Mutual Activation Research Articles

Signaling Pathways (TNF-α-NF-κB, TLR2-TLR4 as well as ROS-MDA) and Cardiac Damages during Cardiac Surgeries (Coronary stenting, Permanent Pacemaker Implantations, Radiofrequency Ablations).

The mutual activations of multiple signaling pathways are the key factors in the development and progression of myocardial cell injuries. This research aimed to compare the different degrees of myocardial injury after coronary stenting, permanent pacemaker implantations, or cardiac radiofrequency ablation and to investigate the effects of the mutual activation of TNF-α/NF-κB, TLR2/TLR4, and ROS/MDA signaling pathways on myocardial injury in elderly patients after coronary stents or permanent pacemakers or radiofrequency ablation. We determined reactive oxygen species (ROS), malondialdehyde (MDA), toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4), nuclear factor kappa B (NF-κB), tumor necrosis factor- α (TNF-α) and high-sensitive cardiac troponin T (hs-cTnT) as a marker of myocardial injury in patients. The levels of ROS, MDA, TLR2, TLR4, NF-κB, TNF-α, and hs-cTnT were increased in patients with permanent pacemaker implantations when compared to patients with cardiac radiofrequency ablation (P < 0.01) at 6 months and were further increased in patients with coronary stenting compared to patients with cardiac radiofrequency ablation and permanent pacemaker implantations at 6 months, respectively (P < 0.01). This research confirmed that ROS, MDA, TLR2, TLR4, NF-κB, and TNF-α predicted myocardial injury severity. Oxidative stress (ROS/MDA signaling pathway) may be linked to immune response (TLR2/TLR4 signaling pathway) and pro-inflammatory response (TNF-α/NF-κB signaling pathway) in myocardial injury, and ROS/MDA signaling may play a dominant role.

Church Conflict, Loneliness, and the Bid to Belong

Loneliness, a perceived sense of isolation, has been demonstrated to have deleterious effects on both physical and mental health. Numerous studies suggest that the prevalence of loneliness is increasing over time and is common in the church, even among pastors. Many researchers now conceptualize the pain of loneliness as an evolutionarily adaptive signal that prompts the lonely person to make social connections. Loneliness, however, can also increase one’s hypervigilance to the threat of rejection, leading to a cycle of behavior and biased interpretation called the “loneliness loop.” Drawing upon Baumeister’s need-to-belong theory, this article is a concept paper that suggests that congregations are places of belonging, and that some church conflict can be understood as stemming from failed bids for social connection, with the conflict being reciprocally maintained by the mutual activation of the loneliness loop. This perspective will be illustrated through two vignettes drawn from the published ministry literature.

ASCT2 Regulates Fatty Acid Metabolism to Trigger Glutamine Addiction in Basal-like Breast Cancer.

As a crucial amino acid, glutamine can provide the nitrogen and carbon sources needed to support cancer cell proliferation, invasion, and metastasis. Interestingly, different types of breast cancer have different dependences on glutamine. This research shows that basal-like breast cancer depends on glutamine, while the other types of breast cancer may be more dependent on glucose. Glutamine transporter ASCT2 is highly expressed in various cancers and significantly promotes the growth of breast cancer. However, the key regulatory mechanism of ASCT2 in promoting basal-like breast cancer progression remains unclear. Our research demonstrates the significant change in fatty acid levels caused by ASCT2, which may be a key factor in glutamine sensitivity. This phenomenon results from the mutual activation between ASCT2-mediated glutamine transport and lipid metabolism via the nuclear receptor PPARα. ASCT2 cooperatively promoted PPARα expression, leading to the upregulation of lipid metabolism. Moreover, we also found that C118P could inhibit lipid metabolism by targeting ASCT2. More importantly, this research identifies a potential avenue of evidence for the prevention and early intervention of basal-like breast cancer by blocking the glutamine-lipid feedback loop.

