Haplotype-resolved 3D chromatin architecture of the hybrid pig.

In diploid mammals, allele-specific three-dimensional (3D) genome architecture may lead to imbalanced gene expression. Through ultradeep in situ Hi-C sequencing of three representative somatic tissues (liver, skeletal muscle, and brain) from hybrid pigs generated by reciprocal crosses of phenotypically and physiologically divergent Berkshire and Tibetan pigs, we uncover extensive chromatin reorganization between homologous chromosomes across multiple scales. Haplotype-based interrogation of multi-omic data revealed the tissue dependence of 3D chromatin conformation, suggesting that parent-of-origin-specific conformation may drive gene imprinting. We quantify the effects of genetic variations and histone modifications on allelic differences of long-range promoter-enhancer contacts, which likely contribute to the phenotypic differences between the parental pig breeds. We also observe the fine structure of somatically paired homologous chromosomes in the pig genome, which has a functional implication genome-wide. This work illustrates how allele-specific chromatin architecture facilitates concomitant shifts in allele-biased gene expression, as well as the possible consequential phenotypic changes in mammals.

Spatio-temporal transcriptome dynamics coordinate rapid transition of core crop functions in 'lactating' pigeon.

Pigeons (Columba livia) are among a select few avian species that have developed a specialized reproductive mode wherein the parents produce a 'milk' in their crop to feed newborn squabs. Nonetheless, the transcriptomic dynamics and role in the rapid transition of core crop functions during 'lactation' remain largely unexplored. Here, we generated a de novo pigeon genome assembly to construct a high resolution spatio-temporal transcriptomic landscape of the crop epithelium across the entire breeding stage. This multi-omics analysis identified a set of 'lactation'-related genes involved in lipid and protein metabolism, which contribute to the rapid functional transitions in the crop. Analysis of in situ high-throughput chromatin conformation capture (Hi-C) sequencing revealed extensive reorganization of promoter-enhancer interactions linked to the dynamic expression of these 'lactation'-related genes between stages. Moreover, their expression is spatially localized in specific epithelial layers, and can be correlated with phenotypic changes in the crop. These results illustrate the preferential de novo synthesis of 'milk' lipids and proteins in the crop, and provides candidate enhancer loci for further investigation of the regulatory elements controlling pigeon 'lactation'.

Cryo-EM structure of human HCN3 channel and its regulation by cAMP.

HCN channels are important for regulating heart rhythm and nerve activity and have been studied as potential drug targets for treating depression, arrhythmia, nerve pain, and epilepsy. Despite possessing unique pharmacological properties, HCN channels share common characteristics in that they are activated by hyperpolarization and modulated by cAMP and other membrane lipids. However, the mechanisms of how these ligands bind and modulate HCN channels are unclear. In this study, we solved structures of full-length human HCN3 using cryo-EM and captured two different states, including a state without any ligand bound and a state with cAMP bound. Our structures reveal the novel binding sites for cholesteryl hemisuccinate in apo state and show how cholesteryl hemisuccinate and cAMP binding cause conformational changes in different states. These findings explain how these small modulators are sensed in mammals at the molecular level. The results of our study could help to design more potent and specific compounds to influence HCN channel activity and offer new therapeutic possibilities for diseases that lack effective treatment.

METTL14-Mediated m6a Modification of CDKN2A Promotes the Development of Retinoblastoma by Inhibiting the p53 Pathway.

The methyltransferase 14, N6-adenosine-methyltransferase subunit (METTL14) and Cyclin-dependent kinase inhibitor 2A (CDKN2A) have been identified as involved in the regulation of various cancer progression, while their mechanism and regulatory effect in retinoblastoma (RB) is still unclear. Cell colony formation, CCK-8 as well as Western blotting were used to evaluate the proliferation, apoptosis as well as p53 protein level of RB cell line. The METTL14 and CDKN2A levels were detected by qRT-PCR or Western blotting when METTL14 was up-regulated or CDKN2A was down-regulated. MeRIP and Pearson analysis were performed to confirm the regulatory relationship between METTL14 among CDKN2A. We found that the levels of CDKN2A and METTL14 were abundant in RB samples, as well as RB cells. METTL14 enhances N6-methyladenosine (m6A) modification of CDKN2A to upregulate its mRNA and protein levels. The proliferation of RB cells can be inhibited by silencing CDKN2A, which promotes apoptosis and p53 protein level. Furthermore, high-expression of METTL14 eliminated the anti-tumor effect of CDKN2A silencing in RB progression in vitro. CDKN2A is mediated by METTL14-m6A modified and restrains p53 pathway activation to accelerate the malignancy of RB. This points to the METTL14-m6A-CDKN2A-p53 pathway axis as a possible prospective target for the future RB treatment.

