Mikania micrantha silver nanoparticles exhibit anticancer activities against human lung adenocarcinoma via caspase-mediated apoptotic cell death.

Green-mediated synthesis of nanoparticles has earned a promising role in the area of nanotechnology due to their biomedical applications. This study describes the synthesis of silver nanoparticles (AgNPs) using Mikania micrantha leaf extract and its functional activities against cancer. The synthesis of AgNPs was confirmed using Ultraviolet-Visible (UV-Vis) spectrum that exhibited an absorption band at 459 nm. The bioactive compounds of M. micrantha leaf extract that functioned as reducing and capping agents were confirmed by a shift in the absorption bands in Fourier Transform Infra-red Spectroscopy (FT-IR). Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) studies validated the spherical shape and size of AgNPs, respectively. Energy Dispersive Spectroscopy (EDS) analysis revealed the presence of elemental silver. The crystalline nature of AgNPs was confirmed by the X-ray Diffraction Analysis (XRD). AgNPs effectively induced cytotoxicity and prevented A549 cell colony formation in a dose-dependent manner. Treatment of A549 cells with AgNPs also increased DNA damage, which was coupled with elevated lipid peroxidation and decreased antioxidant enzymes such as glutathione (GSH), glutathione-s-transferase (GST), and superoxide dismutase (SOD). Following AgNPs treatment, the mRNA expression levels of the pro-apoptotic genes as well as the activities of caspases were significantly elevated in A549 cells while the expression levels of anti-apoptotic genes were downregulated. Our study demonstrates the potential of the synthesised AgNPs for cancer therapy possibly targeting the apoptotic pathway.

Probiotics for the postoperative management of term neonates after gastrointestinal surgery.

BACKGROUND: The intestinal microflora has an essential role in providing a barrier against colonisation of pathogens, facilitating important metabolic functions, stimulating the development of the immune system, and maintaining intestinal motility. Probiotics are live microorganisms that can be administered to supplement the gut flora. Neonates who have undergone gastrointestinal surgery are particularly susceptible to infectious complications in the postoperative period. This may be partly due to a disruption of the integrity of the gut and its intestinal microflora. There may be a role for probiotics in reducing the incidence of sepsis and improving intestinal motility, thus reducing morbidity and mortality and improving enteral feeding in neonates in the postoperative period. OBJECTIVES: To evaluate the efficacy and safety of administering probiotics after gastrointestinal surgery for the postoperative management of neonates born from 35 weeks of gestation. SEARCH METHODS: We searched CENTRAL, MEDLINE, Embase, and trial registries in August 2023. We checked reference lists of included studies and relevant systematic reviews for additional studies. SELECTION CRITERIA: We included randomised controlled trials (RCTs) that investigated the postoperative administration of oral probiotics versus placebo or no treatment in neonates born from 35 weeks of gestation who had one or more gastrointestinal surgical procedures. We applied no restrictions regarding the type or dosage of probiotics or the duration of treatment. DATA COLLECTION AND ANALYSIS: We used standard Cochrane methods, and we used GRADE to assess the certainty of evidence. MAIN RESULTS: We identified one RCT that recruited 61 neonates with a gestational age of 35 weeks or more. All infants were admitted to a neonatal intensive care unit and had surgery for gastrointestinal pathologies. There may be little or no difference in proven sepsis (positive bacterial culture, local or systemic) between infants who receive probiotics compared with those who receive placebo (odds ratio (OR) 0.64, 95% confidence interval (CI) 0.16 to 2.55; 61 infants; low-certainty evidence). Probiotics compared to placebo may have little or no effect on time to full enteral feeds (mean difference (MD) 0.63 days, 95% CI -4.02 to 5.28; 61 infants; low-certainty evidence). There were no reported deaths prior to discharge from hospital in either study arm. Two weeks after supplementation, the infants who received probiotics had a substantially higher relative abundance of non-pathogenic intestinal microflora (Bifidobacteriaceae) than those who received placebo (MD 38.22, 95% CI 28.40 to 48.04; 39 infants; low-certainty evidence). AUTHORS' CONCLUSIONS: This review provides low-certainty evidence from one small RCT that probiotics compared to placebo have little or no effect on the risk of proven sepsis (positive bacterial culture, local or systemic) or time to full-enteral feeds in neonates who have undergone gastrointestinal surgery. Probiotics may substantially increase the abundance of beneficial bacterial in the intestine of these neonates, but the clinical implications of this finding are unknown. There is a need for adequately powered RCTs to assess the role of probiotics in this population. We identified two ongoing studies. As neither reported the gestational age of prospective study participants, we are unsure if they will be eligible for inclusion in this review.

