Anchor Protein Research Articles

Omic markers identification for predicting risks of negative effects in children with elevated copper and nickel contents in blood

Proteomic profiling is a promising procedure for examining and substantiating molecular mechanisms of body reactions occurrence and development as a response to adverse impacts; it allows detecting and examining these reactions at early stages in their development prior to cellular damage and damage to organs. Studies aimed at increasing efficiency of adverse effects prediction are especially vital for solving tasks related to early detection and prevention of consequences associated with exposure to chemical environmental factors, first of all, ambient air. Our research goal was to identify omic-markers for predicting risks of negative effects in children with elevated copper and nickel contents in blood. We performed proteomic blood plasma examination in children and modeled cause-and-effect relations. Children with copper and nickel contents in their blood being 3.5 times higher than physiological standard had approximately 20 protein stains that were authentically different from those detected in children from the reference group. We detected correlations between an increase in relative volume of three protein stains including apolipoprotein A-I, anchor protein of A-kinase 9, vitronectin, and a decrease in relative volume of one protein strain including transthyretin and elevated copper and nickel contents in blood (R2=0.30–0.44; р=0.0001–0.008). All the above-mentioned proteins have predictive significance when it comes down to negative effects related to neuroregulation disorders and endothelial dysfunction. It was proven that there was a risk of predicted negative effects such as greater frequency of nervous and cardiovascular system diseases in case copper and nickel contents in blood were elevated (R2=0.35–0.96; р=0.0001–0.013). The established list of potential target molecules (apolipoprotein A-I, vitronectin, anchor protein of A-kinase 9, and transthyretin) and genes that coded their expression (APOA1, VTN,AKAP9,TTR) was substantiated as omic-markers indicating a possibility that negative effects might occur in the cardiovascular and nervous system.

Выявление омик-маркеров для прогнозирования риска развития негативных эффектов у детей с повышенным содержанием меди и никеля в крови

Proteomic profiling is a promising procedure for examining and substantiating molecular mechanisms of body reactions occurrence and development as a response to adverse impacts; it allows detecting and examining these reactions at early stages in their development prior to cellular damage and damage to organs. Studies aimed at increasing efficiency of adverse effects prediction are especially vital for solving tasks related to early detection and prevention of consequences associated with exposure to chemical environmental factors, first of all, ambient air. Our research goal was to identify omic-markers for predicting risks of negative effects in children with elevated copper and nickel contents in blood. We performed proteomic blood plasma examination in children and modeled cause-and-effect relations. Children with copper and nickel contents in their blood being 3.5 times higher than physiological standard had approximately 20 protein stains that were authentically different from those detected in children from the reference group. We detected correlations between an increase in relative volume of three protein stains including apolipoprotein A-I, anchor protein of A-kinase 9, vitronectin, and a decrease in relative volume of one protein strain including transthyretin and elevated copper and nickel contents in blood (R2=0.30–0.44; р=0.0001–0.008). All the above-mentioned proteins have predictive significance when it comes down to negative effects related to neuroregulation disorders and endothelial dysfunction. It was proven that there was a risk of predicted negative effects such as greater frequency of nervous and cardiovascular system diseases in case copper and nickel contents in blood were elevated (R2=0.35–0.96; р=0.0001–0.013). The established list of potential target molecules (apolipoprotein A-I, vitronectin, anchor protein of A-kinase 9, and transthyretin) and genes that coded their expression (APOA1, VTN,AKAP9,TTR) was substantiated as omic-markers indicating a possibility that negative effects might occur in the cardiovascular and nervous system.

Phylogenetic analysis of the bacterial Pro-Pro-endopeptidase domain reveals a diverse family including secreted and membrane anchored proteins.

