Methylobacterium is a group of methylotrophic microbes associated with soil, fresh water, and particularly the phyllosphere, the aerial part of plants that has been well studied in terms of physiology but whose evolutionary history and taxonomy are unclear. Recent work has suggested that Methylobacterium is much more diverse than thought previously, questioning its status as an ecologically and phylogenetically coherent taxonomic genus. However, taxonomic and evolutionary studies of Methylobacterium have mostly been restricted to model species, often isolated from habitats other than the phyllosphere and have yet to utilize comprehensive phylogenomic methods to examine gene trees, gene content, or synteny. By analyzing 189 Methylobacterium genomes from a wide range of habitats, including the phyllosphere, we inferred a robust phylogenetic tree while explicitly accounting for the impact of horizontal gene transfer (HGT). We showed that Methylobacterium contains four evolutionarily distinct groups of bacteria , characterized by different genome size, GC content, gene content, and genome architecture, revealing the dynamic nature of Methylobacterium genomes. In addition to recovering 59 described species, we identified 45 candidate species, mostly phyllosphere-associated, stressing the significance of plants as a reservoir of Methylobacterium diversity. We inferred an ancient transition from a free-living lifestyle to association with plant roots in Methylobacteriaceae ancestor, followed by phyllosphere association of three of the major groups , whose early branching in Methylobacterium history has been heavily obscured by HGT. Together, our work lays the foundations for a thorough redefinition of Methylobacterium taxonomy, beginning with the abandonment of Methylorubrum. Methylobacterium is an important group of plant-associated bacteria and a model organism in microbiology. Ironically, Methylobacterium diversity and evolution have mostly been studied outside plants. Here, we present the first comprehensive reconstruction of Methylobacterium evolutionary history accounting for gene exchanges typical of Bacteria, and for diversity with known plant association. We demonstrate that Methylobacterium contains four evolutionarily divergent groups of bacteria, also distinguishable by their genome architecture and composition, questioning Methylobacterium taxonomy. We identified 104 Methylobacterium species, of which a large proportion is as of yet undescribed and mostly plant-associated. We also infer an ancient transition in Methylobacterium lifestyle from soil and plant roots, to plant leaves, stressing the significance of plants in Methylobacterium evolution and diversity.For billions of years, bacteria have evolved rapidly through vertical and horizontal gene transmission, mutation, selection, diversification, and extinction. These evolutionary processes allowed bacteria to conquer every biome and living host on Earth and, at the same time, resulted in blurring most traces of their ancient history . In the Methylobacterium is a well-studied group of bacteria that are abundant and widespread in every plant microbiome , a famicrovirga , Enteroverovirga  and Psycaciecola . Easy toaciecola , Methylomulation , 2007, hmulation , protectmulation , sparkinmulation .Methylobacterium, noticing a “greater degree of phenotypic and genotypic heterogeneity than would normally be expected for a single genus.” Accordingly, these authors proposed to split the genus in three distinct taxa corresponding to monophyletic groups in the 16S rRNA ribosomal gene phylogeny . Group A, containing the Methylobacterium type species M. organophilum, was retained as Methylobacterium. For group B, which included the model species M. extorquens, the authors proposed a new genus: Methylorubrum. Finally, the authors suggested that group C, including M. aquaticum and M. nodulans, should constitute a distinct genus, pending future genetic and phenotypic investigations. The Methylobacterium reclassification has been pointed out as problematic, because of the low phylogenetic resolution of the 16S rRNA gene, and because no genus name was proposed for strains that were not retained in Methylorubrum or Methylobacterium, which could potentially render either new genus as paraphyletic  was embedded within Methylobacterium (group A), forming a homogeneous group, and proposed to merge Methylobacterium and Methylorubrum back into a single genus.Recently, phyletic . Accordi species . Alessa Methylobacterium remains poorly resolved for several reasons. First, phylogenetic relationships among and within groups are often inconsistent depending upon the chosen marker gene , whose early branching in Methylobacterium history was heavily obscured by HGT. Finally, our comprehensive phylogenetic analysis of Methylobacterium lays the foundation for a profound redefinition of its taxonomy, beginning with the abandonment of Methylorubrum.Here, we explored Methylobacteriaceae genomes, including 189 Methylobacterium and 24 genomes from related genera as outgroups . Most Methylobacterium (n = 98) and all outgroup genomes (n = 24) came from distinct studies  for a total size (5–7Mb) and average GC content (67–70%) in the expected range for Methylobacterium genomes . We confirmed that the 384 previously identified genes were part of the Methylobacteriaceae core genome and retrieved each core gene nucleotide sequence for at least 26 out of these 29 genomes. Our final dataset consisted of 213 genomes for which we retrieved 327 to 384 core genes nucleotide sequences .We assembled a collection of 213  studies  online. equenced , hence cospheres , and seqospheres , hence e genomes  online. analysis  through analysis  online, ualities  online, re genes  online fl survey . Doing tMethylobacteriaceae from 213 genomes from the 384 core gene nucleotide sequences using three complementary approaches in order to assess the effect of ILS and HGT in the evolutionary history of Methylobacterium. First, we used RAxML to determine a maximum-likelihood (ML) tree  from concatenated alignments of the core gene nucleotide sequences, assuming 57 groups of genes (partitions) with different substitution models , but the same evolutionary tree for all genes, hence not accounting for ILS or HGT (a). Second, we used ASTRAL, a coalescent-based method combining ML trees determined for each core gene independently , accounting for ILS among genes . Group C always formed the most basal group of Methylobacterium, confirming previous observations (Methylorubrum (fig. 1a). Group A included clades A2, A3, A4 and A5 described by We reconstructed the lineage tree of S or HGT a. Secondfig. 1b;  online. rvations . Group Blorubrum  and B2 (lorubrum . Most stlorubrum  were disa). Group D was sister to groups A and B according to ASTRAL  and SVDquartets trees . We evaluated differences between the three lineage tree topologies using the Robinson-Foulds (RF) distance metric in PAUP .Groups A, B and D consistently formed a monophyletic group ; however, phylogenetic relationships among groups A, B and D were more challenging to assess. Groups A, B, and D could not be resolved with the RAxML tree , of which 59 included the type strain for at least one described species , as previously reported (M. populi and M. thiocyanatum type strains were assigned to the same species (PNS range: 98.97%-99.08%), as previously reported , as previously reported . Each group of genomes assigned to the same species was also monophyletic and strongly supported in lineage trees , as well as the proportion of strain isolated from anthropogenic environments (fig. 1d). Group A contained 62 genomes which fell into 41 species, including 17 new species (Methylobacterium sp. 018 to 034). Group B contained 41 genomes which fell into 21 species, including 7 candidate species (Methylobacterium sp. 035 to 041). Group C contained 25 genomes which fell into 19 species, including 4 new candidate species . Species from Microvirga and Enterovirga were mostly isolated from soil samples , often in association with plant roots . Species from Methylobacterium groups B and C were isolated from plants , soil samples (13 and 32%), sediments or water samples (18 and 21%), often in association with anthropogenic environments (29 and 49%). Species from groups A and D were mostly isolated from plants , especially the phyllosphere (51 and 67%). Of the 45 new candidate Methylobacterium species, most were assigned to groups A (n = 17) and D (n = 17); the majority (81%) was isolated from plants, and especially the phyllosphere .In summary, the ronments , fig. 1dere 66%; ; fig. 1dMethylobacterium groups have consistently contrasting genome characteristics (p < 0.001), with group D having smaller genomes , than groups B (5.58 ± 0.49 Mb), A (6.21 ± 0.59 Mb) and C (7.15 ± 0.66 Mb). Groups D and B had a smaller number of annotated genes  than groups A and C . The average number of gene annotation copies per genome was significantly different among groups (p < 0.001) and was smaller for group D (1.31 ± 0.04 copies per annotation) than for group B (1.37 ± 0.05), A (1.46 ± 0.07) and C (1.54 ± 0.07). GC content was significantly lower in groups D and B  than in group A  or group C . We compared the abundance of 10,187 gene annotations  across the four Methylobacterium groups and outgroups (Methylobacterium genomes clustered according to their gene content and abundance and matched the ASTRAL species tree (a). As observed for other genome characteristics, group D had the smaller pan genome size , group A  and group D . On the contrary, group D had a larger core genome size  than groups A (n = 845 ± 79), B (n = 924 ± 65) and C  and 350 ± 32 core genes (fig. 2c).The four main eristics . These futgroups . Methyloies tree a. As obsBC; Hellinger normalization of gene abundance; Methylobacterium species (BC range: 0.044 ± 0.017–0.080 ± 0.023) than among species within Methylobacterium groups (BC range: 0.159 ± 0.031–0.197 ± 0.044) or than among Methylobacterium groups . Groups A, B and D formed a monophyletic group , making group C the most basal group, as observed for lineage . The normalized RF value between the gene content tree and lineage trees  were not fully assembled , and we thus inferred the order or the 384 core genes along the chromosome of draft genomes by aligning their scaffolds to the chromosomes of 36 completely assembled Methylobacteriaceae genomes, while conserving the order of core genes within scaffolds. We compared the order of core genes among genomes using a synteny index (SI) calculated as the proportion of pairs of core genes (links) that were neighbors in two genomes, ranging from 0 (no link conserved) to 1  but was highly reshuffled among species within Methylobacterium groups . Average synteny among Methylobacterium groups A, B, C and D was low (SI range: 0.433 ± 0.025–0.528 ± 0.049) and in the same order of magnitude as synteny between Methylobacterium and Microvirga genomes  as the species having, on average, the highest core genome synteny with other Methylobacterium genomes. Accordingly, we used M. planium as a reference to visualize the conservation of the 384 links identified in its genome across Methylobacterium species . Groups A, B and D formed a monophyletic group , making group C the most basal group, as observed for lineage trees (d). The normalized RF value between the synteny tree and lineage trees ranged from 0.589 to 0.638. As a comparison, normalized RF values between the best synteny tree and its 1,001 replicate trees ranged from 0.235 to 0.390 (RF = 0.310 ± 0.026), indicating that the synteny tree had a significantly different topology than lineage trees. Interestingly, although M. planium and related species previously assigned to clade A2 , these species had on average higher synteny with species from group D (SI = 0.651 ± 0.045) than with other species from group A . Accordingly, we identified 29 links involving 54 core genes that were more often conserved between groups A and D than with other Methylobacterium groups. These links, however, were scattered along the M. planium chromosome   and group D .Recent work has suggested that s status . Here, whylogeny . On the lorubrum , formed planium) . Our anaelatives . Althoug group A , making acterium . AccordiMethylobacterium, as observed in other bacteria , the four groups could also be distinguished upon their gene content. GC content, genome size and gene content are widely accepted as criteria for taxonomic definition in prokaryotes , supports considering them as distinct genera.We observed that the newly defined monophyletic groups  were characterized by distinct genome sizes and GC content, two metrics that were highly correlated with each other in bacteria . With thkaryotes . We alsotaxonomy . The facMethylobacterium lineage tree, assuming the same evolutionary history for each core gene, significantly differed in its topology from lineages tree reconstructions accounting for ILS and/or HGT among core genes (ASTRAL and SVDquartets lineage trees), indicating that both processes were major drivers of Methylobacterium evolution. While the concatenated tree suggested that