The adaptor protein 14-3-3zeta modulates intestinal immunity and aging in Drosophila.

The proteins that coordinate the complex transcriptional networks of aging have not been completely documented. Protein 14-3-3zeta is an adaptor protein that coordinates signaling and transcription factor networks, but its function in aging is not fully understood. Here, we showed that the protein expression of 14-3-3zeta gradually increased during aging. High levels of 14-3-3zeta led to shortened lifespan and imbalance of intestinal immune homeostasis in Drosophila, but the decrease in 14-3-3zeta protein levels by RNAi was able to significantly promote the longevity and intestinal immune homeostasis of fruit flies. Importantly, we demonstrate that adult-onset administration of TIC10, a compound that reduces the aging-related AKT and extracellular signal-regulated kinase (ERK) signaling pathways, rescues the shortened lifespan of 14-3-3zeta-overexpressing flies. This finding suggests that 14-3-3zeta plays a critical role in regulating the aging process. Our study elucidates the role of 14-3-3zeta in natural aging and provides the rationale for subsequent 14-3-3zeta-based antiaging research.

Comparative three-dimensional genome architectures of adipose tissues provide insight into human-specific regulation of metabolic homeostasis.

Elucidating the regulatory mechanisms of human adipose tissues (ATs) evolution is essential for understanding human-specific metabolic regulation, but the functional importance and evolutionary dynamics of three-dimensional (3D) genome organizations of ATs are not well defined. Here, we compared the 3D genome architectures of anatomically distinct ATs from humans and six representative mammalian models. We recognized evolutionarily conserved and human-specific chromatin conformation in ATs at multiple scales, including compartmentalization, topologically associating domain (TAD), and promoter-enhancer interactions (PEI), which have not been described previously. We found PEI are much more evolutionarily dynamic with respect to compartmentalization and topologically associating domain. Compared to conserved PEIs, human-specific PEIs are enriched for human-specific sequence, and the binding motifs of their potential mediators (transcription factors) are less conserved. Our data also demonstrated that genes involved in the evolutionary dynamics of chromatin organization have weaker transcriptional conservation than those associated with conserved chromatin organization. Furthermore, the genes involved in energy metabolism and the maintenance of metabolic homeostasis are enriched in human-specific chromatin organization, while housekeeping genes, health-related genes, and genetic variations are enriched in evolutionarily conserved compared to human-specific chromatin organization. Finally, we showed extensively divergent human-specific 3D genome organizations among one subcutaneous and three visceral ATs. Together, these findings provide a global overview of 3D genome architecture dynamics between ATs from human and mammalian models and new insights into understanding the regulatory evolution of human ATs.

Cryo-EM structure of mouse TRPML2 in lipid nanodiscs.

In mammalians, transient receptor potential mucolipin ion channels (TRPMLs) exhibit variable permeability to cations such as Ca2+, Fe2+, Zn2+, and Na+ and can be activated by the phosphoinositide PI(3,5)P2 in the endolysosomal system. Loss or dysfunction of TRPMLs has been implicated in lysosomal storage disorders, infectious diseases, and metabolic diseases. TRPML2 has recently been identified as a mechanosensitive and hypotonicity-sensitive channel in endolysosomal organelles, which distinguishes it from TRPML1 and TRPML3. However, the molecular and gating mechanism of TRPML2 remains elusive. Here, we present the cryo-EM structure of the full-length mouse TRPML2 in lipid nanodiscs at 3.14 Å resolution. The TRPML2 homotetramer structure at pH 7.4 in the apo state reveals an inactive conformation and some unique features of the extracytosolic/luminal domain and voltage sensor-like domain that have implications for the ion-conducting pathway. This structure enables new comparisons between the different subgroups of TRPML channels with available structures and provides structural insights into the conservation and diversity of TRPML channels. These comparisons have broad implications for understanding a variety of molecular mechanisms of TRPMLs in different pH conditions, including with and without bound agonists and antagonists.

