In vivo photopharmacology with a caged mu opioid receptor agonist drives rapid changes in behavior.

Photoactivatable drugs and peptides can drive quantitative studies into receptor signaling with high spatiotemporal precision, yet few are compatible with behavioral studies in mammals. We developed CNV-Y-DAMGO-a caged derivative of the mu opioid receptor-selective peptide agonist DAMGO. Photoactivation in the mouse ventral tegmental area produced an opioid-dependent increase in locomotion within seconds of illumination. These results demonstrate the power of in vivo photopharmacology for dynamic studies into animal behavior.

The binding affinity-dependent inhibition of cell growth and viability by DNA sulfur-binding domains.

Increasing evidence suggests that DNA phosphorothioate (PT) modification serves several purposes in the bacterial host, and some restriction enzymes specifically target PT-DNA. PT-dependent restriction enzymes (PDREs) bind PT-DNA through their DNA sulfur binding domain (SBD) with dissociation constants (KD) of 5 nM~1 µM. Here, we report that SprMcrA, a PDRE, failed to dissociate from PT-DNA after cleavage due to high binding affinity, resulting in low DNA cleavage efficiency. Expression of SBDs in Escherichia coli cells with PT modification induced a drastic loss of cell viability at 25°C when both DNA strands of a PT site were bound, with one SBD on each DNA strand. However, at this temperature, SBD binding to only one PT DNA strand elicited a severe growth lag rather than lethality. This cell growth inhibition phenotype was alleviated by raising the growth temperature. An in vitro assay mimicking DNA replication and RNA transcription demonstrated that the bound SBD hindered the synthesis of new DNA and RNA when using PT-DNA as the template. Our findings suggest that DNA modification-targeting proteins might regulate cellular processes involved in DNA metabolism in addition to being components of restriction-modification systems and epigenetic readers.

Characterization of a promiscuous DNA sulfur binding domain and application in site-directed RNA base editing.

Phosphorothioate (PT)-modification was discovered in prokaryotes and is involved in many biological functions such as restriction-modification systems. PT-modification can be recognized by the sulfur binding domains (SBDs) of PT-dependent restriction endonucleases, through coordination with the sulfur atom, accompanied by interactions with the DNA backbone and bases. The unique characteristics of PT recognition endow SBDs with the potential to be developed into gene-targeting tools, but previously reported SBDs display sequence-specificity for PT-DNA, which limits their applications. In this work, we identified a novel sequence-promiscuous SBDHga from Hahella ganghwensis. We solved the crystal structure of SBDHga complexed with PT-DNA substrate to 1.8 Å resolution and revealed the recognition mechanism. A shorter L4 loop of SBDHga interacts with the DNA backbone, in contrast with previously reported SBDs, which interact with DNA bases. Furthermore, we explored the feasibility of using SBDHga and a PT-oligonucleotide as targeting tools for site-directed adenosine-to-inosine (A-to-I) RNA editing. A GFP non-sense mutant RNA was repaired at about 60% by harnessing a chimeric SBD-hADAR2DD (deaminase domain of human adenosine deaminase acting on RNA), comparable with currently available RNA editing techniques. This work provides insights into understanding the mechanism of sequence-specificity for SBDs and for developing new tools for gene therapy.

Activated factor X delivered by adeno-associated virus significantly inhibited bleeding and alleviated hemophilic synovitis in hemophilic mice.

In hemophilia, deficiency of factor VIII or IX prevents the activation of the common coagulation pathway, and inhibits the conversion of FX to activated FXa, which is required for thrombin generation. We hypothesized that the direct expressed FXa has the potential to activate the common pathway and restore coagulation in hemophilia patients. In this study, the cassettes that expressed FXa, FXaop and FXa-FVII were packaged into an engineered AAV capsid, AAV843, and were delivered into hemophilia A and B mice by intravenous injection. AAV-FXaop could be stably expressed in vivo and showed the best immediate and prolonged hemostatic effects, similar to those of commercial drugs (Xyntha and Benefix). AAV-FXaop also significantly inhibited bleeding in hemophilia A mice with inhibitors. In addition, FXa expression in joints significantly alleviated the occurrence of hemophilic synovitis. AAV-delivered FXa may be a novel target for treating hemophilic and hemophilic synovitis.

Discovery of Self-Assembled 2D Ru/Si Superlattices Boosting Hydrogen Evolution.