groups A and D formed a monophyletic group, coalescent-based estimations from ASTRAL (ILS + HGT among genes) and SVDquartets (ILS + HGT among sites) rather indicated the earlier divergence of group D from the A/B/D group.The evolution of bacteria is marked by recurrent HGT, gene duplication and loss events, making the reconstruction of bacterial phylogenies challenging. Given that each gene potentially has its own evolutionary history, marked by exchanges among divergent taxa, the evolutionary tree of most bacteria is quite reticulate . The recMethylobacterium groups, our phylogeny reconstructed from core genome architecture suggested the closer relationship between groups A and D, in agreement with the concatenated lineage tree, hence supporting the hypothesis of horizontal core gene exchanges having occurred between groups A and D after the A/B divergence. Accordingly, we observed syntenic islands (groups of neighbor core genes) shared between group D and some basal species of group A . These islands were scattered across the Methylobacterium chromosome, either suggesting that extensive chromosomal rearrangements occurred after HGT between A and D, or that HGT occurred multiple times during their evolutionary history, potentially among divergent lineages. According to a phylogeny reconstructed from gene occurrence in Methylobacterium, M. jeotgalii, M. planium, M. oxalidis and relatives, belonged to different lineages branching at the root of groups A, B and D, supporting the hypothesis of multiple and independent gene exchanges among distinct Methylobacterium lineages after the divergence of the three groups, blurring their phylogenetic relationships.A possible explanation of the divergent topology in the concatenated lineage tree is that shared polymorphism was retained by ILS and/or HGT between groups A and D after the A/B divergence. Interestingly, although supporting the four Methylobacterium is frequently associated with the phyllosphere, yet taxonomic and phylogenomic surveys of its diversity have mostly focused on human-impacted environments such as food factories, contaminated soils, air conditioning systems or even the International Space Station. Here we presented the first comprehensive genomic survey of Methylobacterium diversity in the phyllosphere. By including genomes of strains isolated from the phyllosphere of wheat  with the aerial part of plants. Similarly, we recently showed that groups A and D were the dominant Methylobacterium groups in the phyllosphere of trees from temperate forests . The anagenomes  indicaten plants , among on plants . For insm groups , suggestcrovirga , suggestMethylobacterium adaptation to the phyllosphere. For instance, strains from groups A and D were often identified in the same studies, sometimes isolated from the same plants, indicating that strains from these two groups likely share the same microhabitats on the surface of plant leaves, hence favoring gene exchanges among them and the maintenance of similar molecular pathways and functions. Further identifications of genes exchanges among these groups and the characterization of their functions will be critical to understand evolutionary mechanisms underlying the adaptive role and radiation of Methylobacterium in the phyllosphere.According to our phylogenomic analyses of group A/B/D, group D diverged first, and, like group A, was mostly isolated from the phyllosphere, suggesting that the A/B/D ancestor inhabited the surface of plant leaves. The fact that our analyses support horizontal gene exchanges between groups A, B, and D is also consistent with the hypothesis that these groups lived in the same habitat during their divergence. One can speculate that some horizontally transferred, yet to be discovered, genes may have had shared roles in Methylobacterium revealed the outstanding diversity within this taxon, and the role of HGT in its early evolutionary history. Future genomic and functional studies will be needed to characterize the evolutionary and functional features of Methylobacterium adaptation to the phyllosphere. Finally, our work lays the foundation for a thorough taxonomic redefinition of this genus.Our unprecedented phylogenomic analysis of Methylobacteriaceae genomes, including 189 Methylobacterium and 24 genomes from related genera as outgroups . Most Methylobacterium (n = 98) and all outgroup genomes (n = 24) came from distinct studies  of isolates from a large survey of the temperate forest phyllosphere  were individually annotated using RAST (https://rast.nmpdr.org/rast.cgi) (function in RAST output), excluding Hypothetical proteins, repeat regions and Mobile element proteins (ast.cgi)  with folast.cgi) . For eacproteins  online.Methylobacteriaceae core genome from 184 genomes, excluding 29 genomes that were not yet published nor annotated at the time of the analysis (nucleotide_sequence in RAST output). In order to correct for false gene duplication events that increased consistently with assembly incompleteness (Methylobacteriaceae genomes as references (defined as genomes with N50 > 3 × 106 Mb). For each candidate core gene, we calculated the average expected nucleotide sequence size observed among 36 complete genomes. Then, for each genome (n = 184) and each candidate core gene, we retrieved all nucleotide sequences (0–10 per gene and genome) and calculated their average size normalized (divided) by the average nucleotide sequence size observed in complete genomes. By doing so, we could distinguish between duplication caused by genome incompleteness (single copy genes divided between different scaffolds) and real duplication events . We confirmed that the 384 previously identified core genes were part of the Methylobacteriaceae core genome and retrieved each core gene nucleotide sequence for at least 26 out of 29 genomes. Our final dataset consisted of 213 genomes for which we retrieved 327 to 384 core genes nucleotide sequences  and converted them in sequence fasta files using R package seqinr. We then performed an alignment for each core gene using R packages seqinr and msa. For each gene, nucleotide sequences were translated (function getTrans) and alignments of amino-acid sequences were performed using ClustalW with default parameters in function msa. Sequences were converted back in nucleotides (stop codons excluded) and 5″ and 3″ end codons with more than 90% of missing data (gaps of “Ns”) were trimmed. We also constructed an alignment of concatenated core genes nucleotide sequence alignments. In the concatenated alignment, sequences of genes missing for at least one of the 213 genomes (0–6 genomes missing per gene) were replaced by strings of “Ns”.We performed an alignment for each core gene. For each genome (n = 184 + 29 = 213), we first extracted nucleotide sequences of the 384 core genes  to avoid tree reconstruction, and using the relaxed hierarchical clustering algorithm to reduce the computation burden  and node label , was imported in R as a vector. The gene tree in RAxML format: ((:L1[N1]):L2[N2]) was rewritten so that it could be readable in R (package ape [((:L1)N1:L2)N2. The tree was then reopened in R with function read.tree (package ape) and nodes with < 10% support were collapsed using function collapseUnsupportedEdges (package ips), to optimize accuracy in estimating the lineage and species tree (multiPhylo object), which was used to infer the lineage and the species tree in ASTRAL-III v5.7.7, with default parameters. In ASTRAL trees, branch lengths were measured in coalescent units and nodal support represented local posterior probability  and ASTies tree . All refbability .Methylobacteriaceae core genes by evaluating 2,000,000 random quartets for 100 bootstrap replicates. Phylogenies were estimated under the multispecies coalescent model accounting for ILS and assessing all sites independently to account for recombination within and among loci.Third, we used SVDquartets  as impleMicrovirga and Enterovirga.Lineage trees were displayed in Figtree v1.4.4 and rooted on Methylobacteriaceae genomes in species using PNS on the core genome , similar to average nucleotide identity . We infTRAL-III  and SVDqTRAL-III , as descWe tested for the severity of HGT and ILS in our dataset by measuring the differences in tree topologies estimated using different assumptions. To quantify differences between the topologies obtained under different assumptions, we calculated normalized RF distances , which eMethylobacteriaceae genome characteristics  of the coding sequence for each. We first calculated the number of gene annotations, their total nucleotide size (coding genome size), their GC content, the number of unique annotations , the number of mobile elements, and the average copy number of annotations (Methylobacterium groups (as defined by lineage trees) and outgroups  using a Tukey test.We analyzed otations . For eacMethylobacteriaceae species, per Methylobacterium group and outgroup, ordered according to the ASTRAL species tree (n) of each gene annotation across genomes assigned to the same species, rounded to 0 (n < 0.5), 1 (0.5 ≤ n < 1.5) or 2 copies (n≥2). For gene abundance per group, we calculated the average occurrence of each gene annotation across species assigned to the same group, using the same principle as for species.In a heatmap, we displayed the average abundance of 10,187 gene annotations  per ies tree  online. Methylobacterium group  and in pan genome (any gene present in at least one copy in at least one genome of a given group) over 100 replicates. As pan genome size estimations increased with the number of sampled species per group .We estimated pan genome and core genome sizes per er group  and by fer group  online aer group  online, er group b,c.Methylobacteriaceae based on gene content (d). First, we constructed a matrix of gene occurrence in each genome (0 for absence and 1 for presence) and converted it into a fasta file (one sequence per genome). We inferred an evolutionary tree of based on gene content using with RAxML v. 8.2.8  index of dissimilarity, initially developed in ecology for the comparison of communities based on their species abundance (decostand in R package vegan). We calculated pairwise BC indexes of dissimilarity among normalized gene abundances, using function vegdist in R package vegan.In order to compare bundance  online. bundance . To miniMethylobacteriaceae genomes. All analyses were performed in R (n = 36), each core gene was paired with its two immediate neighbors, based on shortest distance between gene start and stop coordinates, and core genes located on scaffold edges were also paired together, assuming genome circularity. Hence, for each complete genome, each of the 384 core genes was involved in two links (pairs of neighbor core genes), for a total of N = 384 links per genome. For the 177 draft genomes, core genes were located on different scaffolds, so we predicted scaffold order and orientation using complete genomes as references. The draft genome with the highest completeness was reorganized first. Briefly, for each comparison with a reference genome, and for each scaffold of the draft genome, the list of embedded core genes was retrieved, and a score based on gene average order in the reference genome was calculated. Scaffolds of the draft genome were reordered according to these scores (one per scaffold). Then, each scaffold was eventually reoriented (without affecting gene order within scaffolds) to optimize pairing of edge genes (genes located at the edge of a scaffold) as compared to the reference genome. We repeated the operation for comparisons with the 36 reference genomes. Finally, for each of the 36 new configurations of the draft genome, we calculated a synteny conservation index (SI) with each reference genome, as the proportion of links conserved between two genomes. SI ranged from 0 (no link conserved) to 1 (fully conserved synteny). The draft genome configuration with the highest SI value was conserved for further analyses and added to the list of reference genomes. We repeated this operation for each draft genome, ranked according to their decreasing completeness, hence increasing the number of reference genomes and possible configurations for highly fragmented genomes. Finally, we calculated SI for all possible pairwise comparisons between genomes  between med in R . For eac genomes  online, d groups .Methylobacteriaceae chromosome, we used two approaches. First, we realized a heatmap of link conservation per species, along a reference genome . In this network, we represented the 384 core genes as nodes, ordered according to M. planium YIM132548 core genome, and links among neighbor core genes as edges. The network was drawn using 389 links observed in a majority (>50%) of species from a given Methylobacterium group .In order to visualize the spatial organization of core genes along the e genome  online.  v.3.4.0   and converted it into a fasta file. We inferred an ML evolutionary tree of Methylobacteriaceae based on synteny using with RAxML v. 8.2.8 (b).In order to reconstruct the evolution of v. 8.2.8  with a Bv. 8.2.8 b.evac123_Supplementary_DataClick here for additional data file.


