The dengue virus (DENV) has been endemic in Myanmar since 1970, causing outbreaks every 2–3 years. DENV infection symptoms range from mild fever to lethal hemorrhage. Clinical biomarkers must be identified to facilitate patient risk stratification in the early stages of infection. We analyzed 45 cytokines and other factors in serum samples from the acute phase of DENV infection (within 3–5 days of symptom onset) from 167 patients in Yangon, Myanmar, between 2017 and 2019. All of the patients tested positive for serum DENV nonstructural protein 1 antigen (NS1 Ag); 78.4% and 62.9% were positive for immunoglobulin M (IgM) and G (IgG), respectively; and 18.0%, 19.8%, and 11.9% tested positive for serotypes 1, 3, and 4, respectively. Although the DENV-4 viral load was significantly higher than those of DENV-1 or DENV-3, disease severity was not associated with viral load or serotype. Significant correlations were identified between disease severity and CCL5, SCF, PDGF-BB, IL-10, and TNF-α levels; between NS1 Ag and SCF, CCL5, IFN-α, IL-1α, and IL-22 levels; between thrombocytopenia and IL-2, TNF-α, VEGF-D, and IL-6 levels; and between primary or secondary infection and IL-2, IL-6, IL-31, IL-12p70, and MIP-1β levels. These circulating factors may represent leading signatures in acute DENV infections, reflecting the clinical outcomes in the dengue endemic region, Myanmar. The dengue virus (DENV) is a mosquito-borne arbovirus that is endemic in more than 130 countries and represents a significant public health burden worldwide ,3.Flaviviridae family of single-stranded RNA viruses and exists as 4 antigenically distinct serotypes: DENV-1–4. Infection with one serotype does not protect against infection against a heterotypic serotype, and secondary infections carry an elevated risk of developing severe dengue. Depending on the severity of clinical features, symptomatic DENV infection is categorized by WHO as dengue without warning signs (DWoWS), dengue with warning signs (DWWS), and severe dengue (SD) [DENV is a member of the gue (SD) . The devgue (SD) . Dengue gue (SD) ,7.In this study, we investigated the levels of 45 cytokines, chemokines, and growth factors in acute-phase serum samples collected from a cohort of DENV-infected individuals in Myanmar. We then determined whether significant correlations existed between acute-phase cytokine levels and disease severity as well as other relating clinicopathological factors on both viral and host aspects to support our findings.This was a cross-sectional study of children and adults from two hospitals in Yangon, a DENV-endemic region of Myanmar, between June 2017 and December 2019. The sample size was calculated using the following formula:This study was approved by the Ethics Review Committee on Medical Research Including Human Subjects at the Department of Medical Research, Myanmar (Ethics/DMR/2017/068) and the Institute of Tropical Medicine at Nagasaki University in Nagasaki, Japan (170707205).TM DENV NS1 Ag enzyme-linked immunosorbent assay (ELISA) kits  according to the manufacturer’s instructions. NS1 Ag levels are expressed as the ratio of the OD at 450/620 nm of the sample to that of the kit calibrator cutoff value. Ratios of <0.5, 0.5–1.00, and >1.00 were considered negative, equivocal, and positive, respectively. Samples were tested for the presence of anti-DENV antibodies using previously described dengue IgM capture ELISA [All of the serum samples, qualitatively screened for NS1 Ag using the rapid diagnostic test were analyzed using qualitative/semi-quantitative PLATELIAre ELISA  and dengre ELISA ,10,11.2–108 genome copies) were amplified for quantification of viral genome copies [RNA was extracted from 140 μL of serum using a viral RNA mini-kit , according to the manufacturer’s protocol. For quantitative reverse-transcription polymerase chain reaction (qRT-PCR), 5 μL of RNA was used and the envelope gene was amplified in a total of 20 μL of the reaction mixture  using TaqManFast Virus 1-Step MasterMix . Serotype specific primers and probes are described in . Ten-fole copies ,13. The A total of 45 cytokines, chemokines, and growth factors were measured using a Cytokine/Chemokine/Growth Factor 45-Plex Human ProcartaPlex Panel 1 kit  according to the manufacturer’s protocol. Fluorescence signals were detected using the Bio-plex 200 system , and the data were analyzed using the Bio-plex Manager 6.2 software . Results that were outside the lowest range of the standard curve were set as half the lowest value in the set for the respective analyte. The value that was outside the highest range was replaced with the highest value in the set .p–values of < 0.05 were considered as statistically significant. PCA was conducted in R (Version 4.1.2) with plotting tools from the factoextra package.Data were analyzed using SPSS for Windows version 16.0  and GraphPad Prism version 9.0.0 . Continuous variables are presented as the median and interquartile range (IQR) or mean ± standard deviation. Skewness was tested to assess the normal distribution of the variables. As the data did not show normal distribution, we used non-parametric tests. Data from more than two groups were compared using MANCOVA, and between two groups were compared using the Mann–Whitney U test based on distribution of data. The Spearman’s test was used to identify correlations and the results are presented as the coefficients (ρ −1 to +1). n = 106, 63.5%) and aged between 6–12 years . Based on the WHO dengue criteria, 64 (38.3%), 64 (38.3%), and 39 (23.3%) patients were classified as having DWoWS, DWWS, and SD, respectively  were enrolled in the study. Serum samples were collected from all of the participants between 3–5 days after the onset of fever. The majority of patients were male  and abdominal pain  were the most common symptoms, followed by gastrointestinal and bleeding manifestations. Seizures were only observed three patients (p < 0.001) or the DWoWS group  were positive for anti-DENV IgM, of whom 50, 48, and 33 were in the DWoWS, DWWS, and SD groups, respectively. Similarly, 105 (62.9%) patients were positive for anti-DENV IgG, of whom 41, 31, and 33 were in the DWoWS, DWWS, and SD groups, respectively . Of the We detected no significant differences between the DWoWS, DWWS, and SD groups for the IgM P/N ratio A or NS1 Among the 83 patients who were positive for viral RNA as determined using qRT-PCR analysis, 30 (36.1%) were infected with DENV-1, 33 (39.7%9) with DENV-3, and 20 (24.1%) with DENV-4. There were no significant differences in DENV viral load between patients with different disease severity A or thosLevels of 45 cytokines, chemokines, and growth factors in serum samples obtained from the 167 confirmed dengue patients collected between day 3–5 of fever onset, and 10 healthy individuals were analyzed using multiplexed immunoassay. Of the factors evaluated, several were present at significantly different levels between the patient and healthy control groups as well as between the patient severity subgroups while age, gender, days of fever and IgM had no effect on serum cytokines level. For example, levels of SCF, CCL5, and interleukin (IL)-15 were significantly higher in the DWWS group than in the DWoWS group, and chemokine ligand 5 (CCL5) and stem cell factor (SCF) levels were significantly higher in the DWWS, DWoWS, and SD groups than in the control group A–C. Of nIn addition, levels of 16 mediators IL-1α, IL-1β, IL-5, IL-7, IL-8 (CXCL8), IL-13, IL-17A, PIGF1 , IL-23, IP10 (CXCL10), MIP-1β, NGF-β, EGF, vascular endothelial growth factor D (VEGF-D), FGF-2 (fibroblast growth factor) and CCL2, were significantly higher in patients compared with those in healthy subjects, but the levels were not significantly different between the SD, DWoWS, and DWWS groups . Levels A total of 3 principal components were extracted, accounting for 60.8% of variation in the data . In termIn this study, we characterized the acute-phase serological status of 167 children and adults with dengue in Yangon, Myanmar, between 2017–2019, and correlated the findings with disease severity. Nearly 40% of the patients were characterized as having DWWS and DWoWS, whereas only about 20% had SD. The proportion of SD patients in our study is smaller than that detected in another study of DENV-infected patients in Yangon during the same period . The mosWe observed several noteworthy differences in acute-phase parameters between patients with DWWS, DWoWS, and SD. Hematocrit values were significantly higher and platelet counts were significantly lower in the patients in the SD group compared with those in the DWoWS and DWWS groups. Platelet count presentations during DENV were similar to those observed in a population of two widely separated regions in Myanmar in 2015 ,19. PlatThe majority of our patient group (103 (61.6%)) had secondary DENV infections, which is a similar proportion to the previous study of DENV infection in this region, Yangon, Myanmar between 2017–2019 . While nDENV-1, -3, and -4 serotypes were detected in our patient cohort, and the viral load was significantly higher in patients infected with DENV-4 than with DENV-1 or -3; however, we observed serotype difference or viremia level have no effect on severe dengue. All DENV serotypes are known to cause severe clinical features, which have previously been reported to be dependent of the viral serotype and genotype . The NS1The serum cytokine profiles of DENV-infected patients may provide valuable information for characterizing immunological response patterns associated with disease severity, and thus help to identify patient groups at risk for poorer outcomes. DENV infects various immune cells, causing alterations in inflammatory and antiviral cytokine levels, leading to tissue or organ damage . MaintaiWe observed that levels of SCF, CCL5, and IL-15 were significantly higher in patients with DWWS than in those with DWoWS. SCF is expressed by fibroblasts and endothelial cells and plays important role in maintaining hematopoietic stem cells and hematopoiesis . Levels The elevation in VEGF levels is likely linked to its role in angiogenesis and regulation of vascular permeability . CompariThis study was first document of serum cytokine profiles during the acute phase of DENV infection in Myanmar pediatric and adult patients. Understanding changes in cytokine profiles early during DENV infection may be valuable for identifying patients at risk for the development of SD. Indeed, we found that levels of certain cytokines were significantly associated with disease severity; specifically, PDGF-BB, IL-10, and TNF-α levels were higher in patients with SD compared with those in patients with DWWS or DWoWS, suggesting that they may be useful prognostic predictors. Among the 45 cytokines tested, CCL5, IL-15, SCF, PDGF-BB, IL-10, and TNF-α levels were associated with disease outcomes. IL-2, TNF-α, VEGF-D, and IL-6 were correlated with thrombocytopenia and these markers were valuable for predicting bleeding. These acute-phase cytokine signatures may therefore be useful for further investigation and identifying patients at risk for the development of SD, particularly on the recent outbreaks in a dengue endemic region, Myanmar.


