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Gastrointestinal dysfunction is a common peripheral organ complication after traumatic brain injury (TBI), yet the underlying mechanism remains unknown. TBI has been demonstrated to cause gut microbiota dysbiosis in animal models, although the impacts of gut microbiota dysbiosis on gastrointestinal dysfunction were not examined. Bile acids are key metabolites between gut microbiota and host interactions. Therefore, the aim of this study was to investigate the mechanistic links between them by detecting the alterations of gut microbiota and bile acid profile after TBI. For that, we established TBI in mice using a lateral fluid percussion injury model. Gut microbiota was examined by 16S rRNA sequencing, and bile acids were profiled by ultra-performance liquid chromatography-tandem mass spectrometry. Our results showed that TBI caused intestinal inflammation and gut barrier impairment. Alterations of gut microbiota and bile acid profile were observed. The diversity of gut microbiota experienced a time dependent change from 1 h to 7 days post-injury. Levels of bile acids in feces and plasma were decreased after TBI, and the decrease was more significant in secondary bile acids, which may contribute to intestinal inflammation. Specific bacterial taxa such as Staphylococcus and Lachnospiraceae that may contribute to the bile acid metabolic changes were identifed. In conclusion, our study suggested that TBI-induced gut microbiota dysbiosis may contribute to gastrointestinal dysfunction via altering bile acid profile. Gut microbiota may be a potential treatment target for TBI-induced gastrointestinal dysfunction.

The degradation kinetics and migration pattern of chlorfenapyr in celery and soil at Lhasa and Pengzhou were investigated. A simple, rapid analytical method for the quantification of chlorfenapyr in celery and soil was developed using gas chromatography-tandem mass spectrometer. The results indicated that the half-lives of chlorfenapyr in celeries and soils at Lhasa were 6.3 days and 12.8 days. While the half-lives of chlorfenapyr in celeries and soils at Pengzhou were 6.9 days and 20.4 days. The half-lives of chlorfenapyr in celeries and soils at Lhasa were shorter than that at Pengzhou, while the half-lives of chlorfenapyr in soils at Lhasa and Pengzhou were longer than that in celeries at Lhasa and Pengzhou. The final residues of chlorfenapyr in celeries at Lhasa and Pengzhou were 5.074 ± 0.144 mg/kg and 5.981 ± 0.234 mg/kg after 7 days of spraying, respectively. When chlofenapyr was sprayed to soils only, the average root concentration factor of chlorfenapyr were 3.57-4.02, while the average translocation factor of chlorfenapyr in leaves and stems were 0.28-0.38 and 0.20-0.25, respectively. Chlorfenapyr was easy to migrate from soil to the roots of celery, followed by leaves and stems. The limit value of chlorfenapyr in celery has not been specified in China's National MRL standard (GB 2763 in National food safety standard-maximum residue limits for pesticides in food. Standard, Beijing, 2021), this study was useful to draw up the limit values of chlorfenapyr residues in celery at different elevations.

Renal cell carcinoma (RCC) is a lethal malignancy of the genitourinary system. Follistatin-like 3 (FSTL3), which mediates cell differentiation and growth, acts as a biomarker of tumors and participates in cancer development and progression. Presently, the FSTL3’s functions in RCC were investigated. Quantitative reverse transcription PCR (qRT-PCR), Western Blot, and enzyme linked immunosorbent assay (ELISA) were conducted to verify FSTL3 expression in RCC tissues and cell lines. BrdU assay and CCK8 experiment were made to monitor cell proliferation. Transwell was implemented to examine the invasion of the cells. Flow cytometry analyzed cell apoptosis, and Western Blot evaluated the protein levels of E-cadherin, Twist, and Slug. In the meantime, the protein profiles of the GSK-3β, β-catenin, and TGF-β signaling pathways were ascertained. Moreover, the Xenograft tumor model was constructed in nude mice for measuring tumor growth in vivo. The statistics showed that FSTL3 presented an overexpression in RCC, and patients with a lower expression of FSTL3 manifested a better prognosis. Down-regulated FSTL3 hampered the proliferation, invasion, EMT, and tumor growth of RCC cells and caused cell apoptosis. On the contrary, FSTL3 overexpression enhanced the malignant behaviors of RCC cells. Furthermore, FSTL3 knockdown bolstered GSK-3β, suppressed β-catenin, and reduced BMP1-SMAD pathway activation. Inhibited β-catenin substantially mitigated FSTL3-mediated promoting functions in RCC. In short, FSTL3 functions as an oncogene in RCC by modulating the GSK-3β/β-catenin signaling pathway.

Atomically dispersed M-N-C (M refers to transition metals) materials represent the most promising catalyst alternatives to the precious metal Pt for the electrochemical reduction of oxygen (ORR), yet the genuine active sites in M-N-C remain elusive. Here, we develop a two-step approach to fabricate Cu-N-C single-atom catalysts with a uniform and well-defined Cu2+-N4 structure that exhibits comparable activity and superior durability in comparison to Pt/C. By combining operando X-ray absorption spectroscopy with theoretical calculations, we unambiguously identify the dynamic evolution of Cu-N4 to Cu-N3 and further to HO-Cu-N2 under ORR working conditions, which concurrently occurs with reduction of Cu2+ to Cu+ and is driven by the applied potential. The increase in the Cu+/Cu2+ ratio with the reduced potential indicates that the low-coordinated Cu+-N3 is the real active site, which is further supported by DFT calculations showing the lower free energy in each elemental step of the ORR on Cu+-N3 than on Cu2+-N4. These findings provide a new understanding of the dynamic electrochemistry on M-N-C catalysts and may guide the design of more efficient low-cost catalysts.

Complement is known to play a role in ischemia and reperfusion injury (IRI). A general paradigm is that complement is activated by self-reactive natural IgM antibodies (nAbs), after they engage postischemic neoepitopes. However, a role for nAbs in lung transplantation (LTx) has not been explored. Using mouse models of LTx, we investigated the role of two postischemic neoepitopes, modified annexin IV (B4) and a subset of phospholipids (C2), in LTx. Antibody deficient Rag1−/−recipient mice were protected from LTx IRI. Reconstitution with either B4 or C2nAb restored IRI, with C2 significantly more effective than B4 nAb. Based on these information, we developed/ characterized a novel complement inhibitor composed of single-chain antibody (scFv) derived from the C2 nAb linked to Crry (C2scFv-Crry), a murine inhibitor of C3 activation. Using an allogeneic LTx, in which recipients contain a full nAb repertoire, C2scFv-Crry targeted to the LTx, inhibited IRI, and delayed acute rejection. Finally, we demonstrate the expression of the C2 neoepitope in human donor lungs, highlighting the translational potential of this approach.

Qian Zhou,1 Hao Wang,2 Tianxiang Zhan,2 Xiaofeng Yang,1,2 Liang Wen2 1Department of Emergency and Trauma Center, The International Medical Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, 310003, People’s Republic of China; 2Department of Neurosurgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, 310003, People’s Republic of ChinaCorrespondence: Liang WenDepartment of Neurosurgery, The First Affiliated Hospital, Zhejiang University School of Medicine, 79 Qingchun Road, Hangzhou, 310003, Zhejiang Province, People’s Republic of ChinaEmail wenliang@zju.edu.cnXiaofeng YangDepartment of Emergency and Trauma Center, The International Medical Center, The First Affiliated Hospital, Zhejiang University School of Medicine, 79 Qingchun Road, Hangzhou, 310003, Zhejiang Province, People’s Republic of ChinaEmail zjcswk@zju.edu.cnBackground: Central nervous system (CNS) infections caused by multidrug-resistant (MDR) and extensively drug-resistant (XDR) Gram-negative bacillus, including carbapenem-resistant Enterobacteriaceae (CRE) and Pseudomonas aeruginosa, are associated with high mortality rates. Clinical trials of ceftazidime/avibactam (CAZ/AVI) on infections of other systems indicate that they are effective against these infections. However, clinical studies on the efficacies of CAZ/AVI in the treatment of CNS infections have not been done.Case Presentation: We evaluated 3 patients diagnosed with MDR/XDR Gram-negative bacillus-associated CNS infections, and effectively treated with CAZ/AVI. Moreover, we performed literature reviews. Before the onset of CNS infections, the 3 patients were subjected to neurosurgical operations, treated with mechanical ventilation, long-term intensive care unit therapy, and various antibiotics. By intravenously administering CAZ/AVI, combined with another antibiotic, the MDR/XDR K. pneumoniae and P. aeruginosa associated ventriculitis was effectively treated in the 3 patients.Conclusion: CAZ/AVI is a viable treatment option for CNS infections caused by MDR/XDR Gram-negative bacteria.Keywords: ceftazidime–avibactam, extensively drug-resistant Gram-negative bacillus, Klebsiella pneumoniae, Pseudomonas aeruginosa, ventriculitis infection