TMEM63 mechanosensitive ion channels: Activation mechanisms, biological functions and human genetic disorders.

The transmembrane 63 (TMEM63) family of proteins are originally identified as homologs of the osmosensitive calcium-permeable (OSCA) channels in plants. Mechanosensitivity of OSCA and TMEM63 proteins are recently demonstrated in addition to their proposed activation mechanism by hyper/hypo-osmolarity. TMEM63 proteins exist in all animals, with a single member in Drosophila (TMEM63) and three members in mammals (TMEM63 A/B/C). In humans, monoallelic variants of TMEM63A have been reported to cause transient hypomyelination during infancy, or severe hypomyelination and global developmental delay. Heterozygous variants of TMEM63B are found in patients with intellectual disability and abnormal motor function and brain morphology. Biallelic variants of TMEM63C are associated with hereditary spastic paraplegias accompanied by mild or no intellectual disability. Physiological functions of TMEM63 proteins clearly recognized so far include detecting food grittiness and environmental humidity in Drosophila, and supporting hearing in mice by regulating survival of cochlear hair cells. In this review, we summarize current knowledge about the activation mechanisms and biological functions of TMEM63 channels, and provide a concise reference for researchers interested in investigating more physiological and pathogenic roles of this family of proteins with ubiquitous expression in the body.

Bacillus subtilis reduces antibiotic resistance genes of animal sludge in vermicomposting by improving heat stress tolerance of Eisenia foetida and bacterial community adjustment.

Antibiotic resistance genes (ARGs) in livestock industry have been recognized as a kind of pollutant. The effect of Bacillus subtilis (B. subtilis) as an additive for the reduction of ARGs in animal sludge from livestock and poultry wastewater treatment plant during vermicomposting was investigated. We also evaluated the oxidative stress level and growth of earthworms, Eisenia foetida, bacterial community succession, and the quality of the end products. Two treatments were conducted using B. subtilis, one at 18 °C and another at 28 °C. Controls were setup without the bacteria. The results showed that inoculation of B. subtilis promoted the degradation of organics at 28 °C and increased the germination index to 236%. The increased activities of the superoxide dismutase (1.69 U/mg pr) and catalase (8.05 U/mg pr) and the decreased activity of malondialdehyde (0.02 nmol/mg pr) by B. subtilis at 28 °C showed that the earthworms were relieved of heat stress. The addition of B. subtilis reduced the abundance of 32 target ARGs, including integron (intI-1), transposase (IS613) and resistant genes, such as sulfonamide (sul2), quinolone (oprJ), macrolide-lincosamide-streptogramin group B (ermF, ermB), tetracycline (tetL-02, tetX), ß-lactama (blaOXA10-01) and aminoglycoside [strB, aac(6')-Ib(aka aacA4)-01, aac(6')-Ib(aka aacA4)-02]. Organic matter degrading Membranicola, Paludisphaera, Sphingorhabdus and uncultured bacterium belonging to the order Chitinophagales, nitrifying and nitrogen-fixing Singulisphaera and Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, soil remediating Achromobacter, and plant growth promoting Kaistia, Galbibacter and Ilumatobacter were increased significantly (P < 0.05). However, the growth of harmful bacteria such as Burkholderiaceae was inhibited in the vermicompost. In earthworm guts, the probiotic Mesorhizobium was promoted, while the pathogenic uncultured bacterium belonging to the family Enterobacteriaceae was reduced. Besides, B. subtilis enhanced the host relationships between bacteria and ARGs. These findings might be helpful in the removal of ARGs in animal wastes and in understanding the synergy between earthworms and microorganisms.

Nanoarchitecture-based photothermal ablation of cancer: A systematic review.

