High amount of polyclonal free light chains (FLC) are reported in systemic autoimmune diseases (SAD) and we took advantage of the PRECISESADS study to better characterize them. Serum FLC levels were explored in 1979 patients with SAD  and 614 healthy controls. Information regarding clinical parameters, disease activity, medications, autoantibodies (Ab) and the interferon α and/or γ scores were recorded. Among SAD patients, 28.4% had raised total FLC (from 12% in RA to 30% in SLE and APS) with a normal kappa/lambda ratio. Total FLC levels were significantly higher in SAD with inflammation, active disease in SLE and SjS, and an impaired pulmonary functional capacity in SSc, while independent from kidney impairment, infection, cancer and treatment. Total FLC concentrations were positively correlated among the 10/17 (58.8%) autoantibodies (Ab) tested with anti-RNA binding protein Ab , anti-dsDNA/nucleosome Ab, rheumatoid factor and negatively correlated with complement fractions C3/C4. Finally, examination of interferon (IFN) expression as a potential driver of FLC overexpression was tested showing an elevated level of total FLC among patients with a high IFNα and IFNγ Kirou's score, a strong IFN modular score, and the detection in the sera of B-cell IFN dependent factors, such as TNF-R1/TNFRSF1A and CXCL10/IP10. In conclusion, an elevated level of FLC, in association with a strong IFN signature, defines a subgroup of SAD patients, including those without renal affectation, characterized by increased disease activity, autoreactivity, and complement reduction. •Polyclonal FLC elevation characterizes patients with SADs.•Kidney involvement major FLC level elevation.•Elevated FLC levels are associated with disease activity and autoantibody production.•A strong interferon signature is associated with elevated FLC levels. SAD are10.13039/100006600FLC as a biomarker in 10.13039/501100009237SAD is further supported by its correlation with disease activity and autoantibody production.In SAD, B cell homeostasis is disturbed  and B ceIn order to better address the place of FLC in SAD, serum FLC levels were determined in a cross-sectional study including 1979 patients with SAD and 614 healthy controls enrolled in the European's PRECISESAD study. Objectives of the analysis were related to the prevalence of elevated FLC levels in SAD, associations and/or correlations with clinical parameters, disease activity, treatment, autoantibodies and the interplay with the IFN signature in SAD.22.1During the 5-year PRECISESADS project, 1979 SAD patients and 614 healthy controls (HC) were recruited from 18 centers in Europe. According to widely-used classification criteria , 463 patsicca syndrome, gangrene of the fingers, worsening lung function, pulmonary hypertension, pericarditis, erosive arthritis, haemolytic anemia, and systemic hypertension; as well as the following paraclinical markers: inflammation  or erythrocyte sedimentation rate>30 mm in the absence of current infection), leucopenia (white blood cells <3000 cells/mm3 or lymphocytes <1000/mm3 or neutrophils <1500/mm3), abnormal serum creatinemia (>20% N or increase of >50% versus baseline or a glomerular filtration rate <60 ml/min), proteinuria (excess of proteins in urine >500mg/24 h), and autoimmune hemolytic anemia (hemoglobin < N and haptoglobin < N and positive direct Coombs test). In subanalysis, two subgroups of patients were established to exclude (i) patients with chronic/acute infections and/or a history of cancer; and (ii) patients with renal impairment, based on the presence or past detection of proteinuria and/or abnormal serum creatinine.Fifty four relevant bio-clinical parameters were collected for every patient on the PRECISESADS project (supplementary S2 document), and among them 15 were further analyzed based on their association with FLC overexpression in the SAD population: pleural effusions, venous thrombosis, 2, and the ratio FVC/DLCO to predict pulmonary artery hypertension were studied.Disease activity measures included a 0–100 visual analogue scale (VAS) for global disease activity based on the physician's judgement that was done for all SAD patients. As disease specific activity determination was facultative in the PRECISESADS project, available data include SLE disease activity index (SLEDAI) for 321 SLE patients , SjS disClinicalTrials.gov.The Ethical Review Boards of the 18 participating institutions approved the protocol of the study, which adhered to the standards set by the International Conference on Harmonization and Good Clinical Practice (ICH-GCP), and to the Declaration of Helsinki. The study is registered with number NCT02890121 in 2.2FLC kappa (FLC-κ) and lambda (FLC-λ) were determined at a single center  using the Freelite® assay through turbidimetry . FLC-κ, FLC-λ, total FLC (κ+λ), and κ/λ ratio measures were collected for every patient and healthy controls. For cut-off selection and their comparison, the receiver operating characteristic (ROC) curves were calculated, and the Cohen's kappa coefficient was used for inter-reliability.2.3A strategy for determining a broad range of autoantibodies in a single center (Brest) was applied, utilizing two distinct panels. The first panel was performed for all samples and included analysis of anti-ENA, anti-CPP2, anti-β2GPI IgG and IgM, anti-CL IgG and IgM, anti-dsDNA, and anti-centromere autoantibodies using the chemiluminescent immunoanalyser IDS-ISYS . With a positive result from the anti-ENA screening test that includes an autoantigen mixture, a second distinct panel was screened for the corresponding autoantigens: Sm, U1-RNP, SSA 52 kDa, SSA 60 kDa, SSB, and anti-Scl70. In all individuals, an ELISA was used for anti-dsDNA-NcX/chromatin detection , and turbidimetry to determine rheumatoid factors (RF), complement fractions C3 and C4, and in an unselected subgroup for IgG/A/M isotypes (The Binding Site). The cut-off was chosen according to the manufacturer's instructions except for anti-dsDNA (40 UI/mL) and RF (20 UI/mL) based on results of ROC curve analysis. Anti-malondialdehyde (MDA) and anti-phosphorylcholine (PC) IgM were performed as previously described .2.4Cytokines, chemokines and inflammatory mediators were measured in a single center  on an unselected subgroup of serum samples from 866 SAD individuals  and 157 healthy controls using the human pre-mixed multi-analyte Luminex assay  encompassing 12 cytokines and chemokines involved the inflammatory processes: TNF-R1, CXCL10/IP-10, CXCL13/BLC, GDF-15, IL1R2, CCL8/MCP2, CCL13/MCP4, CCL4/MIP-1b, IL-1RA/TNFRSF1A, CCL17/TARC, MMP8, and FASLG/FAS ligand. The concentration of each mediator was calculated from specific standard curves using the software Bio-Plex Manager v6.0 and expressed as pg/ml.2.5homo sapiens hg19 reference genome using STAR v2.52b. RSEM v1.2.31 was used for gene expression quantification. For calculation of IFN scores, the expression of IFN inducible genes was used to evaluate the activation of type I  and type II  IFN pathways according to Kirou's score [IFI44, IFI44L, IFIT1, IFIT3, and MXA), M3.4 ; and M5.12  based on Obermoser's report [Total RNA was extracted from an unselected subgroup of whole blood samples from 1275 SAD individuals  and 406 healthy controls, and sequenced on a HiSeq2500 instrument using a HiSeq SBS kit v4 from Illumina  after quality controls were run. Reads were then aligned to the 's score  and by ts report . IFN sco2.6post hoc comparison. Receiver Operating Characteristics (ROC) curve analysis was performed to determine the area under the curve (AUC) and the optimal cut-off value was chosen at 95% specificity. Cohen's kappa agreement was calculated for side-by-side comparison, in order to determine the best parameter to keep for the following analysis. The strength of Cohen's kappa was interpreted based on the following definitions: less than chance agreement (<0), slight agreement (0.01–0.20), fair agreement (0.21–0.40), moderate agreement (0.41–0.60), substantial agreement (0.61–0.80), and almost perfect agreement (0.81–0.99). For categorical data  and non-categorical data (Spearman rank correlation test), the adjusted false discovery rate values  were calculated and for graphical purpose presented using the –log10 of the q value. Statistical analysis was performed using Prism 7.0  or MS-excel 2007 . Statistical significance was assessed with two-tailed p or q values lower than 0.05.Continuous data are described as mean ± SD. Differences between groups were analyzed by multiple one-way ANOVA and the Tukey's test was used for 33.1A total of 2593 individuals were included in the study with 1979 SAD patients and 614 healthy controls (HC), see −4), while the FLC-κ/λ ratio did not differ from the HC ratio (−5) was further observed, which was not the case with IgA or IgM  levels were evaluated by quantitative turbidimetry and, as illustrated in HC ratio . Next, wHC ratio B, the AUHC ratio , total FA or IgM . Total FA or IgM .Fig. 1FrTo complete such analysis and since FLC elevation in SAD can result from a polyclonal B-cell activation that may result from infection, cancers, and/or from a reduced clearance by impaired renal function, the SAD subpopulation without infection and cancer was evaluated  as well as the population with normal kidney function was individualized . In terms of AUC and cut-off values, the characteristics of the ROC assay was similar for total FLC when considering the SAD subpopulation with normal kidney function instead of all SAD individuals and SAD without infections and/or cancer (3.2−19), inflammation (p = 1.0 × 10−11), proteinuria (p = 5.7 × 10−9), hypertension (p = 4.0 × 10−8), leucopenia (p = 1.4 × 10−6), hemolytic anemia (p = 2.0 × 10−4), erosive arthritis (p = 3.0 × 10−4), pericarditis (p = 1.5 × 10−3), worsening lung function (p = 0.02), gangrene of the fingers (p = 0.02), sicca syndrome (p = 0.03), venous thrombosis (p = 0.03) and pleural effusion (p = 0.03).An excessive total FLC concentration (>37 mg/L) was determined in the 6 SAD subgroups ranging from 12% in RA, 14% in UCTD, 22% in SSc, 24% in SjS, 29% in MCTD and 30% in APS and SLE A. Among −5) and reduced leucopenia (p = 6.0 × 10−5), while moderate associations with systemic hypertension (p = 0.04), erosive arthritis (p = 0.02), pulmonary hypertension (p = 0.03) and worsening lung function (p = 0.04) remained. Altogether this supports the concept that elevated 10.13039/100006600FLC are commonly distributed among 10.13039/501100009237SAD patients regardless of kidney function, and this is predominantly related to the inflammatory status while independent from infection and cancer.However, and as one may argue that such associations resulted predominantly from infections, cancer and/or renal impairment, the analysis was repeated with the SAD subpopulations after exclusion of the individuals presenting infections/cancer and or presenting normal kidney function, which were determined to have an elevated total FLC level in the 6 SAD subgroups ranging respectively from 16 to 17% in RA, 18–20% in UCTD, 30-30% in SSc, 36-36% in SjS, 33-32% in MCTD, 29–30% in APS and 36–37% in SLE C/E. Whil3.3versus 6.7 ± 0.8 in high FLC SLE, p = 0.01), SjS patients , and such associations were not observed when using a global VAS approach.Regarding the association between FLC and disease activity, it appeared that patients with elevated FLC levels had a higher disease activity when considering SLE patients . No differences were observed with regards to DLCO and the ratio FLC/DLCO, two factors known to occur in a more advanced stage of fibrosis and in response to a strong inflammatory process.As a worsening lung function was statistically associated with elevated FLC levels, the lung capacity was explored further in SSc patients, which revealed a median loss of FVC among SSc patients with elevated FLC levels . Interestingly, two groups of autoantibodies can be distinguished according to the