Deciphering iopamidol transformation during the joint disinfection of monochloramine and peroxymonosulfate: A base-catalyzed mutual activation

The joint disinfection of monochloramine (MCA) and peroxymonosulfate (PMS) can combine the advantages of both sides; and the iodine-containing micropollutants are widely present in drinking water. For the first time, the conversion of iopamidol (IPM) was investigated in the MCA/PMS system. For IPM degradation, the observed pseudo-first-order rate constant (kobs) was 0.0076 min−1, and the apparent rate constant (kapp) was 6.14 × 10−7 μM−2.16 min−1. The reaction orders for MCA (m) and PMS (n) were fitted to be 1.14 and 1.02, respectively. The mutual activation of MCA and PMS was a base-catalyzed reaction, which was highly exothermic and thermodynamically favored with a free energy of − 78.74 kcal/mol. It is believed that Cl• and 1O2 contributed to IPM degradation. The kobs value was increased by an alkaline pH and the presence of Cl−, while reduced by the existence of NOM, HCO3− and NH4+. The N in MCA was converted to NH4+-N, NO2−-N and NO3−-N, and 47.0 % of I in IPM was converted to IO3−. In the MCA/PMS system, IPM transformation was mainly via amide hydrolysis, deiodination and substitution reaction by reactive species. The study provides an insight into the fate of iodine-containing micropollutants during the joint disinfection of MCA and PMS.

2,3-Difunctionalization of quinones: a gold-catalyzed cascade approach for trifluoromethyl-amination or sulfoximination.

A one-pot domino protocol employing gold(I) catalysis has been developed for the cascade trifluoromethyl-amination/sulfoximination of quinones. Togni I serves as the trifluoromethyl installing precursor, while amine or sulfoximine serves as the aminating source. Preliminary investigations suggest a mutual activation of Togni I and the amine precursor, facilitating the facile difunctionalization of quinones with excellent regioselectivity. Extensive substrate scope exploration demonstrates moderate to good yields of difunctionalized products. Application to the natural product Juglone highlights its potential for late-stage modifications in medicinal chemistry and drug discovery.

CHARACTERISTICS OF MUTUAL ACTIVATION OF AN INTERGEL SYSTEM BASED ON HYDROGEL POLYMETHACRYL ACID AND POLY-4-VINYL PYRIDINE

The electrochemical properties of mutual activation of polymer networks as a result of remote interaction in an intergel system have been studied. An intergel system consisting of hydrogels of rare crosslinked polymethacrylic acid (hPMAA) and poly-4-vinylpyridine (hP4VP) was chosen as the object of study [1,2]. The purpose of the work is to study the mutual activation features of the intergel system of rare crosslinked hPMAA: hP4VP. The obtained results as the contact time of hydrogels with water increased, zones of maximum and minimum conductivity were observed at the highest points of initial conductivity for different ratios of hPMAA:hP4VP hydrogel systems. In the 6:0 ratio of hPMAA hydrogel, the specific conductivity of the aqueous medium reached its maximum value after one day, and the pH value of the medium decreased relatively to about 4.95. The swelling degree of hPMAA hydrogel was constant at 6:0 different time intervals. In this system, only one polymer, namely hPMAA showed that the hydrogel's water absorption capacity was limited. In the 5:1 ratio of the intergel system, the specific conductivity of the medium reached a high value, and the pH value decreased in the 5:1 ratio. After 2.5 hours of study, a decrease in the conductivity of the aqueous medium was observed at 4:2 hydrogel ratio. After 6 hours, the highest degree of swelling was maintained at 1:5 ratio. In the presence of hP4VP hydrogel at a ratio of 0:6, the pH of the medium increased from 5 to a maximum value of 6.6 within 24 hours. Conclusion. The electrochemical properties of hPMAA:hP4VP hydrogel system were studied by remote interaction. Based on the systematic work, we synthesized the required acidic hydrogel hPMAA:hP4VP and carried out various studies on their properties. To create an intergel system, the obtained hydrogels in seven different proportions were made into an intergel system and their interactions were studied using various physicochemical methods. As a result of the study, the remote interaction of hydrogels in the intergel system leads to conformational changes in their intercellular bonds which undergo additional swelling. As a result of mutual activation, the hydrogels move to a highly ionized state.

