Global and local functions of the Fused kinase ortholog CdaH in intracellular patterning in Tetrahymena.

Ciliates assemble numerous microtubular structures into complex cortical patterns. During ciliate division, the pattern is duplicated by intracellular segmentation that produces a tandem of daughter cells. In Tetrahymena thermophila, the induction and positioning of the division boundary involves two mutually antagonistic factors: posterior CdaA (cyclin E) and anterior CdaI (Hippo kinase). Here, we characterized the related cdaH-1 allele, which confers a pleiotropic patterning phenotype including an absence of the division boundary and an anterior-posterior mispositioning of the new oral apparatus. CdaH is a Fused or Stk36 kinase ortholog that localizes to multiple sites that correlate with the effects of its loss, including the division boundary and the new oral apparatus. CdaH acts downstream of CdaA to induce the division boundary and drives asymmetric cytokinesis at the tip of the posterior daughter. CdaH both maintains the anterior-posterior position of the new oral apparatus and interacts with CdaI to pattern ciliary rows within the oral apparatus. Thus, CdaH acts at multiple scales, from induction and positioning of structures on the cell-wide polarity axis to local organelle-level patterning.

A novel membrane complex is required for docking and regulated exocytosis of lysosome-related organelles in Tetrahymena thermophila.

In the ciliate Tetrahymena thermophila, lysosome-related organelles called mucocysts accumulate at the cell periphery where they secrete their contents in response to extracellular events, a phenomenon called regulated exocytosis. The molecular bases underlying regulated exocytosis have been extensively described in animals but it is not clear whether similar mechanisms exist in ciliates or their sister lineage, the Apicomplexan parasites, which together belong to the ecologically and medically important superphylum Alveolata. Beginning with a T. thermophila mutant in mucocyst exocytosis, we used a forward genetic approach to uncover MDL1 (Mucocyst Discharge with a LamG domain), a novel gene that is essential for regulated exocytosis of mucocysts. Mdl1p is a 40 kDa membrane glycoprotein that localizes to mucocysts, and specifically to a tip domain that contacts the plasma membrane when the mucocyst is docked. This sub-localization of Mdl1p, which occurs prior to docking, underscores a functional asymmetry in mucocysts that is strikingly similar to that of highly polarized secretory organelles in other Alveolates. A mis-sense mutation in the LamG domain results in mucocysts that dock but only undergo inefficient exocytosis. In contrast, complete knockout of MDL1 largely prevents mucocyst docking itself. Mdl1p is physically associated with 9 other proteins, all of them novel and largely restricted to Alveolates, and sedimentation analysis supports the idea that they form a large complex. Analysis of three other members of this putative complex, called MDD (for Mucocyst Docking and Discharge), shows that they also localize to mucocysts. Negative staining of purified MDD complexes revealed distinct particles with a central channel. Our results uncover a novel macromolecular complex whose subunits are conserved within alveolates but not in other lineages, that is essential for regulated exocytosis in T. thermophila.

Dual inhibitors of DNMT and HDAC remodels the immune microenvironment of colorectal cancer and enhances the efficacy of anti-PD-L1 therapy.

Colorectal cancer is the second most prevalent and deadly cancer worldwide. The emergence of immune checkpoint therapy has provided a revolutionary strategy for the treatment of solid tumors. However, less than 5 % of colorectal cancer patients respond to immune checkpoint therapy. Thus, it is of great scientific significance to develop "potentiators" for immune checkpoint therapy. In this study, we found that knocking down different DNMT and HDAC isoforms could increase the expression of IFNs in colorectal cancer cells, which can enhance the effectiveness of immune checkpoint therapy. Therefore, the combined inhibition of DNMT and HDAC cloud synergistically enhance the effect of immunotherapy. We found that dual DNMT and HDAC inhibitors C02S could inhibit tumor growth in immunocompetent mice but not in immunocompromised nude mice, which indicates that C02S exerts its antitumor effects through the immune system. Mechanistically, C02S could increase the expression of ERVs, which generated the intracellular levels of dsRNA in tumor cells, and then promotes the expression of IFNs through the RIG-I/MDA5-MAVS signaling pathway. Moreover, C02S increased the immune infiltration of DCs and T cells in microenvironment, and enhanced the efficacy of anti-PD-L1 therapy in MC38 and CT26 mice model. These results confirmed that C02S can activate IFNs through the RIG-I/MDA5-MAVS signaling pathway, remodel the tumor immune microenvironment and enhance the efficacy of immune checkpoint therapy, which provides new evidence and solutions for the development of "potentiator" for colorectal cancer immunotherapy.

