Massively parallel dissection of RNA in RNA-protein interactions in vivo.

Many of the biological functions performed by RNA are mediated by RNA-binding proteins (RBPs), and understanding the molecular basis of these interactions is fundamental to biology. Here, we present massively parallel RNA assay combined with immunoprecipitation (MPRNA-IP) for in vivo high-throughput dissection of RNA-protein interactions and describe statistical models for identifying RNA domains and parsing the structural contributions of RNA. By using custom pools of tens of thousands of RNA sequences containing systematically designed truncations and mutations, MPRNA-IP is able to identify RNA domains, sequences, and secondary structures necessary and sufficient for protein binding in a single experiment. We show that this approach is successful for multiple RNAs of interest, including the long noncoding RNA NORAD, bacteriophage MS2 RNA, and human telomerase RNA, and we use it to interrogate the hitherto unknown sequence or structural RNA-binding preferences of the DNA-looping factor CTCF. By integrating systematic mutation analysis with crosslinking immunoprecipitation, MPRNA-IP provides a novel high-throughput way to elucidate RNA-based mechanisms behind RNA-protein interactions in vivo.

Zinc oxide nanoparticles exacerbate skin epithelial cell damage by upregulating pro-inflammatory cytokines and exosome secretion in M1 macrophages following UVB irradiation-induced skin injury.

BACKGROUND: Zinc oxide nanoparticles (ZnONPs) are common materials used in skin-related cosmetics and sunscreen products due to their whitening and strong UV light absorption properties. Although the protective effects of ZnONPs against UV light in intact skin have been well demonstrated, the effects of using ZnONPs on damaged or sunburned skin are still unclear. In this study, we aimed to reveal the detailed underlying mechanisms related to keratinocytes and macrophages exposed to UVB and ZnONPs. RESULTS: We demonstrated that ZnONPs exacerbated mouse skin damage after UVB exposure, followed by increased transepidermal water loss (TEWL) levels, cell death and epithelial thickness. In addition, ZnONPs could penetrate through the damaged epithelium, gain access to the dermis cells, and lead to severe inflammation by activation of M1 macrophage. Mechanistic studies indicated that co-exposure of keratinocytes to UVB and ZnONPs lysosomal impairment and autophagy dysfunction, which increased cell exosome release. However, these exosomes could be taken up by macrophages, which accelerated M1 macrophage polarization. Furthermore, ZnONPs also induced a lasting inflammatory response in M1 macrophages and affected epithelial cell repair by regulating the autophagy-mediated NLRP3 inflammasome and macrophage exosome secretion. CONCLUSIONS: Our findings propose a new concept for ZnONP-induced skin toxicity mechanisms and the safety issue of ZnONPs application on vulnerable skin. The process involved an interplay of lysosomal impairment, autophagy-mediated NLRP3 inflammasome and macrophage exosome secretion. The current finding is valuable for evaluating the effects of ZnONPs for cosmetics applications.

Polystyrene microplastic-induced extracellular vesicles cause kidney-related effects in the crosstalk between tubular cells and fibroblasts.

Plastic waste accumulation and its degradation into microplastics (MPs) and nanoplastics (NPs) pose environmental concerns. Previous studies have indicated that polystyrene (PS)-MPs harm living animals. Extracellular vesicles (EVs) are associated with metabolic reprogramming and mitochondrial dysfunction in various kidney diseases. In this article, we evaluated how PS-MPs affected tubular cells and fibroblasts. The results demonstrated that PS-MPs increased EV production in human tubular cells and caused endoplasmic reticulum (ER) stress-related proteins without inducing inflammation-related proteins in human tubular cells. The uptake of PS-MPs and incubation with the conditioned medium of PS-MPs induced reactive oxygen species (ROS) production and ER stress-related proteins in fibroblast cells. The fibroblast cells treated with the conditioned medium of PS-MPs also increased the expression of fibrosis-related proteins. Our findings suggested that the expression of EV-related markers increased in tubular cells via Beclin 1 after PS-MP treatment. In addition, PS-MPs induced ROS production in vitro and in vivo. We found that PS-MPs also altered the expression of EV markers in urine, and CD63 expression was also increased in vitro and in vivo after PS-MP treatment. In conclusion, PS-MP-induced EVs lead to ER stress-related proteins, ROS production and fibrosis-related proteins in tubular cells and fibroblasts.