Dissecting thrombus-directed chemotaxis and random movement in neutrophil near-thrombus motion in flow chambers

BackgroundThromboinflammation is caused by mutual activation of platelets and neutrophils. The site of thromboinflammation is determined by chemoattracting agents release by endothelium, immune cells, and platelets. Impaired neutrophil chemotaxis contributes to the pathogenesis of Shwachman-Diamond syndrome (SDS). In this hereditary disorder, neutrophils are known to have aberrant chemoattractant-induced F-actin properties. Here, we aim to determine whether neutrophil chemotaxis could be analyzed using our previously developed ex vivo assay of the neutrophils crawling among the growing thrombi.MethodsAdult and pediatric healthy donors, alongside with pediatric patients with SDS, were recruited for the study. Thrombus formation and granulocyte movement in hirudinated whole blood were visualized by fluorescent microscopy in fibrillar collagen-coated parallel-plate flow chambers. Alternatively, fibrinogen, fibronectin, vWF, or single tumor cells immobilized on coverslips were used. A computational model of chemokine distribution in flow chamber with a virtual neutrophil moving in it was used to analyze the observed data.ResultsThe movement of healthy donor neutrophils predominantly occurred in the direction and vicinity of thrombi grown on collagen or around tumor cells. For SDS patients or on coatings other than collagen, the movement was characterized by randomness and significantly reduced velocities. Increase in wall shear rates to 300–500 1/s led to an increase in the proportion of rolling neutrophils. A stochastic algorithm simulating leucocyte chemotaxis movement in the calculated chemoattractant field could reproduce the experimental trajectories of moving neutrophils for 72% of cells.ConclusionsIn samples from healthy donors, but not SDS patients, neutrophils move in the direction of large, chemoattractant-releasing platelet thrombi growing on collagen.

Anti-lung cancer synergy of low-dose doxorubicin and PD-L1 blocker co-delivered via mild photothermia-responsive black phosphorus.

We have previously identified a latent interaction mechanism between non-small cell lung cancer cells (NSCLCC) and their associated macrophages (TAM) mediated by mutual paracrine activation of the HMGB1/RAGE/NF-κB signaling. Activation of this mechanism results in TAM stimulation and PD-L1 upregulation in the NSCLCC. In the present work, we found that free DOX at a low concentration that does not cause DNA damage could activate the HMGB1/RAGE/NF-κB/PD-L1 pathway byinducing oxidative stress. It was thus proposed that a combination of low-dose DOX and a PD-L1 blocker delivered in the NSCLC tumor would achieve synergistic TAM stimulation and thereby synergetic anti-tumor potency. To prove this idea, DOX and BMS-202 (a PD-L1 blocker) were loaded to black phosphorus (BP) nanoparticles after dosage titration to yield the BMS-202/DOX@BP composites that rapidly disintegrated and released drug cargo upon mild photothermal heating at 40°C. In vitro experiments then demonstrated that low-dose DOX and BMS-202 delivered via BMS-202/DOX@BP under mild photothermia displayed enhanced tumor cell toxicity with a potent synergism only in the presence of TAM. This enhanced synergism was due to an anti-tumor M1-like TAM phenotype that was synergistically induced by low dose DOX plus BMS-202 only in the presence of the tumor cells, indicating the damaged tumor cells to be the cardinal contributor to the M1-like TAM stimulation. In vivo, BMS-202/DOX@BP under mild photothermia exhibited targeted delivery to NSCLC graft tumors in mice and synergistic anti-tumor efficacy of delivered DOX and BMS-202. In conclusion, low-dose DOX in combination with a PD-L1 blocker is an effective strategy to turn TAM against their host tumor cells exploiting the HMGB1/RAGE/NF-κB/PD-L1 pathway. The synergetic actions involved highlight the value of TAM and the significance of modulating tumor cell-TAM cross-talk in tumor therapy. Photothermia-responsive BP provides an efficient platform to translate this strategy into targeted, efficacious tumor therapy.

Abstract 5638: The BAF complex mediates intertwined activity of YAP1 and β-catenin in synovial sarcoma

