Inhibition of ribosome biogenesis by actinomycin D affects Arabidopsis root development.

Actinomycin D has been reported to selectively inhibit rRNA synthesis and ribosome biogenesis, induce G2 checkpoint of cell cycle arrest in HeLa cells. In Arabidopsis, actinomycin D was also used as agent to preferentially inhibit the ribosome biosynthesis and ribosomal function. However, the function of actinomycin D on Arabidopsis root development remains to be elucidated. In this study, we exposed Arabidopsis seedlings to actinomycin D with the aim of evaluating the effects of ribosome biogenesis on root development. The results demonstrated that actinomycin D inhibited Arabidopsis root growth by reduced meristematic activity in a dose dependent manner. Exposure to actinomycin D decreased the expression of WOX5 and key stem cell niche-defining transcription factors SHR and PLT1, thus the loss function of QC identity and stem cell niche maintenance. In addition, dead cells were observed after actinomycin D treatment in root stele initials and DNA damage response was constitutively activated. Collectively, we propose that ribosome biogenesis plays key role in primary root growth through maintenance of root stem cell niche and DNA damage response in Arabidopsis.

Diagnostic yield and clinical impact of exome sequencing in early-onset scoliosis (EOS).

BACKGROUND: Early-onset scoliosis (EOS), defined by an onset age of scoliosis less than 10 years, conveys significant health risk to affected children. Identification of the molecular aetiology underlying patients with EOS could provide valuable information for both clinical management and prenatal screening. METHODS: In this study, we consecutively recruited a cohort of 447 Chinese patients with operative EOS. We performed exome sequencing (ES) screening on these individuals and their available family members (totaling 670 subjects). Another cohort of 13 patients with idiopathic early-onset scoliosis (IEOS) from the USA who underwent ES was also recruited. RESULTS: After ES data processing and variant interpretation, we detected molecular diagnostic variants in 92 out of 447 (20.6%) Chinese patients with EOS, including 8 patients with molecular confirmation of their clinical diagnosis and 84 patients with molecular diagnoses of previously unrecognised diseases underlying scoliosis. One out of 13 patients with IEOS from the US cohort was molecularly diagnosed. The age at presentation, the number of organ systems involved and the Cobb angle were the three top features predictive of a molecular diagnosis. CONCLUSION: ES enabled the molecular diagnosis/classification of patients with EOS. Specific clinical features/feature pairs are able to indicate the likelihood of gaining a molecular diagnosis through ES.

Fumonisin B1 as a Tool to Explore Sphingolipid Roles in Arabidopsis Primary Root Development.

Fumonisin B1 is a mycotoxin that is structurally analogous to sphinganine and sphingosine and inhibits the biosynthesis of complex sphingolipids by repressing ceramide synthase. Based on the connection between FB1 and sphingolipid metabolism, FB1 has been widely used as a tool to explore the multiple functions of sphingolipids in mammalian and plant cells. The aim of this work was to determine the effect of sphingolipids on primary root development by exposing Arabidopsis (Arabidopsis thaliana) seedlings to FB1. We show that FB1 decreases the expression levels of several PIN-FORMED (PIN) genes and the key stem cell niche (SCN)-defining transcription factor genes WUSCHEL-LIKE HOMEOBOX5 (WOX5) and PLETHORAs (PLTs), resulting in the loss of quiescent center (QC) identity and SCN maintenance, as well as stunted root growth. In addition, FB1 induces cell death at the root apical meristem in a non-cell-type-specific manner. We propose that sphingolipids play a key role in primary root growth through the maintenance of the root SCN and the amelioration of cell death in Arabidopsis.

TBX6 compound inheritance leads to congenital vertebral malformations in humans and mice.