Sucrose non-fermentation-related protein kinase 1 (SnRK1) a Ser/Thr protein kinase, is known to play a crucial role in plants during biotic and abiotic stress responses by activating protein phosphorylation pathways. SnRK1 and some members of the plant-specific SnRK2 and SnRK3 sub-families have been studied in different plant species. However, a comprehensive study of the SnRK gene family in Phaseolus vulgaris is not available. Symbiotic associations of P. vulgaris with Rhizobium and/or mycorrhizae are crucial for the growth and productivity of the crop. In the present study, we identified PvSnRK genes and analysed their expression in response to the presence of the symbiont. A total of 42 PvSnRK genes were identified in P. vulgaris and annotated by comparing their sequence homology to Arabidopsis SnRK genes. Phylogenetic analysis classified the three sub-families into individual clades, and PvSnRK3 was subdivided into two groups. Chromosome localization analysis showed an uneven distribution of PvSnRK genes on 10 of the 11 chromosomes. Gene structural analysis revealed great variation in intron number in the PvSnRK3 sub-family, and motif composition is specific and highly conserved in each sub-family of PvSnRKs. Analysis of cis-acting elements suggested that PvSnRK genes respond to hormones, symbiosis and other abiotic stresses. Furthermore, expression data from databases and transcriptomic analyses revealed differential expression patterns for PvSnRK genes under symbiotic conditions. Finally, an in situ gene interaction network of the PvSnRK gene family with symbiosis-related genes showed direct and indirect interactions. Taken together, the present study contributes fundamental information for a better understanding of the role of the PvSnRK gene family not only in symbiosis but also in other biotic and abiotic interactions in P. vulgaris. Sucrose nonfermenting 1 (SNF-1)-related protein kinase (SnRK1), animal homologue AMP-activated protein kinase (AMPK) and yeast homologue Sucrose Non-Fermenting1 kinase/plant SNF1-related kinase1 (SNF1) are highly conserved serine/threonine protein kinases that function as cellular energy sensors activating catabolism and repress anabolic processes ,3,4. AllSnRKs in plants are classified into SnRK1, SnRK2 and SnRK3 sub-families based on sequence similarity and structural organization [SnRK1 is the homologue of AMPK, and SNF1, SnRK2 and SnRK3 are unique to plants and evolved from the SnRK1 family via gene duplication during plant evolution, and play a key role in the stress, calcium and ABA signalling pathways with epigenetic and metabolic responses [nization . SnRK1 iesponses .SnRK1 sub-family consists of an N-terminal kinase domain, a ubiquitin-associated (UBA) domain and a C-terminal kinase-associated 1 (KA1) domain [SnRK1 gene family is a comparatively smaller sub-family primarily found to be core sensors of energy deficit in plants. The mode of regulation is found to be through downstream phosphorylation and inhibition of the enzymes HMG-CoA reductase (HMGR), sucrose phosphate synthase (SPS), nitrate reductase (NR) and seaweed phosphate synthase 5 (TPS5) [SnRK1 genes are pivotal in various signalling pathways, such as meristem development, cell cycle regulation and pathogen responses [The ) domain . The SnR5 (TPS5) ,10,11 inesponses .SnRK2 sub-family harbours the regulatory C-terminal domain containing acidic amino acids, either Glu or Asp [SnRK2 sub-family involves activation of basic region-leucine zipper (bZIP) transcription factors in association with an epigenetic mechanism [SnRK2 sub-family has been well studied in plants for its involvement in abiotic stress, either ABA mediated or ABA independent. The SnRK2 sub-family in Arabidopsis is classified into three groups: ABA dependent, drought responsive and ABA activated [Arabidopsis and rice, the SnRK2 sub-family is larger than that of SnRK1, with 10 members found to be abiotic stress responsive and activated by ABA and to phosphorylate transcription factors of the ABA-response element-binding protein class (AREBP) [The u or Asp . The regechanism ,14. The ctivated ,16. In A (AREBP) ,19,20,21SnRK3 sub-family, also named CIPKs (CBL-interacting protein kinases), contains two conserved domains at the C-terminus, including NAF  and PPI (protein–protein interacting) domains [SnRK3 sub-family is the largest in all plants studied thus far; these proteins transport Na+, K+ and NO3 ions, along with their binding ability to Ca2+-dependent CBL to regulate downstream genes and enhance abiotic stress tolerance [AtCIPK24 , which is a member of the SnRK3 sub-family in Arabidopsis thaliana. As a Na+/H+ antiporter, it improves plant salt tolerance by maintaining ionic homeostasis [SnRKs represent one of the larger gene families in plants involved in a variety of regulatory mechanisms including metabolism, growth, development, and abiotic stress response.The  domains . The SnRolerance ,23,24. Solerance ,26,27. Teostasis ,29. TakeSnRK genes, the recent years have seen extensive genome-wide analysis in various plant species. Starting form Arabidopsis [Brassica napus [Cucumis sativus [Eucalyptus grandis [Fragaria ananassa [Triticum aestivum [Hordeum vulgare [Oryza sativa [Brachypodium distachyon [SnRK gene family and the highly conserved nature of gene structure and function. Curiously, these studies do not involve any legumes.Due to the varied regulatory roles played by bidopsis  to econoca napus , Cucumis sativus , Eucalyp grandis , Fragariananassa , Triticuaestivum , Hordeum vulgare , Oryza sa sativa  and Bracstachyon . Each ofP. vulgaris, are not considered subjects in the exploration of gene families.Among flowering plants, legumes are the most important agricultural crops after cereals due to their high economic value. Legumes play a central role at the food system level, both for human and animal consumption, as a source of plant proteins . FurtherSnRK with diverse regulatory mechanisms might play a pivotal role in biotic and abiotic interactions of legumes, and greater knowledge of these aspects may help in crop improvement. In the present study, we sought to identify and understand the diversity of the SnRK gene family in P. vulgaris and elucidate their differential expression patterns under rhizobial and mycorrhizal symbiotic conditions.A better understanding of the signalling pathways that affect the productivity and sustainability of such crop systems is particularly important. A gene family such as A. thaliana SnRK gene family were downloaded from NCBI . Homologues of AtSnRKs were searched in the Phytozome  and Legume Information System  databases using BLASTN and BLASTP methods. The respective nucleic acid and peptide sequences were downloaded from the online tool PhytoMine from the plant comparative genomics portal Phytozome v12.1 for further analysis and annotation. The hidden Markov model (HMM) and BLASTP program were applied for preliminary identification of PvSnRK proteins. Local BLASTP  searches were performed based on hidden Markov model (HMM) profiles of PvSnRK gene domains from the Pfam database . The SMART database  was used to confirm PvSnRK gene sequences [The amino acid and nucleotide sequences of the equences , as wereequences  and the equences .PvSnRK homologues based on domain structure [PvSnRK gene family members was verified in the Phytozome v12.1 database, chromosomal images were drawn using the EnsemblPlant tool [PvSnRK proteins were calculated using tools from ExPASy . Subcellular localization of PvSnRK proteins were predicted by ProtCompv.9.0 , BaCelLo  [https://rostlab.org/services/loctree3/, accessed on 10 September 2021) [https://wolfpsort.hgc.jp/, accessed on 10 September 2021) [http://cello.life.nctu.edu.tw/, accessed on 10 September 2021) [MEME and motif discovery tools were employed to filter out tructure . The chrant tool , and theant tool . The numer 2021) , LocTreeer 2021) , WoLF PSer 2021)  and Celler 2021) .PvSnRKs and AtSnRKs was performed using ClustalW. Based on the alignments, phylogenetic analysis of the aligned sequences was carried out using Molecular Evolutionary Genetics Analysis (MEGA XI) with the neighbour-joining (NJ) method and the JTT + I + G substitution model with 1000 bootstrap replicates and default parameters [Multiple sequence alignment of rameters .PvSnRK gene family in P. vulgaris were identified using the Multiple Expectation Maximization for Motif Elicitation (MEME) online program  with the following parameters: number of repetitions = any, maximum number of motifs = 20; and optimum motif length = 6 to 100 residues. The gene structure of the PvSnRK gene family was analysed using the Gene Structure Display Server online program  [Conserved motifs in the er 2021) .PvSnRKs were downloaded from the Phytozome database. The plant transcriptional regulatory map  was used to analyse the promoter sequences.Sequences 2 kb upstream of PvSnRK gene were calculated using the following formula: T = Ks/2λ (λ = 6.56 × 10−9) [To identify putative orthologues between two different species (A and B), each sequence from species A was searched against all sequences from species B using BLASTN; each sequence from species B was also searched against all sequences from species A. The two sequences were defined as orthologues when reciprocal best hits were each within ≥300 bp of the two aligned sequences. The duplication period  and divergence of each  × 10−9) . The cal × 10−9) .https://stringpreview. org/, accessed on 15 March 2022) and visualized using Cytoscape v3.9.1 . Finally, enrichment analysis of Gene Ontology (GO) terms was performed using the online tool AgriGO (v2.0)  [A high-expression candidate gene protein interaction network was analysed using STRING v11.0 (il 2022) .SnRK genes in P. vulgaris tissues under nitrogen treatments and after inoculation with Rhizobium tropici (CIAT899) were obtained from the PvGEA website . Previously, we performed global transcriptome profiling in P. vulgaris L. cv. Negro Jamapa roots colonized by Rhizophagus irregularis spores or Rhizobium tropici strain CIAT899 [PvSnRK family genes under both types of symbiotic conditions. Heatmaps were constructed with fold-change values applying the R package .Data on differential expression of  CIAT899 . The preP. vulgaris L. cv. Negro Jamapa seeds and germinated them as described by Nanjareddy et al. [R. irregularis or R. tropici according to Nanjareddy et al. [To validate the RNA-seq data, we surface-sterilized y et al. . Two-dayy et al. . Total Ry et al. .Arabidopsis SnRKs as a reference, and a total of 42 genes were identified based on conserved domains in each sub-family (PvSnRK1 was identified by the domain KA1 (PF02149), the PvSnRK2 sub-family by the OST domain [PvSnRK3 sub-family by the NAF/FISL domain (PF02149). The genes were named PvSnRK1.1, PvSnRK1.2, PvSnRK2.1-PvSnRK2.11 and PvSnRK3.1-PvSnRK3.29 based on sequence homology with the Arabidopsis SnRK family. The amino acid length of the 42 PvSnRK gene family members ranged from 310 aa (PvSnRK2.11) to 528 aa (PvSnRK1.2), corresponding to molecular weights of 60.54 to 35.67 kDa  ranged from 4.7 to 9.24, with PvSnRK1 sub-family members showing a basic PI, the PvSnRK2 sub-family being mostly acidic (4.7–6.65) and the PvSnRK3 sub-family being slightly acidic to highly basic (6.4–9.37). Subcellular localization analysis was carried out using ProtComp v.9.0, and the results showed PvSnRK1s to localize to the extracellular space; PvSnRK2s mostly localized to the nucleus and plasma membrane and PvSnRK3 sub-family members to the plasma membrane  method with 1000 bootstrap replications , followed by chromosome 8 (eight PvSnRKs). Chromosomes 1, 4, 5 and 7 each have one gene each, as shown in To examine the evolution of Intron–exon analysis was carried out to obtain better insight into the structure of PvSnRK genes. PvSnRK gene family members exhibited a great variation, from 1 to 14 introns, as shown in PvSnRK proteins using the MEME program revealed a total of 25 conserved motifs, named from 1 to 25. The identified motifs were annotated in Pfam, and the details of the putative motifs are shown in PvSnRK3. Ubiquitin-associated domain 20 was found in PvSnRK1 and PvSnRK3. Furthermore, motifs 5 and 13, designated protein superfamily kinase domains, were found only in the PvSnRK1 and PvSnRK3 sub-families  and nonsynonymous (Ka) substitutions per site and their ratio (Ka/Ks) and divergence time of paralogous and orthologous SnRK family genes were calculated for AtSnRK orthologues of PvSnRKs  in the examined regulatory region, with 286 ERF binding sites and 49 MYB and 44 C2H2 sites. PvSnRK 3.20 has 338 TF sites; this was followed by PvSnRK 3.19 with 318 TFs, with 111 TFs being ERF TFs and 56 being bHLH TFs, and PvSnRK 1.2, with 312 TFs, with 100 being NAC TFs, 45 being MYB TFs and 40 being ERF TFs . The expression patterns of all 42 PvSnRK members were analysed in 25 different tissues, including leaves, stems, shoots, pods, seeds, roots (inoculated and uninoculated with Rhizobium) and nodules, at different developmental stages and treatments . Pre-fixing (effective) nodules collected at 5 days after inoculation (PvN5) showed very high expression of only PvSnRK3.5 and PvSnRK3.29 . While predicting interactions among the PvSnRK proteins, we used experimentally proven, co-expressed and co-occurring protein interactions, revealing a total of 40 interacting proteins. The majority of them are phosphoprotein phosphatase (PPP) family proteins, PP2A , PP2CA/PP2CB , PPP2RA/PPP2RB (protein phosphatase 2A 65 kDa regulatory subunit A β isoform) and PPP1C -type proteins. Phosphatases are proteins involved in substrate recognition, plant signalling pathways such as stress regulation, light, pathogen defence and hormonal signalling, the cell cycle, differentiation, metabolism, and plant growth.A protein interaction network was constructed for PvSnRK protein sub-families were found to interact with PPP family members and cyclin B proteins through PKGII, indicating their involvement in various cellular and developmental processes. SnRK2 sub-family members were found to specifically interact with PP2C proteins , RAB proteins, auxin-responsive ARP proteins and many proteins involved in vesicle transport , and through GNBI, they interact with several RPSs  and a variety of symbiosis-related proteins. Network prediction showed that PvSnRK3 sub-family members may be involved in regulating