PERIOD Research Articles

Disruption in the regulation of casein kinase 2 in circadian rhythm leads to pathological states: cancer, diabetes and neurodegenerative disorders.

Circadian rhythm maintains the sleep-wake cycle in biological systems. Various biological activities are regulated and modulated by the circadian rhythm, disruption of which can result in onset of diseases. Robust rhythms of phosphorylation profiles and abundances of PERIOD (PER) proteins are thought to be the master keys that drive circadian clock functions. The role of casein kinase 2 (CK2) in circadian rhythm via its direct interactions with the PER protein has been extensively studied; however, the exact mechanism by which it affects circadian rhythms at the molecular level is not known. Here, we propose an extended circadian rhythm model in Drosophila that incorporates the crosstalk between the PER protein and CK2. We studied the regulatory role of CK2 in the dynamics of PER proteins involved in circadian rhythm using the stochastic simulation algorithm. We observed that variations in the concentration of CK2 in the circadian rhythm model modulates the PER protein dynamics at different cellular states, namely, active, weakly active, and rhythmic death. These oscillatory states may correspond to distinct pathological cellular states of the living system. We find molecular noise at the expression level of CK2 to switch normal circadian rhythm to any of the three above-mentioned circadian oscillatory states. Our results suggest that the concentration levels of CK2 in the system has a strong impact on its dynamics, which is reflected in the time evolution of PER protein. We believe that our findings can contribute towards understanding the molecular mechanisms of circadian dysregulation in pathways driven by the PER mutant genes and their pathological states, including cancer, obesity, diabetes, neurodegenerative disorders, and socio-psychological disease.

Morphological characterization of seeds and germination efficiency of Pinus patula var. longepedunculata and P. pseudostrobus var. oaxacana

Pinus patula and P.pseudostrobus are species of great economic importance for different communities in the Oaxaca State due tothe wood quality and wide use in forest commercial plantations and reforestation. The objective of this research was to evaluate themorphological variation of seeds and their germination efficiency of two pine species under greenhouse conditions. In 2022,selected seeds of outstanding phenotypes was obtained from the Instituto Nacional de Investigaciones Forestales, Agrícolas yPecuarias (INIFAP); three seed samples of 10 g per species were made, the number of seeds was counted, and the equatorial (DE)and polar (DP) diameters and shape coefficient (CF=DE/DP) were measured; sowing was carried out in 49 cavity trays with amixture of mountain forest soil, vermiculite®, agrolite® and Peat Moss® with a 4:1:1:2 ratio. Germination was counted daily andvigor indicators were determined: germinative energy (EG), energy period (PE), germinative speed (VG) and germination speedindex (IVG). Data were analyzed using t-Student tests and regression analysis (α=0.05) in the SAS program. Including days aftersowing (DDS) in the model, we found that between 78 and 84 % of the existing variation in the percentage of accumulatedgermination was explained in both species. P.pseudostrobus presented more spherical (CF=0.41) and heavier seeds (24.27 mg)with an energy period of 69 % at 22 DDS; on the other hand, P.patula has elongated (CF=0.31) and light seeds (8.20 mg) with agreater energy period of 83% at 20 DDS. Both species germinate after 15 days; however, P.patula has a higher germination rateand a higher germination percentage, it is necessary to use germplasm from outstanding phenotypes to guarantee the reproductiveefficiency of the seed and subsequent seedling quality.

The Role of Clock Genes in Maintaining Circadian Rhythm and Rheumatoid Arthritis Pathophysiology.

