Ampelopsis grossedentata, which is commonly known as vine tea, are used widely in the traditional Chinese beverage in southwest China. The leaves contain a large amount of dihydromyricetin, a compound with various biological activities. However, the transcript profiles involved in its biosynthetic pathway in this plant are unknown.Leaves of the medicinal plant We conducted a transcriptome analysis of both young and old leaves of the vine tea plant using Illumina sequencing. Of the transcriptome datasets, a total of 52.47 million and 47.25 million clean reads were obtained from young and old leaves, respectively. Among 471,658 transcripts and 177,422 genes generated, 7768 differentially expressed genes were identified in leaves at these two stages of development. The phenylpropanoid biosynthetic pathway of vine tea was investigated according to the transcriptome profiling analysis. Most of the genes encoding phenylpropanoid biosynthesis enzymes were identified and found to be differentially expressed in different tissues and leaf stages of vine tea and also greatly contributed to the biosynthesis of dihydromyricetin in vine tea.A. grossedentata. The study provides an insight into the expression patterns and differential distribution of genes related to dihydromyricetin biosynthesis in vine tea. The information may pave the way to metabolically engineering plants with higher flavonoid content.To the best of our knowledge, this is the first formal study to explore the transcriptome of  Ampelopsis grossedentata (Hand.-Mazz.) W.T. Wang [Camellia sinensis L.), which contains high levels of catechins [Vine tea (Teng cha) is a famous traditional Chinese tea, made from leaves of .T. Wang . The pla.T. Wang . Vine teatechins , 3, the atechins . DHM, alatechins , anti-inatechins , 6, antiatechins , reno-pratechins , and antatechins , 10, etcatechins . In addiatechins . Vine teatechins .C. sinensis) varied according to geography, developmental stages, and cultivars [C. sinensis, especially at the transcriptional level, using an Illumina HiSeq transcriptome sequencing technique [Secondary metabolites play an essential role in plant defense system and development processes –16. The ultivars , 21, 22.ultivars . The quaultivars . Moreoveechnique –26.Phenylpropanoids are a large group of compounds that play a vital role as secondary metabolites in vine tea. The structural genes in the phenylpropanoid biosynthetic pathway have been well studied –30. In rThe DHM content was quantified in different leaf stages and tissues of vine tea by high-performance liquid chromatography  subsequently generated 157.4 million and 141.8 million reads in total and 52.47 and 47.25 million reads on average, respectively, after removing adapter containing low-quality reads , Hordeum vulgare (2.9%), Picea sitchensis , and Galdieria sulphuraria .A total of 177,422 unigenes were annotated in seven public databases namely the NCBI non-redundant protein sequences database (Nr), NCBI non-redundant nucleotide database (Nt), Protein family database (Pfam), euKaryotic Orthologous Groups (KOG), Gene Ontology (GO), Clusters of Orthologous Groups of protein databases (COG) and Swiss-Prot protein database  enrichment analysis showed that the unigenes were annotated into 56 sub-categories. The highest enriched categories were ‘cellular process’, ‘metabolic process’, ‘binding’ and ‘catalytic activity’, which belong to the ‘biological process’ and ‘molecular function’ categories in vine tea  varies according to the seasons and plant developmental stages [Vine tea is a frequently used herbal tea in south China. The raw material of vine tea is processed by one-step natural fermentation  and the l stages , 38. A sPrevious researchers have reported that the contents of DHM and other flavonoids may vary within different plant parts, developmental stages and seasonal conditions in vine tea plants , 40. EquThe annotation of non-model species by transcriptome analysis is a common strategy for exploring a unique profile. Since it is rare for any chemical compound to make up greater than 10% of a plant part by weight, it is significant that DHM alone makes up nearly one-third of the components of vine tea leaves and over 40% of young leaves by weight. As there are no related data available, we conducted a whole genome transcript analysis to shed light on the biosynthesis of DHM and related flavonoid metabolites in the plant. Using RNAs from the young and old leaves, we performed detailed transcriptome analysis of vine tea and found 7768 differentially expressed unigenes. The best matches for individual unigene searches against the NCBI non-redundant database and KEGG were promoted to assign functional GO annotation under different biological processes. After mapping the unigenes onto KEGG, we found that most of them are involved in phenylpropanoid biosynthesis and starch and sucrose metabolism. The GO classification analysis also revealed that the differentially expressed genes were highly involved in organic substance and primary metabolic processes. The results reveal that the transcripts encoded a large number of genes involved in biosynthesis of flavonoids specifically found in young leaves of vine tea.AgCHI, AgFLS, Ag3GT, AgCHS, AgPAL, AgRT, and AgF3H all have different unigenes with high levels of expression in both young and old leaves. Only one gene, DFR, demonstrated a consistent higher FPKM in old leaves  O. Kuntze) [GmMYB58 and GmMYB205 were demonstrated to be seed-specific activators for isoflavonoid biosynthesis in Glycine max [In addition, it has been demonstrated that many transcription factors (TFs), including MYBs, NACs, basic helix–loop–helix (bHLH), WD40s, and WRKYs, are involved in the regulation of metabolic biosynthesis in plants and the regulatory pattern of these transcription factors is usually tissue-specific –47. MYBs Kuntze) . Additiocine max . Numeroucine max , 54. OurA. grossedentata was performed to explore the putative transcripts involved in the flavonoid biosynthetic pathway of this important tea plant. The differential expression pattern of genes observed in leaves at different stages of development and in different tissues of vine tea suggests tissue-specific synthesis and accumulation of flavonoid metabolites, particularly DHM. This study provides a useful resource for investigating the flavonoid biosynthetic pathway to determine the reasons behind the significant accumulation of and number of transcripts identified for certain flavonoids in vine tea. The information may pave a way to metabolically engineer this plant with increased flavonoid content.In this study, a comparative transcriptome analysis of young and old leaf tissues of A. grossedentata) plants were grown on a private farm in Laifeng County, Enshi Area, Hubei Province, China . The tea was grown in a natural environment. Leaves at different growth stages and tissues of vine tea were collected in the month of June, 2016. The plants were identified using a species identification key by Professor Dr. Xuebo Hu [Vine tea . The quality of mRNA including purity, quantity, and integrity was tested using Nanodrop spectrophotometer , Qubit RNA Assay Kit , and Agilent Bioanalyzer 2100 system . A total amount of 1.5 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina®  following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Finally, the purified double-stranded cDNA samples were further enriched by PCR to construct the final cDNA libraries that were sequenced using Hiseq 2500 (150 bp paired ends) by Novogene Company .A. grossedentata using an RNeasy Plant Mini Kit  TIANGEN RNAprep Pure kit (TIANGEN). For first-strand cDNA synthesis, one μg of high-quality total RNA was used for reverse transcription (RT) with a Takara FastQuant RT Kits . A 20-fold dilution of 20 μL of the resulting cDNA was used as a template for quantitative real-time PCR.The total RNA was isolated from A. grossedentata were analyzed by qRT-PCR analysis. The gene-specific primers were designed to perform qRT-PCR analysis for some major transcripts involved in flavonoid biosynthetic pathway  using the SYBR Premix Ex Taq™ . Each run contained a series of standards and negative control (using water instead of cDNA). The qRT-PCR protocol was as follows: denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 56 °C for 15 s, and elongation at 72 °C for 20 s. The triplicate was conducted for each sample, and the qRT-PCR results were calculated as the mean of 3 replicated treatments.The transcript levels of different samples from standard . Gene exg for 5 min, the supernatant was filtered through a 0.45-μm PTFE syringe filter  and used for metabolites analysis.The samples of vine tea were freeze-dried and ground into powder. Flavonoid extraction was performed as followed. Samples were ground to fine powder, and 0.1 g powdered material was extracted with 1.5 mL of 70% methanol (MeOH). The extraction material was vortexed for 1 min followed by sonication for 0.5 h at 30 °C. After centrifugation at 1000×The HPLC analysis of flavonoids was performed on a Waters HPLC system  equipped with a waters ODS column . The mobile phase consisted of a mixture of (A): acetic acid-water (0.15%) and (B) MeOH. The initial mobile phase composition was 20% solvent B, followed by a linear gradient from 5 to 80% of solvent B. Detection was performed at 285 nm, and the column oven temperature was 30 °C. The flow rate was set at 0.8 mL/min, and the injection volume was ten μL. (+)-Dihydromyricetin (DHM) standard compounds (> 99%) were purchased from Sigma Chemicals Co. . Quantification of the different compounds was calculated as equivalents of representative standard compounds. All contents were expressed as mg/g of dry weight.p < 0.05 were considered statistically significant. Pearson correlation analysis was calculated by SPSS 22.0, and the correlation coefficient was calculated using the means of metabolite concentrations and genes relative expression values. Then cluster analysis and heatmap were drawn by R (http://www.r-project.org/).Data shown in figures was expressed as the mean of 3 independent replicates. Experimental data were processed by analysis of variance (ANOVA), and significant differences among the means were determined by Duncan’s multiple-range test . Values of Additional file 1: Figure S1. The HPLC chromatograms of DHM analysis in young and old leaves of vine tea. A, The standards chromatograms of dihydromyricetin (1), rutin (2), myricetin (3), quercetin (4) and kaempferol (5) at 285 nm; B and C, HPLC chromatograms of young (B) and old (C) leaves of vine tea.Additional file 2: Table S1. Reads annotated with reference database.Additional file 3: Table S2. The distribution of transcripts and genes length based on De novo assembly.Additional file 4: Figure S2. Annotation of all the unigenes from vine tea transcriptome. Number of genes annotated by five representatively databases  was showed in Venn diagram.Additional file 5: Figure S3. Characteristics of unigenes annotated against Nr databases. A, E-value distribution of the top BLAST hits for each unigene ; B, Similarity distribution of the top BLAST hits for each unigene; C, homologous species distribution against Nr database.Additional file 6: Figure S4. Gene Ontology (GO) classification of unigenes of A. grossedentata.Additional file 7: Figure S5. KOG Function classification the transcriptome of A. grossedentata.Additional file 8: Figure S6. KEGG classification of unigenes of A. grossedentata.Additional file 9 Table S3. YL VS OL DEG enriched KEGG Pathway top 20.Additional file 10: Table S4. Transcript abundance of phenylpropanoid biosynthetic pathways genes identified in A. grossedentata transcriptome.Additional file 11: Table S5. The differentially expressed transcription factors (TFs) in vine tea.Additional file 12: Table S6. Nucleotide sequences of primers used for qRT-PCR.