correlation with FLC: one correlated with FLC that includes anti-RNA binding proteins , anti-dsDNA associated or not with histones to form chromatin (p = 6.5 × 10−15 and 1.1 × 10−11) plus RF (p = 3.3 × 10−7) and another group not or modestly correlated with FLC such as RA-associated , SSc-associated , APS-associated , and thrombosis-associated  autoantibodies. Since some of these autoantibodies were more prevalent in SAD patients with kidney involvement , the same analysis was repeated in SAD individuals without infection and cancer (data not shown) and in SAD with normal kidney function , which is consistent with the possible implication of both type I and type II IFNs in FLC production. Such a prediction was further supported by the elevated level of 10.13039/100006600FLC observed in the type I activity module M1.2 and in the type I/II activity modules M3.4 and M5.12. In addition, the number of IFN modules is important since FLC levels were significantly higher in the moderate module (p = 0.01) and even more in the strong module (p < 10−4). For 866 SAD patients, the serum level of 12 cytokine/chemokine was determined showing a significant correlation for 9/12 (75%), such as TNF-RI (p = 2.5 × 10−38) and CXCL10 , which belong to the IFN type I and II, except for CCL18 , according to the Interferome V2.01 database (http://www.interferome.org).The presence of autoantibodies targeting dsDNA and RNA binding proteins  in several cohorts of SAD is associated with the presence of an IFN signature. Accordingly , we next4Systemic autoimmune diseases share common pathophysiological features such as chronic inflammation and an IFN signature leading to the initiation of Th1-type immunity, B cell hyperactivation and differentiation into autoantibody-secreting plasma cells . The conBecause of their rapid turnover, a high level of FLC has been proposed as useful real-time marker for assessing disease activity and B cell hyperactivity, in contrast to total immunoglobulins, which possess a half-life of approximately 3 weeks depending on the isotype . Such asElevated FLC levels were also associated with the occurrence of clinical and biological manifestations and, for such analysis, it's important to consider one group of associations independent from renal impairment that implies inflammation such as pulmonary injury and erosive arthritis; and one group associated with renal impairment such as hypertension, pericarditis, and venous thrombosis that are particularly relevant in SADs. Altogether, elevated FLC levels are associated with the occurrence of clinical manifestations associated with a chronic inflammatory process, in accordance with previous data obtained in SLE, SSc and SjS cohorts ,29,30. TSubsequently, FLCs have also been associated with specific autoantibodies, including anti-RNA binding protein antibodies , as well as anti-DNA-NcX, anti-dsDNA, and RF. In contrast, the other autoantibodies tested  demonstrated poor or no association with increased FLC and the same results were obtained in patients with normal kidney function. One explanation for such a dichotomy is related to the capacity of the FLC-associated autoantibody group to form immune complexes (e.g. with apoptotic cells), which is known to activate the complement pathway and to increase monocyte IFN type-I production that promotes, in turn, plasmablast differentiation and immunoglobulin production ,35. SuchIn addition, an important negative correlation between FLC and complement fractions was noted. An effect of FLC to directly promote complement consumption has been proposed previously, involving the binding of FLC to factor H, which accelerates the decay of the alternative complement compound C3 convertase . This me10.13039/501100007072IFN type I predicted the response to TNFα blockade, but not to rituximab, a B cell depleting monoclonal antibody, supporting the fact that 10.13039/501100007072IFN type I signature in RA induces a high inflammatory activity, but not an elevated autoantibody production by B cells based on the independence observed between 10.13039/100006600FLC levels and anti-10.13039/100005550CCP production [Finally, a strong link between FLC and inflammation has been demonstrated through the analysis of the IFN signature and selected proinflammatory cytokines. Two IFN scores were applied ,24 and boduction . Finallyoduction ,43.10.13039/501100007072IFN, inflammation and 10.13039/100006600FLC synthesis, several inflammatory cytokines belonging to the type 1 and type 2 10.13039/501100007072IFN families were measured and were significantly correlated with higher total 10.13039/100006600FLC levels. Among them, the two most important cytokines retrieved by our study were TNF-RI and CXCL10, both known to be overexpressed in SLE B cells, but such analysis was not exhaustive and needs further exploration.To further support this link between ex-vivo cell culture model of synovial membrane tissue obtained from RA patients, a dual activity between TNF-RI and TNF-RII was observed together with a predominantly pro-inflammatory role played by TNF-RI in RA [The key role played by TNFα in RA was studied extensively through therapeutic use of anti-TNFα monoclonal antibodies like infliximab or the use of fusion proteins trapping soluble TNFα, like etanercept. TNFα can bind to two cell-surface receptors, TNF-RI and TNF-RII. The presence of these receptors in sera of RA patients was shown to be higher as compared to controls and was associated with a higher disease activity score (DAS28) and an abnormal inflammation status . In an eRI in RA . TNF-RI RI in RA , and wasRI in RA . Of noteRI in RA . Less evRI in RA . Last buRI in RA . This isin vivo has recently been demonstrated, highlighting a mechanism for the secondary production of FLC through an inflammatory cytokine related to the IFN signature [On the other hand, CXCL10 is a cytokine responsible for the recruitment and activation of Th1 effectors, predominantly induced by IFNγ. CXCL10, in particular, is involved in the maintenance of chronic pathological inflammation, and abnormal secretion of this cytokine has been described in several SADs. In RA, the expression of CXCL10 was demonstrated to be elevated in the serum and joints of patients . In a muignature . Of noteignature , making Those results are consistent with the participation of IFN type I and type II as well as several proinflammatory cytokines to induce more severe diseases through FLC synthesis. Elevated FLC would then accurately mirror both inflammation as well as pathological B cell activation in SAD patients.5This is the first study to determine the role of FLC on such a large scale of SAD to our knowledge. The major findings are related to the facts that (i) immune stimulation rather than impaired kidney function, infection or cancer increases polyclonal FLC in SAD; (ii) Elevated FLC levels are associated with disease activity, inflammation, and a specific profile of autoantibodies; and (iii) a relationship between elevated FLC levels and a strong IFN signature was further revealed. FLC then account for adaptive immunity by reflecting autoantibody production and B cell activity, but also for innate immunity through its induction by IFN signaling. FLC measurement, especially in patients with stable renal function, would then be interesting to evaluate disease activity in real time with an easy and reliable assay. It would also be interesting to complement these observations with a longitudinal study to evaluate the variation of FLCs over time according to disease activity and inflammatory parameters, and thus define the value of FLC in predicting relapses and/or therapeutic response.EB, PLM, MOB, MAR, YR: Formal analysis and writing original draft; EB, CLG, MM, MOB, GB, ZM, SB, RL: Investigation; MAR, JF: Project administration; and MAR: Coordination of the whole project and the person behind PRECISESADS ideas.All authors declare that they have no conflict of interest.


Tripterygium wilfordii Hook F (TwHF), has been shown to exert therapeutic potential against rheumatoid arthritis (RA). However, its molecular mechanism of action has not been fully elucidated. This study aimed to analyze the potential target of TP based on the discovery of differentially methylated and expressed genes (DMEGs) in RA using methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq). Five RA samples and ten control samples were obtained from China-Japan Friendship Hospital. The various levels of m6A methylation and genes expressed in the RA and control groups were compared by MeRIP-seq and RNA-seq. Bioinformatics explorations were also performed to explore the enriched biological roles and paths of the differentially expressed m6A methylation and genes. Molecular networks between TP target proteins and DMEGs were performed using Ingenuity Pathway Analysis (IPA) software. Potential target of TP was determined with Gene Expression Omnibus (GEO) database mining, molecular docking, and in vitro experiment validation. In total, 583 dysregulated m6A peaks, of which 295 were greatly upregulated and 288 were greatly downregulated, were identified. Similarly, 1,570 differentially expressed genes were identified by RNA-seq, including 539 upregulated and 1,031 downregulated genes. According to the deeper joint exploration, the m6A methylation and mRNA expression degrees of 35 genes varied greatly. Molecular networks between TP target proteins and DMEGs were constructed, and the results revealed that tubulin beta-2A chain (TUBB2A), insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), cytoplasmic dynein 1 intermediate chain 1 (DYNC1I1), and FOS-like 1 (FOSL1) were the most relevant genes that correlated with the target proteins of TP. The results of the GEO database showed that the gene expression of IGF2BP3 was increased in RA synovial tissue and consistent with the trend of our sequencing results of RA PBMCs. Molecular docking and in vitro experiment suggested that TP and IGF2BP3 had a high binding affinity and TP could decrease the mRNA expression of IGF2BP3 in PBMCs and MH7A.This research established a transcriptional map of m6A in RA PBMCs and displayed the hidden association between RNA methylation alterations and associated genes in RA. IGF2BP3 might be a potential therapeutic target of TP during RA treatment.Triptolide (TP), a major active component of the herb  As an autoimmune disease, rheumatoid arthritis (RA) has the characteristics such as chronic inflammatory variations in synovial tissue, bone, and joint cartilage, which lead to the substantial destruction of affected joints . The etiN6-methyladenosine (m6A) is the most abundant internal alteration of mRNA in eukaryotes. This alternation is reversible and regulated by “writers,” “erasers,” and “readers” . 