Rheumatoid arthritis (RA) is a chronic, progressive autoimmune condition that affects up to 1% of the world population and symmetrically affects the joints leading to joint stiffness and decreased mobility. RA patients present with increased pain and chronic inflammation within their joint spaces, which researchers have linked to poorer sleep patterns, including difficulty falling asleep and non-restorative sleep. As such, identifying mediators of poor sleep quality among RA patients may improve their long-term quality of life. More recently, researchers identified an association between chronic inflammation in RA patients and their circadian rhythm. Altered circadian rhythms negatively impact the hypothalamic-pituitary-adrenal (HPA) axis and lead to altered cortisol release. Cortisol has shown to have a strong anti-inflammatory effect; when dysregulated, it may lead to increased pain experienced in RA patients. This literature review aims to provide insight into how chronic inflammation tied to RA pathophysiology may affect clock genes that are involved in maintaining the circadian rhythm. Specifically, this review focused on four common clock genes found dysregulated in RA patients: circadian locomotor output cycles kaput(CLOCK),brain and muscle ARNT like-1(BMAL1),period(PER),and cryptochrome(CRY).Of the four clock genes discussed in this review,BMAL1andPERare the most well-studied of the affected genes. Further knowledge surrounding clock genes and their dysregulated expression in RA may help guide therapy decisions for RA patients. Traditionally, disease-modifying antirheumatic drugs (DMARDs) have been used as first-line therapy for RA patients. Meanwhile, chronotherapy, optimizing drug release in a timed manner, has shown positive results in RA patients as well. Because of the association of altered circadian rhythms with increased symptom severity in RA patients, it seems highly plausible that DMARD therapy with chronotherapy may be an ideal therapeutic regimen for RA.

PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period

PERIOD (PER) and Casein Kinase 1δ regulate circadian rhythms through a phosphoswitch that controls PER stability and repressive activity in the molecular clock. CK1δ phosphorylation of the familial advanced sleep phase (FASP) serine cluster embedded within the Casein Kinase 1 binding domain (CK1BD) of mammalian PER1/2 inhibits its activity on phosphodegrons to stabilize PER and extend circadian period. Here, we show that the phosphorylated FASP region (pFASP) of PER2 directly interacts with and inhibits CK1δ. Co-crystal structures in conjunction with molecular dynamics simulations reveal how pFASP phosphoserines dock into conserved anion binding sites near the active site of CK1δ. Limiting phosphorylation of the FASP serine cluster reduces product inhibition, decreasing PER2 stability and shortening circadian period in human cells. We found that Drosophila PER also regulates CK1δ via feedback inhibition through the phosphorylated PER-Short domain, revealing a conserved mechanism by which PER phosphorylation near the CK1BD regulates CK1 kinase activity.

Timing Is Important—Management of Metabolic Syndrome According to the Circadian Rhythm

Physiological processes occur in accordance with a rhythm regulated by the endogenous biological clock. This clock is programmed at the molecular level and synchronized with the daily light-dark cycle, as well as activities such as feeding, exercise, and social interactions. It consists of the core clock genes, Circadian Locomotor Output Cycles Protein Kaput (CLOCK) and Brain and Muscle Arnt-Like protein 1 (BMAL1), and their products, the period (PER) and cryptochrome (CRY) proteins, as well as an interlocked feedback loop which includes reverse-strand avian erythroblastic leukemia (ERBA) oncogene receptors (REV-ERBs) and retinoic acid-related orphan receptors (RORs). These genes are involved in the regulation of metabolic pathways and hormone release. Therefore, circadian rhythm disruption leads to development of metabolic syndrome (MetS). MetS refers to a cluster of risk factors (RFs), which are not only associated with the development of cardiovascular (CV) disease (CVD), but also with increased all-cause mortality. In this review, we consider the importance of the circadian rhythm in the regulation of metabolic processes, the significance of circadian misalignment in the pathogenesis of MetS, and the management of MetS in relation to the cellular molecular clock.

Analisis Tingkat Perputaran Piutang pada PT.PLN (Persero) Unit Layanan Pelanggan (ULP) Lubuk Alung Tahun 2019

Manage recei vables gi ven, which can be seen from the ratio of receivables activity where this ratio meas ures the effectiveness of the company in collecting recei vables. The recei vable activity ratio consists of recei vables turnover and t he average peri od of collection of recei vables. This type of research is a simple applied research with quantitative descriptive analysis technique that describes the level of receivables turnover and the effectiveness of collecting receivables PT.PLN (Persero) Lubuk Alung Customer Service Unit. The data in this study uses secondary data in the form of an overview report of the balance of receivables obtained indirectly from the object of research. Based on the results of the study, it was found that in 2019, the receivable turnover rate of PT PLN (Persero) Lubuk Alung Customer Service Unit in the first quarter of the receivables turnover was 4 times and the receivables collection period was 10 days, the second quarter the receivables turnover was 3 times and the collection period was 10 days. 12 days, 3rd quarter of receivables collection and collection period of 13 days, 4th quarter of receivables collection 3 times and collection period of 11 days. The effectiveness of the receivables turnover of PT PLN (Persero) Lubuk Alung Customer Service Unit in collecting receivables is quite good because it is still under the conditions determined by the company between 10-15 days.

