Lysine methyltransferases are important regulators of epigenetic signaling and are emerging as a novel drug target for drug discovery. This work demonstrates the positioning of novel 1,5-oxaza spiroquinone scaffold into selective SET and MYND domain-containing proteins 2 methyltransferases inhibitors. Selectivity of the scaffold was identified by epigenetic target screening followed by SAR study for the scaffold. The optimization was performed iteratively by two-step optimization consisting of iterative synthesis and computational studies (docking, metadynamics simulations). Computational binding studies guided the important interactions of the spiro[5.5]undeca scaffold in pocket 1 and Lysine channel and suggested extension of tail length for the improvement of potency (IC50: up to 399 nM). The effective performance of cell proliferation assay for chosen compounds (IC50: up to 11.9 nM) led to further evaluation in xenograft assay. The potent compound 24 demonstrated desirable in vivo efficacy with growth inhibition rate of 77.7% (4 fold decrease of tumor weight and 3 fold decrease of tumor volume). Moreover, mirosomal assay and pharmacokinetic profile suggested further developability of this scaffold through the identification of major metabolites (dealkylation at silyl group, reversible hydration product, the absence of toxic quinone fragments) and enough exposure of the testing compound 24 in plasma. Such spiro[5.5]undeca framework or ring system was neither been reported nor suggested as a modulator of methyltransferases. The chemo-centric target positioning and structural novelty can lead to potential pharmacological benefit.

F-box protein genes have been shown to play vital roles in plant development and stress respones. In Arabidopsis, there are more than 600 F-box proteins, and most of their functions are unclear. The present study shows that the F-box (SKP1-Cullin/CDC53-F-box) gene At5g15710 (Salt and Drought Responsiveness, SDR) is involved in abiotic stress responses in Arabidopsis. SDR is expressed in all tissues of Arabidopsis and is upregulated by salt and heat stresses and ABA treatment but downregulated by drought stress. Subcellular localization analysis shows that the SDR protein colocalizes with the nucleus. 35S:AntiSDR plants are hypersensitive to salt stress, but 35S:SDR plants display a salt-tolerant phenotype. Furthermore, 35S:SDR plants are hypersensitive to drought stress, while 35S:AntiSDR plants are significantly more drought tolerant. Overall, our results suggest that SDR is involved in salt and drought stress responses in Arabidopsis.

Bupleurum chinense DC has a history of using herb in China for more than 2000 years, which can be traced back to the Classic of Shennong Materia Medica in the Han Dynasty. Although Saikosaponin, the main active ingredient of Bupleurum, has the effects of anti-tumor, yet we still do not know the mechanism by total Bupleurum saponin extracts (TBSE) produces this effect on colon cancer.

Osteosarcoma is a prevalent type of bone tumor in children and adolescents, with limited treatment and poor prognosis. Abemaciclib, an inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6), is approved for the treatment of advanced breast cancer as single agent therapy and is currently under investigation in clinical trials for the treatment of several solid tumors.

Because spermatogonia transmit genetic information across generations, their DNA must be protected from environmental damages, including exposure to zinc oxide nanoparticles (ZnO NPs), which are frequently used in modern technology. Here, we used an in vitro system enriched for spermatogonia and exposed them to 10 and 20 μg/ml ZnO NPs for one/seven days. We did not detect any significant cell death, chromosomal instability, or DNA fragmentation in the spermatogonia treated with the ZnO NPs following one-day treatment with 10 or 20 μg/ml ZnO NPs. However, ZnO NPs (both 10 and 20 μg/ml) induced chromosomal instability in the spermatogonia after seven days of treatment. Moreover, one-day exposure to these NPs induced reactive oxygen species (ROS) generation and upregulation of apoptotic pathway-related genes p53, Caspase3 and Il6, as an inflammatory factor. Taken together, our study provides preliminary evidence for possible damages induced by low concentrations of ZnO NPs in spermatogonia. We should pay increased attention when using these NPs because of the silent damages in spermatogonia that can be transmitted to the next generation and cause severe effects. However, more data and validation of these results are required to determine the extent of this concern.