Background: Smad3 is a pivotal intracellular mediator for participating in the activation of multiple immune signal pathway. Objective: The epigenetic regulation mechanism of the positive immune factor Smad3 in cervical cancer remains unknown. Therefore, the epigenetic regulation on Smad3 is investigated in this study. Methods: The methylation status of SMAD3 was detected by Methylation-specific PCR (MS-PCR) and Quantitative Methylation-specific PCR (MS-qPCR) in cervical cancer tissues and cell lines. The underlying molecular mechanisms of SUV39H1-DNMT1-Smad3 regulation was elucidated using cervical cancer cell lines containing siRNA or/and overexpression system. Confirmation of the regulation of DNMT1 by SUV39H1 used Chromatin immunoprecipitation-qPCR (ChIP-qPCR). The statistical methods used for comparing samples between groups were paired t tests and one-way ANOVAs. Results: H3K9me3 protein which regulated by SUV39H1 directly interacts with the DNMT1 promoter region to regulate its expression in cervical cancer cells, resulting in the reduce expression of the downstream target gene DNMT1. In addition, DNMT1 mediates the epigenetic modulation of the SMAD3 gene by directly binding to its promoter region. The depletion of DNMT1 effectively restores the expression of Smad3 in vitro. Moreover, in an in vivo assay, the expression profile of SUV39H1-DNMT1 was found to correlate with Smad3 expression in accordance with the expression at the cellular level. Notably, the promoter region of SMAD3 was hypermethylated in cervical cancer tissues, and this hypermethylation inhibits the subsequent gene expression. Conclusion: These results indicate that SUV39H1-DNMT1 is a crucial Smad3 regulatory axis in cervical cancer. SUV39H1-DNMT1 axis may provide a potential therapeutic target for the treatment of cervical cancer.

Echinococcus granulosus sensu lato has complex defence mechanisms that protect it from the anti-parasitic immune response for long periods. Echinococcus granulosus cyst fluid (EgCF) is involved in the immune escape. Nevertheless, whether and how EgCF modulates the inflammatory response in macrophages remains poorly understood. Here, real-time polymerase chain reaction and enzyme-linked immunosorbent assay revealed that EgCF could markedly attenuate the lipopolysaccharide (LPS)-induced production of pro-inflammatory factors including tumour necrosis factor-α, interleukin (IL)-12 and IL-6 but increase the expression of IL-10 at mRNA and protein levels in mouse peritoneal macrophages and RAW 264.7 cells. Mechanically, western blotting and immunofluorescence assay showed that EgCF abolished the activation of nuclear factor (NF)-κB p65, p38 mitogen-activated protein kinase (MAPK) and ERK1/2 signalling pathways by LPS stimulation in mouse macrophages. EgCF's anti-inflammatory role was at least partly contributed by promoting proteasomal degradation of the critical adaptor TRAF6. Moreover, the EgCF-promoted anti-inflammatory response and TRAF6 proteasomal degradation were conserved in human THP-1 macrophages. These findings collectively reveal a novel mechanism by which EgCF suppresses inflammatory responses by inhibiting TRAF6 and the downstream activation of NF-κB and MAPK signalling in both human and mouse macrophages, providing new insights into the molecular mechanisms underlying the E. granulosus-induced immune evasion.

In this study, we aimed to use ESTIMATE and CIBERSORT computational methods to analyse transcriptional information on COAD and STAD in TCGA. We downloaded transcriptome RNA-seq data of 446 patients with colon cancer from TCGA and estimated the amount of immune and stromal components in the COAD samples using CIBERSORT algorithms. We analysed differentially expressed genes in 446 TCGA samples and 585 Series GSE39582 samples, in high- and low-scoring groups, using Cox regression. The expression of LRRC4C, correlated positively with clinicopathological characteristics and negatively with the survival of patients with COAD. Single-gene survival analysis using Gene Expression Profiling Interactive Analysis 2.0 and Kaplan-Meier plotter revealed an association between high levels of LRRC4C expression and poor prognosis in patients with colon and gastric cancers. Gene set enrichment analysis of COAD and STAD samples indicated that genes in groups expressing high and low LRRC4C levels were mainly enriched in immune-related activities and metabolic pathways, respectively. Difference and correlation analyses of the relationship between LRRC4C expression and tumour-infiltrating immune cells, determined using CIBERSORT algorithms, revealed that monocytes, resting mast cells, and M2 macrophages were positively correlated with LRRC4C expression.

Thousands of Down syndrome cell adhesion molecule (Dscam1) isoforms and ∼60 clustered protocadhrein (cPcdh) proteins are required for establishing neural circuits in insects and vertebrates, respectively. The strict homophilic specificity exhibited by these proteins has been extensively studied and is thought to be critical for their function in neuronal self-avoidance. In contrast, significantly less is known about the Dscam1-related family of ∼100 shortened Dscam (sDscam) proteins in Chelicerata. We report that Chelicerata sDscamα and some sDscamβ protein trans interactions are strictly homophilic, and that the trans interaction is meditated via the first Ig domain through an antiparallel interface. Additionally, different sDscam isoforms interact promiscuously in cis via membrane proximate fibronectin-type III domains. We report that cell–cell interactions depend on the combined identity of all sDscam isoforms expressed. A single mismatched sDscam isoform can interfere with the interactions of cells that otherwise express an identical set of isoforms. Thus, our data support a model by which sDscam association in cis and trans generates a vast repertoire of combinatorial homophilic recognition specificities. We propose that in Chelicerata, sDscam combinatorial specificity is sufficient to provide each neuron with a unique identity for self–nonself discrimination. Surprisingly, while sDscams are related to Drosophila Dscam1, our results mirror the findings reported for the structurally unrelated vertebrate cPcdh. Thus, our findings suggest a remarkable example of convergent evolution for the process of neuronal self-avoidance and provide insight into the basic principles and evolution of metazoan self-avoidance and self–nonself discrimination.

Innate and adaptive immunity participate in and regulate numerous human diseases. Increasing evidence implies that metabolic reprogramming mediates immune cell functional changes during immune responses. In this review, we present and discuss our current understanding of metabolic regulation in different immune cells and their subsets in response to pathological stimuli. An interactive biochemical and molecular model was established to characterize metabolic reprogramming and their functional implication in anti-inflammatory, immune resolution, and proinflammatory responses. We summarize 2 major features of metabolic reprogramming in inflammatory stages in innate and adaptive immune cells: (1) energy production and biosynthesis reprogramming, including increased glycolysis and decreased oxidative phosphorylation, to secure faster ATP production and biosynthesis for defense response and damage repair and (2) epigenetic reprogramming, including enhanced histone acetylation and suppressed DNA methylation, due to altered accessibility of acetyl/methyl group donor and metabolite-modulated enzymatic activity. Finally, we discuss current strategies of metabolic and epigenetic therapy in cardiovascular disease and recommend cell-specific metabolic and gene-targeted site-specific epigenetic alterations for future therapies.

Background: Methylation of histone 3 at lysine 9 (H3K9) and DNA methylation are epigenetic marks correlated with genes silencing. The tumor microenvironment significantly influences therapeutic responses and clinical outcomes. The epigenetic-regulation mechanism of the costimulatory factors Tim-3 and galectin-9 in cervical cancer remains unknown. Methods: The methylation status of HAVCR2 and LGALS9 were detected by MS-PCR in cervical cancer tissues and cell lines. The underlying molecular mechanism of SUV39H1-DNMT3A-Tim-3/galectin-9 regulation was elucidated using cervical cancer cell lines containing siRNA or/and over-expression system. Confirmation of the regulation of DNMT3A by SUV39H1 used ChIP-qPCR.Results: SUV39H1 up-regulates H3K9me3 expression at the DNMT3A promoter region, which in turn induced expression of DNMT3A in cervical cancer. In addition, the mechanistic studies indicate that DNMT3A mediates the epigenetic modulation of the HAVCR2 and LGALS9 genes by directly binding to their promoter regions in vitro. Moreover, in an in vivo assay, the expression profile of SUV39H1 up-regulates the level of H3K9me3 at the DNMT3A promoter region was found to correlate with Tim-3 and galectin-9 cellular expression level. Conclusion: These results indicate that SUV39H1-DNMT3A is a crucial Tim-3 and galectin-9 regulatory axis in cervical cancer.

A common, yet often neglectable, feature of neonatal hypoxic-ischemic brain damage (HIBD) is circadian rhythm disorders resulted from pineal gland dysfunction. Our previous work demonstrated that miRNAs play an important role in regulating key circadian genes in the pineal gland post HIBD [5,21]. In current study, we sought out to extend our investigation by profiling expression changes of pineal long non-coding RNAs (lncRNAs) upon neonatal HIBD using RNA-Seq. After validating lncRNA changes, we showed that one lncRNA: TCONS_00044595 is highly enriched in the pineal gland and exhibits a circadian expression pattern. Next, we performed bioinformatic analysis to predict the lncRNA-miRNA regulatory network and identified 168 miRNAs that potentially targetlncRNA TCONS_00044595. We further validated the bona fide interaction between one candidate miRNA: miR-182, a known factor to regulate pineal Clock expression, and lncRNA TCONS_00044595. Finally, we showed that suppression of lncRNA TCONS_00044595 alleviated the CLOCK activation both in the cultured pinealocytes under OGD conditions and in the pineal gland post HIBD in vivo. Our study thus shed light into novel mechanisms of pathophysiology of pineal dysfunction post neonatal HIBD.

