Genome sequencing and comparative genomics reveal insights into pathogenicity and evolution of Fusarium zanthoxyli, the causal agent of stem canker in prickly ash.

BACKGROUND: Fusarium zanthoxyli is a destructive pathogen causing stem canker in prickly ash, an ecologically and economically important forest tree. However, the genome lack of F. zanthoxyli has hindered research on its interaction with prickly ash and the development of precise control strategies for stem canker. RESULTS: In this study, we sequenced and annotated a relatively high-quality genome of F. zanthoxyli with a size of 43.39 Mb, encoding 11,316 putative genes. Pathogenicity-related factors are predicted, comprising 495 CAZymes, 217 effectors, 156 CYP450s, and 202 enzymes associated with secondary metabolism. Besides, a comparative genomics analysis revealed Fusarium and Colletotrichum diverged from a shared ancestor approximately 141.1 ~ 88.4 million years ago (MYA). Additionally, a phylogenomic investigation of 12 different phytopathogens within Fusarium indicated that F. zanthoxyli originated approximately 34.6 ~ 26.9 MYA, and events of gene expansion and contraction within them were also unveiled. Finally, utilizing conserved domain prediction, the results revealed that among the 59 unique genes, the most enriched domains were PnbA and ULP1. Among the 783 expanded genes, the most enriched domains were PKc_like kinases and those belonging to the APH_ChoK_Like family. CONCLUSION: This study sheds light on the genetic basis of F. zanthoxyli's pathogenicity and evolution which provides valuable information for future research on its molecular interactions with prickly ash and the development of effective strategies to combat stem canker.

Kinase Inhibition via Small Molecule-Induced Intramolecular Protein Cross-Linking.

Remarkable progress has been made in the development of cysteine-targeted covalent inhibitors. In kinase drug discovery, covalent inhibitors capable of targeting other nucleophilic residues (i.e. lysine, or K) have emerged in recent years. Besides a highly conserved catalytic lysine, almost all human protein kinases possess an equally conserved glutamate/aspartate (e.g. E/D) that forms a K-E/D salt bridge within the enzyme's active site. Electrophilic ynamides were previously used as effective peptide coupling reagents and to develop E/D-targeting covalent protein inhibitors/probes. In the present study, we report the first ynamide-based small-molecule inhibitors capable of inducing intramolecular cross-linking of various protein kinases, leading to subsequent irreversible inhibition of kinase activity. Our strategy took advantage of the close distance between the highly conserved catalytic K and E/D residues in a targeted kinase, thus providing a conceptually general approach to achieve irreversible kinase inhibition with high specificity and desirable cellular potency. Finally, this ynamide-facilitated, ligand-induced mechanism leading to intramolecular kinase cross-linking and inhibition was unequivocally established by using recombinant ABL kinase as a representative.

Global Reactivity Profiling of the Catalytic Lysine in Human Kinome for Covalent Inhibitor Development.

Advances in targeted covalent inhibitors (TCIs) have been made by using lysine-reactive chemistries. Few aminophiles possessing balanced reactivity/stability for the development of cell-active TCIs are however available. We report herein lysine-reactive activity-based probes (ABPs; 2-14) based on the chemistry of aryl fluorosulfates (ArOSO2 F) capable of global reactivity profiling of the catalytic lysine in human kinome from mammalian cells. We concurrently developed reversible covalent ABPs (15/16) by installing salicylaldehydes (SA) onto a promiscuous kinase-binding scaffold. The stability and amine reactivity of these probes exhibited a broad range of tunability. X-ray crystallography and mass spectrometry (MS) confirmed the successful covalent engagement between ArOSO2 F on 9 and the catalytic lysine of SRC kinase. Chemoproteomic studies enabled the profiling of >300 endogenous kinases, thus providing a global landscape of ligandable catalytic lysines of the kinome. By further introducing these aminophiles into VX-680 (a noncovalent inhibitor of AURKA kinase), we generated novel lysine-reactive TCIs that exhibited excellent in vitro potency and reasonable cellular activities with prolonged residence time. Our work serves as a general guide for the development of lysine-reactive ArOSO2 F-based TCIs.