2D heterostructuring is a versatile methodology for designing nanoarchitecture catalytic systems that allow for reconstruction and modulation of interfaces and electronic structures. However, catalysts with such structures are extremely scarce due to limited synthetic strategies. Here, a highly ordered 2D Ru/Si/Ru/Si… nano-heterostructures (RSHS) is reported by acid etching of the LaRuSi electride. RSHS shows a superior electrocatalytic activity for hydrogen evolution with an overpotential of 14 mV at 10 mA cm-2 in alkaline media. Both experimental analysis and first-principles calculations demonstrate that the electronic states of Ru can be tuned by strong interactions of the interfacial Ru-Si, leading to an optimized hydrogen adsorption energy. Moreover, due to the synergistic effect of Ru and Si, the energy barrier of water dissociation is significantly reduced. The well-organized superlattice structure will provide a paradigm for construction of efficient catalysts with tunable electronic states and dual active sites.

An approach of gene regulatory network construction using mixed entropy optimizing context-related likelihood mutual information.

MOTIVATION: The question of how to construct gene regulatory networks has long been a focus of biological research. Mutual information can be used to measure nonlinear relationships, and it has been widely used in the construction of gene regulatory networks. However, this method cannot measure indirect regulatory relationships under the influence of multiple genes, which reduces the accuracy of inferring gene regulatory networks. APPROACH: This work proposes a method for constructing gene regulatory networks based on mixed entropy optimizing context-related likelihood mutual information (MEOMI). First, two entropy estimators were combined to calculate the mutual information between genes. Then, distribution optimization was performed using a context-related likelihood algorithm to eliminate some indirect regulatory relationships and obtain the initial gene regulatory network. To obtain the complex interaction between genes and eliminate redundant edges in the network, the initial gene regulatory network was further optimized by calculating the conditional mutual inclusive information (CMI2) between gene pairs under the influence of multiple genes. The network was iteratively updated to reduce the impact of mutual information on the overestimation of the direct regulatory intensity. RESULTS: The experimental results show that the MEOMI method performed better than several other kinds of gene network construction methods on DREAM challenge simulated datasets (DREAM3 and DREAM5), three real Escherichia coli datasets (E.coli SOS pathway network, E.coli SOS DNA repair network and E.coli community network) and two human datasets. AVAILABILITY AND IMPLEMENTATION: Source code and dataset are available at https://github.com/Dalei-Dalei/MEOMI/ and http://122.205.95.139/MEOMI/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

HvBGlu3, a GH1 ß-glucosidase enzyme gene, negatively influences ß-glucan content in barley grains.

KEY MESSAGE: HvBGlu3, a ß-glucosidase enzyme gene, negatively influences ß-glucan content in barley grains by mediating starch and sucrose metabolism in developing grains. Barley grains are rich in ß-glucan, an important factor affecting end-use quality. Previously, we identified several stable marker-trait associations (MTAs) and novel candidate genes associated with ß-glucan content in barley grains using GWAS (Genome Wide Association Study) analysis. The gene HORVU3Hr1G096910, encoding ß-glucosidase 3, named HvBGlu3, is found to be associated with ß-glucan content in barley grains. In this study, conserved domain analysis suggested that HvBGlu3 belongs to glycoside hydrolase family 1 (GH1). Gene knockout assay revealed that HvBGlu3 negatively influenced ß-glucan content in barley grains. Transcriptome analysis of developing grains of hvbglu3 mutant and the wild type indicated that the knockout of the gene led to the increased expression level of genes involved in starch and sucrose metabolism. Glucose metabolism analysis showed that the contents of many sugars in developing grains were significantly changed in hvbglu3 mutants. In conclusion, HvBGlu3 modulates ß-glucan content in barley grains by mediating starch and sucrose metabolism in developing grains. The obtained results may be useful for breeders to breed elite barley cultivars for food use by screening barley lines with loss of function of HvBGlu3 in barley breeding.

T-type voltage-gated channels, Na+/Ca2+-exchanger, and calpain-2 promote photoreceptor cell death in inherited retinal degeneration.

