Dehydroamino acid residues in bioactive natural products.

Covering: 2000 to up to 2023α,ß-Dehydroamino acids (dhAAs) are unsaturated nonproteinogenic amino acids found in a wide array of naturally occurring peptidyl metabolites, predominantly those from bacteria. Other organisms, such as fungi, higher plants and marine invertebrates, have also been found to produce dhAA-containing peptides. The α,ß-unsaturation in dhAAs has profound effects on the properties of these molecules. They display significant synthetic flexibility, readily undergoing reactions such as Michael additions, transition-metal-catalysed cross-couplings, and cycloadditions. These residues in peptides/proteins also exhibit great potential in bioorthogonal applications using click chemistry. Peptides containing contiguous dhAA residues have been extensively investigated in the field of foldamers, self-assembling supermolecules that mimic biomacromolecules such as proteins to fold into well-defined conformations. dhAA residues in these peptidyl materials tend to form a 2.05-helix. As a result, stretches of dhAA residues arrange in an extended conformation. In particular, peptidyl foldamers containing ß-enamino acid units display interesting conformational, electronic, and supramolecular aggregation properties that can be modulated by light-dependent E-Z isomerization. Among approximately 40 dhAAs found in the natural product inventory, dehydroalanine (Dha) and dehydrobutyrine (Dhb) are the most abundant. Dha is the simplest dehydro-α-amino acid, or α-dhAA, without any geometrical isomers, while its re-arranged isomer, 3-aminoacrylic acid (Aaa or ΔßAla), is the simplest dehydro-ß-amino acid, or ß-enamino acid, and displays E/Z isomerism. Dhb is the simplest α-dhAA that exhibits E/Z isomerism. The Z-isomer of Dhb (Z-Dhb) is sterically favourable and is present in the majority of naturally occurring peptides containing Dhb residues. Dha and Z-Dhb motifs are commonly found in ribosomally synthesized and post-translationally modified peptides (RiPPs). In the last decade, the formation of Dha and Dhb motifs in RiPPs has been extensively investigated, which will be briefly discussed in this review. The formation of other dhAA residues in natural products (NPs) is, however, less understood. In this review, we will discuss recent advances in the biosynthesis of peptidyl NPs containing unusual dhAA residues and cryptic dhAA residues. The proposed biosynthetic pathways of these natural products will also be discussed.

Deciphering the molecular and functional basis of TMexCD1: the plasmid-encoded efflux pump of resistance-nodulation-division superfamily.

Horizontal gene transfer has been demonstrated to be an important driver for the emergency of multidrug-resistant pathogens. Recently, a transferable gene cluster tmexCD1-toprJ1 of the resistance-nodulation-division (RND) superfamily was identified in the plasmids of animal-derived Klebsiella pneumoniae strains, with a higher efflux capacity for various drugs than the Escherichia coli AcrAB-TolC homolog system. In this study, we focused on the differences in the inner membrane pump of these two systems and identified some key residues that contribute to the robust efflux activity of the TMexCD1 system. With the aid of homologous modeling and molecular docking, eight residues from the proximal binding pocket (PBP) and nine from the distal binding pocket (DBP) were selected and subjected to site-directed mutagenesis. Several of them, such as S134, I139, D181, and A290, were shown to be important for substrate binding in the DBP region, and all residues in PBP and DBP showed certain substrate preferences. Apart from the conservative switch loop (L613-623TMexD1) previously identified in the E. coli AcrB (EcAcrB), a relatively unconservative loop (L665-675TMexD1) at the bottom of PBP was proposed as a critical element for the robust activity of TMexD1, due to variations at sites E669, G670, N673, and S674 compared to EcAcrAB, and the significantly altered efflux activity due to their mutations. The conservation and flexibility of these key factors can contribute to the evolution of the RND efflux pumps and thus serve as potential targets for developing inhibitors to block the widespread of the TMexCD1 system.

A transmembrane scaffold from CD20 helps recombinant expression of a chimeric claudin 18.2 in an in vitro coupled transcription and translation system.