6A methylome in RA has not been fully characterized, and more in-depth research is rare.As an epigenetic regulatory mechanism, readers” . Recentl obesity  and syst obesity . Many teTripterygium wilfordii Hook F (TwHF), a traditional Chinese medicine, has been used for centuries to treat autoimmune diseases including RA in China. Previous studies have confirmed the effectiveness of TwHF in the treatment of RA  were performed, and differentially methylated peaks within mRNAs were found in RA and control samples. Meanwhile, differentially expressed mRNAs were identified using RNA-seq. In addition, further analysis of the combined MeRIP-seq and RNA-seq information was also performed for the identification of differentially methylated and expressed genes (DMEGs). Furthermore, the molecular network between TP target proteins and DMEGs was constructed using Ingenuity Pathway Analysis (IPA) software. A new potential target of TP was determined with Gene Expression Omnibus (GEO) database mining, molecular docking, and Five patients suffering from RA were obtained from China-Japan Friendship Hospital. All patients had been diagnosed with RA according to the 2010 American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) standards . In thisPBMCs from each subject were separated using Lymphoprep™ with a density of 1.077 ± 0.001 g/ml, according to the manufacturer’s instructions . Total RNA in PBMCs was extracted, purified with TRIzol  according to the manufacturer’s instructions, and stored at −80°C.6A antibody  in IP buffer . Next, SuperScript™ II reverse transcriptase (Invitrogen) was used to reverse-transcribe the IP RNA for the creation of cDNA, which was then used to synthesize U-labeled second-stranded DNAs with E. coli DNA polymerase I , RNase H , and dUTP solution . An A-base was placed on the blunt ends of every strand for ligation to the indexed adapters. There was a T-base overhang for ligating the adapter to the A-tailed-fragmented DNA on each adapter. During the ligation of single- or dual-index adapters to the fragments, size was selected with AMPureXP beads. After treatment of the U-labeled second-stranded DNAs with the heat-labile UDG enzyme , amplification of the ligated products was performed with PCR with 3-min initial denaturation at 95°C, eight cycles of denaturation at 98°C for 15 s, 15 s annealing at 60°C, 30 s extension at 72°C, and 5 min final extension at 72°C. The final cDNA library showed an average insert size of 300 ± 50 bp. Eventually, 2 × 150 bp paired-end sequencing (PE150) was performed on an Illumina Novaseq™ 6000 , according to the vendor’s suggested instructions  was used to quantify the total RNA amount and purity of every sample. A Bioanalyzer 2100  with an RIN > 7.0 was employed to assess the RNA integrity, followed by confirmation by electrophoresis in a denaturing agarose gel. Ribosomal RNA was depleted from approximately more than 25 μg of total RNA from a specific adipose type, based on the instructions of the Epicenter Ribo-Zero Gold Kit . After purification, the 7-min fragmentation of ribosomal-depleted RNA into small pieces was performed with a magnesium RNA fragmentation module  at 86°C. Next, the 2 h incubation of the cleaved RNA fragments was performed at 4°C with the provided mructions .Homo sapiens (Version: v101) with HISAT2  groups detected by RNA-Seq were characterized by Venn analysis. Various colors in the pie diagram show the number of mRNAs with or without overlapping.http://pubchem.ncbi.hlm.nih.gov). Then, the human target proteins and DMEGs were imported into the Ingenuity Pathway Analysis (IPA) platform, and molecular networks were built. In the molecular networks, nodes represent molecules, and an edge (line) shows the biological association between two nodes. At least, one reference from a textbook, literature, or canonical data saved in the IPKB supported all edges. Nodes with various shapes represent the functional class of the gene goods.By using “triptolide” as a key word, we searched for the potential human target proteins of TP in the PubChem platform . TP was docked with IGF2BP3 protein by AutoDock Vina  and fibroblast-like synoviocytes (FLSs) cell line MH7A  were cultured in DMEM medium (GIBCO) with 10% fetal bovine serum (GIBCO) and 1% penicillin–streptomycin in an incubator with 5% CO6 cells/ml and 1 × 105 cells/ml, respectively, followed by 2 h of pretreatment with or without 100 ng/ml of lipopolysaccharides (LPS)  or 20 ng/ml recombinant human tumor necrosis factor (TNF)-α . Then, PBMCs were incubated with 6.25 nM or 12.5 nM TP  for another 24-h, MH7A was incubated with 12.5 nM or 25 nM TP for another 24-h. Finally, the cells were harvested for real-time PCR analysis.PBMCs or MH7A were placed into 6-well plates at a concentration of 1.0 × 10−ΔΔCt method and β-actin was used as an endogenous control. The PrimeScriptTM RT Reagent Kit with gDNA Eraser  was used to reverse-transcribe the extracted RNA into cDNA using the instructions provided by the manufacturer. Quantitative real-time PCR analysis was performed with TB GreenTM Premix Ex TaqTM  and the QuantStudio 5 Real-Time PCR System . The relative RNA expression was quantified with the 2t-test or one-way analysis of variance (ANOVA) according to Tukey’s post hoc analysis. All explorations were performed with GraphPad Prism 8.0 . A p < 0.05 was regarded as statistically significant.The data are shown as the mean ± SEM. The statistical significance for differences between two groups was determined with two-tailed, unpaired Student’s 6A modification patterns in RA PBMCs, MeRIP-seq analysis was performed. According to 6A consensus motif of GGACU was presented with highly enriched in human PBMCs, which conforms to the reported consensus motif RRACH   and the Con group , there were 20,595 and 20,591 common m6A modification peaks in the RA and Con groups, respectively  . In totaectively . In contectively . In addiectively . Accordion peaks . The m6Aal exons . Further6A modification in RA, GO enrichment and KEGG pathway analyses were performed. GO analysis results showed that both hypermethylated and hypomethylated genes in the PBMCs participated in the following functions and pathways: “regulation of transcription, DNA-templated,” “signal transduction,” and “cell differentiation” ; “cytoplasm,” “membrane,” and “nucleus” (ontology: cellular component); and “protein binding,” “nucleotide binding,” “metal ion binding,” and “DNA binding” (ontology: molecular function)  . In KEGGunction) . For KEGunction) . Howeverunction) .p value <0.05 and fold change (FC) ≥ 2, 1,570 genes were shown to be greatly dysregulated compared with the Con group, including 539 upregulated genes and 1,031 downregulated genes . In ed genes . Figure To analyze the biological functions and signaling pathways involved in DEGs, GO and KEGG analyses were performed. The outcomes of the GO analysis suggested the enrichment of these genes in the following functions and pathways: “signal transduction” and “cell adhesion” ; “membrane,” “cytoplasm,” and “integral component of membrane” (ontology: cellular component); and “protein binding” and “DNA binding” (ontology: molecular function). Upregulated and downregulated genes were also involved in “neutrophil degranulation” and “regulation of transcription, DNA-templated” , respectively .In KEGG subclass analyses, upregulated and downregulated genes were involved in the following functions and pathways: “cell motility,” “cellular community-eukaryotes,” “cell growth and death,” and “transport and catabolism” (cellular processes); and “membrane transport,” “signaling molecules and interaction,” and “signal transduction” . In genetic information processing, upregulated and downregulated genes were involved in “folding, sorting, and degradation.” In metabolism, “glycan biosynthesis and metabolism” was associated with the upregulated and downregulated genes. In addition, upregulated and downregulated genes were associated with “immune disease” (human diseases) and “immune system”  . AccordiFurthermore, by the conjoint analysis of MeRIP-seq and RNA-seq data, 35 DMEGs changed significantly, including 13 hypermethylated and upregulated genes (hyper-up), 12 hypermethylated and downregulated genes (hyper-down), five hypomethylated and upregulated genes (hypo-up), and five hypomethylated and downregulated genes (hypo-down) . Table 26A peak distributions of IGF2BP3 between the Con and RA groups are shown in To construct the molecular network between TP target proteins and candidate DMEGs, we first searched and found 169 target proteins of TP  in the P6A, an epigenetic regulatory mechanism, is the most abundant internal modification of mRNA in eukaryotes and modulates the gene expression program at the posttranscriptional level. Variant m6A levels exert various biological functions such as cell differentiation, neuronal signaling, and immune tolerance. m6A modification is involved in various diseases, such as obesity, diabetes, and SLE  signaling is a common hub for many signaling pathways that take part in tolerance mechanisms, and B cells express a large panel of ion channels and transporters that contribute to the production and regulation of Ca2+ signals, which exert a significant effect on B-cell growth and fate (2+ has been shown to be involved in RA pathogenesis. Autoreactive B cells also exert a key effect on the pathogenesis of RA. Defects in Ca2+ influx kinetics and enhancement of basal [Ca2+]cyt have been found in T lymphocytes isolated from RA patients (6A modification may boost the appearance and progression of RA by controlling the calcium signaling pathway or the B-cell receptor signaling pathway. Interestingly, the hypomethylated genes were significantly related to “sphingolipid metabolism.” A previous study reported that sphingolipids are a category of complicated lipids with a backbone of sphingoid bases and that several bioactive sphingolipids are involved in the pathological processes of RA (6A modification may affect RA progression by regulating sphingolipid metabolism. However, the precise functions of sphingolipid metabolism in RA still need to be studied in-depth, and the m6A-altered genes associated with this pathway in RA must be further clarified.Numerous studies have shown that peripheral blood represents the main highway for the RA immune system. PBMCs are immune cells and can initiate the autoimmune inflammatory process directed against target organs. Therefore, the gene expression signatures of PBMCs can shed light on the molecular features of the immune cells in the targeted organs in RA progression . Our res studies . These sfication . In KEGGand fate . Signalipatients . These ses of RA , which iNext, through RNA-seq analyses, 1,570 significantly dysregulated genes were identified, including 539 upregulated genes and 1,031 downregulated genes. These dysregulated genes were significantly involved in “signal transduction,” “cell adhesion” and “neutrophil degranulation.” In KEGG pathway analyses, several canonical pathways associated with RA inflammation were significantly enriched, including “complement and coagulation cascades,” “Jak-STAT signaling pathway” and “IL-17 signaling pathway.” However, the downregulated genes were significantly related to “natural killer cell mediated cytotoxicity.”6A modification in influencing RA development, conjoint explorations of MeRIP-seq and RNA-seq data were performed, and 35 DMEGs were found. The enrichment of genes in “protein transport” and “protein binding” was observed, and KEGG pathway analysiss howed that these DMEGs were enriched primarily in “lysosome.” Lysosomes are membrane-bound organelles involved in the processes of degrading and recycling cellular waste, cellular signaling, and energy metabolism (To elucidate the mechanism of mtabolism . Increastabolism . The earTP is the main active ingredient extracted from TwHF, and it has been considered a promising anti-RA drug , but the6A and TP regulate gene expression is an important direction of our future studies.IGF2BP3 is a highly conserved paralog of IGF2BPs and primarily exerts oncogenic effects in cancer . In rece6A in RA PBMCs and displayed the hidden association between m6A methylation modification and associated genes in RA. The results highlight the importance of m6A modification as a gene regulatory system in RA. IGF2BP3 might be a potential therapeutic target of TP during the treatment of RA. More research based on these epigenetic clues should be conducted in the future to deepen the understanding of the mechanisms of TP and m6A modification in RA.This study established a transcriptional map of m