Inherited retinal degenerations (IRDs) are a group of untreatable and commonly blinding diseases characterized by progressive photoreceptor loss. IRD pathology has been linked to an excessive activation of cyclic nucleotide-gated channels (CNGC) leading to Na+- and Ca2+-influx, subsequent activation of voltage-gated Ca2+-channels (VGCC), and further Ca2+ influx. However, a connection between excessive Ca2+ influx and photoreceptor loss has yet to be proven.Here, we used whole-retina and single-cell RNA-sequencing to compare gene expression between the rd1 mouse model for IRD and wild-type (wt) mice. Differentially expressed genes indicated links to several Ca2+-signalling related pathways. To explore these, rd1 and wt organotypic retinal explant cultures were treated with the intracellular Ca2+-chelator BAPTA-AM or inhibitors of different Ca2+-permeable channels, including CNGC, L-type VGCC, T-type VGCC, Ca2+-release-activated channel (CRAC), and Na+/Ca2+ exchanger (NCX). Moreover, we employed the novel compound NA-184 to selectively inhibit the Ca2+-dependent protease calpain-2. Effects on the retinal activity of poly(ADP-ribose) polymerase (PARP), sirtuin-type histone-deacetylase, calpains, as well as on activation of calpain-1, and - 2 were monitored, cell death was assessed via the TUNEL assay.While rd1 photoreceptor cell death was reduced by BAPTA-AM, Ca2+-channel blockers had divergent effects: While inhibition of T-type VGCC and NCX promoted survival, blocking CNGCs and CRACs did not. The treatment-related activity patterns of calpains and PARPs corresponded to the extent of cell death. Remarkably, sirtuin activity and calpain-1 activation were linked to photoreceptor protection, while calpain-2 activity was related to degeneration. In support of this finding, the calpain-2 inhibitor NA-184 protected rd1 photoreceptors.These results suggest that Ca2+ overload in rd1 photoreceptors may be triggered by T-type VGCCs and NCX. High Ca2+-levels likely suppress protective activity of calpain-1 and promote retinal degeneration via activation of calpain-2. Overall, our study details the complexity of Ca2+-signalling in photoreceptors and emphasizes the importance of targeting degenerative processes specifically to achieve a therapeutic benefit for IRDs. Video Abstract.

Sirt1 inhibits macrophage polarization and inflammation in gouty arthritis by inhibiting the MAPK/NF-κB/AP-1 pathway and activating the Nrf2/HO-1 pathway.

OBJECTIVE AND DESIGN: To elucidate Sirt1's role in gouty arthritis inflammation and its potential mechanisms. MATERIAL: Constructed murine models of gouty arthritis and conducted THP-1 cell experiments. TREATMENT: 1 mg of MSU crystals injected into mice ankle joints for a 72-h intervention. After a 3-h pre-treatment with Sirt1-specific inhibitor (EX527) and agonist (SRT2104), inflammation was induced for 21 h using lipopolysaccharide (LPS) plus MSU crystals. METHODS: We assessed gouty arthritis severity through joint inflammation index, swelling, and hematoxylin and eosin (H&E) staining, and measured CD68 mononuclear macrophages and Sirt1 expression in synovial tissue via immunohistochemistry. ELISA, NO assay, RT-qPCR, Flow cytometry, and Western blot were utilized to examine macrophage inflammatory factors, polarization, reactive oxygen species(ROS), MAPK/NF-κB/AP-1 and Nrf2/HO-1 pathways proteins. RESULTS: Significant joint swelling, synovial tissue edema, and inflammatory cell infiltration were observed. CD68 mononuclear macrophages and Sirt1 expression were elevated in synovium. Sirt1 activation decreased inflammatory factors, M1 polarization, and ROS generation. Sirt1 activation reduced p38/JNK phosphorylation, thereby inhibiting downstream NF-κB p65/AP-1 and enhancing Nrf2/HO-1, thus suppressing inflammation. CONCLUSIONS: Sirt1 alleviates M1 macrophage polarization and inflammation in gouty arthritis by inhibiting the MAPK/NF-κB/AP-1 pathway and activating the Nrf2/HO-1 pathway. Thus, activating Sirt1 may provide a new therapeutic target for gouty arthritis.

Kirenol alleviates inflammation and oxidative stress to improve myocardial ischemia/reperfusion injury in rats.