Non-alcoholic fatty liver disease (NAFLD) was a world-wide health burden. H3K27 acetylation, long non-coding RNA (lncRNA), and miRNA were all implicated in NAFLD regulation, yet the detailed regulatory mechanism was not well understood. LncRNA NEAT1, miR-212-5p, and GRIA3 expression were detected both in high fatty acid-treated hepatocytes cells and NAFLD patients. Lipid droplets were stained and analyzed by oil red O staining. Expression of fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC), and GRIA3 was detected by qRT-PCR and western blot. RNA level of lncRNA NEAT1 and miR-212-5p was analyzed by qRT-PCR. The binding sequences of lncRNA NEAT1/miR-212-5p and miR-212-5p/GRIA3 were predicted bioinformatically and validated through luciferase assay. ChIP was performed to analyze H3K27 acetylation on the promoter of lncRNA NEAT1. LncRNA NEAT1 and GRIA3 was upregulated, while miR-212-5p was downregulated in NAFLD patients. FFA promoted lncRNA NEAT1 and GRIA3 expression while suppressing miR-212-5p and promoted lipid accumulation as indicated by increased oil red O staining and FAS and ACC expression. ChIP indicated enrichment of H3K27 on NEAT1 promoter. Inhibition of H3K27 acetylation suppressed lncRNA NEAT1 level. Luciferase results indicated direct interaction of NEAT1/miR-212-5p (which was confirmed by RIP) and miR-212-5p/GRIA3. LncRNA NEAT1 knockdown upregulated miR-212-5p level and inhibited FFA-induced lipid accumulation while suppressing GRIA3 expression. Such function was antagonized by miR-212-5p inhibition and GRIA3 knockdown counteracted with miR-212-5p inhibition. H3K27 acetylation was enriched within the promoter of lncRNA NEAT1 and promoted lncRNA NEAT1 transcription. LncRNA NEAT1 could then interact with miR-212-5p and suppress its cellular concentration.

Chronotherapy, a promising therapy, may build up the chemotherapy efficacy through thinking about timing of therapy. Here, we observed the roles of period circadian regulator 2 (PER2) on cervical cancer progression and the therapeutic efficacy of cisplatin (DDP) based on the circadian rhythm of PER2.

Pulses of the steroid hormone ecdysone act through transcriptional cascades to direct the major developmental transitions during the Drosophila life cycle. These include the prepupal ecdysone pulse, which occurs 10 ​hours after pupariation and triggers the onset of adult morphogenesis and larval tissue destruction. E93 encodes a transcription factor that is specifically induced by the prepupal pulse of ecdysone, supporting a model proposed by earlier work that it specifies the onset of adult development. Although a number of studies have addressed these functions for E93, little is known about its roles in the salivary gland where the E93 locus was originally identified. Here we show that E93 is required for development through late pupal stages, with mutants displaying defects in adult differentiation and no detectable effect on the destruction of larval salivary glands. RNA-seq analysis demonstrates that E93 regulates genes involved in development and morphogenesis in the salivary glands, but has little effect on cell death gene expression. We also show that E93 is required to direct the proper timing of ecdysone-regulated gene expression in salivary glands, and that it suppresses earlier transcriptional programs that occur during larval and prepupal stages. These studies support the model that the stage-specific induction of E93 in late prepupae provides a critical signal that defines the end of larval development and the onset of adult differentiation.

Pheochromocytomas (PCCs) are rare neuroendocrine tumors derived from adrenal medulla chromaffin cells. Malignancy and recurrence are rare but demand effective treatment. Metformin exerts antiproliferative effects in several cancer cell lines. We thus evaluated the effects of metformin on cell viability and proliferation, cellular respiration and AMPK-AKT-mTOR-HIFA proliferation pathway on a rat PCC cell line (PC12-Adh). We then addressed metformin's effects on the AMPK-AKT-mTOR-HIFA pathway on two human primary cultures: one from a VHL-mutant PCC and other from a sporadic PCC. Metformin (20 mM) inhibited PC12-Adh cell proliferation, and decreased oxygen consumption, ATP production and proton leak, in addition to loss of mitochondrial membrane potential. Further, metformin induced AMPK phosphorylation and impaired AMPK-PI3k-AKT-mTOR pathway activation. The mTOR pathway was also inhibited in human VHL-related PCC cells, however, in an AMPK-independent manner. Metformin-induced decrease of HIF1A levels was likely mediated by proteasomal degradation. Altogether our results suggest that metformin impairs PCC cellular proliferation.