Adiposity, diabetes, and lifestyle factors are linked to gastroesophageal reflux disease (GERD) in observational studies. We conducted a two-sample Mendelian randomization analysis to determine whether those associations are causal. Independent genetic variants associated with body mass index (BMI), waist circumference (with and without adjustment for BMI), type 2 diabetes, smoking, and alcohol, coffee and caffeine consumption at the genome-wide significance level were selected as instrumental variables. Summary-level data for GERD were available from a genome-wide association meta-analysis of 71,522 GERD cases and 261,079 controls of European descent from the UK Biobank and QSkin Sun and Health studies. The odds ratio (OR) of GERD was 1.49 , 1.40–1.60) for one standard deviation (SD) increase in BMI, 1.07  for one-unit increase in log-transformed OR of type 2 diabetes, and 1.41  for one SD increase in prevalence of smoking initiation. There were suggestive associations with GERD for higher genetically predicted waist circumference  and caffeine consumption . Genetically predicted waist circumference adjusted for BMI, alcohol or coffee consumption was not associated GERD. This study suggests causal roles of adiposity, diabetes, and smoking, and a possible role of high caffeine consumption in the development of GERD. Gastroesophageal reflux disease (GERD) is a common gastrointestinal disorder, affecting approximately 13% of the worldwide population and 20% of the adult population in the western countries , diabeteMendelian randomization (MR) design uses genetic variants as instrumental variables for an exposure and can strengthen the causal inference . This deFigure p < 5 × 10–8) were obtained from corresponding GWASs  were used as instrumental variables.Genetic variants  associated with BMI , waist cSummary-level data on the associations of exposure-related SNPs with GERD were obtained from a genome-wide association meta-analysis of the UK Biobank study and QSkin Sun and Health Study including a total of 71 522 GERD cases and 261 079 controls of European descent . GERD cap for intercept < 0.05); however, the model is usually underpowered [p value < 0.007 (0.05/7 exposures) were deemed significant associations, and associations with a p value ≥ 0.007 and ≤ 0.05 were regarded as suggestive associations. All tests were two-sided and performed using the TwoSampleMR [The inverse variance weighted method was used as the main statistical method (the random-effects model for the exposure constructed by ≥ 3 SNPs and the fixed-effect model for the exposure constructed by < 3 SNPs). Several sensitivity analyses, including the weighted median , MR-Eggerpowered . MR-PRESrpowered . The embrpowered . The conrpowered . To asserpowered . AssociaSampleMR , MR-PRESSampleMR  and MendSampleMR  packagesp = 4.09 × 10–33) for one standard deviation (SD) increase in BMI, 1.07  for one-unit increase in log-transformed OR of type 2 diabetes (not diabetes diagnosis), and 1.41  for one SD increase in prevalence of smoking initiation. The association for type 2 diabetes slightly attenuated but remained significant in the multivariable MR analysis with adjustment for genetically predicted BMI . There were suggestive associations for genetically predicted waist circumference  and caffeine consumption    and smoking initiation was associated with an increased risk of GERD  (Table p for distortion test > 0.05)  Table . One to 5) Table .Table 2AThis MR study found that higher genetically predicted BMI and genetic liability to type 2 diabetes and smoking were associated with increased GERD risk. There were suggestive associations of genetically predicted higher waist circumference and caffeine consumption with an increased risk of GERD. The association for waist circumference attenuated after adjustment for genetically predicted BMI. Limited data was observed in support of an association of genetically predicted alcohol and coffee consumption with GERD.Review articles of the association between obesity and GERD have found consistent evidence that overweight and obesity were associated with an ascended risk of GERD , 4, 17. Gastrointestinal symptoms are frequently encountered in patients with type 2 diabetes. The association between type 2 diabetes and GERD was assessed in a meta-analysis including nine observational studies and the OR of GERD was 1.61 for diabetic individuals compared to non-diabetic controls . Our MR Evidence in support of an association between smoking and GERD was generally consistent , 7, 9–12Consumptions of alcohol and coffee, two major beverages, have been associated with GERD risk with inconsistent or weak evidence for alcohol , 13, 15 rg < 0.97) [Limitations need considerations when interpreting our results. The important limitation is the possible effect of horizontal pleiotropy. In this study, we observed significant pleiotropic effects in the analyses of BMI and type 2 diabetes, but not for other exposures. However, the associations for BMI and diabetes were consistent in sensitivity analyses. Anti-diabetes medications may be a pleiotropic source in the analysis of type 2 diabetes . From th < 0.97) , which iIn summary, this MR study suggests causal roles of obesity, diabetes, and smoking in the development of GERD. The possible association between high caffeine consumption and an increased risk of GERD warrants confirmations.


To evaluate the relationship between preoperative primary tumor metabolism and body composition in patients with NSCLC and analyze their effects on DFS.  A retrospective study was conducted on 154 patients with NSCLC. All patients were scanned by baseline 18F-FDG PET/CT. SUVmax  of primary tumor, liver SUVmean , and spleen SUVmean were measured by AW workstation. The skeletal muscle area (SMA), skeletal muscle mass index (SMI), skeletal muscle radiation density (SMD), visceral fat area (VFA), visceral adipose tissue index (VATI), and skeletal muscle visceral fat ratio (SVR) were measured by ImageJ software. Kaplan–Meier survival analysis was used to evaluate the impact of the above parameters on DFS.  Compared with the low SUVmax group of primary tumors, the mean values of SMA, VFA, and VATI in the high SUVmax group were significantly higher. In addition, there were obvious differences in histopathological type, pathological differentiation, AJCC stage, and T stage between the two groups. Univariate analysis of DFS showed that VFA, VATI, pathological differentiation, tumor SUVmax, AJCC stage, tumor T stage, and N stage all affected the DFS of patients except for the parameters reflecting skeletal muscle content. Multivariate regression analysis showed that only VFA and SUVmax were associated with DFS. Kaplan–Meier survival analysis showed that high SUVmax, low VFA, high T stage, and high N stage were related to the decrease of DFS. 18F-FDG PET/CT could comprehensively evaluate the primary tumor SUVmax, skeletal muscle, and visceral fat in patients with NSCLC. The combination of primary tumor SUVmax and visceral fat area can well evaluate the prognosis of patients with NSCLC.：Preoperative  Lung cancer is the leading cause of cancer deaths worldwide, accounting for 1/4 of all cancer deaths . The bod18FFDG PET/CT) scan was performed in our facility 45 days before operation to identify lung lesions. Exclusion criteria were as follows: (a) nonlung adenocarcinoma or squamous cell carcinoma; (B) associated with other cancer types or previous cancers; (C) distant metastases; (D) no PET/CT whole-body scan; (E) patients with tumors larger than 7 cm; (F) patients with other diseases, such as cardiovascular disease, diabetes, or liver or spleen disease; and (F) incomplete follow-up information. A total of 154 patients were selected for the study according to the above inclusion and exclusion criteria.In this retrospective single-center study, patients were selected from the institutional database using the following inclusion/exclusion criteria. Inclusion criteria were as follows: (a) older than 18 years old, less than 90 years old; (B) patients who underwent surgical resection of cancer lesions in lung between 2008 and 2015; (c) fluoro-18 fluorodeoxyglucose positron emission tomography/computed tomography , which is referred to the date of tumor recurrence, tumor-related death, or the date of the last visit (after examination). The follow-up interval of patients was 6 months in the first three years, and then once a year. All patients were followed up for at least 5 years, recurrence or death within 5 years was defined as the occurrence of an event, and the last follow-up was in 2021. This study was approved by the Institutional Ethics Committee.18F-FDG PET/CT image acquisition was carried out according to version 1.0 of the European Association of Nuclear Medicine (EANM) guidelines by an integrated PET/CT scanner . Images were obtained 60 ± 5 minutes after the intravenous injection of 18F-FDG at a dose of 370 MBq/kg. Firstly, a low-dose CT scan without contrast enhancement  was performed for a precise anatomical localization and attenuation correction. Next, a three-dimensional PET scan (thickness of 3.27 mm) was performed from the skull base to the proximal thighs with an acquisition time of 3 min per bed position. The PET data sets were iteratively reconstructed using an ordered-subset expectation maximization (OSEM) algorithm with attenuation correction. All collected images were displayed on the GE Healthcare Xeleris 3.0 to reconstruct the fusion images.Two senior nuclear medicine doctors who did not know the clinical information of the patients evaluated PET/CT images, respectively, both of them had more than 5 years of experiences in PET/CT diagnosis. The size of the tumor was determined on the maximum cross-sectional plane, which was depicted around the tumor outline on CT and PET images. The ROI was carefully placed on different cross sections of the primary tumor to measure the maximum standardized uptake of the primary tumor (SUVmax). Some studies have shown that tumors affect the metabolism of distal organs, so we also included the metabolism of the liver and spleen in this study , 10. We https://imagej.nih.gov/ij/) [2) used a standard threshold of +29 ∼ +150 Hounsfield units (HU) to measure. Skeletal muscle mass index  obtained by dividing the area of skeletal muscle by the square of height. Radiation density of skeletal muscle  draws the ROI in the relevant skeletal muscle area for measurement. Visceral fat area  used a standard threshold of −150∼−50 HU to measure. Visceral adipose tissue index  divided the visceral fat area by the square of height. Using SMA divided by VFA to obtain skeletal muscle and visceral fat ratio (SVR). Based on the previously established gender-specific threshold from healthy adults, low SMI or myopenia is defined as SMI <44.4 cm2/m2 in male and <34.8 cm2/m2 in female, and low SMD or myosteatosis is defined as SMD <34.1 HU in male and <27.2 HU in female. Sarcopenia is defined as the presence of myopenia or myoosteogenesis according to the operational definition of sarcopenia recommended by EWGSOP2 [2) and L-VFA (<100 cm2) [The relevant body components were measured in the baseline CT images of the third vertebral body level (L3) obtained from patients. Each selected CT image is evaluated by two reviewers who do not know the end point. A free program was used in this study, which has been previously verified to provide reliable measurement results  , 13. Ske EWGSOP2 . Accordit test or Mann–Whitney U test was used to estimate the differences between groups according to the normal distribution. Chi-squared test or Fisher's exact test was used to estimate the differences between groups for categorical data. Cumulative survival was plotted using the Kaplan–Meier method, and differences were compared using the log-rank test. Cox proportional-hazards model was used for univariate and multivariate analyses of potential risk factors. Hazard ratios (HRs) together with 95% confidence intervals (CI) were provided. All statistical analyses were performed with the R software, version 4.12 (http://www.r-project.org).Student's The baseline characteristics of the patients are shown in We divided the patients into groups according to the level of SUVmax. Patients with higher SUVmax had higher BMI index, skeletal muscle area, and visceral fat area, as well as less differentiated tumors and a higher proportion of squamous cell carcinoma, while their tumors are larger and their AJCC stages are higher .P < 0.001), visceral fat index , pathological differentiation , and SUVmax ，AJCC stage , T stage , and lymph node staging  can affect the disease-free survival of patients. Multivariate regression analysis showed that only visceral fat area  and SUVmax  were associated with disease-free survival (We evaluated the effect of various indicators on postoperative disease-free survival of patients，Univariate analysis showed that visceral fat area (HR: 0.99, 95%CI, 0.98–0.99, survival .Kaplan–Meier survival analysis showed that high SUVmax, low VFA, high T stage, and high N stage were related to the decrease of disease-free survival . It was Body composition and tumor glucose metabolism are known as prognostic markers for cancer patients. Our study shows the prognostic value of preoperative PET/CT analysis of body composition and tumor glucose metabolism for disease-free survival in patients with non-small-cell lung cancer. In our study, tumor SUVmax, visceral fat area, visceral fat index, tumor differentiation, and various clinical stages were all risk factors for disease-free survival. Combining visceral fat area and tumor SUVmax can better determine the risk of disease-free survival compared with AJCC stage, while there is no relationship between the indexes of muscle and disease-free survival.Glucose metabolism is very important for tumor growth and development. 18F-FDG PET/CT is often used for preoperative evaluation and grading of patients with lung cancer , SUVmax 2 and those with BMI ≤ 18 < 25 kg/m2 [Every year, nearly 500000 of new cancers are caused by obesity. Avoiding obesity can reduce the incidence of cancer. This conclusion has been strongly supported by data in a variety of tumors . However25 kg/m2 . In fact25 kg/m2 –26. One 25 kg/m2 , 27. In 25 kg/m2 , 29. In 25 kg/m2 ; however25 kg/m2 , 32. A r25 kg/m2 , and stu25 kg/m2 , 35, so 25 kg/m2 , 37. The25 kg/m2 , which iPET/CT is recommended to determine the stage of disease in patients with lung cancer before treatment and abdominal and L3 levels are generally selected for the evaluation of fat and muscle, so the results of a whole-body PET/CT scan can make a comprehensive analysis of the patient's body composition and body metabolism without the need for additional examination. Our results also show that the combination of tumor metabolism and visceral fat area can better evaluate the prognosis of patients. We do not distinguish brown adipose tissue in the quantitative evaluation of fat, which may be helpful to study the effect of fat on patients' survival according to some studies, showing that brown adipose tissue plays an important role in tumors , 39.Our study still has some limitations. First of all, our study is a single-center retrospective study with limited sample size, and further multicenter studies are needed to verify our conclusions. In addition, we only studied the effects of body composition combined with tumor glucose metabolism on disease-free survival, and it may be more meaningful when combined with the overall survival.Our study shows that preoperative PET/CT could comprehensively evaluate the metabolic level and body composition of patients with non-small-cell lung cancer, and the combination of tumor glucose metabolism and visceral fat area can better predict the prognosis of patients.


Despite the success, it is highly challenging to battle against pathogenic biofilms-based chronic bacterial infections by conventional antibiotic therapy. Herein, we report a near-infrared (NIR)/acid-induced nanoplatform based on chitosan (CS)-coated indocyanine green /luteolin  nanocomposites (ICG/LUT-CS) as antibacterial and antibiofilm agents for skin wound healing. Initially, the ICG/LUT nanoplatforms are prepared by the supercritical antisolvent technology and coated with the CS layer. The obtained ICG/LUT-CS with ultra-high encapsulation efficiency exhibited more favorable photothermal conversion effects and improved NIR laser/acid dual-induced drug release behavior than individual modalities, achieving exceptional bacteria-killing and biofilm elimination effects. Moreover, the ICG/LUT-CS realized the synergetic effects of chemotherapy and photothermal therapy outcomes for wound healing. Together, our findings provided an appealing strategy for the rapid preparation and future translational application of ICG/LUT-CS as an ideal agent for fighting against biofilm infections.  Pathogenic infections turned into chronic wounds have become critical public health issues associated with high morbidity and mortality . DespiteRecently, several advancements have evidenced novel antimicrobial and antibiofilm therapies, such as photothermal therapy (PTT) , 9, photSeveral therapeutic approaches, such as traditional antibiotic treatment, PDT and chemodynamic therapy with efficient sterilization at a relatively lower temperature, have been integrated as an ideal strategy for achieving synergistic effects . Previou2) is the most frequently used solvent due to excellent characteristic features, including non-toxic, chemically inert, non-combustible, economical and recyclable [2 has been proven to be eco-friendly particle production technology due to moderate critical pressure and temperature, which would be suitable for industrialization [via the supercritical antisolvent (SAS) approach and solution casting method. The innovation lies in the simple, fast and low-cost fabrication of nanocomposites toward addressing the challenges of synthesizing or modifying nanodrugs with high loading efficiency and integrating multiple functions in an “all-in-one” platform [in vivo and in vitro and diminishing the side effects.Although the quorum sensing inhibitor agent has evolved as a promising weapon against infections, LUT suffers from inadequate bactericidal effect and poor solubility . To addrcyclable , 34. Indlization , 36. By platform . A mecha2 was supplied by Xiamen Rihong Co., Ltd. . Mouse embryonic fibroblast (NIH/3T3) cells were obtained from the Type Culture Collection of the Chinese Academy of Sciences . A live/dead bacterial viability kit was purchased from Thermo Fisher Scientific . Staphylococcus aureus (ATCC 29213) was obtained from the China Center of Industrial Culture Collection .ICG (purity of >95%), lysogeny broth (LB), and agar were obtained from Sangon Biotech. Co., Ltd. . LUT was purchased from Dalian Meilun Biotechnology Co. Ltd. . CS , sodium chloride (NaCl) and ortho-nitrophenyl-beta-galactoside (ONPG) were purchased from Aladdin Co. Ltd. . Ethanol (EtOH), acetic acid, dichloromethane (DCM), dimethyl sulfoxide (DMSO) and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sinopharm United Medical Device Co., Ltd . CO2 flow rate = 40 g/min and solvent flow rate = 1 ml/min. After attaining the stable predetermined conditions, the mixture solution was inducted into the SAS apparatus  for particle formation. ICG/LUT constructs were dissolved in water and methanol solution, and the amount of ICG and LUT was analyzed by ultraviolet-visible-NIR (UV-vis-NIR) spectrophotometer at 780 and 361 nm and calculated according to the standard curve.Briefly, the SAS process was initially applied to prepare ICG/LUT, as reported previously . ICG and2O. Afterward, ICG/LUT-CS constructs were immersed in the acetic acid solution for degrading the CS layer. In addition, water and methanol as solvents were used to extract the drug and measure the absorbance. The encapsulation efficiency (EE) of each drug was calculated as:To immobilize CS molecules on the surface of ICG/LUT, 0.1 g of CS powder was dissolved in 100 ml of 2% acetic acid solution. The suspension of ICG/LUT (4 mg) in NaCl solution  was slowly added to the CS solution (5 ml) and stirred for 4 h. The ICG/LUT-CS were purified by centrifugation and washed twice with 1% acetic acid and thrice with dd-Hn = 200) in SEM images were measured using the Nano Measurer (v1.2) software. The surface potential of samples