RESUMEN
The ability of zinc finger antiviral protein (ZAP) to recognize and respond to RNA virus sequences with elevated frequencies of CpG dinucleotides has been proposed as a functional part of the vertebrate innate immune antiviral response. It has been further proposed that ZAP activity shapes compositions of cytoplasmic mRNA sequences to avoid self-recognition, particularly mRNAs for interferons (IFNs) and IFN-stimulated genes (ISGs) expressed during the antiviral state. We investigated whether restriction of the replication of mutants of influenza A virus (IAV) and the echovirus 7 (E7) replicon with high CpG and UpA frequencies varied in different species of mammals and birds. Cell lines from different bird orders showed substantial variability in restriction of CpG-high mutants of IAV and E7 replicons, whereas none restricted UpA-high mutants, in marked contrast to universal restriction of both mutants in mammalian cells. Dinucleotide representation in ISGs and IFN genes was compared with those of cellular transcriptomes to determine whether potential differences in inferred ZAP activity between species shaped dinucleotide compositions of highly expressed genes during the antiviral state. While mammalian type 1 IFN genes typically showed often profound suppression of CpG and UpA frequencies, there was no oversuppression of either in ISGs in any species, irrespective of their ability to restrict CpG- or UpA-high mutants. Similarly, genome sequences of mammalian and avian RNA viruses were compositionally equivalent, as were IAV strains recovered from ducks, chickens and humans. Overall, we found no evidence for host variability in inferred ZAP function shaping host or viral transcriptome compositions.
Asunto(s)
Virus de la Influenza A , Transcriptoma , Animales , Antivirales/farmacología , Pollos/genética , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/metabolismo , Mamíferos/genética , ARN Mensajero , ARN Viral/genética , ARN Viral/metabolismo , Replicación Viral/genéticaRESUMEN
BACKGROUND: Evidence on the distribution of pre-treatment HIV-1 drug resistance (HIVDR) among risk groups is limited in Africa. We assessed the prevalence, trends and transmission dynamics of pre-treatment HIVDR within and between MSM, people who inject drugs (PWID), female sex workers (FSWs), heterosexuals (HETs) and perinatally infected children in Kenya. METHODS: HIV-1 partial pol sequences from antiretroviral-naive individuals collected from multiple sources between 1986 and 2020 were used. Pre-treatment reverse transcriptase inhibitor (RTI), PI and integrase inhibitor (INSTI) mutations were assessed using the Stanford HIVDR database. Phylogenetic methods were used to determine and date transmission clusters. RESULTS: Of 3567 sequences analysed, 550 (15.4%, 95% CI: 14.2-16.6) had at least one pre-treatment HIVDR mutation, which was most prevalent amongst children (41.3%), followed by PWID (31.0%), MSM (19.9%), FSWs (15.1%) and HETs (13.9%). Overall, pre-treatment HIVDR increased consistently, from 6.9% (before 2005) to 24.2% (2016-20). Among HETs, pre-treatment HIVDR increased from 6.6% (before 2005) to 20.2% (2011-15), but dropped to 6.5% (2016-20). Additionally, 32 clusters with shared pre-treatment HIVDR mutations were identified. The majority of clusters had R0 ≥ 1.0, indicating ongoing transmissions. The largest was a K103N cluster involving 16 MSM sequences sampled between 2010 and 2017, with an estimated time to the most recent common ancestor (tMRCA) of 2005 [95% higher posterior density (HPD), 2000-08], indicating propagation over 12 years. CONCLUSIONS: Compared to HETs, children and key populations had higher levels of pre-treatment HIVDR. Introduction of INSTIs after 2017 may have abrogated the increase in pre-treatment RTI mutations, albeit in the HET population only. Taken together, our findings underscore the need for targeted efforts towards equitable access to ART for children and key populations in Kenya.
