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Up to 40% of patients with diabetes mellitus will develop diabetic kidney disease (DKD), characterized pathologically by the accumulation of extracellular matrix proteins, which leads to the loss of kidney function over time. Our previous studies showed that the pan-protease inhibitor alpha 2-macroglobulin (A2M) is increased in DKD and is a critical regulator of the fibrotic response in glomerular mesangial cells (MC), an initial site of injury during DKD development. How A2M is regulated by high glucose (HG) has not yet been elucidated and is the focus of this investigation. Using serial deletions of the full A2M promoter, we identified the -405 bp region as HG-responsive in MC. Site-directed mutagenesis, siRNA, and ChIP studies showed that the transcription factor, nuclear factor of activated T cells 5 (NFAT5), regulated A2M promoter activity and protein expression in response to HG. Forkhead box P1 (FOXP1) served as a cooperative binding partner for NFAT5, required for A2M upregulation. Lastly, we showed that Smad3, known for its role in kidney fibrosis, regulated A2M promoter activity and protein production independently of HG. The importance of NFAT5, FOXP1, and Smad3 in A2M regulation was confirmed in ex vivo studies using isolated glomeruli. In conclusion, Smad3 is required for basal and HG-induced A2M expression, while NFAT5 and FOXP1 cooperatively regulate increased A2M transcription in response to HG. Inhibition of NFAT5/FOXP1 will be further evaluated as a potential therapeutic strategy to inhibit A2M production and attenuate profibrotic signaling in DKD.

The differential diagnosis for neonatal primary lung masses includes developmental anomalies and congenital lung tumors. Fetal lung interstitial tumor (FLIT) is a rare benign mesenchymal lesion which presents either antenatally or within the first 3 months of age. FLIT is a circumscribed solid-cystic mass which histologically resembles the fetal lung during the canalicular stage at 20-24 weeks of gestation. It is composed of immature mesenchymal cells expanding the interstitium and irregular airspace-like structures. Of all published cases, only 1 identified an α2-macroglobulin ( A2M )::anaplastic lymphoma kinase ( ALK ) fusion and all cases underwent surgical resection in the neonatal or infancy period. We present the second case of FLIT with an A2M::ALK fusion diagnosed postnatally in a neonate which partially regressed spontaneously during conservative management with interim resection at 39 months of age, and provide a review of the literature., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Long non-coding RNAs (lncRNAs) have been reported as key regulators of chronic obstructive pulmonary disease (COPD). This study aimed to figure out the regulatory mechanism as well as the effects of lncRNA00612 (LINC00612) in lipopolysaccharide (LPS)-induced inflammation and apoptosis in BEAS-2B cells. LINC00612 and its co-expressed gene alpha-2-macroglobulin (A2M) were strikingly downregulated in the peripheral venous blood of COPD patients. Overexpressed LINC00612 enhances BEAS-2B cells against apoptosis and inflammatory reactions mediated by LPS, however, an A2M knockdown can attenuate the degree of the enhancement. Bioinformatics analysis revealed putative binding sites between LINC00612, signal transducer and activator of transcription 3 (STAT3) and the A2M promoter, while RNA antisense purification and Chromatin immunoprecipitation were performed to confirm the prediction. Knockdown of LINC00612 impaired the binding of p-STAT3 to the promoter of A2M, which meant that LINC00612 was critical for the binding of STAT3 with the A2M promoter. Therefore, it can be concluded that LINC00612 ameliorates LPS-induced cell apoptosis and inflammation via recruiting STAT3 to bind to A2M. This conclusion will serve as a theoretical foundation for the treatment of COPD., Competing Interests: The authors declare that they have no competing interests., (© 2023 Xiao et al.)

Background: Many indigenous peoples are at elevated risk for otitis media, however there is limited information on hearing loss due to OM in these communities. An Indigenous Filipino community that has previously been described with an elevated prevalence of OM that is due to rare A2ML1 variants and a common FUT2 variant underwent additional phenological testing. In this study, we describe the audiologic profiles in A2ML1 - and FUT2 -related otitis media and the validity of otoscopy and genotyping for A2ML1 and FUT2 variants in screening for otitis media and hearing loss. Method: We analyzed A2ML1 and FUT2 genotypes together with demographic, otologic and audiologic data from tympanometry and hearing level assessments of 109 indigenous individuals. Results: We confirmed previous findings of a spectrum of nonsyndromic otitis media as associated with A2ML1 variants. A2ML1 and FUT2 variants were associated with high-frequency hearing loss at 4000 Hz. As expected, young age was associated with flat tympanograms, and eardrum perforations due to chronic otitis media were associated with severe-to-profound hearing loss across frequencies. Adding A2ML1 or FUT2 genotypes improved the validity of otoscopy as a screening test to rule out moderate-to-profound hearing loss. Conclusion: Continued multi-disciplinary management and audiologic follow-up using tympanometry and screening audiometry are needed to document and treat otitis media and prevent permanent hearing loss in the indigenous community.

Background/aims: This study aims to assess the contribution of biallelic CPAMD8 variants in patients with different forms of glaucoma, especially primary open-angle glaucoma (POAG) and primary angle-closure glaucoma (PACG), based on a systematic analysis of exome sequencing (ES)., Methods: Potentially pathogenic CPAMD8 variants were selected from the ES data of 5307 subjects with various eye conditions through multiple bioinformatics analyses. Of the 5307 subjects, 1221 probands had different forms of primary glaucoma. The genotype-phenotype correlation was assessed by a systematic review of biallelic CPAMD8 variants that including our data and data from the literature. The expression profile of CPAMD8 in human tissues was determined at the mRNA and protein levels., Results: Biallelic CPAMD8 variants, including one frameshift and six missense variants, were exclusively present and significantly enriched in patients with glaucoma (one with juvenile open-angle glaucoma (JOAG), two with POAG and two with PACG) compared with none of the 4086 probands with other eye conditions in this cohort (p=4.1E-07). The effect of variants in these patients is relatively mild compared with that reported in patients with anterior segment dysgenesis or primary congenital glaucoma. CPAMD8 mRNA was highly expressed in the optic nerve, ciliary body, retina and iris, whereas the CPAMD8 protein was mainly detected in the nonpigmented epithelium of the iris and ciliary process, determined by immunohistochemistry., Conclusions: The data from this study not only provide further evidence to support the association of biallelic CPAMD8 variants with JOAG but also suggest that biallelic CPAMD8 variants might be associated with POAG and PACG., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Ferroptosis is a newly-discovered cell death mechanism involved in the progression of various tumors, the role of noncoding RNAs (ncRNAs) in it was relatively less explored. This study identified the low levels of a recently studied long noncoding RNA (lncRNA), A2M-AS1, in pancreatic cancer and suggested its positive correlation with the overall survival time of patients with pancreatic cancer. A2M-AS1 was mainly localized in the cytoplasm, inhibiting the cellular proliferation, migration, and invasion as well as the tumor growth of the pancreatic cancer cells. Moreover, the Erastin-induced ferroptosis increased the expression levels of A2M-AS1. The overexpression of A2M-AS1 promoted ferroptosis in the pancreatic cancer, which was inhibited by the silencing of A2M-AS1. Mechanically, A2M-AS1 could directly interact with the poly (rC) binding protein 3 (PCBP3), which plays an important role in the process of iron metabolism, thereby promoting the ferroptosis in pancreatic cancer. In addition, the A2M-AS1/PCBP3 axis could facilitate the p38 activation and inhibit the phosphorylation of the AKT-mTOR signaling pathway; all these participate in regulating ferroptosis. In conclusion, the regulation of ferroptosis by targeting the A2M-AS1/PCBP3 axis might provide a novel target for the treatment of pancreatic cancer in the future., Implications: A2M-AS1 might be a potential novel therapeutic target for patients with pancreatic cancer in the future., (©2022 American Association for Cancer Research.)

Background: Intrahepatic cholangiocarcinoma (ICC) is a type of malignant tumor ranking the second in the incidence of primary liver cancer following hepatocellular carcinoma. Both the morbidity and mortality have been increasing in recent years. Small duct type of ICC has potential therapeutic targets. But overall, the prognosis of patients with ICC is usually very poor., Methods: To search latent therapeutic targets for ICC, we programmatically selected the five most suitable microarray datasets. Then, we made an analysis of these microarray datasets (GSE26566, GSE31370, GSE32958, GSE45001 and GSE76311) collected from the Gene Expression Omnibus (GEO) database. The GEO2R tool was effective to find out differentially expressed genes (DEGs) between ICC and normal tissue. Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were executed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v 6.8. The Search Tool for the Retrieval of Interacting Genes (STRING) database was used to analyze protein-protein interaction of these DEGs and protein-protein interaction of these DEGs was modified by Cytoscape3.8.2. Survival analysis was performed using Gene Expression Profiling Interactive Analysis (GEPIA) online analysis tool., Results: A total of 28 upregulated DEGs and 118 downregulated DEGs were screened out. Then twenty hub genes were selected according to the connectivity degree. The survival analysis results showed that A2M was closely related to the pathogenesis and prognosis of ICC and was a potential therapeutic target for ICC., Conclusions: According to our study, low A2M expression in ICC compared to normal bile duct tissue was an adverse prognostic factor in ICC patients. The value of A2M in the treatment of ICC needs to be further studied., (© 2021. The Author(s).)

