The crystal structure of Vibrio cholerae (6-4) photolyase reveals interactions with cofactors and a DNA-binding region.

Photolyases (PLs) reverse UV-induced DNA damage using blue light as an energy source. Of these PLs, (6-4) PLs repair (6-4)-lesioned photoproducts. We recently identified a gene from Vibrio cholerae (Vc) encoding a (6-4) PL, but structural characterization is needed to elucidate specific interactions with the chromophore cofactors. Here, we determined the crystal structure of Vc (6-4) PL at 2.5 Å resolution. Our high-resolution structure revealed that the two well-known cofactors, flavin adenine dinucleotide and the photoantenna 6,7-dimethyl 8-ribityl-lumazin (DMRL), stably interact with an α-helical and an α/ß domain, respectively. Additionally, the structure has a third cofactor with distinct electron clouds corresponding to a [4Fe-4S] cluster. Moreover, we identified that Asp106 makes a hydrogen bond with water and DMRL, which indicates further stabilization of the photoantenna DMRL within Vc (6-4) PL. Further analysis of the Vc (6-4) PL structure revealed a possible region responsible for DNA binding. The region located between residues 478 to 484 may bind the lesioned DNA, with Arg483 potentially forming a salt bridge with DNA to stabilize further the interaction of Vc (6-4) PL with its substrate. Our comparative analysis revealed that the DNA lesion could not bind to the Vc (6-4) PL in a similar fashion to the Drosophila melanogaster (Dm, (6-4)) PL without a significant conformational change of the protein. The 23rd helix of the bacterial (6-4) PLs seems to have remarkable plasticity, and conformational changes facilitate DNA binding. In conclusion, our structure provides further insight into DNA repair by a (6-4) PL containing three cofactors.

Functional characterization of the CRY2 circadian clock component variant p.Ser420Phe revealed a new degradation pathway for CRY2.

Cryptochromes (CRYs) are essential components of the circadian clock, playing a pivotal role as transcriptional repressors. Despite their significance, the precise mechanisms underlying CRYs' involvement in the circadian clock remain incompletely understood. In this study, we identified a rare CRY2 variant, p.Ser420Phe, from the 1000 Genomes Project and Ensembl database that is located in the functionally important coiled-coil-like helix (CC-helix) region. Functional characterization of this variant at the cellular level revealed that p.Ser420Phe CRY2 had reduced repression activity on CLOCK:BMAL1-driven transcription due to its reduced affinity to the core clock protein PER2 and defective translocation into the nucleus. Intriguingly, the CRY2 variant exhibited an unexpected resistance to degradation via the canonical proteasomal pathway, primarily due to the loss of interactions with E3 ligases (FBXL3 and FBXL21), which suggests Ser-420 of CRY2 is required for the interaction with E3 ligases. Further studies revealed that wild-type and CRY2 variants are degraded by the lysosomal-mediated degradation pathway, a mechanism not previously associated with CRY2. Surprisingly, our complementation study with Cry1-/-Cry2-/- double knockout mouse embryonic fibroblast cells indicated that the CRY2 variant caused a 7 h shorter circadian period length in contrast to the observed prolonged period length in CRY2-/- cell lines. In summary, this study reveals a hitherto unknown degradation pathway for CRY2, shedding new light on the regulation of circadian rhythm period length.

The secondary pocket of cryptochrome 2 is important for the regulation of its stability and localization.

Human clock-gene variations contribute to the phenotypic differences observed in various behavioral and physiological processes, such as diurnal preference, sleep, metabolism, mood regulation, addiction, and fertility. However, little is known about the possible effects of identified variations at the molecular level. In this study, we performed a functional characterization at the cellular level of rare cryptochrome 2 (CRY2) missense variations that were identified from the Ensembl database. Our structural studies revealed that three variations (p.Pro123Leu, p.Asp406His, and p.Ser410Ile) are located at the rim of the secondary pocket of CRY2. We show that these variants were unable to repress CLOCK (circadian locomotor output cycles kaput)/BMAL1 (brain and muscle ARNT-like-1)-driven transcription in a cell-based reporter assay and had reduced affinity to CLOCK-BMAL1. Furthermore, our biochemical studies indicated that the variants were less stable than the WT CRY2, which could be rescued in the presence of period 2 (PER2), another core clock protein. Finally, we found that these variants were unable to properly localize to the nucleus and thereby were unable to rescue the circadian rhythm in a Cry1-/-Cry2-/- double KO mouse embryonic fibroblast cell line. Collectively, our data suggest that the rim of the secondary pocket of CRY2 plays a significant role in its nuclear localization independently of PER2 and in the intact circadian rhythm at the cellular level.