Is obstructive sleep apnea a circadian rhythm disorder?

Obstructive sleep apnea is the most common sleep-related breathing disorder worldwide and remains underdiagnosed. Its multiple associated comorbidities contribute to a decreased quality of life and work performance as well as an increased risk of death. Standard treatment seems to have limited effects on cardiovascular and metabolic aspects of the disease, emphasising the need for early diagnosis and additional therapeutic approaches. Recent evidence suggests that the dysregulation of circadian rhythms, processes with endogenous rhythmicity that are adjusted to the environment through various cues, is involved in the pathogenesis of comorbidities. In patients with obstructive sleep apnea, altered circadian gene expression patterns have been demonstrated. Obstructive respiratory events may promote circadian dysregulation through the effects of sleep disturbance and intermittent hypoxia, with subsequent inflammation and disruption of neural and hormonal homeostasis. In this review, current knowledge on obstructive sleep apnea, circadian rhythm regulation, and circadian rhythm sleep disorders is summarised. Studies that connect obstructive sleep apnea to circadian rhythm abnormalities are critically evaluated. Furthermore, pathogenetic mechanisms that may underlie this association, most notably hypoxia signalling, are presented. A bidirectional relationship between obstructive sleep apnea and circadian rhythm dysregulation is proposed. Approaching obstructive sleep apnea as a circadian rhythm disorder may prove beneficial for the development of new, personalised diagnostic, therapeutic and prognostic tools. However, further studies are needed before the clinical approach to obstructive sleep apnea includes targeting the circadian system.

Contribution of Triassic Tectonomagmatic Activity to the Mineralization of Liziyuan Orogenic Gold Deposits, West Qinling Orogenic Belt, China

The Western Qinling orogenic belt (WQOB) is one of the most important prospective gold districts in China, with widely distributed Indosinian intermediate–acidic intrusions. The Liziyuan Au deposit is a representative orogenic deposit in the northern WQOB, hosting several sections spatially associated with igneous rocks. The Au deposit is hosted by meta-sedimentary volcanic rocks of the Cambrian–Ordovician Liziyuan Group and the Tianzishan monzogranite. Two periods, including five stages of mineralization, are recognized in this area: an early metamorphic mineralization period (PI), including quartz–pyrite (Stage I) and banded quartz–polymetallic sulfide (Stage II) veins, and a later magmatic mineralization period (PII) including quartz–K-feldspar–pyrite–molybdenite veins (Stage III), quartz–polymetallic sulfide–chlorite ± calcite veinlets and stockwork (Stage IV), and late calcite–quartz veinlets (Stage V). Geochronological studies indicate a SHRIMP zircon U-Pb age of 236.1 Ma for the Tianzishan monzogranite, and our published ages of ore-bearing diorite porphyrite of the Suishizi section and granite porphyry of the Jiancaowan section being 213 and 212 Ma, respectively. Pyrites formed in association with PI and PII mineralization have well-defined Rb–Sr ages of 220 ± 7.5, 205.8 ± 8.7, and 199 ± 15 Ma, with close temporospatial coupling between mineralization and magmatism. The δ18O and δD values of fluid inclusions in Stage IV auriferous quartz veins range from −0.03‰ to +5.24‰ and −93‰ to −75‰, respectively, suggesting that mineralizing fluid was likely of magmatic origin. Three distinct ranges of δ34S values are identified in the studied sections (i.e., 7.04‰–9.12‰, −4.95‰ to −2.44‰, and 0.10‰–3.08‰), indicating a source containing multiple sulfur isotopes derived from magmatic and metamorphic fluids. The Liziyuan Au deposit is thus likely an orogenic deposit closely related to magmatism. Geochemical characteristics indicate that Tianzishan monzogranite is adakitic and was derived from thickened lower crust during Triassic orogenesis. The ore-bearing diorite porphyrite and granite porphyry formed in a post-collision extensional setting. Together with previous geological and geochemical data, our results indicate that the Liziyuan orogenic Au deposit was formed by early collisional–compressional metamorphism and late post-collision extensional magmatic fluids related to the evolution of the WQOB.