Background: The previous studies have identified several genes in relation to Alzheimer's disease (AD), such as ABCA7, CR1, etc. A few studies have explored the association between the common variants, mainly in the non-coding regions of these genes, and cerebrospinal fluid (CSF) biomarkers. Fewer studies target the variants in the coding regions., Objective: To illustrate the association between the common variants within or adjacent to the coding regions of AD susceptible genes and CSF biomarkers in AD patients., Methods: 75 sporadic probable AD patients were extracted from the dementia cohort of Peking Union Medical College Hospital. They all had history inquiry, physical examination, blood test, cognitive assessment, brain MRI, CSF testing of Aβ42, 181p-tau, and t-tau, and next-generation DNA sequencing. Sixty-nine common single nucleotide polymorphisms (SNPs) (minor allele frequency > 0.01) within or near the coding region of 13 AD susceptible genes were included in the analysis., Results: The rs7412-CC (APOE) genotype showed lower CSF Aβ42 level and higher p-tau/Aβ42 ratio than the rs7412-CT genotype. The rs3752246-C (ABCA7) allele correlated with lower CSF Aβ42 level. The alternate alleles of six ABCA7 SNPs were related to lower CSF p-tau, including rs3745842, rs3764648, rs3764652, rs4147930, rs4147934 and rs881768. The rs11609582-TT (A2M) genotype showed higher CSF p-tau than the rs11609582-TA genotype. The p-tau/Aβ42 ratio was higher in the rs490460-TT (BACE1) genotype relative to the rs490460-GT genotype., Conclusion: Some common variants within or near the coding regions of APOE, ABCA7, A2M, and BACE1 are associated with CSF Aβ42, p-tau. or p-tau/Aβ42.

Lung adenocarcinoma (LUAD) is a malignant tumor that occurs in the lungs. Numerous reports have substantiated the participation of long non-coding RNAs (lncRNAs) in the tumorigenesis of LUAD. Previously, lncRNA alpha-2-macroglobulin antisense RNA 1 (A2M-AS1) was confirmed to be an important regulator in the biological processes of LUAD and dysregulation of A2M-AS1 was associated with non-small cell lung cancer (NSCLC) progression. However, the precise mechanism of A2M-AS1 in LUAD has not been elucidated. Therefore, our study was designed to investigate the detailed molecular mechanism of A2M-AS1 in LUAD. Herein, the expression of lncRNA A2M-AS1, microRNA (miRNA) miR-587, and bone morphogenetic protein 3 (BMP3) in LUAD cell lines and tissues were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting. The viability, proliferation, migration and invasion of LUAD cells were tested by cell counting kit-8 (CCK-8), colony formation and Transwell assays. In vivo tumor growth was investigated by xenograft animal experiment. Interactions among A2M-AS1, miR-587 and BMP3 were measured by RNA pulldown and luciferase reporter assays. In this study, A2M-AS1 was downregulated in LUAD tissues and cells and related to poor prognosis in LUAD patients. A2M-AS1 overexpression suppressed LUAD cell proliferation, migration and invasion in vitro and inhibited tumor growth in vivo. Mechanistically, A2M-AS1 directly bound with miR-587 to promote BMP3 expression in LUAD cells. Low expression of BMP3 was found in LUAD tissues and cells and was closely correlated with poor prognosis in LUAD patients. BMP3 deficiency reserved the inhibitory influence of A2M-AS1 overexpression on LUAD cell behaviors. Overall, A2M-AS1 inhibits cell growth and aggressiveness via regulating the miR-587/BMP3 axis in LUAD.

Introduction: Gastric ulcer is a multifaceted process and is usually caused by mucosal damage. Herbal medicines have received much attention considering the side effects of chemical drugs. Nowadays, the use of herbal medicines has received much attention considering the side effects of chemical drugs. Quercus brantii Lindl, Cirsium vulgare (Savi) Ten, and Falcaria vulgaris Bernh are plants used as traditional phytomedicine for gastric ulcer diseases., Aim of the Study: This study was aimed to investigate the protective effects of hydroalcoholic extracts of these herbs on ethanol-induced gastric ulceration, in addition, to investigate the antioxidant, anti-inflammatory, and gene expression., Materials and Methods: Thirty Sprague Dawley rats, (200-250 g), were divided into six groups: Control: intact animals; sham: gavaged with distilled water (14 days); negative control: gavaged with 20 mg/kg of omeprazole (14 days); experimental groups I, II, and III: gavaged with 500 mg/kg of the extract of Falcaria vulgaris, Quercus brantii, and Cirsium vulgare, respectively, (14 days). The number of ulcers and pathological parameters were assessed. The serum superoxide dismutase, catalase, glutathione peroxidase, malondialdehyde, total antioxidant capacity, albumin, total protein, haptoglobin, alpha-1-acid glycoprotein, total globulin, alpha-2-macroglobulin, C-fos, C-myc, and Caspase-9 were measured by ELISA and RT-PCR., Results: The extracts significantly reduced gastric ulcer (52.33%). The results showed that the Quercus brantii extract was more effective. There were significant differences between the serum levels of alpha-1-acid glycoprotein and those of alpha-2-macroglobulin. Also, there was a significant difference in the serum level of antioxidant parameters. Changes in the expression of the genes also confirmed the results suggested by other parameters. The expression levels of C-fos, C-myc, and caspase-9 were decreased, but the Bcl-2 expression increased., Conclusion: The hydro-alcoholic extracts revealed various protection and noticeable change in the expression of caspase-9, C-myc, C-fos, and Bcl-2 genes in rats., (© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Epidemiological studies have implied that the nonsteroidal anti-inflammatory drug (NSAID) indomethacin slows the development and progression of Alzheimer's disease (AD). However, the underlying mechanisms are notably understudied. Using a chimeric mouse/human amyloid precursor protein (Mo/HuAPP695swe) and a mutant human presenilin 1 (PS1-dE9) (APP/PS1) expressing transgenic (Tg) mice and neuroblastoma (N) 2a cells as in vivo and in vitro models, we revealed the mechanisms of indomethacin in ameliorating the cognitive decline of AD. By screening AD-associated genes, we observed that a marked increase in the expression of α 2 -macroglobulin (A2M) was markedly induced after treatment with indomethacin. Mechanistically, upregulation of A2M was caused by the inhibition of cyclooxygenase-2 (COX-2) and lipocalin-type prostaglandin D synthase (L-PGDS), which are responsible for the synthesis of prostaglandin (PG)H 2 and PGD 2 , respectively. The reduction in PGD 2 levels induced by indomethacin alleviated the suppression of A2M expression through a PGD 2 receptor 2 (CRTH2)-dependent mechanism. Highly activated A2M not only disrupted the production and aggregation of β-amyloid protein (Aβ) but also induced Aβ efflux from the brain. More interestingly, indomethacin decreased the degradation of the A2M receptor, low-density lipoprotein receptor-related protein 1 (LRP1), which facilitated the brain efflux of Aβ. Through the aforementioned mechanisms, indomethacin ameliorated cognitive decline in APP/PS1 Tg mice by decreasing Aβ production and clearing Aβ from the brains of AD mice.

The RASopathies are a group of clinically and genetically heterogeneous developmental disorders caused by dysregulation of the RAS/MAPK signalling pathway. Variants in several components and regulators of this pathway have been identified as the pathogenetic cause. In 2015, missense variants in A2ML1 were reported in three unrelated families with clinical diagnosis of Noonan syndrome (NS) and a zebrafish model was presented showing heart and craniofacial defects similar to those caused by a NS-associated Shp2 variant. However, a causal role of A2ML1 variants in NS has not been confirmed since. Herein, we report on 15 individuals who underwent screening of RASopathy-associated genes and were found to carry rare variants in A2ML1, including variants previously proposed to be causative for NS. In cases where parental DNA was available, the respective A2ML1 variant was found to be inherited from an unaffected parent. Seven index patients carrying an A2ML1 variant presented with an alternate disease-causing genetic aberration. These findings underscore that current evidence is insufficient to support a causal relation between variants in A2ML1 and NS, questioning the inclusion of A2ML1 screening in diagnostic RASopathy testing.

Fluorescence in situ hybridization (FISH) is a conventional method used to visualize the distribution of DNA elements within a genome. To examine the relationships within the Chrysanthemum genus, ribosomal DNA (rDNA), a popular cytogenetic marker, was utilized as a probe for FISH within this genus. Based on the genome data of Chrysanthemum nankingense, C. seticuspe and its allied genera in the Compositae(Asteraceae), we explored rDNA sequences to design oligonucleotide probes and perform oligonucleotide fluorescence in situ hybridization (Oligo-FISH) in eight Chrysanthemum accessions. The results showed that the majority of 5S rDNA signals were located in subterminal chromosome regions and that the number of 5S rDNA sites might be tightly associated with ploidy. For 45S rDNA sites, the number and intensity of signals differed from those of previously investigated Chrysanthemum resources. These findings may provide an optimally reliable method of examining the chromosome composition and structural variation of Chrysanthemum and its related species and allow researchers to understand the evolutionary history and phylogenetic relationships of Chrysanthemum.