PIGQ) gene. The affected children were all identified by clinical or research exome sequencing. Clinical data, including EEGs and MRIs, was comprehensively reviewed and flow cytometry and transfection experiments were performed to investigate PIGQ function. Pathogenic biallelic PIGQ variants were associated with increased mortality. Epileptic seizures, axial hypotonia, developmental delay and multiple congenital anomalies were consistently observed. Seizure onset occurred between 2.5 months and 7 months of age and varied from treatable seizures to recurrent episodes of status epilepticus. Gastrointestinal issues were common and severe, two affected individuals had midgut volvulus requiring surgical correction. Cardiac anomalies including arrythmias were observed. Flow cytometry using granulocytes and fibroblasts from affected individuals showed reduced expression of glycosylphosphatidylinositol (GPI)‐anchored proteins. Transfection of wildtype PIGQ cDNA into patient fibroblasts rescued this phenotype. We expand the phenotypic spectrum of PIGQ‐related disease and provide the first functional evidence in human cells of defective GPI‐anchoring due to pathogenic variants in PIGQ.We investigated seven children from six families to expand the phenotypic spectrum associated with an early infantile epileptic encephalopathy caused by biallelic pathogenic variants in the phosphatidylinositol glycan anchor biosynthesis class Q ( ALPalkaline phosphataseCHOChinese hamster ovaryDWIdiffusion weighted imagingEEGelectroencephalogramEIMFSepilepsy of infancy with migrating focal seizuresFLAERfluorescein‐labeled proaerolysinGDDglobal developmental delayGPIglycosylphosphatidylinositolGPI‐APGPI anchored proteinIGDinherited GPI disorderMRImagnetic resonance imaginingWTwildtypePIGQ‐related disease and provide the first functional evidence in human cells of defective GPI‐anchoring due to pathogenic variants in PIGQ.We expand the phenotypic spectrum of 1The GPI‐anchor anchors more than 150 proteins to the cell surface.PIGQ (OMIM 605754).PIGQ variants have been described,PIGQ‐deficient Chinese hamster ovary (CHO) cells transfected with human PIGQ cDNA lacking exon 3 did not restore surface expression of GPI‐APs as well as wildtype (WT),PIGQ. Very little is known about the clinical and functional spectrum associated with PIGQ insufficiency.Phosphatidylinositol glycan anchor biosynthesis class Q (PIGQ) is a key co‐enzyme in the N‐acetylglucosamine transferase complex, the first step catalyzing the biosynthesis of the GPI‐anchor,PIGQ cDNA.Herein, we describe and expand the phenotypical and neurophysiological spectrum of PIGQ deficiency in seven affected individuals and show reduced expression of GPI‐APs in patient granulocytes and fibroblast cell lines, with reversibility using 22.1Ethics approvals were obtained from the local institutional review boards and informed consent was obtained from patients' legal guardians. Details of local ethics approvals are available in 2.2DNA was extracted from whole blood, purified, and analyzed using an exome sequencing trio approach when both parents were available. All exomes were aligned to the human reference genome GRCh37/hg19. Detailed protocols for exome capture, sequencing, pipeline and analysis for each patient/family are available in 2.3For St3b, a skin biopsy was taken and sent to the Centre for Applied Genomics , for establishment of a fibroblast cell line. A skin biopsy for St5 was similarly used for establishment of a fibroblast cell line at the Duke Cytogenetics Laboratory. A fibroblast cell line for St2 was established independently at the Children's Hospital of Philadelphia.l‐glutamine , was used for cell‐line maintenance.HyClone DMEM media  supplemented with 10% fetal bovine serum, penicillin‐streptomycin  and 2 mM 2.4Fresh blood samples from St4 and St5, as well as from healthy controls were stained with the GPI‐AP markers: PE‐conjugated anti human CD16 (BioLegend), FITC‐conjugated mouse anti human CD55 and CD59 (BD Pharmingen), or Fluorescein‐labeled proaerolysin (FLAER)‐Alexa 448 (Cedarlane) for 1 hour on ice. Red blood cells were lysed in FACS Lysing Solution (BD Bioscience). Granulocytes from St1 as well as a control were stained with FLAER and analyzed by flow cytometry in a clinical lab.For fibroblasts, cells were harvested at 80% to 90% confluency, stained with FLAER‐Alexa 448, FITC‐conjugated mouse anti human CD73 (BioLegend) or PE‐ conjugated mouse anti human CD109 (BioLegend) for 1 hour on ice in the incubation buffer containing 0.5% BSA, then fixed in 3.7% formaldehyde. For all assays, non‐specific binding was washed off before analyzing by a BD FACSCanto II system (BD Biosciences) followed by the FlowJo software analysis. We use at least three different controls at two different times for fibroblasts (before and after the rescue). Fibroblast characteristics may change with time in culture and with passages, we thus use for our analysis and figures the control sample which displayed similar cell population patterns  as the test's fibroblasts. Fibroblasts established from St2 and a control were stained with FITC‐CD59 and analyzed by flow cytometry in an independent lab see .2.5PIGQ‐Lv105 (NM_004204.3) or an empty‐Lv105 construct (GeneCopoeia) with the presence of packaging plasmids pMD2.G and psPAX2 (AddGene) were produced in HEK293T cells. Fibroblasts (established from St3b and St5) were transduced with the lentiviruses , and selected by Puromycin resistance, creating a stable cell line. Untransduced cells, rescued cells and control cells were subjected to flow cytometry analyses as described above for fibroblasts.Lentiviruses carrying a wildtype 3PIGQ variants from six unrelated families. The cohort comprised of five females and two males, from various ancestries. Detailed clinical summaries can be found in We identified seven affected individuals with biallelic 3.1PIGQ (NM_148920.2) variants predicted to be damaging and one previously identified variant (p.Y400del).We identified seven previously unreported 3.2PIGQ variants. Four of the seven patients died prematurely . A presumed affected older brother of St5 died a few hours after birth, but DNA was not collected so the relationship to the familial variants cannot be confirmed.There was significant morbidity and mortality associated with biallelic Abnormal prenatal findings included polyhydramnios (St2 and St4), prominent kidneys and premature rupture of membranes at 28 weeks (St3a), severe fetal hepatomegaly and hydronephrosis (St4) and enlarged lateral ventricles and hypoplastic cerebellar vermis suggestive of a Dandy Walker anomaly (St5). Neonatal complications were present in six patients, including respiratory distress , jaundice (St3b and St5), heart failure (St3a), atrial ventricular defect (St3b), patent ductus arteriosus and patent foramen ovale (St5), hypoglycemia (St2) and feeding difficulties with irritability and limb hypertonia of the extremities (St2).Age at onset of epileptic seizures in the remaining six patients ranged from 2.5 to 7 months of age. Seizure types included focal tonic seizures , bilateral tonic‐clonic  evolving to status epilepticus (St4 and St6), myoclonic jerks (St3b and St5), epileptic spasms (St4), absence seizures (St1) and migrating focal seizures (St6) Table . SeizureAll six patients had a severe to profound GDD. Axial hypotonia was observed in all patients Table . AppendiVisual problems were diagnosed in all patients that survived the neonatal period. St3b, St4, St5, and St6 were diagnosed with cortical visual impairment. St1 had short fixation/tracking abilities, whereas an electroretinogram for St2 suggested impaired photoreceptor transmission in the retina. St4 had a mild unilateral conductive hearing deficit, whereas the other subjects had normal hearing Table .Six of the seven patients had facial dysmorphisms including coarse facies and macroglossia. Four  had cranial shape anomalies and three  had pectus excavatum. Other findings included inverted nipples (St1), scoliosis (St2), delayed dentition , bilateral pulmonary interstitial emphysema (St3a), an accessory spleen (St3a), increased nuchal redundancy, short stature, hyperextension of the interphalangeal joints and deep palmar and plantar creases (St4), and an enlarged pinna (St5) Table .Cardiac anomalies were observed in six patients, including prolapsed mitral valve (St2), pulmonary stenosis (St5), pulmonary hypertension (St6), heart block and arrythmia , and right ventricular hypertrophy with poor heart function  Table . GenitouEEG findings were in keeping with clinical presentations and ranged from normal findings early in the disease course, to burst‐suppression patterns, background slowing with interictal multifocal sharp waves, hypsarrhythmia and ictal focal spike and slow wave complexes Table .MRI findings showed no initial abnormalities in the cerebral cortex, but subsequent imaging showed progressive cortical volume loss . For CD16, expression was 28% compared to normal for St4, and 20% in St5. CD55 and CD59 in St5 were normal whereas CD55 was decreased to 68% in St4. However, this patient has an increase in CD59 , but recently, in a cohort of 135 patients with EIMFS, two were found with de novo heterozygous pathogenic variants in PIGA, an X‐linked gene involved in GPI‐anchor biosynthesis, implicating this pathway in this particular epileptic syndrome.Seizure type and control were highly variable Tables  and S3. PIGQ, but there is also increased mortality.PIGQ.St1, who was homozygous for the p.(R538Afs*24) variant, seems to have the mildest phenotype in our cohort. She is currently 11 years old and although she has chewing difficulties, she remains the only living patient who is not G‐tube dependent. While she has limited fixing/tracking abilities, she is also the only patient without a cortical visual impairment. While she is homozygous for a frameshift in the last exon, it is unclear if this can explain her milder phenotype, as St6, with a more severe phenotype and who eventually died, had an even later frameshift p.