-to-nitrogen ratio and six biotic and abiotic factors. The mutant obtained produces 159% more astaxanthin (470.1 µg g-1) and 220% more MGG (57.9 mg g-1) than the parental strain (295.8 µg g-1 and 26.2 mg g-1 respectively). Furthermore, we establish that the carotenoids accumulate during the exponential growth phase while MGG accumulates during the stationary phase. The carbon-to-nitrogen ratio affects each metabolite differently, high ratios favoring carotenoid accumulation while low ratios favoring MGG accumulation. Finally, the accumulation of both metabolites is stimulated only by photosynthetically active radiation and low concentrations of hydrogen peroxide. The mutant strain obtained is the first hyper-productive mutant capable of accumulating high concentrations of MGG and astaxanthin described to date. The characterization of how both compounds accumulate during growth and the factors that stimulate their accumulation, are the first steps toward the future commercial exploitation of strains for the simultaneous production of two biotechnologically important metabolites.

Retention of ice-associated amphipods: possible consequences for an ice-free Arctic Ocean.

Recent studies predict that the Arctic Ocean will have ice-free summers within the next 30 years. This poses a significant challenge for the marine organisms associated with the Arctic sea ice, such as marine mammals and, not least, the ice-associated crustaceans generally considered to spend their entire life on the underside of the Arctic sea ice. Based upon unique samples collected within the Arctic Ocean during the polar night, we provide a new conceptual understanding of an intimate connection between these under-ice crustaceans and the deep Arctic Ocean currents. We suggest that downwards vertical migrations, followed by polewards transport in deep ocean currents, are an adaptive trait of ice fauna that both increases survival during ice-free periods of the year and enables re-colonization of sea ice when they ascend within the Arctic Ocean. From an evolutionary perspective, this may have been an adaptation allowing success in a seasonally ice-covered Arctic. Our findings may ultimately change the perception of ice fauna as a biota imminently threatened by the predicted disappearance of perennial sea ice.

Safety and Efficacy of Lemborexant in Patients With Irregular Sleep-Wake Rhythm Disorder and Alzheimer's Disease Dementia: Results From a Phase 2 Randomized Clinical Trial.

BACKGROUND: Irregular sleep-wake rhythm disorder (ISWRD) is a common sleep disorder in individuals with Alzheimer's disease dementia (AD-D). OBJECTIVES: This exploratory phase 2 proof-of-concept and dose-finding clinical trial evaluated the effects of lemborexant compared with placebo on circadian rhythm parameters, nighttime sleep, daytime wakefulness and other clinical measures of ISWRD in individuals with ISWRD and mild to moderate AD-D. DESIGN: Multicenter, randomized, double-blind, placebo-controlled, parallel-group study. SETTING: Sites in the United States, Japan and the United Kingdom. PARTICIPANTS: Men and women 60 to 90 years of age with documentation of diagnosis with AD-D and Mini-Mental State Exam (MMSE) score 10 to 26. INTERVENTION: Subjects were randomized to placebo or one of four lemborexant treatment arms (2.5 mg, 5 mg, 10 mg or 15 mg) once nightly at bedtime for 4 weeks. MEASUREMENTS: An actigraph was used to collect subject rest-activity data, which were used to calculate sleep-related, wake-related and circadian rhythm-related parameters. These parameters included least active 5 hours (L5), relative amplitude of the rest-activity rhythm (RA) and mean duration of sleep bouts (MDSB) during the daytime. The MMSE and the Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog) were used to assess for changes in cognitive function. RESULTS: Sixty-two subjects were randomized and provided data for circadian, daytime and nighttime parameters (placebo, n = 12; lemborexant 2.5 mg [LEM2.5], n = 12; lemborexant 5 mg [LEM5], n = 13, lemborexant 10 mg [LEM10], n = 13 and lemborexant 15 mg [LEM15], n = 12). Mean L5 showed a decrease from baseline to week 4 for LEM2.5, LEM5 and LEM15 that was significantly greater than with placebo (all p < 0.05), suggesting a reduction in restlessness. For RA, LS mean change from baseline to week 4 versus placebo indicated greater distinction between night and day with all dose levels of lemborexant, with significant improvements seen with LEM5 and LEM15 compared with placebo (both p < 0.05). The median percentage change from baseline to week 4 in MDSB during the daytime indicated a numerical decrease in duration for LEM5, LEM10 and LEM15, which was significantly different from placebo for LEM5 and LEM15 (p < 0.01 and p = 0.002, respectively). There were no serious treatment-emergent adverse events or worsening of cognitive function, as assessed by the MMSE and ADAS-Cog. Lemborexant was well tolerated. No subjects discontinued treatment. CONCLUSIONS: This study provides preliminary evidence of the potential utility of lemborexant as a treatment to address both nighttime and daytime symptoms in patients with ISWRD and AD-D.

