Prevalent fast evolution of genes involved in heterochromatin functions.

Heterochromatin is a gene-poor and repeat-rich genomic compartment ubiquitously found in eukaryotes. Despite its low transcriptional activity, heterochromatin plays important roles in maintaining genome stability, organizing chromosomes, and suppressing transposable elements (TEs). Given the importance of these functions, it is expected that the genes involved in heterochromatin regulation would be highly conserved. Yet, a handful of these genes have been found to evolve rapidly. To investigate whether these previous findings are anecdotal or general to genes modulating heterochromatin, we compiled an exhaustive list of 106 candidate genes involved in heterochromatin functions and investigated their evolution over both short and long evolutionary time scales in Drosophila. Our analyses found that these genes exhibit significantly more frequent evolutionary changes, both in the forms of amino acid substitutions and gene copy number variation, when compared to genes involved in Polycomb-based repressive chromatin. While positive selection drives amino acid changes within both structured domains with diverse functions and irregular disordered regions (IDRs), purifying selection may have maintained the proportions of IDRs. Together with the observed negative associations between rates of protein evolution of these genes and genomic TE abundance, we propose an evolutionary model where the fast evolution of genes involved in heterochromatin functions is an inevitable outcome of the unique molecular features of the heterochromatin environment, while the rapid evolution of TEs may be an effect rather than cause. Our study provides an important global view of the evolution of genes involved in this critical cellular domain and provides insights into the factors driving the distinctive evolution of heterochromatin.

In vitro culture of parasitic stages of Haemonchus contortus.

Haemonchus contortus is a constraint to sheep production. Seeking to reduce the use of hosts and produce parasitic stages in large-scale, a 42-day in vitro culture protocol of H. contortus third-stage larvae was optimized using Dulbecco's modified Eagle's medium (DMEM). In cell-free culture, larvae were maintained at 39.6°C, in acidic media (pH 6.1) for 3 or 6 days with Δ4-dafachronic acid followed by DMEM pH 7.4 supplemented or not with Fildes' reagent. In DMEM pH 7.4 at 37°C, supplementation with Caco-2 cells was compared to Fildes. On Day 14, fourth-stage larvae (L4) development rates in acidic media supplemented (86.8-88.4%) or not (74.4-77.8%) with Fildes and in Caco-2 cell co-culture (92.6%) were similar, and superior to DMEM pH 7.4 with Fildes (0.0%). On Day 21, Caco-2 cell co-culture resulted in higher larvae differentiation (25.0%) and lower degeneration (13.9%) compared to acidic media (1.5-8.1% and 48.6-69.9%, respectively). This is the first report of prolonged in vitro culture of H. contortus larvae using commercial media in co-culture with Caco-2 cells. Although no progression to the adult stage, Caco-2 cell co-culture resulted in morphological differentiation of H. contortus L4 and larval viability for up to 28 days.

Moxidectin use in Scottish sheep flocks suggests a need for clearer product labelling and communication of updated SCOPS guidelines.

BACKGROUND: Guidelines for sustainable use of moxidectin were established in 2020. This study aimed to identify how Scottish sheep farmers are using this key endectocide and estimate its effectiveness against gastrointestinal nematodes. METHODS: Questionnaires were distributed to sheep farmers across Scotland, and analysis focused on moxidectin use in relation to Sustainable Control of Parasites in Sheep (SCOPS) guidelines. Farmers using moxidectin in their flock volunteered to submit post-treatment sheep faecal samples, which were analysed for the presence of gastrointestinal nematodes using faecal egg counts with polymerase chain reaction to determine species. RESULTS: Despite 70% of farmers using moxidectin in 2020, knowledge levels varied: 24% of farmers included other anthelmintics when asked about moxidectin use. Moxidectin was used for a wide variety of reasons, and most farmers did not consistently follow SCOPS guidelines. Despite only 2 of 76 farmers reporting failure of moxidectin treatment, gastrointestinal nematodes were found following moxidectin treatment on five out of six farms tested and included Teladorsagia circumcincta, Cooperia curticei, Haemonchus contortus and Nematodirus sp. CONCLUSION: Findings from this project indicate the need for improved anthelmintic product labelling and farmer support to encourage sustainable use. The presence of nematodes in treated animals is suggestive of anthelmintic resistance.