Selection of the Energy Performance Indicator for Hotels Based on ISO 50001: A Case Study

The work deals with the study of the Energy Planning stage according to the ISO 50001:2018 Standard at the Hotel Punta la Cueva in Cienfuegos, Cuba. The current energy management indicators for hotels are well-studied. However, the study aims to incorporate the variable Degree-Days in the Room Days Occupied (RDO) to establish a correct Baseline and Energy Performance Indicator. The methodology followed is based on the energy management standards of energy review stage. The fundamental results were in obtaining the Energy Baseline with a Coefficient of Determination (R2) of 0.97. One of the opportunities for improvement consisted of the replacement of 80 bulbs 15 W with 9 W LED bulbs. It has an Internal Rate of Return (IRR) of 28%, a Present Value Net Income (NPV) of 71.5 USD, and an Investment Recovery Period (PRI) of 3.6 years. The second opportunity is a preliminary project for a Photovoltaic Solar System on the roofs of the buildings, where it is possible to install 1011 photovoltaic and achieve a generation of 384 kilowatt-peak (kWp).

Dynamics of climate changes and its effect on the performance of cereals according to satellite data

Aim. To study the effect of climate changes on the performance of cereals. Methods. Remote, statistical, and analytic methods. Climate changes were determined by the sum of radiation temperatures of the earth’s surface, calculated using the data of the infrared range (10.3–11.3; 11.4–12.4 μm) of the high precision radiometer AVHRR of the meteorological satellites of the Earth, NOAA. The effect of climate changes on the state of vegetation, including the phenological parameters, such as the beginning, the end, and the length of the vegetation peri- od, was determined by the Normalized Difference Vegetation Index (NDVI) obtained using the data of infrared (0.72–1.1 μm) and red (0.58–0.68 μm) ranges of the abovementioned radiometer. The satellite data are openly accessed on the website of STAR NESDIS NOAA – Satellite Applications and Research of NOAA’s National En- vironmental Satellite Data Information Services of the National Ocean and Atmosphere Research Department of the USA – http://www.star.nesdis.noaa.gov/smcd/emb/vci/VH/. The dynamics of СО2 concentration in the air was determined using the satellite data of AIRS/Aqua (https://disc.gsfc.nasa.gov/datasets/AIRS3C2M_005/summary), the precipitation dynamics – using the data of ERA5 ECMWF/Copernicus Climate Change Service (https://devel- opers.google.com/earth-engine/datasets/catalog/ECMWF_ERA5_MONTHLY#description). The association be- tween the performance of cereals and satellite data was found by the method of correlation and regression analysis. Results. Using the satellite data, it was found that in 1982–2021, the sum of radiation temperatures of the earth’s surface reliably increased in the vegetation period (April–September), and the amount of precipitation tended to decrease in the zones of Polissia, Forest-Steppe, and Steppe. The highest tempo of warming was observed in the territory of the Steppe. In general, the warming had a positive effect on the state of vegetation, according to NDVI, and on the performance of cereals. However, in the recent decade (2010–2020), the positive effect of the warming on the vegetation state decreased, and according to the NDVI trend, there has been a tendency towards the negative effect of the further increase in the temperature, which is especially remarkable for the Steppe and Forest-Steppe. On the contrary, the warming in the Polissia zone created the conditions for the increase in the performance of agri- cultural cereals. Conclusions. From 1982 till 2021, there was a strong tendency towards the increase in the sum of radiation temperatures of the earth’s surface in the vegetation period and the decrease in the precipitation amount in Polissia, Forest-Steppe, and Steppe. According to the NDVI data, due to the warming, the length of the vegeta- tion period increased by 14–21 days on average, which had a positive effect on the vegetation. Since 2010, the acceleration in the warming tempo has been observed. The average annual increase in the sum of the radiation tem- peratures of the earth’s surface in the vegetation period is 15–16 °С – for Polissia, 14–18 °С for the Forest-Steppe, and 18–20 °С for the Steppe. According to this tendency, if the current regularity in the temperature increase in 1982–2021 is preserved in the nearest future, the temperature of the vegetation period will additionally increase, and the average sum of temperatures of the earth’s surface will reach 4000 °С in Polissia, which was notable for the Forest-Steppe in 1982–2021, 4440–4600 °С – in the Forest-Steppe, and up to 5800–5900 °С in the Steppe. In 1982–2000, there was a strong regularity of the positive effect of the warming during the vegetation period both on the vegetation state according to the NDVI index, and the performance of cereals in all three natural climatic zones. However, after 2000, due to the negative effect of high summer temperatures on the crops, the effect of the warming on vegetation considerably decreased, which was especially notable for the Steppe where the increase in NDVI compared to the previous period was on average 9.41 % in 1992–2001, and in 2012–2021 – 1.78 %.