Discovery of novel nucleoside derivatives as selective lysine acetyltransferase p300 inhibitors for cancer therapy.

P300 and CBP are two closely related histone acetyltransferases that are important transcriptional coactivators of many cellular processes. Inhibition of the transcriptional regulator p300/CBP is a promising therapeutic approach in oncology. However, there are no reported single selective p300 or CBP inhibitors to date. In this study, we designed and optimized a series of lysine acetyltransferase p300 selective inhibitors bearing a nucleoside scaffold. Most compounds showed excellent inhibitory activity against p300 with IC50 ranging from 0.18 to 9.90 µM, except for J16, J29, J40, and J48. None of the compounds showed inhibitory activity against CBP (inhibition rate < 50 % at 10 µM). Then the cytotoxicity of the compounds against a series of cancer cells were evaluated. Compounds J31 and J32 showed excellent proliferation inhibitory activity on cancer cells T47D and H520 with desirable selectivity profile of p300 over CBP. These compounds could be promising lead compounds for the development of novel epigenetic inhibitors as antitumor agents.

Design, synthesis, and evaluation of VHL-based EZH2 degraders for breast cancer.

EZH2 (enhancer of zeste homolog 2) is one of the most important histone methyltransferases (HMTs), and overexpression of EZH2 can lead to proliferation, migration and angiogenesis of tumor cells. But most of EZH2 inhibitors are only effective against some hematologic malignancies and have poor efficacy against solid tumors. Here, we report the design, synthesis, and evaluation of highly potent proteolysis targeting chimeric (PROTACs) small molecules targeting EZH2. We developed a potent and effective EZH2 degrader P4, which effectively induced EZH2 protein degradation and inhibited breast cancer cell growth. Further studies showed that P4 can significantly decrease the degree of H3K27me3 in MDA-MB-231 cell line, induce apoptosis and G0/G1 phase arrest in Pfeiffer and MDA-MB-231 cell lines. Therefore, P4 is a potential anticancer molecule for breast cancer treatment.

Droplet digital molecular beacon-LAMP assay via pico-injection for ultrasensitive detection of pathogens.

A pico-injection-aided digital droplet detection platform is presented that integrates loop-mediated isothermal amplification (LAMP) with molecular beacons (MBs) for the ultrasensitive and quantitative identification of pathogens, leveraging the sequence-specific detection capabilities of MBs. The microfluidic device contained three distinct functional units including droplet generation, pico-injection, and droplet counting. Utilizing a pico-injector, MBs are introduced into each droplet to specifically identify LAMP amplification products, thereby overcoming issues related to temperature incompatibility. Our methodology has been validated through the quantitative detection of Escherichia coli, achieving a detection limit as low as 9 copies/µL in a model plasmid containing the malB gene and 3 CFU/µL in a spiked milk sample. The total analysis time was less than 1.5 h. The sensitivity and robustness of this platform further demonstrated the potential for rapid pathogen detection and diagnosis, particularly when integrated with cutting-edge microfluidic technologies.

Whole-Bacterium SELEX Aptamer Selection of Fusobacterium nucleatum and Application to Colorectal Cancer Noninvasive Screening in Human Feces.

In terms of cancer diagnoses and cancer-related deaths worldwide, colorectal cancer (CRC) is now the third most common malignancy. The drawbacks of current screening methods are their exorbitant costs, difficult procedures, and lengthy implementation timelines. The benefits of fecal screening for CRC are ease of operation, noninvasiveness, cost-effectiveness, and superior sensitivity. As a result of its enrichment in the malignant tissues and feces of CRC patients, Fusobacterium nucleatum (F. nucleatum) has emerged as a crucial biomarker for the incipient detection, identification, and prognostic prediction of CRC. Here, for the first time, the whole-bacterium SELEX method was used to screen the highly specific and affinity aptamers against F. nucleatum by 13 cycles of selection. The Apt-S-5 linear correlation equation is y = 0.7363x2.8315 (R2 = 0.9864) with a limit of detection (LOD) of 851 CFU/mL. The results of the experiment using fecal samples revealed a substantial disparity between the microorganisms in the CRC patients' feces and those in the feces of healthy individuals and were consistent with those of qPCR. The aptamers may therefore offer a crucial approach to identifying F. nucleatum and hold tremendous promise for CRC diagnosis and prognostic prediction.

