Assembly process and co-occurrence network of microbial community in response to free ammonia gradient distribution.

Microorganisms are crucial components of the aquatic ecosystem due to their immense diversity and abundance. They are vital in sustaining ecological services, especially in maintaining essential biogeochemical cycles. Recent years have seen a substantial increase in surplus nitrogenous pollutants in aquatic ecosystems due to the heightened occurrence of anthropogenic activities. Elevated levels of free ammonia (FA, NH3), stemming from the discharge of excess nitrogenous pollutants, have caused notable fluctuations in aquatic ecosystems, leading to water eutrophication and various ecological challenges. The impact of these oscillations on microbial communities in aquatic ecosystems has not been extensively studied. This study employed 16S rRNA gene amplicon sequencing to systematically investigate the dynamics, co-occurrence networks, and assembly processes of microbial communities and their subcommunities (abundant, moderate, and rare) in the Luanhe River Diversion Project in China. Our findings indicate that NH3 concentration significantly influences the dynamics of microbial communities, with a notable decrease in community Richness and Phylogenetic Distance alongside increased community dissimilarity under higher NH3 conditions. The analysis revealed that certain microbial groups, particularly Actinobacteriaota, were notably more prevalent in environments with elevated NH3 levels, suggesting their potential resilience or adaptive responses to NH3 stress. Additionally, through co-occurrence network analysis, we observed dynamic changes in network topology and increased connectedness under NH3 stress. Key nodes, identified as connectors and module hubs, played crucial roles in maintaining network structure, particularly Cyanobacteria and Actinobacteriaota. Furthermore, stochastic processes, particularly drift and dispersal limitation, predominantly shaped the microbial communities. Within the three subcommunities, the impact of drift became more pronounced as the effect of dispersal limitation diminished. Overall, elucidating the dynamics of microbial communities in aquatic ecosystems exposed to NH3 can enhance our comprehension of the ecological mechanisms of microbial communities and provide new insights into the conservation of microbial community diversity and ecological functions. IMPORTANCE: The research presented in this paper explores how varying concentrations of free ammonia impact microbial communities in aquatic ecosystems. By employing advanced gene sequencing techniques, the study reveals significant changes in microbial diversity and network structures in response to increased ammonia levels. Key findings indicate that high ammonia concentrations lead to a decrease in microbial richness and diversity while increasing community dissimilarity. Notably, certain microbial groups, like Actinobacteria, show resilience to ammonia stress. This research enhances our understanding of how pollution affects microbial ecosystems and underscores the importance of maintaining balanced ammonia levels to preserve microbial diversity and ecosystem health.

Biochar alleviates inhibition effects of humic acid on anaerobic digestion: Insights to performances and mechanisms.

To recover methane from waste activated sludge through anaerobic digestion (AD) is one promising alternative to achieve carbon neutrality for wastewater treatment plants. However, humic acids (HAs) are one of the major compositions in waste activated sludge, and their accumulation performs inhibition effects on AD. This study investigated the potentials of biochar (BC) in alleviating inhibition effects of HAs on AD. Results showed that although the accumulated HAs reduced methane yield by 9.37% compared to control, the highest methane yield, 132.6 mL CH4/g VSS, was obtained after adding BC, which was 45.9% higher than that in HA group. Mechanism analysis showed that BC promoted the activities of hydrolase such as protease and α-glucosidase, which were 69.7% and 29.7% higher than those in HA group, respectively. The conversion of short-chain fatty acids was accelerated. In addition, the evolutions of electroactive microorganisms like Clostridium_sensu_stricto_13 and Methanosaeta were consistent with the activitiies of electron transfer and the contents of cytochrome c. Furthermore, parts of HAs rather than all of them were adsorbed by BC, and the remaining free HAs and BC formed synergistic effects on methanogenesis, then both CO2 reduction and acetoclastic methanogenesis pathways were improved. The findings may provide some solutions to alleviate inhibition effects of HAs on AD.

The Circadian Clock Component RORA Increases Immunosurveillance in Melanoma by Inhibiting PD-L1 Expression.