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.

Transcriptomic and fatty acid analyses of Neochloris aquatica grown under different nitrogen concentration.

In this study, we characterized the fatty acid production in Neochloris aquatica at transcriptomics and biochemical levels under limiting, normal, and excess nitrate concentrations in different growth phases. At the stationary phase, N. aquatica mainly produced saturated fatty acids such as stearic acid under the limiting nitrate concentration, which is suitable for biodiesel production. However, it produced polyunsaturated fatty acids such as α-linolenic acid under the excess nitrate concentration, which has nutritional values as food supplements. In addition, RNA-seq was employed to identify genes and pathways that were being affected in N. aquatica for three growth phases in the presence of the different nitrate amounts. Genes that are responsible for the production of saturated fatty acids were upregulated in the cells grown under a limiting nitrogen amount while genes that are responsible for the production of polyunsaturated fatty acid were upregulated in the cells grown under excess nitrogen amount. Further analysis showed more genes differentially expressed (DEGs) at the logarithmic phase in all conditions while a relatively steady trend was observed during the transition from the logarithmic phase to the stationary phase under limiting and excess nitrogen. Our results provide a foundation for identifying developmentally important genes and understanding the biological processes in the different growth phases of the N. aquatica in terms of biomass and lipid production.

The Arg-293 of Cryptochrome1 is responsible for the allosteric regulation of CLOCK-CRY1 binding in circadian rhythm.

Mammalian circadian clocks are driven by transcription/translation feedback loops composed of positive transcriptional activators (BMAL1 and CLOCK) and negative repressors (CRYPTOCHROMEs (CRYs) and PERIODs (PERs)). CRYs, in complex with PERs, bind to the BMAL1/CLOCK complex and repress E-box-driven transcription of clock-associated genes. There are two individual CRYs, with CRY1 exhibiting higher affinity to the BMAL1/CLOCK complex than CRY2. It is known that this differential binding is regulated by a dynamic serine-rich loop adjacent to the secondary pocket of both CRYs, but the underlying features controlling loop dynamics are not known. Here we report that allosteric regulation of the serine-rich loop is mediated by Arg-293 of CRY1, identified as a rare CRY1 SNP in the Ensembl and 1000 Genomes databases. The p.Arg293His CRY1 variant caused a shortened circadian period in a Cry1-/-Cry2-/- double knockout mouse embryonic fibroblast cell line. Moreover, the variant displayed reduced repressor activity on BMAL1/CLOCK driven transcription, which is explained by reduced affinity to BMAL1/CLOCK in the absence of PER2 compared with CRY1. Molecular dynamics simulations revealed that the p.Arg293His CRY1 variant altered a communication pathway between Arg-293 and the serine loop by reducing its dynamicity. Collectively, this study provides direct evidence that allosterism in CRY1 is critical for the regulation of circadian rhythm.

A CLOCK-binding small molecule disrupts the interaction between CLOCK and BMAL1 and enhances circadian rhythm amplitude.

Proper function of many physiological processes requires a robust circadian clock. Disruptions of the circadian clock can result in metabolic diseases, mood disorders, and accelerated aging. Therefore, identifying small molecules that specifically modulate regulatory core clock proteins may potentially enable better management of these disorders. In this study, we applied a structure-based molecular-docking approach to find small molecules that specifically bind to the core circadian regulator, the transcription factor circadian locomotor output cycles kaput (CLOCK). We identified 100 candidate molecules by virtual screening of ∼2 million small molecules for those predicted to bind closely to the interface in CLOCK that interacts with its transcriptional co-regulator, Brain and muscle Arnt-like protein-1 (BMAL1). Using a mammalian two-hybrid system, real-time monitoring of circadian rhythm in U2OS cells, and various biochemical assays, we tested these compounds experimentally and found one, named CLK8, that specifically bound to and interfered with CLOCK activity. We show that CLK8 disrupts the interaction between CLOCK and BMAL1 and interferes with nuclear translocation of CLOCK both in vivo and in vitro Results from further experiments indicated that CLK8 enhances the amplitude of the cellular circadian rhythm by stabilizing the negative arm of the transcription/translation feedback loop without affecting period length. Our results reveal CLK8 as a tool for further studies of CLOCK's role in circadian rhythm amplitude regulation and as a potential candidate for therapeutic development to manage disorders associated with dampened circadian rhythms.