Pro-Pro-endopeptidases (PPEP, EC 3.4.24.89) are secreted, zinc metalloproteases that have the unusual capacity to cleave a peptide bond between two prolines, a bond that is generally less sensitive to proteolytic cleavage. Two well studied members of the family are PPEP-1 and PPEP-2, produced by Clostridioides difficile, a human pathogen, and Paenibacillus alvei, a bee secondary invader, respectively. Both proteases seem to be involved in mediating bacterial adhesion by cleaving cell surface anchor proteins on the bacterium itself. By using basic alignment and phylogenetic profiling analysis, this work shows that the complete family of proteins that contain a PPEP domain includes proteins from more than 130 species spread over 9 genera. These analyses also suggest that the PPEP domain spread through horizontal gene transfer events between species within the Firmicutes' classes Bacilli and Clostridia. Bacterial species containing PPEP homologs are found in diverse habitats, varying from human pathogens and gut microbiota to free-living bacteria, which were isolated from various environments, including extreme conditions such as hot springs, desert soil and salt lakes. The phylogenetic tree reveals the relationships between family members and suggests that smaller subgroups could share cleavage specificity, substrates and functional similarity. Except for PPEP-1 and PPEP-2, no cleavage specificity, specific physiological target, or function has been assigned for any of the other PPEP-family members. Some PPEP proteins have acquired additional domains that recognize and bind noncovalently to various elements of the bacterial peptidoglycan cell-wall, anchoring these PPEPs. Secreted or anchored to the cell-wall surface PPEP proteins seem to perform various functions.

OsTPR boosts the superior grains through increase in upper secondary rachis branches without incurring a grain quality penalty.

SummaryTo address the future food security in Asia, we need to improve the genetic gain of grain yield while ensuring the consumer acceptance. This study aimed to identify novel genes influencing the number of upper secondary rachis branches (USRB) to elevate superior grains without compromising grain quality by studying the genetic variance of 310 diverse O. sativa var. indica panel using single‐ and multi‐locus genome‐wide association studies (GWAS), gene set analyses and gene regulatory network analysis. GWAS of USRB identified 230 significant (q‐value < 0.05) SNPs from chromosomes 1 and 2. GWAS targets narrowed down using gene set analyses identified large effect association on an important locus LOC_Os02g50790/LOC_Os02g50799 encoding a nuclear‐pore anchor protein (OsTPR). The superior haplotype derived from non‐synonymous SNPs identified in OsTPR was specifically associated with increase in USRB with superior grains being low chalk. Through haplotype mining, we further demonstrated the synergy of offering added yield advantage due to superior allele of OsTPR in elite materials with low glycaemic index (GI) property. We further validated the importance of OsTPR using recombinant inbred lines (RILs) population by introgressing a superior allele of OsTPR into elite materials resulted in raise in productivity in high amylose background. This confirmed a critical role for OsTPR in influencing yield while maintaining grain and nutritional quality.

Computational prediction of the effects of non-synonymous single nucleotide polymorphisms on the GPI-anchor transamidase subunit GPI8p of Plasmodium falciparum

Drug resistance is increasingly evolving in malaria parasites; hence, it is important to discover and establish alternative drug targets. In this context, GPI-anchor transamidase (GPI-T) is a potential drug target primarily of its crucial role in the development and survival of the parasite in the GPI anchor biosynthesis pathway. The present investigation was undertaken to explore the plausible effects of nsSNP on the structure and functions of GPI-T subunit GPI8p of Plasmodium falciparum. The GPI8p (PF3D7_1128700) was analyzed using various sequence-based and structure-based computational tools such as SIFT, PROVEAN, PredictSNP, SNAP2, I-Mutant, MuPro, ConSurf, NetSurfP, MUSTER, COACH server and STRING server. Of the 34 nsSNPs submitted for functional analysis, 18 nsSNPs (R124 L, N143 K, Y145 F, V157I, T195S, K379E, I392 K, I437 T, Y438H, N439D, Y441H, N442D, N448D, N451D, D457A, D457Y, I458 L and N460 K) were predicted to have deleterious effects on the protein GPI8p. Additionally, I-Mutant 2.0 and MuPro both showed a decrease in stability after mutation as a result of these nsSNPs, suggesting the destabilization of protein. ConSurf findings suggest that most of the regions were highly conserved. In addition, COACH server was used to predict the ligand binding sites. It was found that no mutation was present at the predicted ligand binding site. The results of the STRING database showed that the protein GPI8p interacts with those proteins which either involve the biosynthetic process of attaching GPI anchor to protein or GPI anchor. The present study suggested that the GPI8p could be a novel target for anti-malarial drugs, which provides significant details for further experimentation.