Photothermal therapy (PTT) is an emerging non-invasive method used in cancer treatment. In PTT, near-infrared laser light is absorbed by a chromophore and converted into heat within the tumor tissue. PTT for cancer usually combines a variety of interactive plasmonic nanomaterials with laser irradiation. PTT enjoys PT agents with high conversion efficiency to convert light into heat to destroy malignant tissue. In this review, published studies concerned with the use of nanoparticles (NPs) in PTT were collected by a systematic and comprehensive search of PubMed, Cochrane, Embase, and Scopus databases. Gold, silver and iron NPs were the most frequent choice in PTT. The use of surface modified NPs allowed selective delivery and led to a precise controlled increase in the local temperature. The presence of NPs during PTT can increase the reactive generation of oxygen species, damage the DNA and mitochondria, leading to cancer cell death mainly via apoptosis. Many studies recently used core-shell metal NPs, and the effects of the polymer coating or ligands targeted to specific cellular receptors in order to increase PTT efficiency were often reported. The effective parameters (NP type, size, concentration, coated polymers or attached ligands, exposure conditions, cell line or type, and cell death mechanisms) were investigated individually. With the advances in chemical synthesis technology, NPs with different shapes, sizes, and coatings can be prepared with desirable properties, to achieve multimodal cancer treatment with precision and specificity.

A novel self-purified auxiliary protein enhances the lichenase activity towards lichenan for biomass degradation.

Due to the complex composition of lichenan, lichenase alone cannot always hydrolyze it efficiently. Carbohydrate-binding modules (CBMs) and lytic polysaccharide monooxygenases (LPMOs) have been confirmed to increase the hydrolysis efficiency of lichenases. However, their practical application was hampered by the complex and costly preparation procedure, as well as the poor stability of LPMOs. Herein, we discovered a novel and stable auxiliary protein named SCE to boost the hydrolysis efficiency. SCE was composed of SpyCatcher (SC) and elastin-like polypeptides (ELPs) and could be easily and cheaply prepared. Under the optimal conditions, the boosting degree for SCE/lichenase was 1.45, and the reducing sugar yield improved by nearly 45%. The results of high-performance liquid chromatography (HPLC) indicated that SCE had no influence on the hydrolysis pattern of lichenase. Through the experimental verification and bioinformatics analysis, we proposed the role of SCE in promoting the interaction between the lichenase and substrates. These findings endow SC with a novel function in binding to insoluble lichenan, paving the way for biomass degradation and biorefinery. KEY POINTS: • A novel self-purification auxiliary protein that could boost the hydrolysis efficiency of lichenase has been identified. • The protein is highly produced, simple to prepare, well stable, and does not require any external electron donor. • The novel function of SpyCatcher in binding to insoluble lichenan was first demonstrated.

Protocol: identifying policy, system, and environment change interventions to enhance availability of blood for transfusion in Kenya, a mixed-methods study.

BACKGROUND: Safe blood is essential for the care of patients with life-threatening anemia and hemorrhage. Low blood donation rates, inefficient testing procedures, and other supply chain disruptions in blood administration affect patients in low-resource settings across Sub-Saharan countries, including Kenya. Most efforts to improve access to transfusion have been unidimensional, usually focusing on only point along the blood system continuum, and have excluded community stakeholders from early stages of intervention development. Context-appropriate interventions to improve the availability of safe blood at the point of use in low-resource settings are of paramount importance. Thus, this protocol proposes a multifaceted approach to characterize the Kenyan blood supply chain through quantitative and qualitative analyses as well as an industrial engineering approach. METHODS: This study will use a mixed-methods approach in addition to engineering process mapping, modeling and simulation of blood availability in Kenya. It will be guided by a multidimensional three-by-three-by-three matrix: three socioeconomic settings, three components of the blood system continuum, and three levels of urgency of blood transfusion. Qualitative data collection includes one-on-one interviews and focus group discussions with stakeholders across the continuum to characterize ground-level deficits and potential policy, systems, and environment (PSE) interventions. Prospectively-collected quantitative data will be used to estimate blood collection and transfusion of blood. We will create a process map of the blood system continuum to model the response to PSE changes proposed by stakeholders. Lastly, we will identify those PSE changes that may have the greatest impact on blood transfusion availability, accounting for differences across socioeconomic settings and levels of urgency. DISCUSSION: Identifying and prioritizing community-driven interventions to improve blood supply in low-resource settings are of utmost importance. Varied constraints in blood collection, processing, delivery, and use make each socioeconomic setting unique. Using a multifaceted approach to understand the Kenyan blood supply and model the response to stakeholder-proposed PSE changes may lead to identification of contextually appropriate intervention targets to meet the transfusion needs of the population.