While elevations of CSF p-Tau/Aβ40 can occur in amyloid-β (Aβ) negative (Aβ-) individuals, the factors associated with these elevations and their role in neurodegeneration and cognitive decline are unknown. We aim to explore factors associated with elevated tau in CSF, and how these elevated tau are related to neurodegeneration and cognitive decline in the absence of Aβ positivity.We recently reported that CSF phosphorylated tau  as well as vascular risk factors in 149 cognitively unimpaired and 52 impaired individuals who were presumably not on the Alzheimer’s disease (AD) pathway due to negative Aβ status on both CSF and PET. Subgroups had 18F-fluorodeoxyglucose (FDG) PET and adjusted hippocampal volume (aHCV), and longitudinal measures of CSF, aHCV, FDG PET, and cognition data, so we examined CSF p-Tau/Aβ40 associations with these measures as well.We examined relationships between CSF p-Tau/Aβ40 was associated with older age, male sex, greater WMH, and hypertension as well as a pattern of hippocampal atrophy and temporoparietal hypometabolism characteristic of AD. Lower CSF Aβ42/Aβ40, higher WMH, and hypertension but not age, sex, Aβ PET, APOE-ε4 status, body mass index, smoking, and hyperlipidemia at baseline predicted CSF p-Tau/Aβ40 increases over approximately 5 years of follow-up. The relationship between CSF p-Tau/Aβ40 and subsequent cognitive decline was partially or fully explained by neurodegenerative measurements.Elevated CSF p-Tau/AβThese data provide surprising clues as to the etiology and significance of tau pathology in the absence of Aβ. It seems likely that, in addition to age, both cerebrovascular disease and subthreshold levels of Aβ are related to this tau accumulation. Crucially, this phenotype of CSF tau elevation in amyloid-negative individuals share features with AD such as a pattern of metabolic decline and regional brain atrophy.The online version contains supplementary material available at 10.1186/s13195-021-00913-5. Amyloid-β (Aβ) plaques and tau neurofibrillary tangles are the core features of Alzheimer’s disease (AD) . AccordiWhile PET provides spatial information on where tau deposits, CSF measurement of phosphorylated tau (p-Tau) provides complementary, although not interchangeable –21, info40 ratio reduced measurement error likely related to individual differences in CSF production rather than pathology and improved associations with AD biomarkers compared to using CSF p-Tau alone , p = 0.003). None of these significant risk factors  were related to CSF Aβ42/Aβ40 or CSF Aβ40, and only age was related  to the non-ratio CSF p-Tau measurement  and HTN ; HTN and WMH were also positively associated .At baseline, elevated CSF p-Tau/Aβ42/Aβ40 predicted  subsequent ΔCSF p-Tau/Aβ40  and HTN  were associated with subsequent ΔCSF p-Tau/Aβ40  of individuals with diabetes limits interpretation. When all the significant risk factors  were entered into a single model, CSF Aβ42/Aβ40  and HTN  were related to ΔCSF p-Tau/Aβ40  with the non-ratio ΔCSF p-Tau measurement . After FWE correction (p<0.05), significant negative associations remained in bilateral posterior cingulate/precuneus, right inferior temporal, middle temporal, and inferior parietal regions  and ΔPACC   and aHCV . aHCV was correlated with FDG SUVR . Elevated CSF p-Tau/Aβ40 predicted subsequent FDG SUVR decrease , but did not predict change in aHCV . Conversely, FDG SUVR also predicted subsequent aHCV decrease .Among these significant risk factors related to CSF p-Tau/Aβ40, FDG SUVR (metaROIs), aHCV, ΔFDG SUVR, and ΔaHCV after excluding 5 individuals who developed to Aβ+ at follow-up . The results were substantially the same , aHCV, ΔFDG SUVR, and ΔaHCV  than CU individuals (0.09 (0.46)). CSF p-Tau/Aβ40 , FDG SUVR (metaROIs) , and aHCV  all predicted subsequent cognitive decline (ΔPACC). The baseline non-ratio CSF p-Tau and CSF Aβ40 measures were not associated with ΔPACC  annual ΔPACC of CI individuals (−0.56 (1.47)) was faster  related to longitudinal PACC decline when all the predictors were entered into one model , as well as a pattern of neurodegeneration that is characteristic of AD . In longitudinal analyses, lower CSF Aβ42/Aβ40 and CSF p-Tau/Aβ40 at baseline, lower CSF Aβ42/Aβ40 predicted subsequent longitudinal CSF p-Tau/Aβ40 increase but not vice versa. There was thus no countervailing evidence that tau deposition drives changes in Aβ in these data or in a previous report from our laboratory [Although there was no relationship between CSF Aβboratory . These fboratory , 22, andboratory –55, thes40 increases in the absence of abnormal Aβ pathology whereas they had no association with CSF Aβ42/Aβ40 or Aβ40 alone, suggesting vascular risk factors may be closely linked to CSF p-Tau/Aβ40 independent of Aβ pathology. ADNI data have previously been explored to examine some of the relationships between tau and the variables we measured, though usually not in Aβ- individuals. For example, a few studies based on the ADNI cohort reported significant associations of CSF p-Tau or plasma p-Tau with WMH [One of the key findings in this study was that white matter lesions and hypertension were related to both baseline and longitudinal CSF p-Tau/Aβwith WMH , 37, 56 with WMH . In addiwith WMH , global with WMH , and hypwith WMH , 59 werewith WMH  or both with WMH , and higwith WMH  or both with WMH .Age is a well-known risk factor for tau accumulation, and it was also related to these vascular factors in our data. In a multivariable model, age was the only variable associated with tau. However, both WMH and HTN, but not age, were related to longitudinal tau increases. Together, these findings suggest that both age and cerebrovascular risk factors may be related to tau increases in the absence of abnormal Aβ pathology.A surprising result of this study was the association between tau and regional cerebral hypometabolism in a pattern typically associated with AD despite the absence of measurable Aβ. In patients with clinical and biomarker evidence of AD, patterns of tau deposition are highly correlated with glucose metabolism while Aβ deposition is not . Althoug40 predicted metabolic decline but not progressive atrophy). Previous work is inconsistent, with some studies of Aβ- ADNI participants failing to find associations between CSF p-Tau and hippocampal or cortical atrophy [40 ratio [Further evidence that elevated tau is related to neurodegeneration is found in its relationship with hippocampal volume. In fact, all three variables–tau, glucose metabolism in typically affected AD regions, and hippocampal volume–were related to one another at baseline and to a large extent in longitudinal relationships (although baseline p-Tau/Aβ atrophy , 25, whi atrophy . It is d40 ratio  may playIn addition to its relationship with neurodegeneration, tau was also related to cognitive decline, providing further evidence that it is not benign when Aβ is low. These “downstream” pathways appear to be related as tau affects cognition through both glucose metabolism and hippocampal atrophy. Previous studies have not been in agreement that elevated CSF p-Tau in Aβ- individuals is linked to significant cognitive decline , 65, alt181/Aβ40, but it would be helpful to validate the findings in other samples and with other phosphorylation sites [181/Aβ40 ratio introduces a confounder that could account for these associations, we think that this is unlikely since CSF Aβ40 alone was not associated with any of the factors we investigated. Therefore, we believe that these findings reinforce earlier evidence [181 measurements due to individual variability in CSF production and due to the limited dynamic range in this group, and this variability can be attenuated by adjusting for Aβ40. This noise reduction appears to facilitate the observation of subtle associations between tau and other risk factors at early stages of abnormality. An additional limitation of this study was that some Aβ- subgroups we examined were very small, such as APOE-ε4 carriers, which limited our ability to examine covariates and other risk factors. Finally, tau PET is a relatively late addition to ADNI, so we did not include this because of minimal longitudinal tau data. Nevertheless, CSF p-Tau may have been an advantageous measure for the scientific questions in this study because it probably reflects earlier tau pathology than PET [This study has several limitations. Our findings were based on tau measured using CSF p-Tauon sites . Furtherevidence  that thethan PET , 21.The elevation of p-Tau in CSF in our Aβ- subjects is consistent, at least nosologically, with the disorder that has been described as PART; however, in the absence of neuropathology we cannot confirm this diagnosis. These data provide surprising clues as to the etiology and significance of tau pathology in aging. It seems likely that, in addition to age, both cerebrovascular disease and subthreshold levels of Aβ are related to this tau accumulation. Whether cerebrovascular factors alone or in concert with low levels of Aβ can drive tau pathology is uncertain, but the literature is replete with evidence for a relationship between tau and cerebrovascular disease. Crucially, evidence of this tau pathology occurs early, as CSF probably reflects tau abnormalities before PET scans do, and shares features with AD such as a pattern of metabolic decline and regional brain atrophy. Further longitudinal follow-up of these individuals will be critical for determining whether these processes are Alzheimer’s-independent. However, our data indicate that this phenotype of Aβ- CSF tau elevation is similar to AD, which implies that it may represent an amyloid-independent pathway to AD, a pathophysiology that mimics AD, or an important, and very early, interaction between Aβ and vascular disease that underlies neurodegeneration and cognitive decline.Additional file 1. Supplemental material can be found at online.


Many previous studies have estimated the rate of dopaminergic denervation in Parkinson disease (PD) via imaging studies. However, they lack the considerations of onset age, disease duration at onset, gender, and dopaminergic denervation due to normal aging. Herein, using a large prospective cohort, we estimated the rate of dopaminergic denervation in PD patients, compared with an age- and gender-matched normal control group.One hundred forty-one normal controls and 301 PD patients were enrolled. Striatal specific binding ratios (SBRs) of I-123 FP-CIT single positron emission tomography images were analyzed according to the age of onset, gender, and the duration of motor symptoms.P < .05, R2 = 0.02). Moreover, was significantly inversely related to caudate SBRs, but not with putamen SBRs . Patients of different age onsets did not show any significant correlation between symptom durations and striatal SBRs. In the age-matched group, no significant relationship was observed between symptom duration and percent decrease of caudate SBRs, but there was a significant relationship between symptom duration and percent decrease of the putamen SBRs . There was no significant relationship between the symptom duration and the percent decrease of striatal SBRs in the age- and gender-matched group.In the PD group, symptom duration was significantly correlated with caudate SBRs, but with putamen SBRs (R2 values from the regression analysis between symptom duration, age, and dopaminergic denervation are low. This suggests that, contrary to previous knowledge, there is a relatively weak association between dopaminergic denervation and age or symptom duration.The significance and  There is evidence supporting the hypothesis that the progression of pathologic process precedes the development of motor related symptoms, even up to decades. For example, several autonomic features, such as constipation and REM sleep behavior disorders (RBD), may appear 20 years before the onset of tremor and bradykinesia. Therefore, the diagnosis of PD is not usually made until there are profound loss of nigrostriatal neurons, since it is mainly based on the presented motor features. Previous studies have proposed that 50% to 80% of the dopaminergic neurons are already lost during the prodromal stage. ,6 Estimating the duration and degree of this dopaminergic denervation during the disease progression is important to establish effective treatment strategies. While current treatment options for PD are limited to supplementing dopaminergic tones for symptomatic alleviation, multiple treatment strategies with the potential for disease modification are under investigation.  