Drosophila APC2 is a cytoskeletally-associated protein that regulates wingless signaling in the embryonic epidermis.

The tumor suppressor adenomatous polyposis coli (APC) negatively regulates Wingless (Wg)/Wnt signal transduction by helping target the Wnt effector beta-catenin or its Drosophila homologue Armadillo (Arm) for destruction. In cultured mammalian cells, APC localizes to the cell cortex near the ends of microtubules. Drosophila APC (dAPC) negatively regulates Arm signaling, but only in a limited set of tissues. We describe a second fly APC, dAPC2, which binds Arm and is expressed in a broad spectrum of tissues. dAPC2's subcellular localization revealed colocalization with actin in many but not all cellular contexts, and also suggested a possible interaction with astral microtubules. For example, dAPC2 has a striking asymmetric distribution in neuroblasts, and dAPC2 colocalizes with assembling actin filaments at the base of developing larval denticles. We identified a dAPC2 mutation, revealing that dAPC2 is a negative regulator of Wg signaling in the embryonic epidermis. This allele acts genetically downstream of wg, and upstream of arm, dTCF, and, surprisingly, dishevelled. We discuss the implications of our results for Wg signaling, and suggest a role for dAPC2 as a mediator of Wg effects on the cytoskeleton. We also speculate on more general roles that APCs may play in cytoskeletal dynamics.

Non-equivalent roles of Drosophila Frizzled and Dfrizzled2 in embryonic wingless signal transduction.

The highly conserved Wnt family of growth factors is essential for generating embryonic pattern in many animal species [1]. In the fruit fly Drosophila, most Wnt-mediated patterning is performed by a single family member, Wingless (Wg), acting through its receptors Frizzled (Fz) and DFrizzled2 (Dfz2). In the ventral embryonic epidermis, Wg signaling generates two different cell-fate decisions: the production of diverse denticle types and the specification of naked cuticle separating the denticle belts. Mutant alleles of wg disrupt these cellular decisions separately [2], suggesting that some aspect of ligand-receptor affinity influences cell-fate decisions, or that different receptor complexes mediate the distinct cellular responses. Here, we report that overexpression of Dfz2, but not Fz, rescues the mutant phenotype of wgPE2, an allele that produces denticle diversity but no naked cuticle. Fz was able to substitute for Dfz2 only under conditions where the Wg ligand was present in excess. The wgPE2 mutant phenotype was also sensitive to the dosage of glycosaminoglycans, suggesting that the mutant ligand is excluded from the receptor complex when proteoglycans are present. We conclude that wild-type Wg signaling requires efficient interaction between ligand and the Dfz2-proteoglycan receptor complex to promote the naked cuticle cell fate.

Circadian determinants of subjective alertness.

Four healthy male subjects each experienced a temporal isolation experiment lasting several months. During part of each experiment (2-5 weeks), the subject's entire imposed daily routine (including light-dark, rest-activity, and meal routines) was either stretched (two subjects: T = 25.8 hr, 26.0 hr) or reduced (two subjects: T = 22.8 hr, 23.1 hr) to beyond the range of entrainment of the endogenous circadian pacemaker (ECP), which then ran at a different period (tau). Subjective alertness was measured approximately three times per hour (during wakefulness), using a computerized visual analogue scale technique. Circadian rhythms in subjective alertness were then plotted both at tau, the period length of the ECP, and at T, the period length of the imposed sleep-wake cycle (SWC) and light-dark cycle. At tau, the alertness rhythm was closely in phase with the temperature rhythm. At T, the alertness rhythm showed an "inverted-U" function with a peak toward the middle of the subjective day, upon which was superimposed a "postlunch dip" for one subject. Thus, subjective alertness would appear to be under the control fo both ECP and SWC mechanisms, which combine to produce the composite time-of-day function normally observed in a diurnal setting.