Genetic diversity of Toll-like receptor 9 in swamp eels (Monopterus albus).

The swamp eel, Monopterus albus, is an important aquaculture species in Asia (mainly China) whose production has seriously suffered from infectious diseases. In spite of the critical requirement for aquaculture practices, to date there is scant information on its immune defence. Here, the genetic characteristics of Toll-like receptor 9 (TLR9), which plays crucial roles in the initiation of host defence against microbial invasion, were analysed. It exhibits a striking lack of genetic variation resulting from a recent demographic bottleneck. A comparison with the homologue of M. javanensis revealed that replacement but not silent differences have nonrandomly accumulated in the coding sequences at the early stage following their split from a common ancestor. Furthermore, the replacements relevant to the type II functional divergence have mainly occurred in structural motifs mediating ligand recognition and receptor homodimerization. These results provide hints to understand the diversity-based strategy of TLR9 in the arms race against pathogens. Furthermore, the findings reported here give credence to the importance of basic immunology knowledge, especially for the key elements, in genetic engineering and breeding for disease resistance in the eel and other fishes.

The role of 18F-FDG PET/CT in predicting the pathological response to neoadjuvant PD-1 blockade in combination with chemotherapy for resectable esophageal squamous cell carcinoma.

PURPOSE: Accurate assessment of residual disease of tumor and lymph nodes after neoadjuvant immunochemotherapy is crucial in the active surveillance for patients with pathological complete response (pCR) and the optimal extent of lymphadenectomy for patients with non-pCR. This post hoc analysis aimed to evaluate the performance of 18F-FDG PET/CT to predict the pathological response to neoadjuvant immunochemotherapy for esophageal squamous cell carcinoma (ESCC). METHODS: Fifty-eight resectable ESCC patients received two cycles of camrelizumab in combination with chemotherapy and were enrolled in the final analysis. The 18F-FDG PET/CT scans were acquired at baseline (scan-1) and after immunochemotherapy but prior to surgery (scan-2). Maximum standardized uptake value (SUVmax), mean standardized uptake value (SUVmean), tumor-to-blood pool SUVmax ratio (SUVTBR), metabolic tumor volume (MTV), and total lesion glycolysis (TLG) were evaluated for their association with the pathological response to immunochemotherapy. RESULTS: Nineteen patients (32.8%, 19/58) had pCR and thirty-nine patients (67.2%, 39/58) had non-pCR after two doses of camrelizumab and chemotherapy. At scan-2, the SUVmax, SUVmean, SUVTBR, TLG, and MTV were significantly lower in pCR than in non-pCR patients. Decrease in TLG and MTV between scan-2 and scan-1 of the same patient was significantly higher in the pCR than in the non-pCR group. In the receiver operating characteristic curve analysis, SUVmax, SUVmean, SUVTBR, TLG, and MTV in scan-2 showed excellent predictive value for the pCR of primary tumors. Furthermore, SUVmax in scan-2 were higher in positive lymph nodes than in negative ones, suggesting a high negative predictive ability (98.6%) with a cut-off value at 1.4. CONCLUSION: The parameters of 18F-FDG PET/CT have the excellent performance for predicting pCR after the combined neoadjuvant immunochemotherapy in resectable ESCC. TRIAL REGISTRATION: ChiCTR2000028900. Registered on January 6, 2020.

The BLOODSAFE program: Building the future of access to safe blood in Sub-Saharan Africa.

BACKGROUND: The supply of blood in many low- and middle-income nations in Sub-Saharan Africa (SSA) does not meet the patient care needs. Lack and delay of blood transfusion cause harm to patients and slow the rate of progress in other parts of the health system. Recognizing the power of implementation science, the BLOODSAFE Program was initiated which supports three SSA research study teams and one data coordinating center (DCC) with the goal to improve access to safe blood transfusion in SSA. STUDY DESIGN AND METHODS: The study team in Ghana is focusing on studying and decreasing iron deficiency in blood donors and evaluating social engagement of blood donors through different approaches. The study team in Kenya is building a "vein to vein" workflow model to elucidate and devise strategies to overcome barriers to blood donation and improve infrastructural components of blood product production and use. The Malawi team is studying the infectious disease ramifications of blood donation as well as blood donor retention strategies aimed at blood donors who commence their donation career in secondary schools. RESULTS AND DISCUSSION: Together the project teams and the DCC work as a consortium to support each other through a shared study protocol that will study donor motivations, outcomes, and adverse events across all three countries. The BLOODSAFE Program has the potential to lead to generalizable improvement approaches for increasing access to safe blood in SSA as well as mentoring and building the research capacity and careers of many investigators.