Circadian clock perturbation frequently occurs in cancer and facilitates tumor progression by regulating malignant growth and shaping the immune microenvironment. Emerging evidence has indicated that clock genes are disrupted in melanoma and linked to immune escape. Herein, we found that the expression of retinoic acid receptor-related orphan receptor-α (RORA) is downregulated in melanoma patients and that patients with higher RORA expression have a better prognosis after immunotherapy. Additionally, RORA was significantly positively correlated with T-cell infiltration and recruitment. Overexpression or activation of RORA stimulated cytotoxic T-cell-mediated antitumor responses. RORA bound to the CD274 promoter and formed an inhibitory complex with HDAC3 to suppress PD-L1 expression. In contrast, the DEAD-box helicase family member DDX3X competed with HDAC3 for binding to RORA, and DDX3X overexpression promoted RORA release from the suppressive complex and thereby increased PD-L1 expression to generate an inhibitory immune environment. The combination of a RORA agonist with an anti-CTLA4 antibody synergistically increased T-cell antitumor immunity in vivo. A score based on the combined expression of HDAC3, DDX3X, and RORA correlated with immunotherapy response in melanoma patients. Together, this study elucidates a mechanism of clock component-regulated antitumor immunity, which will help inform the use of immunotherapy and lead to improved outcomes for melanoma patients receiving combined therapeutic treatments. Significance: RORA forms a corepressor complex to inhibit PD-L1 expression and activate antitumor T-cell responses, indicating that RORA is a potential target and predictive biomarker to improve immunotherapy response in melanoma patients.

Softening the tumor matrix through cholesterol depletion breaks the physical barrier for enhanced antitumor therapy.

The tumor develops defense tactics, including conversing the mechanical characteristics of tumor cells and their surrounding environment. A recent study reported that cholesterol depletion stiffens tumor cells, which could enhance adaptive T-cell immunotherapy. However, it remains unclear whether reducing the cholesterol in tumor cells contributes to re-educating the stiff tumor matrix, which serves as a physical barrier against drug penetration. Herein, we found that depleting cholesterol from tumor cells can demolish the intratumor physical barrier by disrupting the mechanical signal transduction between tumor cells and the extracellular matrix through the destruction of lipid rafts. This disruption allows nanoparticles (H/S@hNP) to penetrate deeply, resulting in improved photodynamic treatment. Our research also indicates that cholesterol depletion can inhibit the epithelial-mesenchymal transition and repolarize tumor-associated macrophages from M2 to M1, demonstrating the essential role of cholesterol in tumor progression. Overall, this study reveals that a cholesterol-depleted, softened tumor matrix reduces the difficulty of drug penetration, leading to enhanced antitumor therapeutics.

Antigen specific VNAR screening in whitespotted bamboo shark (Chiloscyllium plagiosum) with next generation sequencing.

IgNAR exhibits significant promise in the fields of cancer and anti-virus biotherapies. Notably, the variable regions of IgNAR (VNAR) possess comparable antigen binding affinity with much smaller molecular weight (∼12 kDa) compared to IgNAR. Antigen specific VNAR screening is a changeling work, which limits its application in medicine and therapy fields. Though phage display is a powerful tool for VNAR screening, it has a lot of drawbacks, such as small library coverage, low expression levels, unstable target protein, complicating and time-consuming procedures. Here we report VANR screening with next generation sequencing (NGS) could effectively overcome the limitations of phage display, and we successfully identified approximately 3000 BAFF-specific VNARs in Chiloscyllium plagiosum vaccinated with the BAFF antigen. The results of modelling and molecular dynamics simulation and ELISA assay demonstrated that one out of the top five abundant specific VNARs exhibited higher binding affinity to the BAFF antigen than those obtained through phage display screening. Our data indicates NGS would be an alternative way for VNAR screening with plenty of advantages.

Multifunctional metamaterial device based on VO2 and the equivalent diode.