Chronobiology of aging: temperature, sleep-wake rhythms and entrainment.

Studies were carried out on a group of six young (ages 23-30) and six older (ages 53-70) normal men who lived under conditions of temporal, but not social isolation, from three to eight weeks. During entrained and non-entrained (free-running) conditions, comparative measurements were made of sleep-wake cycles, sleep stages and rectal temperature rhythms for these two age groups. Results demonstrated a reduction in the period and amplitude of the body temperature rhythms during free-running in the older group. Sleep efficiency, total sleep time, REM sleep latency, REM episode length, percent REM in the last 2 hours of sleep, the length and frequency of arousals during sleep, and the terminal wake latency were all age related and dependent on entrainment. The period of the sleep-wake cycle, terminal awakenings from REM and percent REM in the first 3 hours of sleep were not age related but were dependent on entrainment. Sleep stages as percents of total sleep time were found to be age related but independent of entrainment, while sleep latency, mid-REM to mid-REM cycle length and the ratio of sleep to total time were neither age related nor dependent on entrainment. In addition, individual chronobiological differences were prominent in the older group. Changes of period and of the phase relationship of sleep-wake and temperature rhythms occurred in several subjects during the non-entrained condition.

High nocturnal body temperature in premenstrual syndrome and late luteal phase dysphoric disorder.

OBJECTIVE: Because women with late luteal phase dysphoric disorder (LLPDD) experience symptomatic affective states predictably, they can be studied to determine whether there are biological findings related solely to the clinically symptomatic state. The authors sought to answer the question, Does body temperature change with affective state? METHOD: The core body temperature and motor activity patterns of 10 women with premenstrual syndrome (PMS), six of whom also met criteria for LLPDD, and no other psychological or medical illness were compared to those of six women with chronic, noncyclic dysphoria and six asymptomatic comparison women at four phases of the menstrual cycle. RESULTS: The nocturnal temperatures of the women with PMS/LLPDD were significantly higher than those of the comparison subjects across the entire menstrual cycle, but there were no differences in nocturnal activity levels. The women with noncyclic dysphoria had a mean nocturnal temperature in the follicular phase as high as that of the women with PMS/LLPDD. The temperatures of all women were higher in the luteal phase than in the follicular phase. CONCLUSIONS: These findings suggest that in the future investigators should document menstrual cycle phase in all female subjects and, when studying body temperature, should carefully monitor symptomatic state in comparison subjects.

Pediatricians and sleep disorders: training and practice.

OBJECTIVE: A series of studies were conducted to investigate pediatricians' training, knowledge, and practices regarding sleep and sleep disorders in children and adolescents. METHOD AND RESULTS: Study 1, a national survey of 156 pediatric residency programs, found that pediatricians receive a mean of 4.8 hours of instruction on sleep and sleep disorders, although the mode and median hours of instruction is 0 hours. In study 2, 88 pediatricians completing a questionnaire concerning general knowledge about sleep disorders in children and adolescents received a mean score of 71.8% (range, 40% to 93%). Pediatricians appear to know the most about developmental issues and sleep hygiene and the least about specific disorders such as narcolepsy and parasomnias. In the third study, 183 pediatricians were surveyed about their actual beliefs and practices regarding young children's sleep problems. Together, those surveyed reported that approximately 25% of their patients experience some type of sleep problem. Most pediatricians recommend behavioral interventions, although 14.8% of pediatricians report prescribing pharmacological treatments, and 48.9% inform parents that their child is likely to outgrow the problem. CONCLUSIONS: The results of these studies support the need for more education in sleep and sleep disorders in children and adolescents within medical schools, pediatric residency programs, and the practicing pediatric community.

Sex differences in circadian control of LH secretion.