Cluster of differentiation 20 (CD20) is a nonglycosylated, multispanning transmembrane protein specifically integrated by B lymphocytes. Similar to CD20, another four-pass transmembrane protein, claudin 18.2, has attracted attention as an emerging therapeutic target for cancer. However, their poor solubility and toxic nature often hinder downstream applications, such as antibody drug development. Therefore, developing a cost-effective method for producing drug targets with multiple membrane-spanning domains is crucial. In this study, a high yield of recombinant CD20 was achieved through an E. coli-based in vitro coupled transcription-translation system. Surface plasmon resonance results showed that rituximab (an antileukemia drug) has nanomolar affinity with the CD20 protein, which aligns with published results. Notably, a previously hard-to-express claudin 18.2 recombinant protein was successfully expressed in the same reaction system by replacing its membrane-spanning domains with the transmembrane domains of CD20. The folding of the extracellular domain of the chimeric protein was verified using a commercial anti-claudin 18 antibody. This study provides a novel concept for promoting the expression of four-pass transmembrane proteins and lays the foundation for the large-scale industrial production of membrane-associated drug targets, similar to claudin 18.2.

Heterodimers of Aromadendrane Sesquiterpenoid with Benzoquinone from the Chinese Liverwort Mylia nuda.

Mylnudones A-G (1-7), unprecedented 1,10-seco-aromadendrane-benzoquinone-type heterodimers, and a highly rearranged aromadendrane-type sesquiterpenoid (8), along with four known analogs (9-12), were isolated from the liverwort Mylia nuda. Compounds 1-6 and 7, bearing tricyclo[6.2.1.02,7] undecane and tricyclo[5.3.1.02,6] undecane backbones, likely formed via a Diels-Alder reaction and radical cyclization, respectively. Their structures were determined by spectroscopic analysis, computational calculation, and single-crystal X-ray diffraction analysis. Dimeric compounds displayed cytoprotective effects against glutamic acid-induced neurological deficits.

Improved dsDNA recombineering enables versatile multiplex genome engineering of kilobase-scale sequences in diverse bacteria.

Recombineering assisted multiplex genome editing generally uses single-stranded oligonucleotides for site directed mutational changes. It has proven highly efficient for functional screens and to optimize microbial cell factories. However, this approach is limited to relatively small mutational changes. Here, we addressed the challenges involved in the use of double-stranded DNA substrates for multiplex genome engineering. Recombineering is mediated by phage single-strand annealing proteins annealing ssDNAs into the replication fork. We apply this insight to facilitate the generation of ssDNA from the dsDNA substrate and to alter the speed of replication by elevating the available deoxynucleoside triphosphate (dNTP) levels. Intracellular dNTP concentration was elevated by ribonucleotide reductase overexpression or dNTP addition to establish double-stranded DNA Recombineering-assisted Multiplex Genome Engineering (dReaMGE), which enables rapid and flexible insertional and deletional mutagenesis at multiple sites on kilobase scales in diverse bacteria without the generation of double-strand breaks or disturbance of the mismatch repair system. dReaMGE can achieve combinatorial genome engineering works, for example, alterations to multiple biosynthetic pathways, multiple promoter or gene insertions, variations of transcriptional regulator combinations, within a few days. dReaMGE adds to the repertoire of bacterial genome engineering to facilitate discovery, functional genomics, strain optimization and directed evolution of microbial cell factories.

Advances of antitumor drug discovery in traditional Chinese medicine and natural active products by using multi-active components combination.

The antitumor efficacy of Chinese herbal medicines has been widely recognized. Leading compounds such as sterols, glycosides, flavonoids, alkaloids, terpenoids, phenylpropanoids, and polyketides constitute their complex active components. The antitumor monomers derived from Chinese medicine possess an attractive anticancer activity. However, their use was limited by low bioavailability, significant toxicity, and side effects, hindering their clinical applications. Recently, new chemical entities have been designed and synthesized by combining natural drugs with other small drug molecules or active moieties to improve the antitumor activity and selectivity, and reduce side effects. Such a novel conjugated drug that can interact with several vital biological targets in cells may have a more significant or synergistic anticancer activity than a single-molecule drug. In addition, antitumor conjugates could be obtained by combining pharmacophores containing two or more known drugs or leading compounds. Based on these studies, the new drug research and development could be greatly shortened. This study reviews the research progress of conjugates with antitumor activity based on Chinese herbal medicine. It is expected to serve as a valuable reference to antitumor drug research and clinical application of traditional Chinese medicine.

Cell-free protein synthesis of CD1E and B2M protein and in vitro interaction.