(G557Dfs*4). This may be in part because St6 was born from consanguineous parents thus other recessive genes may be contributing, or there may not be a clear genotype‐phenotype correlation. Indeed, even the two siblings in our cohort (St3a and St3b) had different clinical presentations and survived to 2 days and 5 years, respectively. Analysis of further affected individuals may reveal a genotype‐phenotype correlation, and given that PIGQ is part of the N‐acetylglucosamine complex, the protein location of the disease‐causing variants may result in differential impact for the binding of other enzymes in the complex. Compared to other IGDs, there are more often bi‐allelic nonsense and/or frameshift variants in PIGT, another key component to GPI‐biosynthesis, to preferential atrophy of the cerebellar vermis,Previous studies have correlated variants in PIGQ pathogenic variants.Although serum alkaline phosphatase (ALP) has been linked with IGDs, evidence suggests that elevated ALP levels are most often associated with variants in later steps of GPI biosynthesis, whereas earlier steps are associated with ER‐associated degradation of the anchor.PIGM promoter,PIGV, PIGO, and PIGS have been partially treated with vitamin B6 supplementation.FOLR1‐mutations,Treatments remain limited, though butyrate helped treat intractable epilepsy due to variants in the PIGQ variants impact GPI‐AP expression.Given the function of the proteins involved in GPI‐anchor biosynthesis, the gold standard for evaluation of functional defects in this pathway is using flow cytometry to assess the effect of the variants on GPI‐anchored proteins.PIGQ pathogenic variants and show that the impaired expression of GPI‐APs in human cells can be improved in vitro by the transduction of WT PIGQ cDNA. These variants lead to impaired GPI‐biosynthesis and subsequently reduced localization of GPI‐APs, resulting in a broad phenotypic spectrum. While the phenotypic spectrum is broad, the clinical overlap of IGDs is significant, and collectively they may represent as much as 0.15% of all developmental disorders.In summary, we expand the phenotypic spectrum of IGDs related to biallelic Devon L. Johnstone, Thi Tuyet Mai Nguyen, Jessica Zambonin, Kristin Kernohan, Anik St‐Denis, Nissan V. Baratang, Taila Hartley, Michael T. Geraghty, Julie Richer, Jacek Majewski, Eric Bareke, Andrea Guerin, Manuela Pendziwiat, Loren D.M. Pena, Hilde M. H. Braakman, Karen W. Gripp, Andrew C. Edmondson, Miao He, Rebecca C. Spillmann, Erik A. Eklund, Allan Bayat, Hugh J.McMillan, Kym M. Boycott, and Philippe M. Campeau declare that they have no conflict of interest.All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1975, as revised in 2000.Devon L. Johnstone, exome analysis, Sanger sequencing, collection and analysis of clinical data, functional work, drafting of manuscript. Thi Tuyet Mai Nguyen, flow cytometry, experiment planning, drafting of manuscript; Jessica Zambonin, collection and analysis of patient data, revision of supplemental clinical data summaries; Kristin Kernohan, exome analysis, experiment planning, editing of manuscript; Anik St‐Denis, flow cytometry, editing of manuscript; Nissan V. Baratang, flow cytometry, editing of manuscript; Taila Hartley, exome analysis, editing of manuscript; Michael T. Geraghty, collection and analysis of patient data, editing of manuscript; Julie Richer, collection and analysis of patient data, editing of manuscript; Jacek Majewski, exome analysis, editing of manuscript; Eric Bareke, exome analysis, editing of manuscript; Andrea Guerin, collection and analysis of patient data, editing of manuscript; Manuela Pendziwiat, collection and analysis of patient data, editing of manuscript; Loren D.M. Pena, collection and analysis of patient data, editing of manuscript; Hilde M. H. Braakman, collection and analysis of patient data, editing of manuscript; Karen W. Gripp, collection and analysis of patient data, editing of manuscript; Andrew C. Edmondson, collection and analysis of patient data, functional data, editing of manuscript; Miao He, cell culture and flow cytometry, editing of manuscript; Rebecca C. Spillmann, collection and analysis of patient data, editing of manuscript; Erik A. Eklund, collection and analysis of patient data, editing of manuscript; Allan Bayat, collection and analysis of patient data, editing of manuscript; Hugh J. McMillan, collection and analysis of patient data, MRI analysis, editing of manuscript; Kym M. Boycott, co‐senior author; Philippe M. Campeau, co‐senior author.APPENDIXS1: Supporting InformationClick here for additional data file.


Pseudomonas aeruginosa is one of the most important nosocomial pathogens and understanding its virulence is the key to effective control of P. aeruginosa infections. The regulatory network governing virulence factor production in P. aeruginosa is exceptionally complex. Previous studies have shown that the peptide chain release factor methyltransferase PrmC plays an important role in bacterial pathogenicity. Yet, the underlying molecular mechanism is incompletely understood. In this study, we used untargeted liquid and gas chromatography coupled to mass spectrometry to characterise the metabolome of a prmC defective P. aeruginosa PA14 strain in comparison with the corresponding strain complemented with prmC in trans. The comprehensive metabolomics data provided new insight into the influence of prmC on virulence and metabolism. prmC deficiency had broad effects on the endo- and exometabolome of P. aeruginosa PA14, with a marked decrease of the levels of aromatic compounds accompanied by reduced precursor supply from the shikimate pathway. Furthermore, a pronounced decrease of phenazine production was observed as well as lower abundance of alkylquinolones. Unexpectedly, the metabolomics data showed no prmC-dependent effect on rhamnolipid production and an increase in pyochelin levels. A putative virulence biomarker identified in a previous study was significantly less abundant in the prmC deficient strain. Pseudomonas aeruginosa is an important causative agent of infections in predisposed individuals such as those suffering from cystic fibrosis or burn wounds [P. aeruginosa possesses an arsenal of virulence factors that is intricately regulated by quorum sensing systems and activated in response to environmental factors via multiple signalling pathways [S-adenosyl-L-methionine-dependent peptide chain release factor methyltransferase PrmC (synonym: HemK) belongs to the latter group. Its absence results in a reduction of in vivo virulence—specifically the inability to produce pyocyanin—and an impaired adaptation to challenging environments [P. aeruginosa), thereby facilitating translation termination and decreasing the rate of stop-codon readthrough [prmC deficiency in Pseudomonas aeruginosa PA14. The exometabolome, i.e., the metabolites secreted into and consumed from the growth media, was recorded along with the cellular metabolome, i.e., the endometabolome. Furthermore, liquid chromatography–mass spectrometry (LC–MS) and gas chromatography–mass spectrometry (GC–MS) were combined for advanced analytical coverage.The ubiquitous γ-proteobacterium n wounds . Deemed n wounds , it posen wounds ,4. P. aepathways . While aronments . Its oridthrough ,8. Previdthrough ,9. In thP. aeruginosa PA14 prmC transposon mutant from the Harvard PA14 mutant library [prmC and the same strain complemented with the prmC gene on the vector, PA14 tnprmC::prmC. In order to assure that all phenotypic effects are due to the absence or presence of the prmC gene, and to exclude that effects are caused by secondary mutations that might occur in addition to the inactivation of prmC in the transposon mutant, the transposon mutant was not compared to the corresponding wildtype, but to an equivalent, prmC-complemented transposon mutant. Five biological replicates of each strain were cultivated in glass flasks in 60 mL modified BM2 medium supplemented with additional amino acids (7 mM (NH4)2SO4, 40 mM K2HPO4, 22 mM KH2PO4, 2 mM MgSO4, 10 µM FeSO4 with 0.01% casamino acids) in a shaking incubator (approx. 150 rpm) at 37 600 of 1.6–1.7 before harvesting. Each biological replicate was split into two technical replicates for GC–MS analysis and two for LC–MS analysis by transferring 20 mL and 5 mL, respectively, to pre-cooled tubes, which were then immediately centrifuged at 9000× g for 10 min at 4 2. Cell pellets for GC–MS analysis were resuspended in 1.5 mL 75% (v/v) methanol with 2 mg/L ribitol as an internal standard and supplemented with 1.5 mL deionised water and chloroform each, followed by vigorous shaking. The organic phase was discarded, and 1 mL of the aqueous phase was evaporated to dryness. Sample preparation, GC–MS analysis, data pre-processing and metabolite identification were performed as described by Berndt et al. [®, Phenomenex, Torrance, CA, USA) using a temperature gradient from 70 p-values were calculated using Welch’s t-test with Benjamini–Hochberg correction for multiple testing. Four replicates (two of tnprmC samples and two of tnprmC::prmC samples) were lost for data analysis due to a technical problem with the GC–MS instrument. Cell pellets and dried supernatants for LC–MS analysis were extracted using 1 mL and 500 2O plus 0.1% formic acid and acetonitrile plus 0.1% formic acid. The MS data was recorded by a quadrupole time-of-flight mass spectrometer  after electrospray ionisation in positive mode. Pooled samples were additionally subjected to collision-induced dissociation by Bruker’s data-dependent auto-MS/MS functionality to record tandem mass spectra for metabolite identification by comparison to authentic chemical standards and/or metabolite databases. Data pre-processing by XCMS used the same parameters as an earlier study [600 of the respective samples. Isotopes and adducts identified by CAMERA [p-values were calculated as described for the GC–MS data. Throughout data processing, intra- and inter-group variability were assessed with the help of principal component analysis and by monitoring the levels of internal standards.To ensure comparability to previous research, the same bacterial strains as in Krueger et al. were used : The P.  library  carryingd before . The celt et al. : Metabolsoftware  and an iMSonline ,15. In by CAMERA  were alsP. aeruginosa PA14 with and without a functional prmC gene showed various differences in their metabolic profiles , a precursor of shikimate. In the proteomic data of Krueger et al., the only enzyme involved in the shikimate pathway with a significant difference between tnprmC and tnprmC::prmC was 3-phosphoshikimate-1-carboxyvinyl-transferase (PA14_23310) [phzC1 and phzC2 were significantly downregulated in tnprmC [The GC–MS metabolomics data, that generally represent primary and intermediary metabolism, showed few consistent trends for metabolic pathways affected by ectively . Strikinhikimate ,18. Alth4_23310) . This enn tnprmC . The genprmC was also observed in the LC–MS endo- and exometabolomics data  in the absence of ics data . From thprmC deficiency were strongest on phenazine and alkylquinolone (AQ) levels, which was expected from previous studies [prmC was non-functional , a direct precursor of alkyl quinolones. The levels of the short or especially long alkyl chain AQs appeared to be more strongly affected by prmC deficiency, whereas the effect on the medium chain lengths (C6 – C9) was less pronounced. This is in accordance with recent findings on the substrate specificity of the biosynthetic enzyme complex PqsBC, which prefers medium chain activated fatty acids over long and short chain ones [prmC [The metabolomic effects of  studies ,9. It wanctional A. The sanctional B. Consisain ones . KruegerprmC deficiency. In the extracellular metabolome, none of the features that were annotated as a rhamnolipid met the significance criteria . In the endometabolome, only one of the rhamnolipids was significantly differentially abundant. This feature was more abundant in tnprmC. These findings contradict those of Pustelny et al., who found reduced rhamnolipid levels in tnprmC [prmC on rhamnolipid production might be time and/or growth phase-dependent and might not manifest before stationary phase.Rhamnolipids are other important components of pseudomonal small molecule mediated virulence. Members of this class of metabolites were not consistently affected by n tnprmC . The disprmC . Pyochelins are low affinity siderophores of P. aeruginosa, whose production is coordinated with pyoverdine, a high affinity siderophore [pchD transcript as being upregulated in tnprmC [Interestingly, a significantly higher abundance of the two pyochelin isomers was detected in tnerophore . Unfortuerophore ,21,22, am/z of 187.1230 and the putative sum formula C12H15N2, displays a significantly differential abundance with approximately 5-fold lower levels in tnprmC and a corrected p-value of 0.0001 in the cellular metabolomics data . The ‘unknown’ metabolite has been identified as a putative biomarker for virulent phenotypes in clinical P. aeruginosa strains in a previous study [Among the non-annotated features, it is particularly noteworthy that M187T7, a metabolite with an prmC and tnprmC::prmC, suggesting