Significance Low availability of nitrogen (N), mainly nitrate in aerobic soils, is a primary limiting factor for crop production. Most terrestrial plants live in symbiosis with arbuscular mycorrhizal (AM) fungi to increase nutrient uptake, including N, from soil. Research on the AM symbiosis field has focused almost exclusively on ammonium as the form of N transferred to the plants, and there has been no direct evidence of N transfer as nitrate thus far. Here, we report that mycorrhizal rice could receive more than 40% of its N via the mycorrhizal pathway and that the AM-specific nitrate transporter OsNPF4.5 accounted for approximately 45% of the mycorrhizal nitrate uptake. Our work suggests the presence of a mycorrhizal route for nitrate uptake in plants., Low availability of nitrogen (N) is often a major limiting factor to crop yield in most nutrient-poor soils. Arbuscular mycorrhizal (AM) fungi are beneficial symbionts of most land plants that enhance plant nutrient uptake, particularly of phosphate. A growing number of reports point to the substantially increased N accumulation in many mycorrhizal plants; however, the contribution of AM symbiosis to plant N nutrition and the mechanisms underlying the AM-mediated N acquisition are still in the early stages of being understood. Here, we report that inoculation with AM fungus Rhizophagus irregularis remarkably promoted rice (Oryza sativa) growth and N acquisition, and about 42% of the overall N acquired by rice roots could be delivered via the symbiotic route under N-NO3− supply condition. Mycorrhizal colonization strongly induced expression of the putative nitrate transporter gene OsNPF4.5 in rice roots, and its orthologs ZmNPF4.5 in Zea mays and SbNPF4.5 in Sorghum bicolor. OsNPF4.5 is exclusively expressed in the cells containing arbuscules and displayed a low-affinity NO3− transport activity when expressed in Xenopus laevis oocytes. Moreover, knockout of OsNPF4.5 resulted in a 45% decrease in symbiotic N uptake and a significant reduction in arbuscule incidence when NO3− was supplied as an N source. Based on our results, we propose that the NPF4.5 plays a key role in mycorrhizal NO3− acquisition, a symbiotic N uptake route that might be highly conserved in gramineous species.

Periodontal accelerate osteogenesis orthodontics (PAOO) is an extension of described techniques that surgically alter the alveolar bone; however, the specific mechanism underlying the technique is not completely understood. The aim of the present study was to evaluate the roles of microRNA (miR)-21 during PAOO. Sprague-Dawley rats were divided into the following four groups: i) Group tooth movement (TM), underwent TM and were administered normal saline (NS); ii) Group PAOO, underwent PAOO + TM and were administered NS; iii) Group agomiR-21, underwent PAOO + TM and were administered agomiR-21; and iv) Group antagomiR-21, underwent PAOO + TM and were administered antagomiR-21. To validate the rat model of PAOO, morphological analyses were performed and measurements were collected. Reverse transcription-quantitative PCR, western blotting and immunohistochemical staining were performed to examine the expression levels of programmed cell death 4 (PDCD4), activin A receptor type 2B (ACVR2b), receptor activator of NF-κΒ ligand (RANKL) and C-Fos. Dual-luciferase reporter assays were performed to validate PDCD4 as a target of miR-21 in vitro. Following 7 days of treatment, the TM distance of group PAOO was longer compared with groups TM and antagomiR-21 (P

Objectives: To investigate VEGF and Ang-2 level changes in the systemic circulation after laser photocoagulation in premature infants with ROP. Methods: Eleven infants (4 girls and 7 boys) had seru...

Background The GRAS and oleaginous yeast Yarrowia lipolytica (Y. lipolytica) is an attractive cell factory for the production of chemicals and biofuels. The production of many natural products of commercial interest have been investigated in this cell factory by introducing heterologous biosynthetic pathways and by modifying the endogenous pathways. However, since natural products anabolism involves long pathways and complex regulation, re-channelling carbon into the product of target compounds is still a cumbersome work, and often resulting in low production performance. Results In this work, the carotenogenic genes contained carB and bi-functional carRP from Mucor circinelloides and carotenoid cleavage dioxygenase 1 (CCD1) from Petunia hybrida were introduced to Y. lipolytica and led to the low production of β-ionone of 3.5 mg/L. To further improve the β-ionone synthesis, we implemented a modular engineering strategy for the construction and optimization of a biosynthetic pathway for the overproduction of β-ionone in Y. lipolytica. The strategy involved the enhancement of the cytosolic acetyl-CoA supply and the increase of MVA pathway flux, yielding a β-ionone titer of 358 mg/L in shake-flask fermentation and approximately 1 g/L (~ 280-fold higher than the baseline strain) in fed-batch fermentation. Conclusions An efficient β-ionone producing GRAS Y. lipolytica platform was constructed by combining integrated overexpressed of heterologous and native genes. A modular engineering strategy involved the optimization pathway and fermentation condition was investigated in the engineered strain and the highest β-ionone titer reported to date by a cell factory was achieved. This effective strategy can be adapted to enhance the biosynthesis of other terpenoids in Y. lipolytica.

Background Agglomeration of myeloid‐derived suppressor cells (MDSCs) in tumors impedes immunotherapeutic effects. Doxorubicin (DOX) is currently the most specific drug used for the selective removal of MDSCs. Here, we study the feasibility and mechanism of eliminating MDSCs by DOX to improve antigen‐specific cytotoxic T lymphocyte (CTL)‐killing neuroblastoma (NB) cells in vitro. Methods CTL and MDSC were prepared; then, CTLs, NB cells, MDSCs, and DOX were mixed and cultivated in different collocation patterns and divided into different groups. The levels of cluster of differentiation 3ζ chain (CD3ζ) and L‐selectin in CTL in different groups were detected. Thereafter, the killing rate of NB cells and secretion of interleukin‐2 and interferon‐γ were measured and compared. Results By real‐time polymerase chain reaction (PCR) and Western blot test respectively, the proliferation and killing effect of CTLs on NB cells were all inhibited by MDSC through downregulating CD3ζ (p = 0.002; p = 0.001) and L‐selectin (p = 0.006; p 0.05). Conclusions Targeted elimination of MDSCs by DOX can improve Ag‐specific CTL killing of NB cells in vitro by upregulating CD3ζ and L‐selectin. This study provides a novel method to enhance the immunotherapeutic effects of NB., High aggregation of myeloid‐derived suppressor cells (MDSCs) in the tumor microenvironment of neuroblastoma results in immunosuppression and affects its therapeutic effectiveness. Targeted elimination of MDSC by doxorubicin can improve antigen‐specific cytotoxic T lymphocyte killing neuroblastoma cells in vitro by upregulating the levels of cluster of differentiation 3ζ chain and L‐selectin. The present study provided a novel method to enhance immunotherapeutic effects for neuroblastoma.

Tregs play vital roles in suppressing atherogenesis. Pathological conditions reshape Tregs and increase Treg-weakening plasticity. It remains unclear how Tregs preserve their function and how Tregs switch into alternative phenotypes in the environment of atherosclerosis. In this study, we observed a great induction of CD4+Foxp3+ Tregs in the spleen and aorta of ApoE–/– mice, accompanied by a significant increase of plasma IL-35 levels. To determine if IL-35 devotes its role in the rise of Tregs, we generated IL-35 subunit P35–deficient (IL-35P35–deficient) mice on an ApoE–/– background and found Treg reduction in the spleen and aorta compared with ApoE–/– controls. In addition, our RNA sequencing data show the elevation of a set of chemokine receptor transcripts in the ApoE–/– Tregs, and we have validated higher CCR5 expression in ApoE–/– Tregs in the presence of IL-35 than in the absence of IL-35. Furthermore, we observed that CCR5+ Tregs in ApoE–/– have lower Treg-weakening AKT-mTOR signaling, higher expression of inhibitory checkpoint receptors TIGIT and PD-1, and higher expression of IL-10 compared with WT CCR5+ Tregs. In conclusion, IL-35 counteracts hyperlipidemia in maintaining Treg-suppressive function by increasing 3 CCR5-amplified mechanisms, including Treg migration, inhibition of Treg weakening AKT-mTOR signaling, and promotion of TIGIT and PD-1 signaling.