symbiosis in Phaseolus via different signalling pathways (The largest pathways .SnRK gene family, serine/threonine kinases, and its orthologues in animals and yeast are highly conserved. In plants, they have been identified as regulators of abiotic and biotic stress [Phaseolus vulgaris is the most important legume consumed by humans worldwide, as it is an affordable source of proteins, vitamins, minerals, antioxidants, and bioactive compounds [Rhizobium application. Since common bean does not need exogenous N fertilizer application, productivity is cost effective. Any efforts towards the betterment of rhizobial association to improve N fixation in crops such as Phaseolus should be undertaken. The present investigation encompasses the identification, classification, and analysis of the expression patterns of the SnRK gene family under symbiotic conditions.The c stress ,81,82. Iompounds . Climateompounds . The yieSnRK genes have been identified in A. thaliana [Brassica napus [C. sativus [E. grandis [F. ananassa [T. aestivum [H. vulgare [O. sativa [B. distachyon [Phaseolus, we identified 42 SnRK genes with 2 SnRK1s, 11 SnRK2s and 29 SnRK3s with the characteristic domains of each sub-family. As in any other species, the SnRK2 and SnRK3 sub-families in Phaseolus are larger than SnRK1, supporting the view that these two sub-families originated from duplication of SnRK1 [SnRK2 and SnRK3 sub-family members in all reported plant species.Genome-wide identification studies have been carried out, with early reports documenting the presence of various numbers of members in both monocots and dicots. A total of 39, 114, 30, 34, 26, 149, 46, 48 and 44  sativus , E. gran grandis , F. ananananassa , T. aest vulgare , O. sati. sativa  and B. dstachyon , respectof SnRK1 . The nonof SnRK1 ,86 and ePhaseolus, the SnRK gene family is distributed on 10 of 11 chromosomes. This distribution is unlike in other species, in which all chromosomes in the genome contain at least one of these genes [Phaseolus chr3, chr6, chr8 and chr9 show clustering of genes, mostly of the SnRK3 sub-family.In se genes ,34,35,36SnRK sub-families has a characteristic domain; however, the N-terminal kinase domain is highly conserved across species and sub-families. Exon–intron structural diversification and motif composition play an important role in the evolution and function of many gene families [PvSnRK1 sub-family members have 10–11 exons, such as BdSnRK1s, EgrSnRK1s and CsSnRK1s. All PvSnRK2s have nine exons, similar to most HvSnRKs, HcSnRK2s, EgrSnRK2s, VvSnRK2s, AtSnRK2s and BdSnRK2s, indicating the conserved nature of these sub-families. The sub-family PvSnRK3 can also be subdivided into exon-rich and exon-poor types, as can all other species reported thus far. Reports suggest the origin of the SnRK3 sub-family from green algae, and the intron-poor group first appeared in seed plants [SnRK exon–intron numbers are highly conserved during the evolution of each sub-family. The phylogenetic tree and exon–intron structure showed that most paralogous gene pairs contain the same exon number, though some gene pairs have different exon numbers. Motif analysis revealed a close evolutionary relationship within sub-groups due to the conserved nature of motif composition among sub-families. The motif structure of each sub-family might define the biological function in which they are involved. The gene structure and sequence conservation were similar to most of the previously studied species [Each of the families . PvSnRK1d plants . These r species ,34,35,36SnRK gene families, detection of cis-acting elements has focused on abiotic stress conditions in which the presence of hormone-, salt-, temperature-, and drought-specific cis-elements [PvSnRK gene family in symbiosis, we analysed symbiosis-related cis-elements as demonstrated in Medicago and L. japonicus. All PvSnRK gene sub-families contain symbiosis-related cis-elements. Among all cis-elements, ERF and MYB are the most abundant, followed by C2H2 and bHLH.Gene expression is regulated by external factors that are perceived through signalling mechanisms. Such signals activate specific transcription factors that, when combined with cis-acting elements in the promoter regions of genes, alter gene expression. In most genome-wide analysis studies of elements ,34,35,36PvSnRK gene family duplications either occurred slowly or are highly conserved [PvSnRK genes function mostly in molecular and biological processes, specifically in cellular and metabolic processes followed by nucleotide binding activity. When we analysed expression of PvSnRK gene family members in different tissues, the lowest expression of any PvSnRK was found in aerial tissues such as young pods, flowers, seeds, and shoots. Most of the SnRKs were found to be expressed in root or root nodules at some stage of their development. Furthermore, transcriptomic data under early symbiotic conditions showed elevated expression levels of PvSnRK1 s and PvSnRK3s, with some members being more highly induced than others. For some genes, these expression patterns were found under both root nodule and mycorrhizal symbiotic conditions, suggesting a possible role for PvSnRKs in the establishment of symbiotic relationships.Ka/Ks analysis showed that onserved . Gene OnPvSnRKs and PvSnRKs with symbiosis-related genes, we carried out in situ interaction network building using the STRING database and Cytoscape. The results were interesting, as most of the PvSnRKs interact among themselves, and interaction is mediated by master regulators of cellular processes such as TOR and PKGII. Through these proteins, PvSnRKs interact with several protein phosphatases and cell cycle-regulating cyclins. We chose symbiosis-related genes based on a previously published article and identified a total of 200 genes in the Phaseolus genome. PvSnRK1s mostly interact with other major proteins, such as TOR and PI3Ks, which are connected to the NUPs, RAB proteins, ARPs and proteins involved in vesicle transport. On the other hand, PvSnRK2s interact with PKGII, which interact with the cell cycle regulatory proteins cyclins, CDC, and APC. In contrast, PvSNRK2s shows very few symbiotic gene interactions. Of the largest PvSnRK sub-families, PvSnRK3 interacts with NUPs, RPSs and many symbiotic genes.To predict possible interaction networks among the PvSnRK gene family revealed structural conservation of genes across species and possible functional conservation as well. Furthermore, the principle aim of this study was to understand the putative role of PvSnRKs in regulating symbiotic interactions between Phaseolus and Rhizobium or Phaseolus; mycorrhiza are promising, as some genes contain specific cis-elements and showed transcript upregulation in response to symbionts. Finally, the identified in situ protein–protein interactions may help in predicting candidate genes for functional characterization to obtain a clear picture of the regulatory mechanisms involved.Taken together, our extensive analysis of the


The study aimed to estimate the prevalence and associated factors of undiagnosed hypertension (HTN) among adults in Sudan.cross-sectional data were analyzed from 7,226 persons  who participated in the 2016 Sudan STEPS survey, had complete blood pressure measurement and non-pregnant, and responded to a questionnaire, physical measures, and biomedical tests. Logistic regression was used to determine the predictors of undiagnosed HTN.the prevalence of undiagnosed HTN was 26.2% (n=2057) , diagnosed HTN 6.9% (n=690) and total HTN 33.1% (n=2747). In multivariable analysis older age (50-69 years) , obesity , diabetes  and elevated total cholesterol  were positively associated and health care advice  was negatively associated with undiagnosed HTN versus no HTN. Male sex  was positively associated, and older age (50-69 years) , married , urban residence , health care advice , ever cholesterol measured , overweight  and heart attack or stroke  were negatively associated with undiagnosed HTN versus diagnosed HTN.one in four adults in Sudan had undiagnosed HTN  and several associated factors that can help guide interventions were identified. Hypertension (or elevated blood pressure) (HTN) is a major cause of premature death worldwide. An estimated 1.28 billion adults aged 30-79 years worldwide have HTN, most (two-thirds) living in low- and middle-income countries (LMICs) ,5.In sub-Saharan Africa, based on 33 surveys, only 27% were aware of their HTN status before the surveys . For exaUndiagnosed HTN can be conceptualized in problems using health services , using AEnabling/disabling factors associated with undiagnosed HTN include socioeconomic status  in Sudan from February to December in 2016 [ in 2016 . The inc in 2016 .Data collection: following the STEPS survey procedures, socio-demographic and behavioural information was collected in step 1. Physical measurements such as height, weight, and blood pressure were collected in step 2. Biochemical measurements were collected to assess blood glucose and cholesterol levels in step 3 [n step 3 . Blood gn step 3 . The resn step 3 .Outcome variable: undiagnosed HTN was defined as systolic blood pressure (BP) ≥140mmHg and/or diastolic BP ≥90mmHg among people who responded “no” the question “have you ever been told by a doctor or other health worker that you have raised blood pressure or hypertension?” [ension?” ,26. Prioension?” . Predispension?” .2), normal weight (18.5-24.9 kg/m2), overweight (25.0-29.9 kg/m2), and obesity (≥30.0 kg/m2) [Enabling or disabling factors included ever measured cholesterol, education, health care advice, current tobacco use, and physical activity. Health care advice was assessed with the question, “during the past three years, has a doctor or other health worker advised you to maintain a healthy body weight or lose weight?” (yes/no) . Self-reght 18.5-.9 kg/m2,ght 18.5-.9 kg/m2,0 kg/m2)  as: beinEthical considerations: ethics approval was provided by the national ethical committee at Federal Ministry of Health, Sudan. Verbal informed consent was obtained from all participants.Statistical analysis: all statistical analyses were conducted with STATA software version 14.0 . Descriptive statistics are used to describe the sample. Multinomial logistic regression was used to estimate factors associated with undiagnosed HTN and diagnosed HTN (with not having HTN as reference category). Logistic regressions were used to assess the associations with undiagnosed HTN versus diagnosed HTN. Covariates in the logistic regression models included predisposing factors , enabling, or disabling factors  and need factors . Variables significant (at p<0.1) in univariable analyses were subsequently included in the multivariable models. To account for the multi-stage sample design, Taylor linearization methods were utilized. P-values <0.05 were considered significant, and missing values (<4%) were discarded.Sample characteristics: the sample with complete blood pressure measurement, excluding pregnant women, included 7,226 persons , in 2016. The prevalence of undiagnosed HTN was 26.2% (n=2057) , diagnosed HTN 6.9% (n=690) and total HTN 33.1% (n=2747). Further sociodemographic and health characteristics of the sample by HTN status are described in Associations with undiagnosed hypertension versus no hypertension: in univariable analysis, older age, married, ever screened for cholesterol, underweight, overweight, obesity, diabetes and elevated total cholesterol were positively associated, and high physical activity and health care advice were negatively associated with undiagnosed versus no HTN. In multivariable analysis older age (50-69 years) , obesity , diabetes  and elevated total cholesterol  were positively associated and health care advice  was negatively associated with undiagnosed HTN versus no HTN  , married , urban residence , health care advice , ever measured cholesterol , obesity , diabetes , heart attack or stroke , and elevated total cholesterol  were positively associated with diagnosed HTN versus no HTN. Male sex , and underweight  were negatively associated with diagnosed HTN versus no HTN  was positively associated, and older age (50-69 years) , married , urban residence , health care advice , ever cholesterol measured , overweight  and heart attack or stroke  were negatively associated with undiagnosed HTN versus diagnosed HTN  in Sudan. One in four adults in Sudan had undiagnosed HTN . Predisposing factors (older age), enabling factors  and need factors  were associated with undiagnosed HTN versus no HTN, and predisposing factors , enabling factors , and need factors (not overweight and no history of heart attack or stroke) were identified as associated with undiagnosed HTN versus diagnosed HTN.The prevalence of undiagnosed HTN in Sudan , was higher than in a review of 33 surveys in sub-Saharan Africa (73% of HTN)  and highConsistent with some previous research ,13,17,18According to some previous studies ,16,20, eConsistent with some research ,14,18,22The study strengths include the use of a nationally representative sample and standardized STEPS methodology and measures. Some variables were evaluated by self-report, which may have biased responses, and the cross-sectional design precludes causative conclusions between the evaluated variables. The sample only included persons 18-69 years and those who non-institutionalized, while the inclusion of 70 years and older and institutionalized persons would have given different estimates. Furthermore, certain variables, such as knowledge of the symptoms of HTN and a family history of HTN, were not evaluated and should be included in future research.One in four adults in Sudan had undiagnosed HTN . Predisposing factors (older age), enabling factors  and need factors  were associated with undiagnosed HTN versus no HTN, and predisposing factors , enabling factors , and need factors (not overweight and no history of heart attack or stroke) were identified as associated with undiagnosed HTN versus diagnosed HTN, which can be targeted in interventions.Hypertension (or elevated blood pressure) (HTN) is a major cause of premature death;undiagnosed HTN can lead to serious morbidity.One in four adults in Sudan had undiagnosed HTN ;predisposing factors , enabling factors , and need factors (not overweight and no history of heart attack or stroke) were associated with undiagnosed HTN versus diagnosed HTN.


