ENO1 contributes to the gemcitabine resistance of pancreatic cancer through the YAP1 signaling pathway.

Pancreatic cancer (PC), a leading cause of cancer-related deaths, has a 5-year survival rate of approximately 10%. α-Enolase (ENO1) is a junction channel protein involved in tumor cell apoptosis and chemoresistance. However, the role of ENO1 in PC remains unclear. The expression and prognosis of ENO1 levels were determined in PC using public databases based on The Cancer Genome Atlas (TCGA) data sets. Cell viability, half maximal inhibitory concentration (IC50), autophagy, apoptosis, and autophagy markers were examined using cell counting kit-8 (CCK-8), transmission electron microscope, flow cytometry assays, and immunoblot, respectively. Using the Gene Expression Omnibus (GEO) and TCGA data sets, we found that ENO1 was significantly enriched in PC tumor tissues, and high expression levels of ENO1 were associated with an unfavorable prognosis. Whereas ENO1 silencing suppressed proliferation, autophagy, and induced cell apoptosis in PC cells, and inhibited tumor growth in vivo. Mechanistically, knockdown of ENO1 enhanced cellular cytotoxicity of gemcitabine (GEM), as well as reducing the expression of yes-associated protein 1 (YAP1), a major downstream effector of the Hippo pathway in vitro. YAP1 promoted autophagy and protected PC cells from GEM-induced apoptotic cell death. Furthermore, YAP1 overexpression attenuated the inhibition effects of ENO1 silencing. Our results suggest that ENO1 overexpression promotes cell growth and tumor progression by increasing the expression of YAP1 in PC. Further studies are required to understand the detailed mechanisms between ENO1 and YAP1 in PC.

Mg2TiO4 spinel modified by nitrogen doping as a Visible-Light-Active photocatalyst for antibacterial activity.

Nitrogen doped Mg2TiO4 spinel, i.e. Mg2TiO4-xNy, has been synthesized and investigated as a photocatalyst for antibacterial activity. Mg2TiO4-xNy demonstrates superior photocatalytic activity for E. coli disinfection under visible light illumination (λ ≥ 400 nm). Complete disinfection of E. coli at a bacterial cell density of 1.0 × 107 CFU mL-1 can be achieved within merely 60 min. Mg2TiO4-xNy is capable of generating superoxide radicals (•O2 -) under visible light illumination which are the reactive oxygen species (ROSs) for bacteria disinfection. DFT calculations have verified the importance of nitrogen dopants in improving the visible light sensitivity of Mg2TiO4-xNy. The facile synthesis, low cost, good biocompatibility and high disinfection activity of Mg2TiO4-xNy warrant promising applications in the field of water purification and antibacterial products.

Immunomodulatory activity of puerarin in RAW264.7 macrophages and cyclophosphamide-induced immunosuppression mice.

CONTEXT: Puerarin, a natural isoflavone extracted from Radix puerariae, is famous for treating various cardiovascular and cerebrovascular diseases. However, little is known about its direct immunomodulatory activity. OBJECTIVE: This study was designed to investigate the in vitro and in vivo immunomodulatory effects of Radix puerariae by using the murine monocyte-macrophage cell line RAW264.7 and immunosuppressed cyclophosphamide-induced mice. METHODS: MTT and neutral red phagocytosis assays were conducted to evaluate the in vitro immunomodulatory activities of puerarin on cell viability and phagocytosis by measuring the proliferation, phagocytic, nitric oxide (NO) ability, and TNF-α production ability of stimulated and lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Immunosuppressed cyclophosphamide-induced mice were used to evaluate the in vivo immunomodulatory activities of puerarin by measuring IL-4 and IFN-γ, the serum half hemolysis value, spleen and thymus index, and proliferation assay for splenic lymphocytes. RESULTS AND DISCUSSION: Results showed that puerarin improves immunomodulatory activity by increasing cell proliferation, cell phagocytosis, and NO secretion in RAW264.7 macrophages and reduces the abnormal immunologic activity by decreasing cell phagocytosis and NO secretion in LPS-stimulated RAW264.7 macrophages. In addition, puerarin enhanced the immunologic activity of cyclophosphamide-induced immunosuppression mice by increasing the secretion of NO, IFN-γ, and IL-4, the serum half hemolysis value (HC50), the spleen and thymus index, and proliferation for splenic lymphocytes. CONCLUSION: Puerarin exhibited an upregulated immunomodulatory effect on RAW264.7 macrophages and immunosuppression mice. In addition, puerarin had a downregulated immunomodulatory effect on RAW264.7 macrophages. The results suggest that puerarin could be a promising immunomodulator to assist in the treatment of tumors.