Asunto(s)
Fármacos Anti-VIH , Infecciones por VIH , Seropositividad para VIH , VIH-1 , Trabajadores Sexuales , Abuso de Sustancias por Vía Intravenosa , Niño , Humanos , Femenino , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/epidemiología , Kenia/epidemiología , Filogenia , Abuso de Sustancias por Vía Intravenosa/complicaciones , Abuso de Sustancias por Vía Intravenosa/epidemiología , Abuso de Sustancias por Vía Intravenosa/tratamiento farmacológico , Farmacorresistencia Viral/genética , Seropositividad para VIH/tratamiento farmacológico , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Mutación , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/uso terapéuticoRESUMEN
Polyglucosan bodies are accumulations of insoluble glucose polymers and proteins that form intracytoplasmic inclusions in the brain, large numbers of which can be indicative of neurodegenerative diseases such as Lafora disease. Montserrat orioles (Icterus oberi) are an icterid passerine endemic to Montserrat with conservation populations maintained in captivity abroad. We demonstrate that polyglucosan bodies are unusually abundant in the cerebellar molecular and Purkinje cell layers and cerebellar peduncles of captive-bred and wild-caught Montserrat orioles. The bodies are periodic acid-Schiff positive and diastase resistant and label with concanavalin A and for ubiquitin, consistent with those seen in humans. We found no association of the polyglucosan bodies with concurrent neurological lesions or clinical signs, nor with EPM2A and EPM2B gene mutations associated with Lafora disease. We conclude that an abundance of cerebellar polyglucosan bodies may be a normal finding in aged Montserrat orioles and not a threat to the captive breeding population.
RESUMEN
Penguins (Sphenisciformes) are an iconic order of flightless, diving seabirds distributed across a large latitudinal range in the Southern Hemisphere. The extensive area over which penguins are endemic is likely to have fostered variation in pathogen pressure, which in turn will have imposed differential selective pressures on the penguin immune system. At the front line of pathogen detection and response, the Toll-like receptors (TLRs) provide insight into host evolution in the face of microbial challenge. TLRs respond to conserved pathogen-associated molecular patterns and are frequently found to be under positive selection, despite retaining specificity for defined agonist classes. We undertook a comparative immunogenetics analysis of TLRs for all penguin species and found evidence of adaptive evolution that was largely restricted to the cell surface-expressed TLRs, with evidence of positive selection at, or near, key agonist-binding sites in TLR1B, TLR4, and TLR5. Intriguingly, TLR15, which is activated by fungal products, appeared to have been pseudogenized multiple times in the Eudyptes spp., but a full-length form was present as a rare haplotype at the population level. However, in vitro analysis revealed that even the full-length form of Eudyptes TLR15 was nonfunctional, indicating an ancestral cryptic pseudogenization prior to its eventual disruption multiple times in the Eudyptes lineage. This unusual pseudogenization event could provide an insight into immune adaptation to fungal pathogens such as Aspergillus, which is responsible for significant mortality in wild and captive bird populations.
Asunto(s)
Spheniscidae , Animales , Evolución Molecular , Selección Genética , Spheniscidae/genética , Receptores Toll-Like/genéticaRESUMEN
Driven by co-evolution with pathogens, host immunity continuously adapts to optimize defence against pathogens within a given environment. Recent advances in genetics, genomics and transcriptomics have enabled a more detailed investigation into how immunogenetic variation shapes the diversity of immune responses seen across domestic and wild animal species. However, a deeper understanding of the diverse molecular mechanisms that shape immunity within and among species is still needed to gain insight into-and generate evolutionary hypotheses on-the ultimate drivers of immunological differences. Here, we discuss current advances in our understanding of molecular evolution underpinning jawed vertebrate immunity. First, we introduce the immunome concept, a framework for characterizing genes involved in immune defence from a comparative perspective, then we outline how immune genes of interest can be identified. Second, we focus on how different selection modes are observed acting across groups of immune genes and propose hypotheses to explain these differences. We then provide an overview of the approaches used so far to study the evolutionary heterogeneity of immune genes on macro and microevolutionary scales. Finally, we discuss some of the current evidence as to how specific pathogens affect the evolution of different groups of immune genes. This review results from the collective discussion on the current key challenges in evolutionary immunology conducted at the ESEB 2021 Online Satellite Symposium: Molecular evolution of the vertebrate immune system, from the lab to natural populations.