Genomic landscape of drug response reveals mediators of anthelmintic resistance.

Like other pathogens, parasitic helminths can rapidly evolve resistance to drug treatment. Understanding the genetic basis of anthelmintic drug resistance in parasitic nematodes is key to tracking its spread and improving the efficacy and sustainability of parasite control. Here, we use an in vivo genetic cross between drug-susceptible and multi-drug-resistant strains of Haemonchus contortus in a natural host-parasite system to simultaneously map resistance loci for the three major classes of anthelmintics. This approach identifies new alleles for resistance to benzimidazoles and levamisole and implicates the transcription factor cky-1 in ivermectin resistance. This gene is within a locus under selection in ivermectin-resistant populations worldwide; expression analyses and functional validation using knockdown experiments support that cky-1 is associated with ivermectin survival. Our work demonstrates the feasibility of high-resolution forward genetics in a parasitic nematode and identifies variants for the development of molecular diagnostics to combat drug resistance in the field.

Transcriptomic analyses implicate neuronal plasticity and chloride homeostasis in ivermectin resistance and response to treatment in a parasitic nematode.

The antiparasitic drug ivermectin plays an essential role in human and animal health globally. However, ivermectin resistance is widespread in veterinary helminths and there are growing concerns of sub-optimal responses to treatment in related helminths of humans. Despite decades of research, the genetic mechanisms underlying ivermectin resistance are poorly understood in parasitic helminths. This reflects significant uncertainty regarding the mode of action of ivermectin in parasitic helminths, and the genetic complexity of these organisms; parasitic helminths have large, rapidly evolving genomes and differences in evolutionary history and genetic background can confound comparisons between resistant and susceptible populations. We undertook a controlled genetic cross of a multi-drug resistant and a susceptible reference isolate of Haemonchus contortus, an economically important gastrointestinal nematode of sheep, and ivermectin-selected the F2 population for comparison with an untreated F2 control. RNA-seq analyses of male and female adults of all populations identified high transcriptomic differentiation between parental isolates, which was significantly reduced in the F2, allowing differences associated specifically with ivermectin resistance to be identified. In all resistant populations, there was constitutive upregulation of a single gene, HCON_00155390:cky-1, a putative pharyngeal-expressed transcription factor, in a narrow locus on chromosome V previously shown to be under ivermectin selection. In addition, we detected sex-specific differences in gene expression between resistant and susceptible populations, including constitutive upregulation of a P-glycoprotein, HCON_00162780:pgp-11, in resistant males only. After ivermectin selection, we identified differential expression of genes with roles in neuronal function and chloride homeostasis, which is consistent with an adaptive response to ivermectin-induced hyperpolarisation of neuromuscular cells. Overall, we show the utility of a genetic cross to identify differences in gene expression that are specific to ivermectin selection and provide a framework to better understand ivermectin resistance and response to treatment in parasitic helminths.

Label-free screening of biochemical changes in macrophage-like cells following MoS2 exposure using Raman micro-spectroscopy.