an influence of prmC on other parts of pseudomonal metabolism. While the annotated significantly affected metabolites in the endometabolome measured by LC–MS are almost exclusively directly or indirectly associated with virulence as described above, the respective list for the exometabolome also comprises glutathion disulphide with a directional fold change of +7.5 and a corrected p-value of 1.2 × 10‒8. This indicates further consequences of prmC deficiency on the redox regulation of P. aeruginosa, which could potentially be linked to the distorted phenazine production. In the GC–MS metabolomics data, some features stand out by their low p-value and/or prominent fold change, but their connections to prmC cannot be easily established. For instance, succinate is depleted in prmC deficient bacteria , whereas acetylserine is significantly more abundant in tnprmC . These findings show that the complexity of the metabolic consequences of prmC deficiency is considerable and far from being completely understood.There is a substantial number of additional non-annotated features that show highly significant differences in abundances between tnWhile this study consists of a comprehensive metabolomic profiling experiment, it also comes with several limitations. Like many untargeted metabolomics investigations, the data presented here suffers from incomplete metabolite annotation that leaves several interesting pathways underexplored. Furthermore, metabolic fluxes have not been determined which hinders quantitative correlation with transcriptome data. Along with the novel findings outlined above, most results obtained by means of untargeted metabolomics are consistent with previous transcriptomic and proteomic studies ,9 and suprmC in virulence and metabolism of P. aeruginosa. The results support findings from earlier and complementary -omics studies [In conclusion, we present the first metabolomics study of the role of  studies ,9. In ad


Plasma albumin to fibrinogen ratio is involved in human cancer, but its prognostic significance in breast cancer is controversy. In the context of breast invasive ductal carcinoma, this research aims to retrospectively evaluate by preoperative plasma albumin to fibrinogen ratio (AFR) and forecast oncological outcome and recurrence.This retrospective study comprised 230 patients with non-metastatic breast invasive ductal carcinoma who underwent surgery between January 2009 and April 2012 in Fourth Hospital of Hebei Medical University. Patients were categorized base on an optimal value of preoperative plasma fibrinogen (Fib) and albumin. Progression-free and cancer-specific survival were assessed using Kaplan–Meier method. The associations between albumin to fibrinogen ratio and clinical outcomes were assessed with univariate and multivariate analysis. A number of risk factors were used to form nomograms to evaluate survival, and Harrell concordance index (C-index) was used to evaluate the predictive accuracy.P = .03) and overall survival (OS) . Two prediction model of 3-, 5-years OS and DFS based on the AFR was developed.Plasma AFR was significantly associated with diminished disease-free survival (DFS) and overall survival (OS). Multivariate analysis revealed that plasma AFR was an independent prognostic indicator for DFS (HR = 1.346; 95% CI: 1.107–1.636; Elevated preoperative plasma AFR is an independent prognostic factor for oncological outcomes in patients with breast invasive ductal carcinoma. The formulated nomogram showed superior predictive accuracy for DFS and OS. Systemic inflammatory markers have also been reported to have a prognostic association with breast cancer. The systemic inflammatory markers investigated include single markers such as C-reactive protein, Fibrinogen (Fib) is an essential protein for coagulation cascade as well as an acute-phase reaction protein in response to systemic inflammation. Fibrinogen and albumin levels correlated with tumor stage and prognosis in several cancer types. Hence, preoperative albumin to fibrinogen ratio (AFR) level is a useful predictor for severe postoperative complications in elderly gastric cancer subjects after radical laparoscopic gastrectomy.Fibrinogen is a 340 kD glycoprotein consisted of 3 pairs distinct polypeptide chains, α, β, and γ. It is mainly synthesized in liver by hepatocytes and can be degraded by plasmin to form fibrinogen degradation products. Albumin is mainly produced by hepatocytes, however, extrahepatical synthesis by epithelial cells and tumor cells has been described.Whether AFR can serve as a predictor for breast invasive ductal carcinoma patients still remains controversial. The purpose of this study is to further assess the predictive value of AFR in postoperative breast invasive ductal carcinoma and to formulate a new prognostic model through developing a nomogram. Then, according to the formulated nomogram, 2 novel nomogram-based disease-free survival (DFS) and overall survival (OS) was refined.22.1This retrospective study was approved by the Medical Institutional Ethics Committee of Hebei province and the Fourth Hospital of Hebei Medical University hospital (ethics code is 2019MEC111). The study included 230 women who underwent primary surgery at department of breast surgery in the Fourth Hospital of Hebei Medical University from January 2009 to April 2012, without any anticancer therapy before the operation, including radiation therapy, systemic chemotherapy, or hormone therapy.2.2,11 The Ki-67 index was scored as high when 30% or more of the tumor cells were expressed. Analyses for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2) were conducted according to the recommended guidelines of the American Society of Clinical Oncology and College of American Pathologists.,13 Appropriate adjuvant treatments after the surgery were conducted according to the standard guidelines. Most of the hormone receptor (HR) positive patients received the adjuvant hormone therapy for 5 years at least. But none of the patients received the HER2-targeted adjuvant therapy.All patients were followed up after surgery until the date of death or July 2016. The pathologic T: Tumor N: node M: metastasis (TNM) stage was judged by the 7th American Joint Committee on Cancer.2.3The following data were extracted and recorded from our database: clinicopathological features, including age, TNM, lymphovascular invasion nuclear grade, ER, PR, HER2, Ki-67, p53.2.4Fasting blood samples from each participant were before the operation. Blood cell analyses including white blood cell (WBC), red blood cell (RBC), hemoglobin (Hb), platelet (PLT), and albumin, Fib.2.5http://www.r-project.org/). The cutoff value of AFR was determined by median. The OS was compared by Kaplan–Meier curves and analyzed using the log-rank test via Empower Stats. The univariate and multivariate analyses and hazard ratios (HRs) were used by Cox proportional hazards regression model to find its independent prognostic risks, and P < .05 was considered as statistically significant difference. Two novel prognostic nomograms based on AFR for DFS and OS was formulated by Empower Stats. Its predictive performance was measured by concordance index (C-index), calibration curve, and decision curve analysis (DCA) was formulated by R project version.All statistical analyses and random allocation were performed by Empower Stats and R project version 3.3.3 . The median follow-up was 67 months . Means, standard deviations, and ranges of laboratory results for albumin, fibrinogen, and AFR were 41.29 ± 3.39 g/d , 3.21 ± 0.54 g/L , and 13.24 ± 2.43 , respectively. The optimal values for AFR were 13.1 by median . Patients with higher AFR had shorter OS  and a high-risk group . The median OS of the low-risk group and high-risk group is 66.63 months and 62.36 months, respectively. The median DFS of the low-risk group and high-risk group is 65.29 months and 59.97 months, respectively. The DFS and OS were significantly different among the 2 subgroups  and operable soft tissue sarcoma.Few cancer researchers have study AFR. Alb expressions are recommended to be a reliable prognostic factor in patients with cancer.P < .05). In this study, using univariable analysis and subsequent multivariable analysis, we identified WBC, TNM, lymphovascular invasion, nuclear grade, ER/PR(+), P53, and AFR as independent prognostic factors for OS. C-index of the nomogram based on AFR predicted OS with an accuracy of 0.882, and, we identified WBC, TNM, lymphovascular invasion, nuclear grade, P53, Ki-67, and AFR as independent prognostic factors for DFS. C-index of the nomogram based on AFR predicted DFS with an accuracy of 0.922. A value of 0.5 implies that the predictor or model has no discriminatory ability, and a value of 1.0 implies perfect discrimination. A model with an receiver operating characteristic curve of 0.70 to 0.80 is considered good whereas one with an Area Under Curve of 0.80 to 0.90 has excellent discrimination.,22 The nomograms performed well with good discrimination and calibration, identifying this model as a simple and easy tool for estimating OS and DFS of individual patients with breast invasive ductal carcinoma.Our study showed that AFR was a significant and powerful independent prognostic factor in breast cancer. The multivariate Cox regression analysis confirmed the independence of the association between AFR levels and DFS and OS (In this study, we developed 2 nomograms including AFR to improve prognosis prediction for breast invasive ductal carcinoma patients. The nomograms can be used to better predict an individual patient's probability of 3-, and 5-year DFS and OS. The 2 nomograms were performed using calibration plots and the C-index. The nomograms performed well with a good calibration. Furthermore, the C-index for DFS and OS was satisfactory0.922 and 0.884, respectively. indicated preoperative fibrinogen to albumin ratio was a strong and significant independent unfavorable prognosticator of breast cancer, especially in stage II/III and luminal A-like subgroups. Our reach was difference from Hwang's et al. The reason maybe is the difference of the s population. Peoperative AFR level is a useful predictor for severe postoperative complications in elderly gastric cancer subjects after radical laparoscopic gastrectomy. This present study was to indicate preoperative AFR as an independent risk factor for DFS and OS in breast invasive ductal carcinoma patients. The close association between inflammation and breast invasive ductal carcinoma might be a possible mechanism.Hwang's et alThe present study had several limitations. The sample size was still small. Although we create nomograms, we do not validate them, future studies are needed to externally validate the proposed nomograms.5The study is to reveal that breast invasive ductal carcinoma patients with high AFR expression had a higher risk of metastasis and poorer survival than patients with low AFR expression. Our results demonstrated that AFR expression in breast invasive ductal carcinoma was an independent predictor of patient outcomes. The 2 nomograms were developed for predicting the probability of 3-, and 5-year DFS and OS. The model might facilitate both clinician and patient counseling and individualized adjuvant treatment decision-making, as well as follow-up scheduling.Conceptualization: Yunjiang Liu.Data curation: Yaheng Zhao, Yan Yang, Hongsong Zhang.Formal analysis: Feng Liu, Wei LiInvestigation: Yaheng Zhao.Methodology: Lihua Zheng, Peng Liu, Feng Liu.Visualization: Feng Liu.Writing – original draft: Lihua ZhengWriting – review & editing: Lihua Zheng, Feng Liu, Yunjiang Liu.