2-Ethynylbenzaldehyde-Based, Lysine-Targeting Irreversible Covalent Inhibitors for Protein Kinases and Nonkinases.

Lysine-targeting irreversible covalent inhibitors have attracted growing interests in recent years, especially in the fields of kinase research. Despite encouraging progress, few chemistries are available to develop inhibitors that are exclusively lysine-targeting, selective, and cell-active. We report herein a 2-ethynylbenzaldehyde (EBA)-based, lysine-targeting strategy to generate potent and selective small-molecule inhibitors of ABL kinase by selectively targeting the conserved catalytic lysine in the enzyme. We showed the resulting compounds were cell-active, capable of covalently engaging endogenous ABL kinase in K562 cells with long-residence time and few off-targets. We further validated the generality of this strategy by developing EBA-based irreversible inhibitors against EGFR (a kinase) and Mcl-1 (a nonkinase) that covalently reacted with the catalytic and noncatalytic lysine within each target.

Orthogonal Strategies for Profiling Potential Cellular Targets of Anandamide and Cannabidiol.

The human endocannabinoid system regulates a myriad of physiological processes through a complex lipid signaling network involving cannabinoids and their respective receptors, cannabinoid receptor 1 (hCB1 R) and cannabinoid receptor 2 (hCB2 R). Anandamide (AEA) and cannabidiol (CBD) are classical examples of cannabinoids that elicit a variety of effects, both beneficial and detrimental, through these receptors. Mounting evidence suggested the presence of other potential cannabinoid targets that may be responsible for other observable effects. However, prior pharmacological studies on these cannabinoid compounds provided scant evidence of direct engagement to these proposed targets. Moreover, to the best of our knowledge, no chemoproteomic studies have been demonstrated on CBD. Here we showed that, by taking advantage of a recently developed 'label-free' 2D-TPP (2 Dimensional-Thermal Protein Profiling) approach, we have identified several new putative targets of both AEA and CBD. Comparison of these interaction landscapes with those obtained from well-established affinity-based protein profiling (AfBPP) platforms has led to the discovery of both shared and unique protein targets. Subsequent target validation of selected proteins led us to conclude that this 2D-TPP strategy complements well with AfBPP.

A Late-Stage Aryl C-H Olefination Strategy and Its Application Towards Global Proteome Profiling of Δ8 -Tetrahydrocannabinol.

Drugs and bioactive natural products exert their pharmacological effects by engaging numerous cellular targets in our body. Identification of these protein targets is essential for understanding the mechanism-of-action of these compounds, thus contributing to improved drug design in drug discovery programs. Termed "in situ drug profiling", a common strategy for studying these bioactive compounds centralized on the covalent capture of protein targets along with a reporter tag to facilitate downstream proteomic analyses. Though highly successful, such reliance on innate electrophilic traps to facilitate covalent capture restricted its applications to covalent acting compounds. Late-stage C-H functionalization (LSF) may resolve this by substituting biologically inert C-H bonds with desired electrophilic groups. Herein, we demonstrated this concept by arming a diverse range of electron-rich aromatic drugs and natural products with α,ß-unsaturated esters, via late-stage C-H olefination with an arylthio-based carboxylic acid ligand developed by Ibanez and co-workers. We also showed that covalent probes generated from this LSF approach could be applied for "in situ drug profiling" of Δ8 -THC, as exemplified by the successful target engagement of α-4 db, a Δ8 -THC-based probe, to its native target hCB2 R. In combination with AfBP 7, a photoaffinity-based derivative of Δ8 -THC, we identified several novel putative targets that could account for some of the effects in THC consumption. We anticipate our C-H LSF strategy to be widely adopted for future studies of non-covalent drugs.

Rapid detection of SARS-CoV-2 with CRISPR-Cas12a.

The recent outbreak of betacoronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which is responsible for the Coronavirus Disease 2019 (COVID-19) global pandemic, has created great challenges in viral diagnosis. The existing methods for nucleic acid detection are of high sensitivity and specificity, but the need for complex sample manipulation and expensive machinery slow down the disease detection. Thus, there is an urgent demand to develop a rapid, inexpensive, and sensitive diagnostic test to aid point-of-care viral detection for disease monitoring. In this study, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated proteins (Cas) 12a-based diagnostic method that allows the results to be visualized by the naked eye. We also introduced a rapid sample processing method, and when combined with recombinase polymerase amplification (RPA), the sample to result can be achieved in 50 minutes with high sensitivity (1-10 copies per reaction). This accurate and portable detection method holds a great potential for COVID-19 control, especially in areas where specialized equipment is not available.

