Epsilon Gene Research Articles

Influence of Acute Inflammation on the Expression of Clock Genes in the Ovine Pars Tuberalis Under Different Photoperiodic Conditions

The pars tuberalis (PT) plays an important role in the photoperiodic regulation of the secretory activity of the pituitary gland. Additionally, PT secretory activity may be influenced by the animal’s immune status. The melatonin signal processing in PT cells occurs through the presence of melatonin receptors and the expression of molecular clock genes. This study aimed to define the effects of acute inflammation induced by intravenous administration of lipopolysaccharide (LPS) on the expression of clock genes in the PT of ewes under different photoperiodic conditions. Two analogous experiments were conducted in different photoperiods: short-day and long-day. Both experiments included 24 sheep divided into two groups: day (n = 12) and night (n = 12), further subdivided into a control group (n = 6) and a group treated with LPS (n = 6) at a dose of 400 ng/kg. Under short-day conditions, the expression of clock circadian regulator, basic helix-loop-helix ARNT like 1, cryptochrome circadian regulator (CRY) 1, 2, and casein kinase 1 epsilon genes was lower during inflammation. LPS injection increased expression of the period circadian regulator 1 gene during the night. Under long-day conditions, CRY1 mRNA level was lower during the night, while diurnal CRY2 mRNA expression was decreased after LPS injection. Our results showed that inflammation disturbed the expression of molecular clock genes in the PT; however, this influence was partly dependent on photoperiod conditions.

CK1δ/ε kinases regulate TDP-43 phosphorylation and are therapeutic targets for ALS-related TDP-43 hyperphosphorylation

Hyperphosphorylated TAR DNA-binding protein 43 (TDP-43) aggregates in the cytoplasm of neurons is the neuropathological hallmark of amyotrophic lateral sclerosis (ALS) and a group of neurodegenerative diseases collectively referred to as TDP-43 proteinopathies that includes frontotemporal dementia, Alzheimer's disease, and limbic onset age-related TDP-43 encephalopathy. The mechanism of TDP-43 phosphorylation is poorly understood. Previously we reported casein kinase 1 epsilon gene (CSNK1E gene encoding CK1ε protein) as being tightly correlated with phosphorylated TDP-43 (pTDP-43) pathology. Here we pursued studies to investigate in cellular models and in vitro how CK1ε and CK1δ (a closely related family sub-member) mediate TDP-43 phosphorylation in disease. We first validated the binding interaction between TDP-43 and either CK1δ and CK1ε using kinase activity assays and predictive bioinformatic database. We utilized novel inducible cellular models that generated translocated phosphorylated TDP-43 (pTDP-43) and cytoplasmic aggregation. Reducing CK1 kinase activity with siRNA or small molecule chemical inhibitors resulted in significant reduction of pTDP-43, in both soluble and insoluble protein fractions. We also established CK1δ and CK1ε are the primary kinases that phosphorylate TDP-43 compared to CK2α, CDC7, ERK1/2, p38α/MAPK14, and TTBK1, other identified kinases that have been implicated in TDP-43 phosphorylation. Throughout our studies, we were careful to examine both the soluble and insoluble TDP-43 protein fractions, the critical protein fractions related to protein aggregation diseases. These results identify CK1s as critical kinases involved in TDP-43 hyperphosphorylation and aggregation in cellular models and in vitro, and in turn are potential therapeutic targets by way of CK1δ/ε inhibitors.

High prevalence of "non-pathogenic" POLE mutation with poor prognosis in a cohort of endometrial cancer from South India.

The Cancer Genome Atlas (TCGA) project identified favorable prognosis regarding the ultra-mutated endometrial cancer (EC) subtype linked to polymerase epsilon gene (POLE) mutations. This study investigated POLE mutations in EC of Indian patients. This retrospective analytical study was conducted between January 2016 and January 2023 at the Government Medical College, Kozhikode, and the MVR Cancer Center, Kozhikode, Kerala. Sanger sequencing of POLE gene exons 9 and 13 in 151 EC patients was carried out to analyze the relationship between mutations and epidemiological factors, clinicopathologic features, and treatment outcomes. Among 151 cases enrolled, 39 were unique POLE-mutated cases. Significant associations were high-grade tumors, myometrial invasion >50%, and Lymph-vascular space invasion (LVSI). The median follow-up was 40 months (95% confidence interval [CI], 34-46). A lower mean disease-specific survival (DSS) of 51.7 months (95% CI, 43.7-59.6) was noted in the POLE-mutated group compared with 72.11 months (95% CI, 67.60-76.62) for the POLE wild-type. A statistically significant hazard ratio (HR) of 2.683 for DSS in the POLE-mutated group was noted. In advanced stages (FIGO stages II-IV), a nine-fold HR for DSS and overall survival (OS) compared with POLE wild-type was identified. After controlling for treatment effects using Cox proportional HR, advanced-stage POLE-mutated tumors had a significantly higher HR of 8.67 for DSS compared with POLE-wild-type tumors of the same stage. This study identified a unique set of POLE mutations in Indian EC patients associated with poor prognosis, which were particularly pronounced in advanced stages. Advanced stage of presentation, type of POLE mutations, and possibly ethnicity are predictors of adverse outcomes in POLE-mutated EC. The present study highlights ethnicity as a determinant of phenotypic expression of genetic change.