Abstract Introduction: Synovial sarcoma (SySa) is a rare malignant soft tissue tumor characterized by a specific reciprocal translocation t(X;18). The resulting chimeric SS18::SSX fusion protein drives SySa tumorigenesis by integrating into BAF chromatin remodeling complexes, changing their composition and dysregulating gene transcription. The role of BAF complexes in cancer steadily increases with ∼20% of all malignancies having alterations in BAF complex subunits. BAF complexes are multiprotein complexes with several interaction partners determining their localization and functionality. Since previous analyses revealed an induced activation of the transcriptional coregulators YAP1 and β-catenin in SySa, the aim of this study was to analyze if nuclear YAP1 and β-catenin are interacting with BAF complexes and if this has an impact on their transcriptional activity. Experimental procedures: Co-immunoprecipitation experiments on nuclear protein extracts and proximity ligation assays were performed to investigate interactions between YAP1, β-catenin, and BAF complex subunits in SySa cells. Knockdown of common and specific BAF complex subunits was conducted employing RNA interference. To assess changes in YAP1 and β-catenin transcriptional activities, TEAD and TOPFlash luciferase reporter assays were performed, respectively, and protein expression levels were detected by immunoblotting. Results: In SySa, protein-protein interaction experiments revealed nuclear interactions between β-catenin, YAP1, the SS18::SSX fusion protein and various BAF subunits. YAP1 and β-catenin’s transcriptional activities were found to be interdependent as they mutually enhanced each other’s activation. Knockdown of SS18::SSX as well as specific BAF complex subunits was associated with diminished transcriptional activities of these coregulators and, interestingly, substantially affected their mutual activation. This indicates that SS18::SSX-containing BAF complex assembly is crucial for interdependent YAP1 and β-catenin activation in SySa cells. Conclusions: This study reveals a significant role of the SySa-specific fusion protein and BAF chromatin remodeling complexes in mediating intertwined activation of YAP1 and β-catenin. Citation Format: Ilka Isfort, Ruth Berthold, Lorena Heinst, Anna Kuntze, Eva Wardelmann, Marcel Trautmann, Wolfgang Hartmann. The BAF complex mediates intertwined activity of YAP1 and β-catenin in synovial sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5638.

Mutual Activation Mechanism of Cement–GGBS–Steel Slag Ternary System Excited by Sodium Sulfate

To promote the large-scale recycling of solid waste, the hydration characteristics of blended cement with different amounts of GGBS (ground granulated blast-furnace slag) and SS (steel slag) were investigated. The optimum blending amounts of GGBS and SS in cement were 40% and 10% by mass, and the optimum dosage of Na2SO4 in the C50-S40-SS10 (50 wt.% cement–40 wt.% slag–10 wt.% steel slag) system was 2 wt.%. The flexural and compressive strengths of the C50-S40-SS10 system after adding 2 wt.% Na2SO4 are 57.95% and 9.28% higher than that of pure cement at 28 d. XRD, FT-IR and Ca(OH)2 content analysis were chosen to investigate the hydration products of pure cement and blended cement. The results show that GGBS enhanced the hydration of both cement and SS. And GGBS contributed to the generation of calcium silicoaluminate hydrate (C–A–S–H) in the blended cement system. The addition of Na2SO4 promoted the hydration reaction and contributed to the generation of ettringite (AFt) in the ternary system. The hydration heat evolution results showed that GGBS and SS can reduce the hydration heat of cement. Na2SO4 had similar effects and delayed the time of AFt conversion to monosulfide calcium sulphoaluminate (AFm). A mutual activation mechanism of cement–GGBS–SS ternary system mixed with Na2SO4 was proposed in this study.

Single-cell RNA sequencing reveals roles of unique retinal microglia types in early diabetic retinopathy

BackgroundThe pathophysiological mechanisms of diabetic retinopathy (DR), a blinding disease, are intricate. DR was thought to be a microvascular disease previously. However, growing studies have indicated that the retinal microglia-induced inflammation precedes microangiopathy. The binary concept of microglial M1/M2 polarization paradigms during inflammatory activation has been debated. In this study, we confirmed microglia had the most significant changes in early DR using single-cell RNA sequencing.MethodsA total of five retinal specimens were collected from donor SD rats. Changes in various cells of the retina at the early stage of DR were analyzed using single-cell sequencing technology.ResultsWe defined three new microglial subtypes at cellular level, including two M1 types (Egr2+ M1 and Egr2− M1) and one M2 type. We also revealed the anatomical location between these subtypes, the dynamic changes of polarization phenotypes, and the possible activation sequence and mutual activation regulatory mechanism of different cells. Furthermore, we constructed an inflammatory network involving microglia, blood-derived macrophages and other retinal nonneuronal cells. The targeted study of new disease-specific microglial subtypes can shorten the time for drug screening and clinical application, which provided insight for the early control and reversal of DR.ConclusionsWe found that microglia show the most obvious differential expression changes in early DR and reveal the changes in microglia in a high-glucose microenvironment at the single-cell level. Our comprehensive analysis will help achieve early reversal and control the occurrence and progression of DR.