Rapid exosome isolation and in situ multiplexed detection of exosomal surface proteins and microRNAs on microfluidic platform.

Exosomes are considered as promising biomarkers for early cancer diagnosis and prognosis. However, the majority of the research studies focused on a single type of exosomal biomarkers, which cannot comprehensively reflect the state of cancer for accurate diagnosis. To address this problem, we presented a ship-shaped microfluidic device containing a microcolumn array for simultaneous in situ detection of exosomal surface proteins and miRNAs. Exosomes were first captured in the microchannels modified with CD63 protein aptamer. Exosomal surface proteins and miRNAs were simultaneously detected in four parallel channels to avoid the interference of fluorescent signals using specific aptamers labeled by Cy5 and catalytic hairpin assembly (CHA) based signal amplification strategy. The limit of detection for multiplexed markers in exosomes was 83 exosomes per µL, which is comparable to previously reported methods. Through quantitative analysis of two disease-specific surface proteins and miRNAs derived from different cancer cells and clinical serum samples, different cancer subtypes as well as cancer patients and healthy people could be significantly distinguished. These results suggest that this simple, highly sensitive, and more accurate analytical strategy by simultaneous in situ profiling of different types of exosomal biomarkers has potential applications in cancer diagnosis and stage monitoring.

Design, synthesis and biological evaluation of pyrimidine base hydroxamic acid derivatives as dual JMJD3 and HDAC inhibitors.

The Jumonji domain-containing protein demethylase 3 (JMJD3) and histone deacetylase (HADC) are related to various cancers and regard as antitumor targets for drug discovery. In this study, based on rational drug design strategy, we designed and synthesized a series of pyrimidine derivatives with hydroxamic acid as novel dual JMJD3 and HDAC inhibitors for synergistic cancer treatment. Compound A5b exhibited inhibitory potency against JMJD3 and HDAC1/6 simultaneously and favorable cytotoxicity against human cancer cells such as A549 and U937. Furthermore, mechanistic studies showed that A5b treatment in A549 cells increased the hypermethylation of histone H3K27 and hyperacetylation of H3K9, suppressed clonogenicity, migration and invasion of cancer cells. Besides, A5b induced apoptosis via the cleavage of caspase-7 and PARP, and G1 cell cycle arrest via upregulated p21 expression. All these results suggested that A5b was the first dual inhibitor against JMJD3 and HDAC and can be a potential compound for cancer therapy.

Antitumor Activity of s-Triazine Derivatives: A Systematic Review.

1,3,5-triazine derivatives, also called s-triazines, are a series of containing-nitrogen heterocyclic compounds that play an important role in anticancer drug design and development. To date, three s-triazine derivatives, including altretamine, gedatolisib, and enasidenib, have already been approved for refractory ovarian cancer, metastatic breast cancer, and leukemia therapy, respectively, demonstrating that the s-triazine core is a useful scaffold for the discovery of novel anticancer drugs. In this review, we mainly focus on s-triazines targeting topoisomerases, tyrosine kinases, phosphoinositide 3-kinases, NADP+-dependent isocitrate dehydrogenases, and cyclin-dependent kinases in diverse signaling pathways, which have been extensively studied. The medicinal chemistry of s-triazine derivatives as anticancer agents was summarized, including discovery, structure optimization, and biological applications. This review will provide a reference to inspire new and original discoveries.

Total Syntheses of Calyciphylline A-Type Alkaloids (-)-10-Deoxydaphnipaxianine A, (+)-Daphlongamine E and (+)-Calyciphylline R via Late-Stage Divinyl Carbinol Rearrangements.