Human α 2 -macroglobulin (A2M) is the most characterized protease inhibitor in the alpha-macroglobulin (αM) superfamily, but the structure of its native conformation has not been determined. Here, we combined negative stain electron microscopy (EM), small-angle X-ray scattering (SAXS), and cross-linking-mass spectrometry (XL-MS) to investigate native A2M and its collapsed conformations that are obtained through aminolysis of its thiol ester by methylamine or cleavage of its bait region by trypsin. The combined interpretation of these data resulted in a model of the native A2M tetramer and its conformational changes. Native A2M consists of two crescent-shaped disulfide-bridged subunit dimers, which face toward each other and surround a central hollow space. In native A2M, interactions across the disulfide-bridged dimers are minimal, with a single major interface between the linker (LNK) regions of oppositely positioned subunits. Bait region cleavage induces both intrasubunit domain repositioning and an altered configuration of the disulfide-bridged dimer. These changes collapse the tetramer into a more compact conformation, which encloses an interior protease-trapping cavity. A recombinant A2M with a modified bait region was used to map the bait region's position in native A2M by XL-MS. A second recombinant A2M introduced an intersubunit disulfide into the LNK region, demonstrating the predicted interactions between these regions in native A2M. Altogether, our native A2M model provides a structural foundation for understanding A2M's protease-trapping mechanism, its conformation-dependent receptor interactions, and the dissociation of native A2M into dimers due to inflammatory oxidative stress., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Most proteins in the α-macroglobulin (αM) superfamily contain reactive thiol esters that are required for their biological function. Here, we have characterized the human α2-macroglobulin (A2M) and complement component C3 mutants A2M Q975C and C3 Q1013C, which replace the CGEQ thiol ester motifs of the original proteins with the disulfide-forming sequence CGEC. Mass spectrometry showed that the intended disulfide was formed in both proteins. The correct folding and native conformation of A2M Q975C were shown by its assembly to a tetramer, an initially slow electrophoretic mobility with a demonstrable conformational collapse induced by proteolysis, functional protease trapping, and conformation-dependent interactions with low-density lipoprotein receptor-related protein 1. However, A2M Q975C had a decreased capacity to inhibit trypsin and was more susceptible to cleavage by trypsin or thermolysin when compared to wild-type A2M. C3 Q1013C also folded correctly and was initially in a native conformation, as demonstrated by its cation exchange elution profile, electrophoretic mobility, and interaction with complement factor B, although it assumed a conformation that was distinct from native C3, C3b, or C3(H 2 O) when cleaved by trypsin. These results demonstrate that disulfides can substitute thiol esters and maintain the native conformations of A2M and C3. Additionally, they indicate that proteolysis is not the sole factor in the conformational changes of A2M and C3 and that thiol ester lysis also plays a role.

Proteins in the α-macroglobulin (αM) superfamily use thiol esters to form covalent conjugation products upon their proteolytic activation. αM protease inhibitors use theirs to conjugate proteases and preferentially react with primary amines ( e.g. on lysine side chains), whereas those of αM complement components C3 and C4B have an increased hydroxyl reactivity that is conveyed by a conserved histidine residue and allows conjugation to cell surface glycans. Human α 2 -macroglobulin-like protein 1 (A2ML1) is a monomeric protease inhibitor but has the hydroxyl reactivity-conveying histidine residue. Here, we have investigated the role of hydroxyl reactivity in a protease inhibitor by comparing recombinant WT A2ML1 and the A2ML1 H1084N mutant in which this histidine is removed. Both of A2ML1s' thiol esters were reactive toward the amine substrate glycine, but only WT A2ML1 reacted with the hydroxyl substrate glycerol, demonstrating that His-1084 increases the hydroxyl reactivity of A2ML1's thiol ester. Although both A2ML1s conjugated and inhibited thermolysin, His-1084 was required for the conjugation and inhibition of acetylated thermolysin, which lacks primary amines. Using MS, we identified an ester bond formed between a thermolysin serine residue and the A2ML1 thiol ester. These results demonstrate that a histidine-enhanced hydroxyl reactivity can contribute to protease inhibition by an αM protein. His-1084 did not improve A2ML1's protease inhibition at pH 5, indicating that A2ML1's hydroxyl reactivity is not an adaption to its acidic epidermal environment., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Harwood et al.)

Noonan syndrome (NS) is an autosomal-dominant disorder with variable expressivity and locus heterogeneity. Despite several RAS pathway genes were implicated in NS, 20-30% of patients remain without molecular diagnosis, suggesting the involvement of further genes or multiple mechanisms. Eight patients out of 60, negative for conventional NS mutation analysis, with heterogeneous NS phenotype were investigated by means of target resequencing of 26 RAS/MAPK pathway genes. A trio was further characterized by means of whole-exome sequencing. Protein modeling and in silico prediction of protein stability allowed to identify possible pathogenic RAS pathway variants in four NS patients. A new c.355T>C variant in LZTR1 was found in patient 43. Two patients co-inherited variants in LRP1 and LZTR1 (patient 53), or LRP1 and SOS1 genes (patient 67). The forth patient (56) carried a compound heterozygote of RASAL3 gene variants and also an A2ML1 variant. While these subclinical variants are singularly present in healthy parents, they co-segregate in patients, suggesting their addictive effect and supporting a digenic inheritance, as alternative model to a more common monogenic transmission. The ERK1/2 and SAPK/JNK activation state, assessed on immortalized lymphocytes from patients 53 and 67 showed highest phosphorylation levels compared to their asymptomatic parents. These findings together with the lack of their co-occurrence in the 1000Genomes database strengthen the hypothesis of digenic inheritance in a subset of NS patients. This study suggests caution in the exclusion of subclinical variants that might play a pathogenic role providing new insights for alternative hereditary mechanisms.

Abnormal development of the ocular anterior segment may lead to a spectrum of clinical phenotypes ranging from primary congenital glaucoma (PCG) to variable anterior segment dysgenesis (ASD). The main objective of this study was to identify the genetic alterations underlying recessive congenital glaucoma with ASD (CG-ASD). Next-generation DNA sequencing identified rare biallelic CPAMD8 variants in four patients with CG-ASD and in one case with PCG. CPAMD8 is a gene of unknown function and recently associated with ASD. Bioinformatic and in vitro functional evaluation of the variants using quantitative reverse transcription PCR and minigene analysis supported a loss-of-function pathogenic mechanism. Optical and electron microscopy of the trabeculectomy specimen from one of the CG-ASD cases revealed an abnormal anterior chamber angle, with altered extracellular matrix, and apoptotic trabecular meshwork cells. The CPAMD8 protein was immunodetected in adult human ocular fluids and anterior segment tissues involved in glaucoma and ASD (i.e., aqueous humor, non-pigmented ciliary epithelium, and iris muscles), as well as in periocular mesenchyme-like cells of zebrafish embryos. CRISPR/Cas9 disruption of this gene in F0 zebrafish embryos (96 hpf) resulted in varying degrees of gross developmental abnormalities, including microphthalmia, pharyngeal maldevelopment, and pericardial and periocular edemas. Optical and electron microscopy examination of these embryos showed iridocorneal angle hypoplasia (characterized by altered iris stroma cells, reduced anterior chamber, and collagen disorganized corneal stroma extracellular matrix), recapitulating some patients' features. Our data support the notion that CPAMD8 loss-of-function underlies a spectrum of recessive CG-ASD phenotypes associated with extracellular matrix disorganization and provide new insights into the normal and disease roles of this gene.

Chronic systemic inflammation contributes to cardiovascular disease (CVD) and correlates with the abundance of acute phase response (APR) proteins in the liver and plasma. Bromodomain and extraterminal (BET) proteins are epigenetic readers that regulate inflammatory gene transcription. We show that BET inhibition by the small molecule apabetalone reduces APR gene and protein expression in human hepatocytes, mouse models, and plasma from CVD patients. Steady-state expression of serum amyloid P, plasminogen activator inhibitor 1, and ceruloplasmin, APR proteins linked to CVD risk, is reduced by apabetalone in cultured hepatocytes and in humanized mouse liver. In cytokine-stimulated hepatocytes, apabetalone reduces the expression of C-reactive protein (CRP), alpha-2-macroglobulin, and serum amyloid P. The latter two are also reduced by apabetalone in the liver of endotoxemic mice. BET knockdown in vitro also counters cytokine-mediated induction of the CRP gene. Mechanistically, apabetalone reduces the cytokine-driven increase in BRD4 BET occupancy at the CRP promoter, confirming that transcription of CRP is BET-dependent. In patients with stable coronary disease, plasma APR proteins CRP, IL-1 receptor antagonist, and fibrinogen γ decrease after apabetalone treatment versus placebo, resulting in a predicted downregulation of the APR pathway and cytokine targets. We conclude that CRP and components of the APR pathway are regulated by BET proteins and that apabetalone counters chronic cytokine signaling in patients., Competing Interests: All authors are employees and shareholders of Resverlogix Corp., (Copyright © 2020 Sylwia Wasiak et al.)

