Sensitive label-free detection of the biomarker phosphorylated tau-217 protein in Alzheimer's disease using a graphene-based solution-gated field effect transistor.

Alzheimer's disease (AD) is generally diagnosed using advanced imaging, but recent research suggests early screening using biomarkers in peripheral blood is feasible; among them, plasma tau proteins phosphorylated at threonine 231, threonine 181, and threonine 217 (p-tau217) are potential targets. A recent study indicates that the p-tau217 protein is the most efficacious biomarker. However, a clinical study found a pg/ml threshold for AD screening beyond standard detection methods. A biosensor with high sensitivity and specificity p-tau217 detection has not yet been reported. In this study, we developed a label-free solution-gated field effect transistor (SGFET)-based biosensor featuring a graphene oxide/graphene (GO/G) layered composite. The top layer of bilayer graphene grown using chemical vapor deposition was functionalized with oxidative groups serving as active sites for forming covalent bonds with the biorecognition element (antibodies); the bottom G could act as a transducer to respond to the attachment of the target analytes onto the top GO conjugated with the biorecognition element via π-π interactions between the GO and G layers. With this unique atomically layered G composite, we obtained a good linear electrical response in the Dirac point shift to p-tau217 protein concentrations in the range of 10 fg/ml to 100 pg/ml. The biosensor exhibited a high sensitivity of 18.6 mV/decade with a high linearity of 0.991 in phosphate-buffered saline (PBS); in human serum albumin, it showed approximately 90% of the sensitivity (16.7 mV/decade) in PBS, demonstrating high specificity. High stability of the biosensor was also displayed in this study.

Diagnostic Evaluation Using Salivary Gland Ultrasonography in Primary Sjögren's Syndrome.

The purpose of this study is to investigate the clinical manifestations in patients with early primary Sjögren's syndrome (pSS) based on the severity score found by salivary gland ultrasonography. Thirty-five newly diagnosed patients with early pSS were enrolled and divided into mild (score 0-1) and severe (score 2-3) groups according to the salivary gland ultrasonography grade (SGUS) scores at baseline. Clinical evaluation, ESSPRI and ESSDAI index values, sicca symptoms of the mouth, salivary capacity, and serum autoantibodies and cytokines were investigated. The mean age of pSS patients at diagnosis was 49.9 ± 11.9 years, and the mean duration of sicca symptoms was 0.58 years. ESSPRI (EULAR Sjögren's syndrome patient report index) and ESSDAI (EULAR Sjögren's syndrome disease index) scores were 15.97 and 4.77, respectively. Clinical manifestations, including the low production of saliva and autoantibody production, such as antinuclear antibodies, rheumatoid factor, and anti-SSA antibody, were found. A higher prevalence of rheumatoid factor (p = 0.0365) and antinuclear antibody (p = 0.0063) and a higher elevation of total IgG (p = 0.0365) were found in the severe group than in the mild group. In addition, the elevated titer of IL-25 was detected in the severe group than in the mild group. This observation indicated that salivary gland ultrasonography grade (SGUS) scans may help physicians diagnose pSS and the elevated titer of IL-25 in patients may be implicated in the pathogenesis of pSS.

A chemiresistive biosensor for detection of cancer biomarker in biological fluids using CVD-grown bilayer graphene.

A chemiresistive biosensor is described for simple and selective detection of miRNA-21. We developed chemical vapor deposition (CVD) and low-damage plasma treatment (LDPT)-treated bilayer graphene composite of graphene oxide/graphene (GO/GR) for the determination of a reliable biomarker. We have successfully overcome the self-limiting growth mechanism by using CVD method to grow more than one layer of graphene on copper foil. In addition, LDPT can be used to form GO/GR structures for chemiresistive biosensor applications. Due to the direct formation of BLGR (bilayer graphene), the coupling between graphene layers is theoretically superior to that of stacked BLGR, which is also confirmed by the blue shift of the characteristic peak of graphene in Raman spectroscopy. The shift is about double compared with that of stacked BLGR. Based on the results, the limit of detection for the target miRNA-21 was calculated to be 5.20 fM and detection rage is calculated as 100 fM to 10 nM, which is obviously better performance. Compared with previous work, this chemiresistive biosensor has good selectivity, and stability towards detection of miRNA-21. The ability to detect miRNA-21 in different biological fluids was almost identical to that in pH 7.4 phosphate-buffered saline (PBS). Thus, the proposed bilayer GO/GR of modified chemiresistive biosensor may potentially be applied to detect cancer cells in clinical examinations.