Sex differences in the hourly values of serum LH were examined in male and female Syrian hamsters exposed to either long photoperiods (14 h light: 10 darkness; 14L: 10D) or short photoperiods (6L: 18D). In long photoperiods, females exhibited a pro-oestrous surge of LH in response to high levels of circulating oestradiol (1047.36 +/- 90.68 pmol/l), while LH values in male hamsters remained essentially constant. In females housed in short photoperiods oestradiol levels were reduced by fivefold; however, a discrete surge in serum LH, similar to that seen at pro-oestrus in females exposed to long photoperiods, was observed. Although exposure to a short photoperiod eliminates the requirement of a positive feedback of oestrogen for the expression of an afternoon surge in LH, male hamsters housed in short photoperiods showed no indication of an afternoon surge in serum LH. These observations indicate that sex differences in the neural control of LH release are not the result of sex differences in oestrogen sensitivity or responsiveness.

Premenstrual syndrome. Identification and management.

Premenstrual syndrome is a legitimate illness consisting of the cyclic recurrence of symptoms (physical, mental and behavioural) in the late luteal phase of the menstrual cycle of sufficient severity to require treatment. The syndrome has a yet unknown aetiology, an uncertain and variable course, and an unidentified family history. Methods are available for diagnosis including obtaining a reliable history, thorough physical and mental examinations, laboratory tests to exclude other possible causes of symptoms, and daily prospective symptom ratings to confirm symptom patterns. Because there is no known aetiology, treatment consists of symptom management of the specific symptoms experienced, based on prospective symptom ratings.

Removal of temporal constraints in the middle-aged and elderly: effects on sleep and sleepiness.

Ten healthy middle-aged and elderly subjects (44-81 years old; 4 men and 6 women) were studied in time isolation experiments involving: (a) entrainment to the subject's habitual routine (EN condition) and (b) removal of all temporal constraints or "free-running" (FR condition) with the subjects able to choose their own bed and meal times. Subjective day lengths and circadian temperature cycle periods were longer under FR than under EN, but not by an equal amount. Perhaps as a consequence, although time-in-bed (TIB) fractions were greater under FR than under EN, actual sleep fractions were not, reflecting a decline in sleep efficiency. Neither was there an increase in slow-wave sleep (SWS) under FR compared with EN. Daytime subjective alertness did, however, increase upon release into FR, also showing a slightly earlier peak than that observed under the EN condition. The results point to the need to regard sleep and sleepiness as rhythmic, as well as homeostatic, processes.

Cognitive and motor performance of narcoleptic and normal subjects living in temporal isolation.

Six unmedicated narcoleptic subjects and nine normal controls lived in a temporal isolation laboratory for 18-22 days. They were permitted to "free-run" for the last 9-13 days. Brief cognitive and motor performance tests were repeated on average six times per subjective day. They consisted of serial search, complex verbal reasoning tasks and manual dexterity of each hand. Only minor differences in performance were found between the narcoleptic subjects and controls. Narcoleptic subjects showed mild impairment of accuracy on the search task that could be explained by occasional lapses and an afternoon dip in performance. Narcoleptic subjects also tended to perform some tasks more slowly, but the group differences were not significant. Neither speed nor accuracy of performance of narcoleptic subjects decreased over the course of the experiment. By one standard of performance, therefore, all or nearly all of the sleep need of these subjects was met by the sleep they obtained in the laboratory. That amount, in turn, did not exceed the total sleep obtained by the normal controls. Significant time-of-day effects were found in narcoleptic subjects for speed of verbal reasoning (progressive slowing over the course of the day), manual dexterity (fluctuations in speeds) and accuracy of serial search (afternoon dip). These variations in performance could not be attributed to changes in core body temperature or to occurrences of naps or meals.

The Taskforce 2000 survey on medical education in sleep and sleep disorders.

Previous research has shown evidence of a widening gap between scientific research and clinical teaching in sleep and sleep disorders. To address the deficiencies in current medical education in sleep, the Taskforce 2000 was established by the American Sleep Disorders Association. The present study was undertaken to assess the teaching activities, needs and interests of the membership of the two largest professional sleep societies (American Sleep Disorders Association and Sleep Research Society). Survey instruments included a brief, 5-item postcard survey, which was mailed to all members, followed by an in-depth, 34-item questionnaire, which was completed by 158 respondents from the intitial postcard survey (N = 808). Results indicated that the majority of respondents (65.2%) are currently involved in teaching sleep to medical students or postgraduate trainees, although the average amount of teaching time was only 2.1 hours for undergraduate and 4.8 hours for graduate education in sleep. Teaching of sleep laboratory procedures and clinical evaluation of sleep-disordered patients is limited at either an undergraduate or postgraduate level. The major deficiencies noted were the lack of time in the medical curriculum and the need for better resources and teaching facilities. A large majority of respondents indicated their willingness to be involved in sleep education for physicians, and rated this a high priority for the professional organization.