Heat shock proteins (HSPs) are available and/or induced for the survival of all organisms, including eukaryotic, prokaryotic, and plants, from higher temperature stresses. They are the chaperone proteins that protect all cells against heat, as the name implies. In addition to thermal stress, they also protect them from chemical, physical, and other stresses, including exposure to oxidative stress, nutritional deficiencies, ultraviolet radiation, ethanol, viral infection, ischemia-reperfusion injury, and cancer-related stresses. They are classified based on their molecular weights in kDa, such as HSP90 and HSP70. In our label-free, high-throughput, quantitative LC-MS/MS-based proteomic studies of MDA-MB-231, human, triple-negative breast cancer cells, treated with electrical pulses (EP) and cisplatin (CsP), we identified a number of HSPs, such as HSP90AA1, and others to be significantly downregulated in EP + CsP, compared to CsP alone. This indicates that cells will undergo apoptotic cell death and hence could cause effective cancer cure/treatment. Considering that over 2 million new cases and over 600,000 deaths in 2020, of which ~ 15% are TNBC, heat shock proteins could be the untapped resources, available for the next biomarkers and/or inhibitors for new/additional therapies.

Severe pneumonia in children is a group of inflammatory diseases of respiratory tract caused by pathogenic microorganisms. Increasing evidence suggested the crucial effects of microRNA on inflammatory diseases. This study aimed to reveal the expression and role of miR-483-3p in the serum of children with severe pneumonia, and to explore the effect of miR-483-3p on the biological function of lipopolysaccharide (LPS)-induced MRC-5 cells. MRC-5 cells were disposed with LPS to construct an in vitro pneumonia cell model. The relative expression level of miR-483-3p was measured by qRT-PCR. ROC curve was used to evaluate the diagnostic value of miR-483-3p in severe pneumonia. The Kaplan-Meier curve was performed to test the characteristics of survival distribution of different miRNA classifications. Cell viability and apoptosis were performed by CCK-8 assay and flow cytometry. IL-1β, TNF-α, and IL-6 were detected by ELISA. Luciferase reporter gene assay and western blot analysis were performed to detect the interaction between miR-483-3p and IGF-1. The expression of serum miR-483-3p in severe pneumonia patients was higher than in controls. The AUC value of the ROC curve was 0.919, indicating that miR-483-3p had diagnostic value for severe pneumonia. The survival curve showed that patients with high expression of miR-483-3p had higher mortality. Cell viability and apoptosis assay showed that overexpression of miR-483-3p suppressed cell proliferation and promoted apoptosis. And upregulation of miR-483-3p promoted generation of inflammatory cytokines. Luciferase report gene assay and western blot assay both illustrated that IGF-1 might be the target gene of miR-483-3p. Serum miR-483-3p can be used as a biomarker for the diagnosis of severe pneumonia. High expression of miR-483-3p promoted the development of severe pneumonia.

In prostate cancer, calcium homeostasis plays a significant role in the disease's development and progression. Intracellular calcium changes are an important secondary signal, triggered by a variety of extracellular stimuli, that controls many cellular functions. One of the main events affecting calcium is androgen signaling. Indeed, via calcium changes, androgens regulate cell processes like cell growth, differentiation and motility. In the present work we explored the nature of the receptor involved in calcium response induced by membrane-acting testosterone in prostate cancer cells. We report that testosterone, independently of the presence of the classical androgen receptor, can rapidly increase intracellular calcium from calcium stores, through the oxoeicosanoid receptor 1 (OXER1) and a specific signaling cascade that triggers calcium release from the endoplasmic reticulum. These findings reveal for the first time the receptor involved in the rapid calcium changes induced by androgens. Moreover, they further support the notion that androgens, even in the absence of AR, can still exert specific effects that regulate cancer cell fate.