Molecular Determinants for the High-Affinity Blockade of Human Ether-à-go-go-Related Gene K + Channel by Tolterodine.

ABSTRACT: Tolterodine is a first-line antimuscarinic drug used to treat overactive bladder. Adverse cardiac effects including tachycardia and palpitations have been observed, presumably because of its inhibition of the human ether-à-go-go-related gene (hERG) K + channel. However, the molecular mechanism of hERG channel inhibition by tolterodine is largely unclear. In this study, we performed molecular docking to identify potential binding sites of tolterodine in hERG channel, and two-microelectrode voltage-clamp to record the currents of hERG and its mutants expressed in Xenopus oocytes. The results of computational modeling demonstrated that phenylalanine at position 656 (F656) and tyrosine at position 652 (Y652) on the S6 helix of hERG channel are the most favorable binding residues of tolterodine, which was validated by electrophysiological recordings on Y652A and F656A hERG mutants. The Y652A and F656A mutations decreased inhibitory potency of tolterodine 345-fold and 126-fold, respectively. The Y652A mutation significantly altered the voltage dependence of channel inhibition by tolterodine. For both the wild-type and the mutant channels, tolterodine reduced the currents in a time-dependent manner, and the blockade occurred with the channel activated. Tolterodine did not interfere with hERG channel deactivation, whereas channel inactivation greatly impaired its blocking effect. The inhibition of hERG channel by tolterodine is independent of its action on muscarinic acetylcholine receptors. In conclusion, tolterodine is an open-state blocker of hERG K + channel with nanomolar potency. Y652 and F656, 2 aromatic residues on the inner S6 helix, are responsible for the high-affinity binding of tolterodine to hERG channel.

LncRNA FGD5-AS1 enhances the proliferation and stemness of hepatocellular carcinoma cells through targeting miR-223 and regulating the expression of ECT2 and FAT1.

AIM: Hepatocellular carcinoma (HCC) is common and causes many deaths worldwide. The aim of this study is to explore the mechanism by which long non-coding RNA FGD5-AS1 regulates HCC cell proliferation and stemness. METHODS: Tumor and normal adjacent tissues were harvested from HCC patients. Real-time quantitative reverse transcription-PCR was applied to examine the expression of FGD5-AS1, miR-223, Epithelial cell transforming sequence 2 (ECT2) and FAT1. The protein levels of ECT2, FAT1, proliferating cell nuclear antigen (PCNA), OCT4, CD133 and CD90 were analyzed by western blot. The localization of FGD5-AS1 was examined by Fluorescence in situ hybridization. Cell proliferation was analyzed with CCK-8 and colony formation assays. Spheroid formation was used for analyzing cell stemness. Gene interaction was examined by RNA immunoprecipitation and luciferase activity assays. A subcutaneous xenograft mouse model was established to analyze HCC growth and stemness in vivo. Immunohistochemistry staining was used to analyze the expression PCNA and OCT4 in subcutaneous tumors. RESULTS: FGD5-AS1 was upregulated in HCC and its high expression indicated poor prognosis of patients. High expression of FGD5-AS1 enhanced HCC cell proliferation and stemness. Knockdown of FGD5-AS1 restrained tumor growth and stemness in mice. FGD5-AS1 directly sponged miR-223 and promoted the expression of ECT2 and FAT1 in HCC. Both knockdown of miR-223 and overexpression of ECT2 and FAT1 reversed FGD5-AS1 silencing-mediated suppression of HCC cell proliferation and stemness. CONCLUSION: FGD5-AS1 directly sponged miR-223 and promoted the expression of ECT2 and FAT1 in HCC, thus enhancing HCC cell proliferation and stemness. Our study identifies potential prognostic biomarkers and therapeutic targets for HCC.