Background: Stress cardiomyopathy (SCM) is a transient reversible left ventricular dysfunction that more often occurs in women. Symptoms of SCM patients are similar to those of acute coronary syndrome (ACS), but little is known about biomarkers. The goals of this study were to identify the potentially crucial genes and pathways associated with SCM., Methods: We analyzed microarray datasets GSE95368 derived from the Gene Expression Omnibus (GEO) database. Firstly, identify the differentially expressed genes (DEGs) between SCM patients in normal patients. Then, the DEGs were used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Finally, the protein-protein interaction (PPI) network was constructed and Cytoscape was used to find the key genes., Results: In total, 25 DEGs were identified, including 10 upregulated genes and 15 downregulated genes. These DEGs were mainly enriched in ECM-receptor interaction, dilated cardiomyopathy (DCM), human papillomavirus infection, and focal adhesion, whereas in GO function classification, they were mainly enriched in the extracellular region, positive regulation of the multicellular organismal process, establishment of localization, and intracellular vesicle., Conclusion: Seven hub genes contained APOE, MFGE8, ALB, APOB, SAA1, A2M, and C3 identified as hub genes of SCM, which might be used as diagnostic biomarkers or molecular targets for the treatment of SCM., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Xiao-Yu Pan and Zai-Wei Zhang.)

The villous cytotrophoblastic cells have the ability to fuse and differentiate, forming the syncytiotrophoblast (STB). The syncytialisation process is essential for placentation. Nevertheless, the mechanisms involved in cell fusion and differentiation are yet to be fully elucidated. It has been suggested that cell surface glucose-regulated protein 78 (GRP78) was involved in this process. In multiple cancer cells, cell membrane-located GRP78 has been reported to act as a receptor binding to the active form of α 2 -macroglobulin (α 2 M*), activating thus several cellular signalling pathways implicated in cell growth and survival. We hypothesised that GRP78 interaction with α 2 M* may also activate signalling pathways in trophoblastic cells, which, in turn, may promote cell fusion. Here, we observed that α 2 M mRNA is highly expressed in trophoblastic cells, whereas it is not expressed in the choriocarcinoma cell line BeWo. We thus took advantage of forskolin-induced syncytialisation of BeWo cells to study the effect of exogenous α 2 M* on syncytialisation. We first demonstrated that α 2 M* induced trophoblastic cell fusion. This effect is dependent on α 2 M*-GRP78 interaction, ERK1/2 and CREB phosphorylation, and unfolded protein response (UPR) activation. Overall, these data provide novel insights into the signalling molecules and mechanisms regulating trophoblastic cell fusion.

Purpose: Developmental abnormalities of the ocular anterior segment in some cases can lead to ocular hypertension and glaucoma. CPAMD8 is a gene of unknown function recently associated with ocular anterior segment dysgenesis, myopia, and ectopia lentis. We sought to assess the contribution of biallelic CPAMD8 variants to childhood and juvenile open-angle glaucoma., Design: Retrospective, multicenter case series., Participants: A total of 268 probands and their relatives with a diagnosis of childhood or juvenile open-angle glaucoma., Purpose: Developmental abnormalities of the ocular anterior segment in some cases can lead to ocular hypertension and glaucoma. CPAMD8 is a gene of unknown function recently associated with ocular anterior segment dysgenesis, myopia, and ectopia lentis. We sought to assess the contribution of biallelic CPAMD8 variants to childhood and juvenile open-angle glaucoma., Methods: Patients underwent a comprehensive ophthalmic assessment, with DNA from patients and their relatives subjected to genome, exome, or capillary sequencing. CPAMD8 RNA expression analysis was performed on tissues dissected from cadaveric human eyes., Main Outcome Measures: Diagnostic yield within a cohort of childhood and juvenile open-angle glaucoma, prevalence and risk of ophthalmic phenotypes, and relative expression of CPAMD8 in the human eye., Results: We identified rare (allele frequency < 4×10 -5 ) biallelic CPAMD8 variants in 5.7% (5/88) of probands with childhood glaucoma and 2.1% (2/96) of probands with juvenile open-angle glaucoma. When including family members, we identified 11 individuals with biallelic variants in CPAMD8 from 7 unrelated families. Nine of these individuals were diagnosed with glaucoma (9/11, 81.8%), with a mean age at diagnosis of 9.22±14.89 years, and all individuals with glaucoma required 1 or more incisional procedures to control high intraocular pressure. Iris abnormalities were observed in 9 of 11 individuals, cataract was observed in 8 of 11 individuals (72.7%), and retinal detachment was observed in 3 of 11 individuals (27.3%). CPAMD8 expression was highest in neural crest-derived tissues of the adult anterior segment, suggesting that CPAMD8 variation may cause malformation or obstruction of key drainage structures., Conclusions: Biallelic CPAMD8 variation was associated with a highly heterogeneous phenotype and in our cohorts was the second most common inherited cause of childhood glaucoma after CYP1B1 and juvenile open-angle glaucoma after MYOC. CPAMD8 sequencing should be considered in the investigation of both childhood and juvenile open-angle glaucoma, particularly when associated with iris abnormalities, cataract, or retinal detachment., (Copyright © 2020 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Background: Paraneoplastic pemphigus (PNP) is a devastating autoimmune multiorgan syndrome associated with autoantibodies against several autoantigens, including the alpha-2-macroglobulin-like-1 (A2ML1). A2ML1 is recognized by up to 70 % of PNP sera. The currently recommended techniques for serological diagnosis of PNP are inadequate to detect anti-A2ML1 antibodies., Objectives: To develop novel assays which allow to easily and reliably detect anti-A2ML1 autoantibodies in PNP sera., Methods: We produced full-length A2ML1 in fusion with enhanced green fluorescent protein (EGFP-A2ML1) in transfected human embryonic kidney 293 T cells. The recombinant protein was used as fluorescent ligand for immunoprecipitation studies. We further developed an enzyme-linked immunosorbent assay (ELISA) by immobilizing EGFP-A2ML1 on 96-well plates., Results: A2ML1-positive PNP sera were able to immunoprecipitate EGFP-A2ML1. Direct measurement of fluorescence in immunoprecipitates correlates with the relative levels of anti-A2ML1 antibodies in the PNP sera. By the novel ELISA, based on the determined best cut-off value, 61 % of the tested 36 PNP sera were A2ML1 positive with a specificity of 88.9 % and a sensitivity of 95 %. The 20 tested normal sera (NHS) were negative, while 2 (10 %) of 20 pemphigus vulgaris and 3 (15 %) of 20 bullous pemphigoid sera showed borderline values., Conclusions: Our novel immunoassays enable rapid stratification of PNP patients. The novel green fluorescent protein-based ELISA utilizing an active eukaryotic A2ML1 is highly sensitive and reliable and, hence, is useful for a better understanding of the immunological background of PNP. This approach may be easily applied for the rapid detection of antibodies to various other antigens., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2020 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Cataracts are focal to diffuse opacities of the eye lens causing impaired vision or complete blindness. For bilateral congenital cataracts in Red Holsteins a perfectly cosegregating mutation within the CPAMD8 gene (CPAMD8:g.5995966C>T) has been reported. We genotyped the CPAMD8:g.5995966C>T variant in Holstein calves affected by congenital bilateral congenital cataracts, their unaffected relatives and randomly selected herd mates. Ophthalmological examinations were performed in all affected individuals to confirm a congenital cataract. Whole genome sequencing was employed to screen variants in candidate genes for the Morgagnian cataract phenotype. In the present study, 3/35 cases were confirmed as homozygous mutated and 6/14 obligate carriers. Further 7/46 unaffected animals related with these cases were heterozygous mutated for the CPAMD8:g.5995966C>T variant. However 32 cases with a congenital cataract showed the wild type for the CPAMD8 variant. We did not identify variants in the candidate genes CPAMD8 and NID1 or in their close neighborhood as strongly associated with the congenital cataract phenotype in Holstein calves with the CPAMD8 wild type. In conclusion, the CPAMD8:g.5995966C>T variant is insufficient to explain the majority of Morgagnian congenital cataract phenotypes in Holsteins. It is very likely that congenital bilateral cataracts may be genetically heterogeneous and not yet known variants in genes other than CPAMD8 and NID1 are involved., Competing Interests: The authors have declared that no competing interests exist.