Neobalanocarpus heimii (King) Ashton is a highly sought after tree species endemic to the Malay Peninsula. Due to its scarcity and high value, the tree is classified under the IUCN Red List categories of Vulnerable. In this study, we assembled the complete chloroplast (cp) genome of N. heimii using data from high-throughput Illumina sequencing. The Chengal cp genome is 151,191 bp in size and includes two inverted repeat regions of 23,721 bp each, which is separated by a large single copy region of 83,801 bp and a small single copy region of 19,948 bp. A total of 130 genes were predicted, including 37 tRNA, 8 rRNA, and 85 protein-coding genes. Phylogenetic analysis placed N. heimii within the order Malvales.Known for its durable timber quality,  Neobalanocarpus heimii (King) Ashton, is a tropical hardwood found only in the Malay Peninsula. Wood from Chengalis highly durable and is among the strongest in the world  as the seed sequence. The assembled cp genome was annotated using the online annotation tool DOGMA . A voucher specimen (FBL05002) is deposited at the Forest Biotechnology Laboratory in the Faculty of Forestry, UPM. A genomic library with an insert size of ∼400 bp was prepared using a TruSeq DNA Sample Prep Kit  and sequenced on an Illumina HiSeq X Ten platform. Approximately 8 Gb of raw data were generated through pair-end 150 bp sequencing. After removal of adapter sequences, raw reads were fed into the NOVOPlasty  was 151,191 bp in length, consisting of a pair of inverted repeat regions of 23,721 bp each, a large single copy region of 83,801 bp, and a small single copy region of 19,948 bp. A total of 130 genes were annotated, including 37 tRNA, 8 rRNA, and 85 protein-coding genes. The overall GC content of the cp genome was 37.42%.The complete cp genome of N. heimii and eight other complete cp genomes of species from the order Malvales, comprising of six Malvaceae species  and two Thymelaeaceae species (Daphne kiusiana and Aquilaria yunnanensis). Melastoma candidum from the order Myrtales was included as outgroup. The maximum likelihood tree constructed with RAxML (Stamatakis http://www.geneious.com, Kearse et al. N. heimii within the order Malvales (A phylogenetic analysis was carried out with Malvales .


The present study compared the acute effects of aerobic (AER), resistance (RES), and combined (COM) exercises on blood pressure (BP) levels in people with resistant hypertension (RH) and nonresistant hypertension (NON-RH).  Twenty patients (10 RH and 10 NON-RH) were recruited and randomly performed three exercise sessions and a control session. Ambulatory BP was monitored over 24 hours after each experimental session.  Significant reductions on ambulatory BP were found in people with RH after AER, RES, and COM sessions. Notably, ambulatory BP was reduced during awake-time and night-time periods after COM. On the other hand, the effects of AER were more prominent during awake periods, while RES caused greater reductions during the night-time period. In NON-RH, only RES acutely reduced systolic BP, while diastolic BP was reduced after all exercise sessions. However, the longest postexercise ambulatory hypotension was observed after AER (~11 h) in comparison to RES (~8 h) and COM (~4 h) exercises.  Findings of the present study indicate that AER, RES, and COM exercises elicit systolic and diastolic postexercise ambulatory hypotension in RH patients. Notably, longer hypotension periods were observed after COM exercise. In addition, NON-RH and RH people showed different changes on BP after exercise sessions, suggesting that postexercise hypotension is influenced by the pathophysiological bases of hypertension. Hypertension refers to a highly prevalent multifactorial condition characterized by chronic elevations in blood pressure (BP) that is strongly associated with negative health-related outcomes , 2. WhenHowever, approximately 20% of the patients clinically diagnosed with NON-RH may present an inability to achieve BP levels lower than proposed cut-offs, regardless of the optimal dose of 3 or more antihypertensive drugs, including 1 diuretic, a condition denominated as resistant hypertension (RH) –6. In thHence, experts in the field  have sugNotably, the beneficial effects of physical exercise are not restricted to its chronic practice since reduced BP levels may be observed after an acute session of exercise, a phenomenon denominated postexercise hypotension (PEH) , 13, 18.The effects of physical exercise in RH are still poorly explored. A previous seminal study found postexercise ambulatory hypotension after AER exercise in patients with RH . HoweverBased on these premises, the present study investigated the acute effects of AER, RES, and COM on 24-hour ambulatory BP monitoring (ABPM) in people with RH and NON-RH. In addition, postexercise ABPM were compared between RH and NON-RH. Our hypothesis is that AER, RES, and COM might cause different patterns of PEH in RH and NON-RH.This was a randomized controlled crossover trial that investigated the acute effects of AER, RES, and COM exercises on 24-hour ABPM in people with RH and NON-RH. Concealed randomized allocation into one of four experimental sessions was performed by an independent researcher using a simple computer-generated list of random numbers and an allocation ratio of 1 : 1 : 1 : 1. All researchers, including evaluators, exercise supervisors, and those responsible for statistical analysis, knew where participants were allocated. Participants were also aware of exercise session but were blind to study hypothesis.Recruitment was carried out through the Outpatient Resistant Hypertension Clinic of University of Campinas . Twenty patients agreed to participate in the study protocol: 10 subjects with stage 2 hypertension (NON-RH) and 10 subjects clinically diagnosed with RH. RH was defined as an uncontrolled BP despite the use of ≥3 antihypertensive medications at optimal doses, including a diuretic if possible, or patients with controlled BP using ≥4 antihypertensive medications . The diaSample size was estimated using G∗Power version 3.1.9.2. on the basis of the magnitude of the mean differences in SBP levels  among thNCT02987452), and all patients that met the eligibility criteria gave their informed written consent before participation. The investigation was performed according to the Helsinki Declaration of 1975 (as revised in 1983).This study was approved by the Research Ethics Committee of the Faculty of Medical Sciences, University of Campinas   always in the mornings (07:00-12:00 am) in the Laboratory of Cardiovascular Pharmacology of the University of Campinas. Experiments were separated into two distinct phases. In the first phase, participants completed a familiarization period to familiarize them with the proper technique of the physical exercises utilized in the present study. Afterwards, the optimal exercise load to AER, RES, and COM was determined. The familiarization period took place in 4-6 alternate days. In the second phase, participants were requested to come five times to the laboratory after a 12 h overnight fast, including water, energetic beverages, and alcohol consumption, and without performing intense physical activity for 24 h.In the first visit, a Bioimpedance Analyzer 450  was used for anthropometric measurements –33. BioiABPM was recorded for a 24-hour period using the Spacelabs equipment 90217 . Awake-time was considered the interval between the first and tenth hours after the experimental sessions, while night-time referred to the period between the eleventh and eighteenth hours after the experimental sessions. Participants were instructed to maintain and record normal daily activities in a personal diary.Exercise protocols were based on American College of Sports and Medicine (ACSM) guidelines , 29. AERst chair squat, 2nd vertical bench press, 3rd seated knee raise, 4th seated row, 5th dorsiflexion and plantar flexion, and 6th shoulder abduction). A 1-minute interval was adopted between sets and exercises. All exercises were performed in the total range of motion and muscle contractions—concentric and eccentric—and were performed at moderate velocity (2 sec for each). Participants were instructed to avoid the Valsalva maneuver during the entire muscle contraction. COM exercise consisted of a session of AER exercise performed at 50-60% HRmax for 25 minutes plus a session of RES based on 6 exercises with 2 sets of 12 submaximal repetitions at moderate intensity according to the modified Borg scale [A minimum interval of 96 hours was required between exercise sessions. AER exercise was performed in a treadmill for 45 minutes at 50-60% of maximal heart rate (HRmax) obtained from the ergometric stress test. HR was monitored continuously across the exercise session using a cardiac monitor . RES exercise consisted of 6 exercises with 4 sets of 12 submaximal repetitions performed at moderate intensity    method  based onCONT session involved the continuous monitoring of blood pressure to over 60 minutes. This evaluation was considered the baseline values for all