Circadian rhythms, the 24-h oscillations of biological activities guided by the molecular clock, play a pivotal role in regulating various physiological processes in organisms. The intricate relationship between the loss of circadian rhythm and its influence on the tolerability and pharmacokinetic properties of anticancer drugs is poorly understood. In our study, we investigated the effects of oxaliplatin, a commonly used anticancer drug, on Cry1−/− and Cry2−/− mice (Cry DKO mice) under darkness conditions, where they exhibit free-running phenotype. We administered oxaliplatin at a dosage of 12 mg/kg/day at two distinct circadian times, CT8 and CT16, under constant darkness conditions to Cry DKO mice and their wild type littermates. Our results revealed a striking disparity in oxaliplatin tolerance between Cry DKO mice and their wild-type counterparts. Oxaliplatin exhibited severe toxicity in Cry DKO mice at both CT8 and CT16, in contrast to the wild type mice. Pharmacokinetic analyses suggested that such toxicity was a result of high concentrations of oxaliplatin in the serum and liver of Cry DKO mice after repeated dose injections. To understand the molecular basis of such intolerance, we performed RNA-seq studies using mouse livers. Our findings from the RNA-seq analysis highlighted the substantial impact of circadian rhythm disruption on gene expression, particularly affecting genes involved in detoxification and xenobiotic metabolism, such as the Gstm gene family. This dysregulation in detoxification pathways in Cry DKO mice likely contributes to the increased toxicity of oxaliplatin. In conclusion, our study highlights the crucial role of an intact molecular clock in dictating the tolerability of oxaliplatin. These findings emphasize the necessity of considering circadian rhythms in the administration of anticancer drugs, providing valuable insights into optimizing treatment strategies for cancer patients.

Boric acid (BA) is an essential trace element that is required to support the metabolic pathways in plants, humans, and animals. The present study investigates the in vitro development and quality of single-cell mouse embryos in a BA-added culture medium after cryopreservation using the solid-surface vitrification method. For this purpose, the pronuclear-stage embryos derived from superovulated C57Bl/6j mouse strains and the one-cell embryos were then cryopreserved using the solid-surface vitrification (SSV) method. After thawing, the embryos were cultured in a BA-added medium at 37 degrees C in a 5% CO2 environment until the blastocyst stage. The resulting in vitro development rates of the embryos in the control group, SSV group, and SSV + 1.62 x 10(-4) mu M BA group were 68.11% (36/59), 40.16% (16/48), and 64.92% (28/48) respectively, indicating that the BA supported the in vitro development of the embryos cryopreserved using the SSV method. Our results suggest that the addition of boric acid to the culture media increased the development rate of the embryos that were vitrified using the SSV method.

Smart materials are ushering in the era of smart and adaptable products. Hydrogels are a distinct class of smart materials that can be 3D-printed to produce smart and active structures that can be used as sensors and actuators. The development and characterization of a 3D-printable and electrically conductive composite hydrogel, as well as its application in the development of a smart disinfection system, are discussed in this article. The developed composite hydrogel has a maximum electrical conductivity of 145 S.m −1 , is stable up to 200 °C, and has a 3D printable rheology. Virtuous of its electrical conductivity, the composite hydrogel was used to create a smart disinfection system. Various disinfection systems have been adopted for the disinfection of contaminated surfaces; however, most of these systems require human evacuation from the surroundings due to the hazardous nature of the virucide. The proposed system is designed to disinfect contaminated surfaces on common-use equipment and is capable of real-time activation through user interaction. It employs a thermal disinfection process at 60 °C for 5 min and becomes ready for the next user once its temperature drops below 55 °C. This system consumes 1.64 Wh of energy per disinfection cycle and is suitable for scenarios with fewer than 60 user interactions in an 8-h work shift.

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

In order to develop drugs against cancer, which is one of the deadliest diseases in the world, hybridiz-ing structures with known anticancer activity can provide a dual activity for the treatment of cancer cases. For this purpose, a series of novel hybrid compounds combining lonidamine and 7-hydroxy-4-methylcoumarin via different linker modifications have been synthesized in this study. The chemical structures of the novel compound were elucidated with 1 H and 13 C NMR and mass spectrometry (HRMS) methods. All the synthesized compounds were screened for anticancer activity against MCF-7 and A549 cells. Among them, compound 2 appeared to be the most potent compound on MCF-7 lines; and IC50 dose was determined as 33.12 mu M. IC50 of Compound 6 was determined as 139.6 mu M for A549 cells. Con-sidering anticancer activity studies, compounds 2 and 6 have potential to lead to the development of novel hybrid compounds for cancer therapy. Further studies to identify mechanistic explanations of the bioactivity of these molecules are ongoing and will be reported in due course.(c) 2023 Elsevier B.V. All rights reserved.