Knockout of Tmlhe in mice is not associated with autism spectrum disorder phenotypes or motor dysfunction despite low carnitine levels

Deletion of exon 2 of the trimethyllysine hydroxylase epsilon (TMLHE) gene was identified in probands with autism spectrum disorder (ASD). TMLHE encodes the first enzyme in carnitine biosynthesis, N6-trimethyllysine dioxygenase (TMLD). Researchers have suggested that carnitine depletion could be important for the development of ASD and cognitive, locomotor and social dysfunctions, but previous findings have been inconclusive regarding the specific role of endogenous carnitine. We developed a mouse knockout model with constitutive TMLD enzyme inactivation that exhibited a significant decrease in the carnitine by more than 90% compared to wild-type (WT) mice. However, we did not observe any significant social, cognitive, or repetitive-behavior changes associated with ASD in the knockout mice; muscle strength and coordination were also not affected. In addition, the life expectancy of knockout mice was similar to that of WT mice. In conclusion, knockout of Tmlh in mice does not induce an ASD phenotype or motor dysfunction despite extremely low carnitine and gamma-butyrobetaine concentrations. Moreover, inactivation of TMLD does not induce a phenotype similar to previously described primary carnitine deficiency; indeed, our results showed that low levels of carnitine sustained adequate energy production, muscle function and social behavior in mice.

Clinical features and management of atypical hemolytic uremic syndrome patient with DGKE gene variants: a case report.

Atypical hemolytic uremic syndrome (aHUS) with diacylglycerol kinase epsilon (DGKE) gene variant is a rare variant of thrombotic microangiopathy (TMA). The information on the clinical features, management and long-term outcomes of DGKE-aHUS patients have not yet been fully elucidated. The aim of this study was to report a novel variant of the DGKE gene in a Chinese population with aHUS. The present work reports a 7-month-old boy with aHUS, possibly triggered by gastrointestinal infection, without complement activation, with little response to plasma therapy and nephroprotective measures. The patient died during the 8th week of his hospital stay. The causes of death were intracranial hemorrhage and multiorgan dysfunction. Comprehensive WES of peripheral blood-derived DNA revealed two heterozygous variations in the DGKE exon region: NM_003647.2, c.610dup, p.Thr204Asnfs*4 and deletion of exons 4-6. This case suggest that atypical HUS with DGKE gene variant has a poor prognosis with a high mortality rate, which typically manifests in the first year of life and presents as a systemic disease with early-onset HUS with rapidly worsening renal function and chronic proteinuria. There is no specific treatment for DGKE-aHUS. There have an uncertain benefit of plasma therapy for DGKE-aHUS patients. The literature demonstrated that anti-complement therapy showed benefits for DGKE-aHUS with complement activation and autoantibodies during the overt TMA presentation but did not prevent TMA relapses. Early diagnosis and treatment may prevent complications and improve prognosis.

Profile of 5-HT2A receptor involved in signaling cascades associated to intracellular inflammation and apoptosis in hepatocytes and its role in carbon tetrachloride-induced hepatotoxicity