Multiplex Identification of Post-Translational Modifications at Point-of-Care by Deep Learning-Assisted Hydrogel Sensors.

Multiplex detection of protein post-translational modifications (PTMs), especially at point-of-care, is of great significance in cancer diagnosis. Herein, we report a machine learning-assisted photonic crystal hydrogel (PCH) sensor for multiplex detection of PTMs. With closely-related PCH sensors microfabricated on a single chip, our design achieved not only rapid screening of PTMs at specific protein sites by using only naked eyes/cellphone, but also the feasibility of real-time monitoring of phosphorylation reactions. By taking advantage of multiplex sensor chips and a neural network algorithm, accurate prediction of PTMs by both their types and concentrations was enabled. This approach was ultimately used to detect and differentiate up/down regulation of different phosphorylation sites within the same protein in live mammalian cells. Our developed method thus holds potential for POC identification of various PTMs in early-stage diagnosis of protein-related diseases.

Transcriptome analysis and identification of sex pheromone biosynthesis and transport related genes in Atrijuglans hetaohei (Lepidoptera: Gelechioidea).

Atrijuglans hetaohei Yang (Lepidoptera: Gelechioidea) is one of the major pests that can seriously damage the walnut tree, leading to harvest loss. Sex pheromones regulate mating communication and reproduction in insects and provide targets for developing a novel pest control strategy. In this study, by transcriptomic sequencing and analysis of the female pheromone gland (PG) and male genitalia of A. hetaohei, we identified 92 putative genes, of which 7 desaturases (Dess), 8 fatty acyl reductases (FARs), 4 fatty acid synthetases (FASs), 2 aldehyde oxidases (AOXs), 4 acetyltransferases (ACTs), 1 chemosensory protein (CSP), and 2 odorant-binding proteins (OBPs) were predominantly expressed in the female PG, while 5 Dess, 11 FARs, 7 FASs, 6 AOXs, 1 ACT, and 1 CSP showed more robust expression in the male genitalia. Moreover, phylogenetic analysis revealed that 7 Dess and 1 FAR were grouped with genes involved in pheromone synthesis in other Lepidoptera species. Thus, we proposed that these candidate genes are possibly involved in the sex pheromone biosynthetic pathway in A. hetaohei. Our findings will provide a solid genetic basis for further exploring the function of the tissue-biased genes and may be useful to screen potential targets for interfering chemical communication in A. hetaohei.

The role of point defects related with carbon impurity on the kink of logJ-Vin GaN-on-Si epitaxial layers.

Carbon impurity as point defects makes key impact on the leakage in GaN-on-Si structures. GaN-based epitaxial layers with different point defects by changing carbon-doped concentration were used to investigate the point defects behavior. It was found that leakage mechanisms correspond with space-charge-limited current models at low voltages, and after 1st kink, electron injection from silicon to GaN and PF conduction play a key role in the leakage of both point defects case with low carbon and high carbon doped. In addition, high carbon in GaN-on-Si epitaxial layers obtained lower leakage and larger breakdown voltage. The slope of logJ-Vhas two kinks and effective energy barrierEahas two peaks, 0.4247 eV at about 300 V and 0.3485 eV at about 900 V, respectively, which is related to accepted states and donor states related with carbon impurity. While the slope of logJ-Vhas one kink and effective energy barrierEahas one peak, 0.4794 eV at about 400 V of low carbon in GaN-on-Si epitaxial layers, indicating only field-induced accepted ionized makes impact on leakage. The comparative results of more donor trap density in high carbon indicate point defects related with carbon impurity play a key role in the kinks of logJ-Vslope.

Cell-Active, Reversible, and Irreversible Covalent Inhibitors That Selectively Target the Catalytic Lysine of BCR-ABL Kinase.