Circadian rhythm-related factors of PER and CRY family genes function as novel therapeutic targets and prognostic biomarkers in lung adenocarcinoma.

The period (PER) and cryptochrome (CRY) families play critical roles in circadian rhythms. The imbalance of circadian factors may lead to the occurrence of cancer. Expressions of PER and CRY family members decrease in various cancers. Nevertheless, expression levels, genetic variations, and molecular mechanisms of PER and CRY family members in lung adenocarcinoma (LUAD) and their correlations with prognoses and immune infiltration in LUAD patients are still unclear. In this study, to identify their biological functions in LUAD development, comprehensive high-throughput techniques were applied to analyze the relationships of expressions of PER and CRY family members with genetic variations, molecular mechanisms, and immune infiltration. The present results showed that transcription levels of PER1 and CRY2 in LUAD were significantly downregulated. High expression levels of PER2, PER3, CRY1, and CRY2 indicated longer overall survival. Some cancer signaling pathways were related to PER and CRY family members, such as cell-cycle, histidine metabolism, and progesterone-mediated oocyte maturation pathways. Expressions of PER and CRY family members significantly affected the infiltration of different immune cells. In conclusion, our findings may help better understand the molecular basis of LUAD, and provide new perspectives of PER and CRY family members as novel biomarkers for LUAD.

PERIOD Phosphoclusters Control Temperature Compensation of the Drosophila Circadian Clock.

Ambient temperature varies constantly. However, the period of circadian pacemakers is remarkably stable over a wide-range of ecologically- and physiologically-relevant temperatures, even though the kinetics of most biochemical reactions accelerates as temperature rises. This thermal buffering phenomenon, called temperature compensation, is a critical feature of circadian rhythms, but how it is achieved remains elusive. Here, we uncovered the important role played by the Drosophila PERIOD (PER) phosphodegron in temperature compensation. This phosphorylation hotspot is crucial for PER proteasomal degradation and is the functional homolog of mammalian PER2 S478 phosphodegron, which also impacts temperature compensation. Using CRISPR-Cas9, we introduced a series of mutations that altered three Serines of the PER phosphodegron. While all three Serine to Alanine substitutions lengthened period at all temperatures tested, temperature compensation was differentially affected. S44A and S45A substitutions caused undercompensation, while S47A resulted in overcompensation. These results thus reveal unexpected functional heterogeneity of phosphodegron residues in thermal compensation. Furthermore, mutations impairing phosphorylation of the per s phosphocluster showed undercompensation, consistent with its inhibitory role on S47 phosphorylation. We observed that S47A substitution caused increased accumulation of hyper-phosphorylated PER at warmer temperatures. This finding was corroborated by cell culture assays in which S47A slowed down phosphorylation-dependent PER degradation at high temperatures, causing PER degradation to be excessively temperature-compensated. Thus, our results point to a novel role of the PER phosphodegron in temperature compensation through temperature-dependent modulation of the abundance of hyper-phosphorylated PER. Our work reveals interesting mechanistic convergences and differences between mammalian and Drosophila temperature compensation of the circadian clock.