Based on current information, these disease-modifying treatments are most effective when used during the early stages of the disease, that is, at the initiation of dopaminergic denervation. Thus, numerous studies have attempted to estimate the duration and degree of the nigrostriatal denervation during the progression of PD, by either autopsy or functional imaging studies.Parkinson disease (PD) is a neurodegenerative disorder that presents heterogenous symptoms with a highly variable disease progression rate. They were the first to report that patients with even mild Parkinsonism had a proportionately high degree of dopaminergic denervation; however, they did not perform any regression analysis to determine the relationship between the degree of dopaminergic denervation and duration of disease. Another autopsy study by Riederer et al suggested a 68% to 82% decrease of dopamine concentration in the caudate, compared with the age-matched normal control counterparts. Afterwards, numerous imaging studies have also investigated the degree of dopaminergic denervation at the onset of disease, by measuring L-DOPA metabolism (F-18 FDOPA), dopamine transporter binding , and vesicular monoamine transporter binding (C-11 DTBZ), etc  nortropane (I-123 FP-CIT) single photon emission computed tomography (SPECT) images from the Parkinson Progression Markers Initiative (PPMI) database.22.1http://www.ppmi-info.org) in July 2019  stage I or II at baseline, patients with confirmation of dopamine transporter deficit by I-123 FP-CIT SPECT or VMAT-2 PET scans, and patients not expected to require PD medication within 6 months from baseline were enrolled. For normal control subjects, male or female age 30 years or older at screening, without currently active significant neurological disorders, without first degree relatives with PD, and without history of drug administration that could interfere with dopamine transporter imaging were enrolled. Data acquisition of PPMI was performed in accordance with the relevant guidelines and regulations. Written informed consent for all clinical data were obtained of all PPMI participants, and all subjects gave written informed consent in accordance with the 1964 Declaration of Helsinki and its later amendments. The study was approved by all respective Institutional review boards .Data were downloaded from the Parkinson Progression Markers Initiative (PPMI) database  and 301 PD patients . Data of patients and normal control subjects from the PPMI, such as age, gender, subjects’ weight, I-123 FP-CIT SPECT analysis results, previous motor symptom durations at enrollment, H&Y scores, Movement Disorder Society-Unified Parkinson disease rating scale (MDS-UPDRS) II/III scores, and Scale for Outcomes in Parkinson disease-Autonomic (SCOPA-AUT), were adopted.2.2I-123 FP-CIT SPECT scans were performed 4 ± 0.5 hours after I-123 FP-CIT injection (111–185 MBq) upon enrollment. Iterative reconstruction, with no filtering was applied. Each institution was required to receive technical setup visits from the core imaging lab team of PPMI. This team also performed quality controls to maintain uniformity in data quality among the multiple institutions. Images were analyzed with the PMOD software , and the specific binding ratios -1) of each caudate and putamen were acquired with the occipital cortex as a reference tissue. For the healthy control group, minimum SBR values among the bilateral striatal regions were selected; and for the PD group, the dominant side were selected for analysis. To determine the percent decrease of striatal SBRs, ratios of striatal SBRs between PD patients and age-matched normal control patients were acquired.2.3-Bt). Age- and gender-matched groups were brought up by randomization, with a maximum age difference of 2 years. Statistical significance was set at P < .05.Medcalc version 18.9.1  was used for analysis. Demographic factors and striatal SBRs between the groups were compared by one-way ANOVA with Scheffé test for post-hoc, or Kruskal-Wallis test with Conover test for post-hoc. For correlation of SBR values and age or symptom duration, regression analysis was done based on an exponential model (SBR = Ae33.1P < .001). The average symptom duration at enrollment, H&Y scores, MDS-UPDRS II scores, MDS-UPDRS III scores, and SCOPA-AUT of the PD group were 21.7 ± 15.9 months, 1.2 ± 0.8, 6.7 ± 4.1, 20.4 ± 8.6, and 8.7 ± 5.6, respectively. The clinical characteristics of the PD group according to the age of onset were analyzed, with subgroups of symptom onset of 50 years of age or less, 51 to 60 years of age, 61 to 70 years of age, and over 70 years of age  group (n = 141) and the PD patient group n = 301) are presented in Table 01 are pr3.2P < .001) and putamen SBRs (P < .001)  Table .3.3P = .14), but did with the MDS-UPDRS II scores (P < .001), MDS-UPDRS III scores (P < .001), and SCOPA-AUT (P < .01). The putamen SBRs did not show a show a significant correlation with H&Y scores (P = .10) and SCOPA-AUT (P = .02), but did with the MDS-UPDRS II scores (P < .001) and MDS-UPDRS III scores (P < .01)  Table .3.4P = .24), while the putamen SBRs did (P < .05). In PD patients with symptom onset of 50 years of age or less (P = .52), 51 to 60 years of age (P = .46), 61 to 70 years of age (P = .35), and over 70 years of age (P = .52) no significant correlation was observed between caudate SBRs and symptom durations. Furthermore, in PD patients with symptom onset of less than 50 years of age (P = .52), 51 to 60 years of age (P = .13), 61 to 70 years of age (P = .16), and over 70 years of age (P = .56), there was also no significant relationship between putamen SBRs and symptom durations. While the putamen SBRs showed no change as the age increases, the caudate SBRs showed a significant decrease (P < .05) , but there was a significant relationship between the symptom duration and percent decrease of putamen SBRs (P < .01). There was no significant relationship between symptom duration and percent decrease of caudate SBRs (P = .32) and putamen (P = .17) in the age/gender-matched group , the prodromal stage , and the clinical stage (presence of classic motor signs sufficient for diagnosis). A significant portion of the dopaminergic innervation is already lost in the preclinical and prodromal stages.,6 Since the dopaminergic denervation occurs in an exponential manner, the slope of the denervation rate would be higher in the preclinical and prodromal stages than in the clinical stage.,33 An examination of the association between symptom duration and dopaminergic denervation in PD patients in the preclinical and prodromal stages is warranted to fully understand the pattern of dopaminergic denervation. However, the duration of the preclinical stage may be over 20 years before the onset of motor symptoms, making it challenging to conduct such a prospective study with preclinical, prodromal stage patients.In this study, we estimated the rate of dopaminergic denervation in PD patients, in association with symptom durations and aging, and reviewed the possible confounding factors. Unlike numerous previous studies that have demonstrated the relationship or equation between dopaminergic denervation rate and symptom duration, the regression results were not significant or the Our study has some limitations. First, we have not analyzed the correlation between motor severity and dopaminergic denervation. The aim of this study was to review the previous literature and analyze the relationship among symptom duration, age, and the rate of dopaminergic denervation. Second, since the I-123 FP-CIT SPECT images were acquired from multiple institutions, there could be variations of image quality. To maintain uniformity across datasets from the various institutions, the PPMI performs quality control procedures during enrollment of respective institutions. Finally, although I-123 FP-CIT is a presynaptic dopaminergic biomarker, it may not totally reflect the neurodegenerative process. The degree of dopaminergic denervation may vary among different dopamine targeting radioligands.In conclusion, we examined the rate of dopaminergic denervation in PD patients, and analyzed whether it correlated with symptom duration. Though there was a significant correlation between the symptom duration and putamen SBRs, and between the symptom duration and percent decrease of the putamen SBRs, we suggest that the nigrostriatal degeneration involves inherently a greater number of unexplainable variations.Conceptualization: Jeong Won Lee, Yoo Sung Song.Data curation: Jeong Won Lee, Yoo Sung Song.Formal analysis: Jeong Won Lee, Yoo Sung Song, Hyeyun Kim, Bon D. Ku, Won Woo Lee.Funding acquisition: Yoo Sung Song.Methodology: Jeong Won Lee, Yoo Sung Song, Hyeyun Kim, Bon D. Ku, Won Woo Lee.Writing – original draft: Jeong Won Lee, Yoo Sung Song.


The PSI of the isolates ranged between 1.1 to 3.6 mm, with the most efficient and highest PSI being obtained for Bacillus sp. (strain 8) and the lowest for Pseudomonas sp. (strain 15). Six isolates found at all locations were identified at the genus level: Chromobacterium sp., Pseudomonas sp., Bacillus sp., Micrococcus sp., Caulobacter sp., and Aspergillus sp. A correlation was observed between the number of PSMs and the level of soil P availability and moisture content, indicating an increase in soil P availability with a greater abundance of PSMs in soil.Phosphorus (P) is an essential macronutrient for plant growth and is mainly present in agricultural soil in unavailable forms. Phosphate-solubilizing microorganisms (PSMs) increase soil P availability. The objective of the present study was to assess the population and type of PSMs and their relationships with soil characteristics in the agricultural soil of Manokwari. Twenty-one composite soil samples (0–20 cm) were collected at the rhizospheres of plants in the Prafi and Masni Districts. A dilution technique and plate count method on Pikovskayas agar medium were used to examine the PSM population, phosphate-solubilizing index (PSI), and various soil properties. The results obtained showed that the total population of phosphate-solubilizing bacteria ranged between 25×10 Penicillium sp., Mucor sp. and Aspergillus sp. increased plant growth by 5–20% after inoculation . However, their number is not sufficiently high to compete with other microbial species in the rhizosphere . These two areas are fertile, and the major crops are paddy rice, legumes, horticultures, corn, and oil palm. The land use history of this area is mostly food crops (paddy rice and tuber crops) and vegetables as well as some plantation crops. Soil developed from alluvial soil that was dominated by coarse fractions on the surface and fine fractions in the subsoil. There are four soil types in these areas: Entisols, Histosols, Inceptisols, and Ultisols .Soil samples were collected at a depth of 0–20 cm in the rhizosphere at sample points in several locations in the Prafi and Masni Districts of the Manokwari Region in October 2018. A composite soil sample was obtained by mixing 10 sub-samples collected at the rhizosphere of each plant with soil auger (diameter of 2.5 inches). Samples were placed into sterile containers and transported to the laboratory, where they were air-dried and crushed. A portion of each sample was sieved with a 2-mm sieve mesh to remove pebbles and large organic debris, while the remainder was unsieved for a soil biological analysis. Samples were kept in sterile plastics bags and stored in a refrigerator at 4°C for further biological analyses. The coordinates of each sample collection point were obtained using a global positioning system (GPS).Glycine max merril), paddy rice (Oryza sativa), long bean (Vigna sinensis), corn (Zea mays), squash (Luffa