Among the famous Daphniphyllum alkaloids family, the calyciphylline A-type subfamily has triggered particular interest from the organic synthesis community in recent years. Here, we report divergent total syntheses of three calyciphylline A-type alkaloids, namely, (-)-10-deoxydaphnipaxianine A, (+)-daphlongamine E, and (+)-calyciphylline R. Our work highlights an efficient, divergent strategy via late-stage divinyl carbinol rearrangements, including an unprecedented oxidative Nazarov electrocyclization using an unfunctionalized tertiary divinyl carbinol and an unusual allylic alcohol rearrangement. A highly efficient "donor-acceptor" platinum catalyst was used for a critical nitrile hydration step. Moreover, the power of selective amide reductions has also been showcased by novel and classic tactics.

Quinazoline-based hydroxamic acid derivatives as dual histone methylation and deacetylation inhibitors for potential anticancer agents.

Cancer is a common malignant disease with complex signaling networks, which means it is unmanageable to cancer therapy by using single classical targeted drug. Recently, dual- or multitarget drugs have emerged as a promising option for cancer therapies. Although many multifunctional compounds targeting HDAC have been validated, as far as we know, there is no molecule targeting GLP and HDAC synchronously. In the present work, we designed and synthesized a series of quinazoline-based hydroxamic acid derivatives as dual GLP and HDAC inhibitors. These hybrid compounds showed potent enzymatic inhibitory activities against GLP and HDAC1/6 with IC50 values in the nanomolar range of less than 190 nM. Furthermore, most of our compounds displayed significant broad spectrum cytotoxic activities apart from D3 and D8 against all the tested cancer cells with IC50 values less than 50 µM. D1, D6 and D7 showed more potent cytotoxic activities than D2, D4 and D5 in those cancer cells. Especially, compound D7 showed potent inhibitory potency activity against both GLP and HDAC1/6 with IC50 values of 1.3, 89, 13 nM. Besides, D7 exhibited the most potent antiproliferative activity against all the tested cancer cells. Further evaluations indicated that D7 could inhibit the methylation and deacetylation of H3K9 on protein level. Moreover, D7 could induce cancer cell apoptosis, G0/G1 cell cycle arrest, and partly block migration and invasion. All these thorough evaluations warranted D7 as a promising lead compound worth further optimization and development for cancer therapy.

LF4/MOK and a CDK-related kinase regulate the number and length of cilia in Tetrahymena.

The length of cilia is controlled by a poorly understood mechanism that involves members of the conserved RCK kinase group, and among them, the LF4/MOK kinases. The multiciliated protist model, Tetrahymena, carries two types of cilia (oral and locomotory) and the length of the locomotory cilia is dependent on their position with the cell. In Tetrahymena, loss of an LF4/MOK ortholog, LF4A, lengthened the locomotory cilia, but also reduced their number. Without LF4A, cilia assembled faster and showed signs of increased intraflagellar transport (IFT). Consistently, overproduced LF4A shortened cilia and downregulated IFT. GFP-tagged LF4A, expressed in the native locus and imaged by total internal reflection microscopy, was enriched at the basal bodies and distributed along the shafts of cilia. Within cilia, most LF4A-GFP particles were immobile and a few either diffused or moved by IFT. We suggest that the distribution of LF4/MOK along the cilium delivers a uniform dose of inhibition to IFT trains that travel from the base to the tip. In a longer cilium, the IFT machinery may experience a higher cumulative dose of inhibition by LF4/MOK. Thus, LF4/MOK activity could be a readout of cilium length that helps to balance the rate of IFT-driven assembly with the rate of disassembly at steady state. We used a forward genetic screen to identify a CDK-related kinase, CDKR1, whose loss-of-function suppressed the shortening of cilia caused by overexpression of LF4A, by reducing its kinase activity. Loss of CDKR1 alone lengthened both the locomotory and oral cilia. CDKR1 resembles other known ciliary CDK-related kinases: LF2 of Chlamydomonas, mammalian CCRK and DYF-18 of C. elegans, in lacking the cyclin-binding motif and acting upstream of RCKs. The new genetic tools we developed here for Tetrahymena have potential for further dissection of the principles of cilia length regulation in multiciliated cells.

Discovery of the PARP (poly ADP-ribose polymerase) inhibitor 2-(1-(4,4-difluorocyclohexyl)piperidin-4-yl)-1H-benzo[d]imidazole-4-carboxamide for the treatment of cancer.