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.

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, 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 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.

There is an intricate interplay of protein structure-function relationships in the pathogenesis of ne diseases, especially in Ankylosing Spondylitis (AS). the Major Histocompatibility Complex I allele, HLA-B*27, and Endoplasmic Reticulum Associated Aminopeptidase ERAP1 both have pivotal roles in shaping the immune response and contributing to the development of AS.HLA-B*27, a well-established genetic risk factor, undergoes structural changes that influence antigen presentation, ultimately triggering autoimmunity.ERAP1, an aminopeptidase involved in peptide processing, influences the peptide repertoire presented by HLA-B*27, thereby contributing to AS susceptibility.In this talk, I will discuss our recent computational findings based on the arthritogenic peptide presentation on HLA-B*27 and ERAP dimerization.Understanding these dynamic relationships at the structural and molecular level sheds light on the mechanisms underlying autoimmune diseases and opens new avenues for therapeutic strategies.

Cryptochromes are negative transcriptional regulators of the circadian clock in mammals. It is not clear how reducing the level of endogenous CRY1 in mammals will affect circadian rhythm and the relation of such a decrease with apoptosis. Here, we discovered a molecule (M47) that destabilizes Cryptochrome 1 (CRY1) both in vitro and in vivo. The M47 selectively enhanced the degradation rate of CRY1 by increasing its ubiquitination and resulted in increasing the circadian period length of U2OS Bmal1 -d Luc cells. In addition, subcellular fractionation studies from mice liver indicated that M47 increased degradation of the CRY1 in the nucleus. Furthermore, M47-mediated CRY1 reduction enhanced oxaliplatin-induced apoptosis in Ras-transformed p53 null fibroblast cells. Systemic repetitive administration of M47 increased the median lifespan of p53 −/− mice by ~25%. Collectively our data suggest that M47 is a promising molecule to treat forms of cancer depending on the p53 mutation.

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.

Objective: A circadian rhythm in mammals controls the sleep-wake cycle, blood pressure, hormone secretion, metabolism and many other physiological processes. The circadian clock mechanism is regulated by four genes: Bmal1, Clock, Cry, and Per. Mutations in these regulatory genes are associated with sleep and mood disorders, obesity, and cancer. Several PER2 and CRY2 SNPs are associated with advanced sleep phase syndrome. It is, therefore, critical to understand the effect of clock genes’ SNPs on the circadian clock. In this study, we determined “pathogenic” BMAL1 and CLOCK SNPs in the Ensembl database for biochemical characterization. Materials and Methods: BMAL1 and CLOCK SNPs in the Ensemble database were filtered out for only missense mutations. Among the missense mutations, pathogenic ones were determined according to SIFT, PolyPhen, and CADD scores, REVEL, MetalR, Mutation Assessor, I-Mutant, PROVEAN, and FireDock programs. BMAL1 and CLOCK SNPs were visualized by using PyMol. Results: Thousands of BMAL1 and CLOCK missense SNP mutations were reported in the Ensembl database. After the classification of those SNPs according to their SIFT, PolyPhen, and CADD pathogenicity, twelve SNPs for each protein remained as pathogenic. A further analysis with all in silico tools revealed that BMAL1 SNPs causing Ala154Val, Arg166Gln, and Val440Gly mutations; and CLOCK SNPs causing Gly120Val, Asp119Val, Gly120Ser, Ala117Val, and Cys371Gly mutations were predicted as the most “pathogenic” ones. Conclusion: Overall, by using in silico tools, we provided a starting point for experimental studies for determining the effect of pathogenic BMAL1 and CLOCK SNPs on the circadian clock mechanism.