Two types of TNF-α and their receptors in snakehead (Channa argus): Functions in antibacterial innate immunity.

Tumor necrosis factor-α (TNF-α) is a pluripotent mediator of pro-inflammatory and antimicrobial defense mechanisms and a regulator of lymphoid organ development. Although two types of TNF-α have been identified in several teleost species, their functions in pathogen infection remain largely unexplored, especially in pathogen clearance. Herein, we cloned and characterized two types of TNF-α, termed shTNF-α1 and shTNF-α2, and their receptors, shTNFR1 and shTNFR2, from snakehead (Channa argus). These genes were constitutively expressed in all tested tissues, and were induced by Aeromonas schubertii and Nocardia seriolae in head kidney and spleen in vivo, and by lipoteichoic acid (LTA), lipopolysaccharides (LPS), and Polyinosinic-polycytidylic acid [Poly (I:C)] in head kidney leukocytes (HKLs) in vitro. Moreover, recombinant shTNF-α1 and shTNF-α2 upregulated the expression of endogenous shTNF-α1, shTNF-α2, shTNFR1, and shTNFR2, and enhanced intracellular bactericidal activity, with shTNF-α1 having a greater effect than shTNF-α2. These findings suggest important roles of fish TNFα1, TNFα2, and their receptors in bacterial infection and pathogen clearance, and provide a new insight into their function in antibacterial innate immunity.

Bacteria-induced IL-1ß and its receptors in snakehead (Channa argus): Evidence for their involvement in antibacterial innate immunity.

As a central pro-inflammatory cytokine, interleukin-1ß (IL-1ß) plays critical roles in the inflammatory response, pathogen infection, and immunological challenges in mammals. Although fish IL-1ß has been confirmed to participate in inflammatory response to pathogen infection, few studies have been performed to characterize the antibacterial and bactericidal functions of fish IL-1ß. In this study, snakehead (Channa argus) IL-1ß (shIL-1ß) and its receptors, shIL-1R1 and shIL-1R2, were cloned and functionally characterized. ShIL-1ß contained the IL-1 family signature domain, and a potential cutting site at Asp96 that presented in all vertebrate IL-1ß sequences. ShIL-1R1 had three extracellular IG-like domains and one intracellular signal TIR domain, while shIL-1R2 had three extracellular IG-like domain but lacked the intracellular signal TIR domain. ShIL-1ß, shIL-1R1, and shIL-1R2 were constitutively expressed in all tested tissues, and their expressions could be induced by Aeromonas schubertii and Nocardia seriolae in the head kidney and spleen in vivo, and by LTA, LPS, and Poly (I:C) in head kidney leukocytes (HKLs) in vitro. Moreover, recombinant shIL-1ß upregulated the expression of endogenous shIL-1ß, shIL-R1, and shIL-R2 in snakehead HKLs, and enhanced intracellular bactericidal activity. Taken together, this study found that, like IL-1ß and its receptors in mammals, shIL-1ß and its receptors play crucial roles in antibacterial innate immunity. This provides new insight into the evolution of IL-1ß function in vertebrates.