Coronavirus disease 2019 (COVID-19) has caused a crisis to global public health since its outbreak at the end of 2019. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen of COVID-19, appears to efficiently evade the host immune responses, including interferon (IFN) signaling. Several SARS-CoV-2 viral proteins are believed to involve in the inhibition of IFN signaling. In this study, we discovered that ORF3a, an accessory protein of SARS-CoV-2, inhibited IFN-activated Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling via upregulating suppressor of cytokine signaling 1 (SOCS1), a negative regulator of cytokine signaling. ORF3a induced SOCS1 elevation in a dose- and time-dependent manner. RNAi-mediated silencing of SOCS1 efficiently abolished ORF3a-induced blockage of JAK/STAT signaling. Interestingly, we found that ORF3a also promoted the ubiquitin-proteasomal degradation of Janus kinase 2 (JAK2), an important kinase in IFN signaling. Silencing of SOCS1 by siRNA distinctly blocked ORF3a-induced JAK2 ubiquitination and degradation. These results demonstrate that ORF3a dampens IFN signaling via upregulating SOCS1, which suppressed STAT1 phosphorylation and accelerated JAK2 ubiquitin-proteasomal degradation. Furthermore, analysis of ORF3a deletion constructs showed that the middle domain of ORF3a (amino acids 70–130) was responsible for SOCS1 upregulation. These findings contribute to our understanding of the mechanism of SARS-CoV-2 antagonizing host antiviral response.

Traumatic brain injury (TBI) can cause damage to peripheral organ systems, such as digestive organ system, and alterations of gut microbiota in addition to brain injury. Our previous study found that TBI induced gastrointestinal dysfunction accompanied by alterations of bile acid metabolism. Bile acid and its receptors have been reported to play important roles in various neurological diseases. To further examine the changes of bile acid metabolism in TBI patients, we performed a retrospective clinical analysis. In this study, 177 patients were included, and the results showed that TBI patients had more frequent antibiotic use compared with a control group. Regression analysis identified TBI as an independent factor for reduction of serum bile acid level (B = −1.762, p = 0.006), even with antibiotic use taken into a regression model. Sub-group regression analysis of TBI patients showed that antibiotic use was negatively associated with bile acid level, while creatinine and triglyceride were positively associated with bile acid level. In conclusion, these data indicated that TBI could greatly reduce serum bile acid. This study provided preliminary but novel clinical evidence of TBI interfering with bile acid metabolism, and further studies with large sample sizes are needed to validate these findings in the future.

This paper presents an efficient, green and sustainable approach for succinic acid (SA) fermentation, in which glucose-rich hydrolysates from various waste streams were used as the substrate for SA production by Yarrowia lipolytica without pH control for the first time. First, Y. lipolytica PGC202 was found to be able to consume glucose-rich mixed food waste (MFW) hydrolysate for SA production with high efficiency. After medium optimization, a SA titer at 31.7 g/L with a yield of 0.52 g/g and productivity of 0.60 g/L/h was obtained from isFBB fermentation when using 60 g/L glucose-containing MFW hydrolysate as the carbon source supplemented with 3% tryptone. When glucose-rich hydrolysate from other waste streams was used, the highest SA yield of 0.61 g/g via yeast fermentation at low pH was achieved, which demonstrated the extensive substrate adaptability and great potential of Y. lipolytica. By feeding concentrated MFW hydrolysate, SA titer up to 71.6 g/L was achieved from fed-batch fermentation with pH decreased naturally to 2.8. Finally, with the revised recovery method, 68.3% of the total SA with purity between 89.5% and 91.0% was recovered from the acidic fermentation broth with only low input of acid for acidification, which demonstrated the great advantages of SA production at low pH over that under neutral conditions in terms of economic improvement and environmental sustainability. In general, this study overcomes one of the major economic drawbacks for future feasible large-scale fermentative SA production, and it also illustrates a promising prospective for bio-SA production.

Summary Heterogeneous single-atom catalysts (SACs) with atomically dispersed active metal centers represent an intermediary between heterogeneous and homogeneous catalysis. In this work, a porous organic polymer with aminopyridine functionalities was designed to fabricate a stable, atomically dispersed Ir catalyst. This Ir-based SAC exhibits excellent catalytic activity during the liquid-phase hydrogenation of CO2 to formate. The associated turnover number is as high as 25,135, representing the best performance yet for a heterogeneous conversion of CO2 to formate. Spectral characterization and density functional theory modeling demonstrate that the chemical structure of the Ir single-atom active site is analogous to that of a homogeneous mononuclear Ir pincer complex catalyst. As a result, a catalytic mechanism similar to that over a homogeneous Ir catalyst occurs during CO2 hydrogenation with this quasi-homogeneous Ir-based SAC. This work suggests a promising basis for the design of efficient SACs for the once-dominant homogeneous catalytic processes.

Mitochondria are capable of detecting cellular insults and orchestrating inflammatory responses. Mitochondrial reactive oxygen species (mtROS) are intermediates that trigger inflammatory signaling cascades in response to our newly proposed conditional damage associated molecular patterns (DAMP). We recently reported that increased proton leak regulates mtROS generation and thereby exert physiological and pathological activation of endothelial cells. Herein, we report the recent progress in determining the roles of proton leak in regulating mtROS, and highlight several important findings: 1) The majority of mtROS are generated in the complexes I and III of electron transport chain (ETC); 2) Inducible proton leak and mtROS production are mutually regulated; 3) ATP synthase-uncoupled ETC activity and mtROS regulate both physiologica006C and pathological endothelial cell activation and inflammation initiation; 4) Mitochondrial Ca(2+) uniporter and exchanger proteins have an impact on proton leak and mtROS generation; 5) MtROS connect signaling pathways between conditional DAMP-regulated immunometabolism and histone post-translational modifications (PTM) and gene expression. Continuous improvement of our understanding in this aspect of mitochondrial function would provide novel insights and generate novel therapeutic targets for the treatment of sterile inflammatory disorders such as metabolic diseases, cardiovascular diseases and cancers.

To examine whether the expressions of 260 organelle crosstalk regulators (OCRGs) in 16 functional groups are modulated in 23 diseases and 28 tumors, we performed extensive -omics data mining analyses and made a set of significant findings: (1) the ratios of upregulated vs. downregulated OCRGs are 1:2.8 in acute inflammations, 1:1 in metabolic diseases, 1:1.2 in autoimmune diseases, and 1:3.8 in organ failures; (2) sepsis and trauma-upregulated OCRG groups such as vesicle, mitochondrial (MT) fission, and mitophagy but not others, are termed as the cell crisis-handling OCRGs. Similarly, sepsis and trauma plus organ failures upregulated seven OCRG groups including vesicle, MT fission, mitophagy, sarcoplasmic reticulum–MT, MT fusion, autophagosome–lysosome fusion, and autophagosome/endosome–lysosome fusion, classified as the cell failure-handling OCRGs; (3) suppression of autophagosome–lysosome fusion in endothelial and epithelial cells is required for viral replications, which classify this decreased group as the viral replication-suppressed OCRGs; (4) pro-atherogenic damage-associated molecular patterns (DAMPs) such as oxidized low-density lipoprotein (oxLDL), lipopolysaccharide (LPS), oxidized-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (oxPAPC), and interferons (IFNs) totally upregulated 33 OCRGs in endothelial cells (ECs) including vesicle, MT fission, mitophagy, MT fusion, endoplasmic reticulum (ER)–MT contact, ER– plasma membrane (PM) junction, autophagosome/endosome–lysosome fusion, sarcoplasmic reticulum–MT, autophagosome–endosome/lysosome fusion, and ER–Golgi complex (GC) interaction as the 10 EC-activation/inflammation-promoting OCRG groups; (5) the expression of OCRGs is upregulated more than downregulated in regulatory T cells (Tregs) from the lymph nodes, spleen, peripheral blood, intestine, and brown adipose tissue in comparison with that of CD4+CD25− T effector controls; (6) toll-like receptors (TLRs), reactive oxygen species (ROS) regulator nuclear factor erythroid 2-related factor 2 (Nrf2), and inflammasome-activated regulator caspase-1 regulated the expressions of OCRGs in diseases, virus-infected cells, and pro-atherogenic DAMP-treated ECs; (7) OCRG expressions are significantly modulated in all the 28 cancer datasets, and the upregulated OCRGs are correlated with tumor immune infiltrates in some tumors; (8) tumor promoter factor IKK2 and tumor suppressor Tp53 significantly modulate the expressions of OCRGs. Our findings provide novel insights on the roles of upregulated OCRGs in the pathogenesis of inflammatory diseases and cancers, and novel pathways for the future therapeutic interventions for inflammations, sepsis, trauma, organ failures, autoimmune diseases, metabolic cardiovascular diseases (CVDs), and cancers.