Dimocarpus longan), a subtropic fruit tree with high economic value, is sensitive to cold. However, whether the SWEET gene family plays a role in conferring cold tolerance upon longan remains unknown. Here, a total of 20 longan SWEET (DlSWEET) genes were identified, and their phylogenetic relationships, gene structures, cis-acting elements, and tissue-specific expression patterns were systematically analyzed. This family is divided into four clades. Gene structures and motifs analyses indicated that the majority of DlSWEETs in each clade shared similar exon–intron organization and conserved motifs. Tissue-specific gene expression suggested diverse possible functions for DlSWEET genes. Cis-elements analysis and quantitative real-time PCR (qRT-PCR) analysis revealed that DlSWEET1 responded to cold stress. Notably, the overexpression of DlSWEET1 improved cold tolerance in transgenic Arabidopsis, suggesting that DlSWEET1 might play a positive role in D. longan’s responses to cold stress. Together, these results contribute to a better understanding of SWEET genes, which could serve as a foundation for the further functional identification of these genes.Sugars will eventually be exported transporters (SWEET), a group of relatively novel sugar transporters, that play important roles in phloem loading, seed and fruit development, pollen development, and stress response in plants. Longan ( Sugars are not only the predominant carbon and energy sources for both eukaryotes and prokaryotes, but also the essential signaling molecules for diverse physiological processes . In addiThe SWEET families are a recently discovered protein family of carbohydrate transporters in plants and mammals , which fSWEET genes has been comprehensively conducted in several plant species, such as A. thaliana [Oryza sativa) [Litchi chinensis Sonn) [Punica granatum) [SWEET genes are involved in many different functions, such as phloem loading [To date, the genome-wide analysis of thaliana , rice (O sativa) , lychee is Sonn) , and pomranatum) . Biochem loading , nectar  loading , pollen  loading , modulat loading , senesce loading , abiotic loading ,25,26,27 loading ,29,30,31 loading ,33,34.Dimocarpus longan L.), which belongs to the family of Sapindaceae, is an economically important evergreen fruit crop [Longan (uit crop . China iuit crop . Sugar cuit crop . In receSWEET genes in longan (termed DlSWEET) were identified, and their phylogenetic relationship, gene structures, protein motifs, cis-acting elements, and tissue-specific expression patterns were analyzed. We also determined the expression patterns of some selected DlSWEET genes during fruit development stages and in response to different abiotic stress treatments. Additionally, the DlSWEET1 gene was introduced into A. thaliana by Agrobacterium-mediated method to investigate its function. The results will serve to facilitate our understanding of the function of SWEET genes.In the present study, 20 SWEET genes were identified and renamed from DlSWEET1 to DlSWEET16b according to their phylogenetic relationship with 17 AtSWEET , while clade IV had only two members (DlSWEET16a/16b). Clades I and II contained six and four members, respectively.The evolutionary relationships of DlSWEET family members were assessed by constructing an NJ phylogenetic tree . The reshttp://www.cbs.dtu.dk/services/TMHMM-2.0/)  was used to predict the transmembrane domains  contained six exons, while seven DlSWEET genes (DlSWEET7b/7c/9a/9c/9d/10b/15) harbored five exons, two genes (DlSWEET4/9b) displayed four exons, and DlSWEET2b and DlSWEET16b possessed eight and three exons, respectively. Although some similar DlSWEET genes in the same clade shared a similar gene structure, for example, DlSWEET3a/3b/3c contains 6 exons and 5 introns, differences also existed in their length due to the introns.To gain insights into the structure of analyzed . The resDlSWEETs was studied by gathering the 2000 bp upstream regions of 19 DlSWEETs , and the transcriptional response elements of DlSWEETs were predicted using the PLACE database . Additionally, 20 light-responsive cis-elements were identified; the BOX 4 element, in particular, existed in all promoters of DlSWEETs genes, suggesting that the light may play important roles in regulating the expression of DlSWEETs genes. Notably, many plant hormones and stress-responsive cis-elements, such as ABRE, AuxRR-core, LTR, and MBS were present in the promoter region of DlSWEETs, suggesting that some DlSWEET genes may responsed to plant hormone and stress treatments.The regulatory role of database . A serieDlSWEET genes, transcripts abundance of DlSWEET genes in different tissues, including root, stem, leaf, flower, fruit, and seed, were analyzed based on publicly available RNA-seq datasets (GSE84467) [SWEET genes showed different expression patterns in different longan tissues (DlSWEET10b had no record in the datasets). Among the 19 DlSWEETs, 16 were expressed at relatively high levels  in at least one tissue, including 11, 4, 7, 14, 6, and 11 DlSWEETs in root, stem, leaf, flower, fruit, and seed, respectively  and six genes (DlSWEET1/2a/7b/9a/10c/15) showed a higher expression level in leaf and flower, respectively, indicating their potential roles in regulating longan leaf and flower development. Notably, DlSWEET1 showed higher expression levels in flower, fruit and seeds, indicating that it may play an important role in longan growth and development.To understand the tissue-specific-expression patterns of SE84467)  . The concentrations of fructose exhibited a slightly increasing trend as the fruit developed. Additionally, the glucose content increased slightly from 60 DAF to 90 DAF and then slightly decreased at the later stage of ripening. Overall, sucrose is the main component of soluble sugars in mature longan fruit. Considering the potential roles of DlSWEETs in the soluble sugar accumulation, we investigated the transcript abundance of six genes during longan fruit development using the qRT-PCR method (DlSWEET1/10c) contained extremely high expression levels in fruit at 60 DAF, but showed a decrease in transcript abundance throughout fruit development. On the contrary, the other four genes (DlSWEET2a/2b/3a/16a) showed relatively low expression levels at 60 DAF and then increased during fruit development, which are closely connected to the change in sugar content during longan fruit development.As is shown in R method b. Two geDlSWEET genes were induced by different abiotic stresses, qRT-PCR was performed to determine the expression levels of the seven DlSWEET genes responding to cold, heat, salt, and drought treatments in seedlings  were down-regulated by heat and salt stresses, respectively; two genes (DlSWEET2b/3a) were down-regulated by drought stress, whereas two genes (DlSWEET1/10c) were up-regulated by drought stress.To detect whether the eedlings . OverallDlSWEET1 in cold tolerance in plants, the 35S: DlSWEET1: pSAK277 construct was transferred into A. thaliana, and four independent transgenic lines (T1 generation) were obtained based on kanamycin resistance selection and genomic PCR verification. T3 homozygous transgenic lines were used for cold tolerance assessments. Among the T3 generation transformed lines, four lines  presented high expression of DlSWEET1 by qRT-PCR with a wild-type (WT) Arabidopsis line as a negative control  in Annona squamosa L. [DlSWEET16a may be involved in stem growth and development. DlSWEET15 had higher expression levels in flowers and seeds than other tissues, suggesting its function in specialized organs. In Arabidopsis, AtSWEET15 was expressed in both the seed coat and endosperm, and acts as a sucrose transporter in seed coat efflux and seed filling [DlSWEET15 may participate in longan seed development.Plant SWEET proteins play diverse physiological functions and can transport different sugar molecules ,10,13. B flowers , and itsicotiana . The resthe stem , and theamosa L. , suggest filling . These rSWEET gene members were identified in longan, we only detected the transcripts of six DlSWEETs in longan fruit. Among these six DlSWEETs, four genes (DlSWEET1/2a/2b/3a) belong to clade I, in which SWEET proteins mainly transport monosaccharides [DlSWEET1 declined during fruit development, but the transcripts level of the other three genes increased, suggesting that DlSWEET1 may play a role in transporting monosaccharides in the early stages of fruit development, while DlSWEET2a/2b/3a comes to play the roles at the fruit expansion and mature stages. In clade III, SWEETs in Arabidopsis and rice transport disaccharides, mainly sucrose [DlSWEET10c belongs to clade III, its transcript abundance is inconsistent with the sucrose content during fruit development. The cause of the discrepancy may be a rapid conversion of sucrose to glucose or fructose at the early fruit development stage.Sugar content is an important factor for determining fruits’ organoleptic quality . As is scharides . In addi sucrose ,12. AlthSWEET genes were involved in abiotic stress [DlSWEET1/10c) levels were significantly increased. Similar findings were observed in cabbage [DlSWEET1 showed a significantly up-regulated trend. The same result was also observed in tea, in which the expression level of CsSWEET1 increased markedly during cold acclimation [Cis-elements analysis showed that a TC-rich repeats element, which is involved in defense and stress responsiveness, existed in the promoter of DlSWEET1, indicating that DlSWEET1 may play an important role in the longan cold response. Moreover, the overexpression of DlSWEET1 conferred its tolerance to cold stress in transgenic Arabidopsis. However, due to the difference between Arabidopsis and longan, the function of DlSWEET1 in longan cold-response warrants further investigation.Longan is frequently challenged by abiotic stressors, for instance, extreme temperatures, drought, and high salinity. Sugars accumulate in plant cells to reduce the osmotic potential, which is conducive to the normal growth of plants under abiotic stress conditions ,55. Prevc stress ,26,49. T cabbage . Althouglimation . Cis-eleDimocarpus longan L.) cultivar “Qingkebaoyuan”. The trees were grown in the Fruit Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou, China. The fruit samples were randomly picked from three trees, under standard cultivation conditions, at 60, 90, and 120 days after flowering (DAF). Soluble sugars in longan pulp were extracted and measured according to the previously reported protocol [To investigate the function of DlSWEETs involved in longan fruit sugar accumulation, we used fruits of Longan  or PEG6000 (20%) for 4 h at 28 °C, respectively. Each treatment had three replicates of three plants. In addition, three seedlings grown at 28 °C were used as a control. After treatment, the leaf samples of the same leaf position were collected and immediately frozen in liquid nitrogen and stored at −80 °C for further analysis.Arabidopsis SWEETs proteins [−5) (http://gigadb.org/dataset/100276) (accessed on 26 March 2019) [Systematic BLAST homology searches using amino sequences of 17 proteins  were perch 2019) . A phyloch 2019) .http://meme.nbcr.net/meme/cgi-bin/meme.cgi) (accessed on 31 March 2019) was used to analyze the motifs of DlSWEET proteins, with the parameters set to the following: maximum number of motifs 600, motif width 15–60, and number 15 [The MEME (Multiple EM for Motif Elicitation) program (umber 15 .https://www.dna.affrc.go.jp/PLACE/?action=newplace) (accessed on 1 April 2019), and the distribution of four categories of cis-acting elements was visualized in the TBtools toolkit [The exon–intron organization of the DlSWEETs were analyzed and visualized in GSDS 2.0 (gene structure display server) . Further toolkit .DlSWEET genes expression patterns, we utilized the transcriptome data, which included different longan tissues (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE84467) (accessed on 31 March 2019) [To gain insights into the ch 2019) . The gench 2019) .