Transcriptome analysis of the circadian clock gene BMAL1 deletion with opposite carcinogenic effects.

We have previously reported that the deletion of BMAL1 gene has opposite effects in respect to its contribution to the pathways that are effective in the multistage carcinogenesis process. BMAL1 deletion sensitized nearly normal breast epithelial (MCF10A) and invasive breast cancer cells (MDA-MB-231) to cisplatin- and doxorubicin-induced apoptosis, while this deletion also aggravated the invasive potential of MDA-MB-231 cells. However, the mechanistic relationship of the seemingly opposite contribution of BMAL1 deletion to carcinogenesis process is not known at genome-wide level. In this study, an RNA-seq approach was taken to uncover the differentially expressed genes (DEGs) and pathways after treating BMAL1 knockout (KO) or wild-type (WT) MDA-MB-231 cells with cisplatin and doxorubicin to initiate apoptosis. Gene set enrichment analysis with the DEGs demonstrated that enrichment in multiple genes/pathways contributes to sensitization to cisplatin- or doxorubicin-induced apoptosis in BMAL1-dependent manner. Additionally, our DEG analysis suggested that non-coding transcript RNA (such as lncRNA and processed pseudogenes) may have role in cisplatin- or doxorubicin-induced apoptosis. Protein-protein interaction network obtained from common DEGs in cisplatin and doxorubicin treatments revealed that GSK3ß, NACC1, and EGFR are the principal genes regulating the response of the KO cells. Moreover, the analysis of DEGs among untreated BMAL1 KO and WT cells revealed that epithelial-mesenchymal transition genes are up-regulated in KO cells. As a negative control, we have also analyzed the DEGs following treatment with an endoplasmic reticulum (ER) stress-inducing agent, tunicamycin, which was affected by BMAL1 deletion minimally. Collectively, the present study suggests that BMAL1 regulates many genes/pathways of which the alteration in BMAL1 KO cells may shed light on pleotropic phenotype observed.

Biocompatible Quantum Funnels for Neural Photostimulation.

Neural photostimulation has high potential to understand the working principles of complex neural networks and develop novel therapeutic methods for neurological disorders. A key issue in the light-induced cell stimulation is the efficient conversion of light to bioelectrical stimuli. In photosynthetic systems developed in millions of years by nature, the absorbed energy by the photoabsorbers is transported via nonradiative energy transfer to the reaction centers. Inspired by these systems, neural interfaces based on biocompatible quantum funnels are developed that direct the photogenerated charge carriers toward the bionanojunction for effective photostimulation. Funnels are constructed with indium-based rainbow quantum dots that are assembled in a graded energy profile. Implementation of a quantum funnel enhances the generated photoelectrochemical current 215% per unit absorbance in comparison with ungraded energy profile in a wireless and free-standing mode and facilitates optical neuromodulation of a single cell. This study indicates that the control of charge transport at nanoscale can lead to unconventional and effective neural interfaces.

Identification and Characterization of a New Class of (6-4) Photolyase from Vibrio cholerae.

Light is crucial for many biological activities of most organisms, including vision, resetting of circadian rhythm, photosynthesis, and DNA repair. The cryptochrome/photolyase family (CPF) represents an ancient group of UV-A/blue light sensitive proteins that perform different functions such as DNA repair, circadian photoreception, and transcriptional regulation. The CPF is widely distributed throughout all organisms, including marine prokaryotes. The bacterium Vibrio cholerae was previously shown to have a CPD photolyase that repairs UV-induced thymine dimers and two CRY-DASHs that repair UV-induced single-stranded DNA damage. Here, we characterize a hypothetical gene Vca0809 encoding a new member of CPF in this organism. The spectroscopic analysis of the purified protein indicated that this enzyme possessed a catalytic cofactor, FAD, and photoantenna chromophore 6,7-dimethyl 8-ribityl-lumazin. With a slot blot-based DNA repair assay, we showed that it possessed (6-4) photolyase activity. Further phylogenetic and computational analyses enabled us to classify this gene as a member of the family of iron-sulfur bacterial cryptochromes and photolyases (FeS-BCP). Therefore, we named this gene Vc(6-4) FeS-BCP.

Comparative RNA-seq analysis of the drought-sensitive lentil (Lens culinaris) root and leaf under short- and long-term water deficits.