Age-related differences in recovery from simulated jet lag.

Six healthy young men and eight early middle-aged men were isolated from environmental time cues for 15 days. For the first 6-7 days (one or two nights adaptation, four nights baseline), their sleep and meals were scheduled to approximate their habitual patterns. Their daily routines were then shifted 6 hours earlier by terminating the sixth or seventh sleep episode 6 hours early. The new schedules were followed for the next 8 or 9 days. Important age-related differences in adjustment to this single 6-hour schedule shift were found. For the first 4-day interval after the shift, middle-aged subjects had larger increases of waking time during the sleep period and earlier termination of sleep than young subjects. They also reported larger decreases in alertness and well-being and larger increases in sleepiness, weariness and effort required to perform daily functions. The rate of adjustment of the circadian core temperature rhythm to the new schedule did not differ between groups. These results suggest that the symptoms reported by the middle-aged subjects may be due mainly to difficulty maintaining sleep at early times of the circadian day. The compensatory response to sleep deprivation may also be less robust in middle-aged individuals traveling eastbound.

Effects of phototherapy on non-seasonal unipolar and bipolar depressive spectrum disorders.

In a group of 17 patients with non-SAD depressive disorders we compared the response of bipolar spectrum versus unipolar patients to treatment with light therapy. The main hypothesis was that bipolar spectrum depressed patients would preferentially respond to bright light therapy as compared to unipolar depressed patients. All patients were treated with either 400 or 2500 lux phototherapy for 2 h on seven consecutive days. All outcome measures, which included the SIGH-SAD, CGI, and the Anxiety and Depressive Factors of the SCL-90, showed significant improvement in the bipolar vs. the unipolar spectrum patients. Unexpected this occurred regardless of the intensity of the light. These changes were judged to be quite clinically significant. All patients showing response were noted to have maintained their response at a 3-month follow-up.

Response of circadian locomotor activity and the proestrous luteinizing hormone surge to phase shifts of the light-dark cycle in the hamster.

Female hamsters with regular 4 day estrous cycles were exposed to either a 3 hour phase advance or delay of the 14:10 light-dark (LD) cycle on the first, second or third day before proestrus. Blood samples were taken on proestrus to characterize the LH surge, and locomotor activity onset was recorded. Both the LH surge and activity onset phase delayed more quickly than they advanced, which can be explained by the free-running period of the hamster (longer than 24 hours). Higher estradiol levels were correlated with more rapid advances of activity onset.

Diurnal variation in mood and performance in a time-isolated environment.

In order to document circadian rhythmicity in various psychological functions under the chronobiologically 'pure' condition of temporal isolation, a battery of mood and performance tests were administered about 6 times per day to a heterogeneous group of 18 subjects (ages 19-81, 5 female). Each subject spent about 5 days in temporal isolation, entrained to a routine equivalent to his/her own habitual sleep/wake cycle. Average time of day functions were obtained for the mood and performance variables, and compared to rectal temperature data subjected to exactly the same statistical analysis. Significant time of day effects were found in the mood variables of alertness, sleepiness, weariness, effort required, happiness and well-being. Times of 'best' mood were different from the time of peak temperature. Moreover, the minima of sleepiness, weariness and effort occurred earlier in the day than the maximum of alertness. Significant time of day effects were also found in the speed with which search and dexterity tasks were completed. Only the dexterity tasks showed a complete parallelism with the temperature rhythm.

Premenstrual syndrome.

Because researchers are still seeking the cause of PMS, we cannot recommend a single treatment approach that will work satisfactorily with all women who have the syndrome. Investigators have made progress, however, toward defining and diagnosing the condition. We also can help women whose lives are disrupted each month by prescribing specific approaches to symptom management.