The ischemic heart disease gravely threatens human health and even results in death. Kirenol is predominantly derived from the Herba Siegesbeckiae plant species and possesses a wide range of biological effects (such as antibacterial, anti-inflammatory, anti-cancer and cardioprotective). However, the regulatory effects and associated mechanisms of kirenol in myocardial ischemia/reperfusion injury (MI/RI) remain unclear. In this study, firstly, the MI/RI rat model was established. It was demonstrated that kirenol protected against the aggravation of cardiac function in MI/RI rats. In addition, the inflammation was induced by ischemia reperfusion (IR), which was likewise affected by kirenol (5 or 10 mg/kg). Moreover, IR enhanced oxidative stress, a process that was counteracted by kirenol. Next, cell apoptosis was discovered to be heightened after IR, but this effect was neutralized by kirenol. Finally, it was uncovered that kirenol has the ability to block the activation of the NF-κB pathway. In conclusion, it was disclosed that kirenol alleviated inflammation and oxidative stress through modulating the NF-κB pathway to improve MI/RI in rats. This work may offer novel insights for searching useful drugs for treating MI/RI.

A meta-analysis of predictive value of blood biomarkers in gestational trophoblastic neoplasia.

Background: Neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) have been reported to play a diagnostic and predictive role in gestational trophoblastic disease. However, the conclusions are still ambiguous. This meta-analysis aimed to evaluate the combined predictive value of NLR and PLR in the malignant progression of gestational trophoblastic disease. Method: Electronic databases including PubMed, Embase, the Cochrane Library, Web of Science, Chinese National Knowledge Infrastructure, Wanfang and China Biomedical Literature Database were searched for the relevant literature published up to 1 October 2022. Study selection and data extraction were performed independently by two reviewers. All analyses were performed using Revman, MetaDisc and STATA software. Results: A total of 858 patients from five studies were included in this meta-analysis. The pooled sensitivity and specificity of NLR were 0.8 (95% CI: 0.71-0.88) and 0.73 (95% CI: 0.69-0.76), respectively, and the area under curve of the summary receiver operating curve was 0.81. The pooled sensitivity and specificity of PLR were 0.87 (95% CI: 0.75-0.95) and 0.49 (95% CI: 0.44-0.54), respectively, and the area under curve of the summary receiver operating curve was 0.88. I2 statistic and Deek's funnel plot showed no heterogeneity and publication bias. Conclusion: NLR can accurately predict the progression from hydatidiform mole to gestational trophoblastic neoplasia and is a promising biomarker in further follow-up.

Harnessing sulfur-binding domains to separate Sp and Rp isomers of phosphorothioate oligonucleotides.

Chemical synthesis of phosphoromonothioate oligonucleotides (PS-ONs) is not stereo-specific and produces a mixture of Rp and Sp diastereomers, whose disparate reactivity can complicate applications. Although the current methods to separate these diastereomers which rely on chromatography are constantly improving, many Rp and Sp diastereomers are still co-eluted. Here, based on sulfur-binding domains that specifically recognize phosphorothioated DNA and RNA in Rp configuration, we developed a universal separation system for phosphorothioate oligonucleotide isomers using immobilized SBD (SPOIS). With the scalable SPOIS, His-tagged SBD is immobilized onto Ni-nitrilotriacetic acid-coated magnetic beads to form a beads/SBD complex, Rp isomers of the mixture can be completely bound by SBD and separated from Sp isomers unbound in liquid phase, then recovered through suitable elution approach. Using the phosphoromonothioate single-stranded DNA as a model, SPOIS separated PS-ON diastereomers of 4 nt to 50 nt in length at yields of 60-90% of the starting Rp isomers, with PS linkage not locating at 5' or 3' end. Within this length range, PS-ON diastereomers that co-eluted in HPLC could be separated by SPOIS at yields of 84% and 89% for Rp and Sp stereoisomers, respectively. Furthermore, as each Rp phosphorothioate linkage can be bound by SBD, SPOIS allowed the separation of stereoisomers with multiple uniform Sp configurations for multiple phosphorothioate modifications. A second generation of SPOIS was developed using the thermolabile and non-sequence-specific SBDPed, enabling fast and high-yield recovery of PS substrate stereoisomers for the DNAzyme Cd16 and further demonstrating the efficiency of this method. KEY POINTS: • SPOIS allows isomer separations of the Rp and Sp isomers co-eluted on HPLC. • SPOIS can obtain Sp isomers with 5 min and Rp in 20 min from PS-ON diastereomers. • SPOIS was successfully applied to separate isomers of PS substrates of DNAzyme.

Effect of lung metastasis on the treatment and prognosis of patients with gestational trophoblastic neoplasia: A systematic review and meta-analysis.