Роль тромбоцитов в патогенезе инфекционно-воспалительных заболеваний легких у детей (обзор литературы)

At present, a subject of scientific interest is the interaction of immune systems and hemostasis. New data on their synergistic effect in various diseases have been obtained in the last decade, which allowed to introduce the concept of "immunothrombosis". Inflammation and thrombosis factors are capable of mutual activation and enhancement. A special role in these processes belongs to platelets, which are classified as cellular effectors of antimicrobial host defence. These cells are among the first to react to the pathogen when it enters the bloodstream. Platelet activation ensures the direct eradication of pathogens, the immune cells involvement, the formation of primary thrombi to prevent the spread of infection, and the coagulation system process start. However, microthrombosis can become uncontrollable and irreversible during continued activation. Platelet dysfunction may be associated with a hyperergic inflammatory response of the body and a severe disease course. Concomitant endothelial dysfunction intensifies hypercoagulation and inflammatory response, increasing the risk of systemic inflammatory processes. The review examines current data on the platelet role in the pathogenesis of infectious and inflammatory pulmonary diseases in children, including destructive ones. KEYWORDS: hemostasis, platelets, immunity, inflammation, pneumonia, children, neutrophil extracellular traps, microthrombosis. FOR CITATION: Tolstova E.M., Besedina M.V., Zaytseva O.V., Kuzina S.R., Lokshina E.E., Haspekov D.V., Turishchev I.V., Belyaeva T.Yu. Platelets in the pathogenesis of infectious and inflammatory pulmonary diseases in children (literature review). Russian Journal of Woman and Child Health. 2024;7(1):58–63 (in Russ.). DOI: 10.32364/2618-8430-2024-7-1-9.

A Metal-Free and Operationally Simple Radical Trifluoromethylative Borylation of Unactivated Alkenes.

We herein describe a protocol to synthesize trifluoromethylated alkyl boronates from alkenes by the mutual activation of the Togni II and the bis(catecholato)diboron reagents in the absence of any catalyst and additives. This reaction enables synthesizing a series of trifluoromethylated alkyl boronates using unactivated alkenes, including natural products and drug derivatives, in a regioselective manner. Moreover, the synthetic utility of the boronic ester present in the product allows access to a range of trifluoromethyl containing compounds. The radical trapping and gas detection experiments reveal that the more Lewis acidic diboron reagent determines the rapid formation of trifluoromethyl and boron centered radicals.

The Ways of the Virus: Interactions of Platelets and Red Blood Cells with SARS-CoV-2, and Their Potential Pathophysiological Significance in COVID-19.

The hematological effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are important in COVID-19 pathophysiology. However, the interactions of SARS-CoV-2 with platelets and red blood cells are still poorly understood. There are conflicting data regarding the mechanisms and significance of these interactions. The aim of this review is to put together available data and discuss hypotheses, the known and suspected effects of the virus on these blood cells, their pathophysiological and diagnostic significance, and the potential role of platelets and red blood cells in the virus's transport, propagation, and clearance by the immune system. We pay particular attention to the mutual activation of platelets, the immune system, the endothelium, and blood coagulation and how this changes with the evolution of SARS-CoV-2. There is now convincing evidence that platelets, along with platelet and erythroid precursors (but not mature erythrocytes), are frequently infected by SARS-CoV-2 and functionally changed. The mechanisms of infection of these cells and their role are not yet entirely clear. Still, the changes in platelets and red blood cells in COVID-19 are significantly associated with disease severity and are likely to have prognostic and pathophysiological significance in the development of thrombotic and pulmonary complications.