−ΔΔCT method with three biological and three technical replicates [Total RNA was extracted using an RNAprep Pure Plant Kit  with three biological replicates. Then, approximately 1 µg of total RNA per sample was used as the template for reverse-transcription via TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix . The RT-qPCR was performed using SYBR Green I Master Mix  and a LightCycler 96 Real-Time PCR Systems . The relative expression levels were calculated with the formula 2plicates . All priDlSWEET1 was cloned into the pSAK277 vector. The 35S: DlSWEET1 plasmid was transferred into Agrobacterium strain GV3101 by the freeze–thaw method, and transferred into the Arabidopsis plants using the floral dip method. The Arabidopsis ecotype Columbia-0 (col-0) was used as the wild type (WT) in this study. Transformed plants were selected on the basis of their resistance to kanamycin, and 3-weeks-old T3 homozygous plants and WT seedlings were used for further experiments. Two transgenic lines and WT seedlings were transferred to 4 °C growth chambers with the same ambient conditions for 48 h and then were exposed to −4 °C for 12 h, followed by 12 h of darkness at 4 °C, after which they were returned to normal conditions for recovery for 6 days. The plant survival rates and phenotypes were recorded.The full-length cDNA sequence of n = 3). The means are compared using the Student’s t-test at the 0.05 and 0.01 levels of significance.All reported values are presented as mean ± standard error  were identified in the D. longan genome. A phylogenetic analysis showed that DlSWEETs were grouped into four different subfamilies. Tissue-specific expression patterns analysis showed that DlSWEET genes may play various roles in the development of longan tissues. qRT-PCR analyses implied that three and four DlSWEET genes might be involved in abiotic stress response and fruit sugar accumulation, respectively. Furthermore, the overexpression of the DlSWEET1 gene improved cold stress tolerance in transgenic Arabidopsis. This work will contribute to the follow-up study of the functional characteristics of DlSWEET genes and the cultivation of high-quality D. longan varieties. In summary, 20 longan SWEET genes (


Lithium-ion batteries (LIBs)are ubiquitousin all modern portableelectronic devices such as mobile phones and laptops as well as forpowering hybrid electric vehicles and other large-scale devices. Sodium-ionbatteries (NIBs), which possess a similar cell configuration and workingmechanism, have already been proven as ideal alternatives for large-scaleenergy storage systems. The advantages of NIBs are as follows. First,sodium resources are abundantly distributed in the earth’scrust. Second, high-performance NIB cathode materials can be fabricatedby using solely inexpensive and noncritical transition metals suchas manganese and iron, which further reduces the cost of the requiredraw materials. Recently, the unprecedented demand for lithium andother critical minerals has driven the cost of these primary raw materials(which are utilized in LIBs) to a historic high and thus triggeredthe commercialization of NIBs.xTMO2, TM = transitionmetal/s), such as Mn-basedsodium layered oxides, represent an important family of cathode materialswith the potential to reduce costs, increase energy density and cyclingstability, and improve the safety of NIBs for large-scale energy storage.However, these layered oxides face several key challenges, includingirreversible phase transformations during cycling, poor air stability,complex charge-compensation mechanisms, and relatively high cost ofthe full cell compared to LiFePO4-based LIBs. Our workhas focused on the techno-economic analysis, the degradation mechanismof NaxTMO2 upon cycling andair exposure, and the development of effective strategies to improvetheir electrochemical performances and air stability. Correlatingstructure–performance relationships and establishing generaldesign strategies of NaxTMO2 must be considered for the commercialization of NIBs.Sodium layered transition metaloxides intercalation of Na+ ions, followed by discussionsto improve the stability toward ambient air and upon cycling. Thenthe techno-economics are presented, with an emphasis on cathodes withdifferent chemical compositions, cost breakdown of battery packs,and Na deficiency, factors that are critical to the large-scale implementation.Finally, this Account concludes with an overview of the remainingchallenges and new opportunities concerning the practical applicationsof NaxTMO2, with an emphasison the cost, large-scale fabrication capability, and electrochemicalperformance.In thisAccount, we discuss the recent progress in the developmentof air-stable, electrochemically stable, and cost-effective Na ZuoW.; QiuJ.; LiuX.; RenF.; LiuH.; HeH.; LuoC.; LiJ.; OrtizG.F.; DuanH.; LiuJ.; WangM. S.; LiY.; FuR.; YangY.The stability of P2-layered sodium transition metal oxides in ambientatmospheres. Nat. Commun.2020, 11, 354432669558.1This article unveils the comprehensivestructural/chemical degradation mechanisms of P2-NaxTMO2in different ambient atmospheres by using various microscopic/spectroscopiccharacterizations and first-principles calculations. A practical evaluatingrule associated with redox couples has been proposed for designingair-stable NaxTMO2cathodes.LiuX.; ZuoW.; ZhengB.; XiangY.; ZhouK.; XiaoZ.; ShanP.; ShiJ.; LiQ.; ZhongG.; FuR.; YangY.0.67AlxMnx1–O2: cost-effective,stable and high-rate sodium electrodes by suppressing phase transitionsand enhancing Na+ mobilityP2-Na. Angew.Chem., Int. Ed.2019, 58, 18086–18095.2In this paper, we introduce Al into the transitionmetal layers to decrease the number of Mn3+ions and alleviate the phase transformations of Na0.67MnO2. The obtained pure P2-type Na0.67AlxMn1–xO2 materials show good structural stability and promising performance.ZuoW.; LiuX.; QiuJ.; ZhangD.; XiaoZ.; XieJ.; RenF.; WangJ.; LiY.; OrtizG. F.; WenW.; WuS.; WangM. S.; FuR.; YangY.+-layer spacings to stabilize Mn-based layeredcathodes for sodium-ion batteriesEngineering Na. Nat. Commun.2021, 12, 490334385435.3This paper reports a simpleand effective water-mediated strategy to modulate the spacing of Nalayers and alleviate undesirable phase transformations of layeredoxide cathodes for NIBs. The obtained shale-like material exhibitsoutstanding rate capability and cycling stability (>3000 cycles).VaalmaC.; BuchholzD.; WeilM.; PasseriniS.A cost and resourceanalysisof sodium-ion batteries. Nat. Rev. Mater.2018, 3, 18013.4This work uses the BatteryPerformance and Cost (BatPaC) model to undertake a cost analysis ofthe cathodes, anodes, current collector, and complete batteries forNIBs. The detrimental factors for the successful commercializationof NIBs are discussed.16The world’s leadingcountries have proposed carbon neutralityby the mid-21st century to relieve the severe effects of worldwideclimate change. Achieving this objective requires intensive researchefforts in sustainable energy conversion and storage devices. Currently,lithium-ion batteries (LIBs) dominate the energy storage market dueto their high energy density and long cycle life. However, the unevendistribution and low abundance of lithium and cobalt raise cost andsupply concerns, with the increasing market volume of hybrid electricvehicles (EVs) and stationary storage.10 NIBs and LIBs share the same architecture, similar working mechanismsand components, and identical cell fabrication steps, which impliesthat NIBs maintain the core of the roll-to-roll system optimized forLIB manufacturing during the last 30 years.Amongall the proposed alternative concepts, sodium-ion batteries(NIBs) have the great advantage of being essentially a “drop-in”technology.xTMO2), Prussian white, or vanadium phosphate as cathode materials.14To enable theirpractical implementation, advanced NIBs with higherenergy and power densities, better cycle life and safety, and lowercost are needed. These parameters are correlated to the electrodeactive materials’ chemical composition, their structure, andreaction mechanisms. After three decades of development, NIBs areat a critical moment of commercialization. Several companies suchas HiNa and CATL in China, Faradion in the United Kingdom, Tiamatin France, and NATRON ENERGY in the USA, are close to achieving thecommercialization of NIBs, with the aim of employing sodium layeredtransition metal oxides (Na2) arethe most successful cathode materials for commercial LIBs. Similarly,NaxTMO2 are of particular interestfor NIBs due to their high specific capacity, a variety of redox-activeelements, and the possibility for the manufacturers to employ establishedsynthesis processes.18 Other than NaxTMO2, alternativepromising sodium-ion cathode materials are Prussian blue/white,19 phosphates,20 andorganic materials.21 As already mentioned,materials belonging to these classes have already been selected forthe first generation of commercial sodium-ion batteries. Nevertheless,these types of sodium-ion cathodes have the demerits of intrinsicwater/potential toxicity, low specific capacity, and/or low capacityretention. However, the “best choice” for the positiveactive material is still under debate, not least as NaxTMO2-type materials face irreversiblephase transitions, electrolyte decomposition, and high reactivitytoward moisture. Therefore, a comprehensive evaluation of the chemicalcomposition, electrochemical performance, and cost of NaxTMO2 is fundamental to achieve optimalcathodes for commercial NIBs.Lithium layered transition metal oxides intercalation determines the competition betweenNIBs and LIBsinstead of the cost per unit weight , the contribution of various redox-active cations to the energydensity is further evaluated to optimize the chemical compositionsof NaxTMO2. A terminology ofspecific transferred electrons (STE) is defined here as the numberof transferred electrons divided by the atomic mass  tends to be adsorbed on the particles’ surface and/orreact with CO2 to form H2CO3(ad)(2O(ad) and H2CO3(ad) dissociateinto H+, OH–, HCO3–, and CO32–, react with Na+ in the surface layer, and produce NaOH, NaHCO3, and Na2CO3  chemical composition and (ii) structure of the material,(iii) temperature, and (iv) atmospheric conditions .25 Thechemical