Integrating metabolomics and physiological analysis to investigate the toxicological mechanisms of sewage sludge-derived biochars to wheat.

Effects of sewage sludge biochars (SSBCs) on the growth of wheat and the specific toxicological mechanisms were investigated from a metabolic perspective for better ecological risk assessment. We observed that conversion of sludge to biochar remarkably changed the properties, and also caused a significant (p < 0.05) reduction of the toxicity towards wheat. Wheat growth under exposure to SSBCs was influenced by the pyrolysis temperature (300 °C, 500 °C and 700 °C), with root length being promoted by SSBCs prepared at higher temperatures (500 °C and 700 °C). In addition to the contaminants, including polycyclic aromatic hydrocarbons (PAHs) and potentially toxic elements (PTEs) detected in SSBCs, the morphological characteristics of biochars contributed substantially to the wheat growth. Metabolomics analysis revealed the remarkable differences in the metabolic profiles among the control (CK), SS300- and SS700-treated samples. The toxicological mechanisms involved were found to be associated with the regulation of metabolisms pathways of protein, fatty acids and carbohydrates, among which protein metabolism was most affected by SSBCs. This work presents an innovative concept that SSBCs produced at a proper temperature may minimize the toxic effects on plant growth by regulating the metabolic fluxes in vivo.

Structural pharmacological studies on EGFR T790M/C797S.

Drug-resistance is a major challenge in targeted therapy of EGFR mutated non-small cell lung cancers (NSCLCs). The third-generation irreversible inhibitors such as AZD9291, CO-1686 and WZ4002 can overcome EGFR T790M drug-resistance mutant through covalent binding through Cys 797, but ultimately lose their efficacy upon emergence of the new mutation C797S. To develop new reversible inhibitors not relying on covalent binding through Cys 797 is therefore urgently demanded. Gö6976 is a staurosporine-like reversible inhibitor targeting T790M while sparing the wild-type EGFR. In the present work, we reported the complex crystal structures of EGFR T790M/C797S + Gö6976 and T790M + Gö6976, along with enzyme kinetic data of EGFR wild-type, T790M and T790M/C797S. These data showed that the C797S mutation does not significantly alter the structure and function of the EGFR kinase, but increases the local hydrophilicity around residue 797. The complex crystal structures also elucidated the detailed binding mode of Gö6976 to EGFR and explained why this compound prefers binding to T790M mutant. These structural pharmacological data would facilitate future drug development studies.

Functional analysis of insect molting fluid proteins on the protection and regulation of ecdysis.

Molting fluid accumulates between the old and new cuticles during periodical ecdysis in Ecdysozoa. Natural defects in insect ecdysis are frequently associated with melanization (an immunity response) occurring primarily in molting fluids, suggesting that molting fluid may impact immunity as well as affect ecdysis. To address this hypothesis, proteomic analysis of molting fluids from Bombyx mori during three different types of ecdysis was performed. Many proteins were newly identified, including immunity-related proteins, in each molting fluid. Molting fluids inhibited the growth of bacteria in vitro. The entomopathogenic fungi Beauveria bassiana, which can escape immune responses in feeding larvae, is quickly recognized by larvae during ecdysis, followed by melanization in molting fluid and old cuticle. Fungal conidia germination was delayed, and no hyphae were detected in the hemocoels of pharate instar insects. Molting fluids protect the delicate pharate instar insects with extremely thin cuticles against microorganisms. To explore the function of molting fluids in ecdysis regulation, based on protein similarity, 32 genes were selected for analysis in ecdysis regulation through RNAi in Tribolium castaneum, a model commonly used to study integument development because RNAi is difficult to achieve in B. mori. We identified 24 molting proteins that affected ecdysis after knockdown, with different physiological functions, including old cuticle protein recycling, molting fluid pressure balance, detoxification, and signal detection and transfer of molting fluids. We report that insects secrete molting fluid for protection and regulation of ecdysis, which indicates a way to develop new pesticides through interrupting insect ecdysis in the future.