Despite recent interests in developing lysine-targeting covalent inhibitors, no general approach is available to create such compounds. We report herein a general approach to develop cell-active covalent inhibitors of protein kinases by targeting the conserved catalytic lysine residue using key SuFEx and salicylaldehyde-based imine chemistries. We validated the strategy by successfully developing (irreversible and reversible) covalent inhibitors against BCR-ABL kinase. Our lead compounds showed high levels of selectivity in biochemical assays, exhibited nanomolar potency against endogenous ABL kinase in cellular assays, and were active against most drug-resistant ABL mutations. Among them, the salicylaldehyde-containing A5 is the first-ever reversible covalent ABL inhibitor that possessed time-dependent ABL inhibition with prolonged residence time and few cellular off-targets in K562 cells. Bioinformatics further suggested the generality of our strategy against the human kinome.

Cell-Penetrating Mitochondrion-Targeting Ligands for the Universal Delivery of Small Molecules, Proteins and Nanomaterials.

Mitochondria are key organelles that perform vital cellular functions such as those related to cell survival and death. The targeted delivery of different types of cargos to mitochondria is a well-established strategy to study mitochondrial biology and diseases. Of the various existing mitochondrion-transporting vehicles, most suffer from poor cytosolic entry, low delivery efficiency, limited cargo types, and cumbersome preparation protocols, and none was known to be universally applicable for mitochondrial delivery of different types of cargos (small molecules, proteins, and nanomaterials). Herein, two new cell-penetrating, mitochondrion-targeting ligands (named MitoLigand ) that are capable of effectively "tagging" small-molecule drugs, native proteins and nanomaterials are disclosed, as well as their corresponding chemoselective conjugation chemistry. Upon successful cellular delivery and rapid endosome escape, the released native cargos were found to be predominantly localized inside mitochondria. Finally, by successfully delivering doxorubicin, a well-known anticancer drug, to the mitochondria of HeLa cells, we showed that the released drug possessed potent cell cytotoxicity, disrupted the mitochondrial membrane potential and finally led to apoptosis. Our strategy thus paves the way for future mitochondrion-targeted therapy with a variety of biologically active agents.

Biocidal efficacy of tutin and its influence on immune cells and expression of growth-blocking and neuroglian peptides in Mythimna separata (Lepidoptera: Noctuidae).

Mythimna separata Walker (Lepidoptera: Noctuidae) is one of the major pests that can cause severe damage to grain crops. The development of low-toxicity and high-performance botanical insecticides is becoming the focus of new pesticide research to control M. separata. Tutin, a sesquiterpene lactone compound obtained from Coriaria sinica Maxim, a native Chinese poisonous plant, has antifeedant, absorption, and stomach poisoning against a variety of pests. To understand the toxic effect of tutin on M. separata larvae, we set out to determine their antifeedant, mortality, paralysis, weight change, and to examine the spreading of M. separata hemocytes under different concentrations of tutin treatment. Tissue distribution of the immune-associated gene growth-blocking peptide (GBP) and neuroglian peptide (Nrg) was detected by reverse transcription polymerase chain reaction (PCR). Furthermore, real-time quantitative PCR was carried out to determine the expression profiles of GBP and Nrg after different concentrations of tutin stimulation. Our results revealed that tutin exhibited significant antifeedant and insecticidal activities, paralysis, weight loss to M. separata. Besides, tutin significantly influenced on the morphology of hemocytes and enhanced the expression of GBP and Nrg in M. separata.

The adipokinetic hormone signaling system regulates the sensitivity of Bactrocera dorsalis to malathion.

The neuropeptide adipokinetic hormone (AKH) binds to the AKH receptor (AKHR) to regulate carbohydrate and lipid metabolism. It also participates in the insect anti-stress response. We used RT-qPCR to detect the expression levels of 39 neuropeptides in malathion-susceptible (MS) and malathion-resistant (MR) strains of Bactrocera dorsalis. AKH and AKHR were highly expressed in the MR strain. Using a malathion bioassay and RNA interference (RNAi), we demonstrated that AKHR is involved in the susceptibility of B. dorsalis to malathion. We found significantly reduced expression of two detoxification enzyme genes (glutathione-S-transferase, GST and α-esterase, CarE) after AKHR RNAi. Based on our previous data, GSTd10 and CarE6 participate the direct metabolism of malathion in this fly, which is also verified by a malathion metabolism assay by HPLC using the crude enzymes in the current study. These results suggest that AKHR plays an important role in affecting malathion susceptibility via detoxification enzyme genes.