CD1E, one of the most important glycolipid antigens on T cell membranes, is required for glycolipid antigen presentation on the cell surface. Cell-based recombinant expression systems have many limitations for synthesizing transmembrane proteins such as CD1E, including low protein yields and miss folding. To overcome these challenges, here we successfully synthesized high-quality soluble CD1E using an E.coli cell-free protein synthesis system (CFPS) with the aid of detergent. Following purification by Ni2+ affinity chromatography, we were able to obtain CD1E with ≥90% purity. Furthermore, we used the string website to predict the protein interaction network of CD1E and identified a potential binding partner━B2M. Similarly, we synthesized soluble B2M in the E.coli CFPS. Finally, we verified the interaction between CD1E and B2M by using Surface Plasmon Resonance (SPR). Taken together, the methods described here provide an alternative way to obtain active transmembrane protein and may facilitate future structural and functional studies on CD1E.

Construction of a quadruple gene-deleted vaccine confers complete protective immunity against emerging PRV variant challenge in piglets.

BACKGROUND: Pseudorabies virus (PRV) causes Aujeszky's disease or pseudorabies (PR) in pigs worldwide, which leads to heavy economic losses to the swine industry. Pigs are the natural host, meanwhile, animals such as dogs, cats, foxes, rabbits, cattle and sheep are susceptible to infection. In 2011, the emerging PRV variant led to the outbreak of PR in Bartha-K61 vaccinated pigs. The PR outbreaks demonstrated that the Bartha-K61 vaccine did not provide full protection against the emerging PRV variant. It is widely believed that PRV live attenuated vaccine could control PRV infection. METHODS: In this study, we developed a novel PRV live attenuated vaccine by deleting its gI, gE, US9, and US2 genes through CRISPR/Cas9, which was named PRV GDFS-delgI/gE/US9/US2. RESULTS: Safety experiments confirmed that PRV GDFS-delgI/gE/US9/US2 was safe for 5- to 7-day-old suckling piglets. Piglets immunized with the PRV GDFS-delgI/gE/US9/US2 vaccine did not produce PRV gE-specific antibodies but could generate PRV gB-specific antibodies and high neutralizing titers against the PRV GDFS strain (variant PRV strain) or PRV Ea strain (older PRV strain). After challenge with the emerging PRV GDFS variant, none of the piglets immunized with the PRV GDFS-delgI/gE/US9/US2 vaccine showed any clinical signs, and their rectal temperatures were normal. Moreover, the autopsy and histopathological analyses revealed that the piglets in the PRV GDFS-delgI/gE/US9/US2 vaccine group did not show apparent gross or pathological lesions. Furthermore, the piglets in the PRV GDFS-delgI/gE/US9/US2 vaccine groups did not present weight loss. According to the criteria of the OIE terrestrial manual, the results of the experiment confirmed that the PRV GDFS-delgI/gE/US9/US2 vaccine could provide full protection against the emerging PRV variant strain in piglets. CONCLUSIONS: The PRV GDFS-delgI/gE/US9/US2 strain is a potential new live attenuated vaccine against emerging PRV variant strain infections in China.

Hyaluronic Acid-Modified Nanoparticles Self-Assembled from Linoleic Acid-Conjugated Chitosan for the Codelivery of miR34a and Doxorubicin in Resistant Breast Cancer.

In this study, a chitosan-based, self-assembled nanosystem that codelivered microRNA34a (miR34a) and doxorubicin (Dox) with hyaluronic acid (HA) modification (named CCmDH NPs) was developed to reverse the resistance of breast cancer (BCa) cells to Dox. The CCmDH NPs had a diameter of 180 ± 8.3 nm and a ζ potential of 16.5 mV with a slow-release effect for 96 h. The codelivery system could protect miR34a from nuclease and serum degradation and transport miR34a and Dox into drug-resistant MCF-7/A cells. In addition, the CCmDH NPs could inhibit proliferation and promote apoptosis by regulating the protein expression of B-cell lymphoma-2 (Bcl-2) and poly(ADP-ribose) polymerase (PARP) and inhibit invasion, metastasis, and adhesion by regulating E-cadherin, N-cadherin, MMP2, CD44, and Snail molecules. The CCmDH NPs induced a 73.7% tumor reduction in xenograft tumor growth in nude mice in vivo. This study provides evidence for the anticancer activity of CCmDH NPs carrying Dox and miR34a in BCa, especially metastatic Dox-resistant BCa models.