Previously, we found that the 5-HT2A receptor plays a key role in cell injury. However, the mechanism by which the 5-HT2A receptor mediates intracellular processes remains unclear. In this study, we aimed to clarify this intracellular process in hepatocyte LO2 cells and evaluate its role in CCl4-induced hepatotoxicity in mice. In vitro, both the agonist and overexpression of 5-HT2A receptor could promote 5-HT degradation by upregulating the expression of 5-HT synthases and monoamine oxidase-A (MAO-A) to cause overproduction of ROS in mitochondria. We refer to this as the activation of the 5-HT degradation system (5DS) axis, which leads to the phosphorylation of JNK, p38 MAPK, STAT3, and NF-κB; upregulation of Bax, cleaved-caspase3, and cleaved-caspase9; and downregulation of Bcl-2, followed by apoptosis and oversecretion of TNF-α and IL-1β in cells. This phenomenon could be markedly blocked by the 5-HT2A receptor antagonist, MAO-A inhibitor, or gene-silencing MAO-A. Through protein kinases C epsilon (PKCε) agonist treatment and gene silencing of the PKCε and 5-HT2A receptor, we demonstrated that the 5-HT2A receptor controls 5-HT synthases and MAO-A expression via the PKCε pathway in cells. Unexpectedly, we discovered that PKCε-mediated phosphorylation of the AKT/mTOR pathway is also a consequence of the activation of the 5DS axis. Furthermore, we confirmed that the inhibition of the 5DS axis using the 5-HT2A receptor antagonist could prevent hepatotoxicity induced by CCl4 both in vitro and in vivo, inhibiting the aforementioned signaling cascades, inflammation, and apoptosis, and that the 5DS activation area overlapped the necrotic area of mouse liver. Taken together, we revealed a 5DS axis in hepatocytes that controls the signaling cascades associated with inflammation and apoptosis and confirmed its role in CCl4-induced hepatotoxicity.

Tandem duplication of a genomic region encoding glutathione S-transferase epsilon-2 and -4 genes in DDT-resistant Anopheles stephensi strain from India

The glutathione S-transferases (GST) genes are a multigene family of enzymes involved in the metabolism of endogenous and xenobiotic compounds by catalysing the conjugation of the reduced form of glutathione to the substrate. The epsilon class of GST (GSTe), unique to arthropods, is known to be involved in the detoxification process of several classes of insecticides, and GSTe2 in particular is known to have DDT dehydrochlorinase activity. This communication reports a tandem duplication of a genomic region encoding GSTe2 and GSTe4 genes in a laboratory-colonized DDT-resistant Anopheles stephensi. We identified duplication breakpoints and the organization of gene duplication through Sanger sequencing performed on long-PCR products. Manual annotation of sequences revealed a tandemly-arrayed duplication of a 3.62 kb segment of GST epsilon gene clusters comprised of five genes: a partial GSTe1, GSTe2, GSTe2-pseudogene, GSTe4 and partial GSTe5, interconnected by a conserved 2.42 kb DNA insert segment major part of which is homologous to a genomic region located on a different chromosome. The tandemly duplicated array contained a total of two GSTe2 and three GSTe4 functional paralog genes. Read-depth coverage and split-read analysis of Illumina-based whole-genome sequence reads confirmed the presence of duplication in the corresponding region of the genome. The increased gene dose in mosquitoes as a result of the GSTe gene-duplication may be an adaptive process to increase levels of detoxifying enzymes to counter insecticide pressure.

Determination of Type and Molecular Identity of Clostridium perfringens Isolated from Patients with Multiple Sclerosis (MS)

Background & Objectives: Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS). Clostridium perfringens ε-toxin (ETX) can cause emerging complications of MS due to its tendency to the blood-brain barrier. This study aimed to determine the presence of toxin-producing genes in intestinal C.perfringens in patients with multiple sclerosis.
 Materials & Methods: Stool samples were taken from 70 MS patients and 70 without MS individuals. The samples were enriched in cooked meat media, and cultural and biochemical methods separated Clostridium isolates. PCR tested these isolates to identify C. perfringens species. In addition, the presence of alpha, beta, epsilon and iota toxin-producing genes was evaluated in all isolates by Multiplex PCR.
 Results: Isolates containing the etx gene were observed in 10 patients, while no itxA gene was identified in any isolates. The results showed that isolates in 8 patients were type D. Also, the gene encoding toxin type D was identified in 2 isolates obtained from the control group.
 Conclusion: Our findings indicated the high frequency of C. Perfringens in MS patients. In the studied samples with clinical presentations, most of these organisms were type D bacteria that produce ε-toxin.