The emergence of large scale production techniques for 2D particulate materials has dramatically increased their applications potential. Understanding the interactions of biological cells with such particulate material is therefore of paramount importance, both for toxicological assessment and potential biomedical applications. Conventional in-vitro cytological assays commonly record only a single colorimetric end-point, and do not provide an in-depth analysis of how such materials are uptaken and processed within cells. To demonstrate its potential as an alternative, label free approach, confocal Raman micro-spectroscopy has been used to profile the cellular response of macrophage-like immune cells as a result of exposure to a sub-lethal dose of particulate MoS2, as an example novel 2D material. Particles were seen to be uptaken and trafficked in sub-cellular vesicles, and this sensitive technique allows differences in the biochemical composition of the vesicles to be assessed and monitored as a function of time. Untreated macrophage-like cells contain lipidic vesicles which are found to be relatively rich in the membrane lipid sphingomyelin, key to the process of cell membrane regeneration. After exposure to MoS2, the particulate material is seen to be invaginated in similar vesicles, the most prominent of which now, however, have spectroscopic signatures which are dominated by those of phosphatidyl family lipids, consistent with the phagocytotic pathway. The lipidic content of cells is seen to increase at all time-points (4, 24 and 72 h). although vesicles composed of sphingomyelin become more prominent again following a prolonged incubation of 72 h to a sub-lethal dose of MoS2, as the immune cell has processed the particulate material and initiates recovery to a normal/untreated state. This study reveals Raman micro-spectroscopy is an effective method for monitoring cellular responses and evolution of organelle compositions in response to MoS2 exposure. The additional benefit of using this technique is that cells can be monitored as a function of time, while it can also be used for screening other micro/nano materials for toxicology and/or establishing cell responses.

Increasing importance of anthelmintic resistance in European livestock: creation and meta-analysis of an open database.

Helminth infections are ubiquitous in grazing ruminant production systems, and are responsible for significant costs and production losses. Anthelmintic Resistance (AR) in parasites is now widespread throughout Europe, although there are still gaps in our knowledge in some regions and countries. AR is a major threat to the sustainability of modern ruminant livestock production, resulting in reduced productivity, compromised animal health and welfare, and increased greenhouse gas emissions through increased parasitism and farm inputs. A better understanding of the extent of AR in Europe is needed to develop and advocate more sustainable parasite control approaches. A database of European published and unpublished AR research on gastrointestinal nematodes (GIN) and liver fluke (Fasciola hepatica) was collated by members of the European COST Action "COMBAR" (Combatting Anthelmintic Resistance in Ruminants), and combined with data from a previous systematic review of AR in GIN. A total of 197 publications on AR in GIN were available for analysis, representing 535 studies in 22 countries and spanning the period 1980-2020. Reports of AR were present throughout the European continent and some reports indicated high within-country prevalence. Heuristic sample size-weighted estimates of European AR prevalence over the whole study period, stratified by anthelmintic class, varied between 0 and 48%. Estimated regional (country) prevalence was highly heterogeneous, ranging between 0% and 100% depending on livestock sector and anthelmintic class, and generally increased with increasing research effort in a country. In the few countries with adequate longitudinal data, there was a tendency towards increasing AR over time for all anthelmintic classes in GIN: aggregated results in sheep and goats since 2010 reveal an average prevalence of resistance to benzimidazoles (BZ) of 86%, macrocyclic lactones except moxidectin (ML) 52%, levamisole (LEV) 48%, and moxidectin (MOX) 21%. All major GIN genera survived treatment in various studies. In cattle, prevalence of AR varied between anthelmintic classes from 0-100% (BZ and ML), 0-17% (LEV) and 0-73% (MOX), and both Cooperia and Ostertagia survived treatment. Suspected AR in F. hepatica was reported in 21 studies spanning 6 countries. For GIN and particularly F. hepatica, there was a bias towards preferential sampling of individual farms with suspected AR, and research effort was biased towards Western Europe and particularly the United Kingdom. Ongoing capture of future results in the live database, efforts to avoid bias in farm recruitment, more accurate tests for AR, and stronger appreciation of the importance of AR among the agricultural industry and policy makers, will support more sophisticated analyses of factors contributing to AR and effective strategies to slow its spread.