This paper proposes a switchable multifunctional metamaterial device operating in the terahertz (THz) band. The device is loaded with an equivalent diode and utilizes vanadium dioxide (V O 2). The middle layer of the whole device, a metal layer, divides the device into the I side and the II side. When the diode is ON, the I side can achieve dual-band absorption at 1.975 and 4.345 THz. When the diode is OFF, the I side can achieve single-band absorption at 4.28 THz. In the case of V O 2 being insulating, the II side can achieve linear-to-linear (LTL) polarization conversion at 2.342-4.18 THz. In the case of V O 2 being conductive, the II side can realize linear-to-circular (LTC) polarization conversion at 2.105-3.283 THz. The device provides a new strategy for the subsequent combination of multiple functions. The device can be used in electromagnetic stealth, intelligent applications, radiometers, and sensors and has relatively large application potential in miniaturized multifunctional metamaterials and THz band research.

A novel approach to study multi-domain motions in JAK1's activation mechanism based on energy landscape.

The family of Janus Kinases (JAKs) associated with the JAK-signal transducers and activators of transcription signaling pathway plays a vital role in the regulation of various cellular processes. The conformational change of JAKs is the fundamental steps for activation, affecting multiple intracellular signaling pathways. However, the transitional process from inactive to active kinase is still a mystery. This study is aimed at investigating the electrostatic properties and transitional states of JAK1 to a fully activation to a catalytically active enzyme. To achieve this goal, structures of the inhibited/activated full-length JAK1 were modelled and the energies of JAK1 with Tyrosine Kinase (TK) domain at different positions were calculated, and Dijkstra's method was applied to find the energetically smoothest path. Through a comparison of the energetically smoothest paths of kinase inactivating P733L and S703I mutations, an evaluation of the reasons why these mutations lead to negative or positive regulation of JAK1 are provided. Our energy analysis suggests that activation of JAK1 is thermodynamically spontaneous, with the inhibition resulting from an energy barrier at the initial steps of activation, specifically the release of the TK domain from the inhibited Four-point-one, Ezrin, Radixin, Moesin-PK cavity. Overall, this work provides insights into the potential pathway for TK translocation and the activation mechanism of JAK1.

Nitrate-driven anaerobic oxidation of ethane and butane by bacteria.

The short-chain gaseous alkanes (ethane, propane, and butane; SCGAs) are important components of natural gas, yet their fate in environmental systems is poorly understood. Microbially mediated anaerobic oxidation of SCGAs coupled to nitrate reduction has been demonstrated for propane, but is yet to be shown for ethane or butane-despite being energetically feasible. Here we report two independent bacterial enrichments performing anaerobic ethane and butane oxidation, respectively, coupled to nitrate reduction to dinitrogen gas and ammonium. Isotopic 13C- and 15N-labelling experiments, mass and electron balance tests, and metabolite and meta-omics analyses collectively reveal that the recently described propane-oxidizing "Candidatus Alkanivorans nitratireducens" was also responsible for nitrate-dependent anaerobic oxidation of the SCGAs in both these enrichments. The complete genome of this species encodes alkylsuccinate synthase genes for the activation of ethane/butane via fumarate addition. Further substrate range tests confirm that "Ca. A. nitratireducens" is metabolically versatile, being able to degrade ethane, propane, and butane under anoxic conditions. Moreover, our study proves nitrate as an additional electron sink for ethane and butane in anaerobic environments, and for the first time demonstrates the use of the fumarate addition pathway in anaerobic ethane oxidation. These findings contribute to our understanding of microbial metabolism of SCGAs in anaerobic environments.

First-line penpulimab combined with paclitaxel and carboplatin for metastatic squamous non-small-cell lung cancer in China (AK105-302): a multicentre, randomised, double-blind, placebo-controlled phase 3 clinical trial.