Congenital vertebral malformations (CVMs) are associated with human TBX6 compound inheritance that combines a rare null allele and a common hypomorphic allele at the TBX6 locus. Our previous in vitro evidence suggested that this compound inheritance resulted in a TBX6 gene dosage of less than haploinsufficiency (i.e. <50%) as a potential mechanism of TBX6-associated CVMs. To further investigate this pathogenetic model, we ascertained and collected 108 Chinese CVM cases and found that 10 (9.3%) of them carried TBX6 null mutations in combination with common hypomorphic variants at the second TBX6 allele. For in vivo functional verification and genetic analysis of TBX6 compound inheritance, we generated both null and hypomorphic mutations in mouse Tbx6 using the CRISPR-Cas9 method. These Tbx6 mutants are not identical to the patient variants at the DNA sequence level, but instead functionally mimic disease-associated TBX6 variants. Intriguingly, as anticipated by the compound inheritance model, a high penetrance of CVM phenotype was only observed in the mice with combined null and hypomorphic alleles of Tbx6. These findings are consistent with our experimental observations in humans and supported the dosage effect of TBX6 in CVM etiology. In conclusion, our findings in the newly collected human CVM subjects and Tbx6 mouse models consistently support the contention that TBX6 compound inheritance causes CVMs, potentially via a gene dosage-dependent mechanism. Furthermore, mouse Tbx6 mutants mimicking human CVM-associated variants will be useful models for further mechanistic investigations of CVM pathogenesis in the cases associated with TBX6.

Disease activity in patients with synovitis, acne, pustulosis, hyperostosis, and osteitis (SAPHO) syndrome: the utility of the SPARCC MRI scoring system for assessment of axial spine involvement.

OBJECTIVES: The aim of this study is to investigate the relationship between spinal MRI findings with disease activity and other clinical and serological parameters, and to determine the importance of MRI scoring system in evaluating disease activity of SAPHO syndrome. METHODS: Thirty patients with SAPHO syndrome underwent clinical, laboratory and MRI evaluation at baseline, 3 months, 6 months and 1 year. Magnetic resonance images were analysed using modified Spondyloarthritis Research Consortium of Canada (SPARCC) scoring system. Correlations between MRI score and clinical and laboratory parameters were analysed using Spearman's rank correlation test. RESULTS: Persistent improvement was observed after 12 months in terms of total modified SPARCC scores (37(12,59) vs. 23(5,45) at baseline and 12 months, p<0.05). Total modified SPARCC scores showed Spearman correlations with hypersensitive C-reaction protein (hs-CRP), ankylosing spondylitis disease activity score (ASDAS) and bath ankylosing spondylitis metroloty index (BASMI) at baseline, 3 months, 6 months and 12 months (p varied from <0.001 to <0.05, and r varied from 0.418 to 0.601). Modified SPARCC scores of spine joint, as the largest contribution to the total scores with the mean score of 12(5,30) after 12 months vs. 26 (12,40) at baseline. CONCLUSIONS: The modified SPARCC score proposed in this study exhibits promising potential in the evaluation of extensive radiographic damage in SAPHO and the reflection the disease activity. Our study suggests that MRI could be used together with other parameters of disease activity in the assessment of symptomatic SAPHO patients with spine involvement.

TBX6 missense variants expand the mutational spectrum in a non-Mendelian inheritance disease.

Congenital scoliosis (CS) is a birth defect with variable clinical and anatomical manifestations due to spinal malformation. The genetic etiology underlying about 10% of CS cases in the Chinese population is compound inheritance by which the gene dosage is reduced below that of haploinsufficiency. In this genetic model, the trait manifests as a result of the combined effect of a rare variant and common pathogenic variant allele at a locus. From exome sequencing (ES) data of 523 patients in Asia and two patients in Texas, we identified six TBX6 gene-disruptive variants from 11 unrelated CS patients via ES and in vitro functional testing. The in trans mild hypomorphic allele was identified in 10 of the 11 subjects; as anticipated these 10 shared a similar spinal deformity of hemivertebrae. The remaining case has a homozygous variant in TBX6 (c.418C>T) and presents a more severe spinal deformity phenotype. We found decreased transcriptional activity and abnormal cellular localization as the molecular mechanisms for TBX6 missense loss-of-function alleles. Expanding the mutational spectrum of TBX6 pathogenic alleles enabled an increased molecular diagnostic detection rate, provided further evidence for the gene dosage-dependent genetic model underlying CS, and refined clinical classification.