Reconstitution of the Final Steps in the Biosynthesis of Valanimycin Reveals the Origin of Its Characteristic Azoxy Moiety.

Valanimycin is an azoxy-containing natural product isolated from the fermentation broth of Streptomyces viridifaciens MG456-hF10. While the biosynthesis of valanimycin has been partially characterized, how the azoxy group is constructed remains obscure. Herein, the membrane protein VlmO and the putative hydrazine synthetase ForJ from the formycin biosynthetic pathway are demonstrated to catalyze N-N bond formation converting O-(l-seryl)-isobutyl hydroxylamine into N-(isobutylamino)-l-serine. Subsequent installation of the azoxy group is shown to be catalyzed by the non-heme diiron enzyme VlmB in a reaction in which the N-N single bond in the VlmO/ForJ product is oxidized by four electrons to yield the azoxy group. The catalytic cycle of VlmB appears to begin with a resting µ-oxo diferric complex in VlmB, as supported by Mössbauer spectroscopy. This study also identifies N-(isobutylamino)-d-serine as an alternative substrate for VlmB leading to two azoxy regioisomers. The reactions catalyzed by the kinase VlmJ and the lyase VlmK during the final steps of valanimycin biosynthesis are established as well. The biosynthesis of valanimycin was thus fully reconstituted in vitro using the enzymes VlmO/ForJ, VlmB, VlmJ and VlmK. Importantly, the VlmB-catalyzed reaction represents the first example of enzyme-catalyzed azoxy formation and is expected to proceed by an atypical mechanism.

Changing Fates of the Substrate Radicals Generated in the Active Sites of the B12-Dependent Radical SAM Enzymes OxsB and AlsB.

OxsB is a B12-dependent radical SAM enzyme that catalyzes the oxidative ring contraction of 2'-deoxyadenosine 5'-phosphate to the dehydrogenated, oxetane containing precursor of oxetanocin A phosphate. AlsB is a homologue of OxsB that participates in a similar reaction during the biosynthesis of albucidin. Herein, OxsB and AlsB are shown to also catalyze radical mediated, stereoselective C2'-methylation of 2'-deoxyadenosine monophosphate. This reaction proceeds with inversion of configuration such that the resulting product also possesses a C2' hydrogen atom available for abstraction. However, in contrast to methylation, subsequent rounds of catalysis result in C-C dehydrogenation of the newly added methyl group to yield a 2'-methylidene followed by radical addition of a 5'-deoxyadenosyl moiety to produce a heterodimer. These observations expand the scope of reactions catalyzed by B12-dependent radical SAM enzymes and emphasize the susceptibility of radical intermediates to bifurcation along different reaction pathways even within the highly organized active site of an enzyme.

S-Adenosylmethionine: more than just a methyl donor.

Covering: from 2000 up to the very early part of 2023S-Adenosyl-L-methionine (SAM) is a naturally occurring trialkyl sulfonium molecule that is typically associated with biological methyltransfer reactions. However, SAM is also known to donate methylene, aminocarboxypropyl, adenosyl and amino moieties during natural product biosynthetic reactions. The reaction scope is further expanded as SAM itself can be modified prior to the group transfer such that a SAM-derived carboxymethyl or aminopropyl moiety can also be transferred. Moreover, the sulfonium cation in SAM has itself been found to be critical for several other enzymatic transformations. Thus, while many SAM-dependent enzymes are characterized by a methyltransferase fold, not all of them are necessarily methyltransferases. Furthermore, other SAM-dependent enzymes do not possess such a structural feature suggesting diversification along different evolutionary lineages. Despite the biological versatility of SAM, it nevertheless parallels the chemistry of sulfonium compounds used in organic synthesis. The question thus becomes how enzymes catalyze distinct transformations via subtle differences in their active sites. This review summarizes recent advances in the discovery of novel SAM utilizing enzymes that rely on Lewis acid/base chemistry as opposed to radical mechanisms of catalysis. The examples are categorized based on the presence of a methyltransferase fold and the role played by SAM within the context of known sulfonium chemistry.

Engineering Alkaline-Stable Barley Stripe Mosaic Virus-Like Particles for Efficient Surface Modification.