BACKGROUND: Penpulimab is a novel programmed death (PD)-1 inhibitor. This study aimed to establish the efficacy and safety of first line penpulimab plus chemotherapy for advanced squamous non-small-cell lung cancer. METHODS: This multicentre, randomised, double-blind, placebo-controlled, phase 3 clinical trial enrolled patients with locally advanced or metastatic squamous non-small-cell lung cancer from 74 hospitals in China. Eligible participants were aged 18-75 years, had histologically or cytologically confirmed locally advanced (stage IIIb or IIIc) or metastatic (stage IV) squamous non-small-cell lung cancer, were ineligible to complete surgical resection and concurrent or sequential chemoradiotherapy, had an Eastern Cooperative Oncology Group (ECOG) performance status of 0-1, did not have previous systemic chemotherapy for locally advanced or metastatic non-small-cell lung cancer, and had one or more measurable lesions according to RECIST (version 1.1). Participants were randomly assigned (1:1) to receive intravenous penpulimab 200 mg or placebo (excipient of penpulimab injection), plus paclitaxel 175 mg/m2 and carboplatin AUC of 5 intravenously on day 1 every 3 weeks for four cycles, followed by penpulimab or placebo as maintenance therapy. Stratification was done according to the PD-L1 tumour proportion score (<1% vs 1-49% vs ≥50%) and sex (male vs female). The participants, investigators, and other research staff were masked to group assignment. The primary outcome was progression-free survival assessed by the masked Independent Radiology Review Committee in the intention-to-treat population and patients with a PD-L1 tumour proportion score of 1% or more (PD-L1-positive subgroup). The primary analysis was based on the intention-to-treat analysis set (ie, all randomly assigned participants) and the PD-L1-positive subgroup. The safety analysis included all participants who received at least one dose of study drug after enrolment. This trial was registered with ClinicalTrials.gov (NCT03866993). FINDINGS: Between Dec 20, 2018, and Oct 10, 2020, 485 patients were screened, and 350 participants were randomly assigned (175 in the penpulimab group and 175 in the placebo group). Of 350 participants, 324 (93%) were male and 26 (7%) were female, and 347 (99%) were of Han ethnicity. In the final analysis (June 1, 2022; median follow-up, 24·7 months [IQR 0-41·4]), the penpulimab group showed an improved progression-free survival compared with the placebo group, both in the intention-to-treat population (median 7·6 months, 95% CI 6·8--9·6 vs 4·2 months, 95% CI 4·2-4·3; HR 0·43, 95% CI 0·33-0·56; p<0·0001) and in the PD-L1-positive subgroup (8·1 months, 5·7-9·7 vs 4·2 months, 4·1-4·3; HR 0·37, 0·27-0·52, p<0·0001). Grade 3 or worse treatment-emergent adverse events occurred in 120 (69%) 173 patients in the penpulimab group and 119 (68%) of 175 in the placebo group. INTERPRETATION: Penpulimab plus chemotherapy significantly improved progression-free survival in patients with advanced squamous non-small-cell lung cancer compared with chemotherapy alone. The treatment was safe and tolerable. Penpulimab combined with paclitaxel and carboplatin is a new option for first-line treatment in patients with this advanced disease. FUNDING: The National Natural Science Foundation of China, Shanghai Municipal Health Commission, Chia Tai Tianqing Pharmaceutical, Akeso.

Mitochondrial-Targeting Nanotrapper Captured Copper Ions to Alleviate Tumor Hypoxia for Amplified Photoimmunotherapy in Breast Cancer.

Hypoxia is a pervasive feature of solid tumors, which significantly limits the therapeutic effect of photodynamic therapy (PDT) and further influences the immunotherapy efficiency in breast cancer. However, the transient alleviation of tumor hypoxia fails to address the underlying issue of increased oxygen consumption, resulting from the rapid proliferation of tumor cells. At present, studies have found that the reduction of the oxygen consumption rate (OCR) by cytochrome C oxidase (COX) inhibition that induced oxidative phosphorylation (OXHPOS) suppression was able to solve the proposed problem. Herein, we developed a specific mitochondrial-targeting nanotrapper (I@MSN-Im-PEG), which exhibited good copper chelating ability to inhibit COX for reducing the OCR. The results proved that the nanotrapper significantly alleviated the hypoxic tumor microenvironment by copper chelation in mitochondria and enhanced the PDT effect in vitro and in vivo. Meanwhile, the nanotrapper improved photoimmunotherapy through both enhancing PDT-induced immunogenetic cell death (ICD) effects and reversing Treg-mediated immune suppression on 4T1 tumor-bearing mice. The mitochondrial-targeting nanotrapper provided a novel and efficacious strategy to enhance the PDT effect and amplify photoimmunotherapy in breast cancer.