Fecal microbiota changes in NZB/W F1 mice after induction of lupus disease.

The association between the gut microbiota and the development of lupus is unclear. We investigated alterations in the gut microbiota after induction of lupus in a murine model using viral peptide of human cytomegalovirus (HCMV). Three treatment arms for the animals were prepared: intraperitoneal injection of HCMVpp65 peptide, adjuvant alone, and PBS injection. Feces were collected before and after lupus induction biweekly for 16S rRNA sequencing. HCMVpp65 peptide immunization induced lupus-like effects, with higher levels of anti-dsDNA antibodies, creatinine, proteinuria, and glomerular damage, compared with mice treated with nothing or adjuvant only. The Simpson diversity value was higher in mice injected with HCMVpp65 peptide, but there was no difference in ACE or Chao1 among the three groups. Statistical analysis of metagenomic profiles showed a higher abundance of various families (Saccharimonadaceae, Marinifiaceae, and Desulfovibrionaceae) and genera (Candidatus Saccharimonas, Roseburia, Odoribacter, and Desulfovibrio) in HCMVpp65 peptide-treated mice. Significant correlations between increased abundances of related genera (Candidatus Saccharimonas, Roseburia, Odoribacter, and Desulfovibrio) and HCMVpp65 peptide immunization-induced lupus-like effects were observed. This study provides insight into the changes in the gut microbiota after lupus onset in a murine model.

Cytomegalovirus-Associated Autoantibody against TAF9 Protein in Patients with Systemic Lupus Erythematosus.

Background: Evidence indicates a causal link between cytomegalovirus (CMV) infection and the triggering of systemic lupus erythematosus (SLE). Animal studies have revealed that CMV phosphoprotein 65 (pp65) induces autoantibodies against nuclear materials and causes the autoantibody attack of glomeruli. IgG eluted from the glomeruli of CMVpp65-peptide-immunized mice exhibited cross-reactivity against dsDNA and TATA-box-binding protein associated factor 9 (TAF9). Whether the elevation of anti-TAF9 IgG is associated with anti-CMV reactivity in human lupus remains unclear. Methods: The sera from patients with rheumatic diseases, including ankylosing spondylitis (AS), gout, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and Sjögren syndrome (SS) were examined using ELISA for antibodies of CMV, CMVpp65, and TAF9. Results: In total, 83.8% of the rheumatic patients had acquired CMV infections. The SLE patients had a high prevalence of anti-CMV IgM. The highest seropositivity rates for anti-HCMVpp65 and anti-TAF9 IgG were observed in the SLE patients. Purified anti-CMVpp65 IgG from CMVpp65/TAF9 dual-positive SLE sera reacted to both TAF9 and dsDNA. An increased prevalence of proteinuria and low hemoglobin levels were found in CMV IgG- and CMVpp65 IgG-positive SLE patients. Conclusions: This observation suggests that immunity to CMVpp65 is associated with cross-reactivity with TAF9 and dsDNA and that it is involved in the development of clinical manifestations in SLE.

Upregulation of miR-210-5p impairs dead cell clearance by macrophages through the inhibition of Sp1-and HSCARG-dependent NADPH oxidase pathway.