comparisons. Afterwards, the ABPM was removed, and the participants remained seated, but not exercising, in the machines for another 60 minutes.t-test. A two-way ANOVA followed by a Bonferroni post hoc test was performed to identify differences among the different times of evaluations in the experimental sessions. The area under the curve (AUC) was calculated. Peaks less than 10.0% of the distance from minimum to maximum Y were ignored. ANOVA followed by a Bonferroni post hoc test was performed to identify differences among experimental sessions. Categorical variables were presented in frequencies and/or percentages and compared by chi-squared test. All statistics analyses were performed using the GraphPad Prism 6.0 . All statistical methods are two-tailed, P values were calculated, and statistical significance was set at ≤0.05.Normality of data was tested using the Shapiro-Wilk test. Baseline comparisons among groups were performed using unpaired Student's Participant recruitment and experimental sessions were conducted from January 2017 to December 2017. No exclusions or dropouts occurred after randomization, and no patients reported changes in antihypertensive medication during the follow-up examination. Participants completed all experimental sessions.P = 0.03). No other differences were found between the groups.Characteristics of study participants are shown in nd to 7th (Δ = −17.6 mmHg), 12th to 13th (Δ = −15.4 mmHg), and 15th to 16th (Δ = −18.0 mmHg) hours after exercise. A shorter PEH period was observed after RES, given that significant reductions were only found from the 14th to 15th (Δ = −19.5 mmHg) and 17th to 18th (Δ = −15.3 mmHg) hours after exercise. On the other hand, the longest PEH period was observed after COM, so that significant reductions were observed from the 2nd to 6th (Δ = −19.5 mmHg), 13th to 17th (Δ = −18.6 mmHg), and 19th (Δ = −14.3 mmHg) periods. Diastolic BP was also reduced after acute exercise protocols. PEH was observed after AER from the 4th to 6th (Δ = −11.6 mmHg), 11th to 13th (Δ = −12.0 mmHg), and 15th to 17th (Δ = −14.1 mmHg) hours after exercise. RES caused significant PEH from the 14th to 15th (Δ = −11.3 mmHg), 18th (Δ = −8.5 mmHg), and 20th (Δ = −3.2 mmHg) hours after exercise. As observed in systolic BP, COM caused the longest PEH period: from the 3rd to 6th (Δ = −11.5 mmHg) and 10th to 19th (Δ = −11.7 mmHg) hours after exercise. There were no significant differences in systolic and diastolic BPs after CONT.th moment relative to baseline (Δ = −18.0 mmHg). Diastolic BP (th to 20th (Δ = −13.3 mmHg) and 21st to 22nd (Δ = −12.9 mmHg) hours after exercise. A shorter PEH period was observed after RES: from the 12th to 20th (Δ = −12.9 mmHg) periods. COM reduced diastolic BP from the 12th to 16th (Δ = −11.8 mmHg) hours after exercise. There were no significant differences in systolic and diastolic pressures after CONT.tolic BP  was reduThere were no significant differences in systolic and diastolic BPs between RH and NON-RH after acute exercise sessions.There were no harms or other unintended effects of the intervention.The main findings of the present study indicate that an acute session of AER, RES, and COM exercises significantly reduced ambulatory BP in RH patients. Notably, longer reductions in systolic and diastolic BPs were observed after COM (~12 h) relative to AER (~6 h) and RES (~3 h) exercises. According to AUC analysis, COM reduced SBP in both awake-time and night-time periods in comparison to AER and RES. AUC analysis also indicated that the effects of AER exercise were more predominant in the awake-time period, while lower DBP was observed after RES in the night-time period. People with NON-RH showed different BP responses to exercise sessions. Indeed, systolic BP was only significantly reduced after RES. Diastolic BP was reduced after all exercise sessions, but the longest reductions were observed after AER (~11 h) in comparison to RES (~8 h) and COM (~4 h). AUC indicated that the lower diastolic BP values after AER were mainly observed in the awake-time period. Although different BP responses were observed between RH and NON-RH, no significant differences were observed among groups.Findings of the present study are partially supported by Santos et al. , who repThese different results do not seem to occur due to baseline BP levels or designs of AER exercise, given that participants of both studies showed similar baseline ABPM values (~145 mmHg) and performed AER exercise sessions with similar volume and intensity. Hence, a possible explanation for these distinct results may be the amount of exercising muscle mass , 38. In This is the first study that investigated the acute effects of different types of exercise on ABPM. Our findings bring new light to the exercise cardiology field by demonstrating that COM exercise elicits longer reductions in systolic and diastolic BPs compared to AER and RES. COM reduced ABPM over 24 hours, including awake-time and night-time periods. On the other hand, the effects of AER exercise occurred predominantly in the awake-time period, while larger and longer blood pressure reductions were observed in the night-time period after RES. Taken as a whole, these findings suggest that COM exercise might provide further benefits relative to AER or RES exercise alone in RH patients.Earlier studies –28 in noA possible explanation for the greater blood pressure reductions observed after AER exercise is based on the fact that this kind of exercise reduces cardiac output (CO) without changing peripheral vascular resistance (PVR) , 39, 40.On the other hand, RES seems to potentiate postaerobic blood pressure reductions in RH. Only one study investigated the possible mechanisms underlying blood pressure changes after exercise in RH, and researchers  reportedNevertheless, these inferences must be interpreted with caution because they were based on different populations and in mechanisms that were investigated in the first hour after exercise sessions. In addition, CO and PVR have demonstrated an important intersubject variability , which iThe cellular mechanisms underlying blood pressure reduction after exercise sessions were not investigated in the present study. However, many of them may likely have influenced our findings. The role of the activation of H1 and H2 histamine receptors on blood pressure control during  and afteAcute improvements in cardiac autonomic modulation are another possible mechanism mediating exercise-induced blood pressure reduction, although studies have reported conflicting results. Liu et al.  reportedOn the other hand, studies have reported increased sympathetic activity and sympathovagal balance in the first hour after RES in untrained normotensive , 39, 46 Only a few studies investigated the effects of COM on autonomic modulation. Teixeira et al.  and SaccTaken together, this information might suggest that histamine and cardiac autonomic modulation are mediating PEH after COM in RH, while reduction on blood pressure after RES in NON-RH is at least predominantly mediating by a compensatory reflex vasodilation. Nevertheless, generalizations are inappropriate and future studies are needed to confirm our premises.A question that remains unanswered is why RES and AER exercises reduced BP in different periods of the day in patients with RH. The behavior of BP throughout the day after exercise performance is still under debate. According to Nishiguchi et al. , reducedThese findings might indicate that RES and AER exercises affect differently the circadian cycle and the release of possible mechanisms associated with blood pressure reduction  , thus caThe divergent blood pressure responses to exercise sessions between RH and NON-RH may also occur due to antihypertensive medication, given that all patients with RH were under diuretic treatment, while this class of antihypertensive medication was only taken by five participants in the NON-RH group. Indeed, authors have proposed that acute exercise combined with antihypertensive medication may cause greater PEH than exercise alone.Anunciação et al.  observedWe acknowledge some limitations of the present study. First, our findings are based on a small sample size. Second, obesity was not controlled as an eligibility criterion. Third, the possible mechanisms responsible for PEH were not assessed. Fourth, although exercise sessions were equalized by time, different caloric expenditures were likely provided by RES 124 kcal), AER , and COM  exercises 4 kcal, A. HoweverOur findings have practical applications, given that low BP levels after acute exercise may contribute to low cardiovascular risk during the performance of ADL , 24. HenFindings of the present study indicate that AER, RES, and COM exercises elicit systolic and diastolic postexercise ambulatory hypotension in RH patients. Notably, longer hypotension periods were observed after COM exercise. In addition, NON-RH and RH people showed different changes on BP after exercise sessions, suggesting that postexercise hypotension is influenced by the pathophysiological bases of hypertension.