Viruses and virus-like particles are powerful templates for materials synthesis because of their capacity for precise protein engineering and diverse surface functionalization. We recently developed a recombinant bacterial expression system for the production of barley stripe mosaic virus-like particles (BSMV VLPs). However, the applicability of this biotemplate was limited by low stability in alkaline conditions and a lack of chemical handles for ligand attachment. Here, we identify and validate novel residues in the BSMV Caspar carboxylate clusters that mediate virion disassembly through repulsive interactions at high pH. Point mutations of these residues to create attractive interactions that increase rod length ~2 fold, with an average rod length of 91 nm under alkaline conditions. To enable diverse chemical surface functionalization, we also introduce reactive lysine residues at the C-terminus of BSMV coat protein, which is presented on the VLP surface. Chemical conjugation reactions with this lysine proceed more quickly under alkaline conditions. Thus, our alkaline-stable VLP mutants are more suitable for rapid surface functionalization of long nanorods. This work validates novel residues involved in BSMV VLP assembly and demonstrates the feasibility of chemical functionalization of BSMV VLPs for the first time, enabling novel biomedical and chemical applications.

Characterization of the Oxazinomycin Biosynthetic Pathway Revealing the Key Role of a Nonheme Iron-Dependent Mono-oxygenase.

Oxazinomycin is a C-nucleoside natural product with antibacterial and antitumor activities. In addition to the characteristic C-glycosidic linkage shared with other C-nucleosides, oxazinomycin also features a structurally unusual 1,3-oxazine moiety, the biosynthesis of which had previously been unknown. Herein, complete in vitro reconstitution of the oxazinomycin biosynthetic pathway is described. Construction of the C-glycosidic bond between ribose 5-phosphate and an oxygen-labile pyridine heterocycle is catalyzed by the C-glycosidase OzmB and involves formation of an enzyme-substrate Schiff base intermediate. The DUF4243 family protein OzmD is shown to catalyze oxygen insertion and rearrangement of the pyridine C-nucleoside intermediate to generate the 1,3-oxazine moiety along with the elimination of cyanide. Spectroscopic analysis and mutagenesis studies indicate that OzmD is a novel nonheme iron-dependent enzyme in which the catalytic iron center is likely coordinated by four histidine residues. These results provide the first example of 1,3-oxazine biosynthesis catalyzed by an unprecedented iron-dependent mono-oxygenase.

Radical S-Adenosyl Methionine Enzyme BlsE Catalyzes a Radical-Mediated 1,2-Diol Dehydration during the Biosynthesis of Blasticidin S.

The biosynthesis of blasticidin S has drawn attention due to the participation of the radical S-adenosyl methionine (SAM) enzyme BlsE. The original assignment of BlsE as a radical-mediated, redox-neutral decarboxylase is unusual because this reaction appears to serve no biosynthetic purpose and would need to be reversed by a subsequent carboxylation step. Furthermore, with the exception of BlsE, all other radical SAM decarboxylases reported to date are oxidative in nature. Careful analysis of the BlsE reaction, however, demonstrates that BlsE is not a decarboxylase but instead a lyase that catalyzes the dehydration of cytosylglucuronic acid (CGA) to form cytosyl-4'-keto-3'-deoxy-d-glucuronic acid, which can rapidly decarboxylate nonenzymatically in vitro. Analysis of substrate isotopologs, fluorinated analogues, as well as computational models based on X-ray crystal structures of the BlsE·SAM (2.09 Å) and BlsE·SAM·CGA (2.62 Å) complexes suggests that BlsE catalysis likely proceeds via direct elimination of water from the CGA C4' α-hydroxyalkyl radical as opposed to 1,2-migration of the C3'-hydroxyl prior to dehydration. Biosynthetic and mechanistic implications of the revised assignment of BlsE are discussed.

Interfering B cell receptor signaling via SHP-1/p-Lyn axis shows therapeutic potential in diffuse large B-cell lymphoma.