INTRODUCTION: Gestational trophoblastic neoplasia (GTN) is a highly invasive tumor, mainly spreading to the lungs. However, lung metastasis in GTN is usually not considered as an adverse prognostic factor. Therefore, the aim of this study was to summarize the results of previous studies and evaluate the effects of lung metastasis on the treatment and prognosis of GTN. MATERIAL AND METHODS: The study was prospectively registered in PROSPERO (CRD42023372371). Electronic databases including PubMed, Embase, the Cochrane Library, Chinese National Knowledge Infrastructure, Wanfang, and China Biomedical Literature Database were used for a systematical search of relevant studies published up to November 21, 2022. The observational studies reporting the clinical outcomes of GTN patients with and without lung metastasis were selected. The incidences of resistance, relapse, and mortality of GTN patients were extracted and successively grouped based on the presence of lung metastasis. The pooled relative risks (RRs) and 95% confidence interval (95% CI) of the eligible studies were calculated. The qualities of included studies were assessed with the Newcastle-Ottawa Scale and the certainty of evidence was graded based on the GRADE. The meta-analysis was performed using Stata 12.0 and GradePro software. RESULTS: Five publications with 3629 GTN patients were included. The meta-analysis revealed that the GTN with lung metastasis was strongly correlated with first-line chemoresistance (pooled RR = 1.40, 95% CI: 1.22 to 1.61, p < 0.001), recurrence (pooled RR = 3.03, 95% CI: 1.21 to 7.62, p = 0.018), and disease-specific death (pooled RR = 22.11, 95% CI: 3.37 to 145.08, p = 0.001). Ethnicity was also an important factor and Caucasian GTN patients with lung metastasis showed a higher risk of recurrence as revealed by the subgroup analysis (pooled RR = 5.10, 95% CI: 2.38 to 10.94, p < 0.001). CONCLUSIONS: GTN patients with lung metastasis exhibited a higher risk of chemoresistance, relapse, and disease-specific death. Patients with lung metastasis among the Caucasian population had a higher risk of recurrence than Asian populations. Therefore, the presence of lung metastases might be considered as a high-risk factor for prognosis of GTN and deserves more attention in the choice of first-line chemotherapy regimens and follow-up.

A [3Fe-4S] cluster and tRNA-dependent aminoacyltransferase BlsK in the biosynthesis of Blasticidin S.

Blasticidin S is a peptidyl nucleoside antibiotic. Its biosynthesis involves a cryptic leucylation and two leucylated intermediates, LDBS and LBS, have been found in previous studies. Leucylation has been proposed to be a new self-resistance mechanism during blasticidin S biosynthesis, and the leucyl group was found to be important for the methylation of ß-amino group of the arginine side chain. However, the responsible enzyme and its associated mechanism of the leucyl transfer process remain to be elucidated. Here, we report results investigating the leucyl transfer step forming the intermediate LDBS in blasticidin biosynthesis. A hypothetical protein, BlsK, has been characterized by genetic and in vitro biochemical experiments. This enzyme catalyzes the leucyl transfer from leucyl-transfer RNA (leucyl-tRNA) to the ß-amino group on the arginine side chain of DBS. Furthermore, BlsK was found to contain an iron-sulfur cluster that is necessary for activity. These findings provide an example of an iron-sulfur protein that catalyzes an aminoacyl-tRNA (aa-tRNA)-dependent amide bond formation in a natural product biosynthetic pathway.

Natural Compound-Rhein and PROTACs Unleash Potent VEGFR-2 Degraders.

VEGFR-2 is a prominent therapeutic target in antitumor drug research to block tumor angiogenesis. This study focused on the synthesis and optimization of PROTACs based on the natural product rhein, resulting in the successful synthesis of 15 distinct molecules. In A549 cells, D9 exhibited remarkable antitumor efficacy with an IC50 of 5.88±0.50 µM, which was 15-fold higher compared to rhein (IC50=88.45±2.77 µM). An in-depth study of the effect of D9 on the degradation of VEGFR-2 revealed that D9 was able to induce the degradation of VEGFR-2 in A549 cells in a time-dependent manner. The observed effect was reversible, contingent upon the proteasome and ubiquitination system, and demonstrably linked to CRBN. Further experiments revealed that D9 induced apoptosis in A549 cells and led to cell cycle arrest in the G1 phase. Molecular docking simulations validated the binding mode of D9 to VEGFR, establishing the potential of D9 to bind to VEGFR-2 in its natural state. In summary, this study confirms the feasibility of natural product-bound PROTAC technology for the development of a new generation of VEGFR-2 degraders, offering a novel trajectory for the future development of pharmacological agents targeting VEGFR-2.