Human and mouse studies establish TBX6 in Mendelian CAKUT and as a potential driver of kidney defects associated with the 16p11.2 microdeletion syndrome.

Congenital anomalies of the kidney and urinary tract (CAKUTs) are the most common cause of chronic kidney disease in children. Human 16p11.2 deletions have been associated with CAKUT, but the responsible molecular mechanism remains to be illuminated. To explore this, we investigated 102 carriers of 16p11.2 deletion from multi-center cohorts, among which we retrospectively ascertained kidney morphologic and functional data from 37 individuals (12 Chinese and 25 Caucasian/Hispanic). Significantly higher CAKUT rates were observed in 16p11.2 deletion carriers (about 25% in Chinese and 16% in Caucasian/Hispanic) than those found in the non-clinically ascertained general populations (about 1/1000 found at autopsy). Furthermore, we identified seven additional individuals with heterozygous loss-of-function variants in TBX6, a gene that maps to the 16p11.2 region. Four of these seven cases showed obvious CAKUT. To further investigate the role of TBX6 in kidney development, we engineered mice with mutated Tbx6 alleles. The Tbx6 heterozygous null (i.e., loss-of-function) mutant (Tbx6+/‒) resulted in 13% solitary kidneys. Remarkably, this incidence increased to 29% in a compound heterozygous model (Tbx6mh/‒) that reduced Tbx6 gene dosage to below haploinsufficiency, by combining the null allele with a novel mild hypomorphic allele (mh). Renal hypoplasia was also frequently observed in these Tbx6-mutated mouse models. Thus, our findings in patients and mice establish TBX6 as a novel gene involved in CAKUT and its gene dosage insufficiency as a potential driver for kidney defects observed in the 16p11.2 microdeletion syndrome.

Identification of novel FBN1 variations implicated in congenital scoliosis.

Congenital scoliosis (CS) is a form of scoliosis caused by congenital vertebral malformations. Genetic predisposition has been demonstrated in CS. We previously reported that TBX6 loss-of-function causes CS in a compound heterozygous model; however, this model can explain only 10% of CS. Many monogenic and polygenic CS genes remain to be elucidated. In this study, we analyzed exome sequencing (ES) data of 615 Chinese CS from the Deciphering Disorders Involving Scoliosis and COmorbidities (DISCO) project. Cosegregation studies for 103 familial CS identified a novel heterozygous nonsense variant, c.2649G>A (p.Trp883Ter) in FBN1. The association between FBN1 and CS was then analyzed by extracting FBN1 variants from ES data of 574 sporadic CS and 828 controls; 30 novel variants were identified and prioritized for further analyses. A mutational burden test showed that the deleterious FBN1 variants were significantly enriched in CS subjects (OR = 3.9, P = 0.03 by Fisher's exact test). One missense variant, c.2613A>C (p.Leu871Phe) was recurrent in two unrelated CS subjects, and in vitro functional experiments for the variant suggest that FBN1 may contribute to CS by upregulating the transforming growth factor beta (TGF-ß) signaling. Our study expanded the phenotypic spectrum of FBN1, and provided nove insights into the genetic etiology of CS.

Serum IgG4 elevation in SAPHO syndrome: does it unmask a disease activity marker?