BACKGROUND: Diffuse large B cell lymphoma (DLBCL) is an aggressive and molecularly heterogeneous non-Hodgkin's lymphoma. The B cell receptor (BCR) signaling pathway in DLBCL emerges as a new drug target. Protein phosphatase SHP-1 negatively regulates several oncogenic tyrosine kinases and plays a tumor suppressive role. METHODS: The direct SHP-1 agonists were used to evaluate the potential therapeutic implication of SHP-1 in DLBCL. Immunohistochemical staining for SHP-1 was quantified by H-score. The SHP-1 phosphatase activity was determined using tyrosine phosphatase assay. In vitro studies, including MTT, western blot analysis and cell apoptosis, were utilized to examined biological functions of SHP-1. RESULTS: Oral administration of SHP-1 agonist showed the potent anti-tumor effects compared to a selective Bruton's tyrosine kinase (BTK) inhibitor ibrutinib in mice bearing U2932 xenografts. SHP-1 agonist increased SHP-1 activity as well as downregulated p-Lyn in vivo. Here, we demonstrated that immunohistochemical staining for SHP-1 expression was positive in 76% of DLBCL samples. SHP-1 agonist exerted anti-proliferative and apoptotic effects compared with ibrutinib in DLBCL cells. Mechanistically, SHP-1 agonist decreased BCR signaling, especially p-Lyn, and led to apoptosis. CONCLUSIONS: These data suggest that SHP-1 negatively regulates phosphorylation of Lyn, and targeting SHP-1/p-Lyn using SHP-1 agonist has therapeutic potential for treatment of DLBCL.

Skin damage induced by zinc oxide nanoparticles combined with UVB is mediated by activating cell pyroptosis via the NLRP3 inflammasome-autophagy-exosomal pathway.

BACKGROUND: Zinc oxide nanoparticles (ZnONPs) are widely used nanomaterial in personal cosmetics, such as skin creams and sunscreens, due to their whitening properties and strong UV light absorption. However, the safety issues and the hazards of ZnONPs, which can be taken up by the skin and cause skin toxicity, are still unclear. From a chemoprevention point of view, pterostilbene (PT) has been reported to prevent skin damage effectively by its anti-inflammatory and autophagy inducer effect. This study aims to determine the skin toxicity and the potential mechanisms of UVB and ZnONPs exposure and the preventive effect of PT. RESULTS: The co-exposure of UVB and ZnONPs elicit NLRP3 inflammasome activation and pyroptosis in keratinocytes. Furthermore, exposure to both UVB and ZnONPs also disrupts cellular autophagy, which increases cell exosome release. In vivo UVB and ZnONPs exposure triggers skin toxicity, as indicated by increased histological injury, skin thickness and transepidermal water loss. Notably, the NLRP3 inflammasome-mediated pyroptosis are also activated during exposure. Topical application of pterostilbene attenuates NLRP3 inflammasome activation and pyroptosis by decreasing ROS generation and mitochondrial ROS (mtROS) levels. In addition to its antioxidant effect, PT also reversed autophagy abnormalities by restoring normal autophagic flux and decreasing NLRP3 inflammasome-loaded exosome release. CONCLUSIONS: Our findings reveal that ZnONPs induce skin damage in conjunction with UVB exposure. This process involves an interplay of inflammasomes, pyroptosis, autophagy dysfunction, and exosomes in skin toxicity. PT alleviates skin inflammation by regulating the inflammasome-autophagy-exosome pathway, a finding which could prove valuable when further evaluating ZnONPs effects for cosmetic applications.

Use of an in silico knowledge discovery approach to determine mechanistic studies of silver nanoparticles-induced toxicity from in vitro to in vivo.