Neuroblastoma (NB) has high morality rates and is the most common malignant tumor found in children. High aggregation of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment results in immunosuppression and affects therapeutic effectiveness. At present, doxorubicin (DOX) and dopamine (DA) are the specific drugs used to selectively remove or mature MDSCs. The aim of the present study was to explore the feasibility and underlying mechanism of targeting elimination of MDSCs via DOX or DA administration to alleviate tumor immunosuppression in NB. In the present study, a BALB/c tumor-bearing mouse model was established, and mice were grouped into the control, DOX2.5, DOX5 and DA50 mg/kg groups. DOX or DA were injected intravenously on days 7 and 12 after inoculation, following which the parameters related to the signal transducer and activator of transcription (STAT) pathway in MDSCs, the proportion of MDSCs, T cell infiltration, programmed death-1 (PD-1) on the surface of T cells, the number of regulatory T cells (Tregs), polarization of tumor-related macrophages (TAMs) and tumor growth were compared between the groups on days 14, 17 and 23 after inoculation. The results demonstrated that following DOX or DA administration, STAT1/phosphorylated (p)-STAT1 decreased, whereas STAT3/p-STAT3, STAT5/p-STAT5 and STAT6/p-STAT6 increased, which was accompanied by a decrease in the MDSC proportion in each experimental group. Simultaneously, T cell infiltration in tumors was increased, whereas expression of PD-1, the number of Tregs, TAM polarization and tumor growth were inhibited. The most significant findings were observed in the DOX2.5 mg/kg group. To conclude, low dose DOX or DA administration could effectively regulate the STAT pathway to eliminate MDSCs, alleviate immunosuppression and improve the immune response against NB tumor cells.

To characterize transcriptomic changes in endothelial cells (ECs) infected by coronaviruses, and stimulated by DAMPs, the expressions of 1311 innate immune regulatomic genes (IGs) were examined in 28 EC microarray datasets with 7 monocyte datasets as controls. We made the following findings: The majority of IGs are upregulated in the first 12 hours post-infection (PI), and maintained until 48 hours PI in human microvascular EC infected by middle east respiratory syndrome-coronavirus (MERS-CoV) (an EC model for COVID-19). The expressions of IGs are modulated in 21 human EC transcriptomic datasets by various PAMPs/DAMPs, including LPS, LPC, shear stress, hyperlipidemia and oxLDL. Upregulation of many IGs such as nucleic acid sensors are shared between ECs infected by MERS-CoV and those stimulated by PAMPs and DAMPs. Human heart EC and mouse aortic EC express all four types of coronavirus receptors such as ANPEP, CEACAM1, ACE2, DPP4 and virus entry facilitator TMPRSS2 (heart EC); most of coronavirus replication-transcription protein complexes are expressed in HMEC, which contribute to viremia, thromboembolism, and cardiovascular comorbidities of COVID-19. ECs have novel trained immunity (TI), in which subsequent inflammation is enhanced. Upregulated proinflammatory cytokines such as TNFα, IL6, CSF1 and CSF3 and TI marker IL-32 as well as TI metabolic enzymes and epigenetic enzymes indicate TI function in HMEC infected by MERS-CoV, which may drive cytokine storms. Upregulated CSF1 and CSF3 demonstrate a novel function of ECs in promoting myelopoiesis. Mechanistically, the ER stress and ROS, together with decreased mitochondrial OXPHOS complexes, facilitate a proinflammatory response and TI. Additionally, an increase of the regulators of mitotic catastrophe cell death, apoptosis, ferroptosis, inflammasomes-driven pyroptosis in ECs infected with MERS-CoV and the upregulation of pro-thrombogenic factors increase thromboembolism potential. Finally, NRF2-suppressed ROS regulate innate immune responses, TI, thrombosis, EC inflammation and death. These transcriptomic results provide novel insights on the roles of ECs in coronavirus infections such as COVID-19, cardiovascular diseases (CVD), inflammation, transplantation, autoimmune disease and cancers.

We performed a transcriptomic analyses using the strategies we pioneered and made the following findings: 1) Normal lymphoid Tregs, diseased kidney Tregs, splenic Tregs from mice with injured muscle have 3, 17 and 3 specific (S-) pathways, respectively; 2) Tumor splenic Tregs share 12 pathways with tumor Tregs; tumor splenic Tregs and tumor Tregs have 11 and 8 S-pathways, respectively; 3) Normal and non-tumor disease Tregs upregulate some of novel 2641 canonical secretomic genes (SGs) with 24 pathways, and tumor Tregs upregulate canonical secretomes with 17 pathways; 4) Normal and non-tumor disease tissue Tregs upregulate some of novel 6560 exosome SGs with 56 exosome SG pathways (ESP), tumor Treg ESP are more focused than other Tregs; 5) Normal, non-tumor diseased Treg and tumor Tregs upregulate some of novel 961 innate immune caspase-1 SGs and 1223 innate immune caspase-4 SGs to fulfill their tissue/SG-specific and shared functions; 6) Most tissue Treg transcriptomes are controlled by Foxp3; and Tumor Tregs had increased Foxp3 non-collaboration genes with ROS and 17 other pathways; 7) Immune checkpoint receptor PD-1 does, but CTLA-4 does not, play significant roles in promoting Treg upregulated genes in normal and non-tumor disease tissue Tregs; and tumor splenic and tumor Tregs have certain CTLA-4-, and PD-1-, non-collaboration transcriptomic changes with innate immune dominant pathways; 8) Tumor Tregs downregulate more immunometabolic and innate immune memory (trained immunity) genes than Tregs from other groups; and 11) ROS significantly regulate Treg transcriptomes; and ROS-suppressed genes are downregulated more in tumor Tregs than Tregs from other groups. Our results have provided novel insights on the roles of Tregs in normal, injuries, regeneration, tumor conditions and some of canonical and innate immune non-canonical secretomes via ROS-regulatory mechanisms and new therapeutic targets for immunosuppression, tissue repair, cardiovascular diseases, chronic kidney disease, autoimmune diseases, transplantation, and cancers.

Receptors and their ligands are important therapeutic targets for about one third of marketed drugs. Here, we describe an epitope-guided approach for selection of antibodies that modulate cellular signaling of targeted receptors. We chose CXC chemokine receptor 2 (CXCR2) in the G-protein coupled receptor superfamily as receptor and a CXCR2 N-terminal peptide for antibody selection. We obtain a highly selective, tight-binding antibody from a 1011-member antibody library using combinatorial enrichment. Structural and Hydrogen-Deuterium-Exchange mass spectrometry analyses demonstrate antibody interaction with an N-terminal region of CXCR2 that is part of the IL-8 epitope. The antibody strongly inhibits IL-8-induced and CXCR2-mediated neutrophil chemotaxis in vitro and alleviates hCXCR2-dependent experimental autoimmune encephalomyelitis symptoms in mice. As inappropriate neutrophil migration accompanies many diseases including inflammatory bowel disease, glomerulonephritis, allergic asthma, chronic obstructive pulmonary disease, and cancer, this antibody has potential for development as a therapeutic agent, akin to anti-TNF antibodies. However, an important difference here is that the antibody targets the chemokine receptor and competes with natural ligand, rather than targeting the ligand itself., CXCR2 is central to neutrophil chemotaxis and hence to some inflammatory diseases. Here the authors demonstrate the value of an epitope-guided antibody panning method to develop a tight binding anti-hCXCR2 antibody, along with crystal structures of this antibody and antigen, that can block neutrophil chemotaxis and protect mice in an EAE model.

CD4+ T cells are essential for regulating effective immune response to pathogens and immune balance. Recent studies have demonstrated the unique features of T cells in neonate mice, such as more sensitive to antigen response and preference toward T helper 2 (Th2) response and regulatory T cells (Tregs) differentiation. However, the biological characteristics of neonatal age-derived CD4+ T cells following homeostasis remain unclear. Here we utilized a lineage tracing model of TCRδCreERR26ZsGreen to mark neonatal- and adult-derived CD4+ T cells followed by a combination analysis of activation, proliferation, survival, and differentiation. Our results showed that neonatal CD4+ T cells had higher capacity of activation, proliferation, apoptosis, and differentiation toward Th2 and T helper 17 (Th17) lineages, accompanied by a reduced potential for T helper 1 (Th1), T helper 9 (Th9), and Treg lineages. In contrast, tracked neonatal CD4+ T cells exhibited similar characters of above-mentioned of tracked adult cells in adult mice. Therefore, our data support a natural requirement for CD4+ T cells to acquire fully-equipped functional potentials of adult cells.

It has long been thought that aging is a major risk factor for cancer incidence. However, accumulating evidence indicates increased resistance of old animals to tumor growth. A systematic understanding of how old individuals defend against tumor invasion is currently lacking. Here we investigated the differences of age-associated alterations in tumor-infiltrating immune cells between young and old mice using single-cell RNA analysis. Our results showed that a higher proportion of cytotoxic CD8+ T cells, nTregs, cDC, and M1-type macrophages, while a higher percentage of exhausted CD8+ T cells, iTregs, pDC, and M2-type macrophages were found in young mice. Importantly, TCR diversity analysis showed top 10 TCR clones consisted primarily of exhausted CD8+ T cells in young mice whereas tip clones were predominantly cytotoxic CD8+ T cells in old mice. Consistently, trajectory inference demonstrated that CD8+ T cells preferentially differentiated into cytotoxic cells in old mice in contrast to exhausted cells in young mice. Meanwhile, we confirmed the main distinctions between young and old mice by flow cytometry. Collectively, our data revealed that a significantly higher proportion of effector immune cells in old mice defend against tumor progression, providing a framework for the immunotherapy of elderly patients with tumors.