Abstract CT021: PD-1 blockade in solid tumors with defects in polymerase epsilon

Abstract Context: Polymerase epsilon (POLE) gene missense hotspot mutations can generate pathogenic (p) proofreading defects resulting in hypermutated genomic profiles. Aim: Determine the prevalence, genomic consequences and immunotherapy sensitivity of advanced POLE mutated tumors according to mutation site, primary tumor and tumor mutational burden (TMB). Results: Pan-Cancer TCGA & MSKCC databases genomic analyses found a prevalence of non-pathogenic POLE mutations (POLEnp) of 3.4% with median TMB of 11 mutations/Megabase (mt/Mb, IQR 3-34). Pathogenic POLE mutations (POLEp) prevalence was 0.4% with median TMB of 215 mt/Mb (IQR 107-324), predominantly in colorectal and endometrial cancers. Prevalence dropped to 0.1% in metastatic cancers. We assessed prospectively the efficacy of PD-1 blockade in mismatch repair proficient advanced solid tumors harboring POLE missense mutations (phase II ASCe Nivolumab trial; NCT03012581). Variants were categorized prospectively by a molecular board as POLEp, POLEnp or Variants with Unknown significance (VUS). The primary endpoint was the Overall Response Rate (ORR) at 12 weeks according to RECIST 1.1, and secondary endpoints included survival analyses according to POLE variants pathogenicity. Among 61 screened patients, 21 were eligible and 20 received Nivolumab and 19 were assessable for response (table 1). The 12-week ORR was 37% for patients harboring POLEp and VUS and resulted in major survival improvement compared to POLEnp patients (HR=0.1 ; CI95% 0.02-0.7); see results in Table 1. Among patients POLEp tumors, while higher TMB was not predictive of response, higher proportion of POLE-related mutational signature correlated with improved benefit. In silico exonucleasic POLE domain analyses confirmed that all POLEp and 2 VUS clustered in the DNA binding or the Catalytic site. Recategorizing the VUS according to the location within the exonucleasic domain improved the prediction of survival outcomes. Impact: This study gives new insights on how DNA repair defects, mutational burden and signatures sensitize to PD-1 blockade and may offer emerging tumor agnostic biomarkers for benefit to checkpoint blockade. POLE variant pathogenicity All(N=21) POLEnp(N=5) VUS(N=4) POLEp(N=12) Age, years ± SD 57 ± 16 64 ± 10 56 ± 16 54 ± 17 Sex, Male (%) 12 (57) 5 (100) 2 (50) 5 (42) PS (ECOG)=1 (%) 16 (75) 4 (80) 2 (50) 10 (83) Primary tumor Colorectal 9 (43) 2 (40) 2 (50) 5 (42) Endometrial 6 (29) 0 (0) 0 (0) 6 (50) Gastric 2 (9) 2 (40) 0 (0) 0 (0) Glial 1 (5) 0 (0) 0 (0) 1 (8) Biliary tract 1 (5) 0 (0) 1 (25) 0 (0) Pancreas 2 (9) 1 (20) 1 (25) 0 (0) Number of previous treatments 2.4 ± 2 5 ± 2 1.8 ± 1 1.5 ± 1 TMB (mt/Mb, Min-Max)(N=16) 36.2 (2-385) 5 (4-9) 3 (2-4) 114 (25-385) ORR at 12 weeks (CR+PR) 37%(N=7/19) 0%(N=0/5) 50%(N=2/4) 46%(5/10) DCR at 12 weeks (CR+PR+SD) 58%(N=11/19) 0%(N=0/5) 75%(N=3/4) 80%(8/10) Median Progresssion-Free survival (months) 5.6 2.3 10.3vs POLEnp: HR=0.2 IC95% 0.1-0.7 Median Overall Survival (months) 9.1 5.0 Not Reachedvs POLEnp:HR=0.1 IC95% 0.02-0.7 Citation Format: Benoît Rousseau, Ivan Bieche, Eric Pasmant, Nadim Hamzaoui, Nicolas Leulliot, Lucas Michon, Aurelien de Reynies, Mike Foote, Julien Masliah-Planchon, Magali Svrcek, Romain Cohen, Victor Simmet, Paule Augereau, David Malka, Antoine Hollebecque, Damien Pouessel, Carlos Gomez-Roca, Rosine Guimbaud, Amandine Bruyas, Marielle Guillet, Muriel Duluc, Sophie Cousin, Christelle de la Fourchardiere, Frederic Rolland, Sandrine Hiret, Esma Saada-Bouzid, Olivier Bouche, Thierry Andre, Diane Pannier, Farid El Hajbi, Stephane Oudard, Christophe Tournigand, Jean-Charles Soria, Drew Gerber, Dennis Stephens, Michelle Lamandola-Essel, Steven B Maron, Bill Diplas, Guillem Argiles, Asha Krishnan, Neil Segal, Andrea Cercek, Nathalie Hoog-Labouret, Frederic Legrand, Clotide Simon, Assia Lamrani-Ghaouti, Luis A. Diaz, Pierre Saintigny, Sylvie Chevret, Aurelien Marabelle. PD-1 blockade in solid tumors with defects in polymerase epsilon [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT021.