Molecular expression of clock genes in central and peripheral tissues of white-rumped munia (Lonchura striata).

To synchronize with the fluctuating environment, organisms have evolved an endogenous time tracking mechanism referred to as the biological clock(s). This clock machinery has been identified in almost all cells of vertebrates and categorized as central and peripheral clocks. In birds, three independent circadian clocks have been identified in the hypothalamus, the pineal and the retina which interact and generate circadian time at a functional level. However, there is a limited knowledge of molecular clockwork and integration between central and peripheral clocks in birds. Therefore, we studied the daily expression of clock genes (Bmal1, Clock, Per2, Cry1, Npas2, Rev-Erbα, E4bp4, Pparα, Hlf and Tef) in three central circadian clocks (hypothalamus, pineal and retina), other brain areas (cerebellum, optic tectum and telencephalon) and in the peripheral tissues (liver, intestine, muscle and blood) of white-rumped munia. Adult birds were exposed to equinox photoperiod (12 L:12D) for 2 weeks and were then sampled (N = 5 per time point) at six-time points (ZT1, ZT5, ZT9, ZT13, ZT17 and ZT21). Daily expressions of clock genes were studied using qPCR. We observed daily variations and tissue-specific expression patterns for clock genes. These results are consistent with the autoregulatory circadian feedback loop proposed for the mammalian system and thus suggest a conserved tissue-level circadian time generation in white-rumped munia.

Urban environment alter the timing of progression of testicular recrudescence in tree sparrow (Passer montanus).

Urbanization is a rapidly growing phenomenon that affects wildlife. Laboratory studies show the effects of night light on the physiology of the organisms. Limited studies have been conducted on birds in their natural habitat. Here, we studied the effects of the urban environment on reproduction-linked phenomenon and molecules involved in the regulation of seasonal breeding. Birds (N=5/time/site) were procured from urban and rural sites at specific times, i.e., in March (stimulatory phase), June (reproductive phase), September (refractory phase), and December (sensitive phase) of 2018. Immediately after procurement, birds were brought to the laboratory. Bodyweight, bill color, molt in body feathers, and testes size were recorded. The next day, all the birds were sacrificed in the middle of the day. Blood was collected and serum was used for ELISA of corticosterone, triiodothyronine (T3), and thyroxine (T4). mRNA levels of thyroid-stimulating hormone-ß (Tshß), type 2 deiodinase (Dio2), type 3 deiodinase (Dio3), gonadotropin-releasing hormone (GnRh), and gonadotropin inhibitory hormone (GnIh) were measured in hypothalamic tissue. Urban birds showed higher levels of corticosterone during the stimulatory phase. There was a delay in the initiation of testicular growth in urban birds and it was supported by reduced levels of T3 in blood plasma and relatively lower transcription of Dio2 and GnRH mRNA in urban birds. Our findings suggest that the urban environment delays the timing of reproduction in birds and could be the consequence of local environmental conditions.

Season dependent effects of urban environment on circadian clock of tree sparrow (Passer montanus).

Great efforts have been made recently to understand the effect(s) of urban environments on the circadian and seasonal physiology of wild animals, but the mechanisms involved remain largely unknown. Most laboratory studies and a few studies on animals in the wild suggest alterations occur in the physiological functions of organisms in urban habitats. Here, we addressed the effects of the interaction of seasons and urban environments on clock gene expression in three tissues of tree sparrows (Passer montanus). Tree sparrows (N = 30 per site per time of year) were procured from rural and urban habitats during periods corresponding to their three physiological states, i.e., June (longest photoperiod; reproductive phase), September (equinox photoperiod; refractory phase), and December (shortest photoperiod; sensitive phase). Birds (N = 5 per time per site per month) were sampled at six time points; ZT1, ZT5, ZT9, ZT13, ZT17, and ZT21 (ZT0 = sunrise time) and clock gene expression in the hypothalamus, pineal gland, and retina was studied. Our results show that there is persistence of the circadian clock in both rural and urban birds throughout the year. In urban birds Bmal1, Npas2, Per2, and Cry1 acrophases were advanced, compared to rural birds, while Clock acrophase was delayed, depending on the tissue and time of year. This difference could be because of changes in the availability, duration, and intensity of sunlight during different times of the year and/or differential photoreceptor sensitivities, differential physiological states, or a combination of all these factors. These important results reveal, for the first time in any species, season-dependent effects of an urban environment on the molecular machinery of the circadian clock.

