Natural antisense transcripts with coding capacity in Arabidopsis may have a regulatory role that is not linked to double-stranded RNA degradation

BACKGROUND: Overlapping transcripts in antisense orientation have the potential to form double-stranded RNA (dsRNA), a substrate for a number of different RNA-modification pathways. One prominent route for dsRNA is its breakdown by Dicer enzyme complexes into small RNAs, a pathway that is widely exploited by RNA interference technology to inactivate defined genes in transgenic lines. The significance of this pathway for endogenous gene regulation remains unclear. RESULTS: We have examined transcription data for overlapping gene pairs in Arabidopsis thaliana. On the basis of an analysis of transcripts with coding regions, we find the majority of overlapping gene pairs to be convergently overlapping pairs (COPs), with the potential for dsRNA formation. In all tissues, COP transcripts are present at a higher frequency compared to the overall gene pool. The probability that both the sense and antisense copy of a COP are co-transcribed matches the theoretical value for coexpression under the assumption that the expression of one partner does not affect the expression of the other. Among COPs, we observe an over-representation of spliced (intron-containing) genes (90%) and of genes with alternatively spliced transcripts. For loci where antisense transcripts overlap with sense transcript introns, we also find a significant bias in favor of alternative splicing and variation of polyadenylation. CONCLUSION: The results argue against a predominant RNA degradation effect induced by dsRNA formation. Instead, our data support alternative roles for dsRNAs. They suggest that at least for a subgroup of COPs, antisense expression may induce alternative splicing or polyadenylation

T cell immune memory after covid-19 and vaccination

The T cell memory response is a crucial component of adaptive immunity responsible for limiting or preventing viral reinfection. T cell memory after infection with the SARS-CoV-2 virus or vaccination is broad, and spans multiple viral proteins and epitopes, about 20 in each individual. So far the T cell memory response is long lasting and provides a high level of cross reactivity and hence resistance to viral escape by variants of the SARS-CoV-2 virus, such as the omicron variant. All current vaccine regimens tested produce robust T cell memory responses, and heterologous regimens will probably enhance protective responses through increased breadth. T cell memory could have a major role in protecting against severe covid-19 disease through rapid viral clearance and early presentation of epitopes, and the presence of cross reactive T cells might enhance this protection. T cell memory is likely to provide ongoing protection against admission to hospital and death, and the development of a pan-coronovirus vaccine might future proof against new pandemic strains

Mouse labial-like homeobox-containing genes: structure and expression during embryogenesis

While mouse development has been well described at a morphological level, very little is known about how development is regulated. In contrast, the ease of developmental analysis in Drosophila has led to the identification of a large number of developmentally important genes. Molecular characterisation revealed that many of the genes involved specifically in determining the Drosophila body plan contain a conserved sequence called the homeobox. This sequence is highly conserved through evolution and so it can be used to isolate homologous genes in other species. In this way more than 40 homeobox- containing genes have been identified in the mouse. The high level of sequence conservation and the temporally and spatially restricted expression of the mouse genes during development indicate that they are also developmental regulators involved in conferring spatial information within the embryo. Thus, through knowledge of Drosophila development and the techniques of molecular biology, it is now possible to study mouse developmental genes in detail.In this thesis, the characterisation of two mouse homeobox -containing genes, Hox 2.9 and Hox 1.6, is presented. Sequence analysis revealed that these genes are closely related and that, among Drosophila genes, they are most similar to labial in the Antennapedia complex. They are therefore thought to have arisen by duplication of a single ancestral gene. As well as being structurally similar the genes share many features of their expression patterns. Both genes are expressed early in development (71/2 days) and, unlike other known mouse homeobox -containing genes, they are not expressed after 11 days of development. At 8 days the genes share the same anterior boundary of expression in the hindbrain and in the later embryo, with the exception of persistent Hox 2.9 expression in the hindbrain, they have the same anterioposterior restrictions. This indicates that the genes are functionally similar and also that they respond to at least some of the same signals in the embryo.A striking difference between the expression patterns of mouse labial -like genes is the unique expression of Hox 2.9 in a single segmental unit (rhombomere 4) of the hindbrain (from 81/2 days). This expression coincides perfectly with the morphological extent of rhombomere 4 and persists throughout the period that rhombomeres are visible (up to 11 days). It is therefore suggested that Hox 2.9 participates in conferring segment identity. In addition neural crest cells that arise from rhombomere 4 specifically express Hox 2.9 and this supports the idea of neural crest cells being patterned according to their position of origin in the central nervous system. Detailed analysis of the onset of segmental expression of Hox 2.9 and another segmentally expressed gene in the hindbrain, Krox 20, showed that Hox 2.9 expression becomes localised from a broad domain at 81/2 days of development, up to 6 hours before rhombomeres are clearly visible.Retinoic acid is a strong candidate for a natural morphogen in the vertebrate embryo. The effect of in vitro treatment with retinoic acid on segmentation of the mouse hindbrain and on the expression of Hox 2.9 and Krox 20 was therefore analysed. It was found that segmentation in treated embryos is abnormal and that the clear segmental localisation of expression of the two genes is not found. The hindbrain expression domains are shifted rostrally following treatment and while the expression of the two genes remains mutually exclusive there is no longer a single planar boundary between the domains. Instead there is an irregular alternation of cells expressing the two genes at the boundaryTwo differential splicing products of Hox 1.6 were isolated from the developing embryo. A comparison was made of the distribution of these transcripts, only one of which can code for a homeodomain containing protein. It was found that the relative proportion of homeodomain producing message decreases as development proceeds