Interplay between hevin, SPARC, and MDGAs: Modulators of neurexin-neuroligin transsynaptic bridges

Summary Hevin is secreted by astrocytes and its synaptogenic effects are antagonized by the related protein, SPARC. Hevin stabilizes neurexin-neuroligin transsynaptic bridges in vivo. A third protein, membrane-tethered MDGA, blocks these bridges. Here, we reveal the molecular underpinnings of a regulatory network formed by this trio of proteins. The hevin FS-EC structure differs from SPARC, in that the EC domain appears rearranged around a conserved core. The FS domain is structurally conserved and it houses nanomolar affinity binding sites for neurexin and neuroligin. SPARC also binds neurexin and neuroligin, competing with hevin, so its antagonist action is rooted in its shortened N-terminal region. Strikingly, the hevin FS domain competes with MDGA for an overlapping binding site on neuroligin, while the hevin EC domain binds the extracellular matrix protein collagen (like SPARC), so that this trio of proteins can regulate neurexin-neuroligin transsynaptic bridges and also extracellular matrix interactions, impacting synapse formation and ultimately neural circuits.

Roles of A-kinase Anchor Protein 12 in Astrocyte and Oligodendrocyte Precursor Cell in Postnatal Corpus Callosum.

The formation of the corpus callosum in the postnatal period is crucial for normal neurological function, and clinical genetic studies have identified an association of 6q24-25 microdeletion in this process. However, the mechanisms underlying corpus callosum formation and its critical gene(s) are not fully understood or identified. In this study, we examined the roles of AKAP12 in postnatal corpus callosum formation by focusing on the development of glial cells, because AKAP12 is coded on 6q25.1 and has recently been shown to play roles in the regulations of glial function. In mice, the levels of AKAP12 expression was confirmed to be larger in the corpus callosum compared to the cortex, and AKAP12 levels decreased with age both in the corpus callosum and cortex regions. In addition, astrocytes expressed AKAP12 in the corpus callosum after birth, but oligodendrocyte precursor cells (OPCs), another major type of glial cell in the developing corpus callosum, did not. Furthermore, compared to wild types, Akap12 knockout mice showed smaller numbers of both astrocytes and OPCs, along with slower development of corpus callosum after birth. These findings suggest that AKAP12 signaling may be required for postnatal glial formation in the corpus callosum through cell- and non-cell autonomous mechanisms.

Decreased expression of AKAP4 and TyrPho proteins in testis, epididymis, and spermatozoa with low sexual performance of mice induced by modified CUMS.

The molecular mechanism of chronic stress especially reduced motility, a major cause of male infertility, has not been proved. It is known that A-kinase anchor protein 4 (AKAP4) and tyrosine-phosphorylated (TyrPho) proteins are involved in progressive motility. This study aimed to investigate the effect of chronic unpredictable mild stress (CUMS) on sexual behaviours, sperm quality, and expressions of AKAP4 and TyrPho proteins in testis, epididymis, and spermatozoa. Sixteen male mice were divided into control and CUMS groups (n=8/group). Animals were induced by a stressor from twelve stressors for 36days. Sexual behaviours, corticosterone and testosterone, sperm parameters, and histopathology were observed. The expressions of AKAP4 and TyrPho proteins in testis, epididymis, and spermatozoa were examined. Results showed that CUMS significantly increased corticosterone while serum testosterone level was decreased. Sexual behaviours and sperm parameter quality were significantly decreased. CUMS mice showed vacuolisation and pyknotic cells in seminiferous epithelium and less sperm mass was observed within epididymal lumen. CUMS decreased expressions of AKAP4 and TyrPho proteins in testis, epididymis, and spermatozoa. In conclusion, the decreased expression of AKAP4 and TyrPho proteins may be a mechanism associated with low semen qualities particularly decrease of sperm motility in CUMS.

Expression and affinity purification of recombinant mammalian mitochondrial ribosomal small subunit (MRPS) proteins and protein-protein interaction analysis indicate putative role in tumourigenic cellular processes.