Reference symphysis-fundal height (SFH) curves for pregnancies complicated by maternal hyperglycemia are not available.  To build an SFH curve according to gestational age for pregnant women with hyperglycemia-type 2 diabetes (T2DM), gestational diabetes mellitus (GDM), or mild gestational hyperglycemia (MGH) and compare it with three other curves in use in Brazil.  Prospective cohort study of 422 pregnant women with hyperglycemia attending the Perinatal Diabetes Research Center (PDRC) of Botucatu Medical School, São Paulo State University/UNESP. Between 13 and 41 weeks of pregnancy, 2470 SFH measurements were obtained (mean 5.85 per woman). For the assessment of glycemic control, 2074 glucose level measurements were taken and the glycemic mean (GM) at each gestational week was estimated. r2 = 84.6%). At visual analysis, P10 and P90 SFH measurements were higher in the study curve than in the three other curves. Statistical analysis confirmed that SFH median values in this study were higher than those in the reference curve of habitual risk pregnancies, especially after 19 weeks of pregnancy.  GM was adequate (<120 mg/dL) in 94.9% and inadequate (≥120 mg/dL) in 5.1% of the cases. The equation applied for SFH prediction was expressed as SFH = 1.082 + 0.966∗week ( Taking into account that the maternal hyperglycemia was at strict control, our unedited results suggest that the current SFH curve can be a useful tool in prenatal care of T2DM, GDM, and MGH pregnant women. Symphysis-fundal height (SFH) measurement is a simple method to assess fetal growth in relation to gestational age (GA) that can detect twin pregnancies, polyhydramnios, oligohydramnios, and other complications. Ultrasound may be an accurate tool to detect fetal growth restriction (FGR) and macrosomia, with sensitivity at 93% and 90%, respectively. However, the SFH measurement is a current universal practice, and a change from what is usually practiced in a particular setting is not recommended , 2.In the Brazilian Public Health System (SUS), the reference SFH chart was developed by Fescina et al., based on measurements taken from 47 Paraguayan pregnant women , 4.Owing to population differences, some authors advocate that charts should be locally generated for best results, and specific SFH curves were produced from Brazilian pregnant women. Oppermann et al.  construcNotwithstanding their differences in performance, these current standards are unlikely to be a suitable reference for both low-risk and high-risk populations. Gestational diabetes mellitus (GDM) and preeclampsia are common complications of pregnancy that are known to be associated with adverse perinatal outcomes such as macrosomia, fetal growth restriction (FGR), low birthweight, and consequent higher risk of perinatal death –11. HoweThis study was carried out at the Perinatal Diabetes Research Center (PDRC) of Botucatu Medical School, São Paulo State University/UNESP, and approved by Institutional Review Board of the Botucatu Medical School/UNESP (CEP-FMB/UNESP #255/08). Assuming a prevalence of gestational hyperglycemic disorders of 12%, the minimum sample size was estimated at 165 subjects and at least 13 weekly SFH measurements during pregnancy.In the current study, 422 pregnant women were enrolled and 2470 SFH measurements were obtained. Inclusion criteria were as follows: type 2 diabetes mellitus (T2DM) without micro- or macrovascular diseases (classes B and C of Priscilla White), gestational diabetes mellitus (GDM), or mild gestational hyperglycemia (MGH); receive prenatal and labor care at our center; gestational age confirmed by ultrasound before 20 weeks of pregnancy; live singleton pregnancy; and written informed consent. Exclusion criteria were as follows: birth defects detected during pregnancy; type 1 diabetes mellitus (T1DM); and associated consumptive disease. The study flowchart is in 2), smoking status (yes/no), number of previous pregnancies, C-sections and abortions, gestational age at birth, and glycemic mean (GM).Using a pretested protocol designed for the study, data were collected via review of medical records and semistructured interviews and included information on age (years), race (white/nonwhite), body mass index  or 1 h postload plasma glucose equal or above 10.0 mmol/L (180 mg/dL) or 2 h postload plasma glucose between 8.5 and 11.0 mmol/L (153–199 mg/dL) 5 mg/dL o; the cri2, subjects were classified as underweight (<18.5 kg/m2), adequate weight (18.6–24.9 kg/m2), overweight (25–29.9 kg/m2), obese class I (30–34.9 kg/m2), obese class II (35–39.9 kg/m2), or obese class III (≥40 kg/m2)  demonstrated SFH variation only in function of gestational age.It is worthy of note that despite adequate glycemic control, as confirmed the by the GM values lower than 120 mg/dL observed over 24 of the 27 gestational weeks, the pregnant women participating in this study had T2DM, GDM, or MGH and were mostly white (70.9%) and aged ≥25 years (81.7%) with BMI ≥ 25 kg/mThe fact that other studies including populations with characteristics similar to those seen in our subjects are not found in the literature hampers an in-depth analysis of our results. On the other hand, these same characteristics suggest the inadequacy of the currently available SFH curves to the population under study.High sensitivity is a prerequisite for a good screening test. Nonetheless, the sensitivity of the curve of Fescina et al.  to detecGiven our greater interest in detecting fetal macrosomia, which is more common in pregnancies complicated by maternal hyperglycemia, we could keep using the curve of Fescina et al.  or even This study has limitations. The main one is the lack of subsequent FS assessments of the same pregnant woman every week of pregnancy. Due to the prenatal logistics, we chose serial measures in an expanded population but evaluated by a single person, adequately trained and qualified for this. We try to guarantee the greatest number of measures for each pregnant woman, and we reach an average of six evaluations/pregnant woman. In contrast, the gestational age confirmed by ultrasound before 20 weeks , 19 reprFetal growth in high-risk pregnancies should be monitored with serial ultrasound scans by plotting anthropometric measures against international standards . UnfortuIn our study, with regard to the visual comparison of the current SFH curve with the reference ones, it was observed that (i) concerning the Fescina et al. SFH chart , the limIn clinical practice, the Fescina et al. SFH chart  is the BThe unedited results of this study, with strict maternal glucose control, as well as the predictive performance of the current SFH curve, suggest that it may be a useful tool in the prenatal care of T2DM, GDM, and MGH pregnant women.


The atherogenic index of plasma (AIP) is elevated in fatty liver disease, but its value in non-obese people with non-alcoholic fatty liver disease (NAFLD) is unclear. This study aimed to investigate the relationship between AIP and NAFLD as well as to determine whether AIP might be used as an indicator of NAFLD in non-obese individuals.The present study involved non-obese Chinese and Japanese participants. Risk factors are evaluated using univariate and multivariate analysis. The performance of risk factors was compared according to the area under the receiver operating characteristic curve.In the unadjusted model, the odds ratio (OR) for every 1 standard deviation (SD) increase in AIP was 52.30. In adjusted models I and II, the OR for every 1 SD increase in AIP was 36.57 and 50.84, respectively. The area under the receiver operating characteristic curve for AIP was 0.803 and 0.802 in the development and validation groups, respectively. The best cut-off value of AIP for discrimination between NAFLD and non-NAFLD was 0.005 in the Chinese group and − 0.220 in the Japanese group.AIP and NAFLD are positively correlated in Chinese and Japanese populations. Therefore, AIP can be used as a new screening indicator for non-obese people with NAFLD in different nations. Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver disease worldwide. The disease may progress to liver cirrhosis and liver cancer , 2. ObesThe plasma atherosclerosis index (AIP) is a quantitative indicator used to evaluate blood lipid levels. AIP has good predictive value for dyslipidemia diseases such as diabetics, atherosclerosis and heart disease –9. AIP hThis study aimed to investigate the relationship between AIP and NAFLD in non-obese people and to demonstrate that AIP is an independent risk factor for NAFLD in non-obese individuals.www.datadryad.org), which allows users to download raw data free of charge. These data are anonymous. According to the Dryad Terms of Service, researchers may apply these data in secondary analysis without infringing on the authors’ rights. In the study, the Chinese data came from the following source [Data in the study came from the Dryad Digital Repository website  LDL-c > 3.12 mmol/L; and (6) use of antihypertensive agents, antidiabetic agents or lipid-lowing agents. Diagnosis of fatty liver was performed in accordance with the ultrasound diagnostic criteria of the Chinese Liver Disease Association [2, and represented an index of body fat. All biochemical values were analyzed with an automatic measurement analyzer (Abbott) according to standard methods. Among the research population in Japan, a total of 12,932 participants who had undergone medical examination at Murakami Memorial Hospital between 2004 and 2015 were recruited and selected according to the following exclusion criteria: (1) a lack of important data; (2) known liver disease; (3) alcohol intake exceeding 60 g per day for men or 40 g per day for women; (4) drug use; (5) fasting blood glucose ≥6.1 mmol/L; and (6) BMI ≥ 25 kg/m2. Because this study was a secondary study, and the data were anonymous, no informed consent was required. Specific details are given in the original report [For the Chinese study population, participants took part in health examination at the First Affiliated Hospital of Wenzhou Medical University between January 2010 to December 2014. A total of 78,304 participants were recruited and selected on the basis of the following exclusion criteria: (1) a lack of required data; (2) excess alcohol consumption (more than 20 g per day for men or 10 g per day for women); (3) known liver disease; (4) BMI ≥ 25 kg/mociation . AIP wasociation . BMI wasNAFLD is defined by the diffusion enhancement of near-field echo and the gradual attenuation of far-field echo in the liver area (stronger than that in the kidney and spleen area). One of the following conditions must be present: (1) reduced blood flow signal but normal blood flow distribution; (2) mild to moderate hepatomegaly, with round and blunt borders;(3) unclear or incomplete envelope of the right liver lobe and the diaphragm muscle;(4) unclear liver cavity structure. NAFLD is diagnosed with abdominal ultrasonography performed by trained technicians.2) [P-value < 0.05 was considered statistically significant. The R  statistical package and GraphPad Prism  were used for statistical analysis.The overall statistical analysis in this study consisted of five steps. First, in the Chinese study, the population was divided into a development group and validation group in a 7:3 ratio. Continuous variables are evaluated by calculating the means ± standard deviations (SD)  or medians (quartiles) (skewed distribution), and categorical variables are evaluated by calculating frequencies or percentages. Differences across groups were analyzed using One-way ANOVA , Kruskal-Wallis H (skewed distribution) test and chi-square test . Second, risk factors in the development group were analyzed using univariate and multivariate regression analysis. Independent variables were tested for collinearity and were excluded with the variance inflation factor (VIF) ≥ 10. Collinear VIF = 1/(1-R2) . The sub2) , the resAs shown in Table The univariate and multivariate analysis results are shown in Table P <  0.001). After adjustment for age, GGT, ALT, ALB, DBIL, UA, FPG, LDL-C and BMI (adjusted model I), the OR for every 1 SD increase in AIP was 36.57 . After full adjustment for sex, age, GGT, ALT, AST, TP, ALB, GLB, TB, DBIL, BUN, Cr, UA, FPG, TC, TG, LDL-c and BMI (adjusted model II), the OR for every 1 SD increase in AIP was 50.84 . With OR > 1 in three models, AIP was positively correlated with NAFLD, and the results were stable , whereas the tests for interactions were not statistically significant for ALB, GLB, DBIL and LDL-c (P > 0.05). Although all variables were risk factors, they did not affect the correlation between AIP and NAFLD. Compared with that of patients over 60 years old, the AIP in patients under 60 years old was associated with higher risks of NAFLD (OR: 61.03 VS. 25.67). Similar results were found in female and male patients (OR: 92.43 VS. 29.11).The subgroup analysis results are shown in Table The purpose of this study was to analyze the relationship between AIP and NAFLD as well as to verify the diagnostic value of AIP in non-obese patients with NAFLD. AIP has been found to be an independent risk factor for NAFLD in non-obese patients through univariate and multivariate regression analysis, in agreement with results in obese patients . Here, iPrevious studies have focused mainly on obese patients with BMI above 25, thus often causing people with normal BMI to ignore their eating habits . DietaryPrevious research has indicated that TG/HDL-c can be used as predictor of NAFLD in non-obese people . This stThe study has several strengths. First, this research was a large sample, multi-region and multi-ethnic study. Second, this was a retrospective study with many confounding factors, in which strict statistics was used to minimize residual confounding. Third, the authors identified effective cut-off points for AIP in Chinese and Japanese populations, thus providing data support for clinical diagnosis.There were, however, several limitations to this study. First, because it was a secondary study, information on the lifestyles and eating habits of the participating population was not collected. Second, this study focused on the relationship between AIP and NAFLD, without further evaluation of other variables.The results indicate that AIP and NAFLD are positively correlated in Chinese and Japanese populations. Therefore, AIP may be used as a new screening indicator for non-obese people with NAFLD in different nations, thus providing a reference for clinical work.Additional file 1: Table S1. Baseline Characteristics of the Japanese Study Participants. Table S2. Effect of AIP on the incidence of NAFLD in Japanese. Table S3. AUROC analysis for different continuous predictors in Japanese groups. Figure S1. The relationship between AIP and NAFLD. Figure S2. AUROC curve of all risk factors in the Japanese group.