Non-small-cell lung cancer (NSCLC) is one of the main causes of death induced by cancer globally. However, the molecular aberrations in NSCLC patients remain unclearly. In the present study, four messenger RNA microarray datasets (GSE18842, GSE40275, GSE43458, and GSE102287) were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) between NSCLC tissues and adjacent lung tissues were obtained from GEO2R and the overlapping DEGs were identified. Moreover, functional and pathway enrichment were performed by Funrich, while the protein-protein interaction (PPI) network construction were obtained from STRING and hub genes were visualized and identified by Cytoscape software. Furthermore, validation, overall survival (OS) and tumor staging analysis of selected hub genes were performed by GEPIA. A total of 367 DEGs (95 upregulated and 272 downregulated) were obtained through gene integration analysis. The PPI network consisted of 94 nodes and 1036 edges in the upregulated DEGs and 272 nodes and 464 edges in the downregulated DEGs, respectively. The PPI network identified 46 upregulated and 27 downregulated hub genes among the DEGs, and six (such as CENPE, NCAPH, MYH11, LRRK2, HSD17B6, and A2M) of that have not been identified to be associated with NSCLC so far. Moreover, the expression differences of the mentioned hub genes were consistent with that in lung adenocarcinoma and lung squamous cell carcinoma in the TCGA database. Further analysis showed that all the six hub genes were associated with tumor staging except MYH11, while only the upregulated DEG CENPE was associated with the worse OS of patients with NSCLC. In conclusion, the current study showed that CENPE, NCAPH, MYH11, LRRK2, HSD17B6, and A2M might be the key genes contributed to tumorigenesis or tumor progression in NSCLC, further functional study is needed to explore the involved mechanisms., (© 2019 Wiley Periodicals, Inc.)

We reported that breast density (BD) was inversely correlated with the plasma level of DHA in postmenopausal obese, but not in nonobese, women given Lovaza (n-3FA). To identify protein biomarkers for the possible differential effect of n-3FA on BD between obese and nonobese women, an iTRAQ method was performed to analyze plasma from obese and lean women at each time point (baseline, 12 and 24-months, n = 10 per group); 173 proteins with >95% confidence (Unuses Score >1.3 and local false discovery rate estimation <5%) were identified. Comparative analysis between various groups identified several differentially expressed proteins (hemopexin precursor, vitamin D binding protein isoform 1 precursor [VDBP], fibronectin isoform 10 precursor [FN], and α-2 macroglobulin precursor [A2M]). Western blot analysis was performed to verify the differential expression of proteins in the iTRAQ study, and those found to be altered in a tumor protective fashion by an n-3FA rich diet in our previous preclinical study; gelsolin, VDBP, and FN were altered by n-3FA in a manner consistent with reduction in inflammation in obese women. To test the impact of our findings on breast cancer risk reduction by n-3FA, a posthoc analysis revealed that n-3FA administration reduced BD selectively in obese postmenopausal women.

Primary congenital glaucoma is a trabecular meshwork dysgenesis with resultant increased intraocular pressure and ocular damage. CYP1B1 mutations remain the most common identifiable genetic cause. However, important questions about the penetrance of CYP1B1-related congenital glaucoma remain unanswered. Furthermore, mutations in other genes have been described although their exact contribution and potential genetic interaction, if any, with CYP1B1 mutations are not fully explored. In this study, we employed modern genomic approaches to re-examine CYP1B1-related congenital glaucoma. A cohort of 193 patients (136 families) diagnosed with congenital glaucoma. We identified biallelic CYP1B1 mutations in 80.8% (87.5 and 66.1% in familial and sporadic cases, respectively, p < 0.0086). The large family size of the study population allowed us to systematically examine penetrance of all identified alleles. With the exception of c.1103G>A (p.R368H), previously reported pathogenic mutations were highly penetrant (91.2%). We conclude from the very low penetrance and genetic epidemiological analyses that c.1103G>A (p.R368H) is unlikely to be a disease-causing recessive mutation in congenital glaucoma as previously reported. All cases that lacked biallelic CYP1B1 mutations underwent whole exome sequencing. No mutations in LTBP2, MYOC or TEK were encountered. On the other hand, mutations were identified in genes linked to other ophthalmic phenotypes, some inclusive of glaucoma, highlighting conditions that might phenotypically overlap with primary congenital glaucoma (SLC4A4, SLC4A11, CPAMD8, and KERA). We also encountered candidate causal variants in genes not previously linked to human diseases: BCO2, TULP2, and DGKQ. Our results both expand and refine the genetic spectrum of congenital glaucoma with important clinical implications.

Objective: To validate the Genot-PA score, a clinical-genetic logistic regression score that stratifies the thrombolytic therapy safety, in a new cohort of patients with stroke., Methods: We enrolled 1,482 recombinant tissue plasminogen activator (rtPA)-treated patients with stroke in Spain and Finland from 2003 to 2016. Cohorts were analyzed on the basis of ethnicity and therapy: Spanish patients treated with IV rtPA within 4.5 hours of onset (cohort A and B) or rtPA in combination with mechanical thrombectomy within 6 hours of onset (cohort C) and Finnish participants treated with IV rtPA within 4.5 hours of onset (cohort D). The Genot-PA score was calculated, and hemorrhagic transformation (HT) and parenchymal hematoma (PH) risks were determined for each score stratum., Results: Genot-PA score was tested in 1,324 (cohort A, n = 726; B, n = 334; C, n = 54; and D, n = 210) patients who had enough information to complete the score. Of these, 213 (16.1%) participants developed HT and 85 (6.4%) developed PH. In cohorts A, B, and D, HT occurrence was predicted by the score ( p = 2.02 × 10 -6 , p = 0.023, p = 0.033); PH prediction was associated in cohorts A through C ( p = 0.012, p = 0.034, p = 5.32 × 10 -4 ). Increased frequency of PH events from the lowest to the highest risk group was found (cohort A 4%-15.7%, cohort B 1.5%-18.2%, cohort C 0%-100%). The best odds ratio for PH prediction in the highest-risk group was obtained in cohort A (odds ratio 5.16, 95% confidence interval 1.46-18.08, p = 0.009)., Conclusion: The Genot-PA score predicts HT in patients with stroke treated with IV rtPA. Moreover, in an exploratory study, the score was associated with PH risk in mechanical thrombectomy-treated patients., (© 2019 American Academy of Neurology.)

A genetic basis for otitis media is established, however, the role of rare variants in disease etiology is largely unknown. Previously a duplication variant within A2ML1 was identified as a significant risk factor for otitis media in an indigenous Filipino population and in US children. In this report exome and Sanger sequencing was performed using DNA samples from the indigenous Filipino population, Filipino cochlear implantees, US probands, Finnish, and Pakistani families with otitis media. Sixteen novel, damaging A2ML1 variants identified in otitis media patients were rare or low-frequency in population-matched controls. In the indigenous population, both gingivitis and A2ML1 variants including the known duplication variant and the novel splice variant c.4061 + 1 G>C were independently associated with otitis media. Sequencing of salivary RNA samples from indigenous Filipinos demonstrated lower A2ML1 expression according to the carriage of A2ML1 variants. Sequencing of additional salivary RNA samples from US patients with otitis media revealed differentially expressed genes that are highly correlated with A2ML1 expression levels. In particular, RND3 is upregulated in both A2ML1 variant carriers and high-A2ML1 expressors. These findings support a role for A2ML1 in keratinocyte differentiation within the middle ear as part of otitis media pathology and the potential application of ROCK inhibition in otitis media., (© 2019 Wiley Periodicals, Inc.)

Aim: Pancreatic cancer is one of the worst malignant tumors in prognosis. Therefore, to reduce the mortality rate of pancreatic cancer, early diagnosis and prompt treatment are particularly important., Results: We put forward a new feature-selection method that was used to find clinical markers for pancreatic cancer by combination of Support Vector Machine Recursive Feature Elimination (SVM-RFE) and Large Margin Distribution Machine Recursive Feature Elimination (LDM-RFE) algorithms. As a result, seven differentially expressed genes were predicted as specific biomarkers for pancreatic cancer because of their highest accuracy of classification on cancer and normal samples., Conclusion: Three (MMP7, FOS and A2M) out of the seven predicted gene markers were found to encode proteins secreted into urine, providing potential diagnostic evidences for pancreatic cancer.

To date, gene-environment (GxE) interaction studies in depression have been limited to hypothesis-based candidate genes, since genome-wide (GWAS)-based GxE interaction studies would require enormous datasets with genetics, environmental, and clinical variables. We used a novel, cross-species and cross-tissues "omics" approach to identify genes predicting depression in response to stress in GxE interactions. We integrated the transcriptome and miRNome profiles from the hippocampus of adult rats exposed to prenatal stress (PNS) with transcriptome data obtained from blood mRNA of adult humans exposed to early life trauma, using a stringent statistical analyses pathway. Network analysis of the integrated gene lists identified the Forkhead box protein O1 (FoxO1), Alpha-2-Macroglobulin (A2M), and Transforming Growth Factor Beta 1 (TGF-β1) as candidates to be tested for GxE interactions, in two GWAS samples of adults either with a range of childhood traumatic experiences (Grady Study Project, Atlanta, USA) or with separation from parents in childhood only (Helsinki Birth Cohort Study, Finland). After correction for multiple testing, a meta-analysis across both samples confirmed six FoxO1 SNPs showing significant GxE interactions with early life emotional stress in predicting depressive symptoms. Moreover, in vitro experiments in a human hippocampal progenitor cell line confirmed a functional role of FoxO1 in stress responsivity. In secondary analyses, A2M and TGF-β1 showed significant GxE interactions with emotional, physical, and sexual abuse in the Grady Study. We therefore provide a successful 'hypothesis-free' approach for the identification and prioritization of candidate genes for GxE interaction studies that can be investigated in GWAS datasets.