Drought stress is one of the main environmental factors that affects growth and productivity of crop plants, including lentil. To gain insights into the genome-wide transcriptional regulation in lentil root and leaf under short- and long-term drought conditions, we performed RNA-seq on a drought-sensitive lentil cultivar (Lens culinaris Medik. cv. Sultan). After establishing drought conditions, lentil samples were subjected to de novo RNA-seq-based transcriptome analysis. The 207,076 gene transcripts were successfully constructed by de novo assembly from the sequences obtained from root, leaf, and stems. Differentially expressed gene (DEG) analysis on these transcripts indicated that period of drought stress had a greater impact on the transcriptional regulation in lentil root. The numbers of DEGs were 2915 under short-term drought stress while the numbers of DEGs were increased to 18,327 under long-term drought stress condition in the root. Further, Gene Ontology analysis revealed that the following biological processes were differentially regulated in response to long-term drought stress: protein phosphorylation, embryo development seed dormancy, DNA replication, and maintenance of root meristem identity. Additionally, DEGs, which play a role in circadian rhythm and photoreception, were downregulated suggesting that drought stress has a negative effect on the internal oscillators which may have detrimental consequences on plant growth and survival. Collectively, this study provides a detailed comparative transcriptome response of drought-sensitive lentil strain under short- and long-term drought conditions in root and leaf. Our finding suggests that not only the regulation of genes in leaves is important but also genes regulated in roots are important and need to be considered for improving drought tolerance in lentil.

Cryptochrome deletion in p53 mutant mice enhances apoptotic and anti-tumorigenic responses to UV damage at the transcriptome level.

Previous studies have demonstrated that deletion of cryptochrome (Cry) genes protects p53-/- mutant mice from the early onset of cancer and extends their median life-span by about 1.5-fold. Subsequent in vitro studies had revealed that deletion of Crys enhances apoptosis in response to UV damage through activation of p73 and inactivation of GSK3ß. However, it was not known at the transcriptome-wide level how deletion of Crys delays the onset of cancer in p53-/- mutant mice. In this study, the RNA-seq approach was taken to uncover the differentially expressed genes (DEGs) and pathways following UV-induced DNA damage in p53-/- and p53-/-Cry1-/-Cry2-/- mouse skin fibroblasts. Gene set enrichment analysis with the DEGs demonstrated enrichment in immune surveillance-associated genes regulated by IFN-γ and genes involved in TNFα signaling via NF-κB. Furthermore, protein network analysis enabled identification of DEGs p21, Sirt1, and Jun as key players, along with their interacting partners. It was also observed that the DEGs contained a high ratio of non-coding transcripts. Collectively, the present study suggests new genes in NF-κB regulation and IFN-γ response, as well as non-coding RNAs, may contribute to delaying the onset of cancer in p53-/-Cry1-/-Cry2-/- mice and increasing the life-span of these animals compared to p53-/- mice.

Molecular Aspects of Circadian Pharmacology and Relevance for Cancer Chronotherapy.

The circadian timing system (CTS) controls various biological functions in mammals including xenobiotic metabolism and detoxification, immune functions, cell cycle events, apoptosis and angiogenesis. Although the importance of the CTS is well known in the pharmacology of drugs, it is less appreciated at the clinical level. Genome-wide studies highlighted that the majority of drug target genes are controlled by CTS. This suggests that chronotherapeutic approaches should be taken for many drugs to enhance their effectiveness. Currently chronotherapeutic approaches are successfully applied in the treatment of different types of cancers. The chronotherapy approach has improved the tolerability and antitumor efficacy of anticancer drugs both in experimental animals and in cancer patients. Thus, chronobiological studies have been of importance in determining the most appropriate time of administration of anticancer agents to minimize their side effects or toxicity and enhance treatment efficacy, so as to optimize the therapeutic ratio. This review focuses on the underlying mechanisms of the circadian pharmacology i.e., chronopharmacokinetics and chronopharmacodynamics of anticancer agents with the molecular aspects, and provides an overview of chronotherapy in cancer and some of the recent advances in the development of chronopharmaceutics.

RNA-seq analysis of the transcriptional response to blue and red light in the extremophilic red alga, Cyanidioschyzon merolae.