Visual detection of porcine epidemic diarrhea virus by recombinase polymerase amplification combined with lateral flow dipstrip.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) is one of the most important enteric viruses causing diarrhea in pigs. The establishment of a rapid detection method applicable in field conditions will be conducive to early detection of pathogen and implementation of relevant treatment. A novel nucleic acid amplification method, recombinase polymerase amplification (RPA), has been widely used for infectious disease diagnosis. RESULTS: In the present study, a reverse transcription (RT)-RPA assay combined with lateral flow dipstrip (LFD) was established for the visual detection of PEDV by targeting the N gene. The RT-RPA-LFD assay detected as low as 102 copies/µL of PEDV genomic RNA standard. Moreover, the novel RT-RPA-LFD assay did not show cross-reactivity with common swine pathogens, demonstrating high specificity. The performance of the assay for detection of clinical samples was also evaluated. A total number of 86 clinical samples were tested by RT-RPA-LFD and RT-PCR. The detection results of RT-RPA-LFD were compared with those of RT-PCR, with a coincidence rate of 96.5%. CONCLUSION: The newly established RT-RPA-LFD assay in our study had high sensitivity and specificity, with a potential to use in resource-limited areas and countries.

RedEx: a method for seamless DNA insertion and deletion in large multimodular polyketide synthase gene clusters.

Biosynthesis reprograming is an important way to diversify chemical structures. The large repetitive DNA sequences existing in polyketide synthase genes make seamless DNA manipulation of the polyketide biosynthetic gene clusters extremely challenging. In this study, to replace the ethyl group attached to the C-21 of the macrolide insecticide spinosad with a butenyl group by refactoring the 79-kb gene cluster, we developed a RedEx method by combining Redαß mediated linear-circular homologous recombination, ccdB counterselection and exonuclease mediated in vitro annealing to insert an exogenous extension module in the polyketide synthase gene without any extra sequence. RedEx was also applied for seamless deletion of the rhamnose 3'-O-methyltransferase gene in the spinosad gene cluster to produce rhamnosyl-3'-desmethyl derivatives. The advantages of RedEx in seamless mutagenesis will facilitate rational design of complex DNA sequences for diverse purposes.

Latest diagnostic performance of different ultrasonic features for biliary atresia.

BACKGROUND: Early diagnosis of biliary atresia (BA) is an important clinical challenge. PURPOSE: To summarize the latest diagnostic performance of different ultrasonic (US) features for BA. MATERIAL AND METHODS: MeSH terms "biliary atresia" and "ultrasonography" and related hyponyms were used to search PubMed, EMBASE, and the Cochrane Central Register of Controlled Trials. Eligible articles were included and data were retrieved. The methodologic quality was assessed by version 2 of the Quality Assessment of Diagnostic Accuracy Studies tool. Estimated sensitivity and specificity of each US feature were calculated by Stata 14.0. RESULTS: Fifty eligible studies on 5622 patients were included. Respective summary sensitivity and specificity were 77% (95% CI=69-84) and 98% (95% CI=96-99) for triangular cord sign (TCS) in 32 studies, 86% (95% CI=78-92) and 86% (95% CI=72-94) for shear wave elastography (SWE) in seven studies, 75% (95% CI=65-83) and 92% (95% CI=86-95) for gallbladder and biliary system abnormality (GBA) in 25 studies, and 81% (95% CI=69-90) and 79% (95% CI=67-87) for hepatic artery (HA) enlargement in seven studies. The overall US features from 11 studies yielded a summary sensitivity of 84% (95% CI=72-92) and specificity of 86% (95% CI=77-92). CONCLUSION: TCS and GBA were the two most widely accepted US features currently used for differential diagnosis of BA. The newly developed SWE was an objective and convenient method with good diagnostic performance. HA enlargement can be used as an auxiliary sign.

Phytotoxic Metabolites Isolated from Aspergillus sp., an Endophytic Fungus of Crassula arborescens.