BACKGROUND: Silver nanoparticles (AgNPs) are considered a double-edged sword that demonstrates beneficial and harmful effects depending on their dimensions and surface coating types. However, mechanistic understanding of the size- and coating-dependent effects of AgNPs in vitro and in vivo remains elusive. We adopted an in silico decision tree-based knowledge-discovery-in-databases process to prioritize the factors affecting the toxic potential of AgNPs, which included exposure dose, cell type and AgNP type (i.e., size and surface coating), and exposure time. This approach also contributed to effective knowledge integration between cell-based phenomenological observations and in vitro/in vivo mechanistic explorations. RESULTS: The consolidated cell viability assessment results were used to create a tree model for generalizing cytotoxic behavior of the four AgNP types: SCS, LCS, SAS, and LAS. The model ranked the toxicity-related parameters in the following order of importance: exposure dose > cell type > particle size > exposure time ≥ surface coating. Mechanistically, larger AgNPs appeared to provoke greater levels of autophagy in vitro, which occurred during the earlier phase of both subcytotoxic and cytotoxic exposures. Furthermore, apoptosis rather than necrosis majorly accounted for compromised cell survival over the above dosage range. Intriguingly, exposure to non-cytotoxic doses of AgNPs induced G2/M cell cycle arrest and senescence instead. At the organismal level, SCS following a single intraperitoneal injection was found more toxic to BALB/c mice as compared to SAS. Both particles could be deposited in various target organs (e.g., spleen, liver, and kidneys). Morphological observation, along with serum biochemical and histological analyses, indicated that AgNPs could produce pancreatic toxicity, apart from leading to hepatic inflammation. CONCLUSIONS: Our integrated in vitro, in silico, and in vivo study revealed that AgNPs exerted toxicity in dose-, cell/organ type- and particle type-dependent manners. More importantly, a single injection of lethal-dose AgNPs (i.e., SCS and SAS) could incur severe damage to pancreas and raise blood glucose levels at the early phase of exposure.

Comparing different surface modifications of zinc oxide nanoparticles in the developmental toxicity of zebrafish embryos and larvae.

Nanotechnology allows for a greater quality of life, but may also cause environmental and organismic harm. Zinc oxide nanoparticles (ZnONPs) are one of the most commonly used metal oxide nanoparticles for commercial and industrial products. Due to its extensive use in various fields, there has already been much concern raised about the environmental health risks of ZnONPs. Many studies have investigated the toxicological profile of ZnONPs in zebrafish embryonic development; however, the specific characteristics of ZnONPs in zebrafish embryonic/larval developmental damage and their molecular toxic mechanisms of liver development are yet to be fully elucidated. This study aimed to reveal the hazard ranking of different surface modifications of ZnONPs on developing zebrafish and the toxicological mechanisms of these modified ZnONPs in liver tissue. The ~30 nm ZnONPs with amino- (NH2- ZnONPs) or carboxyl- (COOH-ZnONPs) modification were incorporated during the embryonic/larval stage of zebrafish. Severe toxicity was observed in both ZnONP groups, especially NH2-ZnONPs, which presented a higher toxicity in the low concentration groups. After prolonging the exposure time, the long-term toxicity assay showed a greater retardation in body length of zebrafish in the NH2-ZnONP group. Response data from multiple toxicity studies was integrated for the calculation of the EC50 values of bulk ZnO and ZnONPs, and the hazard levels were found to be decreasing in the order of NH2-, COOH-ZnONPs and bulk ZnO. Notably, NH2-ZnONPs induced ROS burden in the developing liver tissue, which activated autophagy-related gene and protein expression and finally induced liver cell apoptosis to reduce liver size. In conclusion, our findings are conducive to understanding the hazard risks of different surface modifications of ZnONPs in aquatic environments and will also be helpful for choosing the type of ZnONPs in future industrial applications.

Contrast Agent Pooling (C.A.P.) sign and imminent cardiac arrest: a retrospective study.

BACKGROUND: The sign of contrast agent pooling (C.A.P.) in dependent part of the venous system were reported in some case reports, which happened in the patients before sudden cardiac arrest. Until now, there is no solid evidence enough to address the importance of the sign. This study aimed to assess the accuracy of the C.A.P. sign in predicting imminent cardiac arrest and the association of the C.A.P. sign with patient's survival. METHODS: This is a retrospective cohort study. The study included all patients who visited the emergency department, who received contrast computed tomography (CT) scan and then experienced cardiac arrest at the emergency department (from January 1, 2016 to December 31, 2018). We evaluated the occurrence of the C.A.P. sign on the chest or abdominal CT scan, patients with ECMO were excluded. With positive C.A.P. sign, the primary outcome is whether in-hospital cardiac arrest happens within an hour; the accuracy of C.A.P. sign was calculated. The secondary outcome is survival to discharge. RESULTS: In the study, 128 patients were included. 8.6% (N = 11) patients had positive C.A.P. sign and 91.4% (N = 117) patients did not. The accuracy of C.A.P. sign in predicting cardiac arrest within 1 h was 85.94%. The C.A.P. sign had a positive association with IHCA within 1 h after the CT scan (adjusted odds ratio 7.35, 95% confidence interval [CI] 1.27 - 42.69). The relative risk (RR) of survival to discharge was 0.90 with positive C.A.P. sign (95% CI 0.85 - 0.96). CONCLUSIONS: The C.A.P. sign can be considered as an alarm for imminent cardiac arrest and poor prognosis. The patients with positive C.A.P. sign were more likely to experience imminent cardiac arrest; in contrast, less likely to survive. TRIAL REGISTRATION: IRB No.108107-E.