Light is one of the main environmental cues that affects the physiology and behavior of many organisms. The effect of light on genome-wide transcriptional regulation has been well-studied in green algae and plants, but not in red algae. Cyanidioschyzon merolae is used as a model red algae, and is suitable for studies on transcriptomics because of its compact genome with a relatively small number of genes. In addition, complete genome sequences of the nucleus, mitochondrion, and chloroplast of this organism have been determined. Together, these attributes make C. merolae an ideal model organism to study the response to light stimuli at the transcriptional and the systems biology levels. Previous studies have shown that light significantly affects cell signaling in this organism, but there are no reports on its blue light- and red light-mediated transcriptional responses. We investigated the direct effects of blue and red light at the transcriptional level using RNA-seq. Blue and red lights were found to regulate 35 % of the total genes in C. merolae. Blue light affected the transcription of genes involved in protein synthesis while red light specifically regulated the transcription of genes involved in photosynthesis and DNA repair. Blue or red light regulated genes involved in carbon metabolism and pigment biosynthesis. Overall, our data showed that red and blue light regulate the majority of the cellular, cell division, and repair processes in C. merolae.

Microcantilever based disposable viscosity sensor for serum and blood plasma measurements.

This paper proposes a novel method for measuring blood plasma and serum viscosity with a microcantilever-based MEMS sensor. MEMS cantilevers are made of electroplated nickel and actuated remotely with magnetic field using an electro-coil. Real-time monitoring of cantilever resonant frequency is performed remotely using diffraction gratings fabricated at the tip of the dynamic cantilevers. Only few nanometer cantilever deflection is sufficient due to interferometric sensitivity of the readout. The resonant frequency of the cantilever is tracked with a phase lock loop (PLL) control circuit. The viscosities of liquid samples are obtained through the measurement of the cantilever's frequency change with respect to a reference measurement taken within a liquid of known viscosity. We performed measurements with glycerol solutions at different temperatures and validated the repeatability of the system by comparing with a reference commercial viscometer. Experimental results are compared with the theoretical predictions based on Sader's theory and agreed reasonably well. Afterwards viscosities of different Fetal Bovine Serum and Bovine Serum Albumin mixtures are measured both at 23°C and 37°C, body temperature. Finally the viscosities of human blood plasma samples taken from healthy donors are measured. The proposed method is capable of measuring viscosities from 0.86 cP to 3.02 cP, which covers human blood plasma viscosity range, with a resolution better than 0.04 cP. The sample volume requirement is less than 150 µl and can be reduced significantly with optimized cartridge design. Both the actuation and sensing are carried out remotely, which allows for disposable sensor cartridges.

Computational and experimental investigation of DNA repair protein photolyase interactions with low molecular weight drugs.

This paper reports the previously unknown interactions between eight low molecular weight commercially available drugs (130-800 Da) and DNA repair protein photolyase using computational docking simulations and surface plasmon resonance (SPR) experiments. Theoretical dissociation constants, K(d), obtained from molecular docking simulations were compared with the values found from SPR experiments. Among the eight drugs analyzed, computational and experimental values showed similar binding affinities between selected drug and protein pairs. We found no significant differences in binding interactions between pure and commercial forms of the drug lornoxicam and DNA photolyase. Among the eight drugs studied, prednisone, desloratadine, and azelastine exhibited the highest binding affinity (K(d) = 1.65, 2.05, and 8.47 µM, respectively) toward DNA photolyase. Results obtained in this study are promising for use in the prediction of unknown interactions of common drugs with specific proteins such as human clock protein cryptochrome.

Dynamic regulation of the serine loop by distant mutations reveals allostery in cryptochrome1.

Cryptochromes (CRYs) are essential components of the molecular clock that generates circadian rhythm. They inhibit BMAL1/CLOCK-driven transcription at the molecular level. There are two CRYs that have differential functions in the circadian clock in mammals. It is not precisely known how they achieve such differential functions. In this study, we performed molecular dynamic simulations on eight CRY mutants that have been experimentally shown to exhibit reduced repressor activities. Our results revealed that mutations in CRY1 affect the dynamic behavior of the serine loop and the availability of the secondary pocket, but not in CRY2. Further analysis of these CRY1 mutants indicated that the differential flexibility of the serine loop leads to changes in the volume of the secondary pocket. We also investigated the weak interactions between the amino acids in the serine loop and those in close proximity. Our findings highlighted the crucial roles of S44 and S45 in the dynamic behavior of the serine loop, specifically through their interactions with E382 in CRY1. Considering the clinical implications of altered CRY1 function, our study opens up new possibilities for the development of drugs that target the allosteric regulation of CRY1.Communicated by Ramaswamy H. Sarma.

Single nucleotide polymorphisms (SNPs) in circadian genes: Impact on gene function and phenotype.