Express and Purify Human CRY2 for Functional Analyses

The circadian rhythm is a biological clock responsible for regulating the daily cycles of essential physiological and metabolic processes, such as sleep wake cycles, by responding to light and dark changes. Every cell has its own circadian rhythm that is synchronized by a master clock in the brain. At the cellular level, circadian rhythms are controlled by a time‐delayed transcriptional‐translational feed‐back loop (TTFL), which consists of several core proteins. These proteins include circadian locomotor output cycles protein kaput (CLOCK), brain and muscle ARNT‐like 1 (BMAL1), cryptochrome (CRY), and period (PER). CLOCK and BMAL1 initiate the TTFL by activating the transcription of several circadian genes including CRYs and PERs that will be translated in the cytosol. With accumulation after a certain time, CRYs/PERs complexes will translocate back to the nucleus to negatively inhibit CLOCK/BMAL1 activities to finish the TTFL. There are two paralogs of CRY within the human genome: CRY1 and CRY2, both of which have essential roles in the negative feedback. The purpose of this project is to study the structure and function of human CRY2. Regardless of many advances in this field including available structure of CRYs, the detailed interaction between CRY2 to other circadian core proteins are still limited. Through this research we would be able to express and purify large quantities of hCRY2 that will be used for the future interaction studies. The results will deepen our understanding of the mechanisms behind circadian rhythm and its associated diseases, facilitating the development of prevention and treatment strategies.

Exploring additional sleep roles for the PERIOD homolog LIN‐42 using the auxin‐inducible degradation system in Caenorhabditis elegans

Sleep has been observed in all animals with a nervous system, however, the neural and molecular mechanisms that regulate it are not fully understood. The genetically tractable nematode Caenorhabditis elegans has a simple and fully mapped nervous system and has emerged as a powerful sleep model. C. eleganssleeps concurrently with each larval molt, called developmentally timed sleep (DTS), which is timed by the PERIOD(PER) homolog LIN‐42. C. elegans also sleep during adulthood in response to cellular damage incurred by stressful stimuli, called stress‐induced sleep (SIS). Based on LIN‐42’s central role in the timing of DTS, we sought to examine its role in the timing of SIS. Null mutations of lin‐42are lethal, so to study the requirement of LIN‐42 during SIS, we used CRISPR/Cas9 to insert the auxin‐induced degron and green fluorescent protein sequence in frame with each isoform of the lin‐42 gene. In the absence of auxin these worms are viable and grow successfully to adulthood. We are currently constructing transgenic strains that will allow us to degrade the LIN‐42 in different subsets of cells. To do this we are expressing the coding sequence for the auxin perceptive F‐box protein TIR1 from different tissue‐specific promoters, including those expressed in all cells, and specific sleep‐regulating neurons. Once these strains are constructed we will degrade LIN‐42 protein and measure the effects on SIS.

Proteome analysis of the circadian clock protein PERIOD2.

Circadian rhythms are a series of endogenous autonomous 24-h oscillations generated by the circadian clock. At the molecular level, the circadian clock is based on a transcription-translation feedback loop, in which BMAL1 and CLOCK transcription factors of the positive arm activate the expression of CRYPTOCHROME (CRY) and PERIOD (PER) genes of the negative arm as well as the circadian clock-regulated genes. There are three PER proteins, of which PER2 shows the strongest oscillation at both stability and cellular localization level. Protein-protein interactions (PPIs) or interactome of the circadian clock proteins have been investigated using classical methods such as two-dimensional gel electrophoresis, immunoprecipitation-coupled mass spectrometry, and yeast-two hybrid assay where the dynamic and weak interactions are difficult to catch. To identify the interactome of PER2 we have adopted proximity-dependent labeling with biotin and mass spectrometry-based identification of labeled proteins (BioID). In addition to known interactions with such as CRY1 and CRY2, we have identified several new PPIs for PER2 and confirmed some of them using co-immunoprecipitation technique. This study characterizes the PER2 protein interactions in depth, and it also implies that using a fast BioID method with miniTurbo or TurboID coupled to other major circadian clock proteins might uncover other interactors in the clock that have yet to be discovered.