TITLE: Importance croissante de la résistance aux anthelminthiques chez les ruminants européens : création et méta-analyse d'une base de données ouverte. ABSTRACT: Les helminthes sont omniprésents dans les systèmes de production de ruminants au pâturage et sont responsables de coûts et de pertes de production importants. La résistance aux anthelminthiques (RA) des parasites est maintenant répandue dans toute l'Europe, bien qu'il existe encore des lacunes dans nos connaissances dans certaines régions et certains pays. La RA est une menace majeure pour la pérennité de la production moderne de ruminants, en diminuant la productivité, en compromettant la santé et le bien-être des animaux, et en augmentant les émissions de gaz à effet de serre au travers d'une augmentation du parasitisme et des intrants agricoles. Une meilleure compréhension de l'étendue de la RA en Europe est nécessaire pour développer et préconiser des approches de lutte antiparasitaire plus durables. Une base de données intégrant des informations publiées et non publiées en Europe concernant la RA des nématodes gastro-intestinaux (NGI) et de la douve du foie (Fasciola hepatica) a été compilée par les membres de l'action européenne COST « COMBAR ¼ (« Combattre la résistance aux anthelminthiques chez les ruminants ¼) et combinée avec les données d'une précédente étude systématique concernant la RA des NGI. Au total, 197 publications sur la RA des NGI étaient disponibles pour analyse, représentant 535 études dans 22 pays et couvrant la période 1980­2020. Des signalements de RA étaient présents sur tout le continent européen et certains rapports indiquaient une forte prévalence nationale. Les estimations heuristiques pondérées par la taille de l'échantillon de la prévalence de la RA en Europe sur toute la période d'étude, stratifiées par classe d'anthelminthiques, variaient de 0 à 48 %. La prévalence régionale (nationale) estimée était très hétérogène, variant entre 0 % et 100 % selon le secteur de l'élevage et la classe d'anthelminthique, et augmentait généralement avec les efforts de recherche dans le pays. Dans les quelques pays disposant de données longitudinales adéquates, il y avait une tendance à l'augmentation de la RA au fil du temps pour toutes les classes d'anthelminthiques des NGI : les résultats agrégés chez les ovins et caprins depuis 2010 révèlent une prévalence moyenne de résistance aux benzimidazoles (BZ) de 86 %, aux lactones macrocycliques sauf moxidectine (ML) de 52 %, au lévamisole (LEV) de 48 % et à la moxidectine (MOX) de 21 %. Tous les genres principaux de NGI ont survécu au traitement dans diverses études. Chez les bovins, la prévalence de la RA variait selon les classes d'anthelminthiques de 0 à 100 % (BZ et ML), 0 à 17 % (LEV) et 0 à 73 % (MOX), et Cooperia et Ostertagia ont survécu aux traitements. Une RA suspectée chez F. hepatica a été signalée dans 21 études portant sur 6 pays. Pour les NGI, et encore plus pour F. hepatica, il y avait un biais d'échantillonnage en faveur des exploitations individuelles suspectées de RA, et l'effort de recherche était biaisé vers l'Europe occidentale et en particulier le Royaume-Uni. La capture continue des résultats futurs dans la base de données, en direct, les efforts pour éviter les biais dans le recrutement des exploitations, des tests plus précis pour la RA et une meilleure appréciation de l'importance de la RA parmi l'industrie agricole et les décideurs politiques, soutiendront des analyses plus sophistiquées des facteurs contribuant à la RA, et des stratégies efficaces pour ralentir sa propagation.

In vitro Label Free Raman Microspectroscopic Analysis to Monitor the Uptake, Fate and Impacts of Nanoparticle Based Materials.