Extracellular Vesicles from Neural Stem Cells Carry microRNA-16-5p to Reduce Corticosterone-induced Neuronal Injury in Depression Rats.

OBJECTIVE: Depression is a common mental illness. Neural stem cell-derived extracellular vesicles (NSC-EVs) are involved in repairing neuronal injury. We estimated the mechanism of miR-16-5p in depression rats. METHODS: EVs were extracted from NSCs. The depression rat model was established by corticosterone (CORT) induction and treated with NSC-EVs. The depression behavioral/pathological changes in rats were assessed using forced swimming test, open field test, sucrose consumption test and western blotting. The neuronal apoptosis in hippocampal tissue were detected. CORT-induced PC12 cell model was established. EV uptake by PC12 cells was measured and PC12 cell apoptosis was detected. The downstream targets of miR-16-5p were predicted and verified. The expressions of miR-16-5p and MYB in rats, PC12 cells, and EVs were measured. Functional rescue experiments were conducted to verify the role of miR-16-5p and MYB in PC12 cell apoptosis. RESULTS: CORT induction increased neuronal apoptosis in hippocampal tissue and induced depression-like behaviors in rats, while NSC-EV treatment improved depression-like behaviors and apoptosis in rats. In PC12 cells, NSC-EVs decreased CORT-induced PC12 cell apoptosis. NSC-EVs carried miR-16-5p into PC12 cells. miR-16-5p knockdown in EVs partially reversed the inhibitory effects of NSC-EVs on CORT-induced PC12 cell apoptosis. miR-16-5p targeted to inhibit MYB to repress CORT-induced PC12 cell apoptosis. In vivo experiments further verified that NSC-EVs reduced neuronal injury in CORT-induced depression rats via the miR-16-5p/MYB axis. CONCLUSION: NSC-EVs-mediated alleviation on neuronal injury by carrying miR-16-5p to target MYB was highly likely one of the mechanisms by which NSC-EVs mediated miR-16-5p in neuroprotection of depression rats.

Biodegradable and Efficient Charge-Migrated Z-Scheme Heterojunction Amplifies Cancer Ferroptosis by Blocking Defensive Redox System.

Ferroptosis is an emerging non-apoptotic death process, mainly involving lipid peroxidation (LPO) caused by iron accumulation, which is potentially lethal to the intrinsically apoptotic-resistant malignant tumor. However, it is still restricted by the inherent antioxidant systems of tumor cells and the poor efficacy of traditional iron-based ferroptosis initiators. Herein, the study develops a novel ferroptosis-inducing agent based on PEGylated Cu+/Cu2+-doped black phosphorus@polypyrrole heterojunction (BP@CPP), which is constructed by utilizing the phosphate on the surface of BP to chelate Cu ions and initiating subsequent in situ polymerization of pyrrole. As a novel Z-scheme heterojunction, BP@CPP possesses an excellent photocatalytic activity in which the separated electron-hole pairs under laser irradiation endow it with powerful oxidizing and reducing capacities, which synergy with Cu+/Cu2+ self-cycling catalyzing Fenton-like reaction to further strengthen reactive oxygen species (ROS) accumulation, glutathione (GSH) depletion, and glutathione peroxidase 4 (GPX4) inactivation, ultimately leading to efficient ferroptosis. Systematic in vitro and in vivo evaluations demonstrate that BP@CPP effectively inhibit tumor growth by inducing desired ferroptosis while maintaining a favorable biosafety in the body. Therefore, the developed BP@CPP-based ferroptosis initiator provides a promising strategy for ferroptosis-like cancer therapy.

