Molecular characterization of porcine genes encoding complement components of the terminal lytic pathway and their association with hemolytic complement activity

Activation of the complement system from three different pathways (classical, alternative and lectin pathway) results in the generation of the C3-convertase enzyme, which plays a key role in formation of the membrane attack complex (C5b-C9) causing the death of target cells. The porcine C3 and C5 complement components were characterized and studied for association with hemolytic complement activity (Kumar et al. 2004, Wimmers et al. 2003). In order to gain understanding for the membrane attack complex action in the innate immune mechanism, in this study it was focussed on the terminal complement components C6, C7, C8, and C9 to characterize their molecular structure, to detect single nucleotide polymorphisms (SNPs), to establish their location on chromosome, and to associate their genetic variation with hemolytic complement activity in both classical and alternative pathway in the pig. The entire length of cDNA sequence of the candidate genes C6, C7, C8A, C8B, C8G and C9 were identified with 3306, 3561, 2146, 2461, 840 and 2536 bp encoding 935, 843, 589, 611, 202 and 543 amino acids, respectively. The porcine deduced protein sequence of the candidate genes showed 67-83% identities with human analogue. Respectively, screening the coding region revealed five, six, seven, nine, and two SNPs in the porcine C6, C7, C8A, C8B, and C9 but non in C8G by polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP). Most of the SNPs belong to the functional protein domains such as TSP1, LDLa, MACPF, CCP and FIMAC. Genotyping for several SNP sites in three porcine breeds German Landrace (LR), Pietrain (PIE) and Muong Khuong (MK) showed that European breeds (LR and PIE) had higher allelic variation than the Asian breed (MK). All genotypic frequencies fit to Hardy-Weinberg equilibrium rule. Using the INRA-Minnesota porcine Radiation Hybrid mapping panel, the porcine C6, C7, and C9 were assigned to the q-arm of chromosome 16 (q1.4) whereas the porcine C8A, and C8B were mapped to chromosome 6 (q3.1-q3.5). Particularly the porcine C8G was located on chromosome 1 (q2.13). Genetic association with hemolytic complement activity in both classical (CH50) and alternative pathway (AH50) was carried out in 417 animals of a F2 DUMI resource population derived from cross between Duroc and Berlin Miniature Pig. Therefore, the F2 DUMI animals were immunized with Mycoplasma hyopneumoniae (Mh), Aujeszky (ADV) and porcine reproductive and respiratory syndrome (PRRSV) vaccine. Sera were isolated from blood samples taken prior and post vaccinations and measurement for CH50 and AH50 was conducted thereafter. For each gene except the porcine C8G, the SNP site with amino acid substitution 862A→G for C6, 881A→G for C7, 1544C→T for C8A, 222C→T for C8B, and 407C→G for C9, segregating in the DUMI, were used for genotyping the F2 animals using PCR-RFLP with the restriction enzymes TaqI, MboII, Hin6I, FnuDII, and HpyCH4III, respectively. The association results illustrated that significant difference in hemolysis among genotypes was found in CH50 for C7 (p=0.0080), and C9 (p=0.0488). However, this was close to significance for C6 (p=0.0853) and C8A (p=0.0650) in CH50. Therefore between homozygous genotypes CC and TT for C8A hemolytic activity showed significant difference (p=0.0522). There was no association of any of the candidate gene with hemolytic complement activity in the alternative pathway. Analyzing the interaction between genotypes and eight different immunization time points in AH50 revealed significant differences for C8A (p=0.0027), C8B (p=0.0231), and C9 (p=0.0340) whereas in CH50 this interaction was found significant for C8B (p=0.0048). Hemolytic complement activity showed the highest values at the fourth day after immunization with ADV vaccine for CH50 whereas linear increment during the experiment was performed for AH50. Along the vaccination program after each of complement stimulation by different vaccines, a short termed increment of complement activity was found, especially with ADV vaccine. Also male animals always performed higher hemolysis than females in both pathways. These results show that hemolytic complement activity depends on the genetic variation, sex, age, kind of vaccine, and interaction of complement components. In summary, the obtained results provide the means for further understanding the role of C6, C7, C8, and C9 in natural immune response of the host against pathogens. It also promotes the porcine C6, C7, C8, and C9 as candidate genes in efforts to genetically improve general animal health, a goal of breeding programmes for food animals