The continued emergence of nanoscale materials for nanoparticle-based therapy, sensing and imaging, as well as their more general adoption in a broad range of industrial applications, has placed increasing demands on the ability to assess their interactions and impacts at a cellular and subcellular level, both in terms of potentially beneficial and detrimental effects. Notably, however, many such materials have been shown to interfere with conventional in vitro cellular assays that record only a single colorimetric end-point, challenging the ability to rapidly screen cytological responses. As an alternative, Raman microspectroscopy can spatially profile the biochemical content of cells, and any changes to it as a result of exogenous agents, such as toxicants or therapeutic agents, in a label free manner. In the confocal mode, analysis can be performed at a subcellular level. The technique has been employed to confirm the cellular uptake and subcellular localization of polystyrene nanoparticles (PSNPs), graphene and molybdenum disulfide micro/nano plates (MoS2), based on their respective characteristic spectroscopic signatures. In the case of PSNPs it was further employed to identify their local subcellular environment in endosomes, lysosomes and endoplasmic reticulum, while for MoS2 particles, it was employed to monitor subcellular degradation as a function of time. For amine functionalized PSNPs, the potential of Raman microspectroscopy to quantitatively characterize the dose and time dependent toxic responses has been explored, in a number of cell lines. Comparing the responses to those of poly (amidoamine) nanoscale polymeric dendrimers, differentiation of apoptotic and necrotic pathways based on the cellular spectroscopic responses was demonstrated. Drawing in particular from the experience of the authors, this paper details the progress to date in the development of applications of Raman microspectroscopy for in vitro, label free analysis of the uptake, fate and impacts of nanoparticle based materials, in vitro, and the prospects for the development of a routine, label free high content spectroscopic analysis technique.

Potential of Raman spectroscopy for the analysis of plasma/serum in the liquid state: recent advances.

There is compelling evidence in the literature to support the application of Raman spectroscopy for analysis of bodily fluids in their native liquid state. Naturally, the strategies described in the literature for Raman spectroscopic analysis of liquid samples have advantages and disadvantages. Herein, recent advances in the analysis of plasma/serum in the liquid state are reviewed. The potential advantages of Raman analysis in the liquid form over the commonly employed infrared absorption analysis in the dried droplet form are initially highlighted. Improvements in measurement protocols based on inverted microscopic geometries, clinically adaptable substrates, data preprocessing and analysis and applications for routine monitoring of patient health as well as therapeutic administration are reviewed. These advances suggest that clinical translation of Raman spectroscopy for rapid biochemical analysis can be a reality. In the future, this method will prove to be highly beneficial to clinicians for rapid screening and monitoring of analytes and drugs in the biological fluids, and to the patients themselves, enabling early treatment, before the disease becomes symptomatic, allowing early recovery.

The dynamics of ovine gastrointestinal nematode infections within ewe and lamb cohorts on three Scottish sheep farms.

Gastrointestinal nematodes (GIN) are a serious concern for sheep producers worldwide. However, there is a paucity of evidence describing the epidemiology of GIN on modern UK sheep farms. The aim of this paper was to understand whether expected seasonal variations of infection are still found in ewes and lambs under varying management strategies in temperate climates. Faecal egg counts (FEC) were conducted on freshly voided samples collected from groups of ewes and lambs every third week for twelve months on three farms in southeast Scotland. The patterns of egg output have been described here in relation to management practices undertaken on the farms. Despite changes in farming practice and climatic conditions, the findings complement historical studies detailing the epidemiology of GIN. Findings include a periparturient rise in ewe FEC on two of the farms, while lambing time treatment appeared to suppress this on the third farm. On the same two farms lamb FEC increased during the summer, reaching a peak in the autumn. The work also highlights how the ad hoc use of anthelmintics does little to impact these patterns.

Raman spectroscopy as a potential tool for label free therapeutic drug monitoring in human serum: the case of busulfan and methotrexate.