In this work, two series of cyclic amine-containing benzimidazole carboxamide derivatives were designed and synthesized as potent anticancer agents. PARP1/2 inhibitory activity assays indicated that most of the compounds showed significant activity. The in vitro antiproliferative activity of these compounds was investigated against four human cancer cell lines (MDA-MB-436, MDA-MB-231, MCF-7 and CAPAN-1), and several compounds exhibited strong cytotoxicity to tumor cells. Among them, 2-(1-(4,4-difluorocyclohexyl)piperidin-4-yl)-1H-benzo[d]imidazole-4-carboxamide (17d) was found to be effective PARP1/2 inhibitors (IC50 = 4.30 and 1.58 nM, respectively). In addition, 17d possessed obvious selective antineoplastic activity and noteworthy microsomal metabolic stability. What's more, further studies revealed that 17d was endowed with an excellent ADME profile. These combined results indicated that 17d could be a promising candidate for the treatment of cancer.

Discovery of triazolo [1,5-a] pyridine derivatives as novel JAK1/2 inhibitors.

Small molecule JAK inhibitors have been demonstrated efficacy in rheumatoid arthritis, inflammatory bowel disease, and psoriasis with the approval of several drugs. Aiming to develop potent JAK1/2 inhibitors, two series of triazolo [1,5-a] pyridine derivatives were designed and synthesized by various strategies. The pharmacological results identified the optimized compounds J-4 and J-6, which exerted high potency against JAK1/2, and selectivity over JAK3 in enzyme assays. Furthermore, J-4 and J-6 effectively suppressed proliferation of JAK1/2 high-expression BaF3 cells accompanied with acceptable metabolic stability in liver microsomes. Therefore, J-4 and J-6 might serve as promising JAK1/2 inhibitors for further investigation.

NEAT1 regulates neuroglial cell mediating Aß clearance via the epigenetic regulation of endocytosis-related genes expression.

The accumulation of intracellular ß-amyloid peptide (Aß) is important pathological characteristic of Alzheimer's disease (AD). However, the exact underlying molecular mechanism remains to be elucidated. Here, we reported that Nuclear Paraspeckle Assembly Transcript 1 (NEAT1), a long n on-coding RNA, exhibits repressed expression in the early stage of AD and its down-regulation declines neuroglial cell mediating Aß clearance via inhibiting expression of endocytosis-related genes. We find that NEAT1 is associated with P300/CBP complex and its inhibition affects H3K27 acetylation (H3K27Ac) and H3K27 crotonylation (H3K27Cro) located nearby to the transcription start site of many genes, including endocytosis-related genes. Interestingly, NEAT1 inhibition down-regulates H3K27Ac but up-regulates H3K27Cro through repression of acetyl-CoA generation. NEAT1 also mediates the binding between STAT3 and H3K27Ac but not H3K27Cro. Therefore, the decrease of H3K27Ac and/or the increase of H3K27Cro declines expression of multiple related genes. Collectively, this study first reveals the different roles of H3K27Ac and H3K27Cro in regulation of gene expression and provides the insight of the epigenetic regulatory mechanism of NEAT1 in gene expression and AD pathology.

Therapeutic activity of DCC-2036, a novel tyrosine kinase inhibitor, against triple-negative breast cancer patient-derived xenografts by targeting AXL/MET.

Triple-negative breast cancer (TNBC) is insensitive to endocrine therapies and targeted therapies to human epidermal growth factor receptor-2 (HER2), estrogen receptor (ER) and progesterone receptor (PR). New targets and new targeted therapeutic drugs for TNBC are desperately needed. Our study confirmed that DCC-2036 inhibited the proliferation, invasion, migration and epithelial-mesenchymal transition (EMT) of TNBC cells as well as induced apoptosis. Moreover, the antiproliferative activity of DCC-2036 was more efficient than that of most clinical drugs. In addition, the combination of DCC-2036 and cisplatin or lapatinib had synergistic effects on TNBC cells. Mechanistically, DCC-2036 targeted AXL/MET, especially AXL, and regulated the downstream PI3K/Akt-NFκB signaling to exert its antitumor effect in TNBC. DCC-2036 also inhibited the growth and metastasis of xenografted MDA-MB-231 cells (AXL/MET-high TNBC cells) but not MDA-MB-468 cells (AXL-low TNBC cells) in NSG mice in vivo. Furthermore, DCC-2036 significantly inhibited tumor growth and invasion of AXL/MET-high TNBC PDX tumors but not AXL/MET-low TNBC PDX tumors. These results highlighted the roles of AXL/MET in cancer growth and metastasis and further verified that the critical targets of DCC-2036 are AXL and MET, especially AXL. In addition, there was no significant toxicity of DCC-2036 even at a high dosage. Therefore, DCC-2036 may be a potential compound to treat TNBC, especially for tumors with AXL/MET overexpression.