Functional Identification of HhUGT74AG11-A Key Glycosyltransferase Involved in Biosynthesis of Oleanane-Type Saponins in Hedera helix.

Hedera helix is a traditional medicinal plant. Its primary active ingredients are oleanane-type saponins, which have extensive pharmacological effects such as gastric mucosal protection, autophagy regulation actions, and antiviral properties. However, the glycosylation-modifying enzymes responsible for catalyzing oleanane-type saponin biosynthesis remain unidentified. Through transcriptome, cluster analysis, and PSPG structural domain, this study preliminarily screened four candidate UDP-glycosyltransferases (UGTs), including Unigene26859, Unigene31717, CL11391.Contig2, and CL144.Contig9. In in vitro enzymatic reactions, it has been observed that Unigene26859 (HhUGT74AG11) has the ability to facilitate the conversion of oleanolic acid, resulting in the production of oleanolic acid 28-O-glucopyranosyl ester. Moreover, HhUGT74AG11 exhibits extensive substrate hybridity and specific stereoselectivity and can transfer glycosyl donors to the C-28 site of various oleanane-type triterpenoids (hederagenin and calenduloside E) and the C-7 site of flavonoids (tectorigenin). Cluster analysis found that HhUGT74AG11 is clustered together with functionally identified genes AeUGT74AG6, CaUGT74AG2, and PgUGT74AE2, further verifying the possible reason for HhUGT74AG11 catalyzing substrate generalization. In this study, a novel glycosyltransferase, HhUGT74AG11, was characterized that plays a role in oleanane-type saponins biosynthesis in H. helix, providing a theoretical basis for the production of rare and valuable triterpenoid saponins.

Carbon Dots: A Versatile Platform for Cu2+ Detection, Anti-Counterfeiting, and Bioimaging.

Carbon dots (CDs) have garnered extensive interest in basic physical chemistry as well as in biomedical applications due to their low cost, good biocompatibility, and great aqueous solubility. However, the synthesis of multi-functional carbon dots has always been a challenge for researchers. Here, we synthesized novel CDs with a high quantum yield of 28.2% through the straightforward hydrothermal method using Diaminomaleonitrile and Boc-D-2, 3-diaminopropionic acid. The size, chemical functional group, and photophysical properties of the CDs were characterized by TEM, FTIR, XPS, UV, and fluorescence. It was demonstrated in this study that the prepared CDs have a high quantum yield, excellent photostability, and low cytotoxicity. Regarding the highly water-soluble property of CDs, they were proven to possess selective and sensitive behavior against Cu2+ ions (linear range = 0-9 µM and limit of detection = 1.34 µM). Moreover, the CDs were utilized in fluorescent ink in anti-counterfeiting measures. Because of their low cytotoxicity and good biocompatibility, the CDs were also successfully utilized in cell imaging. Therefore, the as-prepared CDs have great potential in fluorescence sensing, anti-counterfeiting, and bioimaging.

A Biomimetic C-Terminal Extension Strategy for Photocaging Amidated Neuropeptides.

Photoactivatable neuropeptides offer a robust stimulus-response relationship that can drive mechanistic studies into the physiological mechanisms of neuropeptidergic transmission. The majority of neuropeptides contain a C-terminal amide, which offers a potentially general site for installation of a C-terminal caging group. Here, we report a biomimetic caging strategy in which the neuropeptide C-terminus is extended via a photocleavable amino acid to mimic the proneuropeptides found in large dense-core vesicles. We explored this approach with four prominent neuropeptides: gastrin-releasing peptide (GRP), oxytocin (OT), substance P (SP), and cholecystokinin (CCK). C-terminus extension greatly reduced the activity of all four peptides at heterologously expressed receptors. In cell type-specific electrophysiological recordings from acute brain slices, subsecond flashes of ultraviolet light produced rapidly activating membrane currents via activation of endogenous G protein-coupled receptors. Subsequent mechanistic studies with caged CCK revealed a role for extracellular proteases in shaping the temporal dynamics of CCK signaling, and a striking switch-like, cell-autonomous anti-opioid effect of transient CCK signaling in hippocampal parvalbumin interneurons. These results suggest that C-terminus extension with a photocleavable linker may be a general strategy for photocaging amidated neuropeptides and demonstrate how photocaged neuropeptides can provide mechanistic insights into neuropeptide signaling that are inaccessible using conventional approaches.