Cryptochrome proteins regulate the circadian intracellular behavior and localization of PER2 in mouse suprachiasmatic nucleus neurons

The ∼20,000 cells of the suprachiasmatic nucleus (SCN), the master circadian clock of the mammalian brain, coordinate subordinate cellular clocks across the organism, driving adaptive daily rhythms of physiology and behavior. The canonical model for SCN timekeeping pivots around transcriptional/translational feedback loops (TTFL) whereby PERIOD (PER) and CRYPTOCHROME (CRY) clock proteins associate and translocate to the nucleus to inhibit their own expression. The fundamental individual and interactive behaviors of PER and CRY in the SCN cellular environment and the mechanisms that regulate them are poorly understood. We therefore used confocal imaging to explore the behavior of endogenous PER2 in the SCN of PER2::Venus reporter mice, transduced with viral vectors expressing various forms of CRY1 and CRY2. In contrast to nuclear localization in wild-type SCN, in the absence of CRY proteins, PER2 was predominantly cytoplasmic and more mobile, as measured by fluorescence recovery after photobleaching. Virally expressed CRY1 or CRY2 relocalized PER2 to the nucleus, initiated SCN circadian rhythms, and determined their period. We used translational switching to control CRY1 cellular abundance and found that low levels of CRY1 resulted in minimal relocalization of PER2, but yet, remarkably, were sufficient to initiate and maintain circadian rhythmicity. Importantly, the C-terminal tail was necessary for CRY1 to localize PER2 to the nucleus and to initiate SCN rhythms. In CRY1-null SCN, CRY1Δtail opposed PER2 nuclear localization and correspondingly shortened SCN period. Through manipulation of CRY proteins, we have obtained insights into the spatiotemporal behaviors of PER and CRY sitting at the heart of the TTFL molecular mechanism.

Dysregulation of PER3 clock gene and its only pseudogene in colorectal cancer and type 2 diabetes

The period (PER) family genes (PER1, PER2, and PER3) play a fundamental role in regulating the day/night cycle. PER3 has a pseudogene variant, PER3P1 or PER4, whose role and expression pattern is unclear in human health and diseases. This study was performed to evaluate the expression levels of normal PER family members and the PER3P1 pseudogene in colorectal cancer (CRC) and type 2 diabetes (T2D). Blood samples were taken from 50 diabetic patients and analyzed using real-time PCR for quantification of PER3 and PER3P1 expression. Colorectal tumor tissues of 50 individuals were also used to evaluate the expression of PER members. All PER members, including PER3P1, were found to be downregulated in colorectal tumor samples. Blood samples collected from diabetic subjects revealed an opposite expression pattern; both PER3 and its pseudogene were found to be upregulated when compared to the control group. Our results reveal coordination between the expression pattern of PER3P1 and normal PER family genes. Based on our findings and the pathological importance of this pseudogene, it can be suggested that PER3P1 may be one of the key regulators of the molecular clock network and PER family expression. This hypothesis needs to be confirmed by further studies.

Loss of Timeless Underlies an Evolutionary Transition within the Circadian Clock.

Most organisms possess time-keeping devices called circadian clocks. At the molecular level, circadian clocks consist of transcription–translation feedback loops (TTFLs). Although some components of the negative TTFL are conserved across the animals, important differences exist between typical models, such as mouse and the fruit fly. In Drosophila, the key components are PERIOD (PER) and TIMELESS (TIM-d) proteins, whereas the mammalian clock relies on PER and CRYPTOCHROME (CRY-m). Importantly, how the clock has maintained functionality during evolutionary transitions between different states remains elusive. Therefore, we systematically described the circadian clock gene setup in major bilaterian lineages and identified marked lineage-specific differences in their clock constitution. Then we performed a thorough functional analysis of the linden bug Pyrrhocoris apterus, an insect species comprising features characteristic of both the Drosophila and the mammalian clocks. Unexpectedly, the knockout of timeless-d, a gene essential for the clock ticking in Drosophila, did not compromise rhythmicity in P. apterus, it only accelerated its pace. Furthermore, silencing timeless-m, the ancestral timeless type ubiquitously present across animals, resulted in a mild gradual loss of rhythmicity, supporting its possible participation in the linden bug clock, which is consistent with timeless-m role suggested by research on mammalian models. The dispensability of timeless-d in P. apterus allows drawing a scenario in which the clock has remained functional at each step of transition from an ancestral state to the TIM-d-independent PER + CRY-m system operating in extant vertebrates, including humans.