Ornithine transcarbamylase deficiency (OTCD) is the most common X-linked hereditary disorder of urea cycle disorders that is caused by neonatal hyperammonemia. OTC gene sequence variations are common causes of OTCD. The current study presents a 28-month-old baby girl proband with phenotypical characteristics of OTCD such as irritability, somnolence, intermittent vomiting, and high levels of serum ammonium. Whole-exome sequencing revealed a de novo c.275G>A p.(Arg92Gln) variant within the OTC gene. In silico analysis revealed a possible differential affinity between wild-type and mutant OTCase, while Arg92Gln decreases the binding ability of OTCase to the substrate, which can disrupt the urea cycle and explains the molecular pathogenicity of clinical hyperammonemia. In light of the fact that the genotype and phenotype correlation of OTCD is still uncertain, the present in silico analysis outcome can enhance our knowledge on this complicated, rare, and severe genetic disorder.

The circadian rhythms are physiological, biochemical, and behavioral oscillations that cycle every 24 hours to anticipate the daily changes in the external environment. Disruption of the circadian clock in mammals results in increased susceptibility to different types of diseases such as metabolic, mood and sleep disorders and cancer. To this end, different approaches have been taken to find small molecules that have the potential to correct the disrupted circadian clock. In this review, we highlight the recent developments in identifying novel molecules that regulate the activities of the core clock proteins. Finally, we discuss virtual screening-based methods using the crystal structures of core clock proteins for the discovery of small molecules that regulate the circadian rhythm.

Heterogeneity in symptoms associated with COVID-19 in infected patients remains unclear. ACE2 and TMPRSS2 gene variants are considered possible risk factors for COVID-19. In this study, a retrospective comparative genome analysis of the ACE2 and TMPRSS2 variants from 946 whole-exome sequencing data was conducted. Allele frequencies of all variants were calculated and filtered to remove variants with allele frequencies lower than 0.003 and to prioritize functional coding variants. The majority of detected variants were intronic, only two ACE2 and three TMPRSS2 nonsynonymous variants were detected in the analyzed cohort. The main ACE2 variants that putatively have a protective or susceptibility effect on SARS-CoV-2 have not yet been determined in the Turkish population. The Turkish genetic makeup likely lacks any ACE2 variant that increases susceptibility to SARS-CoV-2 infection. TMPRSS2 rs75603675 and rs12329760 variants that were previously defined as common variants that have different allele frequencies among populations and may have a role in SARS-CoV-2 attachment to host cells were determined in the population. Overall, these data will contribute to the formation of a national variation database and may also contribute to further studies of ACE2 and TMPRSS2 in the Turkish population and differences in SARS-CoV-2 infection among other populations.

Birt-Hogg-Dube syndrome (BHDS) is a rare disorder characterized by the triad of cutaneous lesions, renal tumors, lung cysts and inactivation of the gene folliculin (FLCN). Here, we present three female patients diagnosed with BHDS. First case a 55-year-old female had flesh moles histopathology compatible with angiofibroma, multiple cysts in the lung and kidneys, FLCN gene mutations ('c.1285dupC [p.His429Profs*]' 11th exon and 'c.653G>A [p.Arg258His]' 7th exon). The second case a 76-year-old female had trichodiscoma on her skin, multiple cysts in the lung, spontaneous pneumothorax, FLCN gene mutation 'c.1285dupC (p.His429Profs*27) 11th exon' and, her son had renal carcinoma history under 50 years of age. Our third case, also the daughter of case 2, had dermal papules histopathology compatible with trichodiscoma, spontaneous pneumothorax, FLCN gene mutation 'c.1285dupC (p.His429Profs*27) 11th exon' and, parotid oncocytoma. Through our cases, we document the first case of two mutations ('c.1285dupC [p.His429Profs*]' 11th exon and 'c.653G>A [p.Arg258His]' 7th exon) in the same FLCN gene and the 11th known case of parotid oncocytoma associated with BHDS in the light of the literature.

Circadian rhythms are part of the body's clock, which regulates several physiological and biochemical variables according to the 24-h cycle. Ample evidence indicated disturbance of the circadian clock leads to an increased susceptibility to several diseases. Therefore, a great effort has been made to find small molecules that regulate circadian rhythm by high-throughput methods. Having crystal structures of core clock proteins, makes them amenable to structure-based drug design studies. Here, we describe virtual screening methods that can be utilized for the identification of small molecules regulating the activity of core clock protein Cryptochrome 1.