Mitochondrial ribosomal small subunit (MRPS) group of proteins is structural constituents of the small subunit of mitoribosomes involved in translation. Recent studies indicate role in tumourigenic process, however, unlike cytosolic ribosomal proteins, knowledge on the role of MRPS proteins in alternate cellular processes is very limited. Mapping protein-protein interactions (PPIs) onto known cellular processes can be a valuable tool to identify novel protein functions. In this study, to identify PPIs of MRPS proteins, we have constructed 31 glutathione-S-transferase (GST)/MRPS fusion clones. GST/MRPS fusion proteins were confirmed by MALDI-TOF analysis. GST pull-downs were performed using eight GST/MRPS proteins (MRPS9, MRPS10, MRPS11, MRPS18B, MRPS31, MRPS33, MRPS38 and MRPS39), GST alone as pull-down control and HEK293 cell lysate as the source for anchor proteins followed by nLC/MS/MS analysis and probable PPIs of eight MRPS proteins were identified. Three PPIs from GST pull-downs and interaction between six MRPS proteins and p53 previously reported in PPI database were validated. The PPI network analysis revealed putative role in cellular processes with implications for tumourigenesis. Gene expression screening of a cancer cell line panel indicated overexpression of MRPS10 and MRPS31 in breast cancer. Co-expression module identification tool analysis of breast cancer gene expression and MRPS10 and MRPS31 PPIs revealed putative role for PPI with acyl-CoA dehydrogenase in fatty acid oxidation process regulated by brain-derived neurotrophic factor signalling pathway.

Structural information and membrane binding of truncated RGS9-1 Anchor Protein and its C-terminal hydrophobic segment

Visual phototransduction takes place in photoreceptor cells. Light absorption by rhodopsin leads to the activation of transducin as a result of the exchange of its GDP for GTP. The GTP-bound ⍺-subunit of transducin then activates phosphodiesterase (PDE), which in turn hydrolyzes cGMP leading to photoreceptor hyperpolarization. Photoreceptors return to the dark state upon inactivation of these proteins. In particular, PDE is inactivated by the protein complex R9AP/RGS9-1/Gβ5. R9AP (RGS9-1 anchor protein) is responsible for the membrane anchoring of this protein complex to photoreceptor outer segment disk membranes most likely by the combined involvement of its C-terminal hydrophobic domain as well as other types of interactions. This study thus aimed to gather information on the structure and membrane binding of the C-terminal hydrophobic segment of R9AP as well as of truncated R9AP (without its C-terminal domain, R9AP∆TM). Circular dichroism and infrared spectroscopic measurements revealed that the secondary structure of R9AP∆TM mainly includes ⍺-helical structural elements. Moreover, intrinsic fluorescence measurements of native R9AP∆TM and individual mutants lacking one tryptophan demonstrated that W79 is more buried than W173 but that they are both located in a hydrophobic environment. This method also revealed that membrane binding of R9AP∆TM does not involve regions near its tryptophan residues, while infrared spectroscopy validated its binding to lipid vesicles. Additional fluorescence measurements showed that the C-terminal segment of R9AP is membrane embedded. Maximum insertion pressure and synergy data using Langmuir monolayers suggest that interactions with specific phospholipids could be involved in the membrane binding of R9AP∆TM.

Phenotypic characterization of PIGN–associated paroxysmal dyskinesia in Soft-coated wheaten terriers and preliminary response to acetazolamide therapy