acutangula), cacao (Theobrema cacao), chili (Capsicum anuum), kangkong (Ipomoea aquatic), eggplant (Solanum tuberosum), cassava (Manihot utilisina), and peanut (Arachis hypogea).Twenty-one samples were collected from the rhizospheres of agricultural plants, mainly soybean  containing insoluble tricalcium phosphate, followed by an incubation at 27–30°C for 7 days. The composition of the medium was 5 g Ca3(PO4), 0.5 g (NH4)2SO4, 0.2 g NaCl, 0.1 g MgSO4.7H2O, 0.2 g KCl, 10 g glucose, 0.5 g yeast extract, 20 g agar, small amounts of MnSO4 and FeSO4, and 1,000 mL distilled water (–1 (CFU g–1)=(number of colonies)×(dilution factor)  . Colonie factor) .i.e. the clearance zone, including bacterial growth, and the diameter of the colony.Halozone and colony diameters were successively measured during the incubation period to assess the PSI of PSMs. The PSI is the ratio of the total diameter, 2O 1:5), available phosphorus (mg kg–1) , C-organic (g kg–1) , and total nitrogen (g kg–1) , while the soil moisture content (%) (gravimetric method) was measured using a fresh soil sample. Data on the population of PSMs and PSI were analyzed using the t-test. The relationships between PSMs and soil characteristics were examined using Pearson’s correlation (SPSS).Composite soil samples from all locations were air-dried, crushed, and passed through a 2-mm sieve and then analyzed for soil properties, such as pH related to soil-water 1:5 (w/v) , followed by chili (Capsicum anuum) and corn (Zea mays) in Masni District, while the lowest was detected in the rhizosphere of squash (Luffa acutangula) in Prafi District. The total population of PSF was low in most rhizosphere samples. In comparisons between Prafi and Masni Districts, the total populations of PSB and PSF were low in the former. Prafi District is known as the center of agricultural production in Manokwari, most of the lands are cultivated with agricultural crops, and, thus, the use of chemical fertilizers and pesticides is more intense in this area.A significant difference was observed in the population of PSMs between plant rhizospheres in agricultural soil . The totChromobacterium sp. (3 strains), Pseudomonas sp. (17 strains), Bacillus sp. (8 strains), Micrococcus sp. (5 strains), Caulobacter sp. (3 strains), and Aspergillus sp. (1 strain). The representative PSM types found in the agricultural soils of Prafi and Masni Districts are shown in Colony shapes were mostly circular/round (31 isolates) and whitish in color (32 isolates), while cell shapes were mainly rods (coccobacillus) (32 isolates) that were Gram negative (24 isolates) or Gram positive (12 isolates), and the majority of isolates were motile . Six genBacillus sp. (strain 8) and the lowest for Pseudomonas sp. (strain 15).Approximately 58 PSM isolates were found in all sampling locations; however, only 37 PSM isolates, which considered as best PSM isolates, were identified and tested for their PSI . The PSIPseudomonas sp., six showed higher PSI than the others.However, among the isolates of In the present study, an increase in the halo zone was not associated with a larger colony diameter.–1 (low) and 2.95 g kg–1 (medium) in Prafi District, while that at all sampling locations in Masni District was low (0.14–0.16 g kg–1). The phosphorus content in all areas was low to medium (6.00–9.90 mg kg–1), while the carbon organic content was very low to low (0.10 to 0.28 g kg–1) in Prafi District and low (1.20 to 1.91 g kg–1) in Masni District. Prafi and Masni Districts are the main centers of agricultural crop production for vegetables and food crops in Manokwari region and Papua Barat Province; therefore, most of the agricultural land is highly dependent on chemical fertilizers and pesticides, the high and continuous use of which without the application of organic matter has affected the physical, chemical, and biological properties of soil.An analysis of soil properties at each location examined  revealedCorrelations were observed between the number of PSMs and soil P availability and moisture content, indicating an increase in soil P availability with a greater abundance of PSMs . However4 and 106 CFU g–1. Although PSMs are found in all soils, their number depended on the soil climate as well as cropping history . In contrast, a correlation was found between the number of PSB and total nitrogen and organic matter  Population Distribution of Phosphate-solubilizing Microorganisms in Agricultural Soil. https://doi.org/10.1264/jsme2.ME21041


Diagnosis of mature-onset diabetes of the young (MODY), a non-autoimmune monogenic form of diabetes mellitus, is confirmed by genetic testing. However, a positive genetic diagnosis is achieved in only around 50% of patients with clinical characteristics of this disease.HADH expression was significantly lower in patients versus controls. Among patients with suspected MODY, GLIS3 expression was higher in obese versus normal-weight patients, and in patients aged < 25 versus > 25 years at diabetes onset. Significant alteration with respect to controls of any gene was observed in 57.9% of patients.We evaluated the diagnostic utility of transcriptomic analysis in patients with clinical suspicion of MODY but a negative genetic diagnosis. Using Nanostring nCounter technology, we conducted transcriptomic analysis of 19 MODY-associated genes in peripheral blood samples from 19 patients and 8 healthy controls. Normalized gene expression was compared between patients and controls and correlated with each patient’s biochemical and clinical variables. Z-scores were calculated to identify significant changes in gene expression in patients versus controls. Only 7 of the genes analyzed were detected in peripheral blood. Although blood does not seem to be a suitable sample for transcriptomic analysis of patients with suspected MODY, in our study, we detected expression alterations in some of the genes studied in almost 58% of patients. That opens the door for future studies that can clarify the molecular cause of the clinic of these patients and thus be able to maintain a more specific follow-up and treatment in each case.The online version contains supplementary material available at 10.1186/s13023-022-02263-3. Maturity-onset diabetes of the young  is a non-autoimmune monogenic form of diabetes mellitus with characteristic destruction of pancreatic β cells and impaired insulin biosynthesis . MODY clDistinguishing MODY from the two most common types of DM (types 1 and 2) represents an interesting challenge for so-called personalized or precision medicine, as it entails selecting a treatment based on the patient’s etiology. Insulin and metformin administration are the main treatments for DM types 1 and 2, respectively. Hepatocyte nuclear factor-1 alpha (HNF1A)-MODY (MIM#142410) and, in some cases, HNF4A-MODY MIM#600281) are treated with low-dose sulfonylureas, a low-cost oral treatment , 6. Gluc00281 areABCC8, APPL1, BLK, CEL, GCK, HNF1A, HNF1B, HNF4A, INS, KCNJ11, KLF11, NEUROD1, PAX4, and PDX1  and islet antigen-2 [IA-2] antibodies), the preservation of endogenous insulin secretion and at least one of the following: (1) early age of diagnosis (≤ 35 years); (2) a positive, multigenerational family history of diabetes following an autosomal dominant inheritance pattern . The stuThe following variables were recorded for each patient: family history of DM; sex; age at recruitment and at clinical onset; anthropometric characteristics including weight, height, and body mass index (BMI); DM-related blood biochemistry parameters , and C-peptide); and pharmacological treatment. The presence or absence of secondary complications, both microvascular  and macrovascular , was also recorded, in addition to gene expression data obtained using the Nanostring nCounter platform. Clinical data such as the family history of DM, age at clinical onset, pharmacological treatment, and the presence or absence of secondary complications were collected from the patient’s electronic medical records.2) = weight (kg)/height2. Patients were classified according to World Health Organization (WHO) criteria as underweight (BMI < 18.5), normal weight (BMI 18.5–24.99), overweight (BMI 25–29.99), or obese (BMI ≥ 30) [Patients were weighed in the morning before eating. Qualified personnel measured standing height using a wall-mounted stadiometer and body weight using a digital scales. Nutritional status was assessed by calculating BMI using the following formula: BMI (mMI ≥ 30) .® Centaur XP immunoassay system (Siemens). The ADVIA® Chemistry XPT was used to measure glucose levels, and HbA1c was measured using the D-100 TM hemoglobin testing system (Bio-Rad). Reference ranges for the biochemical markers were as follows: glucose 74–105 mg/dL, Hb1Ac < 5.7%, and C-peptide 0.81– 2.85 ng/mL.C-peptide levels were measured using the ADVIA® tubes (Qiagen) to guarantee RNA stability. RNA from each sample was obtained using the PAXgene® Blood RNA Kit (Qiagen), following the manufacturer’s instructions. To assess both the quantity and quality of RNA, the samples were analyzed with the Agilent 2200 TapeStation system, ensuring a RIN (RNA integrity number) > 8 in each case.Blood samples were received from each of the participating centers within 48 h of extraction. These samples were collected in PAXgene® (Nanostring) platform was used to quantify the number of copies of candidate genes for diagnosis of MODY. This assay consists of hybridization, without the need for amplification, of specific probes of each transcript: one with biotin at the 3′ end that allows detection and another reporter probe containing a unique color code at the 5′ end that identifies the gene. After hybridization and removal of excess probes, the probe-target complexes are bound, immobilized, and aligned on the nCounter cartridge [ABCC8, APPL1, BLK, CEL, GCK, GLIS3, HADH, HNF1A, HNF1B, HNF4A, IER3IP1, INS, KCNJ11, KLF11, NEUROD1, PAX4, PDX1, PLAGL1, and UCP2. The transcripts quantified and the specific probe and its position are detailed in Additional file ABCF1, ALAS1, EEF1G, G6PD, GAPDH, GUSB, HPRT1, and TBP. After confirming that all technical parameters fulfilled Nanostring requirements, technical normalization was carried out using the panel negative controls, establishing a background threshold (mean + 2 standard deviations); and using the positive controls to establish a normalization factor to correct for differences due to variations in hybridization efficiency. Finally, we normalized our data based on RNA content, using the GeNorm algorithm to select the optimal number of the most stable reference genes. Ultimately, ABCF1, ALAS1, GAPDH, GUSB, TBP were chosen as reference genes.The nCounterartridge . A speciZ-scores were calculated to identify significant changes in gene expression in patients versus controls. For this, the following formula was used: z-score = (x − µ)/σ, where “x” is the sample normalized counts; “µ”, the mean of normalized counts of controls; and “σ” the standard deviation of normalized counts in the control population.Appropriate statistical analyses were performed using GraphPad Prism software (version 6). Differences between groups means were analyzed using an unpaired t-test or, in cases in which the data did not follow a normal distribution, a non-parametric Mann–Whitney U-test.Pearson’s correlation coefficient was used to evaluate correlations between gene expression data and biochemical and anthropometric parameters, except in cases in which normal distribution could not be assumed, in which case Spearman’s non-parametric correlation was used.p value < 0.05 was considered significant.Data are presented as the mean ± SD. A Additional file 1: Table S1. Presents information on the genes analyzed using the Nanostring nCounter platform, including the corresponding reference sequence and target position.Additional file 2: Table S2 Presents housekeeping genes included in the panel for nCounter-Nanostring analysis.