The antitumor activity of Meconopsis horridula Hook, a traditional Tibetan medical plant, in murine leukemia L1210 cells.

BACKGROUND: Meconopsis horridula Hook (M. horridula) has been used as a traditional Tibetan medicine to relieve heat and pain as well as mobilize static blood, and it is recognized as a good treatment for bruises. This study is the first trial to evaluate the tumor inhibitory activity of M. horridula extract and its underlying mechanism in the hope of providing evidence to support the anticancer function of M. horridula. METHODS AND RESULTS: M. horridula extract was cytotoxic to L1210 cells in a dose- and time-dependent manner. SEM (scanning electron microscope) observation revealed obvious morphological changes in L1210 cells after M. horridula treatment. Flow cytometry analysis demonstrated that the extract dose-dependently induced early apoptosis. Additional apoptosis parameters, such as alterations in nuclear morphology and DNA damage, were also observed. Furthermore, M. horridula treatment induced G2/M arrest. M. horridula treatment significantly increased reactive oxygen species (ROS) production, suggesting that ROS are a key factor in M. horridula-induced apoptosis. Volatile constituent detection found 15 abundant chemicals in M. horridula, which may contribute to its anticancer effect. CONCLUSION: In conclusion, M. horridula extract induced L1210 cell apoptosis and inhibited proliferation through G2/M phase arrest, and ROS were involved in the process.

Thermolysin damages animal life through degradation of plasma proteins enhanced by rapid cleavage of serpins and activation of proteases.

Thermolysin, a metallopeptidase secreted by pathogenic microbes, is concluded as an important virulence factor due to cleaving purified host proteins in vitro. Using the silkworm Bombyx mori as a model system, we found that thermolysin injection into larvae induces the destruction of the coagulation response and the activation of hemolymph melanization, which results in larval death. Thermolysin triggers the rapid degradation of insect and mammalian plasma proteins at a level that is considerably greater than expected in vitro and/or in vivo. To more specifically explore the mechanism, thermolysin-induced changes to key proteins belonging to the insect melanization pathway were assessed as a window for observing plasma protein cleavage. The application of thermolysin induced the rapid cleavage of the melanization negative regulator serpin-3, but did not directly activate the melanization rate-limiting enzyme prophenoloxidase (PPO) or the terminal serine proteases responsible for PPO activation. Terminal serine proteases of melanization are activated indirectly after thermolysin exposure. We hypothesize that thermolysin induces the rapid degradation of serpins and the activation of proteases directly or indirectly, boosting uncontrolled plasma protein degradation in insects and mammalians.

Induction of Mitochondrial Dependent Apoptosis in Human Leukemia K562 Cells by Meconopsis integrifolia: A Species from Traditional Tibetan Medicine.

OBJECTIVES: Meconopsis integrifolia (M. integrifolia) is one of the most popular members in Traditional Tibetan Medicine. This study aimed to investigate the anticancer effect of M. integrifolia and to detect the underlying mechanisms of these effects. METHODS: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and trypan blue assay were used to evaluate the cytotoxicity of M. integrifolia. Changes in cell nuclear morphology and reactive oxygen species (ROS) level were observed by fluorescent microscopy. Apoptosis ratio, DNA damage and mitochondrial membrane potential (MMP) loss were analyzed by flow cytometry. Western blotting assay was adopted to detect the proteins related to apoptosis. Immunofluorescence was used to observe the release of cytochrome C. RESULTS: The obtained data revealed that M. integrifolia could significantly inhibit K562 cell viability, mainly by targeting apoptosis induction and cell cycle arrest in G2/M phase. Collapse in cell morphology, chromatin condensation, DNA damage and ROS accumulation were observed. Further mechanism detection revealed that mitochondrion might be a key factor in M. integrifolia-induced apoptosis. CONCLUSIONS: M. integrifolia could induce mitochondria mediated apoptosis and cell cycle arrest in G2/M phase with little damage to normal cells, suggesting that M. integrifolia might be a potential and efficient anticancer agent that deserves further investigation.