A hereditary movement disorder in Soft coated wheaten terriers (SCWT) has been associated with a mutation in PIGN which encodes an enzyme involved in synthesis of glycosylphosphatidylinositol (GPI). The objective of this study was to describe and classify the clinical phenotype and assess therapeutic response.Twenty-five SCWT and related dogs homozygous for PIGN:c.398C>T with paroxysmal dyskinesia were available for inclusion. Medical records and video recordings of 17 dogs were evaluated in a retrospective case series. Affected dogs had episodes of involuntary, hyperkinetic movements and dystonia. Median age of onset was 2.5 years. A typical episode consisted of rapid, irregular hyperflexion and extension of the pelvic limbs with some degree of truncal dystonia. A mild episode consisted of spontaneous flexion of one pelvic limb while walking which could resemble a lameness. Episodes lasted several minutes to several hours and occurred up to 10 times/day or more. They were not associated with exercise or fasting but were sometimes triggered by excitement or stress. Acetazolamide therapy improved nine of 11 dogs, in seven cases abolishing episodes. Five of 17 dogs treated with other agents had mild improvement with clonazepam (n = 2), levetiracetam (n = 1), or phenobarbital (n = 2). Paroxysmal dyskinesias must be differentiated from seizure disorders since they often respond to different therapies. The SCWT phenotype consisted predominantly of hyperkinesia, and can respond dramatically to acetazolamide. GPI anchors proteins to the cell surface including carbonic anhydrase IV which modulates synaptic pH in the brain. Altered activity of this enzyme may be the target of acetazolamide therapy.

The importance of flow cytometry in the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria

ABSTRACT Introduction: The paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disease of the hematopoietic stem cells, and it is clinically characterized by chronic intravascular hemolysis, bone marrow failure and hypercoagulability leading to thrombosis. It is a rare disorder of the hematopoietic stem cells that occurs due to a somatic mutation in the gene phosphatidylinositol glycan class A (PIG-A). Objective: Here we reviewed the importance of screening and monitoring of individuals with high risk of developing PNH, since the early diagnosis of the disease is essential for better prognostic and treatment choice for the patient. Method: A review was carried out with great focus on the pathophysiology and diagnosis of PNH, mainly with the use of flow cytometry technique to detect the disease. Results: This gene codifies an enzyme essential to the formation of glycosylphosphatidylinositol (GPI), which acts as a molecular anchor for many membrane proteins. The alteration of GPI synthesis promotes a partial or complete loss of proteins that needs this molecular anchor to bind to the cell surface. Among these proteins are the CD55 and the CD59, which control the activation of the complement cascade. Conclusion: The immunophenotyping exam with flow cytometry is considered the reference test for PNH diagnosis, since the technique is highly sensitive and specific, presenting advantages as the quantitative identification of small populations of cells with PNH phenotype and the capacity to distinguish cells with partial or total deficiency of GPI-anchored proteins.

The Glycosylphosphatidylinositol Anchor Regulates T Cell Antigen Receptor Induced IL-2 Production

Differential contributions of the glycosylphosphatidylinositol (GPI)-anchor and GPI-anchored proteins (GPI-AP) to signalling remain poorly understood. Here we show that GPI-AP deficient murine clones produce on average 18 and 181-fold more IL-2 mRNA and protein, respectively, upon T cell receptor (TCR) stimulation, in a cell-intrinsic fashion. This phenotype is formally attributed to a mutation within the transferase complex that predicates the initial step in GPI-anchor biosynthesis. Conditional disruption of the transferase complex enabled the generation of primary GPI-AP deficient CD4+ T cells, which produce on average 10- and 23-fold more IL-2 mRNA and protein, respectively, upon TCR stimulation. Conditional disruption of the transamidase complex yields GPI-sufficient, GPI-AP deficient primary CD4+ T cells. TCR stimulation of these cells yields levels of IL-2 mRNA and protein ranging from 1 - 3 and 3-fold, respectively, of controls. These results provide the first evidence of a profound impact of GPI in the regulation of TCR signalling.

Generation and oriented evaluation of nitrocellulosebinding achor protein fused protein A and its application