Aspergillus sp., an endophytic fungus isolated from Crassula arborescens, displayed potent inhibitory activity against the seed germination of Arabidopsis thaliana. The bioactivity-guided fractionation of the culture extract of Aspergillus sp. MJ01 led to the isolation of nine compounds, including one previously undescribed furanone, namely aspertamarinoic acid (1), and eight known compounds, (-)-dihydrocanadensolide (2), kojic acid (3), citreoisocoumarin (4), astellolide A (5), astellolide B (6), astellolide G (7), cyclo-N-methylphenylalanyltryptophenyl (8) and (-)-ditryptophenaline (9). In the evaluation of the phytotoxic activities of compounds 1-9, the results suggested that 1 and 5 showed significant inhibitory activity on the seed germination of A. thaliana. This is the first report to disclose the phytotoxic activity of these compounds.

APB-13 improves the adverse outcomes caused by TGEV infection by correcting the intestinal microbial disorders in piglets.

Porcine transmissible gastroenteritis virus (TGEV) is an enteric coronavirus that has caused high morbidity and mortality of piglets worldwide. Previous studies have shown that the TGEV can lead to severe diarrhoea, vomiting and dehydration in 2-week-old piglets and weaned piglets, resulting in a large number of piglet deaths. Antimicrobial peptides have broad-spectrum antimicrobial activity and a strong killing effect on bacteria, especially on the drug-resistant pathogenic bacteria, and it has attracted broad concern. However, there are very few reports on the effect of APB-13 (an antimicrobial peptide) on the intestinal microbes of piglets infected with TGEV. In this study, 16S rRNA gene sequencing was used to compare the microbial phylum and the genus of piglet's enteric microorganism in different experimental groups, and to predict the metabolic function of the microbial flora. At the same time, the apparent digestibility of nutrients, digestive enzyme activity, daily weight gain and survival rate were also measured. TGEV infection could cause the imbalance of intestinal microbes in piglets, and increase of the relative abundance of Proteobacteria, and decrease of the relative abundance of Firmicutes, Bacteroidetes and Actinobacteri. With the addition of APB-13, this problem can be alleviated, which can reduce the relative abundance of Proteobacteria and improve the balance of intestinal microorganisms. At the microbial genus level, after adding APB-13, the relative abundance of Catenibacterium, Enterobacter and Streptococcus in the intestinal tract of piglets infected with TGEV showed significant decrease, while the relative abundance of Lactobacillus and Ruminococcus increased. Finally, we found that APB-13 can significantly increase the activity of digestive enzyme in the intestinal tract of piglet, thereby improving the apparent digestibility of nutrients and the growth performance of piglets. This study demonstrates that APB-13 can alleviate the adverse outcomes caused by TGEV infection by correcting the intestinal microbial disorders.

Challenges and potential for improving the druggability of podophyllotoxin-derived drugs in cancer chemotherapy.

Covering: up to 2020As a main bioactive component of the Chinese, Indian, and American Podophyllum species, the herbal medicine, podophyllotoxin (PTOX) exhibits broad spectrum pharmacological activity, such as superior antitumor activity and against multiple viruses. PTOX derivatives (PTOXs) could arrest the cell cycle, block the transitorily generated DNA/RNA breaks, and blunt the growth-stimulation by targeting topoisomerase II, tubulin, or insulin-like growth factor 1 receptor. Since 1983, etoposide (VP-16) is being used in frontline cancer therapy against various cancer types, such as small cell lung cancer and testicular cancer. Surprisingly, VP-16 (ClinicalTrials NTC04356690) was also redeveloped to treat the cytokine storm in coronavirus disease 2019 (COVID-19) in phase II in April 2020. The treatment aims at dampening the cytokine storm and is based on etoposide in the case of central nervous system. However, the initial version of PTOX was far from perfect. Almost all podophyllotoxin derivatives, including the FDA-approved drugs VP-16 and teniposide, were seriously limited in clinical therapy due to systemic toxicity, drug resistance, and low bioavailability. To meet this challenge, scientists have devoted continuous efforts to discover new candidate drugs and have developed drug strategies. This review focuses on the current clinical treatment of PTOXs and the prospective analysis for improving druggability in the rational design of new generation PTOX-derived drugs.

Fibroblasts in cancer dormancy: foe or friend?

