22,265 research outputs found

    Multiple functions and regulatory network of miR-150 in B lymphocyte-related diseases

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    MicroRNAs (miRNAs) play vital roles in the post-transcriptional regulation of gene expression. Previous studies have shown that miR-150 is a crucial regulator of B cell proliferation, differentiation, metabolism, and apoptosis. miR-150 regulates the immune homeostasis during the development of obesity and is aberrantly expressed in multiple B-cell-related malignant tumors. Additionally, the altered expression of MIR-150 is a diagnostic biomarker of various autoimmune diseases. Furthermore, exosome-derived miR-150 is considered as prognostic tool in B cell lymphoma, autoimmune diseases and immune-mediated disorders, suggesting miR-150 plays a vital role in disease onset and progression. In this review, we summarized the miR-150-dependent regulation of B cell function in B cell-related immune diseases

    OLIG2 neural progenitor cell development and fate in Down syndrome

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    Down syndrome (DS) is caused by triplication of human chromosome 21 (HSA21) and is the most common genetic form of intellectual disability. It is unknown precisely how triplication of HSA21 results in the intellectual disability, but it is thought that the global transcriptional dysregulation caused by trisomy 21 perturbs multiple aspects of neurodevelopment that cumulatively contribute to its etiology. While the characteristics associated with DS can arise from any of the genes triplicated on HSA21, in this work we focus on oligodendrocyte transcription factor 2 (OLIG2). The progeny of neural progenitor cells (NPCs) expressing OLIG2 are likely to be involved in many of the cellular changes underlying the intellectual disability in DS. To explore the fate of OLIG2+ neural progenitors, we took advantage of two distinct models of DS, the Ts65Dn mouse model and induced pluripotent stem cells (iPSCs) derived from individuals with DS. Our results from these two systems identified multiple perturbations in development in the cellular progeny of OLIG2+ NPCs. In Ts65Dn, we identified alterations in neurons and glia derived from the OLIG2 expressing progenitor domain in the ventral spinal cord. There were significant differences in the number of motor neurons and interneurons present in the trisomic lumbar spinal cord depending on age of the animal pointing both to a neurodevelopment and a neurodegeneration phenotype in the Ts65Dn mice. Of particular note, we identified changes in oligodendrocyte (OL) maturation in the trisomic mice that are dependent on spatial location and developmental origin. In the dorsal corticospinal tract, there were significantly fewer mature OLs in the trisomic mice, and in the lateral funiculus we observed the opposite phenotype with more mature OLs being present in the trisomic animals. We then transitioned our studies into iPSCs where we were able to pattern OLIG2+ NPCs to either a spinal cord-like or a brain-like identity and study the OL lineage that differentiated from each progenitor pool. Similar to the region-specific dysregulation found in the Ts65Dn spinal cord, we identified perturbations in trisomic OLs that were dependent on whether the NPCs had been patterned to a brain-like or spinal cord-like fate. In the spinal cord-like NPCs, there was no difference in the proportion of cells expressing either OLIG2 or NKX2.2, the two transcription factors whose co-expression is essential for OL differentiation. Conversely, in the brain-like NPCs, there was a significant increase in OLIG2+ cells in the trisomic culture and a decrease in NKX2.2 mRNA expression. We identified a sonic hedgehog (SHH) signaling based mechanism underlying these changes in OLIG2 and NKX2.2 expression in the brain-like NPCs and normalized the proportion of trisomic cells expressing the transcription factors to euploid levels by modulating the activity of the SHH pathway. Finally, we continued the differentiation of the brain-like and spinal cord-like NPCs to committed OL precursor cells (OPCs) and allowed them to mature. We identified an increase in OPC production in the spinal cord-like trisomic culture which was not present in the brain-like OPCs. Conversely, we identified a maturation deficit in the brain-like trisomic OLs that was not present in the spinal cord-like OPCs. These results underscore the importance of regional patterning in characterizing changes in cell differentiation and fate in DS. Together, the findings presented in this work contribute to the understanding of the cellular and molecular etiology of the intellectual disability in DS and in particular the contribution of cells differentiated from OLIG2+ progenitors