Characteristics and time points to inhibit ferroptosis in human osteoarthritis.

Ferroptosis is a form of cell death that is triggered by iron-dependent lipid peroxidation and is closely associated with osteoarthritis. The primary interventions for inhibiting ferroptosis in osteoarthritis are anti-lipid peroxidation and iron chelation. The objective of our study is to investigate the characteristics of ferroptosis in osteoarthritis and identify the optimal time points for inhibiting ferroptosis to alleviate disease progression. Ferroptosis-related alterations and markers of OA were analyzed in paired intact and damaged cartilages from OA patients by immunofluorescence, qRT-PCR, mitochondrial membrane potential and immunohistochemistry. We also compared Ferroptosis-related alterations in cartilage of mild, moderate, and severe OA (according to the modified Mankin score). In addition, we compared the effect of Fer-1 on ferroptosis and the protection of chondrocytes by detecting markers of both ferroptosis and OA by immunofluorescence, CCK8 and qRT-PCR. Ferroptosis-related alterations (GPX4 downregulation, ACSL4 upregulation, MDA, LPO accumulation, Mitochondrial membrane potential decreased) in the damaged area cartilage were more severe than those in the intact area and increased with the progression of OA. Compared with mild OA group, the activity of chondrocytes treated with Fer-1 (a ferroptosis inhibitor) was increased, mitochondrial function was improved, and ferroptosis was reduced (GPX4 upregulation, SLC7A11 upregulation, ACSL4 downregulation,), and promoted the expression of COL2A1 and inhibited the expression of MMP13. However, these changes were not observed in moderate and severe OA chondrocytes. Ferroptosis occurs in a region-specific manner and is exacerbated with the progression of human OA cartilage degeneration. Inhibition of ferroptosis might had a therapeutic effect on chondrocytes with mild OA but had no significant therapeutic effect on chondrocytes with moderate to severe OA.

Bound ion effects: Using machine learning method to study the kinesin Ncd's binding with microtubule.

Drosophila Ncd proteins are motor proteins that play important roles in spindle organization. Ncd and the tubulin dimer are highly charged. Thus, it is crucial to investigate Ncd-tubulin dimer interactions in the presence of ions, especially ions that are bound or restricted at the Ncd-tubulin dimer binding interfaces. To consider the ion effects, widely used implicit solvent models treat ions implicitly in the continuous solvent environment without focusing on the individual ions' effects. But highly charged biomolecules such as the Ncd and tubulin dimer may capture some ions at highly charged regions as bound ions. Such bound ions are restricted to their binding sites; thus, they can be treated as part of the biomolecules. By applying multiscale computational methods, including the machine-learning-based Hybridizing Ions Treatment-2 program, molecular dynamics simulations, DelPhi, and DelPhiForce, we studied the interaction between the Ncd motor domain and the tubulin dimer using a hybrid solvent model, which considers the bound ions explicitly and the other ions implicitly in the solvent environment. To identify the importance of treating bound ions explicitly, we also performed calculations using the implicit solvent model without considering the individual bound ions. We found that the calculations of the electrostatic features differ significantly between those of the hybrid solvent model and the pure implicit solvent model. The analyses show that treating bound ions at highly charged regions explicitly is crucial for electrostatic calculations. This work proposes a machine-learning-based approach to handle the bound ions using the hybrid solvent model. Such an approach is not only capable of handling kinesin-tubulin complexes but is also appropriate for other highly charged biomolecules, such as DNA/RNA, viral capsid proteins, etc.

Recent Progress of Natural and Recombinant Phycobiliproteins as Fluorescent Probes.