The protective mechanisms of blood-brain barrier (BBB) prohibiting entry of pathogens into central nervous system (CNS) are critical for maintenance of brain homeostasis. These include various intracellular defense mechanisms that are vital to block transcytosis of neurotropic pathogens into the CNS. However, mechanistic details of coordination between these defense pathways remain unexplored. In this study, we established that BBB-driven ubiquitination acts as a major intracellular defense mechanism for clearance of Streptococcus pneumoniae, a critical neurotropic pathogen, during transit through BBB. Our findings suggest that the BBB employs differential ubiquitination with either K48- or K63-ubiquitin (Ub) chain topologies as an effective strategy to target S. pneumoniae toward diverse killing pathways. While K63-Ub decoration triggers autophagic killing, K48-Ub directs S. pneumoniae exclusively toward proteasomes. Time-lapse fluorescence imaging involving proteasomal marker LMP2 revealed that in the BBB, the majority of the ubiquitinated S. pneumoniae was cleared by proteasome. Fittingly, inhibition of proteasome and autophagy pathway led to accumulation of K48-Ub- and K63-Ub-marked S. pneumoniae, respectively, and triggered significant increases in intracellular S. pneumoniae burden. Moreover, genetic impairment of either K48- or K63-Ub chain formation demonstrated that although both chain types are key in disposal of intracellular S. pneumoniae, K48-Ub chains and subsequent proteasomal degradation have more pronounced contributions to intracellular S. pneumoniae killing in the BBB. Collectively, these observations, for the first time, illustrated a pivotal role of differential ubiquitination deployed by BBB in orchestrating a symphony of intracellular defense mechanisms for interception and degradation of S. pneumoniae, blocking its entry into the brain, which could be exploited to prevent bacterial CNS infections. IMPORTANCE The blood-brain barrier (BBB) represents a unique cellular barrier that provides structural integrity and protection to the CNS from pathogen invasion. Recently, ubiquitination, which is key for cellular homeostasis, was shown to be involved in pathogen clearance. In this study, we deciphered that the BBB deploys differential ubiquitination as an effective strategy to prevent S. pneumoniae trafficking into the brain. The different ubiquitin chain topologies formed on S. pneumoniae dictated the selection of downstream degradative pathways, namely, autophagy and proteasomes, among which the contribution of the proteasomal system in S. pneumoniae killing is more pronounced. Overall our study revealed how the BBB deploys differential ubiquitination as a strategy for synchronization of various intracellular defense pathways, which work in tandem to ensure the brain's identity as an immunologically privileged site.

Cells that cannot synthesize one of the DNA precursors, dTTP, due to thyA mutation or metabolic poisoning, undergo thymineless death (TLD), a chromosome-based phenomenon of unclear mechanisms. In Escherichia coli, thymineless death is caused either by denying thyA mutants thymidine supplementation or by treating wild-type cells with trimethoprim. Two recent reports promised a potential breakthrough in TLD understanding, suggesting significant oxidative damage during thymine starvation. Oxidative damage in vivo comes from Fenton's reaction when hydrogen peroxide meets ferrous iron to produce hydroxyl radical. Therefore, TLD could kill via irreparable double-strand breaks behind replication forks when starvation-caused single-strand DNA gaps are attacked by hydroxyl radicals. We tested the proposed Fenton-TLD connection in both thyA mutants denied thymidine, as well as in trimethoprim-treated wild-type (WT) cells, under the following three conditions: (i) intracellular iron chelation, (ii) mutational inactivation of hydrogen peroxide (HP) scavenging, and (iii) acute treatment with sublethal HP concentrations. We found that TLD kinetics are affected by neither iron chelation nor HP stabilization in cultures, indicating no induction of oxidative damage during thymine starvation. Moreover, acute exogenous HP treatments completely block TLD, apparently by blocking cell division, which may be a novel TLD prerequisite. Separately, the acute trimethoprim sensitivity of the rffC and recBCD mutants demonstrates how bactericidal power of this antibiotic could be amplified by inhibiting the corresponding enzymes. IMPORTANCE Mysterious thymineless death strikes cells that are starved for thymine and therefore replicating their chromosomal DNA without dTTP. After 67 years of experiments testing various obvious and not so obvious explanations, thymineless death is still without a mechanism. Recently, oxidative damage via in vivo Fenton's reaction was proposed as a critical contributor to the irreparable chromosome damage during thymine starvation. We have tested this idea by either blocking in vivo Fenton's reaction (expecting no thymineless death) or by amplifying oxidative damage (expecting hyperthymineless death). Instead, we found that blocking Fenton's reaction has no influence on thymineless death, while amplifying oxidative damage prevents thymineless death altogether. Thus, oxidative damage does not contribute to thymineless death, while the latter remains enigmatic.

Lycium barbarum L., a classical traditional Chinese Medicine, has long been used to treat ocular diseases. Lycium barbarum polysaccharides (LBP) is an effective component of Lycium barbarum L. with a wide range of pharmacological activities. This research aims to investigate the inhibition of high glucose-induced angiogenesis by LBP in RF/6A cells.