DSS), disease activity index (p = 0.0025 vs. DSS) and histological damage score (p = 0.023 vs. DSS). No significant effects were obtained for the expression of TJ proteins (claudin-2 and claudin-3) after diosmectite treatment. Characterization of the microbial composition by 16S amplicon NGS showed that diosmectite treatment modified the DSS-associated dysbiosis. Thus, diosmectites are promising candidates for therapeutic approaches to target intestinal inflammation and to identify possible underlying mechanisms of diosmectites in further studies.Natural smectites have demonstrated efficacy in the treatment of diarrhea. The present study evaluated the prophylactic effect of a diosmectite (FI5pp) on the clinical course, colon damage, expression of tight junction (TJ) proteins and the composition of the gut microbiota in dextran sulfate sodium (DSS) colitis. Diosmectite was administered daily to Balb/c mice from day 1 to 7 by oral gavage, followed by induction of acute DSS-colitis from day 8 to 14 . Mice were sacrificed on day 21. Clinical symptoms  were checked daily after colitis induction. Colon tissue was collected for histological damage scoring and quantification of tight junction protein expression. Stool samples were collected for microbiome analysis. Our study revealed prophylactic diosmectite treatment attenuated the severity of DSS colitis, which was apparent by significantly reduced weight loss ( Inflammatory bowel disease (IBD) is a chronic and relapsing inflammatory disease of the intestine with severe, occasionally bloody diarrhea with high-frequency abdominal pain, weight loss, anemia, fever and general physical weakness . Severe Natural clay minerals and their healing properties were already mentioned by Aristotle 384–322 BC) and their external and internal use has been documented over several centuries . Clay mi4–322 BC For the current study we hypothesized that a prophylactic smectite treatment attenuates the clinical course of dextran sulphate sodium (DSS) colitis. Herein, diosmectite FI5pp from Friedland (Germany) was administered before induction of acute DSS colitis in Balb/c mice. Besides clinical parameters and histological alteration of colonic tissue, the effect of diosmectite on the expression of tight junction (TJ) proteins within the colon as well as the gut microbiome, was investigated. p = 0.043 and p = 0.022 vs. DSS, respectively) (p = 0.0025 vs. DSS). Mice without DSS-induced colitis showed an increase in body weight during the experiment A. In conctively) A. Monitoctively) B. The maHistological analysis of the colon in control animals showed a normal morphology of crypts and no signs of inflammation. The colon of DSS mice without treatment revealed multifocal crypt distortion, infiltration of inflammatory cells, extensive epithelial damage , which was not apparent after FI5pp treatment. The claudin-2 level in proximal colons was only slightly enhanced after DSS-treatment but did not reach a significant different value. Similar but less pronounced effects were observed for claudin-2 expression in mice treated with FI5pp. We studied the expression of selected TJ proteins such as claudin-2 and -3 in proximal and distal colon tissue A,B. The Bacteroidales S27-7 group ge and Bacteroides, two commensals in mouse stool, were the most abundant genera in all three groups. We observed a strong decrease of Bacteroidales S27-7 group ge in “DSS” and “FI5pp + DSS” compared to the “Control” group. Bacteroides increased in “DSS”, but slightly decreased in “FI5pp + DSS”. Lachnospiraceae unclassified decreased in “DSS” and “FI5pp + DSS”, compared to the “Control” group. The Lachnospiraceae NK4A136 group was at the same level in all three groups. Prevotellacea UCG-001 was most abundant in the “DSS” group and had the lowest abundance in the “Control” group. Akkermansia and Alistipes were exclusively present in an extremely low relative abundance in the “Control” group. Akkermansia showed the strongest presence in the “DSS” group and Alistipes in the “FI5pp + DSS” group , “DSS” (n = 6) and “FI5pp + DSS” (n = 5) samples. Of those genera, 93 have a relative abundance of below 1% in all 14 samples across the analyzed groups. S” group A. The rarefaction curve based on the number of observed operational taxonomic units (OTU) for the different samples shows no difference among the three groups B. PrinciBacteroidales S27-7group ge represented with 22 distinct OTUs the most common genus in the 100 most abundant OTUs. Eleven of these showed a significantly different expression across all three groups and primarily confirm the higher abundance of Bacteroidales S27-7group ge in “Control” compared to “DSS” and “FI5pp + DSS”. The only significant change for the Bacteroides genus, represented by three OTUs, is the lower amount of reads for OTU0001 in “DSS” than in “FI5pp + DSS”. Of the ten OTUs present, only two showed significant changes for the Lachnospiraceae NK4A136 group. OTU0041 was increased in “Control” versus. “FI5pp + DSS” and OTU0072 in “Control” versus. “DSS”. Both OTUs represented only a small fraction of the total number of reads and confirm the overall same relative abundance for the Lachnospiraceae NK4A136 group. Lachnospiraceae unclassified genus was distributed over 17 OTUs and 7 of those OTUs showed a significant different abundance. While we saw some evidence for the changes on genus level, the total change is not explained by the 100 OTUs with the highest total abundance alone, but due to the large amount of smaller changes outside the top 100. Prevotellacea UCG-001 and Akkermansia rendered as a single OTU . Both were significantly increased in “DSS” compared to “Control” and “FI5pp + DSS”. Additionally, Akkermansia was significantly increased in “FI5pp + DSS” versus. “Control”. Alistipes (OTU0005) was significantly increased in “FI5pp + DSS” compared to “Control” and “DSS”. A second OTU for the genus Alistipes (OTU0015) was increased in “DSS” and “FI5pp + DSS” compared to “Control”.To verify the changes of the seven genera described above on OTU level, we analyzed the differences between the three groups with Metastats . While aDepending on their mineralogical structure, natural silicate minerals are characterized by a large surface area and a strong ion-exchange capacity, which allows high adsorption potential of water, toxins and bacteria. Due to these properties, natural silicate minerals have been used for decades in the treatment of diarrheal conditions and reflux diseases ,15,16. IIn this in vivo study, we found that the clinical course of DSS-induced colitis, characterized by weight lost and increased DAI, was significantly attenuated by prophylactic treatment with FI5pp compared to the untreated DSS-group. The histological examination of the colon remarkably confirmed these findings. DSS-treated mice showed marked morphological epithelial damages and cell infiltration into colon tissue resulting in a high overall histological score and colon shortening. In contrast, FI5pp treatment prevented DSS-induced morphological changes and significantly reduced the histological score. However, it failed to prevent shortening of the colon. These data correspond with the findings in our previous studies, where the application of a diosmectite also decreased histological alteration and improved the overall histological score . AnotherIt is well known that IBD is associated with a disturbed intestinal epithelial barrier, which is due to pathologically altered expression of tight junction proteins. As a consequence, an uncontrolled translocation of luminal substances throughout the epithelium results in an overactive mucosal immune response . The desAkkermansia genus, Alistipes spp., Bacteroidales S24-7, Lachnospiraceae NK4A136 group and Prevotellaceae UCG-001. The Akkermansia genus is part of the Verrucomicrobia phylum and has currently two members (A. glycaniphila and A. muciniphila), which are both strictly anaerobic mucin-degrading bacteria [A. glycaniphila was isolated from python faeces while A. muciniphila is commonly reported in mouse feces and gut. Multiple studies associated an increased Akkermansia population with a lower disease severity [A. muciniphila as a pathobiont which promotes inflammation and colitis [Akkermansia population in our DSS group suggests the latter case. The nearly complete absence of Alistipes spp. in the control samples seems atypical and remains unexplained. A reduction of Alistipes in DSS group compared to FI5pp + DSS treated mice was also observed in patients with IBD, UC and CD [Alistipes finegoldi attenuated DSS-induced colitis in mice [Bacteroidales S24-7 is a family of mostly uncultured bacteria which are dominant in the mouse gut and other mammals [Bacteroidales S24-7 has the highest overall abundance in our data for all three groups and was considerably reduced in the two DSS groups. Multiple Bacteroides spp. have been reported to induce or enhance colitis in mice [Bacteroidales S24-7 contributes to the gut homeostasis in mice.In mammals, the gut microbiome is associated with physiology, human developmental processes and has an important role in human health and disease status ,30. Therbacteria ,36. A. gseverity ,38, whil colitis . The incC and CD . Further in mice . Bactero mammals ,43. Cons in mice ,44, but Lachnospiraceae NK4A136 group and Prevotellaceae UCG-001 leads, in most cases, to uncultured bacteria in the SILVA ribosomal RNA gene database, which restricts conclusions for the genus and function of these OTUs in the mouse gut. The Lachnospiraceae family, represented by the Lachnospiraceae unclassified and Lachnospiraceae NK4A136 group, is a strong producer of butyrate and other short-chain fatty acids [Lachnospiraceae in FI5pp + DSS treated mice, compared to the DSS group, is an indication for a normalization of the gut microbiome to a level shown in animals of the control group. The classification of the OTUs within the ty acids  which arty acids . A reducty acids . We concPrevotella spp. in fecal and colon samples from mice with IBD [Prevotella in the mucosa-associated flora and in fecal samples from human patients with UC or IBD [Prevotella spp. has been negatively [Prevotella intestinalis-induced decrease in interleukin 18 exacerbates colonic inflammation [Prevotellaceae UCG-001 in the FI5pp + DSS group compared to the DSS group in our microbiome analysis could be an explanation for the weaker inflammatory response of the colon.While some studies report a decrease in with IBD ,49, otheC or IBD ,51. Simigatively  and posigatively ,53 assocOverall, our study shows that prophylactic diosmectite treatment has beneficial effects on ulcerative colitis development in mice. We found positive effects on the clinical symptoms associated with DSS-colitis, such as weight loss, stool consistency and occult blood. Furthermore, the positive impact of diosmectite on the histological score, as observed in a previous in vivo study, could be confirmed . Analysiad libitum. Mice aged 15 weeks (average body weight 26 g) were randomly assigned to different experimental groups. All animal experiments were performed in compliance with the German animal protection law and were approved by the local animal care and use committee (7221.3-1.1-029/13).Male Balb/c mice obtained from Charles River Laboratories  were grouped housed with 3–8 mice per cage, under controlled environmental conditions regarding temperature and humidity, with a 12:12 h light:dark cycle. They received standard food and water w/v). Healthy control animals  and DSS-treated animals  received a solution of saline  in a final volume of 125-150 µL (depending on weight). Acute colitis was induced for 7 days (days 8–14) by 4.5% DSS  dissolved in drinking water. Fresh DSS solution was prepared every second day. Mice were euthanized 7 days after the last DSS application (day 21) by an overdose of intraperitoneally injected ketamine-xylazine (90/25 mg/kg BW). The whole colon was removed and the length was measured. Small sections of the proximal and distal colon were collected and stored in liquid nitrogen for protein isolation. The remaining colon was opened along its long axis, unrolled and was fixed in a 10% formalin solution and embedded in paraffin for hematoxylin and eosin (H&E) staining. Prophylactic treatment  was initThe experiment was repeated twice (experiment 1 and experiment 2) with 3–8 mice per group, in total 6 “control” mice, 14 “DSS” mice and 11 “FI5pp + DSS” mice. Four mice from the DSS group had to be euthanized prematurely because they reached the predetermined humane endpoint criteria and had to be excluded from the final evaluation. This resulted in a total number of 10 mice for the DSS group. Data from separate experiments  were combined for the evaluation. Changes in body weight, stool texture/consistency and fecal blood were scored every day to determine the DAI. The DAI was determined according to Cooper et al. with minor modifications, as detailed in After fixing and embedding the tissue in paraffin, thin sections (5 µm) were cut and stained with H&E using standard methods. Pathological changes in colon sections were evaluated using a modified pathology score for evidence of inflammatory damages as described previously  and as dg for 5 min at 4 °C and the supernatant was collected. Total protein concentrations