Anthropogenic controls over soil organic carbon distribution from the cultivated lands in Northeast China

Both natural and anthropogenic variables affect soil C distribution and its pool, however studies about anthropogenic influence on soil C distribution are very limited in the literature. This study investigated anthropogenic effects on soil organic carbon (SOC) changes in the cultivated lands of Northeast China. A total of 196 topsoil samples (0–30 cm) were collected, and analyzed for SOC content, and 12 environmental variables (natural and anthropogenic) were selected as SOC predictors. Natural factors included elevation, slope gradient, slope aspect (SA), topographic wetness index (TWI), mean annual temperature, mean annual precipitation, and normalized difference vegetation index, while population (POP), gross domestic product (GDP), distance to the socioeconomic center, distance to roads, and reclamation period (PER) represented anthropogenic variables. Three different boosted-regression trees models with different combination of SOC predictors were constructed, and the model performance was evaluated with 10-fold cross-validation. We found that the model that included all predictors had the best performance, followed by the model with topography and climate variables, and the model with only anthropogenic variables. However, adding the anthropogenic variables in the model greatly improved its performance. Results showed that PER, POP and GDP were the key environmental variables affecting SOC content in the topsoil agroecosystems in Northeast China. This study suggests that anthropogenic variables should be selected as the main environmental variable in predicting of SOC content in agroecosystem with a higher human influence. We believe that the accurate prediction and mapping of SOC content in the topsoil agroecosystem will help formulate farmland soil management policies and promote soil carbon sequestration.

TAMI-44. ASSOCIATION OF CIRCADIAN CLOCK GENE EXPRESSION WITH GLIOMA TUMOR MICROENVIRONMENT AND PATIENT SURVIVAL

Abstract Circadian clock genes have been linked to differences in clinical outcomes in cancer, including gliomas. However, these studies have not accounted for established prognostic markers, including mutations in Isocitrate Dehydrogenase (IDH). To study the connection between circadian clock genes and glioma outcomes while accounting for IDH mutational status, we analyzed multiple publicly available gene expression datasets. Unsupervised clustering of 13 clock gene transcriptomic signatures from The Cancer Genome Atlas resulted in four distinct transcriptomic clusters, two clusters were enriched for IDH mutant (Circadian 1-2) and the others for IDH wild-type gliomas (Circadian 3-4). Within these clusters we observed differential prognosis of the patients by Kaplan–Meier analysis (Circadian 1-2, p=0.0001; Circadian 3-4, p=0.0002) suggesting that these transcriptomic circadian subtypes might reflect different disease states. Further analyses using Cox Proportional Hazards Regression showed that lower Period (PER) gene expression was associated with worse prognosis (increasing PER expression HR=0.655, p=0.007) independent of IDH wild-type status (HR=5.312, p< 0.001) and increasing age (HR = 1.04, p< 0.001). Lower PER expression was associated with enrichment of a number of immune signaling pathways. These findings prompted the exploration of the relationship between microenvironment and clock genes using the Ivy GAP dataset to explore tumor location-specific differences and single cell RNA sequencing data from Darmanis (accession: GSE84465) to explore cell-specific differences. Circadian clock genes were found to be differentially expressed across anatomical tumor locations and cell types, including microglia. In ongoing studies we are examining the role of the microenvironment and PER2 expression on tumor growth by disrupting PER2 expression in tumor cells and microglia using IDH mutant and wild-type in vitro models. Clock gene expression is a potential prognostic biomarker in glioma and further studies to elucidate the importance of circadian rhythms in other cell types beyond the tumor are warranted.