BackgroundMurine monocytes (MC) are classified into Ly6Chigh and Ly6Clow MC. Ly6Chigh MC is the pro-inflammatory subset and the counterpart of human CD14++CD16+ intermediate MC which contributes to systemic and tissue inflammation in various metabolic disorders, including hyperhomocysteinemia (HHcy). This study aims to explore molecule signaling mediating MC subset differentiation in HHcy and control mice.MethodsRNA-seq was performed in blood Ly6Chigh and Ly6Clow MC sorted by flow cytometry from control and HHcy cystathionine β-synthase gene-deficient (Cbs-/-) mice. Transcriptome data were analyzed by comparing Ly6Chigh vs. Ly6Clow in control mice, Ly6Chigh vs. Ly6Clow in Cbs-/- mice, Cbs-/- Ly6Chigh vs. control Ly6Chigh MC and Cbs-/- Ly6Clow vs. control Ly6Clow MC by using intensive bioinformatic strategies. Significantly differentially expressed (SDE) immunological genes and transcription factor (TF) were selected for functional pathways and transcriptional signaling identification.ResultsA total of 7,928 SDE genes and 46 canonical pathways derived from it were identified. Ly6Chigh MC exhibited activated neutrophil degranulation, lysosome, cytokine production/receptor interaction and myeloid cell activation pathways, and Ly6Clow MC presented features of lymphocyte immunity pathways in both mice. Twenty-four potential transcriptional regulatory pathways were identified based on SDE TFs matched with their corresponding SDE immunological genes. Ly6Chigh MC presented downregulated co-stimulatory receptors (CD2, GITR, and TIM1) which direct immune cell proliferation, and upregulated co-stimulatory ligands (LIGHT and SEMA4A) which trigger antigen priming and differentiation. Ly6Chigh MC expressed higher levels of macrophage (MΦ) markers, whereas, Ly6Clow MC highly expressed lymphocyte markers in both mice. HHcy in Cbs-/- mice reinforced inflammatory features in Ly6Chigh MC by upregulating inflammatory TFs (Ets1 and Tbx21) and strengthened lymphocytes functional adaptation in Ly6Clow MC by increased expression of CD3, DR3, ICOS, and Fos. Finally, we established 3 groups of transcriptional models to describe Ly6Chigh to Ly6Clow MC subset differentiation, immune checkpoint regulation, Ly6Chigh MC to MΦ subset differentiation and Ly6Clow MC to lymphocyte functional adaptation.ConclusionsLy6Chigh MC displayed enriched inflammatory pathways and favored to be differentiated into MΦ. Ly6Clow MC manifested activated T-cell signaling pathways and potentially can adapt the function of lymphocytes. HHcy reinforced inflammatory feature in Ly6Chigh MC and strengthened lymphocytes functional adaptation in Ly6Clow MC.

We used functional -omics angles and examined transcriptomic heterogeneity in CD4+Foxp3+ regulatory T cells (Treg) from spleen (s-Treg), lymph nodes (LN-Treg), intestine (int-Treg), and visceral adipose tissue (VAT-Treg), and made significant findings: 1) Five new shared Treg genes including NIBAN, TNFRSF1b, DUSP4,VAV2, and KLRG1, and 68 new signatures are identified. Among 27 signaling pathways shared in four tissue Treg, 22 pathways are innate immune pathways (81.5%); 2) s-Treg, LN-Treg, int-Treg, and VAT-Treg have zero, 49, 45, and 116 upregulated pathways, respectively; 3) 12, 7, and 15 out of 373 CD markers are identified as specific for LN-Treg, int-Treg, and VAT-Treg, respectively, which may initiate innate immune signaling; 4) 7, 49, 44, and 79 increased cytokines out of 1176 cytokines are identified for four Treg, respectively, suggesting that Treg have much more secretory proteins/cytokines than IL-10, TGF-β, and IL-35; 5) LN-Treg, int-Treg, and VAT-Treg have 13 additional secretory functions more than s-Treg, found by analyzing 1,706 secretomic genes; 6) 2, 20, 25, and 43 increased transcription factors (TFs) out of 1,496 TFs are identified four Treg, respectively; 7) LN-Treg and int-Treg have increased pyroptosis regulators but VAT-Treg have increased apoptosis regulators; 8) 1, 15, 19, and 31 increased kinases out of 661 kinome are identified for s-Treg, LN-Treg, int-Treg, and VAT-Treg, respectively; 9) comparing with that of s-Treg, LN-Treg, int-Treg, and VAT-Treg increase activated cluster (clusters 1–3) markers; and decrease resting cluster (clusters 4–6) markers; and 10) Treg promote tissue repair by sharing secretomes and TFs AHR, ETV5, EGR1, and KLF4 with stem cells, which partially promote upregulation of all the groups of Treg genes. These results suggest that stem cell-shared master genes make tissue Treg as the first T cell type using a Treg niche to maintain their Treg-ness with 80% innate immune pathways, and triple functions of immunosuppression, tissue repair, and homeostasis maintenance. Our results have provided novel insights on the roles of innate immune pathways on Treg heterogeneity and new therapeutic targets for immunosuppression, tissue repair, cardiovascular diseases, chronic kidney disease, autoimmune diseases, transplantation, and cancers.

Background. The immune mechanisms underlying low-intensity ultrasound- (LIUS-) mediated suppression of inflammation and tumorigenesis remain poorly determined. Methods. We used microarray datasets from the NCBI GEO DataSet repository and conducted comprehensive data-mining analyses, where we examined the gene expression of 1376 innate immune regulators (innatome genes (IGs) in cells treated with LIUS. Results. We made the following findings: (1) LIUS upregulates proinflammatory IGs and downregulates metastasis genes in cancer cells, and LIUS upregulates adaptive immunity pathways but inhibits danger-sensing and inflammation pathways and promote tolerogenic differentiation in bone marrow (BM) cells. (2) LIUS upregulates IGs encoded for proteins localized in the cytoplasm, extracellular space, and others, but downregulates IG proteins localized in nuclear and plasma membranes, and LIUS downregulates phosphatases. (3) LIUS-modulated IGs act partially via several important pathways of reactive oxygen species (ROS), reverse signaling of immune checkpoint receptors B7-H4 and BTNL2, inflammatory cytokines, and static or oscillatory shear stress and heat generation, among which ROS is a dominant mechanism. (4) LIUS upregulates trained immunity enzymes in lymphoma cells and downregulates trained immunity enzymes and presumably establishes trained tolerance in BM cells. (5) LIUS modulates chromatin long-range interactions to differentially regulate IGs expression in cancer cells and noncancer cells. Conclusions. Our analysis suggests novel molecular mechanisms that are utilized by LIUS to induce tumor suppression and inflammation inhibition. Our findings may lead to development of new treatment protocols for cancers and chronic inflammation.

Background Post-transplant ischemia reperfusion injury (IRI) is a recognized risk factor for subsequent organ dysfunction, alloresponsiveness, and rejection. The complement system is known to play a role in IRI and represents a therapeutic target. Complement is activated in transplanted grafts when circulating IgM antibodies bind to exposed ischemia-induced neoepitopes upon reperfusion, and we investigated the targeting of a human complement inhibitor , CR1, to a post-transplant ischemia-induced neoepitope. Methods A fragment of human CR1 was linked to a single chain antibody construct (C2 scFv) recognizing an injury-specific neoepitope to yield C2-CR1. This construct, along with a soluble untargeted counterpart, was characterized in a cardiac allograft transplantation model of IRI in terms of efficacy and safety. Results CR1 was similarly effective against mouse and human complement. C2-CR1 provided effective protection against cardiac IRI at a lower dose than untargeted CR1. The increased efficacy of C2-CR1 relative to CR1 correlated with decreased C3 deposition, and C2-CR1, but not CR1, targeted to cardiac allografts . At a dose necessary to reduce IRI, C2-CR1 had minimal impact on serum complement activity, in contrast to CR1 which resulted in a high level of systemic inhibition. The circulatory half-life of CR1 was markedly longer than that of C2-CR1, and whereas a minimum therapeutic dose of CR1 severely impaired host susceptibility to infection, C2-CR1 had no impact. Conclusion We show the translational potential of a human complement inhibitor targeted to a universal ischemia-induced graft-specific epitope, and demonstrate advantages compared to an untargeted counterpart in terms of efficacy and safety.