were determined using a BCA Protein Assay Kit . A total of 25 µg protein/sample was used with three biological replicates each. Proteins were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-Page) and the gels were then transferred onto a nitrocellulose filter membrane or polyvinylidene difluoride (PVDF) . Membranes were subsequently blocked in 5% skim milk and incubated (overnight at 4 °C) with primary antibodies against claudin-2 , claudin-3  and ß-actin . The membranes were subsequently washed and incubated (90 min at room temperature) with horseradish peroxidase-conjugated secondary antibodies. The immunoblots were developed with the Western Blot Luminescence Reagent and exposed by a gel documentation system . Densiometric analyses were performed using Fusion-Capt. ß-actin was used as loading control for normalization.Colon proteins were extracted using RIPA buffer supplemented with protease inhibitors . Briefly, frozen proximal and distal samples were powdered using a homogenizer PeqLab precellys24 . The homogenized samples were centrifuged at 10,000× In order to identify differential influences of the treatments on the gut microbiome at the endpoint of the experiment, the bacterial diversity in the mice stool samples was assessed. In experiment 2, stool samples were collected from mice on day 21 . Bacterial genomic DNA from fecal sample (~35 g) was extracted using a QUIAamp DNA Stool Mini Kit  according to the manufacturer’s instructions.www.illumina.com).The isolated DNA was amplified with the bacterial 16S ribosomal RNA (rRNA) gene primers Bakt_341F (CCTACGGGNGGCWGCAG) and Bakt_805R (GACTACHVGGGTATCTAATCC). The PCR amplicon and the PCR index, a quantity and a quality control, and the sequencing of the individual libraries as a pool in one Illumina MiSeq run were performed as described in the Illumina “16S Metagenomic Sequencing Library Preparation” protocol  . Paired p < 0.05 was considered the criterion for statistical significance. A probability level of p < 0.01 was considered very significant.All data were statistically analyzed using GraphPad Prism 6 . Data are expressed as mean ± SEM or mean ± SD. Data from separate experiments  were combined for statistical analyses and presentation in figures and tables. Group and time effects of clinical parameters (weight loss and DAI) were analyzed by two-way ANOVA for repeated measurements. To analyze the impact of DSS-colitis, we first compared “control” and “DSS” groups. To analyze the influence of the treatment during course of colitis, comparison was made between “DSS” and “FI5pp + DSS”. Histological score and protein expressions were analyzed by Mann-Whitney U test. https://www.ncbi.nlm.nih.gov/sra/PRJNA694316) last accessed date 6 August 2021.Microbiome sequencing data have been submitted to the NCBI Short Read Archive repository under the BioProject accession number PRJNA694316 (


Here, we exploited InAs nanowires (NWs) to engineer antenna-coupled THz photodetectors that operated as efficient bolometers or photo thermoelectric receivers at room temperature. We controlled the core detection mechanism by design, through the different architectures of an on-chip resonant antenna, or dynamically, by varying the NW carrier density through electrostatic gating. Noise equivalent powers as low as 670 pWHz Photodetectors that operate in the terahertz (THz) frequency range  have attracted increasing attention in the last two decades in a broad range of application fields, such as astronomy , medicin−19 WHz−1/2) receivers operating at low temperatures for lab- [−11 WHz−1/2), room-temperature (RT), single-pixel devices  or quanfor lab- ; fast  for THzy diodes ,16 for tIn the last decade, a broad research effort has been oriented towards the understanding of the relevant physical mechanisms that govern the detection of far-infrared light in low-dimensional materials. For example, low-dimensional materials, such as graphene ,18,19,20Semiconductor nanowire (NW) FETs also represent a very promising platform for the development of THz-frequency light detectors, owing to their typically low shunt capacitance, in the order of few aF , and to n~1016 cm−3), and the photo-thermoelectric effect (PTE), activated at high carrier densities (n ≳ 1017 cm−3). The PTE effect has also been studied in quantum dots, defined in axially heterostructured InAs/InAs0.3P0.7 NWs [Importantly, the versatility of the NW-FET platform allows it to be easily integrated in THz architectures with tailored detection properties ,35,36, aP0.7 NWs  under exP0.7 NWs .In this work, taking advantage of the physical understanding of the core detection dynamics in semiconductor nanowire FETs, retrieved via near-field THz nanoscopy , we desi17 cm−3 in the InAs NWs. Selenium incorporation has been demonstrated to improve NW mobility and contact resistance [Selenium-doped InAs NWs were grown by chemical beam epitaxy (CBE) in a Riber Compact-21 system by Au-assisted growth ,41 on Insistance ,42. The 2/intrinsic silicon host wafer. Subsequently, source (S), drain (D) and gate (G) electrodes were defined by means of an aligned electron-beam lithography (EBL) procedure, Cr/Au (10/100 nm) thermal evaporation and lift-off. The detectors were then mounted and wire bonded on dual inline packages.Homogeneous Se-doped InAs NWs were integrated within lateral dual gated FETs, as shown in In order to activate different detection mechanisms, we designed two distinct device geometries: a symmetric FET configuration s-FET, c, where In s-FETs, the THz energy was symmetrically driven to the NW, whose temperature was expected to rise homogeneously as a consequence of free-carrier absorption . Insteaddc voltage generators  to drive the source-drain bias (VSD) and gate voltage , while monitoring the current (ISD) through the NW channel. In the employed electrical setup, the heat sink temperature (T) could be set and monitored by the combination of a heater and a temperature sensor, which allowed the control of the device temperature during operation. SD vs. VG curve collected for the two samples in the two different antenna configurations, recorded at two different temperatures, 298 K and 330 K. We then estimated the transconductance (g) curve as the first derivative of the ISD vs. VG characteristic, from which we could retrieve the field-effect mobility (μFE) and the pristine carrier density (n0) of the individual NWs. Indeed, μFE could be estimated from the maximum transconductance (gm) using the formula [μFE = gmwG2/(CwGVSD), where wG is the gate width and CwG is the gate-to-channel capacitance; n0 could be calculated as n0= CwGVth/(eπr2wG) [th is the NW-FET threshold voltage, e is the elementary charge and πr2 is the cross-sectional area of the NW, approximated as a cylinder. Importantly, the estimation of both parameters required knowledge of CwG. For the two different architectures, CwG was simulated using a commercial software . We obtained CwG = 12.3 aF for the s-FET and CwG = 18.1 aF for the a-FET.The transport characteristics of the designed NW-FETs were measured with two  formula  μFE = gm(eπr2wG) , where Vn0 ≳ 5 × 1017 cm−3, μFE decreased with increasing T and at large and positive values of VG. The conductivity (σ) decreased with increasing T, i.e., beyond a specific VG the NW behaved as a degenerate semiconductor. For samples with n0 ≲ 5 × 1017 cm−3, μFE was instead slowly varying with T. In both cases, n0 increased with T as a consequence of the thermal activation of surface donors [e donors .μFE and n0 for the devised NW-FETs, at 298 K. μFE ranged from 80 to 650 cm2 V−1s−1, while n0 ranged from 0.8 to 20 × 1017 cm−3. This latter spread in the carrier density was a combined effect of ambient pressure exposure, processing-related factors and different nanowire morphologies .o ~1.1 mW, calibrated with a power meter (Ophir 3A-P-THz). The divergent QCL beam was first collimated by a TPX (polymethylpentene polymer) lens with 1′ focal length and then focused by a TPX lens with 2′ focal length in a circular focal spot of radius ~200 μm , as retrieved by the measured beam profile in the focal point , azimuthal (φ) and elevation (θ) angles.The optical characterization of the investigated NW-FETs was performed by employing the experimental setup shown in al point b. The QCu), recorded at the D electrode while keeping S grounded, was then pre-amplified with a voltage preamplifier  and sent to a lock-in (SR5210). We used as lock-in reference a modulation frequency of 1.333 kHz, which was also used as a square-wave envelope for the QCL pulses. Δu could be inferred from the lock-in reading (VLI) via the relation Δu = (π√2/2) × VLI/G [o, demonstrating the NW-FET linearity.The measured photovoltage  and θ (E-plane). D0 = Δu/⟨Δu⟩ = 3.75, where ⟨…⟩ represents the average photovoltage over an angle of 4π, calculated as a series approximation, assuming that the variations over θ and φ as separable [We evaluated the antenna directivity eparable .v), defined as the ratio between Δu and the optical power (Pa) impinging on the detector. Pa is related to the intensity in the focal point through the detector effective area (Aeff), as Pa = Io × Aeff. We calculated Aeff as the diffraction limited area [eff = λ2/4 = 2800 μm2, where λ is the free-space wavelength. We note that, from the knowledge of D0, it is possible to evaluate the effective area using a different formalism [eff = D0λ2/4π = 3300 μm2. Thus, in the present geometry, there is a <20% difference in the estimation of Aeff between the diffraction-limited method and the antenna-directivity method.One of the most important figures of merit for a THz photodetector is the voltage responsivity  at different temperatures, it is possible to extrapolate β(VG). The expected trend of ΔuB(VG) is reported in v(VG). Interestingly, the bolometric effect could explain the photoresponse only for VG < 3 V, whereas at higher VG another physical mechanism seemed to dominate. To verify this conclusion, we extrapolated the detector photocurrent by quantifying the change of the ISD vs. VSD characteristic between the illuminated state (THz-on) and the dark state (THz-off), while keeping S grounded and while sweeping VSD. At VG = −2 V  thermal gradient between the D (cold) and S (hot) electrodes, resulting in ISD = σ(VSD–SB∇T) [SD corresponds to a negative electrostatic voltage gradient from D to S). Importantly, for VG > 3 V, the BE was still observable in a negative Δσ/σ = −5%, in agreement with the expected trend of ΔuB(VG).The plot of Rd a-FETs a, displae change . Thus, fG = −2 V b, the THSD–SB∇T)  (here a G < −6 V. This discrepancy can be attributed to two main factors: (i) the loading effect, which is a general phenomenon that affects the responsivity of FETs [G sweeps, especially if the NW is operated at ambient pressure and temperature.We note that the experimentally measured responsivity departed from the expected bolometric response for V of FETs  and (ii) of FETs , which ms-FETs  over the whole gate voltage sweep. This indicated that in symmetric architectures, the photoresponse was mainly driven by the BE. SD vs. VSD characteristic between the illuminated and dark states, testifying a huge change in the NW conductivity upon illumination, Δσ/σ = 25%.A different behaviour was observed in s-FETs d. In thiv. We measured the root mean square of NSD by connecting the detectors to a lock-in amplifier and employing an internal oscillator frequency (f) sweep technique [f < 4 kHz, whereas it flattened close to the thermal noise floor (Johnson-Nyquist noise NJ) at higher frequencies. At the modulation frequency employed in our experiments f = 1.333 kHz, the NSD was ~2 NJ = 2 × (4 kBRT)1/2, where kB is the Boltzmann constant and R is the NW resistance. From the knowledge of NSD we found a minimum NEP = 2 nWHz−1/2 among the tested a-FETs and a minimum NEP = 670 pWHz−1/2 among the tested s-FETs.To assess the detector sensitivity, we evaluated NEP as the ratio between the noise spectral density (NSD) and Rechnique . The ampout = P1 + Vpeak × exp(−(t−P2)/τ), where Vout is the voltage read by the oscilloscope, t is the time independent variable, P1, Vpeak and P2 are fitting parameters and τ was the detector response time. In the whole batch of samples, we extracted τ~1 μs.Finally, we characterized the THz detection speed of the NW-FETs by recording the time trace of the photovoltage with a fast oscilloscope. For this measurement, we used a THz pulse duration of 1.6 μs and we connected the detector output (D electrode) to a high-bandwidth (200 MHz) voltage preamplifier  before the oscilloscope. The recorded waveform is depicted in −1/2) and response times of (1 µs), suitable for real-time sensing, security and imaging applications in the far infrared, opening realistic perspectives for the development of potential nanoarrays for multi-pixel image reconstruction at high (>2 THz) THz frequencies.We engineered semiconductor nanowire field-effect transistors operating as sensitive bolometers or phototermoelectric receivers at 2.8 THz, at room temperature. We selected and controlled the dominant detection dynamics via the symmetry of a lithographically patterned on-chip resonant antenna and through electrostatic gating, respectively. The devised detectors showed state-of-the-art room temperature noise equivalent powers (0.67–2 nWHz