The deficiency of dead cell clearance is a prominent pathogenic factor in systemic lupus erythematosus (SLE). In this study, the overexpression of miR-210-5p resulted in the accumulation of secondary necrotic cells (SNECs) in macrophages through the reduction of protein degradation. The upreguation of miR-210-5p inhibited NADPH oxidase (NOX) activation, reactive oxygen species (ROS) generation, and SNEC clearance. miR-210-5p overexpression suppressed Sp1 and HSCARG expression, and the knockdown of SP1 and HSCARG inhibited NOX expression and superoxide production in macrophages. Furthermore, patients with active SLE expressed a higher level of miR-210-5p and lower expression of SP1 and HSCARG in peripheral blood mononuclear cells. In summary, our findings indicate that the upregulation of miR-210-5p increases the accumulation of SNECs through a decrease in the Sp1-and HSCARG-mediated NOX activity and ROS generation in macrophages. Our results also suggest that targeting miR-210-5p may have therapeutic potential for SLE.

Human cytomegalovirus pp65 peptide-induced autoantibodies cross-reacts with TAF9 protein and induces lupus-like autoimmunity in BALB/c mice.

Human cytomegalovirus (HCMV) has been linked to the triggering of systemic lupus erythematosus (SLE). We proposed that B cell epitope region of HCMV phosphoprotein 65 (HCMVpp65)422-439 mimics an endogenous antigen and initiates lupus-like autoimmunity. Amino acid homology between HCMVpp65422-439 and TAF9134-144 (TATA-box binding protein associated factor 9, TAF9) was investigated using a similarity search in NCBI protein BLAST program (BLASTP). A murine model was used to confirm their antigenicity and ability to induce lupus-like symptoms. HCMVpp65422-439 induced immune responses with the presence of specific antibodies against HCMVpp65422-439 and TAF9134-144, as well as anti-nuclear and anti-double-stranded (ds)DNA antibodies that are characteristic of SLE. In addition, the majority of HCMVpp65422-439 and TAF9134-144 immunized mice developed proteinuria, and their renal pathology revealed glomerulonephritis with typical abnormalities, such as mesangial hypercellularity and immune complex deposition. Immunoglobulin eluted from the glomeruli of HCMVpp65422-439 immunized mice showed cross-reactivity with TAF9134-144 and dsDNA. Increased anti-TAF9 antibody activity was also observed in the sera from SLE patients compared with healthy people and disease controls. Molecular mimicry between HCMVpp65 peptide and host protein has the potential to drive lupus-like autoimmunity. This proof-of-concept study highlights the mechanisms underlying the link between HCMV infection and the induction of SLE.

Impact of urate-lowering drugs on the progression and recovery from chronic kidney disease among gout patients.

BACKGROUND: This study investigates the association between exposure to urate-lowering drugs (ULDs) and progression and recovery from chronic kidney disease (CKD). METHODS: We identified 5860 incident gout patients at Chang Gung Memorial Hospital from 2012 to 2015. Propensity score (PS)-weighted Cox proportional hazards model was used to estimate hazard ratios (HRs) for CKD progression and improvement. A separate analysis was conducted to assess the HR for CKD progression and CKD recovery among those with worsening CKD. RESULTS: The incidence of CKD progression among allopurinol, febuxostat and uricosuric agent users were 1.98, 1.88 and 1.64 per 1000 person-days. Compared with allopurinol users, the PS-weighted HR (95% confidence intervals [CIs]) was 1.77 (0.85-1.76) for febuxostat users and 1.37 (0.71-1.37) for uricosuric agent users for CKD progression and 1.43 (1.26-1.62) for febuxostat users and 1.00 (0.88-1.14) for uricosuric agent users for CKD improvement. Compared to allopurinol users, the HRs for CKD progression were 1.14 (0.80-1.66) for febuxostat users and 0.92 (0.67-1.31) for uricosuric agent users. Among 741 patients who had CKD progression, the incidence of CKD recovery was 1.33, 6.21 and 3.53 per 1000 person-days for allopurinol, febuxostat and uricosuric agent users. The HRs (95% CIs) for recovery in febuxostat and uricosuric agent users were 2.17 (1.40-3.47) and 1.80 (1.20-2.83) compared to allopurinol users. CONCLUSIONS: CKD progression and recovery are common in gout patients using ULDs. Febuxostat and benzbromarone were associated with a similar risk of CKD progression with allopurinol, which has a poorer recovery compared with other ULDs.