Isotope Labeling Strategies for Acylcarnitines Profile in Biological Samples by Liquid Chromatography-Mass Spectrometry.

Acylcarnitines are closely related to many metabolic diseases and likely to be good biomarkers for clinical diagnosis. Studies on acylcarnitines may help deepen the understanding of their functions associated with disease processes. However, the diversity of their structures and lack of commercial standards make them difficult to be fully detected. In this work, a highly efficient isotope labeling strategy was developed to detect acylcarnitines in biological samples using liquid chromatography-mass spectrometry (LC-MS). A pair of reagents with or without deuterium labeling tags containing both a positive charge and a primary amine group were synthesized, which were used to label the carboxyl group of acylcarnitines. The reaction was performed under mild conditions to avoid hydrolysis of acylcarnitines. The reaction products had two positive charges, forming a double charge peak in MS spectra and with mass-to-charge ratio difference (Δ m/ z) of 4.0251 Da between the light- and heavy-labeled products. The feature made it possible to distinguish signals of acylcarnitines from other carboxyl metabolites with Δ m/ z of 8.0502 Da between their light- and heavy-labeled products. On the basis of the characteristics and the tandem mass spectrometry (MS/MS) spectra, a total of 108 acylcarnitines were discovered and identified in urine samples. Our established approach will be very helpful for the studies of diseases associated with acylcarnitines metabolism.

A protein network descriptor server and its use in studying protein, disease, metabolic and drug targeted networks.

The genetic, proteomic, disease and pharmacological studies have generated rich data in protein interaction, disease regulation and drug activities useful for systems-level study of the biological, disease and drug therapeutic processes. These studies are facilitated by the established and the emerging computational methods. More recently, the network descriptors developed in other disciplines have become more increasingly used for studying the protein-protein, gene regulation, metabolic, disease networks. There is an inadequate coverage of these useful network features in the public web servers. We therefore introduced upto 313 literature-reported network descriptors in PROFEAT web server, for describing the topological, connectivity and complexity characteristics of undirected unweighted (uniform binding constants and molecular levels), undirected edge-weighted (varying binding constants), undirected node-weighted (varying molecular levels), undirected edge-node-weighted (varying binding constants and molecular levels) and directed unweighted (oriented process) networks. The usefulness of the PROFEAT computed network descriptors is illustrated by their literature-reported applications in studying the protein-protein, gene regulatory, gene co-expression, protein-drug and metabolic networks. PROFEAT is accessible free of charge at http://bidd2.nus.edu.sg/cgi-bin/profeat2016/main.cgi.

Recent advances in single cell manipulation and biochemical analysis on microfluidics.

Single cell analysis has become of great interest with unprecedented capabilities for the systematic investigation of cell-to-cell variation in large populations. Rapid and multi-parametric analysis of intercellular biomolecules at the single-cell level is imperative for the improvement of early disease diagnosis and personalized medicine. However, the small size of cells and the low concentration levels of target biomolecules are critical challenges for single cell analysis. In recent years, microfluidic platforms capable of handling small-volume fluid have been demonstrated to be powerful tools for single cell analysis. In addition, microfluidic techniques allow for precise control of the localized microenvironment, which yield more accurate outcomes. Many different microfluidic techniques have been greatly improved for highly efficient single-cell manipulation and highly sensitive detection over the past few decades. To date, microfluidics-based single cell analysis has become the hot research topic in this field. In this review, we particularly highlight the advances in this field during the past three years in the following three aspects: (1) microfluidic single cell manipulation based on microwells, micropatterns, droplets, traps and flow cytometric methods; (2) detection methods based on fluorescence, mass spectrometry, electrochemical, and polymerase chain reaction-based analysis; (3) applications in the fields of small molecule detection, protein analysis, multidrug resistance analysis, and single cell sequencing with droplet microfluidics. We also discuss future research opportunities by focusing on key performances of throughput, multiparametric target detection and data processing.