Background: We previously identified ovostatin 2 (OVOS2) as a new candidate gene for cutaneous malignant melanoma (CMM) in a Chinese population. In this study, we aimed to investigate the exact role of OVOS2 in cell proliferation, invasion, and tumorigenesis of melanoma A375 cells., Methods: The downregulation of OVOS2 expression was performed using lentiviral vectors with specific shRNA. The effects of OVOS2 expression on cell proliferation, cell cycle, cell migration, cell invasion, and potential of tumorigenesis were further investigated., Results: The downregulation of OVOS2 significantly suppressed the proliferation of A375 cells and led to a G2/M phase block. The transwell cell migration assay showed that the reduced expression of OVOS2 also significantly inhibited the transmigration of A375 cells. The western blot results showed downregulated expression of p-FAK, p-AKT, and p-ERK. This was accompanied by the upregulated epithelial phenotypes E-cadherin and β-catenin, and downregulated expression of mesenchymal phenotype N-cadherin after OVOS2 knockdown. The transplantation tumor experiment in BALB/C nude mouse showed that after an observation period of 32 days, the growth speed and weight of the transplanted tumors were significantly suppressed in the BALB/c nude mice subcutaneously injected with OVOS2 knocked-down A375 cells., Conclusion: The inhibition of OVOS2 had significant suppressive effects on the proliferation, motility, and migration capabilities of A375 cells, suggesting a crucial promotive role of OVOS2 in the pathogenesis and progression of CMM. The involved mechanisms are at least partly associated with the overactivation of FAK/MAPK/ERK and FAK/PI3K/AKT signals.

RASopathies are a group of heterogeneous conditions caused by germline mutations in RAS/MAPK signalling pathway genes. With next-generation sequencing (NGS), sequencing capacity is no longer a limitation to molecular diagnosis. Instead, the rising number of variants of unknown significance (VUSs) poses challenges to clinical interpretation and genetic counselling. We investigated the potential of an integrated pipeline combining NGS and the functional assessment of variants for the diagnosis of RASopathies. We included 63 Chinese patients with RASopathies that had previously tested negative for PTPN11 and HRAS mutations. In these patients, we performed a genetic analysis of genes associated with RASopathies using a multigene NGS panel and Sanger sequencing. For the VUSs, we evaluated evidence from genetic, bioinformatic and functional data. Twenty disease-causing mutations were identified in the 63 patients, providing a primary diagnostic yield of 31.7%. Four VUSs were identified in five patients. The functional assessment supported the pathogenicity of the RAF1 and RIT1 VUSs, while the significance of two VUSs in A2ML1 remained unclear. In summary, functional analysis improved the diagnostic yield from 31.7% to 36.5%. Although technically demanding and time-consuming, a functional genetic diagnostic analysis can ease the clinical translation of these findings to aid bedside interpretation.

Importance: Alteration of ex vivo magnetic resonance imaging transverse relaxation is associated with late-life cognitive decline even after controlling for common neuropathologic conditions. However, the underlying neurobiology of this association is unknown., Objective: To investigate the association between brain gene expression, DNA methylation, and alteration of magnetic resonance imaging transverse relaxation in late-life cognitive decline., Design, Setting, and Participants: Data came from 2 community-based longitudinal cohort studies of aging and dementia, the Religious Orders Study, which began in 1993, and the Rush Memory and Aging Project, which began in 1997. All participants agreed to undergo annual clinical evaluations and to donate their brains after death. By October 24, 2016, a total of 1358 individuals had died and had brain autopsies that were approved by board-certified neuropathologists. Of those, 552 had undergone ex vivo imaging. The gene expression analysis was limited to 174 individuals with both imaging and brain RNA sequencing data. The DNA methylation analysis was limited to 225 individuals with both imaging and brain methylation data., Main Outcomes and Measures: Maps of ex vivo magnetic resonance imaging transverse relaxation were generated using fast spin echo imaging. The target was a composite measure of the transverse relaxation rate (R2) that was associated with cognitive decline after controlling for common neuropathologic conditions. Next-generation RNA sequencing and DNA methylation data were generated using frozen tissue from the dorsolateral prefrontal cortex. Genome-wide association analysis was used to investigate gene expression and, separately, DNA methylation for signals associated with the R2 measure., Results: Of the 552 individuals with ex vivo imaging data, 394 were women and 158 were men, and the mean (SD) age at death was 90.4 (6.0) years. Four co-expressed genes (PADI2 [Ensembl ENSG00000117115], ZNF385A [Ensembl ENSG00000161642], PSD2 [Ensembl ENSG00000146005], and A2ML1 [Ensembl ENSG00000166535]) were identified, of which higher expressions were associated with slower R2. The association of R2 with cognitive decline was attenuated when the gene expression signals were added to the model, such that the mean (SE) coefficient of association was reduced from 0.028 (0.008) (P < .001) to 0.019 (0.009) (P = .03). The DNA methylation scan did not detect a genome-wide significant signal, but it revealed an anticorrelation between R2 and DNA methylation in many of the cytosine-guanine dinucleotides., Conclusions and Relevance: Brain gene expression and DNA methylation dysregulations are implicated in the alteration of brain tissue properties associated with late-life cognitive decline above and beyond the influence of common neuropathologic conditions.

We report 2 infantile cases of pulmonary tumor carrying a chimeric A2M-ALK gene. A2M-ALK is a newly identified anaplastic lymphoma kinase (ALK)-related chimeric gene from a tumor diagnosed as fetal lung interstitial tumor (FLIT). FLIT is a recently recognized infantile pulmonary lesion defined as a mass-like lesion that morphologically resembles the fetal lung. Grossly, FLIT characteristically appears as a well-circumscribed spongy mass, whereas the tumors in these patients were solid and firm. Histologically, the tumors showed intrapulmonary lesions composed of densely proliferating polygonal or spindle-shaped mesenchymal cells with diffuse and dense infiltrations of inflammatory cells forming microcystic or micropapillary structures lined by thyroid transcription factor 1-positive pneumocytes, favoring inflammatory myofibroblastic tumor rather than FLIT. The proliferating cells were immunoreactive for ALK, and A2M-ALK was identified in both tumors with reverse-transcription polymerase chain reaction. The dense infiltration of inflammatory cells, immunoreactivity for ALK, and identification of an ALK-related chimeric gene suggested a diagnosis of inflammatory myofibroblastic tumor. Histologically, most reported FLITs show sparse inflammatory infiltrates and a relatively low density of interstitial cells in the septa, although prominent infiltration of inflammatory cells and high cellularity of interstitial cells are seen in some FLITs. The present cases suggest that ALK rearrangements, including the chimeric A2M-ALK gene, may be present in these infantile pulmonary lesions, especially those with inflammatory cell infiltration. We propose that these infantile pulmonary lesions containing a chimeric A2M-ALK gene be categorized as a specific type of inflammatory myofibroblastic tumor that develops exclusively in neonates and infants., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The shrimp multifunctional protein alpha-2-macroglobulin (A2M) is abundantly expressed in plasma, highly up-regulated upon microbial infection and involved in several immune pathways such as blood clotting system, phagocytosis and melanization. Herein, the function of LvA2M from Litopenaeus vannamei on the prophenoloxidase (proPO) system is reported. The recombinant (r)LvA2M produced strongly and specifically inhibited trypsin and the PO activity in shrimp plasma in a dose-dependent manner. Silencing of LvA2M led to an increase in the PO activity in shrimp plasma although the expression of proPO-associated genes, proPO-activating enzyme (PPAE) and prophenoloxidase (proPO) but not the proPO-activating factor (PPAF) was down-regulated. In Vibrio parahaemolyticus AHPND-infected shrimp, the LvA2M activity was suppressed in an early phase of infection while the PO activity was increased. Thus, the proPO-activating system was regulated by the LvA2M., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The assessment of non-genotoxic hepatocarcinogens (NGHCs) is currently relying on two-year rodent bioassays. Toxicogenomics biomarkers provide a potential alternative method for the prioritization of NGHCs that could be useful for risk assessment. However, previous studies using inconsistently classified chemicals as the training set and a single microarray dataset concluded no consensus biomarkers. In this study, 4 consensus biomarkers of A2m, Ca3, Cxcl1, and Cyp8b1 were identified from four large-scale microarray datasets of the one-day single maximum tolerated dose and a large set of chemicals without inconsistent classifications. Machine learning techniques were subsequently applied to develop prediction models for NGHCs. The final bagging decision tree models were constructed with an average AUC performance of 0.803 for an independent test. A set of 16 chemicals with controversial classifications were reclassified according to the consensus biomarkers. The developed prediction models and identified consensus biomarkers are expected to be potential alternative methods for prioritization of NGHCs for further experimental validation.