Standardised neonatal parenteral nutrition formulations - Australasian neonatal parenteral nutrition consensus update 2017.

BACKGROUND: The first consensus standardised neonatal parenteral nutrition formulations were implemented in many neonatal units in Australia in 2012. The current update involving 49 units from Australia, New Zealand, Singapore, Malaysia and India was conducted between September 2015 and December 2017 with the aim to review and update the 2012 formulations and guidelines. METHODS: A systematic review of available evidence for each parenteral nutrient was undertaken and new standardised formulations and guidelines were developed. RESULTS: Five existing preterm Amino acid-Dextrose formulations have been modified and two new concentrated Amino acid-Dextrose formulations added to optimise amino acid and nutrient intake according to gestation. Organic phosphate has replaced inorganic phosphate allowing for an increase in calcium and phosphate content, and acetate reduced. Lipid emulsions are unchanged, with both SMOFlipid (Fresenius Kabi, Australia) and ClinOleic (Baxter Healthcare, Australia) preparations included. The physicochemical compatibility and stability of all formulations have been tested and confirmed. Guidelines to standardise the parenteral nutrition clinical practice across facilities have also been developed. CONCLUSIONS: The 2017 PN formulations and guidelines developed by the 2017 Neonatal Parenteral Nutrition Consensus Group offer concise and practical instructions to clinicians on how to implement current and up-to-date evidence based PN to the NICU population.

Hypothalamus but not liver retains daily expression of clock genes during hibernation in terai tree frog (Polypedates teraiensis).

Circadian clock(s) allow an organism to be in synchrony with the surrounding environment and perform daily and seasonal physiological processes, including hibernation, migration, and reproduction. To cope with adverse environmental conditions, organisms have evolved various strategies. Insects undergo diapause, while some higher animals either migrate or hibernate/aestivate during unfavorable environmental conditions. Hibernation is an energy conservation strategy used to cope with adverse environmental conditions. Limited knowledge is available on the physiology of hibernation in non-mammalian vertebrates. Some studies suggest that metabolism is altered during amphibian hibernation, but nothing is known about the circadian clock. In the present study, we investigated daily oscillation of clock genes in the brain and liver of the terai tree frog (Polypedates teraiensis) during two annual phases of life: breeding and hibernation. Adult male terai tree frogs were procured from their natural habitat on the Mizoram University campus (23°N 92°E) during their breeding and hibernation phases. Body mass and testes weight were recorded. Animals were sacrificed at six time points: ZT1, ZT5, ZT9, ZT13, ZT17, and ZT21 (ZT0, zeitgeber time 0, indicates the sunrise time at the respective time of the year; N = 5 frogs per time point). Quantitative real-time polymerase chain reaction (qPCR) was performed for clock genes (Bmal1, Clock, Per2, and Cry2) in the hypothalamus and liver. Our results showed that body and testes weights decreased during hibernation. Further, the hypothalamus retained daily clock gene oscillations during breeding and hibernation. However, the liver lost this daily oscillation during hibernation. The maintained rhythm in hypothalamus in contrast to other hibernating animals might be the result of the fact that these animals hibernate at a higher temperature and might be more alert. As the animals have no food intake during their hibernation season which might be the reason the animals loose their rhythm in liver clock genes. These results suggest that retaining daily clock gene oscillations in the hypothalamic clock could be important for internal time tracking and post-hibernation emergence.

Effect of urban environment on pineal machinery and clock genes expression of tree sparrow (Passer montanus).