composition and structure determine the working potentialand formation energy of the materials, which further influence thefeasibility of Na loss in moisture and therefore the air stability.1Our research suggests that the air stability ofNaxTMO2 materialswith lower working potentials during the first charge process aremore vulnerable to Na loss and hydration  exhibits little structural change except for thebreath effect aroused by Mn valence change and shows excellent electrochemicalreversibility  force lead to phase transitions from P-type to O-type structures3b.3 The sibility 3h.338 the intralayer migration of TM ions leads to the formation of O2 and bulk holes. Second, at high working potentials, the surfaceoxygen ions of NaxTMO2 becomehighly active, react with the electrolyte, and generate O2 gas.Oxygen loss is a well-knowninstability issue that originates fromtwo mechanisms: First, like Li-rich oxides,292.46 and NaClO4 as Na salts are the mostwidely used liquid electrolytes for NIBs.40 Despite the acceptable electrochemical performance, the continuousgrowth of the cathode–electrolyte interphase (CEI) in carbonateelectrolytes upon cycling impoverishes the electrochemical propertiesof NaxTMO2, as our group demonstratedpreviously in different NaxTMO2 chemistries.43 For example, the inorganic-rich carbonate and alkylcarbonate CEI is highly soluble in the electrolyte due to their mildLewis acidity,41 which leads to batterydegradation.43Carbonate-based solventswith NaFP44 Our group tested the physicochemicalproperties of a variety of fluorinated-IL electrolytes. As shown in 45 the IL electrolytes sodium bis(fluorosulfonyl)imide (NaFSI)in N-methyl-N-propylpyrrolidiniumbis(fluorosulfonyl)imide (Pyr13FSI), denoted as F-13F,and NaFSI in N-butyl-N-methylpyrrolidiniumbis(fluorosulfonyl)imide (Pyr14FSI), denoted as F-14F,exhibit a very low tendency to crystallize at room/subambient temperatureand a high ionic conductivity above 10–3 S cm–1. Moreover, as shown in 45 F-13F exhibitssuperior anodic stability of 4.89 V. In contrast, the other studiedchemistries show inferior anodic stabilities of 4.72, 4.97, and 4.95V for F-14F, F-14T, and F-14FT, respectively. Among various ILs, theF-14F (10 mol % NaFSI in Pyr14FSI) electrolyte enabledoutstanding electrochemical performance in NaxTMO2 cathodes -based electrolytes have a wider electrochemical stabilitywindow, superior thermal stability, and negligible flammability andvolatility.cathodes 4d,45 sug3xTMO2 cathodes should lead to a much lower cost forNIBs.6 However, the previous cost analysismade by our group on both materials and complete batteries opposedthis assumption.4 Recently, the price ofthe main raw materials for LIBs has risen drastically ,47 P2-type Na0.66[Ni0.26Zn0.06Mn0.67]O2 (NZNMO),48 O3-type Na[Li0.10Ni0.35Mn0.55]O2 (NLNMO),35 O3-type Na0.9[Cu0.22Fe0.30Mn0.48]O2 (NCFMO),49 and O3-type Na[Fe0.40Ni0.30Mn0.30]O2 (NFNMO),50 compared to LIBs based on Li[Ni0.5Mn0.3Co0.2]O2 (NMC 532), Li[Ni0.8Co0.15Al0.05]O2 (NCA),LiMn2O4 (LMO), and LiFePO4 (LFP)cathodes coupled with graphite. The initial discharge curves of allcathodes are presented in As discussed in the above section,the most favorable TM elementsfor commercial Na3.1Table S1 show the mass/energy costs of the evaluatedcathodes. In 2020, the mass costs of the evaluated Na cathodes followthe order NLNMO (12.48 $ kg–1) > NZNMO > NFNMO>NCFMO > NAFMO (10.22 $ kg–1), which are comparableto those of LFP (12.46 $ kg–1) and LMO (11.16 $kg–1). Two years after, the cost difference betweenthese cathode materials increased substantially. The costs of Li cathodesincreased by at least 143%, while those of NAFMO and NCFMO only increasedby 16% and 14%, respectively. The mass cost increment of NLNMO is62%, which is the highest among the Na cathodes due to the presenceof Li. As a result, the mass costs of Li cathodes have become significantlyhigher than those of Na cathodes. Note that the content of Co of theconsidered Li cathodes is very low; otherwise, the difference in costwould be greater.–1 among Na cathodes, respectively,which are higher than those of LFP (24.21 $ kWh–1) and LMO (23.43 $ kWh–1). Two years later, theenergy costs of the five Na cathodes follow the order NLNMO (54.56$ kWh–1) > NZNMO (41.11 $ kWh–1) > NFNMO (40.42 $ kWh–1) > NCFMO (39.77$ kWh–1) > NAFMO (29.67 $ kWh–1), whichare lower than those of LFP (58.91 $ kWh–1) andLMO (59.94 $ kWh–1), demonstrating that the currentNaxTMO2 cathodes have an overwhelmingcost advantage over Li cathodes.Similar trends are observed in energy cost.In 2020, NAFMO and NLNMO showed the lowest and highest energy costsof 25.34 and 33.76 $ kWhTable S2), i.e., adomestic battery pack of 7 kW and 11.5 kWh, a plug-in hybrid EV (pHEV)pack of 110 kW and 15 kWh, a high-end EV pack of 150 kW and 100 kWh,and a grid-storage pack of 250 kW and 500 kWh. LFP-/LMO-based packsshow the lowest costs, followed by NMC-/NCA-based batteries. Withincreased pack energy, the energy cost of LIBs decreases due to economyof scale effects and the lower impact of the hardware costs . The energy costs ofall NIBs are higher than those of LIBs. NZNMO-/NFNMO-based batterypacks for grid storage show the lowest energy cost among the NIB packs,but it is still 33% higher than that of LFP-/LMO-based grid storagepacks. NAFMO has the lowest mass and energy cost among the consideredNa cathodes. However, the pack costs of NAFMO-based NIBs are muchhigher than those of NZNMO, NCFMO, and NFNMO because of a lower workingpotential and increased positive electrode porosity due to the presenceof the sacrificial salt. NLNMO has the highest cost among the Na cathodes,and NLNMO-based batteries show the highest pack costs, demonstratingthat high ratio of Li, such as 10%, should be avoided in cost-effectiveNa cathodes.3.2Tables S3 and S4).To clarify the cost gap between NIBs and LIBs, cost breakdowns ofgrid-storage packs are analyzed more in detail.The current raw material costs of Na cathodes are substantially lowerthan those of Li cathodes. However, the pack costs of NIBs are stillhigher than those of LIBs (As presentedin N-methyl-2-pyrrolidone (NMP) to water-soluble binders due to the costadvantages of water over NMP (70–200 times), water-solubleand F-free polymers over PVdF (2–5 times), and the lower energyneeded for the electrode drying process.52 The challenge of incorporating water-soluble binders lies in thehigh reactivity of NaxTMO2 towardmoisture.25 However, our group recently reported superiorcycling stability and (de)sodiation kinetics for P2-NaxTMO2 materials due to the removal of carbonatesurface species during the electrode processing.3 Furthermore, better surface coverage of the active materialparticles by the binder, suppressing electrolyte decomposition, andenhanced mechanical integrity of such electrodes could be achievedvia the interaction with the hydroxyl/carboxyl groups in such water-solublepolymers as well as the reduced swelling with the electrolyte.54With the higher amount of positive active material,the cost ofthe NIB packs might be significantly reduced by transitioning frompoly(vinylidene difluoride) (PVdF) dissolved in LFP-based LIBs and NZNMO-based NIBs are further selected tocomparethe cost proportions of pack components 6b. With xTMO2-basedNIBs have cost advantages for the positive active material and negativecurrent collector, which are outbalanced by the higher costs of otherbattery components.Insummary, Na3.3xTMO2 is in the range of 0.6 ≤ x ≤ 1.0,19 and after-synthesistreatments or air exposure can bring this initial sodium content toan even lower value.3 In commercial setups, the cathodeis the only Na source for the whole battery, and low Na content inNaxTMO2 might lead to severeNa deficiency in full cells. Moreover, the low initial Coulombic efficiency(ICE) of hard carbon further decreases the quantity of available sodium,making the issue of Na deficiency more complicated. In a work of ourgroup on bio-based hard carbons, the ICE of a peanut-shell-derivedhard carbon was around 70%, and the best commercial hard carbons achieveslightly more than 80%, while the graphite of LIB anodes can easilyreach 90%.56The stoichiometry of sodium in Na0.67Ni0.33Mn0.67O2 is a Na-sufficientcathode , anode , and battery (De) in QC,ic  and QA,ic  are the initial charge (discharge) capacitiesof the cathode and anode, respectively.Na cathodes can be classifiedinto Na-deficient 7a, Na-sucathode 7b,even De, De 57 is addedin the cathode, the pack cost increment is decreased by 3–5times compared to the case without sacrificial salt. As shown in De of 10%, 30%, and 50%, respectively.When De is below 30%,the cost increments are similar for different NIBs, while at higherNa-deficient states, the cost increment is correlated to the workingpotential and energy density of the cathode material. For example,at 50% De, the cost incrementsof NAFMO-, NZNMO-, NLNMO-, NCFMO-, and NFNMO-based NIBs are 15%, 10%,13%, 17%, and 16%, respectively.When a stoichiometric amountof sacrificial salt xTMO2, theirsupply risk and turbulent price substantially offset the cost advantageof NIBs. For example, the addition of 10 atom % Li in the active material,i.e., Na[Li0.10Ni0.35Mn0.55]O2, results in the highest cost among the simulated Na cathodes.This will be further increased when considering the battery scale.Expensive and scarce elements, inparticular Li and Co, should be carefully considered before utilization.Although Li and Co improve the structural stability and reversiblecapacity of Na(2)Developing high-voltageNa cathodesis essential for cost-effective NIBs. Mass cost, working potential,and specific/volumetric capacity might contribute equally to the costof cathode materials. However, working potential ranks as the highestto further improve the competitiveness of NIBs. First, for a givenrequired power density, a higher current density is necessary forthe batteries with lower operational voltage. Second, the higher averageworking potential of the hard carbon anode makes it harder to fullyutilize the capacity of cathodes in the low state-of-charge region.(3)Three primary directionsare highlightedto further optimize the cost of NIBs. The first is to pursue higher-energy-densitycathodes, which not only decreases the mass of active material butalso saves electrode-preparation compounds , current collectors, andelectrolyte. Another critical direction is to reduce the need andcost of anode, binder, and electrolyte, which could be achieved respectivelyby lowering the unit cost and volume of electrolyte, transitioningfrom PVdF to bioderived water-soluble binders, and adopting advancedhard carbons with higher energy density, improved ICE, and cost-effectiveprecursors. Third, adopting strategies to compensate for the Na deficiencyin full cells and increasing the starting sodium content of cathodeswill be important for the practical implementation of NIBs.In this Account,we have reviewed the properties, challenges, andcosts of Na