Cancer dormancy is defined that the residual cancer cells could enter into a state of quiescence and patients remain asymptomatic for years or even decades after anti-tumor therapies. Fibroblasts, which represent a predominant cell type in tumor microenvironment, play a pivotal role in determining the ultimate fate of tumor cells. This review recapitulates the pleiotropic roles of fibroblasts which are divided into normal, senescent, cancer-associated fibroblasts (CAFs) and circulation CAFs in tumor dormancy, relapse, metastasis and resistance to therapy to help the treatment of cancer metastasis.

Fatty acid oxidation: driver of lymph node metastasis.

Fatty acid oxidation (FAO) is the emerging hallmark of cancer metabolism because certain tumor cells preferentially utilize fatty acids for energy. Lymph node metastasis, the most common way of tumor metastasis, is much indispensable for grasping tumor progression, formulating therapy measure and evaluating tumor prognosis. There is a plethora of studies showing different ways how tumor cells metastasize to the lymph nodes, but the role of FAO in lymph node metastasis remains largely unknown. Here, we summarize recent findings and update the current understanding that FAO may enable lymph node metastasis formation. Afterward, it will open innovative possibilities to present a distinct therapy of targeting FAO, the metabolic rewiring of cancer to terminal cancer patients.

Genomics-Driven Activation of Silent Biosynthetic Gene Clusters in Burkholderia gladioli by Screening Recombineering System.

The Burkholderia genus possesses ecological and metabolic diversities. A large number of silent biosynthetic gene clusters (BGCs) in the Burkholderia genome remain uncharacterized and represent a promising resource for new natural product discovery. However, exploitation of the metabolomic potential of Burkholderia is limited by the absence of efficient genetic manipulation tools. Here, we screened a bacteriophage recombinase system Redγ-BAS, which was functional for genome modification in the plant pathogen Burkholderia gladioli ATCC 10248. By using this recombineering tool, the constitutive promoters were precisely inserted in the genome, leading to activation of two silent nonribosomal peptide synthetase gene clusters (bgdd and hgdd) and production of corresponding new classes of lipopeptides, burriogladiodins A-H (1-8) and haereogladiodins A-B (9-10). Structure elucidation revealed an unnatural amino acid Z- dehydrobutyrine (Dhb) in 1-8 and an E-Dhb in 9-10. Notably, compounds 2-4 and 9 feature an unusual threonine tag that is longer than the predicted collinearity assembly lines. The structural diversity of burriogladiodins was derived from the relaxed substrate specificity of the fifth adenylation domain as well as chain termination conducted by water or threonine. The recombinase-mediating genome editing system is not only applicable in B. gladioli, but also possesses great potential for mining meaningful silent gene clusters from other Burkholderia species.

Biosynthesis of Chuangxinmycin Featuring a Deubiquitinase-like Sulfurtransferase.

The knowledge on sulfur incorporation mechanism involved in sulfur-containing molecule biosynthesis remains limited. Chuangxinmycin is a sulfur-containing antibiotic with a unique thiopyrano[4,3,2-cd]indole (TPI) skeleton and selective inhibitory activity against bacterial tryptophanyl-tRNA synthetase. Despite the previously reported biosynthetic gene clusters and the recent functional characterization of a P450 enzyme responsible for C-S bond formation, the enzymatic mechanism for sulfur incorporation remains unknown. Here, we resolve this central biosynthetic problem by in vitro biochemical characterization of the key enzymes and reconstitute the TPI skeleton in a one-pot enzymatic reaction. We reveal that the JAMM/MPN+ protein Cxm3 functions as a deubiquitinase-like sulfurtransferase to catalyze a non-classical sulfur-transfer reaction by interacting with the ubiquitin-like sulfur carrier protein Cxm4GG. This finding adds a new mechanism for sulfurtransferase in nature.

What makes leader cells arise: Intrinsic properties and support from neighboring cells.

Cancer cells collectively invading as a cohesive and polarized group is termed collective invasion, which is a fundamental property of many types of cancers. In this multicellular unit, cancer cells are heterogeneous, consisting of two morphologically and functionally distinct subpopulations, leader cells and follower cells. Leader cells at the invasive front are responsible for exploring the microenvironment, paving the way, and transmitting information to follower cells. Here, in this review, we will describe the important role of leader cells in collective invasion and the emerging underlying mechanisms of leader cell formation including intrinsic properties and the support from neighboring cells. It will help us to elucidate the essence of collective invasion and provide new anticancer therapeutic clues.