Anterior segment dysgeneses (ASDs) comprise a spectrum of developmental disorders affecting the anterior segment of the eye. Here, we describe three unrelated families affected by a previously unclassified form of ASD. Shared ocular manifestations include bilateral iris hypoplasia, ectopia lentis, corectopia, ectropion uveae, and cataracts. Whole-exome sequencing and targeted Sanger sequencing identified mutations in CPAMD8 (C3 and PZP-like alpha-2-macroglobulin domain-containing protein 8) as the cause of recessive ASD in all three families. A homozygous missense mutation in the evolutionarily conserved alpha-2-macroglobulin (A2M) domain of CPAMD8, c.4351T>C (p. Ser1451Pro), was identified in family 1. In family 2, compound heterozygous frameshift, c.2352&#95;2353insC (p.Arg785Glnfs ∗ 23), and splice-site, c.4549-1G>A, mutations were identified. Two affected siblings in the third family were compound heterozygous for splice-site mutations c.700+1G>T and c.4002+1G>A. CPAMD8 splice-site mutations caused aberrant pre-mRNA splicing in vivo or in vitro. Intriguingly, our phylogenetic analysis revealed rodent lineage-specific CPAMD8 deletion, precluding a developmental expression study in mice. We therefore investigated the spatiotemporal expression of CPAMD8 in the developing human eye. RT-PCR and in situ hybridization revealed CPAMD8 expression in the lens, iris, cornea, and retina early in development, including strong expression in the distal tips of the retinal neuroepithelium that form the iris and ciliary body, thus correlating CPAMD8 expression with the affected tissues. Our study delineates a unique form of recessive ASD and defines a role for CPAMD8, a protein of unknown function, in anterior segment development, implying another pathway for the pathogenicity of ASD., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Previously rare A2ML1 variants were identified to confer otitis media susceptibility in an indigenous Filipino community and in otitis-prone US children. The goal of this study is to describe differences in the middle ear microbiome between carriers and non-carriers of an A2ML1 duplication variant that increases risk for chronic otitis media among indigenous Filipinos with poor health care access., Methods: Ear swabs were obtained from 16 indigenous Filipino individuals with chronic otitis media, of whom 11 carry the A2ML1 duplication variant. Ear swabs were submitted for 16S rRNA gene sequencing., Results: Genotype-based differences in microbial richness, structure, and composition were identified, but were not statistically significant. Taxonomic analysis revealed that the relative abundance of the phyla Fusobacteria and Bacteroidetes, and genus Fusobacterium were nominally increased in carriers compared to non-carriers, but were non-significant after correction for multiple testing. We also detected rare bacteria including Oligella that was reported only once in the middle ear., Conclusions: These findings suggest that A2ML1-related otitis media susceptibility may be mediated by changes in the middle ear microbiome. Knowledge of middle ear microbial profiles according to genetic background can be potentially useful for therapeutic and prophylactic interventions for otitis media and can guide public health interventions towards decreasing otitis media prevalence within the indigenous Filipino community.

Objective: To identify genetic and environmental risk factors for otitis media in an indigenous Filipino population., Study Design: Cross-sectional study., Setting: Indigenous Filipino community., Subjects and Methods: Clinical history and information on breastfeeding, tobacco smoke exposure, and swimming were obtained from community members. Heads of households were interviewed for family history and personal beliefs on ear health. Height and weight were measured. Otoscopic findings were described for the presence and character of perforation or discharge. An A2ML1 duplication variant that confers otitis media susceptibility was Sanger sequenced in all DNA samples. Co-occurrence of middle ear bacteria detected by 16S rRNA gene sequencing was determined according to A2ML1 genotype and social cluster., Results: The indigenous Filipino population has a ~50% prevalence of otitis media. Young age was associated with otitis media (4 age strata; P = .004); however, age was nonsignificant as a bistratal or continuous variable. There was no association between otitis media and sex, body mass index, breastfeeding, tobacco exposure, or deep swimming. In multivariate analyses, A2ML1 genotype is the strongest predictor of otitis media, with an odds ratio of 3.7 (95% confidence interval: 1.3-10.8; P = .005). When otitis media diagnoses were plotted across ages, otitis media was observed within the first year of life, and chronic otitis media persisted up to adulthood, particularly in A2ML1-variant carriers., Conclusion: Among indigenous Filipinos, A2ML1 genotype is the primary risk factor for otitis media and main determinant of disease progression, although age, the middle ear microbiome, and social clusters might modulate the effect of the A2ML1 genotype., Competing Interests: Competing interests: None. Sponsorships: None. Funding source: This study was funded by grants from the University of the Philippines Manila – National Institutes of Health (to G.T.A.); the Albert and Margaret Alkek Foundation (to J.F.P.); the National Institute on Deafness and Other Communication Disorders at the United States National Institutes of Health (grants R01 DC011651 and R01 DC003594 to S.M.L. and R01 DC015004 to R.L.P.S.C.); and the National Organization for Hearing Research, Action for Hearing Loss and the Hearing Health Foundation (to R.L.P.S.C.)., (© American Academy of Otolaryngology—Head and Neck Surgery Foundation 2016.)

Alzheimer's disease (AD) is a polygenic and multifactorial disease with a complex inheritance caused by the formation of amyloid plaques and neurofibrillary tangles in the brain. Increasing evidence indicates that many genes including interleukin-6 (IL-6) and alpha 2-macroglobulin (A2M) may contribute to the pathogenesis of AD. The A2M gene encodes α2-macroglobulin which specifically binds with the beta-amyloid peptides and prevents fibril formation. Protein of the IL-6 gene linked to beta-amyloid (βA) aggregation was detected in βA plaques in the brain of AD patients. The aim of the present study is to investigate the relationship of the IL-6 and A2M gene polymorphisms with AD and also the impact of rivastigmine on AD patients regarding their genotypes on IL-6 and A2M genes in 150 Iranian AD patients under rivastigmine therapy and 150 matched healthy controls. The results indicated that IL-6 G and C alleles had significant positive and negative association with AD, respectively, (P = 0.0001, relative risks (RR) = 1.39) and frequency of AD patients carrying IL-6 GG genotype was significantly in higher proportion in familial Alzheimer's disease (FAD) patients compared to controls (P = 0.02, RR = 2.25), and the IL-6 CC genotype was significantly protective against AD (P = 0.0003, RR = 0.65). Genotype analysis of A2M gene showed a significant positive correlation between A2M AA genotype and the AD patients (sporadic Alzheimer's disease (SAD) and FAD) (P = 0.001, RR = 1.56), proposing it as a possible risk factor for AD. Drug response from pharmacogenetic viewpoint after 3-year follow-up of AD patients and Clinical Dementia Rating (CDR) analysis demonstrated that AD patients carrying bigenic genotype IL-6 CC-A2M AG (ΔCDR = 4.5) and male patients with IL-6 CC genotype (ΔCDR = 3.83) provided the best response and the A2M GG genotype (ΔCDR = 7.97) and bigenic genotype IL-6 GG-A2M GG (ΔCDR = 8.5) conferred the worst response to the rivastigmine, suggesting likely involvement of genotype-specific response to rivastigmine therapy in AD patients. The results also propose that in view of the fact that C and G alleles created by nucleotide changes in the promoter region of IL-6 gene and this may affect the expression of the IL-6 gene and, hence, susceptible and protective role of GG and CC genotype in AD might be caused by higher and lower expression of IL-6 cytokine, respectively.