    Deciphering Regulation in Escherichia coli: From Genes to Genomes

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    Advances in DNA sequencing have revolutionized our ability to read genomes. However, even in the most well-studied of organisms, the bacterium Escherichia coli, for ≈ 65% of promoters we remain ignorant of their regulation. Until we crack this regulatory Rosetta Stone, efforts to read and write genomes will remain haphazard. We introduce a new method, Reg-Seq, that links massively-parallel reporter assays with mass spectrometry to produce a base pair resolution dissection of more than 100 E. coli promoters in 12 growth conditions. We demonstrate that the method recapitulates known regulatory information. Then, we examine regulatory architectures for more than 80 promoters which previously had no known regulatory information. In many cases, we also identify which transcription factors mediate their regulation. This method clears a path for highly multiplexed investigations of the regulatory genome of model organisms, with the potential of moving to an array of microbes of ecological and medical relevance.</p

    RNA pull-down-confocal nanoscanning (RP-CONA), a novel method for studying RNA/protein interactions in cell extracts that detected potential drugs for Parkinson’s disease targeting RNA/HuR complexes

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    MicroRNAs (miRNAs, miRs) are a class of small non-coding RNAs that regulate gene expression through specific base-pair targeting. The functional mature miRNAs usually undergo a two-step cleavage from primary miRNAs (pri-miRs), then precursor miRNAs (pre-miRs). The biogenesis of miRNAs is tightly controlled by different RNA-binding proteins (RBPs). The dysregulation of miRNAs is closely related to a plethora of diseases. Targeting miRNA biogenesis is becoming a promising therapeutic strategy. HuR and MSI2 are both RBPs. MiR-7 is post-transcriptionally inhibited by the HuR/MSI2 complex, through a direct interaction between HuR and the conserved terminal loop (CTL) of pri-miR-7-1. Small molecules dissociating pri-miR-7/HuR interaction may induce miR-7 production. Importantly, the miR-7 levels are negatively correlated with Parkinson’s disease (PD). PD is a common, incurable neurodegenerative disease causing serious motor deficits. A hallmark of PD is the presence of Lewy bodies in the human brain, which are inclusion bodies mainly composed of an aberrantly aggregated protein named α-synuclein (α-syn). Decreasing α-syn levels or preventing α-syn aggregation are under investigation as PD treatments. Notably, α-syn is negatively regulated by several miRNAs, including miR-7, miR-153, miR-133b and others. One hypothesis is that elevating these miRNA levels can inhibit α-syn expression and ameliorate PD pathologies. In this project, we identified miR-7 as the most effective α-syn inhibitor, among the miRNAs that are downregulated in PD, and with α-syn targeting potentials. We also observed potential post-transcriptional inhibition on miR-153 biogenesis in neuroblastoma, which may help to uncover novel therapeutic targets towards PD. To identify miR-7 inducers that benefit PD treatment by repressing α-syn expression, we developed a novel technique RNA Pull-down Confocal Nanoscaning (RP-CONA) to monitor the binding events between pri-miR-7 and HuR. By attaching FITC-pri-miR-7-1-CTL-biotin to streptavidin-coated agarose beads and incubating them in human cultured cell lysates containing overexpressed mCherry-HuR, the bound RNA and protein can be visualised as quantifiable fluorescent rings in corresponding channels in a confocal high-content image system. A pri-miR-7/HuR inhibitor can decrease the relative mCherry/FITC intensity ratio in RP-CONA. With this technique, we performed several small-scale screenings and identified that a bioflavonoid, quercetin can largely dissociate the pri-miR-7/HuR interaction. Further studies proved that quercetin was an effective miR-7 inducer as well as α-syn inhibitor in HeLa cells. To understand the mechanism of quercetin mediated α-syn inhibition, we tested the effects of quercetin treatment with miR-7-1 and HuR knockout HeLa cells. We found that HuR was essential in this pathway, while miR-7 hardly contributed to the α-syn inhibition. HuR can directly bind an AU-rich element (ARE) at the 3’ untranslated region (3’-UTR) of α-syn mRNA and promote translation. We believe quercetin mainly disrupts the ARE/HuR interaction and disables the HuR-induced α-syn expression. In conclusion, we developed and optimised RP-CONA, an on-bead, lysate-based technique detecting RNA/protein interactions, as well as identifying RNA/protein modulators. With RP-CONA, we found quercetin inducing miR-7 biogenesis, and inhibiting α-syn expression. With these beneficial effects, quercetin has great potential to be applied in the clinic of PD treatment. Finally, RP-CONA can be used in many other RNA/protein interactions studies