Insight into the Phylogeny and Binding Ability of WRKY Transcription Factors.

WRKY transcription factors (TFs), which make up one of the largest families of TFs in the plant kingdom, are key players in modulating gene expression relating to embryogenesis, senescence, pathogen resistance, and abiotic stress responses. However, the phylogeny and grouping of WRKY TFs and how their binding ability is affected by the flanking regions of W-box sequences remain unclear. In this study, we reconstructed the phylogeny of WRKY across the plant kingdom and characterized the DNA-binding profile of Arabidopsis thaliana WRKY (WRKY54) based on its W-box recognition sequence. We found that WRKY TFs could be separated into five clades, and that the functional zinc-finger motif at the C-terminal of WRKY appeared after several nucleotide substitutions had occurred at the 3'-end of the zinc-finger region in chlorophytes. In addition, we found that W-box flanking regions affect the binding ability of WRKY54 based on the results of a fluorescence-based electrophoretic mobility shift assay (fEMSA) and quartz crystal microbalance (QCM) analysis. The great abundance of WRKY TFs in plants implicates their involvement in diverse molecular regulatory networks, and the flanking regions of W-box sequences may contribute to their molecular recognition mechanism. This phylogeny and our findings on the molecular recognition mechanism of WRKY TFs should be helpful for further research in this area.

Two Radical SAM Enzymes Are Necessary and Sufficient for the In Vitro Production of the Oxetane Nucleoside Antiviral Agent Albucidin.

Oxetanocin A and albucidin are two oxetane natural products. While the biosynthesis of oxetanocin A has been described, less is known about albucidin. In this work, the albucidin biosynthetic gene cluster is identified in Streptomyces. Heterologous expression in a nonproducing strain demonstrates that the genes alsA and alsB are necessary and sufficient for albucidin biosynthesis confirming a previous study (Myronovskyi et al. Microorganisms 2020, 8, 237). A two-step construction of albucidin 4'-phosphate from 2'-deoxyadenosine monophosphate (2'-dAMP) is shown to be catalyzed in vitro by the cobalamin dependent radical S-adenosyl-l-methionine (SAM) enzyme AlsB, which catalyzes a ring contraction, and the radical SAM enzyme AlsA, which catalyzes elimination of a one-carbon fragment. Isotope labelling studies show that AlsB catalysis begins with stereospecific H-atom transfer of the C2'-pro-R hydrogen from 2'-dAMP to 5'-deoxyadenosine, and that the eliminated one-carbon fragment originates from C3' of 2'-dAMP.

Biosynthesis of Oxetanocin-A Includes a B12-Dependent Radical SAM Enzyme That Can Catalyze both Oxidative Ring Contraction and the Demethylation of SAM.

Oxetanocin-A is an antitumor, antiviral, and antibacterial nucleoside. It is biosynthesized via the oxidative ring contraction of a purine nucleoside co-opted from primary metabolism. This reaction is catalyzed by a B12-dependent radical S-adenosyl-l-methionine (SAM) enzyme, OxsB, and a phosphohydrolase, OxsA. Previous experiments showed that the product of the OxsB/OxsA-catalyzed reaction is an oxetane aldehyde produced alongside an uncharacterized byproduct. Experiments reported herein reveal that OxsB/OxsA complex formation is crucial for the ring contraction reaction and that reduction of the aldehyde intermediate is catalyzed by a nonspecific dehydrogenase from the general cellular pool. In addition, the byproduct is identified as a 1,3-thiazinane adduct between the aldehyde and l-homocysteine. While homocysteine was never included in the OxsB/OxsA assays, the data suggest that it can be generated from SAM via S-adenosyl-l-homocysteine (SAH). Further study revealed that conversion of SAM to SAH is facilitated by OxsB; however, the subsequent conversion of SAH to homocysteine is due to protein contaminants that co-purify with OxsA. Nevertheless, the observed demethylation of SAM to SAH suggests possible methyltransferase activity of OxsB, and substrate methylation was indeed detected in the OxsB-catalyzed reaction. This work is significant because it not only completes the description of the oxetanocin-A biosynthetic pathway but also suggests that OxsB may be capable of methyltransferase activity.