Zhuyu pill (ZYP), an effective prescription of traditional Chinese medicine, is composed of Coptis chinensis Franch. and Tetradium ruticarpum (A. Jussieu) T. G. Hartley and has shown potential anticholestatic effects. However, its mechanism of action in treating cholestasis remains unclear. Since post-transcriptional control of mRNA by micro-RNAs (miRNAs) represents an important mechanism of gene regulation, it is promising to explore this in relation to ZYP and cholestasis.

To characterise splicing machinery (SM) alterations in leucocytes of patients with rheumatoid arthritis (RA), and to assess its influence on their clinical profile and therapeutic response.

Bisphenol A (BPA) is an endocrine disrupting chemical that promotes obesity. It acts on the hypothalamus by increasing expression of the orexigenic neuropeptides, Npy and Agrp. Exactly how BPA dysregulates energy homeostasis is not completely clear. Since microRNAs (miRNA) have emerged as crucial weight regulators, the question of whether BPA could alter hypothalamic miRNA profiles was examined. Treatment of the mHypoA-59 cell line with 100 μM BPA altered a specific subset of miRNAs, and the most upregulated was miR-708-5p. BPA was found to increase the levels of miR-708-5p, and its parent gene Odz4, through the ER stress-related protein Chop. Overexpression of an miR-708-5p mimic resulted in a reduction of neuronatin, a proteolipid whose loss of expression is associated with obesity, and an increase in orexigenic Npy expression, thus potentially increasing feeding through converging regulatory pathways. Therefore, hypothalamic exposure to BPA can increase miR-708-5p that controls neuropeptides directly linked to obesity.

Burkitt lymphoma (BL) is an aggressive lymphoma type that is currently treated by intensive chemoimmunotherapy. Despite the favorable clinical outcome for most patients with BL, chemotherapy-related toxicity and disease relapse remain major clinical challenges, emphasizing the need for innovative therapies. Using genome-scale CRISPR-Cas9 screens, we identified B-cell receptor (BCR) signaling, specific transcriptional regulators, and one-carbon metabolism as vulnerabilities in BL. We focused on serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in one-carbon metabolism. Inhibition of SHMT2 by either knockdown or pharmacological compounds induced anti-BL effects in vitro and in vivo. Mechanistically, SHMT2 inhibition led to a significant reduction of intracellular glycine and formate levels, which inhibited the mTOR pathway and thereby triggered autophagic degradation of the oncogenic transcription factor TCF3. Consequently, this led to a collapse of tonic BCR signaling, which is controlled by TCF3 and is essential for BL cell survival. In terms of clinical translation, we also identified drugs such as methotrexate that synergized with SHMT inhibitors. Overall, our study has uncovered the dependency landscape in BL, identified and validated SHMT2 as a drug target, and revealed a mechanistic link between SHMT2 and the transcriptional master regulator TCF3, opening up new perspectives for innovative therapies.

Small molecules that present complex absorption, distribution, metabolism, and elimination (ADME) properties can be challenging to investigate as potential therapeutics. Acquiring data through standard methods can yield results that are insufficient to describe the in vivo situation, which can affect downstream development decisions. Implementing in vitro-in vivo-in silico strategies throughout the drug development process is effective in identifying and mitigating risks while speeding up their development. Risdiplam (Evrysdi)-an orally bioavailable, small molecule approved by the US Food and Drug Administration and more recently by the European Medicines Agency for the treatment of patients ≥2 months of age with spinal muscular atrophy-is presented here as a case study. Risdiplam is a low-turnover compound whose metabolism is mediated through a non-cytochrome P450 enzymatic pathway. Four main challenges of risdiplam are discussed: predicting in vivo hepatic clearance, determining in vitro metabolites with regard to metabolites in safety testing guidelines, elucidating enzymes responsible for clearance, and estimating potential drug-drug interactions. A combination of in vitro and in vivo results was successfully extrapolated and used to develop a robust physiologically based pharmacokinetic model of risdiplam. These results were verified through early clinical studies, further strengthening the understanding of the ADME properties of risdiplam in humans. These approaches can be applied to other compounds with similar ADME profiles, which may be difficult to investigate using standard methods. SIGNIFICANCE STATEMENT: Risdiplam is the first approved, small-molecule, survival of motor neuron 2 mRNA splicing modifier for the treatment of spinal muscular atrophy. The approach taken to characterize the absorption, distribution, metabolism, and excretion (ADME) properties of risdiplam during clinical development incorporated in vitro-in vivo-in silico techniques, which may be applicable to other small molecules with challenging ADME. These strategies may be useful in improving the speed at which future drug molecules can be developed.