Urate-lowering treatment and risk of total joint replacement in patients with gout.

Objectives: To examine whether gout is an independent risk factor for total joint replacement (TJR) and whether urate-lowering treatment (ULT) reduces this risk. Methods: Using the Taiwan National Health Insurance database and the UK Clinical Practice Research Datalink, 74 560 Taiwan patients and 34 505 UK patients with incident gout were identified and age and sex matched to people without gout. Cox proportional hazards models and condition logistic regression were used to examine the risk of TJR in gout patients and the association between cumulative defined daily dose (cDDD) of ULT and TJR. Results: The prevalence rates of TJR in the patients at the time of diagnosis of gout and in people without gout were 1.16% vs 0.82% in Taiwan and 2.61% vs 1.76% in the UK. After a gout diagnosis, the incidence of TJR was higher in the patients with gout compared with those without (3.23 vs 1.91 cases/1000 person-years in Taiwan and 6.87 vs 4.61 cases/1000 person-years in the UK), with adjusted HRs of 1.56 (95% CI 1.45, 1.68) in Taiwan and 1.14 (1.05, 1.22) in the UK. Compared with patients with gout with <28 cDDD ULT, the adjusted ORs for TJR were 0.89 (95% CI 0.77, 1.03) for 28-90 cDDD, 1.03 (0.85, 1.24) for 90-180 cDDD and 1.12 (0.94, 1.34) for >180 cDDD ULT in Taiwan. In the UK, the respective ORs were 1.09 (0.83, 1.42), 0.93 (0.68, 1.27) and 1.08 (0.94, 1.24). Conclusion: This population-based study provides evidence from two nation populations that gout confers significant TJR risk, which was not reduced by current ULT.

Fragment of tegument protein pp65 of human cytomegalovirus induces autoantibodies in BALB/c mice.

INTRODUCTION: Human cytomegalovirus (HCMV) infection has been implicated in the development of autoimmunity, including systemic lupus erythematosus (SLE). Previously we reported that HCMV phosphoprotein 65 (pp65) could induce early onset of autoantibody and glomerulonephritis on lupus-prone NZB/W mice. This study further examined whether the B cell epitope(s) in pp65 is able to drive the development of autoantibody. METHODS: Sera from SLE patients or HCMVpp65-immunized mice were analyzed for anti-nuclear antibody by immunoblotting, enzyme-linked immunosorbent assay (ELISA), immunofluorescent stain and Crithidia luciliae stain. The deposition of immunoglobulin to the kidney was also examined by immunofluorescent stain. The interactions between pp65 sub-fragment to cellular proteins were revealed by yeast two-hybrid analyses. RESULTS: Our results showed that most SLE patients possessed antibodies to the C-terminal half of the HCMVpp65 antigen. Of these positive sera, 73% were also positive to the pp65336-439 sub-fragment. The immunization of pp65336-439 induced formation of multiple anti-nuclear antibodies, including anti-chromatin, anti-centriole, anti-mitotic spindle type I/II (MSA I/II) and a significant elevation of anti-double-stranded DNA (anti-dsDNA) antibodies on BALB/c mice. Yeast two-hybrid analyses revealed the binding of pp65336-439 sub-fragment to cellular proteins. Immunoglobulin deposition on glomeruli was also detected on pp65336-439-immunized mice. CONCLUSIONS: Our data suggested that HCMVpp65336-439 sub-fragment may induce cross-reactive antibodies to several nuclear antigens, which could contribute to the development of autoimmunity in genetic-suspected individuals.