Point-of-care testing diagnostics are current interest in research and development. Among them, Lateral Flow Test (LFT) is an on-site test strip with wide range of applications. Therefore, optimizing the sensitivity of rapid test strip is a necessary requirement. Immobilizing antibodies onto the nitrocellulose membrane is an important step to increase the sensitivity of the LFT strip for detecting pathogenic antigens. In this research, the fusion protein between nitrocellulose binding anchor protein 3-helix, a protein that has a strong affinity to nitrocellulose membrane and protein A, a protein that specifically binds to the Fc tail of IgG antibody was generated. This fusion protein was expected to help IgG antibodies strongly binding onto the nitrocellulose membrane and orientating the Fab of immobilized antibodies outwardly, thereby increasing the ability to capture the target antigen. The recombinant vector pET22b-proA and pET22b-proA-3-helix encoded for protein A and A-3-helix, respectively were subsequently cloned. Protein A and A-3-helix was induced to express using IPTG, and confirmed by SDS-PAGE and Western blot. These proteins were purified by immobilized metal affinity chromatography with the purity of more than 90%. The purified proteins were used to evaluate orientation binding on nitrocellulose membranes by the lateral flow test and antibidy capture as well. Results showed that protein A 3 helix bound to nitrocellulose membrane better than that of protein A. The former proteins increased the antibody binding and stereochemical immobilizing onto nitrocellulose membrane compared to its protein A counterpart. The present results laid the foundation for the application of fusion proteins in the directed immobilization of antibodies in rapid test strips.

Constitutive cell surface expression of ZZ domain for the easy preparation of yeast-based immunosorbents.

We describe a novel expression cassette that enables efficient and constitutive expression of the ZZ domain derived from Staphylococcus aureus protein A on the yeast cell surface to easily prepare yeast-based immunosorbents. Using this expression cassette containing the PGK1 promoter, a secretion signal derived from α-factor, and a Flo1-derived anchor protein, we successfully created a yeast-based immunosorbent for human serum albumin.

Cross-protective immunity of the haemagglutinin stalk domain presented on the surface of Lactococcus lactis against divergent influenza viruses in mice

ABSTRACT Most of the current approaches to influenza vaccine design focus on antibodies against influenza (HA). However, these influenza vaccines typically provide strain-specific protection against mostly homologous subtypes. There is an urgent need to develop a universal vaccine that confers cross-protection against influenza viruses. Of note, the HA stalk domain (HAsd) is a promising target for such an influenza vaccine. In this study, we generated recombinant Lactococcus lactis (L. lactis)/pNZ8150-phosphatidylglycerophosphate synthetase A (pgsA)-HAsd, in which pgsA was used as an anchor protein, and investigated the immunogenicity of HAsd in a mouse model by oral administration without the use of a mucosal adjuvant. Compared with L. lactis/pNZ8150-pgsA, mice were orally vaccinated with L. lactis/pNZ8150-pgsA-HAsd and then produced strong humoral and mucosal immune responses. Importantly, L. lactis/pNZ8150-pgsA-HAsd provided cross-protection against H5N1, H3N2 and H1N1 virus infections. Our data support the hypothesis that HAsd presented on the surface of L. lactis can provide cross-protective immunity against divergent influenza A viruses. Taken together, these findings suggest that L. lactis/pNZ8150-pgsA-HAsd can be considered an alternative approach to developing a novel universal vaccine during an influenza A pandemic. Abbreviations: HA, HAsd, HA stalk domain; L. lactis, Lactococcus lactis; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; IFA, immunofluorescence assay; PBS, phosphate-buffered saline; pgsA, phosphatidylglycerophosphate synthetase A; SPF, specific pathogen-free; CFU, colony-forming unit; BSL-3, biosafety level-3 laboratory; TCID50, 50% tissue culture infective dose; ELISA, enzyme-linked immunosorbent assay; OD, optical density; LTB, liable enterotoxin B subunit; CTB, cholera toxin B subunit.

Screening for Genetic Mutations for the Early Diagnosis of Common Variable Immunodeficiency in Children With Refractory Immune Thrombocytopenia: A Retrospective Data Analysis From a Tertiary Children's Center.