    Innate immunity and metabolism in the bovine ovarian follicle

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    Postpartum uterine disease in dairy cows is associated with reduced fertility. One of the first and most prevalent bacteria associated with uterine disease is Escherichia coli. The bacterial endotoxin, lipopolysaccharide (LPS), accumulates in the ovarian follicular fluid of animals with uterine disease. The granulosa cells of the ovarian follicle respond to LPS by secreting pro-inflammatory cytokines, such as interleukin (IL)-1a, IL-1b and IL-8, and oocyte health is perturbed. Dairy cows also experience metabolic energy stress in the postpartum period, which is associated with an increased risk of developing uterine disease and ovarian dysfunction. This thesis explored the crosstalk between innate immunity and metabolic energy stress in bovine granulosa cells and cumulus-oocyte complex. Firstly, we found that glycolysis, AMP-activated protein kinase and the mechanistic target of rapamycin, regulate the innate immune responses to LPS in granulosa cells isolated from bovine ovarian follicles. Activation of AMP-activated protein kinase decreased the LPS-induced secretion of IL-1a, IL-1b, and IL8, and was associated with shortened duration of ERK1/2 and JNK phosphorylation. Next, we found that decreasing the availability of cholesterol or inhibiting cholesterol biosynthesis using short-interfering RNA impaired the LPS-induced secretion of IL-1a and IL-1b by granulosa cells. Furthermore, metabolic energy stress or inhibiting cholesterol biosynthesis in the bovine cumulus-oocyte complex modulated the innate immune responses to LPS, and perturbed meiotic progression during in vitro maturation. Finally, we explored an in vivo model of uterine disease in heifers, using RNAseq to investigate alterations to the transcriptome of the reproductive tract. We found that uterine disease altered the transcriptome of the endometrium, oviduct, granulosa cells and oocyte, several months after bacterial infusion; these changes were most evident in the granulosa cells and oocyte of the ovarian follicle. The findings from this thesis imply that there is crosstalk between innate immunity and metabolism in the bovine ovarian follicle

    Desenvolvimento de testes genéticos por PCR em tempo-real para diagnóstico rápido de LHON e surdez