Strategic Design of Catalytic Lysine-Targeting Reversible Covalent BCR-ABL Inhibitors*.

Targeted covalent inhibitors have re-emerged as validated drugs to overcome acquired resistance in cancer treatment. Herein, by using a carbonyl boronic acid (CBA) warhead, we report the structure-based design of BCR-ABL inhibitors via reversible covalent targeting of the catalytic lysine with improved potency against both wild-type and mutant ABL kinases, especially ABLT315I bearing the gatekeeper residue mutation. We show the evolutionarily conserved lysine can be targeted selectively, and the selectivity depends largely on molecular recognition of the non-covalent pharmacophore in this class of inhibitors, probably due to the moderate reactivity of the warhead. We report the first co-crystal structures of covalent inhibitor-ABL kinase domain complexes, providing insights into the interaction of this warhead with the catalytic lysine. We also employed label-free mass spectrometry to evaluate off-targets of our compounds at proteome-wide level in different mammalian cells.

Chemoproteomics Reveals the Antiproliferative Potential of Parkinson's Disease Kinase Inhibitor LRRK2-IN-1 by Targeting PCNA Protein.

LRRK2-IN-1, one of the first selective inhibitors of leucine-rich repeat kinase 2 (LRRK2), was serendipitously found to exhibit potent antiproliferative activity in several types of human cancer cells. In this study, we employed a chemoproteomic strategy utilizing a photoaffinity probe to identify the cellular target(s) of LRRK2-IN-1 underlying its anticancer activity. LRRK2-IN-1 was found to induce cell cycle arrest as well as cancer cell death by specifically binding to human proliferating cell nuclear antigen (PCNA) in cancer cells. Our current findings suggest the potential of LRRK2-IN-1 as a novel pharmacological molecule for scrutinizing cell physiology and furnish a logical foundation for the future development of therapeutic reagents for cancer.

Characterization of the Artemisinin Binding Site for Translationally Controlled Tumor Protein (TCTP) by Bioorthogonal Click Chemistry.

Despite the fact that multiple artemisinin-alkylated proteins in Plasmodium falciparum have been identified in recent studies, the alkylation mechanism and accurate binding site of artemisinin-protein interaction have remained elusive. Here, we report the chemical-probe-based enrichment of the artemisinin-binding peptide and characterization of the artemisinin-binding site of P. falciparum translationally controlled tumor protein (TCTP). A peptide fragment within the N-terminal region of TCTP was enriched and found to be alkylated by an artemisinin-derived probe. MS2 fragments showed that artemisinin could alkylate multiple amino acids from Phe12 to Tyr22 of TCTP, which was supported by labeling experiments upon site-directed mutagenesis and computational modeling studies. Taken together, the "capture-and-release" strategy affords consolidated advantages previously unavailable in artemisinin-protein binding site studies, and our results deepened the understanding of the mechanism of protein alkylation via heme-activated artemisinin.

Aquaporin-4: A Potential Therapeutic Target for Cerebral Edema.

Aquaporin-4 (AQP4) is a family member of water-channel proteins and is dominantly expressed in the foot process of glial cells surrounding capillaries. The predominant expression at the boundaries between cerebral parenchyma and major fluid compartments suggests the function of aquaporin-4 in water transfer into and out of the brain parenchyma. Accumulating evidences have suggested that the dysregulation of aquaporin-4 relates to the brain edema resulting from a variety of neuro-disorders, such as ischemic or hemorrhagic stroke, trauma, etc. During edema formation in the brain, aquaporin-4 has been shown to contribute to the astrocytic swelling, while in the resolution phase, it has been seen to facilitate the reabsorption of extracellular fluid. In addition, aquaporin-4-deficient mice are protected from cytotoxic edema produced by water intoxication and brain ischemia. However, aquaporin-4 deletion exacerbates vasogenic edema in the brain of different pathological disorders. Recently, our published data showed that the upregulation of aquaporin-4 in astrocytes probably contributes to the transition from cytotoxic edema to vasogenic edema. In this review, apart from the traditional knowledge, we also introduce our latest findings about the effects of mesenchymal stem cells (MSCs) and microRNA-29b on aquaporin-4, which could provide powerful intervention tools targeting aquaporin-4.