Aim: This study aimed to identify common variable immunodeficiency (CVID) by high-throughput next-generation sequencing (NGS) in children with refractory immune thrombocytopenia (RITP) to facilitate early diagnosis.Methods: CVID-related genetic mutations were explored in patients with RITP during 2016–2019. They were tested consecutively through NGS by the ITP team of the tertiary children hospital in China. An evaluation system was devised based on the phenotype, genetic rule, and serum immunoglobulins (Igs) of all patients with RITP. The patients were divided into highly suspicious, suspicious, and negative groups using the evaluation system.Results: Among 176 patients with RITP, 16 (9.1%) harbored CVID-related genetic mutations: 8 (4.5%) were highly suspicious of CVIDs. Five had mutations in tumor necrosis factor receptor superfamily 13B (TNFRSF13B), one in lipopolysaccharide responsive beige-like anchor protein (LRBA), one in nuclear factor kappa-B2 (NF-κB2), and one in caspase recruitment domain11 (CARD11). Others were classified into the suspicious group because the clinical phenotype and pedigree were suggestive, yet insufficient, for diagnosis. Repeated infection existed in all patients. Two had an allergic disease. Positive autoimmune serologies were noted in 62.5%. Five had a definite positive family history. The median serum immunoglobulin (Ig)A, IgG, and IgM levels were 0.3875, 6.14, and 0.522 g/L, respectively. Nearly 85.7% of patients had insufficient serum IgA levels, while 37.5% had low IgG and IgM levels.Conclusions: High-throughput NGS and a thorough review of the medical history are beneficial for the early diagnosis of patients without any significant clinical characteristics, distinguishing them from those with primary pediatric ITP. The cases suspicious of CVID need further investigation and follow-up to avoid deterioration.

AKAP12 Supports Blood-Brain Barrier Integrity against Ischemic Stroke.

A-kinase anchor protein 12 (AKAP12) is a scaffolding protein that associates with intracellular molecules to regulate multiple signal transductions. Although the roles of AKAP12 in the central nervous system are still relatively understudied, it was previously shown that AKAP12 regulates blood-retinal barrier formation. In this study, we asked whether AKAP12 also supports the function and integrity of the blood-brain barrier (BBB). In a mouse model of focal ischemia, the expression level of AKAP12 in cerebral endothelial cells was upregulated during the acute phase of stroke. Also, in cultured cerebral endothelial cells, oxygen-glucose deprivation induced the upregulation of AKAP12. When AKAP12 expression was suppressed by an siRNA approach in cultured endothelial cells, endothelial permeability was increased along with the dysregulation of ZO-1/Claudin 5 expression. In addition, the loss of AKAP12 expression caused an upregulation/activation of the Rho kinase pathway, and treatment of Rho kinase inhibitor Y-27632 mitigated the increase of endothelial permeability in AKAP12-deficient endothelial cell cultures. These in vitro findings were confirmed by our in vivo experiments using Akap12 knockout mice. Compared to wild-type mice, Akap12 knockout mice showed a larger extent of BBB damage after stroke. However, the inhibition of rho kinase by Y-27632 tightened the BBB in Akap12 knockout mice. These data may suggest that endogenous AKAP12 works to alleviate the damage and dysfunction of the BBB caused by ischemic stress. Therefore, the AKAP12-rho-kinase signaling pathway represents a novel therapeutic target for stroke.

Patterns of Immune Dysregulation in Primary Immunodeficiencies: A Systematic Review

Immune dysregulation is as important as susceptibility to infection in defining primary immunodeficiencies (PIDs). Because of the variability and nonspecificity of the symptoms of PIDs, diagnosis can be delayed-especially if a patient presents with immune dysregulation. Diagnosis is then based on certain combinations of symptoms and relies on the clinician's ability to recognize a pattern. So far there is no large report linking patterns of immune dysregulations to the underlying genetic defects. To identify immune dysregulatory patterns associated with PIDs and to help clinicians to detect an underlying PID in certain patients with noninfectious inflammatory diseases. A systematic literature review was performed. We included 186 articles that reported on n= 745 patients. The most common immune dysregulation category was "autoimmunity" (62%, n= 463), followed by "intestinal disease" (38%, n= 283) and "lymphoproliferation" (36%, n=268). Most patients (67%) had 1 or more symptoms of immune dysregulation. Autoimmune hemolytic anemia, the most common autoimmune phenotype, was most frequently reported in patients with LPS responsive beige-like anchor protein deficiency (when combined with hypogammaglobulinemia or gastrointestinal symptoms), activation-induced cytidine deaminase deficiency (when combined with autoimmune hepatitis), or RAG1 deficiency (when it was the only symptom of immune dysregulation). Eczema, allergies, and asthma were reported in 34%, 4%, and 4% of the patients, respectively. Patterns of immune dysregulation may help the physician to recognize specific PIDs. This systematic review provides clinicians with an overview to better assess patients with immune dysregulation.