Phycobiliproteins (PBPs) are natural water-soluble pigment proteins, which constitute light-collecting antennae, and function in algae photosynthesis, existing in cyanobacteria, red algae, and cryptomonads. They are special pigment-protein complexes in algae with a unique structure and function. According to their spectral properties, PBPs can be mainly divided into three types: allophycocyanin, phycocyanin, and PE. At present, there are two main sources of PBPs: one is natural PBPs extracted from algae and the other way is recombinant PBPs which are produced in engineered microorganisms. The covalent connection between PBP and streptavidin was realized by gene fusion. The bridge cascade reaction not only improved the sensitivity of PBP as a fluorescent probe but also saved the preparation time of the probe, which expands the application range of PBPs as fluorescent probes. In addition to its function as a light-collecting antenna in photosynthesis, PBPs also have the functions of biological detection, ion detection, and fluorescence imaging. Notably, increasing studies have designed novel PBP-based far-red fluorescent proteins, which enable the tracking of gene expression and cell fate.

Advances in Intercellular Communication Mediated by Exosomal ncRNAs in Cardiovascular Disease.

Cardiovascular diseases are a leading cause of worldwide mortality, and exosomes have recently gained attention as key mediators of intercellular communication in these diseases. Exosomes are double-layered lipid vesicles that can carry biomolecules such as miRNAs, lncRNAs, and circRNAs, and the content of exosomes is dependent on the cell they originated from. They can be involved in the pathophysiological processes of cardiovascular diseases and hold potential as diagnostic and monitoring tools. Exosomes mediate intercellular communication, stimulate or inhibit the activity of target cells, and affect myocardial hypertrophy, injury and infarction, ventricular remodeling, angiogenesis, and atherosclerosis. Exosomes can be released from various types of cells, including endothelial cells, smooth muscle cells, cardiomyocytes, fibroblasts, platelets, adipocytes, immune cells, and stem cells. In this review, we highlight the communication between different cell-derived exosomes and cardiovascular cells, with a focus on the roles of RNAs. This provides new insights for further exploring targeted therapies in the clinical management of cardiovascular diseases.

Structural Analysis of Janus Tyrosine Kinase Variants in Hematological Malignancies: Implications for Drug Development and Opportunities for Novel Therapeutic Strategies.

Janus tyrosine kinase (JAK) variants are known drivers for hematological disorders. With the full-length structure of mouse JAK1 being recently resolved, new observations on the localization of variants within closed, open, and dimerized JAK structures are possible. Full-length homology models of human wild-type JAK family members were developed using the Glassman et al. reported mouse JAK1 containing the V658F structure as a template. Many mutational sites related to proliferative hematological disorders reside in the JH2 pseudokinase domains facing the region important in dimerization of JAKs in both closed and open states. More than half of all JAK gain of function (GoF) variants are changes in polarity, while only 1.2% are associated with a change in charge. Within a JAK1-JAK3 homodimer model, IFNLR1 (PDB ID7T6F) and the IL-2 common gamma chain subunit (IL2Rγc) were aligned with the respective dimer implementing SWISS-MODEL coupled with ChimeraX. JAK3 variants were observed to encircle the catalytic site of the kinase domain, while mutations in the pseudokinase domain align along the JAK-JAK dimerization axis. FERM domains of JAK1 and JAK3 are identified as a hot spot for hematologic malignancies. Herein, we propose new allosteric surfaces for targeting hyperactive JAK dimers.

JAK3 Y841 Autophosphorylation Is Critical for STAT5B Activation, Kinase Domain Stability and Dimer Formation.

Janus tyrosine kinase 3 (JAK3) is primarily expressed in immune cells and is needed for signaling by the common gamma chain (γc) family of cytokines. Abnormal JAK3 signal transduction can manifest as hematological disorders, e.g., leukemia, severe combined immunodeficiency (SCID) and autoimmune disease states. While regulatory JAK3 phosphosites have been well studied, here a functional proteomics approach coupling a JAK3 autokinase assay to mass spectrometry revealed ten previously unreported autophosphorylation sites (Y105, Y190, Y238, Y399, Y633, Y637, Y738, Y762, Y824, and Y841). Of interest, Y841 was determined to be evolutionarily conserved across multiple species and JAK family members, suggesting a broader role for this residue. Phospho-substitution mutants confirmed that Y841 is also required for STAT5 tyrosine phosphorylation. The homologous JAK1 residue Y894 elicited a similar response to mutagenesis, indicating the shared importance for this site in JAK family members. Phospho-specific Y841-JAK3 antibodies recognized activated kinase from various T-cell lines and transforming JAK3 mutants. Computational biophysics analysis linked Y841 phosphorylation to enhanced JAK3 JH1 domain stability across pH environments, as well as to facilitated complementary electrostatic JH1 dimer formation. Interestingly, Y841 is not limited to tyrosine kinases, suggesting it represents a conserved ubiquitous enzymatic function that may hold therapeutic potential across multiple kinase families.