Alpha-2 macroglobulin (α2M) is a ubiquitous protease inhibitor and considered to be an evolutionarily conserved constituent of innate host defence system. Here, an α2M gene (designated as Pfα2M) was obtained from the pearl oyster Pinctada fucata by RT-PCR, PCR walking and rapid amplification of cDNA ends (RACE). The Pfα2M cDNA consists of 6394 bp with an open reading frame (ORF) of 5745 bp encoding a protein of 1914 amino acids with a 19 residues signal peptide. Pfα2M sequence contains three putative functional domains, including a bait region, a thiol ester domain and a receptor-binding domain. Phylogenetic analysis revealed that Pfα2M is closely related to the α2Ms from other molluscs. Pfα2M was expressed in all tested tissues including digestive gland, gill, adductor muscle, mantle and foot, while the highest expression was found in hemocytes. Following challenge with Vibrio alginolyticus, Pfα2M expression in hemocytes was significantly up-regulated at 2 h and then returned to the original level at 48 h. Knockdown of Pfα2M by RNA interference significantly reduced the phagocytosis of V. alginolyticus by hemocytes in vivo, and similar results were obtained upon chemical inactivation of the reactive thioester bond in Pfα2M by methylamine treatment. Taken together, it is suggested that Pfα2M is an immune-relevant molecule and involved in phagocytosis of V. alginolyticus by P. fucata hemocytes, and the function of Pfα2M in phagocytosis is dependent on the active thioester bond., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The non-specific protease inhibitor alpha-2-macroglobulin (A2M) is a key macromolecular glycoprotein that involved in host immune defense against pathogens in vertebrates and invertebrates. However, no research regarding A2M has been developed in echinoderms to date. In this study, the full-length cDNA of A2M was cloned from the sea cucumber (Holothuria atra), which is a tropical species widely distributed along the coasts of the South China Sea and designated HaA2M. HaA2M possesses all three conserved functional domains of known A2M proteins, including the bait region domain, thioester domain and receptor-binding domain. Compared to fish and shrimp A2Ms, the histidine residue from the catalytical regions is well conserved in HaA2M. HaA2M mRNA was predominantly expressed in coelomocytes and, to a lesser extent, in the body wall, intestine and respiratory tree. A2M activity was detected in the coelomic fluids of H. atra. The mRNA expression and activity levels were investigated in the major immune tissues and coelomic fluids of H. atra after challenge with inactivated Vibrio alginolyticus or polyriboinosinic polyribocytidylic acid [Poly (I: C)]. RNA interference (RNAi)-mediated knockdown of HaA2M resulted in a significant reduction of HaA2M gene transcript level (86%). RNAi-mediated silencing of HaA2M gene significantly decreased the A2M activity (38%) and increased the number of viable bacteria (2.8-fold) in the coelomic fluids of H. atra infected by V. alginolyticus. Our study, as a whole, supplied the evidences for HaA2M as an immune-relevant molecule and it might have multiple functions in the innate immune system of H. atra., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Activated α2-macroglobulin (α2M*) signals predominantly through cell surface GRP78 (CS-GRP78) to promote proliferation and survival of cancer cells; however, the molecular mechanism remains obscure. c-MYC is an essential transcriptional regulator that controls cell proliferation. We hypothesize that α2M*/CS-GRP78-evoked key signaling events are required for transcriptional activation of c-MYC target genes. Activation of CS-GRP78 by α2M* requires ligation of the GRP78 primary amino acid sequence (Leu(98)-Leu(115)). After stimulation with α2M*, CS-GRP78 signaling activates 3-phosphoinositide-dependent protein kinase-1 (PDK1) to induce phosphorylation of PLK1, which in turn induces c-MYC transcription. We demonstrate that PLK1 binds directly to c-MYC and promotes its transcriptional activity by phosphorylating Ser(62) Moreover, activated c-MYC is recruited to the E-boxes of target genes FOSL1 and ID2 by phosphorylating histone H3 at Ser(10) In addition, targeting the carboxyl-terminal domain of CS-GRP78 with a mAb suppresses transcriptional activation of c-MYC target genes and impairs cell proliferation. This work demonstrates that α2M*/CS-GRP78 acts as an upstream regulator of the PDK1/PLK1 signaling axis to modulate c-MYC transcription and its target genes, suggesting a therapeutic strategy for targeting c-MYC-associated malignant progression., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The study of genes associated with host defense mechanisms, such as the A2M gene, plays a critical role in preventing diseases that reduce milk yield and its constituents. The aim of this study was to identify polymorphisms in the A2M gene in Murrah buffaloes (Bubalus bubalis), and investigate their associations with milk yield, fat and protein production, fat and protein percentages, and somatic cell count. Hair follicle samples of 136 animals were collected for DNA extraction, and polymorphisms were identified by polymerase chain reactions and sequencing. Statistical analyses were performed to ascertain the allelic and genotypic frequencies, the Hardy-Weinberg equilibrium, and association analysis was conducted between the polymorphisms and the traits studied. Comparative analysis between buffalo and bovine sequences revealed seven nucleotide substitutions. Alignments among the buffalo sequences identified three single nucleotide polymorphisms (SNPs), including one in exon 29, g.241A>G, which was used in subsequent statistical analyses. A Hardy-Weinberg test indicated that this SNP was in equilibrium in this population, and was significantly associated (P < 0.05) with fat production and fat and protein percentages. Therefore, this SNP can be used as a molecular marker for these traits.

Vibrio alginolyticus is a virulent pathogen that affects crab aquacultures. In the present study, the immune responses and antioxidant status of big and small (based on body weight and size) 80-, 100- and 120-day-old specimens of Portunus trituberculatus, challenged for 72 h with Vibrio alginolyticus, were studied. The total hemocyte count (THC), and phagocytic, prophenoloxidase and phenoloxidase activities, of the big individuals (BIs) were higher than those of the small individuals (SIs) (P < 0.05). The antioxidant status of the organisms showed a similar pattern: superoxide dismutase (SOD) activity and glutathione/oxidized glutathione (GSH/GSSG) in the cell-free hemolymph and hepatopancreases of the BIs were higher than in the SIs (P < 0.05). There were no significant differences in α2-macroglobulin (α2-M), antibacterial and bacteriolytic activities in the cell-free hemolymph, or glutathione peroxidase activity in the cell-free hemolymph or hepatopancreas between the BIs and SIs. The α2-M and crustin gene expression levels in the hemocytes, and SOD expression in the hemocytes and hepatopancreas, were also significantly higher in the BIs. The results suggest that, compared with the SIs, the BIs possessed a higher resistance to V. alginolyticus infection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In fish, a series of maternal derived immune components have been identified in their eggs or embryos at very early stages, which are proposed to provide protections to themselves against pathogenic attacks from hostile environment. The phenomenon of maternal immunity has been also recorded in several invertebrate species, however, so far, very limited information about the maternal immune molecules are available. In this study, it was demonstrated maternal alpha2 macroglobulin (A2m) protein, an important innate immune factor, exists in the fertilized eggs of amphioxus Branchiostoma japonicum, an invertebrate chordate. Maternal mRNA of A2m was also detected in amphioxus embryos at very early developing stages. In addition, it was recorded that the egg lysate prepared from the newly fertilized eggs can inhibit the growth of both Gram-negative bacterium Escherichia coli and Gram-positive bacterium Staphylococcus aureus in a concentration dependent manner. The bacteriostatic activity can be reduced notably after precipitated A2m with anti-A2m antibody. Thus maternal A2m is partly attributed to the bacteriostatic activity. It was further demonstrated that recombinant A2m can bind to E. coli cells directly. All these points come to a result that A2m is a maternal immune factor existing in eggs of invertebrate chordate, which may be involved in defense their embryos against harmful microbes' attacks., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Purpose: Due to the controversial results in assessment of the association between α-2-Macroglobulin gene (A2M) polymorphisms and Parkinson's disease (PD), we performed a meta-analysis to evaluate the relationship between two A2M variants (rs669 and rs3832852) and PD., Methods: All eligible studies were retrieved in PubMed, Web of Science, Embase and PDGene databases. Data were extracted by two investigators independently. All the four genetic models were used for rs669 and rs3832852., Results: A total of 877 PD patients and 1296 controls from six studies were included for rs669 polymorphisms. The combined odds ratio (OR) indicated that rs669 polymorphisms were likely associated increased risk of PD only in dominant genetic models (OR = 1.41, CI = 1.03-1.92), especially in Asian subgroup (OR = 1.97, CI = 1.03-3.75). Five studies containing 772 PD patients and 1178 controls were identified for rs3832852 polymorphisms. The result suggested that rs3832852 polymorphisms were not associated with PD in all genetic models., Conclusions: Our data indicate that the rs669 (A/G) polymorphisms in A2M gene are associated with increased risk in PD. To further validate the association of A2M polymorphisms with PD, more studies with larger samples are required.

A duplication variant within the middle ear-specific gene A2ML1 cosegregates with otitis media in an indigenous Filipino pedigree (LOD score = 7.5 at reduced penetrance) and lies within a founder haplotype that is also shared by 3 otitis-prone European-American and Hispanic-American children but is absent in non-otitis-prone children and >62,000 next-generation sequences. We identified seven additional A2ML1 variants in six otitis-prone children. Collectively, our studies support a role for A2ML1 in the pathophysiology of otitis media.

The survival of commensal bacteria requires them to evade host peptidases. Gram-negative bacteria from the human gut microbiome encode a relative of the human endopeptidase inhibitor, α2-macroglobulin (α2M). Escherichia coli α2M (ECAM) is a ∼ 180-kDa multidomain membrane-anchored pan-peptidase inhibitor, which is cleaved by host endopeptidases in an accessible bait region. Structural studies by electron microscopy and crystallography reveal that this cleavage causes major structural rearrangement of more than half the 13-domain structure from a native to a compact induced form. It also exposes a reactive thioester bond, which covalently traps the peptidase. Subsequently, peptidase-laden ECAM is shed from the membrane and may dimerize. Trapped peptidases are still active except against very large substrates, so inhibition potentially prevents damage of large cell envelope components, but not host digestion. Mechanistically, these results document a novel monomeric "snap trap."