Mesenchymal stem cells maintain blood-brain barrier integrity by inhibiting aquaporin-4 upregulation after cerebral ischemia.

RATIONALE: Cerebral ischemia upregulates aquaporin-4 expression, increases blood-brain barrier (BBB) permeability, and induces brain edema. Mesenchymal stem cells (MSCs) can repress inflammatory cytokines and show great potential for ischemic stroke therapy. However, the effect of MSCs regarding the protection of ischemia-induced BBB break down is unknown. OBJECTIVE: We test whether MSCs therapy protects BBB integrity and explore the molecular mechanisms of aquaporin-4 on BBB integrity. METHODS AND RESULTS: Two hundred and twenty-eight adult CD1 male mice underwent 90 minutes transient middle cerebral artery occlusion and received 2 × 10(5) MSCs intracranial transplantation. The neurological severity score was improved and both ischemia-induced brain edema and BBB leakage were reduced in MSC-treated mice. MSCs therapy reduced astrocyte apoptosis and inhibited ischemia-induced aquaporin-4 upregulation. In addition, small-interfering RNA knockdown of aquaporin-4 after cerebral ischemia effectively reduced aquaporin-4 expression, brain edema, BBB leakage, and astrocyte apoptosis. Conditional medium from lipopolysaccharide (LPS)-activated microglia enhanced aquaporin-4 expression, p38 and JNK phosphorylation, and apoptosis of cultured astrocytes. MSC treatment reduced the expression of inflammatory cytokines in LPS-activated microglia, and subsequently reduced aquaporin-4 expression and apoptosis of astrocytes. Knockdown of aquaporin-4 in cultured astrocytes also reduced apoptosis. Treatment with p38 and JNK inhibitors showed that p38, but not the JNK signaling pathway, was responsible for the aquaporin-4 upregulation. CONCLUSION: MSCs protected BBB integrity by reducing the apoptosis of astrocytes after ischemic attack, which was due to the attenuation of inflammatory response and downregulation of aquaporin-4 expression via p38 signaling pathway.

Effect of HMGB1 on the paracrine action of EPC promotes post-ischemic neovascularization in mice.

Transplantation of endothelial progenitor cells (EPCs) leads to better outcomes in experimental stroke, but the mechanism remains unclear. It was reported that astrocytic-high mobility group box1 (HMGB1) promoted endogenous EPC-mediated neurovascular remodeling during stroke recovery. It is unclear whether HMGB1 involves in exogenous EPC-mediated stroke recovery. In this study, we aim to explore whether microglial HMGB1 contributes to human peripheral blood-derived (hPB)-EPCs-mediated neurovascular remodeling by modulating the paracrine function of exogenous hPB-EPCs. Coculturing hPB-EPCs with lipopolysaccharides stimulated BV2 cells upregulated Interleukin-8 expression in hPB-EPCs; this was blocked by treating BV2 cells with HMGB1 inhibitor Glycyrrhizin. Conditioned medium (CM) of hPB-EPCs cocultured with BV2 cells promoted the viability and tube formation of human umbilical cord vein cells. Inhibiting either HMGB1 or IL-8 could block the effect of hPB-EPCs CM. In vivo study showed hPB-EPCs transplantation improved neurobehavioral outcomes, reduced brain atrophy volume, and enhanced neovascularization in transient middle cerebral artery occlusion (tMCAO) mice. Intraperitoneally administration of HMGB1 inhibitor glycyrrhizin blocked the beneficial effect of hPB-EPC transplantation. We did not observe the integration of green fluorescent protein-labeled hPB-EPCs with microvessels in peri-infarct areas at day-14 after tMCAO. In summary, the result suggested that HMGB1 upregulation in postischemic brain could promote exogenous hPB-EPC-mediated stroke recovery by modulating paracrine function of hPB-EPCs.