Asunto(s)
Inmunidad Adaptativa , Evolución Biológica , Animales , Inmunidad Adaptativa/genética , Vertebrados/genética , Evolución Molecular , Inmunidad Innata/genéticaRESUMEN
Animal experimentation has been integral to drug discovery and development and safety assessment for many years, since it provides insights into the mechanisms of drug efficacy and toxicity (e.g. pharmacology, pharmacokinetics and pharmacodynamics). However, due to species differences in physiology, metabolism and sensitivity to drugs, the animal models can often fail to replicate the effects of drugs and chemicals in human patients, workers and consumers. Researchers across the globe are increasingly applying the Three Rs principles by employing innovative methods in research and testing. The Three Rs concept focuses on: the replacement of animal models (e.g. with in vitro and in silico models or human studies), on the reduction of the number of animals required to achieve research objectives, and on the refinement of existing experimental practices (e.g. eliminating distress and enhancing animal wellbeing). For the last two years, Oncoseek Bio-Acasta Health, a 3-D cell culture-based cutting-edge translational biotechnology company, has organised an annual International Conference on 3Rs Research and Progress. This series of global conferences aims to bring together researchers with diverse expertise and interests, and provides a platform where they can share and discuss their research to promote practices according to the Three Rs principles. In November 2022, the 3rd international conference, Advances in Animal Models and Cutting-Edge Research in Alternatives, took place at the GITAM University in Vishakhapatnam (AP, India) in a hybrid format (i.e. online and in-person). These conference proceedings provide details of the presentations, which were categorised under five different topic sessions. It also describes a special interactive session on in silico strategies for preclinical research in oncology, which was held at the end of the first day.
Asunto(s)
Experimentación Animal , Animales , Humanos , Modelos Animales , Descubrimiento de Drogas , India , Alternativas a las Pruebas en AnimalesRESUMEN
As the size and complexity of high-dimensional (HD) cytometry data continue to expand, comprehensive, scalable, and methodical computational analysis approaches are essential. Yet, contemporary clustering and dimensionality reduction tools alone are insufficient to analyze or reproduce analyses across large numbers of samples, batches, or experiments. Moreover, approaches that allow for the integration of data across batches or experiments are not well incorporated into computational toolkits to allow for streamlined workflows. Here we present Spectre, an R package that enables comprehensive end-to-end integration and analysis of HD cytometry data from different batches or experiments. Spectre streamlines the analytical stages of raw data pre-processing, batch alignment, data integration, clustering, dimensionality reduction, visualization, and population labelling, as well as quantitative and statistical analysis. Critically, the fundamental data structures used within Spectre, along with the implementation of machine learning classifiers, allow for the scalable analysis of very large HD datasets, generated by flow cytometry, mass cytometry, or spectral cytometry. Using open and flexible data structures, Spectre can also be used to analyze data generated by single-cell RNA sequencing or HD imaging technologies, such as Imaging Mass Cytometry. The simple, clear, and modular design of analysis workflows allow these tools to be used by bioinformaticians and laboratory scientists alike. Spectre is available as an R package or Docker container. R code is available on Github (https://github.com/immunedynamics/spectre).