OBJECTIVES: SAPHO syndrome is a rare inflammatory disorder with multiple phenotypes, including synovitis, acne, pustulosis, hyperostosis, and osteitis. IgG4 is a subclass of immunoglobulin G, and the elevation of IgG4 has been found in different autoimmune diseases. In the present study, we explored the clinical significance of serum IgG4 levels in patients with SAPHO syndrome. METHODS: Fifty-two patients who met the classification criteria of SAPHO syndrome were included in this study. Clinical data and disease activity markers were collected including erythrocyte sedimentation rate (ESR), high sensitivity C-reactive protein (hsCRP), pain visual analogue scale (VAS), Bath Ankylosing Spondylitis Metrology Index (BASMI), Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI) and Ankylosing Spondylitis Disease Activity Score (ASDAS). Serum immunoglobin (IgA, IgM, and IgG) and IgG subclass (IgG1, IgG2, IgG3, and IgG4) levels were determined using the immunonephelometric assay. RESULTS: Raised serum IgG4 levels (>1400 mg/dL) were detected in 23% (12/52) of patients. Patients with elevated sIgG4 levels had significantly higher pain VAS (5.42±2.76 vs. 3.08±1.78, p=0.02), BASMI (1.80±1.64 vs. 0.38±0.94, p=0.03) and ASDAS (3.20±0.65 vs. 1.74±0.58, p<0.001) levels compared with patients with normal sIgG4 levels. This difference was also observed for ESR (38.2 vs. 22.2 mm/h, p=0.01) and serum CRP (21.0 vs. 2.2 mg/L, p=0.04) levels, which also positively correlated with sIgG4 levels. We also included 4 patients whose IgG4 levels decreased and correlated with the decrease in hsCRP and ESR levels after treatment. CONCLUSIONS: Elevated sIgG4 levels are common in patients with SAPHO syndrome and are associated with high disease activity. Further investigations are needed for this phenomenon.

TBX6-associated congenital scoliosis (TACS) as a clinically distinguishable subtype of congenital scoliosis: further evidence supporting the compound inheritance and TBX6 gene dosage model.

PURPOSE: To characterize clinically measurable endophenotypes, implicating the TBX6 compound inheritance model. METHODS: Patients with congenital scoliosis (CS) from China(N = 345, cohort 1), Japan (N = 142, cohort 2), and the United States (N = 10, cohort 3) were studied. Clinically measurable endophenotypes were compared according to the TBX6 genotypes. A mouse model for Tbx6 compound inheritance (N = 52) was investigated by micro computed tomography (micro-CT). A clinical diagnostic algorithm (TACScore) was developed to assist in clinical recognition of TBX6-associated CS (TACS). RESULTS: In cohort 1, TACS patients (N = 33) were significantly younger at onset than the remaining CS patients (P = 0.02), presented with one or more hemivertebrae/butterfly vertebrae (P = 4.9 × 10‒8), and exhibited vertebral malformations involving the lower part of the spine (T8-S5, P = 4.4 × 10‒3); observations were confirmed in two replication cohorts. Simple rib anomalies were prevalent in TACS patients (P = 3.1 × 10‒7), while intraspinal anomalies were uncommon (P = 7.0 × 10‒7). A clinically usable TACScore was developed with an area under the curve (AUC) of 0.9 (P = 1.6 × 10‒15). A Tbx6-/mh (mild-hypomorphic) mouse model supported that a gene dosage effect underlies the TACS phenotype. CONCLUSION: TACS is a clinically distinguishable entity with consistent clinically measurable endophenotypes. The type and distribution of vertebral column abnormalities in TBX6/Tbx6 compound inheritance implicate subtle perturbations in gene dosage as a cause of spine developmental birth defects responsible for about 10% of CS.

Three patterns of osteoarticular involvement in SAPHO syndrome: a cluster analysis based on whole body bone scintigraphy of 157 patients.