Transcription Factors HNF1A, HNF4A, and FOXA2 Regulate Hepatic Cell Protein N-Glycosylation

Hepatocyte nuclear factor 1 alpha (HNF1A), hepatocyte nuclear factor 4 alpha (HNF4A), and forkhead box protein A2 (FOXA2) are key transcription factors that regulate a complex gene network in the liver, creating a regulatory transcriptional loop. The Encode and ChIP-Atlas databases identify the recognition sites of these transcription factors in many glycosyltransferase genes. Our in silico analysis of HNF1A, HNF4A, and FOXA2 binding to the ten candidate glyco-genes studied in this work confirms a significant enrichment of these transcription factors specifically in the liver. Our previous studies identified HNF1A as a master regulator of fucosylation, glycan branching, and galactosylation of plasma glycoproteins. Here, we aimed to functionally validate the role of the three transcription factors on downstream glyco-gene transcriptional expression and the possible effect on glycan phenotype. We used the state-of-the-art clustered regularly interspaced short palindromic repeats/dead Cas9 (CRISPR/dCas9) molecular tool for the downregulation of the HNF1A, HNF4A, and FOXA2 genes in HepG2 cells—a human liver cancer cell line. The results show that the downregulation of all three genes individually and in pairs affects the transcriptional activity of many glyco-genes, although downregulation of glyco-genes was not always followed by an unambiguous change in the corresponding glycan structures. The effect is better seen as an overall change in the total HepG2 N-glycome, primarily due to the extension of biantennary glycans. We propose an alternative way to evaluate the N-glycome composition via estimating the overall complexity of the glycome by quantifying the number of monomers in each glycan structure. We also propose a model showing feedback loops with the mutual activation of HNF1A–FOXA2 and HNF4A–FOXA2 affecting glyco-genes and protein glycosylation in HepG2 cells.

Crosstalk enables mutual activation of coupled quorum sensing pathways through “jump-start” and “push-start” mechanisms

Many quorum sensing microbes produce more than one chemical signal and detect them using interconnected pathways that crosstalk with each other. While there are many hypotheses for the advantages of sensing multiple signals, the prevalence and functional significance of crosstalk between pathways are much less understood. We explore the effect of intracellular signal crosstalk using a simple model that captures key features of typical quorum sensing pathways: multiple pathways in a hierarchical configuration, operating with positive feedback, with crosstalk at the receptor and promoter levels. We find that crosstalk enables activation or inhibition of one output by the non-cognate signal, broadens the dynamic range of the outputs, and allows one pathway to modulate the feedback circuit of the other. Our findings show how crosstalk between quorum sensing pathways can be viewed not as a detriment to the processing of information, but as a mechanism that enhances the functional range of the full regulatory system. When positive feedback systems are coupled through crosstalk, several new modes of activation or deactivation become possible.

The novel RNA-RNA activation of H19 on MyoD transcripts promoting myogenic differentiation of goat muscle satellite cells

The elaborate interplay of coding and noncoding factors governs muscle growth and development. Here, we reported a mutual activation between long noncoding RNA (lncRNA) H19 and MyoD (myogenic determination gene number 1) in the muscle process. We successfully cloned the two isoforms of goat H19, which were significantly enriched and positively correlated with MyoD transcripts in skeletal muscles or differentiating muscle satellite cells (MuSCs). To systematically screen genes altered by H19, we performed RNA-seq using cDNA libraries of differentiating H19-deficiency MuSCs and consequently anchored MyoD as the critical genes in mediating H19 function. Intriguingly, some transcripts of MyoD and H19 overlapped in the cytoplasm, which was dramatically damaged when the core complementary nucleotides were mutated. Meanwhile, MyoD RNA successfully pulled down H19 in MS2-RIP experiments. Furthermore, HuR could bind both H19 and MyoD transcripts, while H19 or its truncated mutants successfully stabilized MyoD mRNA, with or without HuR deficiency. In turn, novel functional MyoD protein-binding sites were identified in the promoter and exons of the H19 gene. Our results suggest that MyoD activates H19 transcriptionally, and RNA-RNA hybridization is critical for H19-promoted MyoD expression, which extends our knowledge of the hierarchy of regulatory networks in muscle growth.