In This Issue

In This Issue| June 02 2022 In This Issue Author & Article Information Online Issn: 2159-8290 Print Issn: 2159-8274 ©2022 American Association for Cancer Research.2022American Association for Cancer Research. Cancer Discov (2022) 12 (6): 1397–1399. https://doi.org/10.1158/2159-8290.CD-12-6-ITI Related Content A commentary has been published: Characterization of INCB086550: A Potent and Novel Small-Molecule PD-L1 Inhibitor A commentary has been published: Identification of Functional Heterogeneity of Carcinoma-Associated Fibroblasts with Distinct IL6-Mediated Therapy Resistance in Pancreatic Cancer A commentary has been published: Epigenetic Activation of Plasmacytoid DCs Drives IFNAR-Dependent Therapeutic Differentiation of AML View more A commentary has been published: Integrated Multiomic Profiling Identifies the Epigenetic Regulator PRC2 as a Therapeutic Target to Counteract Leukemia Immune Escape and Relapse A commentary has been published: The Spatial Landscape of Progression and Immunoediting in Primary Melanoma at Single-Cell Resolution A commentary has been published: Precision Combination Therapies Based on Recurrent Oncogenic Coalterations A commentary has been published: PD-1 Blockade in Solid Tumors with Defects in Polymerase Epsilon A commentary has been published: Reverse Transcriptase Inhibition Disrupts Repeat Element Life Cycle in Colorectal Cancer A commentary has been published: Discovery, Preclinical Characterization, and Early Clinical Activity of JDQ443, a Structurally Novel, Potent, and Selective Covalent Oral Inhibitor of KRASG12C View less Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Get Permissions Cite Icon Cite Search Site Article Versions Icon Versions Version of Record June 2 2022 Citation In This Issue. Cancer Discov 1 June 2022; 12 (6): 1397–1399. https://doi.org/10.1158/2159-8290.CD-12-6-ITI Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest Search Advanced Search Proofreading defects produced from mutations in the DNA polymerase epsilon (POLE) gene contribute to a hypermutated genome that can potentially sensitize tumors to immune checkpoint blockade. Despite this, many POLE-mutant tumors do not demonstrate a robust response to this type of therapy. Rousseau and colleagues conducted a multicenter clinical trial in patients with POLE-mutated solid tumors and showed that only mutations in the DNA binding or catalytic site of the exonuclease domain of POLE contributed to high tumor burden and subsequent increases to T-cell infiltration and response rates to anti–PD-1 therapy, suggesting their use as a biomarker of anti–PD-1 response. See article, p. 1435. Escape from the “graft-versus-tumor” effect represents the main driver of leukemia relapses after allogeneic hematopoietic cell transplantation. In up to 40% of cases, the expression of HLA class II molecules is lost from the surface of leukemic cells, without evidence of... You do not currently have access to this content.

Development of a Novel Reference Material for Tumor Mutational Burden Measurement Based on CRISPR/Cas9 Technology.

As a biomarker that affects treatment decisions of immune checkpoint inhibitors, the accuracy, reliability, and comparability of tumor mutational burden (TMB) estimation is of paramount importance. To improve the consistency and reliability of these tests, qualified reference materials providing ground-truth data are crucial. In this study, we developed a set of formalin-fixed and paraffin-embedded (FFPE) samples with different TMB values as the novel reference materials for TMB estimation. By introducing several clinically relevant variants in MutS Homolog 2 (MSH2) gene and DNA polymerase epsilon (POLE) gene into human cell lines using CRISPR/Cas9 technology, we first constructed four typical cell lines which verified with hypermutator or ultramutator phenotype. Followed by cell mixing and paraffin embedding, the novel FFPE samples were prepared. It was confirmed that our novel FFPE samples have sufficient quantity of cells, high reproducibility, and they can provide matched wild type sample as the genetic background. The double-platform whole exome sequencing validation showed that our FFPE samples were also highly flexible as they containing different TMB values spanning a clinically relevant range (2.0–106.1 mut/Mb). Without limitations on production and TMB values, our novel FFPE samples based on CRISPR/Cas9 editing are suitable as candidate reference materials. From a practical point of view, these samples can be used for the validation, verification, internal quality control, and proficiency testing of TMB assessment.