OBJECTIVES: To explore the patterns of osteoarticular involvement in SAPHO syndrome. METHODS: Baseline clinical characteristics and imaging data of 99mTc-MDP whole-body bone scintigraphy (WBBS) were collected from 157 out of 164 patients diagnosed with SAPHO syndrome. The twelve most frequently involved osteoarticular sites were analysed by hierarchical cluster analysis with the Ward minimum-variance method. RESULTS: Three distinctive patterns of osteoarticular involvement were identified: the spinal type (70 patients, 44.6%), with predominantly thoracic, lumbar or sacral vertebral lesions; the costal type (52 patients, 33.1%), with lesions of anterior ribs, particularly the first ribs; and the sternoclavicular type (35 patients, 22.3%), with predominantly sternal and bilateral sternoclavicular lesions, characterized by the typical bullhead sign. Notably, a total of 77 (49%) patients exhibited lesions of ribs on WBBS, of which 61.3% involved the first ribs. Interestingly, patients of spinal type were older at onset of cutaneous manifestations than those of sternoclavicular type (P = 0.036) and costal type (P = 0.035). The disease course was remarkably longer in sternoclavicular type than costal type (P = 0.001) and spinal type (P < 0.001). CONCLUSION: The osteoarticular involvement in SAPHO syndrome can be categorized as three distinct patterns with different corresponding clinical features. The costal involvement in SAPHO syndrome, which was under-recognized previously, may define a distinct sub-type of the disease.

Efficacy of bisphosphonates in patients with synovitis, acne, pustulosis, hyperostosis, and osteitis syndrome: a prospective open study.

OBJECTIVES: To evaluate the clinical efficacy of bisphosphonates treatment for spinal bone marrow oedema (BME) in patients with synovitis, acne, pustulosis, hyperostosis, and osteitis (SAPHO) syndrome. METHODS: SAPHO syndrome patients presenting to Peking Union Medical College Hospital from 2015 to 2016 were recruited. Patients were administered pamidronate disodium 1 mg/kg/d intravenously, for 3 days, at baseline and 3 months later. The symptoms were evaluated using the Visual Analog Score (VAS) for pain, and other clinical measures including, spinal BME scores, ß-crosslaps, osteocalcin, and inflammatory factors, were collected. RESULTS: A total of 30 patients (20 women and 10 men) with a median age of 47.2 (interquartile range 8.8) years were recruited. In a short time, the patients showed a significant decrease in VAS (before vs. after; first treatment: 5.70±1.62 vs. 2.30±1.29 cm, second treatment: 4.03±1.88 vs. 2.17±1.23 cm) and ß-crosslaps (first treatment: 0.4441±0.1923 vs. 0.0859±0.0374 pg/ml, second treatment: 0.2891±0.1983 vs. 0.0962±0.0324 pg/ml) (all p<0.05). At 12-month follow-up, compared with the baseline, we noticed a significant drop in the VAS (5.70±1.62 vs. 2.43±1.25 cm), erythrocyte sedimentation rate (28.87±25.26 vs. 18.00±18.65 mm/h), high-sensitivity C-reactive protein level (11.76±10.19 vs. 5.84±5.88 mg/L), osteocalcin (2.30±1.27 vs. 1.65±0.80 ng/ml), and BME (30.50±24.09 vs. 22.13±27.79) (all p<0.05). No one had serious adverse events. CONCLUSIONS: Bisphosphonates can significantly and rapidly relieve symptoms in patients with SAPHO syndrome and have a long-term effect on inflammation and spinal BME. We suggest that bisphosphonates could be used as the first-line therapeutic drug for SAPHO syndrome, especially in patients with spinal BME.

Arabidopsis COG Complex Subunits COG3 and COG8 Modulate Golgi Morphology, Vesicle Trafficking Homeostasis and Are Essential for Pollen Tube Growth.

Spatially and temporally regulated membrane trafficking events incorporate membrane and cell wall materials into the pollen tube apex and are believed to underlie the rapid pollen tube growth. In plants, the molecular mechanisms and physiological functions of intra-Golgi transport and Golgi integrity maintenance remain largely unclear. The conserved oligomeric Golgi (COG) complex has been implicated in tethering of retrograde intra-Golgi vesicles in yeast and mammalian cells. Using genetic and cytologic approaches, we demonstrate that T-DNA insertions in Arabidopsis COG complex subunits, COG3 and COG8, cause an absolute, male-specific transmission defect that can be complemented by expression of COG3 and COG8 from the LAT52 pollen promoter, respectively. No obvious abnormalities in the microgametogenesis of the two mutants are observed, but in vitro and in vivo pollen tube growth are defective. COG3 or COG8 proteins fused to green fluorescent protein (GFP) label the Golgi apparatus. In pollen of both mutants, Golgi bodies exhibit altered morphology. Moreover, γ-COP and EMP12 proteins lose their tight association with the Golgi. These defects lead to the incorrect deposition of cell wall components and proteins during pollen tube growth. COG3 and COG8 interact directly with each other, and a structural model of the Arabidopsis COG complex is proposed. We believe that the COG complex helps to modulate Golgi morphology and vesicle trafficking homeostasis during pollen tube tip growth.