Asunto(s)
Algoritmos , Análisis de la Célula Individual , Análisis por Conglomerados , Citometría de Flujo/métodos , Programas InformáticosRESUMEN
Climate shifts are key drivers of ecosystem change. Despite the critical importance of Antarctica and the Southern Ocean for global climate, the extent of climate-driven ecological change in this region remains controversial. In particular, the biological effects of changing sea ice conditions are poorly understood. We hypothesize that rapid postglacial reductions in sea ice drove biological shifts across multiple widespread Southern Ocean species. We test for demographic shifts driven by climate events over recent millennia by analyzing population genomic datasets spanning 3 penguin genera (Eudyptes, Pygoscelis, and Aptenodytes). Demographic analyses for multiple species (macaroni/royal, eastern rockhopper, Adélie, gentoo, king, and emperor) currently inhabiting southern coastlines affected by heavy sea ice conditions during the Last Glacial Maximum (LGM) yielded genetic signatures of near-simultaneous population expansions associated with postglacial warming. Populations of the ice-adapted emperor penguin are inferred to have expanded slightly earlier than those of species requiring ice-free terrain. These concerted high-latitude expansion events contrast with relatively stable or declining demographic histories inferred for 4 penguin species (northern rockhopper, western rockhopper, Fiordland crested, and Snares crested) that apparently persisted throughout the LGM in ice-free habitats. Limited genetic structure detected in all ice-affected species across the vast Southern Ocean may reflect both rapid postglacial colonization of subantarctic and Antarctic shores, in addition to recent genetic exchange among populations. Together, these analyses highlight dramatic, ecosystem-wide responses to past Southern Ocean climate change and suggest potential for further shifts as warming continues.
RESUMEN
BACKGROUND: Despite increasing interest in γδ T cells and their non-classical behaviour, most studies focus on animals with low numbers of circulating γδ T cells, such as mice and humans. Arguably, γδ T cell functions might be more prominent in chickens where these cells form a higher proportion of the circulatory T cell compartment. The TCR repertoire defines different subsets of γδ T cells, and such analysis is facilitated by well-annotated TCR loci. γδ T cells are considered at the cusp of innate and adaptive immunity but most functions have been identified in γδ low species. A deeper understanding of TCR repertoire biology in γδ high and γδ low animals is critical for defining the evolution of the function of γδ T cells. Repertoire dynamics will reveal populations that can be classified as innate-like or adaptive-like as well as those that straddle this definition. RESULTS: Here, a recent discrepancy in the structure of the chicken TCR gamma locus is resolved, demonstrating that tandem duplication events have shaped the evolution of this locus. Importantly, repertoire sequencing revealed large differences in the usage of individual TRGV genes, a pattern conserved across multiple tissues, including thymus, spleen and the gut. A single TRGV gene, TRGV3.3, with a highly diverse private CDR3 repertoire dominated every tissue in all birds. TRGV usage patterns were partly explained by the TRGV-associated recombination signal sequences. Public CDR3 clonotypes represented varying proportions of the repertoire of TCRs utilising different TRGVs, with one TRGV dominated by super-public clones present in all birds. CONCLUSIONS: The application of repertoire analysis enabled functional annotation of the TCRG locus in a species with a high circulating γδ phenotype. This revealed variable usage of TCRGV genes across multiple tissues, a pattern quite different to that found in γδ low species (human and mouse). Defining the repertoire biology of avian γδ T cells will be key to understanding the evolution and functional diversity of these enigmatic lymphocytes in an animal that is numerically more reliant on them. Practically, this will reveal novel ways in which these cells can be exploited to improve health in medical and veterinary contexts.
Asunto(s)
Pollos , Genoma , Receptores de Antígenos de Linfocitos T gamma-delta , Animales , Pollos/genética , Genómica , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Linfocitos TRESUMEN
Over evolutionary time, pathogen challenge shapes the immune phenotype of the host to better respond to an incipient threat. The extent and direction of this selection pressure depend on the local pathogen composition, which is in turn determined by biotic and abiotic features of the environment. However, little is known about adaptation to local pathogen threats in wild animals. The Gentoo penguin (Pygoscelis papua) is a species complex that lends itself to the study of immune adaptation because of its circumpolar distribution over a large latitudinal range, with little or no admixture between different clades. In this study, we examine the diversity in a key family of innate immune genes-the Toll-like receptors (TLRs)-across the range of the Gentoo penguin. The three TLRs that we investigated present varying levels of diversity, with TLR4 and TLR5 greatly exceeding the diversity of TLR7. We present evidence of positive selection in TLR4 and TLR5, which points to pathogen-driven adaptation to the local pathogen milieu. Finally, we demonstrate that two positively selected cosegregating sites in TLR5 are sufficient to alter the responsiveness of the receptor to its bacterial ligand, flagellin. Taken together, these results suggest that Gentoo penguins have experienced distinct pathogen-driven selection pressures in different environments, which may be important given the role of the Gentoo penguin as a sentinel species in some of the world's most rapidly changing environments.