Evaluations of two glutathione S-transferase epsilon genes for their contributions to metabolism of three selected insecticides in Locusta migratoria

The insect-specific epsilon class of glutathione S-transferases (GSTEs) plays important roles in insecticide detoxification in insects. In our previous work, five GSTEs were identified in Locusta migratoria, and two recombinant GSTEs, rLmGSTE1 and rLmGSTE4, showed high catalytic activity when 1-chloro-2,4-dinitrobenzene (CDNB) was used as a substrate. In this work, we further investigated whether these two GSTEs could metabolize three insecticides including malathion, deltamethrin and DDT. Using ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC/MS) method, we found that rLmGSTE4, but not rLmGSTE1, can metabolize malathion and DDT. Malathion bioassays of L.migratoria after the expression of LmGSTE4 was suppressed by RNA interference (RNAi) showed increased insect mortality from 33.8% to 68.9%. However, no changes in mortality were observed in deltamethrin- or DDT-treated L.migratoria after the expression of LmGSTE4 was suppressed by RNAi. Our results provided direct evidences that LmGSTE4 participates in malathion detoxification in L.migratoria. These findings are important for understanding the mechanisms of insecticide resistance in L.migratoria and developing new strategies for managing the insect populations in the field.

Thrombotic microangiopathy in children.

The syndrome of thrombotic microangiopathy (TMA) is a clinical-pathological entity characterized by microangiopathic hemolytic anemia, thrombocytopenia, and end organ involvement. It comprises a spectrum of underlying etiologies that may differ in children and adults. In children, apart from ruling out shigatoxin-associated hemolytic uremic syndrome (HUS) and other infection-associated TMA like Streptococcus pneumoniae-HUS, rare inherited causes including complement-associated HUS, cobalamin defects, and mutations in diacylglycerol kinase epsilon gene must be investigated. TMA should also be considered in the setting of solid organ or hematopoietic stem cell transplantation. In this review, acquired and inherited causes of TMA are described with a focus on particularities of the main causes of TMA in children. A pragmatic approach that may help the clinician tailor evaluation and management is provided. The described approach will allow for early initiation of treatment while waiting for the definitive diagnosis of the underlying TMA.

803P An histopathological algorithm to help the search for POLE mutated endometrial cancers

Endometrial cancers (EC) mutated in the exonuclease domain of the polymerase epsilon gene (POLE mut) accounts for 8-10% of EC and have an excellent outcome. POLE sequencing is not routinely performed but emerging guidelines suggest that POLE mut stage I–II EC may spare adjuvant treatment while advanced POLE mut EC are eligible for a check point inhibitor. Given its therapeutic impact, we aimed at identifying surrogate pathological markers suggesting POLE mut.

Genomic features of humoral immunity support tolerance model in Egyptian rousette bats.

Bats asymptomatically harbor many viruses that can cause severe human diseases. The Egyptian rousette bat (ERB) is the only known reservoir for Marburgviruses and Sosuga virus, making it an exceptional animal model to study antiviral mechanisms in an asymptomatic host. With this goal in mind, we constructed and annotated the immunoglobulin heavy chain locus, finding an expansion on immunoglobulin variable genes associated with protective human antibodies to different viruses. We also annotated two functional and distinct immunoglobulin epsilon genes and four distinctive functional immunoglobulin gamma genes. We described the Fc receptor repertoire in ERBs, including features that may affect activation potential, and discovered the lack of evolutionary conserved short pentraxins. These findings reinforce the hypothesis that a differential threshold of regulation and/or absence of key immune mediators may promote tolerance and decrease inflammation in ERBs.

A patient with a homozygous diacylglycerol kinase epsilon (DGKE) gene mutation with atypical haemolytic uraemic syndrome and low C3 responded well to eculizumab: a case report

BackgroundAtypical haemolytic uraemic syndrome (aHUS) is a rare systemic syndrome characterized by non-immune haemolytic anaemia, thrombocytopenia, and kidney injury. In most cases, alternative complement pathway dysregulation is the identifying cause. Recently, other genetic causes have been identified, including a mutation in the diacylglycerol kinase epsilon (DGKE) gene, which theoretically affect the coagulation pathway and does not affect the complement pathway. Data about the management of these patients are limited. Ideal management and definitive treatment protocols have not yet been established.Case presentationA three-year-old boy presented with features of atypical haemolytic uraemic syndrome (aHUS) and low complement C3. He was presumed to have complement-mediated aHUS and was managed empirically with eculizumab. Two weeks after starting eculizumab, his haemoglobin levels, platelet count, and complement C3 level normalized but he continued to have non-nephrotic range proteinuria. His genetic testing revealed a homozygous DGKE mutation, with no other mutation detected. Six months after presentation, the patient was still in remission with no features of aHUS, a trial of weaning eculizumab by increasing dose interval was followed by nephrotic range proteinuria and severe oedema. His proteinuria improved and his oedema resolved after resuming his recommended eculizumab dose.ConclusionsDGKE gene mutation can lead to aHUS with theoretically no complement dysregulation. However, some patients with this mutation show alternative complement pathway activation. This case report describes a patient with aHUS due to a DGKE gene mutation and low C3 levels who responded to eculizumab, adding to the previously reported cases of patients with DGKE gene mutations who had complete remission with no relapse with C5 blockers and/or plasma exchange. A randomized controlled study on patients with DGKE mutations might be beneficial in understanding the disease and generating a management protocol.