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    Mitochondrial cytopathies are a set of diseases caused by a disturbance in the cell energy production. Mitochondrial dysfunction impairs efficiency of the mitochondrial respiratory chain (MRC) and ATP production, affecting the organism’s energetic equilibrium. Pathogenic sequence variants in mitochondrial DNA (mtDNA) that lead to these pathologies are more frequent in tissues that need higher energy levels to function. The presented work looks into two such diseases: Leber’s Hereditary Optic Neuropathy and mitochondrial non-syndromic Hearing Loss (MNSHL). LHON is characterized by presence of genetic alterations in mtDNA, with three main primary pathogenic sequence variants existing, which represent 90-95% of LHON cases with an identified genetic cause: m.3460G>A, in ND1 subunit gene; m.11778G>A, in ND4 subunit gene; and m.14484T>C, in ND6 subunit gene. All of these are subunits of the MRC’s complex I. These mtDNA variations lead to mitochondrial dysfunction in complex I, creating ATP depletion, reactive oxygen species (ROS) increase and oxidative stress. LHON is commonly characterized by a sequential vision loss and, within 1 year of symptoms starting, 97% of patients with vision loss in one eye develop loss in the second. Therapy administration yields good outcomes, if done in a short-time span after first vision loss. It is essential to quickly and reliably scan for pathogenic sequence variants, in order to act timely and rescue function. Mitochondrial non-syndromic hearing loss and deafness (MNSHL) is characterized by sensorineural hearing loss (SNHL). This type of hearing loss, particularly when induced by aminoglycosides, has also three primary pathogenic sequence variants associated with ototoxicity: m.1494C>T and m.1555A>G, both in the MTRNR1 gene, and m.7445A>G, in the MTCO1 and MTTS1 genes. These are responsible for ATP depletion, an increase of ROS and oxidative stress, due to alterations in the mitochondrial ribosome or tRNA. In MNSHL, the cochlea is the affected tissue. With this disorder the principal modifier factor is the administration of aminoglycosides, a type of antibiotics, which trigger a cascade, that leads the individual permanently deaf. The best course of action is prevention, and to ensure clinical action is not dramatically slowed down, results that show whether administration is safe or not need to be quick. The aim of this work, for both diseases, is the development of a screening method characterized by fast and reliable approach for genetic assessment, to be used for clinical guidance, particularly in therapeutics. For LHON, the screening method is based on real-time PCR with High-Resolution Melting (HRM) analysis, for detection of the TOP-3 pathogenic sequence variants, by assessing the amplicon’s Tm. In this case, 94 samples were analyzed, including LHON suspected patients, relatives, other mitochondrial disease patients and healthy controls. All samples were previously classified by another method, having then been blinded before the performance of this work. For analysis, Real-Time PCR was run in triplicates, to allow for a more robust HRM analysis. The software had the ability to classify samples as different variants, wild-type or mutant; information which was then crossed with the previous classification of the sample to assess the success of the software classification. Samples were correctly assigned. This approach provides results in a quick fashion that guides clinical action in a timely fashion. The presence of other polymorphisms in the amplicons might be a hindrance to the robustness of the results provided by this technique and their effect on variant classification needs to be considered. For this, a predictive in-silico analysis was performed, regarding all described variants’ presence in the sequences in analysis. Accordingly, an additional complementary method may be necessary for assurance of result’s specificity. For MNSHL, the screening method was also real-time PCR based, but this one was performed with Amplification-Refractory Mutation System (ARMS) primers, designed for the pathogenic sequence variants previously associated in literature for the MNSHL. Discrimination of results was done based on amplification in positive cases and lack of it in negative cases. This approach analyzed 32 samples, including MNSHL suspected patients, their relatives, other mitochondrial disease patients and healthy controls, but only results concerning the m.1555A>G were obtained timely. All samples were previously classified by another method, having then been blinded before performance of this work. For optimization, Real-Time PCR was run in duplicates, to increase robustness of analysis. The Real-Time software showed if samples amplified as wild-type or mutant, with classification following. This data was crossed with previous known classification of the samples to assess the success of the approach. All analyzed samples were correctly identified with this approach. However, two of the three pathogenic sequence variants did not achieve implementation within the timeframe necessary for their inclusion, namely m.1494C>T and m.7445A>G. The optimization of their screening was not possible and further work is necessary to optimize and implement the approach concerning the analysis for these variants. In conclusion, it was possible to implement an analysis method for LHON’s TOP-3 pathogenic sequence variants within 24h, which represents a big step in precision medicine for diagnosis of this disease. On the other hand, although the implementation was not concluded, a similar approach was started for MNSHL – that, when concluded, will have an enormous impact in preventing aminoglycoside induced HL. This work represents a high impact scientific contribution in reverse translational research.As citopatias mitocondriais são um conjunto de doenças causadas por um distúrbio na produção de energia celular. A disfunção mitocondrial prejudica a eficiência da cadeia respiratória mitocondrial (CRM) e a produção de ATP, afetando o equilíbrio energético do organismo. As variações de sequência patogénicas no DNA mitocondrial (mtDNA) que