Dysphagia with Blood Disorder? an Unusual Presentation for Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired hemolytic disorder of the multipotent myeloid stem cells, due to underexpression of surface complement inhibitory proteins CD55 and CD59, affecting 0.13/100,000 patients per year. Besides anemia and thrombosis, PNH can also cause smooth muscle dystonias owing to various mechanisms in the body. Studies have described smooth muscle dystonias presenting as recurrent abdominal pain, lethargy and erectile dysfunction, but intermittent dysphagia is rare with PNH. We describe a case of dysphagia with negative work up as initial presentation of PNH. 16-year-old female with no significant past medical history presented to the clinic with 1 week of ongoing fatigue and difficulty swallowing. She never had any similar complaints in the past nor in her family. The dysphagia was progressively worsening, initially for solid, then with liquids, accompanied by nausea, vomiting and globus sensation in the middle chest. No prior psychiatric history, binging-purging behavior, chest pain, shortness of breath, heat/cold intolerance, weight changes, bladder or bowel dysfunction. She also denied any recent travels, allergies, transfusions, sick contacts, or over-the-counter medication use. Initial labwork showed Hb 11.2 g/dL, MCV 80 fL, WBC 9 x103 K/uL, and platelet 219,000 K/uL. Metabolic panel, troponin, BNP, ANA, rheumatoid factor, lyme serology, vitamin B12, folate and iron studies were within normal limits. Mentzer index &amp;lt;13, serum protein electrophoresis ruled out Thalassemia. LDH was elevated and Haptoglobin decreased. Direct and indirect Coomb's antibody test were negative. Peripheral smear showed normocytic normochromic anemia with normal morphology of RBCs. Flow cytometry showed lack of CD55 and CD59. Esophagogastroduodenoscopy, done for dysphagia, to rule out luminal obstruction was unremarkable. CT chest with contrast ruled out external compression on esophagus. Videofluoroscopic swallow study revealed segmental, dynamic obstruction to both solids and liquids in the lower third of the esophagus. Dysphagia was attributed to PNH and patient was started on eculizumab. She responded to the medication in 3 weeks, during which outpatient symptomatic therapy was given for her anemia along with soft semisolid diet. In PNH, the RBCs may have varying degree of CD59 and glycosyl phosphatidylinositol (GPI) anchor protein deficiency, with unaffected cells described as type I, partially affected cells called type II, and complete lack of the proteins deemed type III red cells. PNH red cell type III and type II red cells are 15 to 25 times and 3 to 5 times more sensitive to complement-mediated lysis than the type I normal red cells. This variability in the severity of the deficiency, as well as in the proportion of the cell population affected, defines the extent of the clinical manifestations of the disease. Approximately 40% of patients have a combination of types I, II and III PNH cells With hemolysis, after exceeding haptoglobin's binding capacity, the free hemoglobin binds any available nitrous oxide (NO) in the blood. Hemolysis also reduces endogenous NO generation by conversion of l-arginine, which is a component in NO generation, to ornithine, thereby further reducing the systemic availability of NO. NO is endogenous smooth muscle relaxor via activation of cGMP. Decreases in NO levels are hypothesized to cause smooth muscle contraction, manifesting as abdominal pain, erectile dysfunction and rarely, dysphagia in the lower two-third of the esophagus which is predominantly smooth muscle. These symptoms occur more commonly in patients with large PNH clone sizes and, therefore, higher hemolytic rates. In this case study, our focus was to recognize PNH among differentials for new onset dysphagia due to complement-mediated intravascular hemolysis causing gastrointestinal dysmotility. In doing so, invasive investigations may be avoided. Advance studies may be needed to completely understand the causes and correlate between PNH, intravascular hemolysis and dysphagia. Disclosures No relevant conflicts of interest to declare.