Comparative analysis of serum proteome in congenital scoliosis patients with TBX6 haploinsufficiency - a first report pointing to lipid metabolism.

Congenital scoliosis (CS) is a three-dimensional deformity of the spine affecting quality of life. We have demonstrated TBX6 haploinsufficiency is the most important contributor to CS. However, the pathophysiology at the protein level remains unclear. Therefore, this study was to explore the differential proteome in serum of CS patients with TBX6 haploinsufficiency. Sera from nine CS patients with TBX6 haploinsufficiency and nine age- and gender-matched healthy controls were collected and analysed by isobaric tagged relative and absolute quantification (iTRAQ) labelling coupled with mass spectrometry (MS). In total, 277 proteins were detected and 20 proteins were designated as differentially expressed proteins, which were submitted to subsequent bioinformatics analysis. Gene Ontology classification analysis showed the biological process was primarily related to 'cellular process', molecular function 'structural molecule activity' and cellular component 'extracellular region'. IPA analysis revealed 'LXR/RXR activation' was the top pathway, which is a crucial pathway in lipid metabolism. Hierarchical clustering analysis generated two clusters. In summary, this study is the first proteomic research to delineate the total and differential serum proteins in TBX6 haploinsufficiency-caused CS. The proteins discovered in this experiment may serve as potential biomarkers for CS, and lipid metabolism might play important roles in the pathogenesis of CS.

The coexistence of copy number variations (CNVs) and single nucleotide polymorphisms (SNPs) at a locus can result in distorted calculations of the significance in associating SNPs to disease.

With the recent advance in genome-wide association studies (GWAS), disease-associated single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) have been extensively reported. Accordingly, the issue of incorrect identification of recombination events that can induce the distortion of multi-allelic or hemizygous variants has received more attention. However, the potential distorted calculation bias or significance of a detected association in a GWAS due to the coexistence of CNVs and SNPs in the same genomic region may remain under-recognized. Here we performed the association study within a congenital scoliosis (CS) cohort whose genetic etiology was recently elucidated as a compound inheritance model, including mostly one rare variant deletion CNV null allele and one common variant non-coding hypomorphic haplotype of the TBX6 gene. We demonstrated that the existence of a deletion in TBX6 led to an overestimation of the contribution of the SNPs on the hypomorphic allele. Furthermore, we generalized a model to explain the calculation bias, or distorted significance calculation for an association study, that can be 'induced' by CNVs at a locus. Meanwhile, overlapping between the disease-associated SNPs from published GWAS and common CNVs (overlap 10%) and pathogenic/likely pathogenic CNVs (overlap 99.69%) was significantly higher than the random distribution (p < 1 × 10-6 and p = 0.034, respectively), indicating that such co-existence of CNV and SNV alleles might generally influence data interpretation and potential outcomes of a GWAS. We also verified and assessed the influence of colocalizing CNVs to the detection sensitivity of disease-associated SNP variant alleles in another adolescent idiopathic scoliosis (AIS) genome-wide association study. We proposed that detecting co-existent CNVs when evaluating the association signals between SNPs and disease traits could improve genetic model analyses and better integrate GWAS with robust Mendelian principles.

Arabidopsis EXO70A1 recruits Patellin3 to the cell membrane independent of its role as an exocyst subunit.