Caffeine was administered at 50 mg/kg p.o. The effects of HFSCD+CCl4 and caffeine on the liver were evaluated using biochemical, ultrastructural, histological, and molecular biological approaches. The HFSCD+CCl4-treated rats showed fat accumulation in the liver, elevated levels of inflammatory mediators, NLRP3 inflammasome activation, antioxidant dysregulation, and liver fibrosis. Caffeine reduced necrosis, cholestasis, oxidative stress, and fibrosis. Caffeine exhibited anti-inflammatory effects by attenuating NLRP3 inflammasome activation. Moreover, caffeine prevented increases in toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) protein levels and mitigated the phosphorylation of mitogen-activated protein kinase (MAPK). Importantly, caffeine prevented the activation of hepatic stellate cells. This study is the first to report that caffeine ameliorates NASH by inhibiting NLRP3 inflammasome activation through the suppression of the TLR4/MAPK/NF-κB signaling pathway.Caffeine elicits protective effects against liver diseases, such as NASH; however, its mechanism of action involving the pyrin domain-containing-3 (NLRP3) inflammasome signaling pathway remains to be elucidated. This study aimed to evaluate the effect of caffeine on the NLRP3 inflammasome signaling pathway in a rat model of NASH. NASH was induced by feeding rats a high-fat, -sucrose, and -cholesterol diet (HFSCD) for 15 weeks along with a weekly low dose  of CCl Non-alcoholic steatohepatitis (NASH) is a common liver disease that is primarily caused by the consumption of high-fat, cholesterol, and sugar diets and a sedentary lifestyle ,2. NASH Fat accumulation triggers reactive oxygen species (ROS) production in the liver and consequently induces necrosis and apoptosis . InjuredNF-κB promotes the synthesis of NLRP3, pro-IL-1β, and other proinflammatory cytokines. Cholesterol crystals, ROS, and fatty acids may activate inflammasomes, which function as damage-associated molecular patterns (DAMPs) ,10. ThesWhen the inflammasome is activated, NLRP3, the effector pro-caspase-1, and the adapter protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) form a complex that leads to the activation of caspase-1 and the maturation of IL-1β, a cytokine that contributes to the maintenance of inflammatory and fibrogenic responses ,9,10. In4 doses.Caffeine is a natural compound with beneficial effects on the liver ,13. Caff4 and HFSCD+CCl4+caffeine groups turned yellowish-white  results in plasma concentrations of 0.5 to 2.6 µg/mL of caffeine in humans ,21,22. MIt is known that the development of NAFLD and NASH is closely related to metabolic syndrome, which includes disorders such as high blood pressure, high blood sugar, excess body fat around the waist, abnormal blood lipid levels, and alteration in the gut micro-biome. In this sense, previous investigations have shown that no associations were found between caffeine concentrations with total cholesterol and low-density lipoprotein levels either in caffeine drug users or nonusers , while aOur results are consistent with previous findings that caffeine protects against liver damage ,32,33,344-treated rats. Notably, attenuation of oxidative stress may decrease inflammation and fibrosis induction [Oxidative stress plays an important role in the progression of liver damage during NASH pathology ,32. In tThe NLRP3 inflammasome signaling pathway plays a crucial role in liver diseases ,38. Evid4 treatment. On the other hand, TLR4, a receptor that stimulates several downstream pathways, including the MAPK and NF-κB pathways [4-treated rats. The MAPK signaling pathway is also involved in the activation of the NF-κB pathway and upregulation of IL-1β [4-induced JNK, ERK, and p38 phosphorylation. These results are consistent with those of previous studies using different experimental models and tissues [4 treatment. Taken together, our findings indicate that caffeine prevents NLRP3 inflammasome activation and inhibits IL-1β upregulation by interfering with TLR4 receptor-mediated NF-κB and MAPK activities, resulting in the suppression of inflammation and fibrosis during NASH progression. Our results provide new evidence for the therapeutic potential of caffeine in the treatment of chronic liver diseases, particularly NASH alongside fibrosis. The main limitation of this study is that caffeine was administered along with HFSCD and CCl4. Therefore, further studies need to be done to investigate the ability of caffeine to reverse experimental NASH. Although our investigation shows clear evidence of the role of caffeine in preventing NASH-associated alterations, additional preclinical and clinical studies are warranted before caffeine can be prescribed as a treatment for NASH.Previous studies have shown that DAMPs, such as ROS, trigger NF-κB and MAPK activation to upregulate IL-1β ,10,40. Apathways , acts aspathways . The preof IL-1β ,51. In t tissues ,53,54. F tissues , thereby4 at 400 mg/kg once a week to induce NASH. The rats were divided into four groups (n = 8 per group): control group, fed a standard diet ad libitum ; HFSCD+CCl4 group, subjected to HFSCD plus CCl4 treatment; HFSCD+CCl4+caffeine group, subjected to HFSCD+CCl4 treatment plus caffeine at 50 mg/kg body weight daily via gavage; and caffeine group, subjected to caffeine alone. All treatments were administered for 15 weeks. The rats were housed in polycarbonate cages under controlled conditions  and water ad libitum. At the end of the experiment, the animals were anesthetized with ketamine and xylazine and euthanized by exsanguination. Blood was collected by heart puncture and then centrifuged at 12,000× g to obtain serum and hepatic samples, which were stored at −75 °C. The study complies with the Cinvestav’s guidelines, the Mexican official regulation (NOM-062-ZOO-1999), and technical specifications for the production, care, and handling of laboratory animals and follows the Guide for the Care and Use of Laboratory Animals . The Ethics Committee of the Animal Lab Facility of Cinvestav approved the study (protocol number 281-19).Rats were obtained from the animal production and experimentation unit of the Center for Research and Advanced Studies of the National Polytechnic Institute . Thirty-two Wistar male rats weighing 100–120 g were fed a high-fat, -sucrose, and -cholesterol diet (HFSCD) ad libitum  and inje4 was obtained from J.T. Backer . Gloria® brand unsalted butter  and rennet casein powder 30 mesh size  were used in this study.Caffeine, cholesterol, sucrose, and cholate were purchased from Sigma-Aldrich . CCl®, St. Louis, MO, USA) as previously described [Alanine aminotransferase (ALT) , gamma-gescribed . Liver cescribed . Proteinescribed . The livescribed .®, Tokyo, Japan).Hepatic tissue was fixed in 4% paraformaldehyde for 48 h, dehydrated, embedded in paraffin, and then cut into 5-µm-thick sections. The sections were deparaffinized in xylene for 45 min, rehydrated in a graded alcohol series, and then prepared for Masson trichrome, red oil O (ORO), hematoxylin and eosin (H&E) staining, or IHC analysis. The sections were sealed with neutral resin, and images were captured under a light microscope  [® software  [Hepatic sections were dewaxed, hydrated with xylene and alcohol, immersed in PBS, autoclaved with 0.10 N citrate buffer, and then washed again with PBS. Endogenous peroxidase was blocked with methanol peroxidase, and the samples were washed with PBS. Then, 5% skim milk in PBS was used to block nonspecific binding. The sections were incubated overnight with primary antibodies  diluted , Japan) , and posMD, USA) .v/v) glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) and then washed with 0.1 M sodium cacodylate. The samples were post-fixed with 2% (w/v) osmium tetroxide in 0.2 M sodium cacodylate, washed with 0.1 M sodium cacodylate, dehydrated using increasing concentrations of ethanol, and then embedded in epoxy resin. Semi-thin sections were stained with toluidine blue. Thin sections were contrasted with uranyl acetate and lead citrate and examined under a JEOL JEM-1011 transmission electron microscope  [Fresh liver samples were fixed with 2.5%  .The tissue sections were subjected to double-labeling IF of NLRP3/caspase 1 and NLRP3/α-SMA as previously described  and then®, New York, NY, USA) and then measured through densitometric scanning using the ImageJ® software  [Protein analysis via western blotting was performed as previously described . The proMD, USA) .® software  [The activity of MMPs was assessed on gelatin-substrate gels as previously described (33). Digitalized images were used to quantify band intensity using the ImageJMD, USA) .® software  and one-way ANOVA were used to perform multiple comparisons and compare multiple groups, respectively. Tukey’s test was performed, and statistical significance was considered at p ≤ 0.05.Results are expressed as the mean ± standard error of the mean (SEM). The GraphPad by Dotmatics Prism4 doses. Thus, this study describes a novel mechanism by which caffeine exerts its hepatoprotective effects against NASH and supports previous suggestions that this alkaloid is effective in treating human NASH.Cumulative evidence indicates that caffeine exerts protective effects against liver disease. However, to the best of our knowledge, the mechanism by which caffeine, as an anti-inflammatory drug, targets the NLRP3 inflammasome in NASH remains to be elucidated. Results showed that caffeine significantly reduced the NLRP3 protein level, NLRP3 inflammasome activation, and TLR4/MAPK/NF-κB signaling pathways in a rat model of NASH induced by an atherogenic diet and low CCl


Chronic rhinosinusitis with nasal polyps (CRSwNP) is a complicated inflammatory disease, and the underlying mechanism remains unclear. While some reactive oxygen/nitrogen species-related gene products are reported to participate in CRSwNP, a systemic and full analysis of oxidative-stress-associated genes in CRSwNP has not been extensively studied. Therefore, this study sought to catalog the gene-expression patterns related to oxidative stress and antioxidant defense in control and CRSwNP patients. In total, 25 control and 25 CRSwNP patients were recruited. The distribution and expression of 4-hydroxynonenal and 3-nitrotyrosine as markers of oxidative stress—which is represented by lipid peroxidation and the protein nitration of tyrosine residues in CRSwNP nasal polyps (NPs)—were more apparently increased than those found in the control nasal mucosae, as determined by immunohistochemistry (IHC). The expression of 84 oxidative-stress-related genes in nasal mucosae and NP tissues was analyzed via real-time PCR, which showed that 19 genes and 4 genes were significantly up- and downregulated, respectively; among them, inducible nitric oxide synthase (iNOS) and heme oxygenase 1 (HO-1) were notably upregulated, whereas lactoperoxidase (LPO), myeloperoxidase (MPO), and superoxide dismutase 3 (SOD3) were highly downregulated. Changes in the mRNA and protein levels of these redox proteins were confirmed with a customized, real-time PCR array and RT-PCR analysis, as well as Western blotting and IHC assays. A receiver operating characteristic curve analysis further suggested that LPO, MPO, SOD3, HO-1, and iNOS are possible endotype predictors of CRSwNP development. Collectively, we present an oxidative-stress-related