levam a estas patologias são mais frequentes em tecidos que necessitam de maiores níveis de energia para funcionar. O presente trabalho explora duas dessas doenças: Neuropatia ótica hereditária de Leber (LHON) e Surdez mitocondrial induzida por aminoglicosídeos. A LHON é caracterizada pela presença de alterações genéticas do mtDNA, existindo três variações de sequência patogénicas primárias principais, que representam 90-95% de casos de LHON com identificação da causa genética: m.3460G>A, no gene que codifica a subunidade ND1; m.11778G>A, no gene que codifica a subunidade ND4; e m.14484T>C, no gene que codifica a subunidade ND6. Todas estas subunidades pertencem ao complexo I da CRM. Estas alterações no mtDNA levam a disfunção mitocondrial no complexo I, criando depleção de ATP, aumento de espécies reativas de oxigénio (ROS) e stresse oxidativo. A LHON é comummente caracterizada pela perda sequencial de visão e, 1 ano após o início dos sintomas, 97% dos casos com perda de visão num olho desenvolvem perda de visão no segundo. A administração de terapia produz bons resultados, quando realizada num curto período de tempo após a primeira perda de visão. Assim, é essencial pesquisar variações de sequência patogénicas genéticas de forma rápida e fiável, para atuar rapidamente e recuperar a função visual. A Surdez mitocondrial não-sindrómica (MNSHL), em particular a induzida por aminoglicosídeos, tem também três mutações principais associadas à perda de audição: m.1494C>T e m.1555A>G, ambas no gene MTRNR1, e m.7445A>G, nos genes MTCO1 e MTTS1. Estas são responsáveis pela depleção de ATP, aumento de ROS e stresse oxidativo, devido a alterações no ribossoma ou no tRNA mitocondrial. Aqui, o tecido afetado é a cóclea. Nesta doença, o fator modificador em destaque é a administração de antibióticos de tipo aminoglicosídeos, que despoletam uma cascata de acontecimentos, levando à surdez permanente. A melhor estratégia passa pela prevenção, enquanto ao mesmo tempo se garante que a ação clínica não sofre atrasos. Desta forma, são necessários resultados rápidos, que demonstrem se a administração será segura ou não. O objetivo deste trabalho, para ambas as doenças, é o desenvolvimento de um método de screening, caracterizado por uma abordagem rápida e fiável, usado para guiar a decisão clínica, particularmente na terapêutica. Para a LHON, o método de screening é baseado em PCR em tempo-real com análise de High-Resolution Melting (HRM), para deteção das variantes patogénicas TOP-3, avaliando as Tm dos amplicons. Neste caso, foram analisadas 94 amostras, incluindo doentes com suspeita de LHON, familiares, outros doentes com suspeita de outra doença mitocondrial e controlos saudáveis. Todas as amostras foram previamente classificadas por outro método, tendo sido sujeitas a anonimização antes da realização do trabalho. Para a análise, a PCR em tempo-real foi realizada em triplicados, para permitir uma análise de HRM mais robusta. O software teve a capacidade de classificar amostras como diferentes variantes, ou seja, normal ou mutante. Esta informação foi cruzada com as classificações previamente existentes para avaliar o sucesso da classificação pelo software. As amostras foram corretamente classificadas. Esta abordagem fornece resultados de forma rápida, podendo guiar a ação clínica em tempo útil. A presença de outros polimorfismos nos amplicons poderão obstruir a robustez dos resultados fornecidos por esta técnica e o seu efeito na classificação de variantes precisa de ser considerado. Por esta razão, foi realizada uma análise de previsão in-silico, considerando a presença de todas as variantes descritas. Nesse sentido, pode ser necessário um método complementar de análise para assegurar a especificidade dos resultados. Para a Surdez mitocondrial não-sindrómica, o método de screening baseou-se também na PCR em tempo-real, mas foi realizada com primers de Amplification-Refractory mutation system (ARMS), desenhados para as variantes de sequência patogénicas associadas à MNSHL induzida por aminoglicosídeos, previamente descritas na literatura para esta doença. A discriminação de resultados foi feita com base na presença/ausência de amplificação para cada variante. Foram analisadas 32 amostras com esta abordagem, incluindo doentes com suspeita de MNSHL, seus familiares, doentes com suspeita de outra doença mitocondrial e controlos saudáveis, mas apenas foram obtidos resultados em tempo útil para a m.1555A>G. Todas as amostras tinham sido previamente classificadas por outro método, tendo sido anonimizadas antes da realização do trabalho. Para a otimização, a PCR em tempo-real foi realizada em duplicados, aumentando a robustez da análise. O software de tempo-real mostrou quais as amostras que amplificaram como normais ou mutantes, permitindo a classificação das mesmas. Os dados foram comparados com as classificações previamente conhecidas, para avaliar o sucesso da abordagem em estudo. Todas as amostras em análise foram corretamente identificadas. No entanto, duas das três variantes patogénicas não foram implementadas em tempo útil para inclusão neste trabalho. Para a m.1494C>T e a m.7445A>G, a otimização não foi possível, e será necessário trabalho adicional no futuro, para a implementação da análise destas variantes. Em conclusão, foi possível implementar um método da análise das variantes genéticas TOP-3 da LHON em 24h, o que representa um grande passo na medicina de precisão para diagnóstico desta doença. Por outro lado, apesar de não ter sido concluída a implementação, iniciou-se uma abordagem semelhante para a MNSHL – que, quando for concluída, terá um enorme impacto para evitar a perda auditiva por exposição a aminoglicosídeos. Este trabalho representa uma contribuição científica de alto impacto na investigação translacional reversa.O Laboratório de Biomedicina Mitocondrial e Teranóstica recebeu apoio financeiro da Santhera Pharmaceuticals que permitiu implementação do projeto nacional “Investigação Translacional Epidemiológica, Bigenómica e Funcional nas Atrofias Ópticas” (IP Professora Doutora Manuela Grazina). Apoio financeiro do CNC.IBILI no âmbito do Plano Estratégico UID/NEU/04539/2019.Mestrado em Biologia Aplicad