The exocyst is a well-known complex which tethers vesicles at the cell membrane before fusion. Whether an individual subunit can execute a unique function is largely unknown. Using yeast-two-hybrid (Y2H) analysis, we found that EXO70A1 interacted with the GOLD domain of Patellin3 (PATL3). The direct EXO70A1-PATL3 interaction was supported by in vitro and in vivo experiments. In Arabidopsis, PATL3-GFP colocalized with EXO70A1 predominantly at the cell membrane, and PATL3 localization was insensitive to BFA and TryA23. Remarkably, in the exo70a1 mutant, PATL3 proteins accumulated as punctate structures within the cytosol, which did not colocalize with several endomembrane compartment markers, and was insensitive to BFA. Furthermore, PATL3 localization was not changed in the exo70e2, PRsec6 or exo84b mutants. These data suggested that EXO70A1, but not other exocyst subunits, was responsible for PATL3 localization, which is independent of its role in secretory/recycling vesicle-tethering/fusion. Both EXO70A1 and PATL3 were shown to bind PI4P and PI(4,5)P2 in vitro. Evidence was obtained that the other four members of the PATL family bound to EXO70A1 as well, and shared a similar localization pattern as PATL3. These findings offered new insights into exocyst subunit-specific function, and provided data and tools for further characterization of PATL family proteins.

Quadruple valve replacement for patient with infective endocarditis 16 years after Fallot's Tetralogy Repair procedure: A case report.

A 33-year-old man, who had previously undergone repair for Tetralogy of Fallot, presented with extensive infective endocarditis. Following thorough preoperative preparation and evaluation, we performed a simultaneous quadruple valve replacement alongside the repatching of the remaining defect. We posit that this comprehensive one-stage surgical intervention not only enhanced the patient's quality of life but also reduced the necessity for future reoperations. Our approach offers valuable insights for managing adult patients with repaired congenital heart diseases and multiple valve pathologies.

Functional annotation and enrichment analysis of differentially expressed serum proteins in patients with type 2 diabetes after dapagliflozin.

BACKGROUND: Only 50% of patients with type 2 diabetes mellitus (T2DM) can control their blood glucose levels. Dapagliflozin is a selective inhibitor of sodium-glucose co-transporter 2 (SGLT-2) that improves the insulin sensitivity of the liver and peripheral tissues. Many studies confirmed that SGLT2 inhibitors reduce blood glucose and have multiple beneficial effects such as weight loss, lipid regulation, and kidney protection. Nevertheless, the mechanisms of the renal and cardiovascular protective effects of dapagliflozin from the perspective of differentially expressed proteins in the serum of T2DM patients have not been intensively explored so far. AIM: To identify differentially expressed proteins associated with dapagliflozin treatment in patients with T2DM. METHODS: Twenty T2DM patients [hemoglobin A1c (HbA1c) 7.0%-10.0%] were enrolled at The Affiliated Hospital of Inner Mongolia Medical University between January 1, 2017 and December 1, 2018. They received dapagliflozin (10 mg/d) for 3 mo, and the HbA1c < 7.0% target was achieved. The changes in clinical indexes were compared before and after treatments. Label-free quantitative proteomics was used to identify differentially expressed proteins using the serum samples of five patients. The identified differentially expressed proteins were analyzed using various bioinformatics tools. RESULTS: Dapagliflozin significantly improved the clinical manifestation of the patients. There were 18 downregulated proteins and one upregulated protein in the serum samples of patients after dapagliflozin administration. Bioinformatics analyses, including subcellular localization, EuKaryotic Orthologous Groups, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes annotations, were used to profile the biological characteristics of the 19 differentially expressed proteins. Based on the literature and function enrichment analysis, two downregulated proteins, myeloperoxidase (MPO) and alpha II B integrin (ITGA2B), and one upregulated protein, podocalyxin (PCX), were selected for enzyme linked immunosorbent assay validation. These validated differentially expressed proteins had multiple correlations with clinical indexes, including HbAc1 and fasting C-peptide. CONCLUSION: Dapagliflozin has hypoglycemic effects and regulates the serum expressions of MPO, ITGA2B, and PCX, possibly contributing to the effects of dapagliflozin on oxidative stress, insulin resistance, and lipid metabolism.