Few studies have been designed to evaluate the short-term outcomes between robotic-assisted total gastrectomy (RATG) and laparoscopy-assisted total gastrectomy (LATG) for advanced gastric cancer (AGC). The purpose of this study was to assess the short-term outcomes of RATG compared with LATG for AGC.We retrospectively evaluated 126 and 257 patients who underwent RATG or LATG, respectively. In addition, we performed propensity score matching (PSM) analysis between RATG and LATG for clinicopathological characteristics to reduce bias and compared short-term surgical outcomes.p = 0.003), less intraoperative bleeding  and more N2 tier RLNs  than the LATG group. Additionally, the total RLNs of the RATG group were almost significantly different compared to that of the LATG group . Moreover, no significant differences were found between the two groups in terms of the length of incision, proximal resection margin, distal resection margin, residual disease and postoperative hospital stay. There was no significant difference in the overall complication rate between the RATG and LATG groups after PSM . Grade II complications accounted for most of the complications in the two cohorts after PSM. The conversion rates were 4.55 and 8.54% in the RATG and LATG groups, respectively, with no significant difference (p = 0.145), and the ratio of splenectomy were 1.59 and 0.39% (p = 0.253). The mortality rates were 0.8 and 0.4% for the RATG and LATG groups, respectively (p = 1.000).After PSM, the RATG group had a longer mean operation time (291.14 ± 59.18 vs. 270.34 ± 52.22 min, This study demonstrates that RATG is comparable to LATG in terms of short-term surgical outcomes. Gastric cancer (GC) is the fifth most common cancer and the third leading cause of cancer-related death worldwide . Its incPatients diagnosed with GC by means of gastroscopy, biopsy and histopathological assessment who underwent total gastrectomy were screened from the prospectively maintained gastric cancer database at the Department of General Surgery, Southwest Hospital, Army Medical University from March 2010 to December 2017. Data from 573 consecutive patients who underwent RATG or LATG for gastric cancer were collected. The inclusion criteria of the study were defined as follows: (1) age between 18 and 80 years old; (2) no preoperative chemotherapy or radiation therapy performed before surgery; (3) depth of invasion confined to pT2, pT3, or pT4a; (4) no distant metastasis or invasion to adjacent organs; (5) receiving LATG or RATG with D2 lymphadenectomy. Patients who underwent RATG were matched to those who underwent LATG at a 1:1 ratio by using a propensity score matching (PSM) method to reduce the effect of bias due to the imbalanced clinicopathological features of the two groups. The matched variables included age, sex, body mass index (BMI), American Society of Anesthesiologists (ASA) grade, T stage, N stage, tumour-node-metastasis classification (TNM), tumour size, tumour location, Borrmann type, differentiation and comorbidities. Postoperative complications were recorded and classified according to the Clavien-Dindo classification system , 26. PatAll patients underwent standard radical total gastrectomy with D2 lymphadenectomy according to the Guidelines of the Japanese Gastric Cancer Association , 28. Thet test, Mann–Whitney test and chi-square test were used for continuous variables or categorical variables. Continuous variables are presented as the mean ± standard deviation (SD). A value of p < 0.05 was considered statistically significant, and all p values were two-sided.SPSS version 22.0 for Windows  was used for statistical analysis. R version 3.5.2 for Windows was used for PSM by using the MatchIt package. The independent sample n = 3), had early gastric cancer (n = 33), received palliative surgery (n = 75), received neoadjuvant chemotherapy before surgery (n = 21), underwent combined organ resection (n = 23), underwent D2+ lymphadenectomy (n = 5). The statistical analyses were performed in the remaining 413 patients undergoing radical total gastrectomy, of whom 132 underwent RATG and 281 underwent LATG  in age, sex, BMI, ASA grade, Borrmann type, N stage, TNM stage, or medical comorbidities , except T stage, tumour differentiation and abdominal surgery history (p < 0.05). However, those biases were reduced after PSM, and the clinicopathological characteristics were better matched between the two groups.A total of 160 patients were excluded for the following reasons: patients were over 80 years old (p = 0.003), less intraoperative bleeding , more total RLNs , and more N2 tier RLNs  than the LATG group. After PSM, the RATG group still had a longer mean operation time , less intraoperative bleeding  and more N2 tier RLNs  than the LATG group. Additionally, the total RLNs of the RATG group were almost significantly different compared to that of the LATG group . Moreover, no significant differences were found between the two groups in terms of the length of incision, proximal resection margin, distal resection margin, residual disease and postoperative hospital stay.The postoperative clinical outcomes before and after PSM are detailed in Table p = 0.145). In the robotic group, 2 patients encountered uncontrollable bleeding, 2 caused by tight adhesion and 2 had the left gastric artery surrounded by lymph nodes. In the laparoscopic group, 13 patients had tight adhesion, 4 had the left gastric artery surrounded by lymph nodes, 2 caused by enlarged lymph nodes, 1 caused by the tumour surrounding the artery, 2 caused by a giant tumour, 1 encountered bleeding of a short gastric vessel, and 1 encountered mechanical failure of the stapler. Furthermore, two patients underwent splenectomy in the robotic group, and one underwent splenectomy in the laparoscopic group because of the tight adhesion of the spleen hilum .Six patients underwent conversion to laparotomy in the robotic group and 24 in the laparoscopic group  and after PSM . Grade II complications accounted for most of the complications in the two cohorts both before and after PSM. Moreover, no significant differences were noted in the major complications (Clavien-Dindo grade ≥ IIIa) among all complications between the two cohorts before PSM  and after PSM . One patient in the RATG died of MODS after anastomotic leakage who received a second surgical procedure. One patient in the LATG died of MODS after pulmonary failure. The mortality rates were 0.8 and 0.4% for the RATG and LATG groups, respectively (p = 1.000).The postoperative complications before and after PSM are shown in Table . Subgroup analysis of tumour location (Table ). RATG had less intraoperative bleeding and more N2 tier RLNs compared with the LATG group in patients with age younger than 65 years old, while there was no significant difference between them in patients older than 65 years old . Although the mean difference of approximately 30 mL of blood loss between the two minimally invasive groups may not provide much clinical benefit for every individual patient, this may show that the robot can operate more accurately to reduce bleeding. However, the present study demonstrated that the operative time of RATG was significantly longer than that of LATG after PSM, which was consistent with the findings of previous studies . Our study shown that RATG can retrieve more N2 tier RLNs (p = 0.007 vs. p = 0.016) than LATG both before and after PSM. Nevertheless, the difference in RLNs between the two methods was not clinically significant after PSM. Moreover, the study by Shen et al., which included 23 robotic and 75 laparoscopic total gastrectomy procedures, reported that the RAG and LAG groups had no significant difference in the number of harvested lymph nodes . Therefore, it is still controversial whether robotic total gastrectomy can retrieve more lymph nodes. Thus further studies of robotic total gastrectomy, especially RCTs, should be conducted to focus on this issue.D2 lymphadenectomy is an indispensable process for the application of minimally invasive surgery for AGC . The disph nodes . Li et ap = 0.268) and after PSM . Not surprisingly, pulmonary complications obviously accounted for most of the complications in our study. Upper abdominal surgery combined with pneumoperitoneum and postoperative pain affect the activity of the diaphragm and lead to micro-atelectasis, which in turn causes pulmonary dysfunction. More importantly, total gastrectomy was an independent risk factor for pulmonary complications [p = 0.689). The ratio of anastomosis-related complications in the present study was similar with that in previous studies.Postoperative complications are an important factor to evaluate the safety and feasibility of a surgical procedure. We evaluated postoperative complications according to the Clavien-Dindo classification system, which is applicable in most parts of the world . Previouications . Moreoveications . The Japications . Of the Since total gastrectomy was the most common treatment choice for upper gastric cancer, which includes tumours in the proximal third of the stomach and EGJ –8, we coHowever, this study has several limitations. First, the results were based on a retrospective analysis from a single-clinic institution. Second, the present study lacks a detailed comparative analysis of the cost-effectiveness and gastrointestinal function recovery index between robotic and laparoscopic gastric surgery. Third, although the five surgeons who performed the surgeries received robotic surgery certification and were experienced in both minimally invasive surgeries, different surgeons can still cause some bias and further influence the results. Despite this study having some limitations, our findings provide evidence for minimally invasive surgery of total gastrectomy for AGC. Further well-designed studies, especially RCTs or prospective trials, are needed to assess the impact of RATG and LATG.This retrospective study demonstrates that RATG is comparable to LATG in terms of short-term surgical outcomes. With longer operation time, less estimated blood loss, more N2 tier RLNs and similar complication rate after PSM, RATG is a safe, reliable and promising approach compared with LATG for the treatment of AGC. Well-designed and randomized controlled trials are needed to further compare RATG with LATG.