Asunto(s)
Selección Genética , Spheniscidae/genética , Receptores Toll-Like/genética , Animales , Flagelina/inmunología , Variación Genética , Filogeografía , Spheniscidae/inmunologíaRESUMEN
The COVID-19 pandemic has brought biosafety to the forefront of many life sciences. The outbreak has compelled research institutions to re-evaluate biosafety practices and potential at-risk areas within research laboratories and more specifically within Shared Resource Laboratories (SRLs). In flow cytometry facilities, biological safety assessment encompasses known hazards based on the biological sample and associated risk group, as well as potential or unknown hazards, such as aerosol generation and instrument "failure modes." Cell sorting procedures undergo clearly defined biological safety assessments and adhere to well-established biosafety guidelines that help to protect SRL staff and users against aerosol exposure. Conversely, benchtop analyzers are considered low risk due to their low sample pressure and enclosed fluidic systems, although there is little empirical evidence to support this assumption of low risk. To investigate this, we evaluated several regions on analyzers using the Cyclex-d microsphere assay, a recently established method for cell sorter aerosol containment testing. We found that aerosol and/or droplet hazards were detected on all benchtop analyzers predominantly during operation in "failure modes." These results indicate that benchtop analytical cytometers present a more complicated set of risks than are commonly appreciated.
Asunto(s)
COVID-19/prevención & control , Separación Celular/instrumentación , Contención de Riesgos Biológicos , Contaminación de Equipos/prevención & control , Citometría de Flujo/instrumentación , Personal de Laboratorio , Exposición Profesional/efectos adversos , Salud Laboral , Aerosoles , COVID-19/transmisión , Humanos , Medición de Riesgo , Factores de RiesgoRESUMEN
Biosafety has always been an important aspect of daily work in any research institution, particularly for cytometry Shared Resources Laboratories (SRLs). SRLs are common-use spaces that facilitate the sharing of knowledge, expertise, and ideas. This sharing inescapably involves contact and interaction of all those within this working environment on a daily basis. The current pandemic caused by SARS-CoV-2 has prompted the re-evaluation of many policies governing the operations of SRLs. Here we identify and review the unique challenges SRLs face in maintaining biosafety standards, highlighting the potential risks associated with not only cytometry instrumentation and samples, but also the people working with them. We propose possible solutions to safety issues raised by the COVID-19 pandemic and provide tools for facilities to adapt to evolving guidelines and future challenges.