The three-way relationship of polymorphisms of porcine genes encoding terminal complement components, their differential expression, and health-related phenotypes

<p>Abstract</p> <p>Background</p> <p>The complement system is an evolutionary ancient mechanism that plays an essential role in innate immunity and contributes to the acquired immune response. Three modes of activation, known as classical, alternative and lectin pathway, lead to the initiation of a common terminal lytic pathway. The terminal complement components (TCCs: C6, C7, C8A, C8B, and C9) are encoded by the genes <it>C6</it>, <it>C7</it>, <it>C8A</it>, <it>C8B</it>, <it>C8G</it>, and <it>C9</it>. We aimed at experimentally testing the porcine genes encoding TCCs as candidate genes for immune competence and disease resistance by addressing the three-way relationship of genotype, health related phenotype, and mRNA expression.</p> <p>Results</p> <p>Comparative sequencing of cDNAs of animals of the breeds German Landrace, Piétrain, Hampshire, Duroc, Vietnamese Potbelly Pig, and Berlin Miniature Pig (BMP) revealed 30 SNPs (21 in protein domains, 12 with AA exchange). The promoter regions (each ~1.5 kb upstream the transcription start sites) of <it>C6</it>, <it>C7</it>, <it>C8A</it>, <it>C8G</it>, and <it>C9</it> exhibited 29 SNPs. Significant effects of the TCC encoding genes on hemolytic complement activity were shown in a cross of Duroc and BMP after vaccination against Mycoplasma hyopneumoniae, Aujeszky disease virus and PRRSV by analysis of variance using repeated measures mixed models. Family based association tests (FBAT) confirmed the associations. The promoter SNPs were associated with the relative abundance of TCC transcripts obtained by real time RT-PCR of 311 liver samples of commercial slaughter pigs. Complement gene expression showed significant relationship with the prevalence of acute and chronic lung lesions.</p> <p>Conclusions</p> <p>The analyses point to considerable variation of the porcine TCC genes and promote the genes as candidate genes for disease resistance.</p

FIRST - Flexible interactive retrieval SysTem for visual lifelog exploration at LSC 2020

Lifelog can provide useful insights of our daily activities. It is essential to provide a flexible way for users to retrieve certain events or moments of interest, corresponding to a wide variation of query types. This motivates us to develop FIRST, a Flexible Interactive Retrieval SysTem, to help users to combine or integrate various query components in a flexible manner to handle different query scenarios, such as visual clustering data based on color histogram, visual similarity, GPS location, or scene attributes. We also employ personalized concept detection and image captioning to enhance image understanding from visual lifelog data, and develop an autoencoderlike approach for query text and image feature mapping. Furthermore, we refine the user interface of the retrieval system to better assist users in query expansion and verifying sequential events in a flexible temporal resolution to control the navigation speed through sequences of images

Safety and efficacy of fluoxetine on functional outcome after acute stroke (AFFINITY): a randomised, double-blind, placebo-controlled trial

Background Trials of fluoxetine for recovery after stroke report conflicting results. The Assessment oF FluoxetINe In sTroke recoverY (AFFINITY) trial aimed to show if daily oral fluoxetine for 6 months after stroke improves functional outcome in an ethnically diverse population. Methods AFFINITY was a randomised, parallel-group, double-blind, placebo-controlled trial done in 43 hospital stroke units in Australia (n=29), New Zealand (four), and Vietnam (ten). Eligible patients were adults (aged ≥18 years) with a clinical diagnosis of acute stroke in the previous 2–15 days, brain imaging consistent with ischaemic or haemorrhagic stroke, and a persisting neurological deficit that produced a modified Rankin Scale (mRS) score of 1 or more. Patients were randomly assigned 1:1 via a web-based system using a minimisation algorithm to once daily, oral fluoxetine 20 mg capsules or matching placebo for 6 months. Patients, carers, investigators, and outcome assessors were masked to the treatment allocation. The primary outcome was functional status, measured by the mRS, at 6 months. The primary analysis was an ordinal logistic regression of the mRS at 6 months, adjusted for minimisation variables. Primary and safety analyses were done according to the patient's treatment allocation. The trial is registered with the Australian New Zealand Clinical Trials Registry, ACTRN12611000774921. Findings Between Jan 11, 2013, and June 30, 2019, 1280 patients were recruited in Australia (n=532), New Zealand (n=42), and Vietnam (n=706), of whom 642 were randomly assigned to fluoxetine and 638 were randomly assigned to placebo. Mean duration of trial treatment was 167 days (SD 48·1). At 6 months, mRS data were available in 624 (97%) patients in the fluoxetine group and 632 (99%) in the placebo group. The distribution of mRS categories was similar in the fluoxetine and placebo groups (adjusted common odds ratio 0·94, 95% CI 0·76–1·15; p=0·53). Compared with patients in the placebo group, patients in the fluoxetine group had more falls (20 [3%] vs seven [1%]; p=0·018), bone fractures (19 [3%] vs six [1%]; p=0·014), and epileptic seizures (ten [2%] vs two [<1%]; p=0·038) at 6 months. Interpretation Oral fluoxetine 20 mg daily for 6 months after acute stroke did not improve functional outcome and increased the risk of falls, bone fractures, and epileptic seizures. These results do not support the use of fluoxetine to improve functional outcome after stroke