A methodology is proposed, based on Raman spectroscopy coupled with multivariate analysis, to determine the Limit of Detection (LOD) and Limit of Quantification (LOQ) for therapeutic drug monitoring in human serum, using the examples of Busulfan, a cell cycle non-specific alkylating antineoplastic agent, and, Methotrexate, a chemotherapeutic agent and immune system suppressant. In this study, ultrafiltration is employed to fractionate spiked human pooled serum to efficiently recover the drug in the filtrate prior to performing Raman analysis. The drug concentration ranges were chosen to encompass the recommended therapeutic ranges and toxic levels in patients. Raman spectra were collected from the filtrates in the liquid form, using an inverted backscattering microscopic geometry, using 532 nm as source. Finally, prediction models were built by using Partial Least Squares Regression (PLSR) and LOD and LOQ were calculated directly from the linear prediction models. The LOD calculated for Busulfan is 0.0002 ± 0.0001 mg mL-1, 30-40 times lower than the level of toxicity, enabling the application of this method in target dose adjustment of Busulfan for patients undergoing, for example, bone marrow transplantation. The LOD and LOQ calculated for Methotrexate are 7.8 ± 5 µM and 26 ± 5 µM, respectively, potentially enabling high dose monitoring. The promising results obtained from this study suggest the potential of Raman spectroscopy for therapeutic drug monitoring of drugs in bodily fluids.

Raman spectroscopic screening of high and low molecular weight fractions of human serum.

This study explores the suitability of Raman spectroscopy as a bioanalytical tool, when coupled with ultra-filtration and multivariate analysis, to detect imbalances in both high molecular weight (total protein content, γ globulins and albumin) and low molecular weight (urea and glucose) fractions of the same samples of human patient serum, in the native liquid form. Ultracentrifugation was employed to separate and concentrate the high and low molecular weight fractions of the serum. Initially, aqueous solutions of the respective molecular species, covering physiologically relevant concentration ranges, were analysed to optimise the measurement protocols. An adapted Extended Multiplicative Signal Correction (EMSC) algorithm was applied to raw spectra to remove water background signal and spectral interferents (ß-carotene). Using a validated partial least squares regression modelling method, R2 values, Root Mean Square Error of Cross Validation (RMSECV) and standard deviations were established for the quantification of γ globulin, total protein, albumin, urea and glucose content of the patient serum samples. The study demonstrates that Raman spectroscopy in the liquid form is a viable alternative and/or adjunct to current clinical practice for the parallel analysis of high and low molecular weight fractions, and simultaneous analysis of multiple analytes in the low molecular weight fraction, of human serum for diagnostic applications.

Refugia and anthelmintic resistance: Concepts and challenges.

Anthelmintic resistance is a threat to global food security. In order to alleviate the selection pressure for resistance and maintain drug efficacy, management strategies increasingly aim to preserve a proportion of the parasite population in 'refugia', unexposed to treatment. While persuasive in its logic, and widely advocated as best practice, evidence for the ability of refugia-based approaches to slow the development of drug resistance in parasitic helminths is currently limited. Moreover, the conditions needed for refugia to work, or how transferable those are between parasite-host systems, are not known. This review, born of an international workshop, seeks to deconstruct the concept of refugia and examine its assumptions and applicability in different situations. We conclude that factors potentially important to refugia, such as the fitness cost of drug resistance, the degree of mixing between parasite sub-populations selected through treatment or not, and the impact of parasite life-history, genetics and environment on the population dynamics of resistance, vary widely between systems. The success of attempts to generate refugia to limit anthelmintic drug resistance are therefore likely to be highly dependent on the system in hand. Additional research is needed on the concept of refugia and the underlying principles for its application across systems, as well as empirical studies within systems that prove and optimise its usefulness.

Analysis of bodily fluids using vibrational spectroscopy: a direct comparison of Raman scattering and infrared absorption techniques for the case of glucose in blood serum.