Increasing urbanisation is altering the physiology of wild animals and the mechanisms involved are largely unknown. We hypothesised that altering the physiology of urban organisms is due to the effect of extra light at night on the circadian clock by modulating the expression of pineal machinery and clock genes. Two experiments were performed. In Experiment 1, immediately after being procured from their respective sites (urban and rural sites), birds were released individually in LLdim light conditions. Circadian rhythm period, activity duration, and total activity count were calculated and did not differ between urban and rural birds. In Experiment 2, birds (from urban and rural habitats) were sampled at six time points at regular 4-h intervals, beginning 1 h after sunrise. We measured daily variations in plasma melatonin levels. We also analysed the expression levels of Aanat, Mel1A and Mel1B as an indicator of melatonin biosynthesis and action machinery. Clock and clock-controlled genes (Bmal1, Clock, Per2, Per3, Cry1 and Npas2) were studied in the hypothalamus, the pineal gland, and retina to investigate the effects of urban habitats on the circadian clock. Our results show that there is a lower expression of Aanat in the pineal gland and relatively low plasma melatonin levels in urban birds. Further, clock genes are also differentially expressed in all three central tissues of urban birds. We propose that alterations in the melatonin biosynthesis machinery and the expression of clock genes could result in miscalculations in the internal timing of the organism, with environmental timings leading to altered physiology in urban wild animals.

Temporal expression of genes coding for aryl-alkamine-N-acetyltransferase and melatonin receptors in circadian clock tissues: Circadian rhythm dependent role of melatonin in seasonal responses.

We investigated at the transcriptional level the role of daily rhythm in melatonin secretion in seasonal responses in the migratory blackheaded bunting (Emberiza melanocephala), which when exposed to short (SP) and long (LP) photoperiods exhibits distinct seasonal life-history states (LHSs). We reproduced the seasonal LHS by subjecting buntings to SP (8 h light: 16 h darkness, 8 L:16D), which maintained the nonmigratory/ nonbreeding phenotype, and to LP (16 L:8D), which induced the premigratory/ prebreeding, migratory/ breeding and nonmigratory/ postbreeding phenotypes. Plasma melatonin measured at 4 h intervals showed loss of the daily rhythm in the LP-induced premigratory/ prebreeding and migratory/ breeding LHSs. Subsequently, mRNA expression of genes coding for the aryl-alkamine-N-acetyltransferase (AANAT; the rate-liming enzyme of melatonin biosynthesis) and for the receptors for melatonin (Mel1A, Mel1B and Mel1C) was examined in the retina, pineal and hypothalamus; the interacting independent circadian clocks comprising the songbird circadian timing system. Except AANAT that was not amplified in the hypothalamus, we found significant alterations in both, the level and persistence of 24 h rhythm in mRNA expression of all genes, albeit with photoperiod and seasonal differences between three circadian clock tissues. Particularly, 24 h mRNA expression pattern of all genes, except retinal Mel1A, lacked a significant daily rhythm in the LP-induced migratory/ breeding LHS. These results underscore the overall importance of the circadian rhythm in the role of melatonin in photoperiodically-controlled seasonal responses in migratory songbirds.

Temperature alters the hypothalamic transcription of photoperiod responsive genes in induction of seasonal response in migratory redheaded buntings.

We investigated the temperature effects on hypothalamic transcription of genes involved in the induction of photoperiodic response in redheaded buntings. Birds were exposed at 22 and 38 °C to 13-h long photoperiods (LP), with controls at 22 °C on 8-h short photoperiods (SP). At 22 °C, compared to SP, we found higher tshb, eya3 and dio2 and low dio3 and gnih mRNA expressions after a week of LP; concomitant with testis recrudescence this confirmed buntings' responsiveness to LP-induced photostimulation. tshb, dio2 and gnrh mRNA levels were further increased by 2.5 weeks of LP at 38 °C. Temperature sensitive trpm8, but not trpv4, bdnf or adcyap1 also showed LP-induced expression at 22 °C. Concomitant changes in dnmt3b and tet2 mRNA expressions further suggested epigenetic modification of temperature influence on photoperiodic responses. These results demonstrate the role of temperature in hypothalamic molecular regulation of the photoperiodic gonadal response in seasonally breeding birds.

Daily expression of clock genes in central and peripheral tissues of tree sparrow (Passer montanus).