Asunto(s)
COVID-19/epidemiología , Contención de Riesgos Biológicos/tendencias , Laboratorios/tendencias , COVID-19/prevención & control , COVID-19/transmisión , Contención de Riesgos Biológicos/normas , Citometría de Flujo , Humanos , Laboratorios/normas , Medición de Riesgo/normas , Medición de Riesgo/tendenciasRESUMEN
PURPOSE: To investigate the sensitivity of visual read (VR) to detect early amyloid pathology and the overall utility of regional VR. METHODS: [18F]Flutemetamol PET images of 497 subjects (ALFA+ N = 352; ADC N = 145) were included. Scans were visually assessed according to product guidelines, recording the number of positive regions (0-5) and a final negative/positive classification. Scans were quantified using the standard and regional Centiloid (CL) method. The agreement between VR-based classification and published CL-based cut-offs for early (CL = 12) and established (CL = 30) pathology was determined. An optimal CL cut-off maximizing Youden's index was derived. Global and regional CL quantification was compared to VR. Finally, 28 post-mortem cases from the [18F]flutemetamol phase III trial were included to assess the percentage agreement between VR and neuropathological classification of neuritic plaque density. RESULTS: VR showed excellent agreement against CL = 12 (κ = .89, 95.2%) and CL = 30 (κ = .88, 95.4%) cut-offs. ROC analysis resulted in an optimal CL = 17 cut-off against VR (sensitivity = 97.9%, specificity = 97.8%). Each additional positive VR region corresponded to a clear increase in global CL. Regional VR was also associated with regional CL quantification. Compared to mCERADSOT-based classification (i.e., any region mCERADSOT > 1.5), VR was in agreement in 89.3% of cases, with 13 true negatives, 12 true positives, and 3 false positives (FP). Regional sparse-to-moderate neuritic and substantial diffuse Aß plaque was observed in all FP cases. Regional VR was also associated with regional plaque density. CONCLUSION: VR is an appropriate method for assessing early amyloid pathology and that grading the extent of visual amyloid positivity could present clinical value.
Asunto(s)
Enfermedad de Alzheimer , Enfermedad de Alzheimer/diagnóstico por imagen , Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Compuestos de Anilina , Benzotiazoles , Encéfalo/metabolismo , Humanos , Tomografía de Emisión de PositronesRESUMEN
INTRODUCTION: Programmes that merge management of Human Immunodeficiency Virus (HIV) and tuberculosis (TB) aim to improve HIV/TB co-infected patients' access to comprehensive treatment. However, several reports from sub-Saharan Africa (SSA) indicate suboptimal uptake of antiretroviral therapy (ART) even after integration of HIV and TB treatment. This study assessed ART uptake, its barriers and enablers in programmes integrating TB and HIV treatment in SSA. METHOD: A systematic review was performed. Seven databases were searched for eligible quantitative, qualitative and mixed-methods studies published from March 2004 through July 2019. Random-effects meta-analysis was used to obtain pooled estimates of ART uptake. A thematic approach was used to analyse and synthesise data on barriers and enablers. RESULTS: Of 5139 references identified, 27 were included in the review: 23/27 estimated ART uptake and 10/27 assessed barriers to and/or enablers of ART uptake. The pooled ART uptake was 53% (95% CI: 42, 63%) and between-study heterogeneity was high (I2 = 99.71%, p < 0.001). WHO guideline on collaborative TB/HIV activities and sample size were associated with heterogeneity. There were statistically significant subgroup effects with high heterogeneity after subgroup analyses by region, guideline on collaborative TB/HIV activities, study design, and sample size. The most frequently described socioeconomic and individual level barriers to ART uptake were stigma, low income, and younger age group. The most frequently reported health system-related barriers were limited staff capacity, shortages in medical supplies, lack of infrastructure, and poor adherence to or lack of treatment guidelines. Clinical barriers included intolerance to anti-TB drugs, fear of drug toxicity, and contraindications to antiretrovirals. Health system enablers included good management of the procurement, supply, and dispensation chain; convenience and accessibility of treatment services; and strong staff capacity. Availability of psychosocial support was the most frequently reported enabler of uptake at the community level. CONCLUSIONS: In SSA, programmes integrating treatment of TB and HIV do not, in general, achieve high ART uptake but we observe a net improvement in uptake after WHO issued the 2012 guidelines on collaborative TB/HIV activities. The recurrence of specific modifiable system-level and patient-level factors in the literature reveals key intervention points to improve ART uptake in these programmes. Systematic review registration: CRD42019131933.