Analysis of biomarkers present in the blood stream can potentially deliver crucial information on patient health and indicate the presence of numerous pathologies. The potential of vibrational spectroscopic analysis of human serum for diagnostic purposes has been widely investigated and, in recent times, infrared absorption spectroscopy, coupled with ultra-filtration and multivariate analysis techniques, has attracted increasing attention, both clinical and commercial. However, such methods commonly employ a drying step, which may hinder the clinical work flow and thus hamper their clinical deployment. As an alternative, this study explores the use of Raman spectroscopy, similarly coupled with ultra-filtration and multivariate analysis techniques, to quantitatively monitor diagnostically relevant changes of glucose in liquid serum samples, and compares the results with similar analysis protocols using infrared spectroscopy of dried samples. The analysis protocols to detect the imbalances in glucose using Raman spectroscopy are first demonstrated for aqueous solutions and spiked serum samples. As in the case of infrared absorption studies, centrifugal filtration is utilised to deplete abundant analytes and to reveal the spectral features of Low Molecular Weight Fraction analytes in order to improve spectral sensitivity and detection limits. Improved Root Mean Square Error of Cross Validation (RMSECV) was observed for Raman prediction models, whereas slightly higher R2 values were reported for infrared absorption prediction models. Summarising, it is demonstrated that the Raman analysis protocol can yield accuracies which are comparable with those reported using infrared absorption based measurements of dried serum, without the need for additional drying steps.

Graphene Nanoflake Uptake Mediated by Scavenger Receptors.

The biological interactions of graphene have been extensively investigated over the last 10 years. However, very little is known about graphene interactions with the cell surface and how the graphene internalization process is driven and mediated by specific recognition sites at the interface with the cell. In this work, we propose a methodology to investigate direct molecular correlations between the biomolecular corona of graphene and specific cell receptors, showing that key protein recognition motifs, presented on the nanomaterial surface, can engage selectively with specific cell receptors. We consider the case of apolipoprotein A-I, found to be very abundant in the graphene protein corona, and observe that the uptake of graphene nanoflakes is somewhat increased in cells with greatly elevated expression of scavenger receptors B1, suggesting a possible mechanism of endogenous interaction. The uptake results, obtained by flow cytometry, have been confirmed using Raman microspectroscopic mapping, exploiting the strong Raman signature of graphene.

Raman spectroscopic analysis of high molecular weight proteins in solution - considerations for sample analysis and data pre-processing.

This study explores the potential of Raman spectroscopy, coupled with multivariate regression techniques and a protein separation technique (ion exchange chromatography), to quantitatively monitor diagnostically relevant changes in high molecular weight proteins in liquid plasma. Measurement protocols to detect the imbalances in plasma proteins as an indicator of various diseases using Raman spectroscopy are optimised, such that strategic clinical applications for early stage disease diagnostics can be evaluated. In a simulated plasma protein mixture, concentrations of two proteins of identified diagnostic potential (albumin and fibrinogen) were systematically varied within physiologically relevant ranges. Scattering from the poorly soluble fibrinogen fraction is identified as a significant impediment to the accuracy of measurement of mixed proteins in solution, although careful consideration of pre-processing methods allows construction of an accurate multivariate regression prediction model for detecting subtle changes in the protein concentration. Furthermore, ion exchange chromatography is utilised to separate fibrinogen from the rest of the proteins and mild sonication is used to improve the dispersion and therefore quality of the prediction. The proposed approach can be expeditiously employed for early detection of pathological disorders associated with high or low plasma/serum proteins.

Advancing Raman microspectroscopy for cellular and subcellular analysis: towards in vitro high-content spectralomic analysis.

In the confocal mode, Raman microspectroscopy can profile the biochemical content of biological cells at a subcellular level, and any changes to it by exogenous agents, such as therapeutic drugs or toxicants. As an exploration of the potential of the technique as a high-content, label-free analysis technique, this report reviews work to monitor the spectroscopic signatures associated with the uptake and response pathways of commercial chemotherapeutic agents and polymeric nanoparticles by human lung cells. It is demonstrated that the signatures are reproducible and characteristic of the cellular event, and can be used, for example, to identify the mode of action of the agent as well as the subsequent cell death pathway, and even mechanisms of cellular resistance. Data mining approaches are discussed and a spectralomics approach is proposed.