Acid tolerance of microorganism is a desirable phenotype for many industrial fermentation applications. In Escherichia coli, the stress response sigma factor RpoS is a promising target for engineering acid tolerant phenotypes. However, the simple overexpression of RpoS alone is insufficient to confer these phenotypes. In this study, we showed that the simultaneous overexpression of the noncoding small RNA (sRNA) DsrA and the sRNA chaperone Hfq, which act as RpoS activators, significantly increased the acid tolerance, in terms of cell growth under modest acidic pH, as well as cell survival upon extreme acid shock. Directed evolution of the DsrA-Hfq module further improved the acid tolerance, with the best mutants showing a 51%-72% increase in growth performance at pH 4.5 compared with the starting strain MG1655. Further analyses found that the improved acid tolerance of these DsrA-Hfq strains coincided with activation of genes associated with the proton-consuming acid resistance system 2 (AR2), protein chaperone HdeB, and reactive oxygen species (ROS) removal in the exponential phase. This study illustrated that the fine-tuning of sRNAs and their chaperones, can be a novel strategy for improving the acid tolerance of E. coli. Importance Many of the traditional studies on bacterial acid tolerance generally focused on improving cell survival under extreme pH conditions, but cell growth under less harsh acidic conditions is more relevant to industrial applications. Under normal conditions, the general stress response sigma factor RpoS is maintained at low levels in the growth phase through a number of mechanisms. This study showed that RpoS can be activated prior to the stationary phase via engineering its activators, the sRNA DsrA and the sRNA chaperone Hfq, resulting in significantly improved cell growth at modest acidic pH. This work suggests that the sigma factors and likely other transcription factors, can be re-tuned or re-timed by manipulating the respective regulatory sRNAs along with the sufficient supply of the respective sRNA chaperones (i.e., Hfq). This provides a novel avenue for strain engineering of microbes.

Summary: Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam1) can generate 38,016 different isoforms through largely stochastic, yet highly biased, alternative splicing. These isoforms are required for nervous functions. However, the functional significance of splicing bias remains unknown. Here, we provide evidence that Dscam1 splicing bias is required for mushroom body (MB) axonal wiring. We generate mutant flies with normal overall protein levels and an identical number but global changes in exon 4 and 9 isoform bias (DscamΔ4D−/− and DscamΔ9D−/−), respectively. In contrast to DscamΔ4D−/−, DscamΔ9D−/− exhibits remarkable MB defects, suggesting a variable domain-specific requirement for isoform bias. Importantly, changes in isoform bias cause axonal defects but do not influence the self-avoidance of axonal branches. We conclude that, in contrast to the isoform number that provides the molecular basis for neurite self-avoidance, isoform bias may play a role in MB axonal wiring by influencing non-repulsive signaling.

In aged individuals, age-related changes in immune cells, especially T cell deficiency, are associated with an increased incidence of infection, tumor, and autoimmune disease, as well as an impaired response to vaccination. However, the features of gene expression levels in aged T cells are still unknown. Our previous study successfully tracked aged T cells generated from one wave of developing thymocytes of young age by a lineage-specific and inducible Cre-controlled reporter (TCRδCreERR26ZsGreen mouse strain). In this study, we utilized this model and genome-wide transcriptomic analysis to examine changes in gene expression in aged naïve and memory T cell populations during the aging process. We identified profound gene alterations in aged CD4 and CD8 T cells. Both aged CD4+ and CD8+ naïve T cells showed significantly decreased organelle function. Importantly, genes associated with lymphocyte activation and function demonstrated a significant increase in aged memory T cells, accompanied by upregulation of immunosuppressive markers and immune checkpoints, revealing an abnormal T cell function in aged cells. Furthermore, aging significantly affects T cell survival and death signaling. While aged CD4 memory T cells exhibited pro-apoptotic gene signatures, aged CD8 memory T cells expressed anti-apoptotic genes. Thus, the transcriptional analysis of gene expression and signaling pathways in aged T cell subsets shed light on our understanding of altered immune function with aging, which will have great potential for clinical interventions for older adults.

Background Prostate cancer (PCa) is a kind of malignancy occurring in the prostate gland. Substantial researches have proved the major role of long noncoding RNAs (lncRNAs) in PCa. However, the role of long intergenic non-protein coding RNA 1006 (LINC01006) in PCa has not been investigated yet. Methods RT-qPCR was used to examine the expression levels of LINC01006 and its downstream targets. The function of LINC01006 in PCa was tested by in vitro and in vivo assays. With application of RNA pull down, RNA immunoprecipitation (RIP) and luciferase reporter assays, the interaction among LINC01006, miR-34a-5p and disheveled associated activator of morphogenesis 1 (DAAM1) were verified. Results LINC01006 expression presented high in PCa cell lines. LINC01006 silencing suppressed cell proliferative, migratory, invasive capacities while accelerated apoptotic rate. Besides, LINC01006 knockdown also suppressed tumor growth and metastasis in vivo. Furthermore, miR-34a-5p, a tumor suppressor in PCa, was sponged by LINC01006. Moreover, DAAM1 was targeted by miR-34a-5p and promoted PCa progression. More intriguingly, rescue assays suggested that the inhibitory effect of LINC01006 knockdown on PCa development was offset by DAAM1 overexpression. Conclusions LINC01006 promoted PCa progression by sponging miR-34a-5p to up-regulate DAAM1, providing a novel target for PCa therapy.

The mechanisms that underlie various inflammation paradoxes, metabolically healthy obesity, and increased inflammations after inflammatory cytokine blockades and deficiencies remain poorly determined. We performed an extensive –omics database mining, determined the expressions of 1367 innate immune regulators in 18 microarrays after deficiencies of 15 proinflammatory cytokines/regulators and eight microarray datasets of patients receiving Mab therapies, and made a set of significant findings: 1) proinflammatory cytokines/regulators suppress the expressions of innate immune regulators; 2) upregulations of innate immune regulators in the deficiencies of IFNγ/IFNγR1, IL-17A, STAT3 and miR155 are more than that after deficiencies of TNFα, IL-1β, IL-6, IL-18, STAT1, NF-kB, and miR221; 3) IFNγ, IFNγR and IL-17RA inhibit 10, 59 and 39 proinflammatory cytokine/regulator pathways, respectively; in contrast, TNFα, IL-6 and IL-18 each inhibits only four to five pathways; 4) The IFNγ-promoted and -suppressed innate immune regulators have four shared pathways; the IFNγR1-promoted and -suppressed innate immune regulators have 11 shared pathways; and the miR155-promoted and -suppressed innate immune regulators have 13 shared pathways, suggesting negative-feedback mechanisms in their conserved regulatory pathways for innate immune regulators; 5) Deficiencies of proinflammatory cytokine/regulator-suppressed, promoted programs share signaling pathways and increase the likelihood of developing 11 diseases including cardiovascular disease; 6) There are the shared innate immune regulators and pathways between deficiency of TNFα in mice and anti-TNF therapy in clinical patients; 7) Mechanistically, up-regulated reactive oxygen species regulators such as myeloperoxidase caused by suppression of proinflammatory cytokines/regulators can drive the upregulation of suppressed innate immune regulators. Our findings have provided novel insights on various inflammation paradoxes and proinflammatory cytokines regulation of innate immune regulators; and may re-shape new therapeutic strategies for cardiovascular disease and other inflammatory diseases.

Reactive oxygen species (ROS) are critical for the progression of cardiovascular diseases, inflammations and tumors. However, the mechanisms of how ROS sense metabolic stress, regulate metabolic pathways and initiate proliferation, inflammation and cell death responses remain poorly characterized. In this analytic review, we concluded that: 1) Based on different features and functions, eleven types of ROS can be classified into seven functional groups: metabolic stress-sensing, chemical connecting, organelle communication, stress branch-out, inflammasome-activating, dual functions and triple functions ROS. 2) Among the ROS generation systems, mitochondria consume the most amount of oxygen; and nine types of ROS are generated; thus, mitochondrial ROS systems serve as the central hub for connecting ROS with inflammasome activation, trained immunity and immunometabolic pathways. 3) Increased nuclear ROS production significantly promotes cell death in comparison to that in other organelles. Nuclear ROS systems serve as a convergent hub and decision-makers to connect unbearable and alarming metabolic stresses to inflammation and cell death. 4) Balanced ROS levels indicate physiological homeostasis of various metabolic processes in subcellular organelles and cytosol, while imbalanced ROS levels present alarms for pathological organelle stresses in metabolic processes. Based on these analyses, we propose a working model that ROS systems are a new integrated network for sensing homeostasis and alarming stress in metabolic processes in various subcellular organelles. Our model provides novel insights on the roles of the ROS systems in bridging metabolic stress to inflammation, cell death and tumorigenesis; and provide novel therapeutic targets for treating those diseases. (Word count: 246).