Circadian rhythm is an endogenous timing system that allows an organism to anticipate and adapt to daily changes and regulate various physiological variables such as the sleep-wake cycle. This rhythm is governed by a molecular circadian clock mechanism, generated by a transcriptional and translational feedback loop (TTFL) mechanism. In mammals, TTFL is determined by the interaction of four main clock proteins: BMAL1, CLOCK, Cryptochromes (CRY), and Periods (PER). BMAL1 and CLOCK form dimers and initiate the transcription of clock-controlled genes (CCG) by binding an E-box element with the promotor genes. Among CCGs, PERs and CRYs accumulate in the cytosol and translocate into the nucleus, where they interact with the BMAL1/CLOCK dimer and inhibit its activity. Several epidemiological and genetic studies have revealed that circadian rhythm disruption causes various types of disease. In this chapter, we summarize the effect of core clock gene SNPs on circadian rhythm and diseases in humans.

Diurnal Changes in Capecitabine Clock-Controlled Metabolism Enzymes Are Responsible for Its Pharmacokinetics in Male Mice.

The circadian timing system controls absorption, distribution, metabolism, and elimination processes of drug pharmacokinetics over a 24-h period. Exposure of target tissues to the active form of the drug and cytotoxicity display variations depending on the chronopharmacokinetics. For anticancer drugs with narrow therapeutic ranges and dose-limiting side effects, it is particularly important to know the temporal changes in pharmacokinetics. A previous study indicated that pharmacokinetic profile of capecitabine was different depending on dosing time in rat. However, it is not known how such difference is attributed with respect to diurnal rhythm. Therefore, in this study, we evaluated capecitabine-metabolizing enzymes in a diurnal rhythm-dependent manner. To this end, C57BL/6J male mice were orally treated with 500 mg/kg capecitabine at ZT1, ZT7, ZT13, or ZT19. We then determined pharmacokinetics of capecitabine and its metabolites, 5'-deoxy-5-fluorocytidine (5'DFCR), 5'-deoxy-5-fluorouridine (5'DFUR), 5-fluorouracil (5-FU), in plasma and liver. Results revealed that plasma Cmax and AUC0-6h (area under the plasma concentration-time curve from 0 to 6 h) values of capecitabine, 5'DFUR, and 5-FU were higher during the rest phase (ZT1 and ZT7) than the activity phase (ZT13 and ZT19) (p < 0.05). Similarly, Cmax and AUC0-6h values of 5'DFUR and 5-FU in liver were higher during the rest phase than activity phase (p < 0.05), while there was no significant difference in liver concentrations of capecitabine and 5'DFCR. We determined the level of the enzymes responsible for the conversion of capecitabine and its metabolites at each ZT. Results indicated the levels of carboxylesterase 1 and 2, cytidine deaminase, uridine phosphorylase 2, and dihydropyrimidine dehydrogenase (p < 0.05) are being rhythmically regulated and, in turn, attributed different pharmacokinetics profiles of capecitabine and its metabolism. This study highlights the importance of capecitabine administration time to increase the efficacy with minimum adverse effects.

TW68, cryptochromes stabilizer, regulates fasting blood glucose levels in diabetic ob/ob and high fat-diet-induced obese mice.

Cryptochromes (CRYs), transcriptional repressors of the circadian clock in mammals, inhibit cAMP production when glucagon activates G-protein coupled receptors. Therefore, molecules that modulate CRYs have the potential to regulate gluconeogenesis. In this study, we discovered a new molecule called TW68 that interacts with the primary pockets of mammalian CRY1/2, leading to reduced ubiquitination levels and increased stability. In cell-based circadian rhythm assays using U2OS Bmal1-dLuc cells, TW68 extended the period length of the circadian rhythm. Additionally, TW68 decreased the transcriptional levels of two genes, Phosphoenolpyruvate carboxykinase 1 (PCK1) and Glucose-6-phosphatase (G6PC), which play crucial roles in glucose biosynthesis during glucagon-induced gluconeogenesis in HepG2 cells. Oral administration of TW68 in mice showed good tolerance, a good pharmacokinetic profile, and remarkable bioavailability. Finally, when administered to fasting diabetic animals from ob/ob and HFD-fed obese mice, TW68 reduced blood glucose levels by enhancing CRY stabilization and subsequently decreasing the transcriptional levels of Pck1 and G6pc. These findings collectively demonstrate the antidiabetic efficacy of TW68 in vivo, suggesting its therapeutic potential for controlling fasting glucose levels in the treatment of type 2 diabetes mellitus.