gene profile of CRSwNP NP tissues, providing evidence that the systemic changes in oxidative stress and the antioxidative defense system, including novel iNOS, heme peroxidases, and other genes, are closely linked to CRSwNP pathology, development, and progression. Chronic rhinosinusitis (CRS) affects people of all ages and is one of the most prevalent chronic illnesses in the United States and countries worldwide. The disease affects 12.5% of the population worldwide and more than about 10% of the Asian population . CRS canOxidative stress refers to the excessive production of reactive oxygen/nitrogen species (ROS/RNS) in cells and tissues, in which an antioxidant system cannot neutralize them . The acc2O2 generation in the airway epithelium, their expression closely correlates with 2O2 release and key inflammatoryH cytokine production in CRSsNP and -wNP patients [phox NADPH oxidase mRNA and protein, as well as 4-hydroxynonenal (4-HNE), are upregulated in NP tissue [It was reported that the levels of oxidative stress in nasal secretions, in the forms of total antioxidant status, malondialdehyde, and nitric oxide (NO), correlate with the severity of the nasal obstruction and congestion in CRS nasal polyposis in Turkish people . In addipatients . On the patients . More reP tissue . LipopolP tissue . InteresP tissue , and bacP tissue . While sIn this study, we hypothesized that oxidative stress may be involved in the development and progression of CRSwNP. Therefore, the main goals of this study were to understand how nasal sinus mucosae and polyp tissues imbalance oxidant/antioxidant defense in CRSwNP development and severity by using a systemic transcriptomic analysis approach. Our results showed that an apparent increase in oxidative stress, including lipid peroxidation and protein nitrotyrosination, occurred in CRSwNP NP tissues, as compared to the controls. Of the 84 oxidative stress genes tested, 23 were differentially and significantly expressed, including 19 overexpressed and 4 underexpressed. The results were confirmed by a customized real-time PCR array and RT-PCR analysis as well as Western blotting. Further analysis suggested that LPO, MPO, SOD3, HO-1, and iNOS are possible endotype predictors for CRSwNP development.The Abs raised against NOS2 (iNOS) was obtained from BD Biosciences . The heme oxygenase-1  and superoxide dismutase 3 (SOD3) Ab were from Abcam . The Ab for GAPDH was purchased from GeneTex, Inc. .This study was approved by the Ethics Committee of the Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan (Permission No: 20161210R) and conducted with the written informed consent of the patients. A total of 25 patients with CRSwNP and a control group of 25 patients with nasolacrimal duct obstructions were recruited. Briefly, CRSwNP was diagnosed based on criteria from EPOS 2020 [2 Reaction Ready First Strand Synthesis Kit (Qiagen) and was analyzed by using the human oxidative stress PCR array (Qiagen). The real-time PCR microarrays used an RT2 SYBR Green Fluor qPCR Mastermix (Qiagen) on a 7300 Real-Time PCR System . Data were normalized by using housekeeping genes , β2-microglobulin (B2M), ribosomal protein, large, p0 (RPLP0)) and analyzed by comparing the 2−ΔΔCt of the normalized sample.For balance in the sample availability, data acquisition, and statistical demand, seven randomly selected control and CRSwNP nasal tissue samples were used for real-time PCR microarray to catalog the full oxidative-stress-related gene expression. Total RNAs of human control nasal mucosae and NP tissues were isolated by using an RNeasy Mini kit . The cDNA was transcribed by using an RT−ΔΔCt of the normalized sample. In total, 18 additional human nasal tissue samples per group (control and CRSwNP group) were used for confirmation and validation.To confirm and validate the differentially expressed genes observed in the microarrays, a customized human oxidative stress PCR array (Qiagen) was performed. Briefly, the randomly selected genes from the results of the first-round analysis, including unchanged GPX2, PRDX4, and DUSP1 genes and significantly regulated genes such as LPO, MPO, SOD3, NOS2, GCLM, HMOX1, and AKR1C2, were confirmed and verified. The specific primers for detecting the above gene expression and two housekeeping genes (β-actin and RPLP0) were custom-coated on the plate, and a similar procedure was performed as described in the real-time PCR microarray section. Data were normalized by using multiple housekeeping genes and analyzed by comparing the 2The 4-hydroxynonenal (4-HNE) and 3-nitrotyrosine modifications and SOD1 expression in the control nasal mucosae and CRSwNP NPs were determined via immunohistochemistry (IHC), as previously described, with minor modifications . BrieflyChanges in the mRNA expression levels in some of the significantly and differentially expressed genes observed in the microarrays were examined by using RT-PCR for individual transcripts. The tissues were processed as homogenates, and the primers used are listed in Nasal tissue lysates were prepared as previously described . Total pt-test. Differences were considered to be statistically significant at p < 0.05. Logistic regression analysis and receiver operating characteristic (ROC) curves were performed to determine the ability of oxidative gene expression to distinguish the development of CRSwNP. The cutoff value was obtained based on the ROC curve analysis. The statistical analyses were performed using SPSS statistics software version 19.0 .All data are expressed as the mean ± SEM unless otherwise indicated. Differences between groups were compared with an unpaired Student’s Polyunsaturated fatty acids are susceptible to peroxidation, and they yield various degradation products, including main α, β-unsaturated hydroxyalkenal, and 4-hydroxy-2,3-trans-nonenal (4-HNE), in oxidative stress . In addip-value of >0.05. As detailed in To investigate the impact of the changes observed in the NPs of patients with CRSwNP on the transcriptional profile of genes involved in oxidative stress, we applied quantitative real-time PCR microarrays to catalog gene-expression levels in a total of 84 genes in the control nasal mucosae and the NPs of patients with CRSwNP. Genes were considered to be expressed differentially and significantly in the NPs of patients with CRSwNP if the expressions were greater than a two-fold change, with a p-value > 0.05 could be easily observed in a volcano plot , NCOA7 (2-fold increase), HMOX1  (1.633-fold increase), GPX3 (2.1-fold decrease), SOD3 (1.5-fold decrease), and MPO (1.5-fold decrease) in the CRSwNP NPs .p-values for six of the seven validated genes were smaller than 0.001. Based on the results shown in The significantly and differentially changed genes observed in the microarrays were further confirmed and validated using a customized real-time PCR array analysis on the 18 additional patients’ nasal tissue samples using probes targeting significantly and non-significantly changed genes. Three non-significantly changed genes, including GPX2, PRDX4, and DUSP1, were selected, whereas seven significantly changed genes, including LPO, MPO, SOD3, NOS2, GCLM, HMOX1, and AKR1C2, were chosen from We confirmed the mRNA levels in selected changed genes in the CRSwNP NPs, including iNOS, NCOA7, HMOX1, GPX3, SOD3, and MPO . To furtThe binary logistic regression analysis for the significant genes shown in the results of 2) is mandatory for energy production in human cells, it may also contribute to the production of oxidizing free radicals [As oxygen  synthase 2, also known as inducible NO synthase (iNOS), was the most significantly upregulated gene. NOS2 is well known for catalyzing L-arginine to produce nitric oxide (NO), which is a free radical that acts as a cellular signaling molecule in many different biochemical processes . In the 2O2 and, subsequently, H2O via catalase [2•- in extracellular space. However, an increase in cellular SOD1 may have an inverse effect on the dismutation of superoxide in intracellular cytoplasm. In this regard, it has been reported that SOD-1 and -2 decrease and that SOD-3 is unchanged in (non)eosinophilic CRSwNP. Moreover, SOD activity decreases [Members of the oxidative stress-responsive gene family, including eosinophil peroxidase (EPX/EPO), glutamate–cysteine ligase, modifier subunit (GCLM), ferritin, heavy polypeptide 1 (FTH1), heme oxygenase (decycling) 1 (HMOX1), PDZ and LIM domain 1 (PDLIM1), superoxide dismutase 1, soluble (SOD1), and NAD(P)H dehydrogenase (NQO1) were all upregulated in the real-time PCR array assay. NQO1, also known as NAD(P)H quinone oxidoreductase, was the most significantly upregulated gene identified by the microarray analysis. It is an antioxidant flavoprotein that reduces quinones to hydroquinones and indirectly prevents the one-electron reduction of quinone to the semiquinone free radical ,35. The catalase . Interesecreases . Since tecreases . Therefo2O2 from dual oxidases (DUOX) to produce antimicrobial substances. LPO uses H2O2 and thiocyanate (SCN−) substrate to generate antimicrobial hypothiocyanite (OSCN−) [In this study, one of the striking findings was the identification of a significant and marked reduction in LPO and MPO expression in CRSwNP. LPO, MPO, and EPX/EPO all belong to heme peroxidases, which contribute to immune protection on epithelial surfaces and in secretions . LPO is  (OSCN−) ,42. Ther (OSCN−)  also der (OSCN−) ,44. It w (OSCN−) . Thus, i (OSCN−) ,47. Thus (OSCN−) . Moreove (OSCN−) . Taken t2•− accumulation and a decrease in H2O2 in extracellular space. Moreover, an apparent decrease in LPO and MPO and a lack of H2O2 sources may lead to a reduction in OSCN− production, which may compromise the microbicidal ability of the nasal epithelium. Meanwhile, the reaction between O2•− and nitric oxide (NO•) may produce ONOO−, whose decomposition, in turn, gives rise to some highly oxidizing intermediates, including NO2•, OH•, and CO3•−, as well as, ultimately, stable NO3− [•), ozone (O3), nitrogen oxide (NO) and dioxide (NO2), and sulfur dioxide (SO2) [2•− and NO production. We also found that the increase in free radicals was accompanied by an increase in antioxidant systems such as NOQ1, preventing the one-electron reduction of quinone to the semiquinone free radical GCLM (known as gamma-glutamylcysteine synthetase) from participating in the first-rate limiting step of glutathione synthesis, as well as preventing GSTP1 from detoxifying reduced glutathione.Regarding the functional consequence of changed genes in CRSwNP, this results in the enhancement of oxidative stress, leading to the formation and production of protein tyrosine nitrosylation and 4-HNE in NPs , which cble NO3− ,50. The ble NO3− , which wde (SO2) . This maPrevious studies generally focused on the relationship between control and CRS nasal tissues with respect to selected oxidative-stress-related genes, such as iNOS, HO-1, SOD, PRDX, and others ,15,39,52Some researchers have suggested that antioxidants, such as glutathione, N-acetylcysteine, polyphenols, vitamin C, etc., could be used to treat upper airway inflammation ,55. The