Almost all organisms live in a fluctuating environment. To achieve synchrony with the fluctuating environment, organisms have evolved with time-tracking mechanism commonly known as biological clocks. This circadian clock machinery has been identified in almost all cells of vertebrates and categorized as central and peripheral clocks. In birds, three independent circadian clocks reside within the nervous tissues in the hypothalamus, pineal and retina, which interact with each other and produce circadian time at a functional level. There is limited knowledge available of the molecular clockwork, and of integration between central and peripheral clocks in birds. Here, we studied daily expression of canonical clock genes (Bmal1, Clock, Per2, Per3, Cry1 and Cry2) and clock-controlled gene (Npas2) in all three central tissues (hypothalamus, pineal and retina) and in peripheral tissues (liver, intestine and muscle). Wild caught adult male tree sparrows were exposed to equinox photoperiod (12L:12D) for 2 weeks and after that birds were sacrificed (N = 5 per time point) at six time points (ZT1, ZT5, ZT9, ZT13, ZT17 and ZT21; ZT0 is lights on). Daily expression of clock genes was studied using qPCR. Bmal1, Clock, Per2, Per3, Cry1, Cry2 and Npas2 showed daily oscillation in all tissues except Cry2 in hypothalamus, pineal and intestine. We observed tissue-specific expression pattern for all clock and clock-controlled genes. Bmal1 transcripts expressed during early phase of night. Clock acrophase was observed during middle or late day time in the central clock while during early-to-middle phase of night in peripheral tissues. Npas2 expression pattern was similar to Bmal1. Per genes peaked either late at night or early during day time. However, Cry genes were peaked either at late day time (Cry1in retina, liver and intestine; Cry2 in liver and intestine) or at early night phase (Cry1 in hypothalamus, pineal and muscle; Cry2 in hypothalamus, pineal, retina and muscle). Our results are consistent with the autoregulatory circadian feedback loop, and suggest a conserved tissue-level circadian time generation in tree sparrows. Change in peak expression timing of these genes in different tissues implicates tissue-specific contribution of individual clock genes in the circadian time generation.

Pinealectomy abolishes circadian behavior and interferes with circadian clock gene oscillations in brain and liver but not retina in a migratory songbird.

In songbirds, the pineal gland is part of the multi-oscillatory circadian timing system, with participating component oscillators in the eyes and hypothalamus. This study investigated the role of the pineal gland in development of the nighttime migratory restlessness (Zugunruhe) and generation of circadian gene oscillations in the retina, brain and liver tissues in migratory redheaded buntings (Emberiza bruniceps). Pinealectomized (pinx) and sham-operated buntings entrained to short days (8h light: 16h darkness, 8L:16D) were sequentially exposed for 10days each to stimulatory long days (13L: 11D) and constant dim light (LLdim; a condition that tested circadian rhythm persistence). Whereas activity-rest pattern was monitored continuously, the mRNA expressions of clock genes (bmal1, clock, npas2, per2, cry1, rorα, reverα) were measured in the retina, hypothalamus, telencephalon, optic tectum and liver tissues at circadian times, CT, 1, 6, 13, 17 and 21 (CT 0, activity onset) on day 11 of the LLdim. The absence of the pineal gland did not affect the development of long-day induced Zugunruhe but caused decay of the circadian rhythm in Zugunruhe as well as the clock gene oscillations in the hypothalamus, but not in the retina. Further, there were variable effects of pinealectomy in the peripheral brain and liver tissue circadian gene oscillations, notably the persistence of per 2 and cry1 (optic tectum), rorα (telencephalon) and npas2 (liver) mRNA oscillations in pinx birds. We suggest the pineal gland dependence of the generation of circadian gene oscillations in the hypothalamus, not retina, and peripheral brain and liver tissues in migratory redheaded buntings.

Circadian timing in central and peripheral tissues in a migratory songbird: dependence on annual life-history states.

Predictable seasonal change in photoperiod triggers a sequential change in the daily activity-rest pattern, adaptive for migration in several bird species. The night-migratory black-headed bunting (Emberiza melanocephala) is day active under short photoperiods (8 h light:16 h dark, short day sensitive). Under long photoperiods (16 h light:8 h dark), the buntings are initially day active (long day premigratory) but subsequently become intensely night active (long day migratory) and after few weeks again return to a day active pattern (long day refractory). However, it is unclear how the daily expression of circadian genes changes during photoperiod-induced seasonal life-history states (LHSs). We measured period 2 (Per2), cryptochrome 1 (Cry1), brain and muscle arnt-like protein 1 (Bmal1), and circadian locomotor output cycles kaput (Clock) mRNA expressions in various neural and peripheral tissues of buntings in different LHSs and discovered differences of ∼2 to 6 h in the phase and 2- to 4-fold in amplitude of circadian oscillations of Per2, Cry1, and Bmal1 between photoperiod-induced LHSs. Phase relationship in mRNA oscillations was altered between oscillator components in the circadian pacemaker system (retina, pineal, hypothalamus) as well as in the peripheral (liver, muscle) tissues. These results show for the first time altered waveforms of clock gene expressions in all tissues in parallel with behavioral shifts and suggest the involvement of circadian system in photoperiod induction of seasonal LHSs in a migratory species.