zu Bonn

Molecular characterization of porcine genes encoding complement components of the terminal lytic pathway and their association with hemolytic complement activit

The three-way relationship of polymorphisms of porcine genes encoding terminal complement components, their differential expression, and health-related phenotypes

BACKGROUND The complement system is an evolutionary ancient mechanism that plays an essential role in innate immunity and contributes to the acquired immune response. Three modes of activation, known as classical, alternative and lectin pathway, lead to the initiation of a common terminal lytic pathway. The terminal complement components (TCCs: C6, C7, C8A, C8B, and C9) are encoded by the genes C6, C7, C8A, C8B, C8G, and C9. We aimed at experimentally testing the porcine genes encoding TCCs as candidate genes for immune competence and disease resistance by addressing the three-way relationship of genotype, health related phenotype, and mRNA expression. RESULTS Comparative sequencing of cDNAs of animals of the breeds German Landrace, Piétrain, Hampshire, Duroc, Vietnamese Potbelly Pig, and Berlin Miniature Pig (BMP) revealed 30 SNPs (21 in protein domains, 12 with AA exchange). The promoter regions (each ~1.5 kb upstream the transcription start sites) of C6, C7, C8A, C8G, and C9 exhibited 29 SNPs. Significant effects of the TCC encoding genes on hemolytic complement activity were shown in a cross of Duroc and BMP after vaccination against Mycoplasma hyopneumoniae, Aujeszky disease virus and PRRSV by analysis of variance using repeated measures mixed models. Family based association tests (FBAT) confirmed the associations. The promoter SNPs were associated with the relative abundance of TCC transcripts obtained by real time RT-PCR of 311 liver samples of commercial slaughter pigs. Complement gene expression showed significant relationship with the prevalence of acute and chronic lung lesions. CONCLUSIONS The analyses point to considerable variation of the porcine TCC genes and promote the genes as candidate genes for disease resistance

Multimodal analysis of genome-wide methylation, copy number aberrations, and end motif signatures enhances detection of early-stage breast cancer

IntroductionBreast cancer causes the most cancer-related death in women and is the costliest cancer in the US regarding medical service and prescription drug expenses. Breast cancer screening is recommended by health authorities in the US, but current screening efforts are often compromised by high false positive rates. Liquid biopsy based on circulating tumor DNA (ctDNA) has emerged as a potential approach to screen for cancer. However, the detection of breast cancer, particularly in early stages, is challenging due to the low amount of ctDNA and heterogeneity of molecular subtypes.MethodsHere, we employed a multimodal approach, namely Screen for the Presence of Tumor by DNA Methylation and Size (SPOT-MAS), to simultaneously analyze multiple signatures of cell free DNA (cfDNA) in plasma samples of 239 nonmetastatic breast cancer patients and 278 healthy subjects.ResultsWe identified distinct profiles of genome-wide methylation changes (GWM), copy number alterations (CNA), and 4-nucleotide oligomer (4-mer) end motifs (EM) in cfDNA of breast cancer patients. We further used all three signatures to construct a multi-featured machine learning model and showed that the combination model outperformed base models built from individual features, achieving an AUC of 0.91 (95% CI: 0.87-0.95), a sensitivity of 65% at 96% specificity.DiscussionOur findings showed that a multimodal liquid biopsy assay based on analysis of cfDNA methylation, CNA and EM could enhance the accuracy for the detection of early- stage breast cancer