A Cross-Sectional Analysis of APOE Gene Polymorphism and the Risk of Cognitive Impairments in the Alzheimer's Disease Neuroimaging Initiative Study.

It is inconclusive on how apolipoprotein epsilon (APOE) gene polymorphism is associated with the risk of having mild cognitive impairment (MCI) or Alzheimer's disease (AD). To investigate how APOE genotype is associated with the risk of MCI or AD using the data collected from the Alzheimer's Disease Neuroimaging Initiative (ADNI) participants. A cross-sectional design was used to analyze the baseline data collected from the 1,720 ADNI participants. APOE gene polymorphism was analyzed on how they are related to the risk of cognitive impairments of either MCI or AD using a percent yield (PY) method. Then cognitive functions were compared among six different APOE genotypes using a two-way ANCOVA by controlling possible confounding factors. The prevalence of six APOE genotypes in 1,720 participants is as following: e2/e2 (0.3%), e2/e3 (7.4%), e3/e3 (45.4%), e2/e4 (2%), e3/e4 (35%) and e4/e4 (9.9%). The e2/e2 and e4/e4 genotypes were associated with the lowest and the highest risk respectively for cognitive impairments of either MCI or AD. Further, a worse cognitive diagnosis was associated with an increasing number of APOE e4 allele in a dose dependent manner. Participants with genotype e3/e3 had a better memory measure than those with the genotype of e3/e4. APOE gene polymorphism is associated with different level of risks for cognitive impairments. The heterozygous genotype e3/e4 is associated with a worse memory function compared to the genotype of e3/e3. Further investigations are needed to intervene the cognitive deteriorations in those with at risk APOE genotypes.

Expression of Epsilon Toxin from Clostridium perfringens Type D in E. coli Rosetta (DE3): Study on a New Vaccine Production

Clostridium perfringens is a Gram-positive anaerobic bacterium which is divided based on major toxins into five types (A-E). C. perfringens type D causes fatal enterotoxaemia (pulpy kidney) in sheep and goats that causes heavy economic losses in domestic animals. However current enterotoxaemia vaccine has been effective in controlling of disease; Strategies for the development of effective vaccine can be achieved by the production of new generation experimental vaccines. The aim of this study was cloning and expression of epsilon toxin of C. perfringens type D in E. coli Rosetta (DE3) to improve of the immunity. Epsilon toxin gene was cloned into pJET1.2/blunt vector and pET22b (+) expression vector and finally transformed into E. coli Rosetta competent cells (DE3). The result showed that the epsilon gene of C. perfringens type D can be cloned and expressed in E. coli RosettaTM (DE3) successfully. E. coli was suitable host for the expression of C. perfringens epsilon toxin. Clearly, future research is open for further refinement of the study.