Hidden in plain sight - Multiple resistant species within a strongyle community.

Ovine parasitic gastroenteritis is a complex disease routinely treated using anthelmintics. Although many different strongyle species may contribute to parasitic gastroenteritis, not all are equally pathogenic: in temperate regions, the primary pathogen is Teladorsagia circumcincta. In this study we investigated benzimidazole and ivermectin resistance on a commercial sheep farm in southeast Scotland. We assessed the impact of species diversity on the diagnosis of resistance using the faecal egg count reduction test and in vitro bioassays, and correlated the results with the frequency of benzimidazole resistance-associated genotypes measured in the T. circumcincta population by pyrosequencing of the ß-tubulin isotype-1 gene. Faecal egg count reduction test results showed efficacies of 65% for albendazole and 77% for ivermectin, indicating moderate resistance levels on the farm. However, PCR speciation of the same populations pre- and post-treatment revealed that removal of susceptible species had masked the presence of a highly resistant population of T. circumcincta. Less than 25% of individuals in the pre-treatment populations were T. circumcincta, the remainder consisting of Cooperia curticei, Chabertia ovina, Oesophagostomum venulosum and Trichostrongylus spp. In contrast, post-treatment with albendazole or ivermectin, the majority (88% and 100% respectively) of the populations consisted of T. circumcincta. The egg hatch test for benzimidazole resistance and the larval development test for ivermectin resistance were carried out using eggs obtained from the same populations and the results were broadly consistent with the faecal egg count reduction test. Thirty individual T. circumcincta from each sampling time point were assessed for benzimidazole resistance by pyrosequencing, revealing a high frequency and diversity of resistance-associated mutations, including within the population sampled post-ivermectin treatment. These results highlight the potential diversity of parasite species present on UK farms, and their importance in the diagnosis of anthelmintic resistance. On this particular farm, we demonstrate the presence of a highly dual-resistant population of T. circumcincta, which was strongly selected by treatment with either benzimidazoles or ivermectin, while other potentially less pathogenic species were removed.

Label-free discrimination analysis of de-differentiated vascular smooth muscle cells, mesenchymal stem cells and their vascular and osteogenic progeny using vibrational spectroscopy.

The accumulation of vascular smooth muscle (SMC)-like cells and stem cell-derived myogenic and osteogenic progeny contributes significantly to arteriosclerotic disease. This study established whether label-free vibrational spectroscopy can discriminate de-differentiated 'synthetic' SMCs from undifferentiated stem cells and their myogenic and osteogenic progeny in vitro, compared with conventional immunocytochemical and genetic analyses. TGF-ß1- and Jagged1-induced myogenic differentiation of CD44+ mesenchymal stem cells was confirmed in vitro by immunocytochemical analysis of specific SMC differentiation marker expression (α-actin, calponin and myosin heavy chain 11), an epigenetic histone mark (H3K4me2) at the myosin heavy chain 11 locus, promoter transactivation and mRNA transcript levels. Osteogenic differentiation was confirmed by alizarin red staining of calcium deposition. Fourier Transform Infrared (FTIR) maps facilitated initial screening and discrimination while Raman spectroscopy of individual cell nuclei revealed specific spectral signatures of each cell type in vitro, using Principal Components Analysis (PCA). PCA fed Linear Discriminant Analysis (LDA) enabled quantification of this discrimination and the sensitivity and specificity value was determined for all cell populations based on a leave-one-out cross validation method and revealed that de-differentiated SMCs and stem-cell derived myogenic progeny in culture shared the greatest similarity. FTIR and Raman spectroscopy discriminated undifferentiated stem cells from both their myogenic and osteogenic progeny. The ability to detect stem cell-derived myogenic progeny using label-free platforms in situ may facilitate interrogation of these important phenotypes during vascular disease progression.