The genetics of familial hypercholesterolaemia and establishing familial hypercholeserolaemia genetic testing as a clinical diagnostic service

Familial hypercholesterolaemia (FH) is a monogenically inherited disorder of lipoprotein metabolism caused by a mutation in the low density lipoprotein receptor gene (LDLR). However in a few individuals, the defect lies in the gene for apolipoprotein B (APOB), the ligand for the LDL-receptor, and this is called familial defective apolipoprotein B-100 (FDB), while in others the receptor function is apparently normal and the defect must lie elsewhere. Mutation studies are the most practical way in which one can identify the cause of heterozygous hypercholesterolaemia. A genetic diagnostic service for FH has been established and the various aspects of setting up are described, with unusual cases being reported. The mutations identified are described and mutation detection rates were calculated for groups of paediatric and adult probands from the UK. The feasibility of altemative mutation screening methods and the specificity and sensitivity of reducing the number of tests has been assessed from the results obtained over the last four years. A quantitative fluorescent multiplex PCR screen was adapted to analyse LDLR rearrangements which would improve the genetic diagnosis of FH individuals. One assay based on exons 1, 8, 10, 12 and 16 were optimised and tested on known major rearrangements. A group of FH probands from the USA were then analysed with this multiplex assay. The inter and intra-assay variation were very wide, so a second method was designed to overcome these problems, universal primer quantitative fluorescent multiplex PCR (UPQFM-PCR). The multiplex set developed analysed exons 3, 5, 8, 14, and 17 of LDLR, and the method could also be used to detect major rearrangements in other genes. The method was evaluated by conducting a trial on 15 reported deletions and duplications. Two groups of FH patients from the UK were screened with this UPQFM-PCR assay. The influence of LDLR & APOB mutations on the cholesterol-lowering response of the HMG-CoA reductase inhibitor simvastatin was investigated in patients with heterozygous FH. Data suggest that there may be a difference in cholesterol-lowering between 'severe' and 'mild' LDLR mutations. Future developments and transferring the findings into a clinical genetic service are discussed

Cross-reactivity of B and T cells: desired in influenza vaccine responses, feared in autoimmune diseases

Innate immune mechanisms are very efficient at mounting rapid immune responses at the site of infection. Complete clearance of a pathogen and long-lasting protection through memory formation requires the adaptive immune system. To be able to cope with the large variety of pathogens we encounter, T and B cells acquire an almost infinite number of specificities by VDJ-recombination and somatic hypermutation. However, not all recombinations are equally likely to occur and the majority of lymphocyte clones will never be released from the thymus or bone marrow due to negative selection. T cells also need to recognize host HLA-proteins, adding further constraints. Therefore, immune cell diversity is more restricted than theoretically possible. A certain redundancy is induced by the fact that a T or B cell clone may recognize multiple epitopes, albeit with different affinities, a feature termed cross-reactivity. In a vaccine against a genetically diverse pathogen, cross-reactivity of vaccine-induced immune cells is desirable. An ideal vaccine enables the host to mount an immune response not only against the vaccine strain but also against naturally occurring variants that may be antigenically different. Influenza virus is one of the most prevalent human pathogens and of high economic relevance. The ‘success’ of influenza virus is tightly linked to its extraordinary ability to evolve – that is, evading the host’s immune system – while still maintaining its integrity and virulence. Annually updated influenza vaccines provide some protection against infection. However, vaccine efficacy is strongly reduced when there is an antigenic mismatch between vaccine strain and predominant circulating virus. We hypothesized that the cross-reactivity of the influenza vaccine response is affected by the individual B cell repertoire and wanted to test whether low cross-reactivity associates with a narrow repertoire. A narrow antibody repertoire could be related to the previous infection history or to repetitive vaccination with very similar influenza vaccine strains. Consequently, this may lead to higher susceptibility to emerging viral variants. The breadth and degree of antigen-specificity of the B cell receptor (BCR) repertoire can be assessed by sequencing the immunoglobulin heavy chains before and after vaccination. We tested this hypothesis by analyzing samples from a previous cohort of influenza-vaccinated healthy subjects and aimed to extend our findings by conducting a prospective clinical influenza vaccination study in individuals with known vaccination history. Since the composition of the influenza vaccine is an active debate in the field, our results could inform on both strain selection and better vaccination strategies. Cross-reactivity can be beneficial in the case of vaccination but may be harmful if cross-reactive lymphocytes target self-structures, as it is the case in autoimmunity. While B cells recognize native macromolecular structures, T cells mainly respond towards peptides displayed on MHC of antigen-presenting cells (APC). In Giant Cell Arteritis (GCA), a disease affecting medium-sized and large arteries, considerable infiltration of CD4+ T cells is found in the affected vessels. Several lines of evidence suggest that these T cells are not just merely attracted to a site of inflammation, but rather might recognize a specific antigen. Whether this is a primary response against a microbial or self-protein or infection-induced cross-reactivity to self-proteins is currently unknown. In order to investigate antigen involvement in GCA pathogenesis, we used an antigen-centered approach to screen for T cell reactivity against self- and candidate viral antigens. Complementary, we used a T cell receptor (TCR)-based approach in order to investigate expanded clones and enriched CDR3-motifs in the affected arteries. Finally, taking advantage of our prospective GCA cohort study at the University Hospital Basel, we tested the antibody reactivity in newly diagnosed GCA patients against a self-protein proposed by others to be important in GCA pathogenesis. These results will help us to better understand the (early) disease pathogenesis and to find possible druggable pathways