High Hemoglobin F in Sickle Cell Disease: Waning Protection with Age

Background: Sickle cell disease (SCD) is a monogenetic inherited red cell disorder with pleomorphic clinical manifestations. Hemoglobin F (HbF) concentration is the major genetic modifier of clinical expression and levels between 10% and 20% have been found to improve survival and decrease vaso-occlusive complications (VOCs). As survival for patients with SCD has increased dramatically in the past 20 years, we hypothesized that SCD-related complications may increase with age, even in "high F" individuals. Recent data suggest that individuals with the Arab-Indian haplotype and/or HbS-δβ thalassemia, who have HbF of 15% to 25%, still suffer from VOCs. SCD patients with HBS and deletional hereditary persistence of fetal hemoglobin (HPFH) have high levels of HbF (>30%) in a pancellular distribution, and absence of symptoms as suggested by prior studies. Here, we report on a cohort of patients with HbSS/Sβ0, with HbF >14%, either hydroxyurea naïve or treated for ≤ 6 months. Methods: We report on a cohort of 11 adults with SCD and HbF levels > 14%, who had never been on hydroxyurea, or were treated for ≤6 months. Charts were reviewed for VOCs, opioid therapy, acute chest syndrome (ACS), avascular necrosis (AVN), retinopathy, pneumonia, leg ulcers, splenomegaly/infarction, stroke, pulmonary hypertension, cardiomyopathy/infarction, chronic kidney disease, priapism and miscarriage. All events were confirmed by supportive laboratory or imaging data. HbF levels for each patient are represented as lifetime average values. In 10/11 patients, cellular distribution of HbF by flow cytometry (CytoFlex, Beckman Coulter, CA) was performed following fixation, permeabilization, and incubation of red blood cells with a monoclonal antibody against Hb-F, directly conjugated with FITC (Cat # MHFH01, Life Technologies, MD). Beta globin dosage genetic analysis was performed on a subset of patients (8/11) which used multiplex-polymerase chain amplification of fragments specific for portions of the epsilon, gammaG, gammaA, delta, and beta globin genes, which is commonly associated with deletional variant of HPFH (Quest Diagnostics, VA). Results: Our cohort consisted of 11 patients, 3 males, age range 23 -72 years, one of which (age 72) is deceased. Genotypes included 10 HbSS, four with alpha thalassemia trait, and one HbS/β0. Mean HbF levels ranged from 14 % to 29.50%, SD ± 4.76. Table 1 shows patients ordered in ascending age with clinical complications for each patient. Notably, all patients experienced VOCs regardless of age or HbF level, 91% were prescribed opioids, 46% of whom were on chronic opioid therapy, 40 % had leg ulcers 36% had AVN and 27% had ACS . The number of complications per patient increased with age, despite maintenance of high HbF. Available flow cytometry and genetic data revealed heterocellular distribution of HbF in 8 patients, and pancellular distribution in 2 patients: patient (#2) and patient (#10). Patient (#2) is positive for deletional HPFH with genetic confirmation of heterozygous positive deletion in the beta globin gene cluster. Genetic data is not available for patient (#10) at this time. Interestingly, both patients still exhibited VOCs, complicated by yearly hospitalizations, required intermittent opioid therapy and with age, developed AVN. Laboratory data are notable for minimal levels of hemolysis. Discussion: Most individuals with SCD living in high resources countries now reach adulthood. Therefore the disease profile that was previously studied and reported mostly in the pediatric population is evolving. High levels of HbF appear to delay the onset of complications, but do not completely prevent them, perhaps due to a heterocellular distribution of HbF or non-deletional HPFH. In our cohort, two patients with confirmed pancellular distribution of HbF, one of which with confirmed deletional HPFH, were not fully protected from the clinical complications of SCD, suggesting alternative pathogenic mechanisms for VOCs may be at play. Therefore, individuals with HbF concentrations of as high as ~30 % may still suffer from pain, hospitalizations for VOC's, and vasculopathic changes, such as leg ulcers. The number of complications increases with age despite maintenance of high HbF. Therefore, additional therapy and close vigilance is necessary when treating sickle cell patients with high levels of HbF, especially with increasing age. Disclosures Reyes Gil: Albert Einstein College of Medicine: Patents & Royalties: Patent application pending for NETs AI software. Ogu:Vertex Pharmaceuticals: Consultancy. Minniti:Doris Duke Foundation: Research Funding.

Molecular docking and molecular dynamics simulation studies on PLCE1 encoded protein

Phospholipase C epsilon gene (PLCE1) is encoded the 1-phosphatidylinositol 4,5- bisphosphate phosphodiesterase ɛ-1 protein. This protein is characterized by the fetal onset of enormous proteinuria following by severe steroid resistance nephrotic syndrome (SRNS). The mutation position is located on the PIPLC_X domain of PLCE1 encoded protein. Molecular docking study was carried out between aegeline, berberine, diterpenoid, roseoside phytochemicals with wild type and mutant of the PIPLC_X domain of the PLCE1 gene encoded protein. From the molecular docking study, it was found that, due to mutation the binding energy was decreased for aegeline, berberine diterpenoid and roseoside with mutant PLCE1 at S1484L compared to wildtype. To achieve the depth analysis, we extended our study by performing the molecular dynamic simulation of 100 ns on wild type and the mutant (S1484L) model of above said domain of the studied protein. In the MD simulation study, we analyzed the result obtained from root mean square deviation, root mean square fluctuation, radius of gyration, salvation accessible surface area, hydrogen bond number and principal component analysis. These dynamical behaviour results of wild type protein show similar behaviour with mutant protein.