Innate and adaptive immune cell activation and infiltration is the key characteristic of tissue inflammation. The innate immune system is the front line of host defense in which innate immune cells are activated by danger signals, including pathogen- and danger-associated molecular pattern, and metabolite-associated danger signal. Innate immunity activation can directly contribute to tissue inflammation or immune resolution by phagocytosis and secretion of biologically active molecules, or indirectly via antigen-presenting cell (APC) activation-mediated adaptive immune responses. This review article describes the cellular and molecular interplay of innate-adaptive immune systems. Three major mechanisms are emphasized in this article for their role in facilitating innate-adaptive immunity interplay. 1) APC can be formed from classical and conditional innate immune cells to bridge innate-adaptive immune response. 2) Immune checkpoint molecular pairs connect innate and adaptive immune cells to direct one-way and two-way immune checkpoint reactions. 3) Metabolic reprogramming during immune responses leads to excessive cytosolic and mitochondrial reactive oxygen species (ROS) production. Increased NADPH oxidase-derived extracellular and intracellular ROS are mostly responsible for oxidative stress, which contributes to functional changes in immune cells. Further understanding of innate-adaptive immunity interplay and its underlying molecular basis would lead to the identification of therapeutic targets for immunological and inflammatory disease.

The mutations in the SARS-CoV-2 virus genome during COVID-19 dissemination are unclear. In 788 COVID-19 patients from Zhejiang province, we observed decreased rate of severe/critical cases compared with patients in Wuhan. For mechanisms exploration, we isolated one strain of SARS-CoV-2 (ZJ01) from a mild COVID-19 patient. Thirty-five specific gene mutations were identified. Phylogenetic and relative synonymous codon usage analysis suggested that ZJ01 may be a potential evolutionary branch of SARS-CoV-2. We classified 54 global virus strains based on the base (C or T) at positions 8824 and 28247 while ZJ01 has T at both sites. The prediction of the Furin cleavage site (FCS) and sequence alignment indicated that the FCS may be an important site of coronavirus evolution. ZJ01 mutations identified near the FCS (F1-2) caused changes in the structure and electrostatic distribution of the S surface protein, further affecting the binding capacity of Furin. Single-cell sequencing and ACE2-Furin co-expression results confirmed that the Furin expression was especially higher in glands, liver, kidneys, and colon. The evolutionary pattern of SARS-CoV-2 towards FCS formation may result in its clinical symptom becoming closer to HKU-1 and OC43 caused mild flu-like symptoms, further showing its potential in differentiating into mild COVID-19 subtypes.

Background: High-Risk Human Papillomavirus (HR-HPV) persistent infection is the main cause of cervical cancer and its precancerous lesions. A previous study showed that HPV16 and HPV58 infections were the most common infection types in the local region. Some studies also declared that HPV58 E7 variants increased the risk of cervical cancer among Asian populations. Objective: This study aimed to determine whether the HPV58 E7 T20I (C632T) variant promotes the malignant behavior of cervical cancer cells and the underlying mechanism of the HR-HPV E7 oncoprotein involved in the development of cervical cancer. Methods: CCK-8 and clone formation assays were used to detect cell proliferation ability. Transwell assays and cell wound healing assays were used to evaluate cell migration ability. Targeted knockdown of E2F1 expression using specific siRNA, RT-qPCR and Western blot were performed to assess gene expression changes. A chromatin immunoprecipitation assay was used to verify that E2F1 interacted with the TOP2A promoter region. Results: HPV58 E7 and HPV58 E7M oncoproteins increased the proliferation and migration ability of cervical cancer cells. However, the HPV58 E7 T20I variant did not promote malignant behaviors compared with wildtype HPV58 E7. HPV E7 and E7M oncoproteins increased the expression of TOP2A, BIRC5 and E2F1, and knockdown of HPV E7 decreased their expression. Low E2F1 expression reduced the expression of TOP2A and BIRC5 and inhibited the proliferation and migration ability of cervical cancer cells. E2F1 interacted with the TOP2A gene promoter region to promote its transcriptional expression. Conclusions: The HPV58 E7 T20I variant did not promote malignant behaviors compared with wild-type HPV58 E7. The HR-HPV E7 oncoprotein enhanced the proliferation and migration of cervical cancer cells, which was considered to be due to the HPV E7 oncoprotein, increasing the expression of BIRC5 and TOP2A by upregulating the transcription factor E2F1.

In addition to the roles of endothelial cells (ECs) in physiological processes, ECs actively participate in both innate and adaptive immune responses. We previously reported that, in comparison to macrophages, a prototypic innate immune cell type, ECs have many innate immune functions that macrophages carry out, including cytokine secretion, phagocytic function, antigen presentation, pathogen-associated molecular patterns-, and danger-associated molecular patterns-sensing, proinflammatory, immune-enhancing, anti-inflammatory, immunosuppression, migration, heterogeneity, and plasticity. In this highlight, we introduce recent advances published in both ATVB and many other journals: (1) several significant characters classify ECs as novel immune cells not only in infections and allograft transplantation but also in metabolic diseases; (2) several new receptor systems including conditional danger-associated molecular pattern receptors, nonpattern receptors, and homeostasis associated molecular patterns receptors contribute to innate immune functions of ECs; (3) immunometabolism and innate immune memory determine the innate immune functions of ECs; (4) a great induction of the immune checkpoint receptors in ECs during inflammations suggests the immune tolerogenic functions of ECs; and (5) association of immune checkpoint inhibitors with cardiovascular adverse events and cardio-oncology indicates the potential contributions of ECs as innate immune cells.

Site-directed protein immobilization allows the homogeneous orientation of proteins with high retention of activity, which is advantageous for many applications. Here, we report a facile, specific, and efficient strategy based on the SpyTag-SpyCatcher chemistry. Two SpyTag-fused model proteins, that is, the monomeric red fluorescent protein (RFP) and the oligomeric glutaryl-7-aminocephalosporanic acid acylase, were easily immobilized onto a SpyCatcher-modified resin directly from cell lysates, with activity recoveries in the range of 85-91%. This strategy was further adapted to protein purification, which proceeded through the selective capture of the SpyCatcher-fused target proteins by a SpyTag-modified resin, with the aid of an intein to generate authentic N-termini. For two model proteins, that is, RFP and a variable domain of a heavy chain antibody, the yields were ∼3-7 mg/L culture with >90% purities. This approach could provide a versatile tool for producing high-performance immobilized protein devices and proteins for industrial and therapeutic uses.

The outbreak of COVID-19 become enormous threat to human beings, showing unclear virus mutation during dissemination. We found, in our 788 confirmed COVID-19 patients, the decreased rate of severe/critical type, increased liver/kidney damage and prolonged period of nuclear acid positivity, when compared with Wuhan. To investigate underlining mechanisms, we isolated one strain of SARS-CoV-2 (ZJ01) in mild COVID-19 patient and found the existence of 35 specific gene mutation by gene alignment. Further phylogenetic analysis and RSCU heat map results suggested that ZJ01 may be a potential evolutionary branch of SARS-CoV-2. We classified 54 strains of viruses worldwide (C/T type) based on the base (C or T) at positions 8824 and 28247. ZJ01 has both T at those sites, becoming the only TT type currently identified in the world. The prediction of Furin cleavage site (FCS) and the sequence alignment of virus family indicated that FCS may be an important site of coronavirus evolution. ZJ01 had mutations near FCS (F1-2), which caused changes in the structure and electrostatic distribution of S protein surface, further affecting the binding capacity of Furin. Single cell sequencing and ACE2-Furin co-expression results confirmed that Furin level was higher in the whole body, especially in glands, liver, kidney and colon while FCS may help SARS-CoV-2 infect these organs. The evolutionary pattern of SARS-CoV-2 towards FCS formation may result in its clinical symptom becoming closer to HKU-1 and OC43 (the source of FCS sequence-PRRA) caused influenza, further showing potential in differentiating into mild COVID-19 subtypes.

PurposeGenome sequencing (GS) is potentially the most suitable diagnostic tools for fetal CNS structural anomalies. However, its efficacy hasn’t been proved in large cohort of fetal CNS structural anomalies.MethodsPatients were enrolled by a multiple-level referral system when fetal CNS structure anomalies were found by ultrasonography. Samples from fetuses were subjected to GS.ResultsData of 162 fetuses with 11 frequent types of CNS anomalies was collected. The overall diagnosis yield of GS was 38.9%. 36(20.3%) fetuses were detected with chromosomal anomalies and pathogenic CNVs. Pathogenic or likely pathogenic single-gene variants and intragenic CNVs were found in 24 and three fetuses, contributing 14.8% and 1.9% diagnostic yield respectively. The diagnostic rate in 41 fetuses with CNS malformation combined with anomalies out of brain was as high as 73.3%. Malformations of the posterior cerebral fossa, abnormal neuronal proliferation and migration have the highest diagnostic rates. NTDs had the second lowest diagnostic rates of 14.7% and none pathogenic variants were found in ultrasound anomalies that suggested destructive cerebral lesions.ConclusionGS is an efficient genetic testing tool with the diagnostic power compared to current CMA plus ES procedure in fetal CNS anomalies evaluation.