Biochar: an effective amendment for remediating contaminated soil.

Biochar is a carbon-rich material derived from incomplete combustion of biomass.Applying biochar as an amendment to treat contaminated soils is receiving increasing attention, and is a promising way to improve soil quality. Heavy metals are persistent and are not environmentally biodegradable. However, they can be stabilized in soil by adding biochar. Moreover, biochar is considered to be a predominant sorptive agent for organic pollutants, having a removal efficiency of about 1 order of magnitude higher than does soil/sediment organic matter or their precursor substances alone.When trying to stabilize organic and inorganic pollutants in soil, several features of biochar' s sorption capacity should be considered, viz., the nature of the pollutants to be remediated, how the biochar is prepared, and the complexity of the soil systemin which biochar may be used. In addition, a significant portion of the biochar or some of its components that are used to remediate soils do change over time through abiotic oxidation and microbial decomposition. This change process is commonly referred to as "aging:" Biochar "aging" in nature is inevitable, and aged biochar exhibits an effect that is totally different than non-aged biochar on stabilizing heavy metals and organic contaminants in soils.Studies that have been performed to date on the use of biochar to remediate contaminated soil are insufficient to allow its use for wide-scale field application.Therefore, considerable new data are necessary to expand both our understanding of how biochar performs in the field, and where it can be best used in the future for soil remediation. For example, how biochar and soil biota (microbial and faunal communities)interact in soils is still poorly understood. Moreover, studies are needed on how to best remove new species of heavy metals, and on how biochar aging affects sorption capacity are also needed.

Tribbles pseudokinase 3 promoted renal fibrosis by regulating the expression of DNA damage-inducible transcript 3 in diabetic nephropathy.

Diabetic nephropathy (DN) is a severe complication of prolonged diabetes, impacting millions worldwide with an increasing incidence. This study investigates the role of tribbles pseudokinase 3 (TRIB3), a protein implicated in the progression of DN, focusing on its mechanisms underlying glomerular damage. Through analysis of the Gene Expression Omnibus (GEO) database, we identified TRIB3 among differentially expressed genes in streptozotocin (STZ)-treated C57BL/6J mice. Both in vitro and in vivo experiments were conducted to examine the effects of TRIB3 inhibition on high glucose (HG)-induced damage in podocytes and DN mouse models. The results demonstrated that TRIB3 inhibition reduced inflammatory responses and extracellular matrix (ECM) production in MPC5 cells, mediated by the downregulation of DNA damage-inducible transcript 3 (DDIT3) - a critical regulator of proinflammatory cytokine secretion and ECM synthesis. Inhibiting TRIB3 decreased inflammatory factors and ECM deposition in diabetic mice in vivo, confirming its pivotal role in DN pathogenesis. These findings indicate that TRIB3 and its interaction with DDIT3 contribute significantly to DN by promoting inflammatory cascades and ECM accumulation, presenting potential therapeutic targets for managing the disease.

Edge-oriented phosphatizing engineering of 2D Ni-MOFs with a tailored d-band center for boosting catalytic activity.

Metal-organic framework (MOF)-based heterostructures have aroused widespread interest owing to their extensive compositional tunability and interesting catalytic properties. However, the precise edge-oriented growth of transition metal compounds at the edges of 2D MOFs to construct edge mode heterostructures remains a great challenge due to their inherent thermodynamic instability. Here, edge-oriented growth of Ni2P at the edges of a 2D Ni-MOF was achieved for the first time by precisely tuning the phosphorus source content and phosphating temperature. Owing to the formation of the edge mode Ni-MOF/Ni2P heterostructure, the as-prepared heterostructure showed upregulated d-band center, more robust 4-nitrophenol (4-NP) adsorption capacity, lowered energy barrier of the rate-determining step (RDS), and higher specific surface area, resulting in the best performance of the hydrogenation reduction of 4-NP to 4-aminophenol (4-AP) in the presence of non-precious metal catalysts.