was measured by a zeta potential analyzer . UV-vis-NIR spectra of different samples were recorded on a spectrophotometer over a wavelength range of 300–1100 nm . Fourier transform infrared (FT-IR) spectra of samples were chronicled by attenuated total internal reflectance FT-IR  with a wave number range from 4000 to 400 cm−1. Powder X-ray diffractometer (PXRD) recordings were performed by Smar/SmartLa  to evaluate the crystalline change after SAS process.The surface morphologies of samples were measured by field emission-scanning electron microscopy at 5.0 kV . To further calculate the average size and particle size distribution, the diameters of randomly selected nanoparticles  was irradiated by an NIR laser for 5 min. Meanwhile, the temperature increase behaviors of samples were recorded each 15 s interval by an NIR thermal imaging instrument .2 was irradiated. The mixture was kept be a shaker at around 150 rpm at 37°C for 48 h. One milliliter of supernatant was collected by centrifugation at the predetermined time and then supplied with 1 ml of the fresh medium. The concentrations of LUT were quantified at 351 nm against the corresponding standard curves of the drugs to determine the released amount of the drug.ICG/LUT-CS nanocomposites were initially dispersed in the PBS solution with different pH values (5.5 and 7.4). Furthermore, an NIR laser with 1.5 W/cmStaphylococcus aureus strain, a typical bacteria, was used to investigate the synergistic PTT–chemotherapy antibacterial and antibiofilm activity. The bacteria were inoculated on LB and cultured in a 37°C shaker at 150 rpm for 12 h. Furthermore, part of the colony was suspended in the fresh broth and incubation for 8 h. To analyze the antibacterial efficacy of photoexcited ICG/LUT-CS, S.aureus was adjusted to 107 CFU/ml with LB. Then, ICG/LUT and ICG/LUT-CS at the same concentration  were mixed with the bacterial solution. After irradiation for 5 min using an NIR light source with a power density of 1.5 W/cm2, the bacterial solution was incubated at 37°C for another 6 h. Subsequently, the treated bacterial solution was diluted by PBS, transferred and sprayed onto the LB nutrient agar plates. Visible colonies were then enumerated and photographed after incubation for 18 h at 37°C. The bacteria culture with LB treatment was used as a control. The colonies on the plate were counted using the Image J software.600 = 1) to obtain final concentrations of ICG  into a 96-well microtiter plate and irradiation for 5 min using NIR. The biofilm inhibition efficacy of different treatments was evaluated at the corresponding time intervals by the crystal violet (CV) method. In brief, the residual biofilm on the plate surface was obtained after washing twice with PBS, fixed with 95% EtOH, stained with 0.1% (w/v) CV solution for 10 min, washed and dried. The CV was dissolved using EtOH, and OD550 values were recorded with a microplate reader .To investigate the biofilm inhibition activity of ICG/LUT-CS in comparison with ICG/LUT, the suspensions of nanoparticles were mixed with the bacterial suspension  suspension was incubated for 48 h at 96-well plates. Then, the adherent bacterial biofilms grown on the surface were treated with different groups for 4 h. Afterward, the biofilms were irradiated by an NIR laser and then incubated at 37°C. The corresponding antibiofilm efficiencies of different groups and kinetic processes of antibiofilm were also evaluated by the CV staining method.To access the antibiofilm eradication activity, Staphylococcus aureus biofilms were cultured on the cover glass, placed in a 24-well microtiter plate and treated with different groups, including bacteria treated with PBS as control. After treatment with NIR laser, residual biofilms were washed with PBS twice and stained with a live/dead bacterial viability kit for 20 min. Furthermore, the fluorescent images of bacteria in biofilms were captured using fluorescence microscopy , and ablation effects were also observed using FE-SEM. In brief, the residual biofilms were treated as described above, washed with PBS, fixed with 2.5% glutaraldehyde at 4°C overnight, then dried with different ethanol grades , respectively. Finally, the morphological transformation of S.aureus in biofilms was characterized according to the standard specification.S.aureus strain in the biofilms was mixed with ONPG solution and incubated for 24 h. After treatment, OD420 values were recorded with a microplate reader.The ONPG hydrolysis assay was performed to characterize the leakage of the bacterial membrane. After being irradiated by NIR, in vivo antibacterial assays, male Balb/c mice (8–10 weeks) with an average weight of 25 g were used to establish the wound infection model. All animal studies were performed under the guideline approved by the Institutional Animal Care and Use Committee of Huaqiao University and following the Administration of Affairs Concerning Experimental Animals of China. Mice were randomly divided into five groups: (i) PBS, (ii) ICG/LUT, (iii) ICG/LUT+NIR, (iv) ICG/LUT-CS and (v) ICG/LUT-CS+NIR for the wound-healing experiments. Full-thickness wounds with 0.5 cm diameter were grafted on the back of the mice, and S.aureus suspension  was inoculated on the wound area for 24 h.For 2). The real-time temperature of the infection wound was captured by an NIR thermal imaging camera. In addition, the effect of treatment was evaluated every 2 days by recording the wound area using a digital camera. The wound area was measured by tracing around the margin via Image J software. The percentage of wound closure was calculated as:The dispersions of different samples  were sprayed carefully on the infected wounds and irradiated for 5 min with an NIR laser  embedded in paraffin. The wound tissues were also stained with Masson’s trichrome for wound-healing evaluation. All histological images were captured using an optical microscope.in vitro. 1 × 104 cells were seeded in the 96-well plates and then cultured at 37°C for 24 h in dulbecco’s modified eagle medium containing 10% fetal bovine serum. Then, after incubation with different samples (same concentration gradient) for 30 min, an NIR laser at an intensity of 1.5 W/cm2 was irradiated for 5 min and incubated for 24 h. After that, cells were washed with PBS and treated with a medium containing MTT  for 4 h. Then, the working solution was removed and added with DMSO to dissolve the formazan crystals. Finally, OD570 values were determined by a microplate reader. NIH/3T3 cells treated with a culture medium were used as control. The cell survival was calculated as:The NIH/3T3 cells were used to evaluate the biocompatibility of materials in vitro, the ICG/LUT-CS was mixed with 4% (v/v) human erythrocyte suspension to final concentrations at 30, 60, 120 and 240 μg/ml and incubated at 37°C for 30 min. Then, the mixture was centrifuged, and the absorbance of supernatants at 570 nm was determined using a UV-Vis spectrophotometer. The hemolysis percentage of ICG/LUT-CS in vitro was calculated as :For testing the hemolysis rate in vivo, the body weights were measured every 2 days. The blood samples from the sacrificed mice were subjected to analyze liver and kidney function tests. In addition, the major organs were dissected, fixed, embedded and stained with H&E.To test biocompatibility To further demonstrate the successful preparation of the ICG/LUT, the morphologies of particles were characterized by FE-SEM. Accordingly, the drug ratio significantly influenced the SAS-based particle formation process . The SEM3 Minitab-based factorial method was applied as a suitable tool for calculating and producing ICG/LUT. The surface morphology of ICG/LUT under different operation conditions was obtained by SEM  from the central heterocyclic ring signals could be observed in ICG/LUT, verifying the existence of ICG and LUT in the coprecipitates [−1 could be observed, attributing to the O−H stretching vibration and the C−H stretching of CS, respectively [Due to intrinsic bacteria-killing ability, CS-based nanomaterials driven by electrostatic interactions or hydrogen bond linkages have been used extensively in wound dressings and antibacterial applications . In thisipitates . After cectively , 47.Moreover, the UV-vis-NIR spectra  revealedin vitro under different conditions  in vitro. As shown in S.aureus growth on plates compared to the control treatment group . Different drugs showed concentration-dependent bacteria-killing efficiency. The SAS-assisted ICG/LUT composite displayed limited bactericidal outcome in the absence of NIR, which might be due to the partly bacteriostatic activity of LUT at the therapeutic range. Interestingly, the subsequent ICG/LUT-CS composites treatment group showed 50% bacteria reduction at a concentration of 10 µg/ml. Remarkably, in the presence of the NIR treatment, the bactericidal effects of ICG/LUT and ICG/LUT-CS against S.aureus were significantly increased. Notably, the antibacterial rate of ICG/LUT-CS could reach almost 100% at a concentration of 40 µg/ml, indicating the strengthened PTT-based bactericidal effect.Initially, we evaluated the antibacterial efficacy of different samples against typical, highly infectious gram-negative bacteria  compared with the PBS+NIR group (3.7%), ICG/LUT (40.2%), ICG/LUT-CS (48.0%) and ICG/LUT+NIR (64.78%). These experimental results showed synergistic antibiofilm effects in hyperthermia, nanoparticle–biofilms interaction and responsive release of LUT. Furthermore, the biofilm inhibiting effects with various treatments for different incubation times were also quantitatively measured by CV staining assay , and the unsurpassed antibacterial activity may benefit from the enhanced chemotherapy/PTT  compared with the wounds treated with other groups  was developed for the synergistic treatment of REGBIO online.This work was supported by the National Key Research & Development Program of China (2019YFE0113600); the National Natural Science Foundation of China ; and Program for Innovative Research Team in Science, Scientific Research Funds of Huaqiao University (21BS113).Conflicts of interest statement. None declared.rbac072_Supplementary_DataClick here for additional data file.