Gout-associated monosodium urate crystal-induced necrosis is independent of NLRP3 activity but can be suppressed by combined inhibitors for multiple signaling pathways.

Monosodium urate (MSU) crystals, the etiological agent of gout, are formed in joints and periarticular tissues due to long-lasting hyperuricemia. Although MSU crystal-triggered NLRP3 inflammasome activation and interleukin 1ß (IL-1ß) release are known to have key roles in gouty arthritis, recent studies revealed that MSU crystal-induced necrosis also plays a critical role in this process. However, it remains unknown what forms of necrosis have been induced and whether combined cell death inhibitors can block such necrosis. Here, we showed that MSU crystal-induced necrosis in murine macrophages was not dependent on NLRP3 inflammasome activation, as neither genetic deletion nor pharmacological blockade of the NLRP3 pathway inhibited the necrosis. Although many cell death pathways (such as ferroptosis and pyroptosis) inhibitors or reactive oxygen species inhibitors did not have any suppressive effects, necroptosis pathway inhibitors GSK'872 (RIPK3 inhibitor), and GW806742X (MLKL inhibitor) dose-dependently inhibited MSU crystal-induced necrosis. Moreover, a triple combination of GSK'872, GW806742X, and IDN-6556 (pan-caspase inhibitor) displayed enhanced inhibition of the necrosis, which was further fortified by the addition of MCC950 (NLRP3 inhibitor), suggesting that multiple cell death pathways might have been triggered by MSU crystals. Baicalin, a previously identified inhibitor of NLRP3, inhibited MSU crystal-induced inflammasome activation and suppressed the necrosis in macrophages. Besides, baicalin gavage significantly ameliorated MSU crystal-induced peritonitis in mice. Altogether, our data indicate that MSU crystals induce NLRP3-independent necrosis, which can be inhibited by combined inhibitors for multiple signaling pathways, highlighting a new avenue for the treatment of gouty arthritis.

Reply to: 'comments on 'effects of refractive accommodation on subfoveal choroidal thickness in silicone oil-filled eyes".

In this article, we answered the questions in Lei Gao et al.'s comments on the "effects of refractive accommodation on subfoveal choroidal thickness in silicone oil-filled eyes" one by one.

Induction of multiple subroutines of regulated necrosis in murine macrophages by natural BH3-mimetic gossypol.

Macrophages are critical sentinel cells armed with multiple regulated necrosis pathways, including pyroptosis, apoptosis followed by secondary necrosis, and necroptosis, and are poised to undergo distinct form(s) of necrosis for tackling dangers of pathogenic infection or toxic exposure. The natural BH3-mimetic gossypol is a toxic phytochemical that can induce apoptosis and/or pyroptotic-like cell death, but what exact forms of regulated necrosis are induced remains largely unknown. Here we demonstrated that gossypol induces pyroptotic-like cell death in both unprimed and lipopolysaccharide-primed mouse bone marrow-derived macrophages (BMDMs), as evidenced by membrane swelling and ballooning accompanied by propidium iodide incorporation and lactic acid dehydrogenase release. Notably, gossypol simultaneously induces the activation of both pyroptotic and apoptotic (followed by secondary necrosis) pathways but only weakly activates the necroptosis pathway. Unexpectedly, gossypol-induced necrosis is independent of nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, as neither inhibitor for the NLRP3 pathway nor NLRP3 deficiency protects the macrophages from the necrosis. Furthermore, necrotic inhibitors or even pan-caspase inhibitor alone does not or only partly inhibit such necrosis. Instead, a combination of inhibitors composed of pan-caspase inhibitor IDN-6556, RIPK3 inhibitor GSK'872 and NADPH oxidase inhibitor GKT137831 not only markedly inhibits the necrosis, with all apoptotic and pyroptotic pathways being blocked, but also attenuates gossypol-induced peritonitis in mice. Lastly, the activation of the NLRP3 pathway and apoptotic caspase-3 appears to be independent of each other. Collectively, gossypol simultaneously induces the activation of multiple subroutines of regulated necrosis in macrophages depending on both apoptotic and inflammatory caspases.