Structure of Arabidopsis HISTONE DEACETYLASE15.

Mammalian histone deacetylases (HDACs) undergo phosphorylation to regulate their localization, activity, and function. However, little is known about the regulation of plant HDAC function and activity by phosphorylation. Here, we report the crystal structure of the Reduced Potassium Dependency3/Histone Deacetylase1 (RPD3/HDA1) type class II histone deacetylase HDA15 in Arabidopsis (Arabidopsis thaliana). The histone deacetylase domain of HDA15 (HDA15HD) assembles as tetrameric forms with each monomer composed of 12 α-helices and 9 ß-sheets. The L1 loop and ß2 sheet of HDA15HD are the essential interfaces for the tetramer formation. The N-terminal zinc finger domain enhances HDA15HD dimerization and increases its enzymatic activity. Furthermore, HDA15 can also be phosphorylated at Ser-448 and Ser-452 in etiolated seedlings. The HDA15 phosphorylation status determines its subnuclear localization and oligomerization. Phosphomimetics of HDA15 partially disrupt its oligomerization and cause loss of enzymatic activity and translocation from the nucleolus into nucleoplasm. Together, these data indicate that phosphorylation plays a critical role in regulating the structure and function of HDA15.

Carbon monoxide-triggered health effects: the important role of the inflammasome and its possible crosstalk with autophagy and exosomes.

Carbon monoxide (CO) has long been known as a "silent killer" because of its ability to bind hemoglobin (Hb), leading to reduced oxygen carrying capacity of Hb, which is the main cause of CO poisoning (COP) in humans. Emerging studies suggest that mitochondria is a key target of CO action that can impact key biological processes, including apoptosis, cellular proliferation, inflammation, and autophagy. Despite its toxicity at high concentrations, CO also exhibits cyto- and tissue-protective effects at low concentrations in animal models of organ injury and disease. Specifically, CO modulates the production of pro- or anti-inflammatory cytokines and mediators by regulating the NLRP3 inflammasome. Given that human diseases are strongly associated with inflammation, a deep understanding of the exact mechanism is helpful for treatment. Autophagic factors and inflammasomes interact in various situations, including inflammatory disease, and exosomes might function as the bridge between the inflammasome and autophagy activation. Thus, the interplay among autophagy, mitochondrial dysfunction, exosomes, and the inflammasome may play pivotal roles in the health effects of CO. In this review, we summarize the latest research on the beneficial and toxic effects of CO and their underlying mechanisms, focusing on the important role of the inflammasome and its possible crosstalk with autophagy and exosomes. This knowledge may lead to the development of new therapies for inflammation-related diseases and is essential for the development of new therapeutic strategies and biomarkers of COP.

Modulation of Innate Immune Toxicity by Silver Nanoparticle Exposure and the Preventive Effects of Pterostilbene.

Silver nanoparticles pose a potential risk to ecosystems and living organisms due to their widespread use in various fields and subsequent gradual release into the environment. Only a few studies have investigated the effects of silver nanoparticles (AgNPs) toxicity on immunological functions. Furthermore, these toxic effects have not been fully explored. Recent studies have indicated that zebrafish are considered a good alternative model for testing toxicity and for evaluating immunological toxicity. Therefore, the purpose of this study was to investigate the toxicity effects of AgNPs on innate immunity using a zebrafish model and to investigate whether the natural compound pterostilbene (PTE) could provide protection against AgNPs-induced immunotoxicity. Wild type and neutrophil- and macrophage-transgenic zebrafish lines were used in the experiments. The results indicated that the exposure to AgNPs induced toxic effects including death, malformation and the innate immune toxicity of zebrafish. In addition, AgNPs affect the number and function of neutrophils and macrophages. The expression of immune-related cytokines and chemokines was also affected. Notably, the addition of PTE could activate immune cells and promote their accumulation in injured areas in zebrafish, thereby reducing the damage caused by AgNPs. In conclusion, AgNPs may induce innate immune toxicity and PTE could ameliorate this toxicity.