Rational Design of Covalent Kinase Inhibitors by an Integrated Computational Workflow (Kin-Cov).

Covalent kinase inhibitors (CKIs) hold great promise for drug development. However, examples of computationally guided design of CKIs are still scarce. Here, we present an integrated computational workflow (Kin-Cov) for rational design of CKIs. The design of the first covalent leucine-zipper and sterile-α motif kinase (ZAK) inhibitor was presented as an example to showcase the power of computational workflow for CKI design. The two representative compounds, 7 and 8, inhibited ZAK kinase with half-maximal inhibitory concentration (IC50) values of 9.1 and 11.5 nM, respectively. Compound 8 displayed an excellent ZAK target specificity in Kinome profiling against 378 wild-type kinases. Structural biology and cell-based Western blot washout assays validated the irreversible binding characteristics of the compounds. Our study presents a rational approach for the design of CKIs based on the reactivity and accessibility of nucleophilic amino acid residues in a kinase. The workflow is generalizable and can be applied to facilitate CKI-based drug design.

The ultrathin palladium nanosheets for sensitive and visual Hg2+ detection in the food chain.

The detection of mercury, one of the ten most dangerous chemicals, is significant to provide helpful information for assessing mercury toxicity and health risks. However, it is a challenge to explore simple, sensitive, accurate, and cheap Hg2+ detection methods. Noble metal nanomaterials are used for Hg2+ detection by the colorimetric method widely. Still, the pure noble metal materials' detection limit of Hg2+ is high, and sensitivity enhancement usually requires further complex modification. Here, we use a facile one-step route to synthesize ultra-thin two-dimensional palladium nanosheets (PdNS), which have high selectivity and sensitivity for Hg2+ detection by colorimetric method with a low detection limit (0.55 ppb). The detection of Hg2+ by PdNS involves multiple mechanisms, including the formation of amalgam and PdO to improve the peroxidase-mimic activity of PdNS and PdNS motor function to increase its collision probability with the detection reactant. The PdNS can be used to detect Hg2+ in various actual samples. The detection results are highly consistent with the data obtained by the atomic fluorescence spectrometer (AFS). Then, we developed a Hg2+ detection kit, which can realize simple, sensitive, and accurate Hg2+ detection by naked eye or cellphone at a meager cost (0.3 dollars each sample).

Effect of temperature on PTEs deportment and ecological risks of the biochars obtained from sewage sludge.

Sewage sludge-derived biochars (SSBCs) were obtained at temperatures of 300, 500, and 700 °C to investigate the potentially toxic elements (PTEs) behaviors and assess the environmental acceptability for the possible application in the environment. Results indicated that PTEs exhibited diversely in the distribution of chemical speciation, while all elements tended to be immobilized in biochar matrix and the total amount elevated during the pyrolysis. The risk assessment of biochars implied a low degree of environmental risk for the utilization of SSBCs prepared at high temperatures. In addition, higher pyrolysis temperature alleviated the inhibition on the early seedling growth of Triticum aestivum L., with root elongation more sensitive to the biochar addition. PTEs, especially Cr, contributed much to the phytotoxicity of biochars as revealed by the principle component analysis (PCA) and leaner correlation analysis. Findings from this work illustrated that SSBCs prepared at higher temperatures might be more conductive to a wide range of applications with acceptable environmental risk.

The influence of modified molecular (D/L-serine) chirality on the theragnostics of PAMAM-based nanomedicine for acute kidney injury.