Distant metastasis is the major cause of treatment failure in patients with nasopharyngeal carcinoma (NPC). Thus, the identification of the molecular mechanisms and the development of novel therapeutic strategies are important. Previous studies suggest that PNCK promotes tumor growth by suppressing PI3K/AKT/mTOR signaling in NPC. However, the underlying regulatory mechanism of PNCK for NPC invasion and metastasis remains unclear.  The PNCK expression level was evaluated in nonmetastatic and metastatic NPC specimens by mRNA sequencing and immunohistochemistry. In vitro migration and invasion and in vivo nude mouse metastasis model and zebrafish model were used to evaluate the effects of PNCK ectopic expression on the metastatic ability of NPC cells. Gene set enrichment and western blot analyses were used to investigate the PNCK downstream signaling pathway. κB/VEGF signaling pathway.  Human metastatic NPC samples showed elevated PNCK expression at both mRNA and protein levels. Upregulated PNCK promoted in vitro NPC cell migration, invasion, and the formation of lung metastases; the vascular-labeled fluorescence signal increased in the in vivo zebrafish model. Mechanistically, pathway analysis showed that the upregulation of PNCK may promote cell metastasis by activating the NF- These findings revealed the specific critical role of PNCK in promoting NPC metastasis and angiogenesis, which suggested that PNCK may have implications as a potential therapeutic target for individualized NPC treatment. Nasopharyngeal carcinoma (NPC) is a common malignancy originating in the epithelium of the nasopharynx. Its incidence has a unique geographical distribution; high incidences are reported in southeast Asia and southern China , 2. Due κB (NF-κB) is a family of transcription factors that plays a critical role in inflammation, immunity, cell proliferation, tumor-associated angiogenesis, and metastasis [κB pathway is of particular interest in cancer metastasis, as it can bind the transcription of several target genes that promote invasion and metastasis [κB promotes cancer invasion and metastasis; it has been observed in many tumors, including breast cancer and colorectal cancer [κB signaling pathway in NPC metastasis remains poorly understood. Thus, the identification of new upstream regulators of NF-κB signaling is of great importance for prognostic prediction and therapy.Nuclear factor-tastasis , 8 The Ntastasis . Aberranl cancer , 11. HowPregnancy upregulated nonubiquitous calmodulin (CaM) kinase (PNCK) is a unique member of the CaM kinase I family. It impacts various biological functions and plays an important role in human malignancies –15. SomeκB pathway and promoted NPC angiogenesis. Taken together, this study elucidates the mechanism of PNCK overexpression in promoting metastasis in NPC cells and identifies a potential prognostic biomarker and target for individualized NPC treatment.The PNCK expression was evaluated in metastatic and nonmetastatic NPC tissues and it was found that PNCK expression was significantly high in the metastatic tissue. By in vitro and in vivo studies, it was found that upregulated PNCK significantly promoted the metastatic ability of NPC cells. Furthermore, it was demonstrated that PNCK facilitated the metastasis in NPC cells by the NF-A total of 5-freshly frozen metastatic and 5-non-metastatic NPC tissue were collected by biopsy from the Fujian Cancer Hospital between January 2015 and March 2015. Gene expression analysis was performed using 132 paraffin-embedded NPC specimens obtained from patients diagnosed with primary NPC and who did not undergo any treatments before the biopsy , 113 patient data were included as per the following selection criteria: (a) definite histologically diagnosis of NPC; (b) availability of RNA-seq data  from the Gene Expression Omnibus (GEO)  in CNE-2 and SUNE-1 cells were introduced  were obtained from the Shanghai Institutes for Biological Sciences. All cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL streptomycin and maintained at 37°C in a 5% COP value (FDR P) < 0.05 was used as a cutoff for statistical significance. Parameters for defining significant DEGs were ≥2-fold change ) in the transcript data, and P ≤ 0.05. The DEGs were annotated based on the information acquired from the ENSEMBL, NCBI, UniProt, GO, and KEGG databases. The biological processes associated with the DEGs were identified using the Ingenuity Pathway Analysis (IPA) Software . Volcano plots were generated with the “pheatmap” and “ggplot2” packages in R.The freshly frozen tissues and CNE-2 cells were used for mRNA sequencing. Total RNA was extracted with TRIzol (Invitrogen), and its quality was assessed with Agilent 2100 BioAnalyzer  and Qubit Fluorometer (Invitrogen). hg38 was used as the reference assembly for the human genome. The sequencing quality was assessed using the quality assessment software, FastQC (v0.11.5); the low-quality reads were removed using NGSQC (v2.3.3) . The cleThe paraffin-embedded NPC tissue sections were analyzed by IHC using the anti-human PNCK antibody . Following the DAB treatment, HRP-linked secondary antibodies  were used. Stained cells were blindly scored based on the staining intensity , and the abundance of positive cells  was validated by two senior pathologists. To obtain the final score, the intensity score (0-3) was multiplied by the extent score (0-5) to determine the levels of PNCK expression.κB P65 , NF-κB p-P65 Ser536 , p-IκB Ser32 , PNCK , VEGFA , Histone-H3 , and GAPDH .PCR and WB were performed as previously described . We usedGSEA was used to compare the levels of expression enrichment between an a priori defined set of genes and the high- and low-PNCK expression groups using the MSigDB Collection (c2.cp.kegg and c5.go.bp. v7.2. symbols.gmt). High- and low-PNCK were used as the phenotype label, and the gene set permutations were performed 1000 times for each analysis. False discovery rate (FDR) and normalized enrichment score (NES) were used to classify the GO and KEGG pathways enriched in differential phenotypes.5 cells/well) were seeded into six-well plates and cultured till they attained 80% confluence. Then, the monolayers were wounded by dragging a 1 mL pipette tip across the cells. The cells were then washed to remove debris and were allowed to migrate for 48 h. Following wounding, cells were imaged at 0 h and 48 h using a DMI 6000 inverted microscope. Cell migration was quantified using the ImageJ software as the wound healing index, i.e., the wound area healed by the cells at 48 h after scratching relative to the wound area at 0 h.Transfected CNE-2 and SUNE-1 cells (~1 × 10μL of the cell suspension was seeded (1 × 106 cells/mL) into the upper chambers of an 8 μm transwell filter (Millipore), precoated with 1 : 3 diluted Matrigel (BD Biosciences). RPMI 1640 supplemented with 10% FBS (700 μL) was added to the lower chamber. Subsequently, the plate was incubated for 24 h at 37°C and 5% CO2. The migration and invasive ability of the cells were assessed based on the penetration of the membrane and gel membrane of the matrix, respectively. Following 24 h incubation, the residual cells in the upper chamber were removed with cotton swabs and dried. Then, migrated and invaded cells were fixed with 4% paraformaldehyde for 15 min at room temperature, washed with PBS, and stained with 0.1% crystal violet for 10 min at room temperature. Cells were counted in at least 5 randomly selected fields of view observed under a DMI 6000 inverted microscope (×200).Transfected cells were digested and resuspended in serum-free RPMI 1640. Then, 200 Male BALB/c (nu/nu) nude mice (4-6 weeks of age) were purchased from Wushi Experimental Animal Supply Co., Ltd. . Metastasis of PNCK-overexpressing CNE-2 cells and wild-type cells was injected intravenously in the tail vein. The experiment was terminated 28 days after tumor cell implantation. Then, the lungs were embedded in paraffin and H&E staining was performed after euthanizing the animals. Metastatic nodules in the lungs of mice were counted.μL) and gRNA (100 ng/μL). After 48 hours of injection, the overall fluorescence signal was measured using a stereoscopic fluorescence microscope (SMZ800N). The ImageJ software was used to evaluate the formation of vascular structure and length of intersegmental vessels (ISVs). Moreover, the antiangiogenic or angiogenesis activity was identified based on the total inhibition of ISV growth and incomplete sprouting of the ISVs from the dorsal aorta (DA) to the dorsal longitudinal anastomotic vessel (DLAV).For PNCK overexpression, the one-cell-stage embryos of Tg (fli1a: EGFP) transgenic line with vascular fluorescent labeling, were microinjected with CMV-derived human PNCK gene overexpression vector CMV-hPNCK (about 200-300 pg/embryo) using a microinjection apparatus . For knockdown of PNCK, RNA-targeted editing technology through the CRISPR-RfxCas13d (CasRx) system was used . Brieflyt-test and ANOVA were used to compare the differences between the groups. Spearman's rho test was used to analyze the correlation among the qualitative parameters. All differences in data were determined as statistically significant at P < 0.05.SPSS 17.0 and R software (Version 3.4.4) were used for all statistical analyses. The quantitative data from at least three independent experiments are presented as mean ± SD. Student's P < 0.05 and |log2 FC| > 1.0 threshold values . However, PNCK levels were significantly correlated with distant metastasis .Stably overexpressing PNCK-NPC cell lines (oePNCK cells) were generated using CNE-2 and SUNE-1 cells to assess the PNCK-mediated metastatic abilities in NPC. The mRNA and protein levels were confirmed by real-time PCR and western blot analysis Figures . It was P < 0.001). Similarly, the numbers and volumes of micrometastases in the lungs of mice injected with SUNE-1 cells with overexpressing PNCK were significantly high  by tail vein injection. Then, the number of metastatic lesions in the lungs after 4 weeks was evaluated. The metastatic foci on the lung surface were significantly higher in the oePNCK group than those in the oeVec group, as shown in Figures tly high . These rμmol/L sunitinib; embryos with a high level of PNCK had upregulated angiogenesis in ISVs at 48 hpf , at 10 ng/mL for 30 min, P65 was rapidly phosphorylated in both nuclei and cytoplasm of PNCK-overexpressed CNE-2 cells. Furthermore, phosphorylated IκB was accumulated in PNCK-overexpressed cells after TNF-α stimulation . VEGFA is a key facilitator of angiogenesis in cancer [r = 0.4, P < 0.001, κB/VEGF signaling pathway in NPC.Angiogenesis, the recruitment of new blood vessels, is essential in the metastatic pathway . Tumor a pathway . Althougn cancer . GEO datκB/VEGF signaling pathway in NPC metastasis.NPC is a malignancy with a high potential of invasion and metastasis; distant metastasis is a major cause of treatment failure , 34. HenPNCK is located on chromosome Xq28. It is a member of the CaM kinase family and has the enzymatic activity of serine/threonine kinases . It is iκB/VEGF signaling pathway. NF-κB is critical for the regulation of tumor cell metastasis [κB pathway inhibition [2+ activates PNCK, which in turn phosphorylates IκBα at Ser32 and triggers Ca2+-dependent NF-κB signaling activity under hypoxic conditions [2+-induced NF-κB pathway promotes tumor progression by increasing the transcription of VEGF and thus promoting angiogenesis and cancer development [κB pathway was significantly enriched in the high PNCK group. Upregulation of PNCK may induce the phosphorylation of IκBα at Ser32, which further induced IκBα degradation and NF-κB activation in NPC. The underlying mechanisms of these roles need to be further studied. Moreover, the zebrafish model was used to validate the potential role of PNCK-mediated angiogenesis in NPC in the present study. Interestingly, it was found that increased PNCK expression promoted the outgrowth of ISVs of zebrafish, which indicated positive regulation of NPC angiogenesis.Invasion and metastasis are the major contributors to treatment failures for most cancers . Distanttastasis . Liang ehibition , 38. Howhibition . Oxygen hibition . Previounditions . The actelopment . In thisκB/VEGF signaling pathways play a critical role in NPC metastasis, but the detailed mechanism of regulation by PNCK and the corresponding upstream and downstream targets remain unclear. Therefore, further studies are needed to elucidate the mechanisms underlying the interactions between PNCK and the NF-κB/VEGF axis in NPC metastasis.Nevertheless, there are certain limitations of the current study. First, the sample size of the sequencing cohort was small. However, at the protein level, a larger tissue sample size was included. Second, due to the lack of access to the complete clinical data, the evaluation was limited to the association with PNCK expression and progression-free survival only. Third, it was found that NF-κB/VEGF signaling pathway. These data provide new insights for PNCK as a useful indicator to predict future metastasis and as an efficient antiangiogenic target for NPC treatment. Further studies are needed to better understand the molecular mechanisms underlying the interactions between PNCK and NF-κB/VEGF axis in NPC metastasis.In conclusion, it was confirmed that PNCK expression was high in metastatic tissues and was associated with PFS in NPC. PNCK promoted NPC cell migration, invasion, and angiogenesis by the activation of the NF-


Non-SMC condensin I complex subunit H (NCAPH) plays a regulatory role in various cancers. However, its role in prognosis and immune infiltrates in lung adenocarcinoma (LUAD) remains unclear. This study examined the expression of NCAPH in tumor tissues and its association with immune infiltrates and prognostic roles in LUAD patients. Patients characteristics were obtained from The Cancer Genome Atlas (TCGA). Integrated analysis of TCGA showed that NCAPH was overexpressed across cancers, including LUAD. NCAPH expression was verified by quantitative polymerase chain reaction and western blotting in 20 LUAD matched tissues. High NCAPH expression was significantly related to T, N, M, pathologic stage, primary therapy outcome and smoking status according to the Wilcoxon rank sum test. Cox and Kaplan–Meier analyses showed that the NCAPH-high group was associated with shorter OS. The PFI and DSS in the NCAPH-high group were significantly decreased. Multivariate analysis showed that NCAPH was an independent predictive factor for poor prognosis. Gene set enrichment analysis demonstrated that the G2/M checkpoint, ncRNA metabolic, memory B cells, KRAS, E2F targets and MIER1 process were significantly associated with NCAPH expression. Single-sample Gene Set Enrichment Analysis indicated that NCAPH expression was associated with levels of Th2 and mast cells. The impact of NCAPH on malignant phenotypes was evaluated by MTT, transwell, cell cycle and apoptosis assays in vitro. The malignant phenotype of LUAD cells was inhibited if NCAPH was knocked down. In conclusion, this research indicates that NCAPH could be a potential factor for predicting prognosis and a new biomarker in LUAD. Non-small-cell lung cancer (NSCLC) accounts for almost 85% of lung cancers. LUAD is the most common type of NSCLC and accounts for approximately 40% of NSCLC cases. In recent decades, with the advent of small molecule tyrosine kinase inhibitors and immunotherapy, the survival of selected LUAD patients has been significantly prolonged2. However, the overall cure and survival rates for lung adenocarcinoma remain limited depending on stage and regional differences3. LUAD is a highly heterogeneous malignancy. Current biomarkers, such as EGFR mutations, BRAF mutations, HER2 amplification and microsatellite instability, cannot completely explain the different prognoses or therapeutic responses of LUAD4. Since the molecular mechanisms of LUAD tumorigenesis and development are still not fully clear, numerous studies of targeted therapies and experiments have tried to elucidate the pathogenesis and improve the prognosis of LUAD patients5. Therefore, the development of novel biomarkers for diagnosis and treatment targets in LUAD is critical.Lung cancer is the leading cause of cancer mortality worldwide, with more than 1,760,000 deaths each year6. NCAPH was revealed to be important for viability and to play a key role in mitotic chromosomal architecture and segregation in previous studies8. Recent studies demonstrated that NCAPH was involved in various malignancies. In microarray analysis, NCAPH was found to be highly expressed and to contribute to carboplatin resistance in serous ovarian cancer patients9. In hepatocellular carcinoma, NCAPH was highly expressed in tumor tissues compared to normal noncancerous tissues and was associated with poor prognosis. In addition, the overexpression of NCAPH promoted tumor proliferation, migration and invasion in vitro and in vivo10. These findings suggest that NCAPH has notable roles in cancer progression and development. However, the functions and mechanisms of NCAPH in LUAD have not been fully explored. Moreover, we found that NCAPH may play an oncogenic role in LUAD through bioinformatic analysis. Therefore, we investigated the impact of NCAPH on LUAD development.Non-SMC condensin I complex subunit H (NCAPH) is encoded by a gene located on chromosome 2q11.2. It belongs to the barr gene family and is a regulatory subunit of the condensin complexTo better explore the role of NCAPH in LUAD progression, we applied RNA-seq data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. Statistical and bioinformatics methods, such as differentially expressed gene (DEG) analysis, Kaplan–Meier (KM) survival analysis, Cox and logistic regression analysis, nomogram, Gene Ontology (GO) analysis, Gene Set Enrichment Analysis (GSEA) and single-sample Gene Set Enrichment Analysis (ssGSEA) were utilized. Moreover, NCAPH was knocked down in vitro to determine how it affected LUAD proliferation, invasion and migration.https://portal.gdc.cancer.gov/). The exclusion criteria were OS  less than 30 days and normal tissues. Then, HTSeq-FPKM information of level 3 was transformed into transcripts per million (TPM); then, the TPM information of 513 lung adenocarcinoma samples was applied for the next analyses. Twenty-two samples were excluded due to a lack of clinical variables.LUAD patients’ clinical information and gene expression data  were obtained from TCGA  as a variable, scatter plots and boxplots were generated to estimate different expression levels of NCAPH. Receiver operating characteristic (ROC) curves were generated to estimate the diagnostic value of NCAPH. NCAPH expression above or below the median value was defined as NCAPH-high or NCAPH-low, respectively.P value < 0.05 and an absolute FC larger than 1.5 were considered to be statistically significant. All significant DEGs are presented in volcano plots and heatmaps, which were constructed using R software.Analysis of the differential expression of genes between the NCAPH-high and NCAPH-low patients from TCGA LUAD datasets was conducted using DESeq2 (4.0 package). Genes with an adjusted http://metasape.org). Those with an enrichment factor > 1.5, a minimum count of three and P < 0.01 were regarded as statistically significant. By using GSEA, we investigated the differences in the signaling pathways between the NCAPH-high and NCAPH-low groups to predict NCAPH-related phenotypes and signaling pathways. The significantly changed pathways were identified by permutation testing 1000 times. A false discovery rate (FDR) < 0.25 and adjusted P < 0.01 were recognized as significantly associated genes. The R package cluster Profiler (4.0) was used for analysis and graphical plotting11. The relative tumor infiltration levels of 24 immune cell types were analyzed by ssGSEA to research the expression levels of genes in published signature gene lists12. The signatures included multiple sets of innate and adaptive immune-related cell types and comprised 509 genes in total. To evaluate the association between the infiltration levels of immune cells and NCAPH, Spearman correlation and Wilcoxon tests were used.Enrichment of NCAPH-related DEGs by pathway and process was analyzed by Metascape . The calibration curves were pictorially evaluated by drafting the nomogram measuring likelihood versus actual occurrence, and the 45-degree line indicated the best predicting values. A concordance index (C-index) was calculated and used to evaluate the discrimination of the model using a bootstrap method with 1,000 resamples. The C-index was used to assess the prognostic features and predictive accuracy of the nomogram.All statistical analyses were performed using an R package (V3.6.2). Using logistic regression and the Wilcoxon signed-rank sum test, the link between clinicopathological features and NCAPH was investigated. The clinical-pathological variables linked to 10-year OS, disease-specific survival (DSS) and progression-free interval (PFI) in TCGA database were analyzed by using the Kaplan–Meier method and Cox regression. Univariate and multivariate Cox analyses were utilized to investigate the effect of NCAPH levels on survival and other clinical variables. The cutoff value for NCAPH expression was determined as the median level. -ΔΔCt method. The following primers were used: sense: 5’-ATGTTGCTGATGGAAGTG-3’ and antisense: 5’-GTTCTGCTCA ATAGTTCTGT-3’ for NCAPH; sense: 5’-AGGCGCGCAAATTACCCAATCC-3’ and antisense: 5’-GCCCTCCAATTGTTCCTCGTTAAG-3’ for 18S rRNA.From January 2018 to January 2019, 20 matched LUAD tissues and neighboring noncancerous tissues were taken from patients who received surgery at the National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital & Shenzhen Hospital. All cases were pathologically confirmed. The current study was approved by the Ethics Committee of the National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital & Shenzhen Hospital and conducted according to the Declaration of Helsinki. All patients signed informed consent forms. Total RNA was extracted from tissues by using TRIzol reagent  according to the manufacturer’s protocol. The extracted RNA was reverse-transcribed into cDNA by using the Takara PrimeScript RT Reagent Kit . RT-PCR was performed by using a LightCycler 480 Real-time PCR System . To normalize NCAPH, 18S rRNA was used as an internal reference. The relative expression of NCAPH mRNA was calculated with the 2Total protein was extracted from frozen tissues. Protein concentrations were tested by a BCA protein assay kit. Protein samples were separated on a 10% sodium dodecyl (lauryl) sulfate–polyacrylamide gel electrophoresis gel. The separated proteins were transferred to an Immun-Blot polyvinylidene fluoride membrane (Bio–Rad) using a wet transfer system (Bio–Rad) and then incubated with primary antibody at 4 °C overnight, followed by incubation with horseradish peroxidase-linked anti-rabbit immunoglobulin G (Merck Millipore) at a dilution of 1:10,000 for 1 h at room temperature. The following antibodies were applied in the experiment: NCAPH  and β-actin . Relative NCAPH protein expression levels were normalized to β-actin.2.H2122 and H3122 cell lines were purchased from the IMMOCELL company . The H2122 and H3122 cell lines were cultured in RPMI 1640 medium (GIBCO) supplemented with 10% fetal bovine serum (HyClone). Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO4 cells in 200 μL of medium without serum were trypsinized, suspended and seeded in the upper chamber . Subsequently, medium (600 μL) containing 20% FBS was added to the bottom compartment of the chamber. Then, the chamber was placed in an incubator at 37 °C. After incubation for 48 h, the cells were fixed with methanol and stained with 0.1% crystal violet (Sigma–Aldrich). Then, the nonmigrated cells were removed by scraping. Finally, migrated cells were counted by using a microscope . The Transwell invasion assay was similar to the migration assay. The difference was that the upper chamber of the Transwell invasion assay was covered with Matrigel matrix. These experiments were repeated three times. Relative migration or invasion (%) was calculated by the average number of migrated (invaded) cells in the transfection group/average number of migrated (invaded) cells in the control group × 100%.For the cell migration assay, 1 × 10Cell cycle distribution and apoptosis assays were conducted by flow cytometry. H2122 and H3122 cells were digested with trypsin, resuspended in phosphate-buffered saline (PBS) and then fixed with 70% ethanol. Cells were washed with PBS and treated with 100 μg/ml RNase for 30 min. Then, DNA was stained with propidium iodide (50 μg/ml) and analyzed on a FACS Calibur flow cytometer . Apoptosis was assessed by using an Annexin V FITC/PI apoptosis kit . The samples were measured and analyzed by using a flow cytometer .P < 0.05 were considered statistically significant.Statistical analysis was performed with Student’s two-tailed t test using SPSS (version 22). Values of The current study was approved by the Ethics Committee of the National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital and Shenzhen Hospital. Signed informed consent was obtained from all patients.P < 0.05)     combined with TCGA and matched normal samples. NCAPH was abnormally expressed in bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), esophageal carcinoma (ESCA), head and neck squamous cell carcinoma (HNSC), renal chromophobe cell carcinoma (KICH), renal clear cell carcinoma (KIRC), renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), pancreatic cancer (PAAD), prostate cancer (PRAD), rectum adenocarcinoma (READ), gastric cancer (STAD), thyroid cancer (THCA), and endometrial cancer (UCEC) (05) Fig. A,B. The 01) Fig. . The qPCP value < 0.05, log2-fold change > 1.5) . A total of 1592 DEGs were identified, including 1167 upregulated genes and 425 downregulated genes , cellular compositions (CCs) and molecular functions (MFs), including distal axon, neuron projection terminus, axon terminus, integrator complex, premiRNA processing, RNA 3'-end processing and miRNA catabolic process Fig. A.Figure P < 0.05, FDR P value < 0.25) . According to the normalized enrichment score (NES), the top significantly enriched pathways associated with high NCAPH expression were selected. The G2/M checkpoint, ncRNA metabolic process, memory B cells, KRAS signaling, E2F targets and MIER1 process were significantly enriched in patients with NCAPH ] and negatively correlated with the abundance of innate immunocytes [mast cells ]  cells ] Fig. A–E.FigurP < 0.001), N stage , M stage , pathologic stage , primary therapy outcome  and smoking status . Furthermore, we conducted univariate analysis to investigate whether NCAPH expression is a dependent variable associated with poor prognostic clinicopathological characteristics (Table P < 0.05). High expression of NCAPH in LUAD was negatively correlated with primary therapy outcome .Data from 513 patients and NCAPH expression data were collected from TCGA to explore the relationship between NCAPH expression and clinicopathologic parameters. Table Univariate logistic regression was used to explore the role of NCAPH in the prognosis of LUAD patients. The OS of LUAD patients with high NCAPH expression was significantly shorter, with a median of 41.2 months versus 77.3 months in patients with low NCAPH expression  , siRNA showed the most significant inhibition ratio and was selected for further experiments. The MTT assay data indicated that the siRNA targeting NCAPH significantly reduced cell growth rates. Transwell assays revealed that NCAPH-targeted siRNA transfection notably reduced migration and invasion in both cell lines. By using flow cytometry analysis, the cell cycle distribution of the NCAPH siRNA-transfected cells demonstrated an increase in the G13. Condensin I and II are two forms of condensin complexes found that are in many eukaryotic cells, and both share the identical pair of the structural maintenance of chromosome (SMC) 2 and 4 subunits. The condensin I complex comprises SMC2-SMC4 proteins and three non-SMC proteins, including subunits H (NCAPH), G (NCAPG) and D2 (NCAPD2)14. Previous research showed that phosphorylation of NCAPH at Ser70 by Aurora B kinase was indispensable in the recruitment of condensin I to mitotic chromosomes15. Intriguingly, bioinformatics analyses of potential molecular mechanisms have shown that NCAPH is a key gene involved in lung and prostate tumorigenesis17. Although a previous study found that overexpression of NCAPH was associated with LUAD pathogenesis, the function and molecular mechanism remain unclear. Herein, we attempt to elucidate the oncogenic impact of NCAPH in LUAD development.Condensin is a highly conserved multiprotein complex that regulates chromosomal assembly and separation during mitosisAccording to our findings, the expression levels and prognostic value of NCAPH were assessed. We found that NCAPH expression was increased in various tumors, including LUAD, in databases. Furthermore, NCAPH may serve as a good biomarker for high ROC scores with an AUC of 0.967 for LUAD. Generally, NCAPH was expressed differently in tumor and normal samples. Further studies are needed to fully research the diagnostic value of NCAPH in LUAD.To further study the functional enrichment information of NCAPH-related genes, Metascape was utilized for GO enrichment analysis. NCAPH-related genes were involved in many BPs, CCs and MFs, including distal axon, neuron projection terminus, axon terminus, integrator complex, premiRNA processing, RNA 3' − end processing and miRNA catabolic process.18. This study directly supports our results. The KRAS signaling pathway and E2F were proven to play an important role in the progression and development of LUAD21. These studies and our results indicate that NCAPH might contribute to LUAD initiation and development by modulating E2F, the cell cycle and the KRAS pathway. The associations of NCAPH expression with memory B cells, ncRNA metabolism and MIER1 process were first reported. The molecular mechanisms need to be further researched.We also revealed that the G2/M checkpoint, ncRNA metabolic process, memory B cells, KRAS signaling, E2F targets and MIER1 process were significantly associated with NCAPH expression. In a previous in vitro study, cell proliferation, cell cycle, colony formation, migration and invasion were inhibited when NCAPH was knocked down24. Strong infiltration of TILs was associated with a positive clinical outcome in several cancers, including lung cancer25. Our study showed that NCAPH was positively associated with the level of acquired immunocytes (Th2 cells) and negatively correlated with the abundance of innate immunocytes (mast cells). Th2 cells are defined by the expression of their signature cytokines IL-4, IL-5, and IL-13, which are important components in the defense against extracellular pathogens26. Cytokines, such as IFN-γ, TNF-α and IL-2, produced by Th1 cells were found to be vital factors in the inhibition of tumor growth28. In contrast, the cytokines IL-10, IL-4 and TGF-β from Th2 cells were proven to promote tumor cell dissemination and metastasis in various cancers29. Therefore, maintaining the Th1/Th2 immune cell balance is considered to be critical. Enhancing the Th1 response and inhibiting the Th2 effect may help to prevent disseminated cancer cells, recurrence and metastasis30. Consistent with these findings, our results revealed that NCAPH may be associated with the Th2 immune response in LUAD. In several studies, mast cells were found to be a predictor of poor outcome33. However, a study of 175 patients with NSCLC also demonstrated that mast cell presence was a good prognostic factor34. The prognostic role of mast cells is still uncertain. However, the association of NCAPH expression with mast cells was the first to be reported.Previous clinical studies found that tumor-infiltrating lymphocytes (TILs) had a vital impact on several cancersOur research indicated that high NCAPH expression is associated with clinical pathological characteristics and poor prognosis in LUAD. NCAPH expression was significantly related to T stage, N stage, M stage, pathological stage and smoking status. In univariate logistic regression, the OS of LUAD patients with high NCAPH expression was significantly shorter. After adjusting for clinicopathological factors, our study found that NCAPH could act as an independent predictive factor for a poor prognosis of LUAD. Then, we constructed a clinical nomogram with NCAPH expression and other clinical factors. Based on the calibration plot, there was a favorable consistency between the actual and predicted values for 1-year OS. Our model could be a new method to estimate prognosis in the future.We also investigated the function of NCAPH in the proliferation, invasion, migration, cell cycle progression and apoptosis of LUAD cells in vitro. The malignant phenotype of LUAD cells was inhibited when NCAPH was knocked down.In this study, we first reported that high NCAPH expression was significantly associated with poor survival and immune infiltration in LUAD, which might promote tumorigenesis through abnormal inflammation and immune responses. NCAPH may be a potential factor for predicting prognosis and a new biomarker. The in vitro study demonstrates that NCAPH may function as an oncogene in LUAD.Supplementary Figure 1.Supplementary Figure 2.Supplementary Information 3.