    The development of ALICE-tRNA-sequencing and its use in exploring the role of tRNAs in translational control

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    Sustaining proliferative signalling and loss of translational control is arguably the most fundamental trait of cancer cells, enabling tumour growth and metastatic dissemination. Transfer RNAs (tRNAs) have long been considered abundant “housekeeping” RNAs, functioning to decipher the universal genetic code. However, exhaustive analyses have implicated tRNA participation in a host of regulatory networks including the cellular stress response and protein synthesis. Recent findings suggest that the expression of tRNAs for synonymous codon usage is dependent on the differentiation/proliferation status of the cell and are coordinated with changes in translation. Although the molecular mechanisms that govern these changes are yet to be elucidated, cellular tRNA composition potentially introduces an additional layer of translational control. tRNAs are the most post-transcriptionally modified RNA species, with well over 50 unique modifications identified in eukaryotes. Consequently, isoacceptor identification and the measuring of the tRNA pool using next generation sequencing has long been an area of interest, with many attempts being made in literature. Using the Escherichia coli dealkylating enzyme AlkB and the novel tRNA high throughput sequencing methodology ALICE-tRNA-seq, we have developed a methodology that can accurately measure relative tRNA pools in vitro and in vivo. We show how other published tRNA sequencing protocols show bias towards tRNA sub populations, with our method showing a more realistic distribution across all tRNAs. We also show relative distribution changes in cellular and genetically modified mouse models of cancer, opening up a high resolution approach to establish the role of tRNAs in translational control and cell fate decisions

    Investigating the mechanism of human beta defensin-2-mediated protection of skin barrier in vitro

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    The human skin barrier is a biological imperative. Chronic inflammatory skin diseases, such as Atopic Dermatitis (AD), are characterised by a reduction in skin barrier function and an increased number of secondary infections. Staphyloccocus aureus (S. aureus) has an increased presence on AD lesional skin and contributes significantly to AD pathology. It was previously demonstrated that the damage induced by a virulence factor of S. aureus, V8 protease, which causes further breakdown in skin barrier function, can be reduced by induction of human β- defensin (HBD)2 (by IL-1β) or exogenous HBD2 application. Induction of this defensin is impaired in AD skin. This thesis examines the mechanism of HBD2-mediated barrier protection in vitro; demonstrating that in this system, HBD2 was not providing protection through direct protease inhibition, nor was it altering keratinocyte proliferation or migration, or exhibiting specific localisation within the monolayer. Proteomics data demonstrated that HBD2 did not induce expression of known antiproteases but suggested that HBD2 stimulation may function by modulating expression of extracellular matrix proteins, specifically collagen- IVα2 and Laminin-β-1. Alternative pathways of protection initiated by IL-1β and TNFα stimulation were also investigated, as well as their influence over generalised wound healing. Finally, novel 3D human skin epidermal models were used to better recapitulate the structure of human epidermis and examine alterations to skin barrier function in a more physiological system. These data validate the barrier-protective properties of HBD2 and extended our knowledge of the consequences of exposure to this peptide in this context

    The mechanisms of antibody generation in the llama

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    The llama is able to generate a unique class of antibody. The heavy chain immunoglobulins consist only of two heavy chain polypeptides and bind antigen specifically through single protein domains. Although the mechanisms by which such an antibody interacts with antigen has been studied at some length the manner in which the heavy chain antibody is generated within the llama is unknown. In this study a number of components of the llama immune system have been characterised. The isolation of genes encoding the variable domain of the heavy chain antibody indicates that specific genetic elements within the llama genome are responsible for the generation of the heavy chain antibody. The discovery of constant region genes that encode the heavy chain antibody provides an explanation for the absence of a major immunoglobulin domain from the final, secreted gene product. The lack of this domain within the expressed antibody is believed to be the result of a single nucleotide splice site mutation. In order to investigate the process of llama antibody generation further additional components of the llama immune system, the recombination activating genes (rag) were isolated. One such llama rag gene (rag-i) was cloned, expressed and utilised in an in vitro assay system to investigate recombination events taking place during antibody generation. This assay involved the use of specific signal sequences derived from variable domain gene sequence data and represents, to our knowledge, the first examination of non-murine RAG activity. Through the use of this system distinct differences between llama and mouse recombination signal sequences (RSSs) were uncovered. These differences, located within a specific region of the RSS known as the coding flank, may play an important role in llama antibody generation. These results have led to the proposal of a number of models for the mechanisms involved in llama antibody generation
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