Acute kidney injury (AKI) is a severe clinical disease with extremely high morbidity and mortality. It is challenging to find a simple method for early detection of AKI and monitoring the treatment results. Renal tubular damage and inflammation are early events in AKI. Renal tubular damage is conducive to the accumulation of small-sized nanoparticles in the kidney, and inflammation is related to the excessive production of H2O2. Recent studies proved that chiral molecule modification of nanomaterials is a powerful strategy to regulate their biodistribution. Thus, L-serine and D-serine modified poly(amidoamine) (PAMAM) dendrimers were synthesized and used as fluorescent probe (NPSH) carriers to obtain L-SPH and D-SPH, respectively. D-SPH has a strong accumulation capability in the kidney of AKI mice. Then, the H2O2 fluorescent probe can detect the excessively produced H2O2 to generate fluorescence to diagnose AKI. Subsequently, the anti-inflammatory drug manganese pentacarbonyl bromide (CORM) was loaded in D-SPH to obtain D-SPHC with AKI theragnostic functions. Simultaneously, the D-SPHC fluorescence signal intensity change during the treatment can be used to monitor the recovery process. This study is the first report of chiral materials used in the diagnosis and treatment of AKI.

MEKK3 in hybrid snakehead (Channa maculate ♀ ×Channa argus ♂): Molecular characterization and immune response to infection with Nocardia seriolae and Aeromonas schubertii.

Mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 3 (MEKK3) is a serine/threonine protein kinase that acts as a key regulator and is widely involved in various innate and acquired immune signaling pathways. In this study, we first cloned the complete open reading frame (ORF) of the MEKK3 gene (named CcMEKK3) in a hybrid snakehead (Channa maculate ♀ × Channa argus ♂). The full-length ORF of CcMEKK3 is 1851 bp, and encodes a putative protein of 616 amino acids containing a serine/threonine kinase catalytic (S-TKc) domain and a Phox and Bem1p (PB1) domain. A sequence alignment and phylogenetic tree analysis showed that CcMEKK3 is highly conserved relative to the MEKK3 proteins of other teleost species. CcMEKK3 was constitutively expressed in all the healthy hybrid snakehead tissues tested, with greatest expression in the immune tissues, such as the head kidney and spleen. The expression of CcMEKK3 was usually upregulated in the head kidney, spleen, and liver at different time points after infection with Nocardia seriolae or Aeromonas schubertii. Similarly, the dynamic expression levels of CcMEKK3 in head kidney leukocytes after stimulation revealed that CcMEKK3 was induced by LTA, LPS, and poly(I:C). In the subcellular localization analysis, CcMEKK3 was evenly distributed in the cytoplasm of HEK293T cells, and its overexpression significantly promoted the activities of NF-κB and AP-1. These results suggest that CcMEKK3 is involved in the immune defense against these two pathogens, and plays a crucial role in activating the NF-κB and MAPK signaling pathways.

HPF1 remodels the active site of PARP1 to enable the serine ADP-ribosylation of histones.

Upon binding to DNA breaks, poly(ADP-ribose) polymerase 1 (PARP1) ADP-ribosylates itself and other factors to initiate DNA repair. Serine is the major residue for ADP-ribosylation upon DNA damage, which strictly depends on HPF1. Here, we report the crystal structures of human HPF1/PARP1-CAT ΔHD complex at 1.98 Å resolution, and mouse and human HPF1 at 1.71 Å and 1.57 Å resolution, respectively. Our structures and mutagenesis data confirm that the structural insights obtained in a recent HPF1/PARP2 study by Suskiewicz et al. apply to PARP1. Moreover, we quantitatively characterize the key residues necessary for HPF1/PARP1 binding. Our data show that through salt-bridging to Glu284/Asp286, Arg239 positions Glu284 to catalyze serine ADP-ribosylation, maintains the local conformation of HPF1 to limit PARP1 automodification, and facilitates HPF1/PARP1 binding by neutralizing the negative charge of Glu284. These findings, along with the high-resolution structural data, may facilitate drug discovery targeting PARP1.