Ultrasensitive quantitation of Paraquat based on a small molecule-induced dual-cycle amplification strategy

Paraquat (PQ) is a typical biotoxic small molecule. Knowledge of how to directly introduce it into cyclic amplification rather than transform it into a secondary target is lacking in current analytical methods. Considering the urgent need for trace pesticide residue detection and the inherent defects of small molecule analysis, a CRISPR/Cas12a-driven small molecule-induced dual-cycle strategy was developed based on the immune competition method. The key to signal amplification is the mutual activation and acceleration between Cycle 1 triggered by the small molecule and Cycle 2 driven by CRISPR/Cas12a. Impressively, small molecules have been successfully incorporated into the dual-cycle strategy, which achieves a low detection limit (3.1 pg/mL) and a wide linear range (from 10 pg/mL to 50 μg/mL). Moreover, the designed biosensor was successfully employed to evaluate the PQ residual level in real samples and showed effective implementation for the bioanalysis of small molecule targets and pesticide residue-related food safety.

The autoactivation of human single-chain urokinase-type plasminogen activator (uPA)

Most serine proteases are synthesized as inactive zymogens that are activated by cleavage by another protease in a tightly regulated mechanism. The urokinase-type plasminogen activator (uPA) and plasmin cleave and activate each other, constituting a positive feedback loop. How this mutual activation cycle begins has remained a mystery. We used hydrogen deuterium exchange mass spectrometry to characterize the dynamic differences between the inactive single-chain uPA (scuPA) and its active form two-chain uPA (tcuPA). The results show that the C-terminal β-barrel and the area around the new N terminus have significantly reduced dynamics in tcuPA as compared with scuPA. We also show that the zymogen scuPA is inactive but can, upon storage, become active in the absence of external proteases. In addition to plasmin, the tcuPA can activate scuPA by cleavage at K158, a process called autoactivation. Unexpectedly, tcuPA can cleave at position 158 even when this site is mutated. TcuPA can also cleave scuPA after K135 or K136 in the disordered linker, which generates the soluble protease domain of uPA. Plasmin cleaves scuPA exclusively after K158 and at a faster rate than tcuPA. We propose a mechanism by which the uPA receptor dimerization could promote autoactivation of scuPA on cell surfaces. These results resolve long-standing controversies in the literature surrounding the mechanism of uPA activation.

The crescent-like Golgi ribbon is shaped by the Ajuba/PRMT5/Aurora-A complex-modified HURP

BackgroundGolgi apparatus (GA) is assembled as a crescent-like ribbon in mammalian cells under immunofluorescence microscope without knowing the shaping mechanisms. It is estimated that roughly 1/5 of the genes encoding kinases or phosphatases in human genome participate in the assembly of Golgi ribbon, reflecting protein modifications play major roles in building Golgi ribbon.MethodsTo explore how Golgi ribbon is shaped as a crescent-like structure under the guidance of protein modifications, we identified a protein complex containing the scaffold proteins Ajuba, two known GA regulators including the protein kinase Aurora-A and the protein arginine methyltransferase PRMT5, and the common substrate of Aurora-A and PRMT5, HURP. Mutual modifications and activation of PRMT5 and Aurora-A in the complex leads to methylation and in turn phosphorylation of HURP, thereby producing HURP p725. The HURP p725 localizes to GA vicinity and its distribution pattern looks like GA morphology. Correlation study of the HURP p725 statuses and GA structure, site-directed mutagenesis and knockdown-rescue experiments were employed to identify the modified HURP as a key regulator assembling GA as a crescent ribbon.ResultsThe cells containing no or extended distribution of HURP p725 have dispersed GA membranes or longer GA. Knockdown of HURP fragmentized GA and HURP wild type could, while its phosphorylation deficiency mutant 725A could not, restore crescent Golgi ribbon in HURP depleted cells, collectively indicating a crescent GA-constructing activity of HURP p725. HURP p725 is transported, by GA membrane-associated ARF1, Dynein and its cargo adaptor Golgin-160, to cell center where HURP p725 forms crescent fibers, binds and stabilizes Golgi assembly factors (GAFs) including TRIP11, GRASP65 and GM130, thereby dictating the formation of crescent Golgi ribbon at nuclear periphery.ConclusionsThe Ajuba/PRMT5/Aurora-A complex integrates the signals of protein methylation and phosphorylation to HURP, and the HURP p725 organizes GA by stabilizing and recruiting GAFs to its crescent-like structure, therefore shaping GA as a crescent ribbon. Therefore, the HURP p725 fiber serves a template to construct GA according to its shape.6goRpNfQL_y2Q765kUtnH_Video