Hyperthermia-triggered NO release based on Cu-doped polypyrrole for synergistic catalytic/gas cancer therapy.

Nitric oxide (NO) is a crucial gaseous medium for tumor growth and progression, but it may also cause mitochondrial disorder and DNA damage by drastically increasing its concentration in tumor. Due to its challenging administration and unpredictable release, NO based gas therapy is difficult to eliminate malignant tumor at low safe doses. To address these issues, herein, we develop a multifunctional nanocatalyst called Cu-doped polypyrrole (CuP) as an intelligent nanoplatform (CuP-B@P) to deliver the NO precursor BNN6 and specifically release NO in tumors. Under the aberrant metabolic environment of tumors, CuP-B@P catalyzes the conversion of antioxidant GSH into GSSG and excess H2O2 into ·OH through Cu+/Cu2+ cycle, which results in oxidative damage to tumor cells and the concomitant release of cargo BNN6. More importantly, after laser exposure, nanocatalyst CuP can absorb and convert photons into hyperthermia, which in turn, accelerates the aforesaid catalytic efficiency and pyrolyzes BNN6 into NO. Under the synergistic effect of hyperthermia, oxidative damage, and NO burst, almost complete tumor elimination is achieved in vivo with negligible toxicity to body. Such an ingenious combination of NO prodrug and nanocatalytic medicine provides a new insight into the development of NO based therapeutic strategies. STATEMENT OF SIGNIFICANCE: A hyperthermia-responsive NO delivery nanoplatform (CuP-B@P) based on Cu-doped polypyrrole was designed and fabricated, in which CuP catalyzed the conversion of H2O2 and GSH into ·OH and GSSG to induce intratumoral oxidative damage. After laser irradiation, hyperthermia ablation and responsive release of NO further coupled with oxidative damage to eliminate malignant tumors. This versatile nanoplatform provides new insights into the combined application of catalytic medicine and gas therapy.

Phosphorylation of Tyrosine 841 Plays a Significant Role in JAK3 Activation.

Janus Kinase 3 (JAK3) plays a key role in the development, proliferation, and differentiation of various immune cells. It regulates gene expression by phosphorylation of Signal Transducers and Activators of Transcriptions (STATs) via the JAK/STAT pathway. Recently, we found a new JAK3 phosphorylation site, tyrosine 841 (Y841). The results showed that pY841 helps the kinase domain flip around the pseudo kinase domain, which may cause JAK3 conformational changes. It also reduces the size of the cleft between the N-lobe and the C-lobe of the JAK3 kinase domain. However, pY841 was found to enlarge the cleft when ATP/ADP was bound to the kinase. The increase in the cleft size suggested that pY841 enhanced the elasticity of the kinase domain. For unphosphorylated JAK3 (JAK3-Y841), the binding forces between the kinase domain and ATP or ADP were similar. After phosphorylation of Y841, JAK3-pY841 exhibited more salt bridges and hydrogen bonds between ATP and the kinase than between ADP and the kinase. Consequently, the electrostatic binding force between ATP and the kinase was higher than that between ADP and the kinase. The result was that compared to ADP, ATP was more attractive to JAK3 when Y841 was phosphorylated. Therefore, JAK3-pY841 tended to bind ATP rather than ADP. This work provides new insights into the role of phosphorylation in kinase activation and ATP hydrolysis and sheds light on the importance of understanding the molecular mechanisms that regulate the kinase function.