Combined inhibition of surface CD51 and γ-secretase-mediated CD51 cleavage improves therapeutic efficacy in experimental metastatic hepatocellular carcinoma.

BACKGROUND & AIMS: Integrin αv (ITGAV, CD51) is regarded as a key component in multiple stages of tumor progression. However, the clinical failure of cilengitide, a specific inhibitor targeting surface CD51, suggests the importance of yet-unknown mechanisms by which CD51 promotes tumor progression. METHODS: In this study, we used several hepatocellular carcinoma (HCC) cell lines and murine hepatoma cell lines. To investigate the role of CD51 on HCC progression, we used a 3D invasion assay and in vivo bioluminescence imaging. We used periostin-knockout transgenic mice to uncover the role of the tumor microenvironment on CD51 cleavage. Moreover, we used several clinically relevant HCC models, including patient-derived organoids and patient-derived xenografts, to evaluate the therapeutic efficacy of cilengitide in combination with the γ-secretase inhibitor LY3039478. RESULTS: We found that CD51 could undergo transmembrane cleavage by γ-secretase to produce a functional intracellular domain (CD51-ICD). The cleaved CD51-ICD facilitated HCC invasion and metastasis by promoting the transcription of oxidative phosphorylation-related genes. Furthermore, we identified cancer-associated fibroblast-derived periostin as the major driver of CD51 cleavage. Lastly, we showed that cilengitide-based therapy led to a dramatic therapeutic effect when supplemented with LY3039478 in both patient-derived organoid and xenograft models. CONCLUSIONS: In summary, we revealed previously unrecognized mechanisms by which CD51 is involved in HCC progression and uncovered the underlying cause of cilengitide treatment failure, as well as providing evidence supporting the translational prospects of combined CD51-targeted therapy in the clinic. IMPACT AND IMPLICATIONS: Integrin αv (CD51) is a widely recognized pro-tumoral molecule that plays a crucial role in various stages of tumor progression, making it a promising therapeutic target. However, despite early promising results, cilengitide, a specific antagonist of CD51, failed in a phase III clinical trial. This prompted further investigation into the underlying mechanisms of CD51's effects. This study reveals that the γ-secretase complex directly cleaves CD51 to produce an intracellular domain (CD51-ICD), which functions as a pro-tumoral transcriptional regulator and can bypass the inhibitory effects of cilengitide by entering the nucleus. Furthermore, the localization of CD51 in the nucleus is significantly associated with the prognosis of patients with HCC. These findings provide a theoretical basis for re-evaluating cilengitide in clinical settings and highlight the importance of identifying a more precise patient subpopulation for future clinical trials targeting CD51.

The CAP superfamily protein PsCAP1 secreted by Phytophthora triggers immune responses in Nicotiana benthamiana through a leucine-rich repeat receptor-like protein.

The role of cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 (CAP) superfamily proteins in the innate immune responses of mammals is well characterized. However, the biological function of CAP superfamily proteins in plant-microbe interactions is poorly understood. We used proteomics and transcriptome analyses to dissect the apoplastic effectors secreted by the oomycete Phytophthora sojae during early infection of soybean leaves. By transiently expressing these effectors in Nicotiana benthamiana, we identified PsCAP1, a novel type of secreted CAP protein that triggers immune responses in multiple solanaceous plants including N. benthamiana. This secreted CAP protein is conserved among oomycetes, and multiple PsCAP1 homologs can be recognized by N. benthamiana. PsCAP1-triggered immune responses depend on the N-terminal immunogenic fragment (aa 27-151). Pretreatment of N. benthamiana with PsCAP1 or the immunogenic fragment increases plant resistance against Phytophthora. The recognition of PsCAP1 and different homologs requires the leucine-rich repeat receptor-like protein RCAP1, which associates with two central receptor-like kinases BRI1-associated receptor kinase 1 (BAK1) and suppressor of BIR1-1 (SOBIR1) in planta. These findings suggest that the CAP-type apoplastic effectors act as an important player in plant-microbe interactions that can be perceived by plant membrane-localized receptor to activate plant resistance.