Asunto(s)
Infecciones por VIH , Tuberculosis , Antirretrovirales/uso terapéutico , Antituberculosos/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/epidemiología , Humanos , Estigma Social , Tuberculosis/tratamiento farmacológicoRESUMEN
Microbiomes associated with human skin and the oral cavity are uniquely exposed to personal care regimes. Changes in the composition and activities of the microbial communities in these environments can be utilized to promote consumer health benefits, for example, by reducing the numbers, composition, or activities of microbes implicated in conditions such as acne, axillary odor, dandruff, and oral diseases. It is, however, important to ensure that innovative approaches for microbiome manipulation do not unsafely disrupt the microbiome or compromise health, and where major changes in the composition or activities of the microbiome may occur, these require evaluation to ensure that critical biological functions are unaffected. This article is based on a 2-day workshop held at SEAC Unilever, Sharnbrook, United Kingdom, involving 31 specialists in microbial risk assessment, skin and oral microbiome research, microbial ecology, bioinformatics, mathematical modeling, and immunology. The first day focused on understanding the potential implications of skin and oral microbiome perturbation, while approaches to characterize those perturbations were discussed during the second day. This article discusses the factors that the panel recommends be considered for personal care products that target the microbiomes of the skin and the oral cavity.
Asunto(s)
Seguridad de Productos para el Consumidor , Cosméticos/normas , Microbiota/fisiología , Boca/microbiología , Piel/microbiología , Educación , HumanosRESUMEN
In conventional fluorescence cytometry, each fluorophore present in a panel is measured in a target detector, through the use of wide band-pass optical filters. In contrast, spectral cytometry uses a large number of detectors with narrow band-pass filters to measure a fluorophore's signal across the spectrum, creating a more detailed fluorescent signature for each fluorophore. The spectral approach shows promise in adding flexibility to panel design and improving the measurement of fluorescent signal. However, few comparisons between conventional and spectral systems have been reported to date. We therefore sought to compare a modern conventional cytometry system with a modern spectral system, and to assess the quality of resulting datasets from the point of view of a flow cytometry user. Signal intensity, spread, and resolution were compared between the systems. Subsequently, the different methods of separating fluorophore signals were compared, where compensation mathematically separates multiple overlapping fluorophores and unmixing relies on creating a detailed fluorescent signature across the spectrum to separate the fluorophores. Within the spectral data set, signal spread and resolution were comparable between compensation and unmixing. However, for some highly overlapping fluorophores, unmixing resolved the two fluorescence signals where compensation did not. Finally, data from mid- to large-size panels were acquired and were found to have comparable resolution for many fluorophores on both instruments, but reduced levels of spreading error on our spectral system improved signal resolution for a number of fluorophores, compared with our conventional system. Furthermore, autofluorescence extraction on the spectral system allowed for greater population resolution in highly autofluorescent samples. Overall, the implementation of a spectral cytometry approach resulted in data that are comparable to that generated on conventional systems, with a number of potential advantages afforded by the larger number of detectors, and the integration of the spectral unmixing approach. © 2020 International Society for Advancement of Cytometry.
Asunto(s)
Colorantes Fluorescentes , Virosis , Citometría de Flujo , HumanosRESUMEN
Throughout history, humans have been afflicted by parasitic worms, and eggs are readily detected in archaeological deposits. This study integrated parasitological and ancient DNA methods with a large sample set dating between Neolithic and Early Modern periods to explore the utility of molecular archaeoparasitology as a new approach to study the past. Molecular analyses provided unequivocal species-level parasite identification and revealed location-specific epidemiological signatures. Faecal-oral transmitted nematodes (Ascaris lumbricoides and Trichuris trichiura) were ubiquitous across time and space. By contrast, high numbers of food-associated cestodes (Diphyllobothrium latum and Taenia saginata) were restricted to medieval Lübeck. The presence of these cestodes and changes in their prevalence at approximately 1300 CE indicate substantial alterations in diet or parasite availability. Trichuris trichiura ITS-1 sequences grouped into two clades; one ubiquitous and one restricted to medieval Lübeck and Bristol. The high sequence diversity of T.tITS-1 detected in Lübeck is consistent with its importance as a Hanseatic trading centre. Collectively, these results introduce molecular archaeoparasitology as an artefact-independent source of historical evidence.