Circadian synchronization determines critical day length for seasonal responses.

A photoperiodic species initiates fat deposition (in migrants) and gonadal recrudescence in response to a specific duration of natural daylight, called critical day length (CD), when light extends in the inductive phase of the endogenous circadian rhythm of photoinducibility (CRP). The molecular basis of species-specificCD, determined by the entrainment of the CRP, has been poorly understood. To investigate this, we measured expression levels of genes implicated in the photoperiod-induced changes in reproduction (EYA3, TSH beta, DIO2, DIO3, GNRH and GNIH) and metabolism (SIRT1, HMGCR, FASN and PPAR alpha) in photosensitive redheaded buntings subjected to light-dark cycles of varying period lengths (T-photocycles). Buntings were exposed to six T22, T24 or T26 photocycles, with 1h additional light at night falling at different phases of the entrained CRP (T2211L=6L:4D:1L:11D; T2411L=6L:4D:1L:13D,T2412L=6L:5D:1L:12D, T2413L=6L:6D:1L:11D; T2612L=6L:5D:1L:14D). Photoinduction at genetic and phenotypic levels in T2412L and T2413L, not T2411L, groups confirmed CD being close to 12h in buntings under T24. Compared to T24, exposure to T22 advanced CD by 1h, as evidenced by photoinduction in the T2211L, not T226L, group. Similarly, CD appeared to be delayed under T26, with no photoinduction in the T2612L group. Further, to show that induction of response under a T-photocycle was because of the interaction of inductive phase of the CRP with 1h during the dark period in each cycle, not with the 6h main light periods falling 2h earlier each successive 24hday in a T22 paradigm, a group of buntings was exposed to 6L:16D (T226L), to which they did not respond. The mRNA expression of genes, particularly TSH beta, DIO2, DIO3 and PPAR alpha, was significantly correlated with changes in reproductive and metabolic phenotypes. These results suggest CRP-entrainment based genetic regulation of the CD, and extend the idea that synchronization with environment is a critical measure in a seasonal species for its temporal adaptation in the wild.

Annual life history-dependent gene expression in the hypothalamus and liver of a migratory songbird: insights into the molecular regulation of seasonal metabolism.

Birds seasonally switch from one life history state (LHS) to another to maximize their fitness. Accordingly, they exhibit distinct differences in their physiological and behavioral phenotypes between seasons. Possible molecular mechanisms underlying changes through the seasons have scarcely been examined in migratory birds. The present study measured key genes suggested to be involved in the metabolic regulation of 4 photoperiodically induced seasonal LHSs in a long-distance migratory songbird, the blackheaded bunting (Emberiza melanocephala). Buntings were held under short days (8 h light:16 h darkness, 8L:16D), during which they maintained the winter nonmigratory phenotype. Then they were exposed for several weeks to long days (13L:11D). Differences in the activity-rest pattern, body fattening and weight gain, testis size, organ (heart, intestine) weights, and blood glucose and triglyceride levels confirmed that buntings sequentially exhibited spring migration-linked premigratory, migratory, and postmigratory LHSs under long days. The mRNA levels of circadian genes involved in metabolism (Bmal1, Clock, Npas2, Rorα, and Rev-erbα) and of genes that encode for proteins/enzymes involved in the regulation of glucose (Sirt1, FoxO1, Glut1, and Pygl) and lipids (Hmg-CoA; Pparα, Pparγ; Fasn and Acaca) showed LHS-dependent changes in their light-dark expression patterns in the hypothalamus and liver. These initial results on genetic regulation of metabolism in a migratory species extend the idea that the transitions between LHSs in a seasonal species are accomplished by changes at multiple regulatory levels. Thus, these findings promise new insights into the mechanism(s) of adaptation to seasons in higher vertebrates.