Small-cell lung cancer (SCLC), an aggressive neuroendocrine tumor with early dissemination and dismal prognosis, accounts for 15-20% of lung cancer cases and ∼200,000 deaths each year. Most cases are ...inoperable, and biopsies to investigate SCLC biology are rarely obtainable. Circulating tumor cells (CTCs), which are prevalent in SCLC, present a readily accessible 'liquid biopsy'. Here we show that CTCs from patients with either chemosensitive or chemorefractory SCLC are tumorigenic in immune-compromised mice, and the resultant CTC-derived explants (CDXs) mirror the donor patient's response to platinum and etoposide chemotherapy. Genomic analysis of isolated CTCs revealed considerable similarity to the corresponding CDX. Most marked differences were observed between CDXs from patients with different clinical outcomes. These data demonstrate that CTC molecular analysis via serial blood sampling could facilitate delivery of personalized medicine for SCLC. CDXs are readily passaged, and these unique mouse models provide tractable systems for therapy testing and understanding drug resistance mechanisms.
African bovine trypanosomiasis caused by Trypanosoma sp., is a major constraint on cattle productivity in sub-Saharan Africa. Some African Bos taurus breeds are highly tolerant of infection, but the ...potentially more productive Bos indicus zebu breeds are much more susceptible. Zebu cattle are well adapted for plowing and haulage, and increasing their tolerance of trypanosomiasis could have a major impact on crop cultivation as well as dairy and beef production. We used three strategies to obtain short lists of candidate genes within QTL that were previously shown to regulate response to infection. We analyzed the transcriptomes of trypanotolerant N'Dama and susceptible Boran cattle after infection with Trypanosoma congolense. We sequenced EST libraries from these two breeds to identify polymorphisms that might underlie previously identified quantitative trait loci (QTL), and we assessed QTL regions and candidate loci for evidence of selective sweeps. The scan of the EST sequences identified a previously undescribed polymorphism in ARHGAP15 in the Bta2 trypanotolerance QTL. The polymorphism affects gene function in vitro and could contribute to the observed differences in expression of the MAPK pathway in vivo. The expression data showed that TLR and MAPK pathways responded to infection, and the former contained TICAM1, which is within a QTL on Bta7. Genetic analyses showed that selective sweeps had occurred at TICAM1 and ARHGAP15 loci in African taurine cattle, making them strong candidates for the genes underlying the QTL. Candidate QTL genes were identified in other QTL by their expression profile and the pathways in which they participate.
Diagnostic use of gene panel next-generation sequencing (NGS) techniques is commonplace for individuals with inherited retinal dystrophies (IRDs), a highly genetically heterogeneous group of ...disorders. However, these techniques have often failed to capture the complete spectrum of genomic variation causing IRD, including CNVs. This study assessed the applicability of introducing CNV surveillance into first-tier diagnostic gene panel NGS services for IRD.
Three read-depth algorithms were applied to gene panel NGS data sets for 550 referred individuals, and informatics strategies used for quality assurance and CNV filtering. CNV events were confirmed and reported to referring clinicians through an accredited diagnostic laboratory.
We confirmed the presence of 33 deletions and 11 duplications, determining these findings to contribute to the confirmed or provisional molecular diagnosis of IRD for 25 individuals. We show that at least 7% of individuals referred for diagnostic testing for IRD have a CNV within genes relevant to their clinical diagnosis, and determined a positive predictive value of 79% for the employed CNV filtering techniques.
Incorporation of CNV analysis increases diagnostic yield of gene panel NGS diagnostic tests for IRD, increases clarity in diagnostic reporting and expands the spectrum of known disease-causing mutations.
Although profiling of RNA in single cells has broadened our understanding of development, cancer biology and mechanisms of disease dissemination, it requires the development of reliable and flexible ...methods. Here we demonstrate that the EpiStem RNA-Amp™ methodology reproducibly generates microgram amounts of cDNA suitable for RNA-Seq, RT-qPCR arrays and Microarray analysis.
Initial experiments compared amplified cDNA generated by three commercial RNA-Amplification protocols (Miltenyi μMACS™ SuperAmp™, NuGEN Ovation® One-Direct System and EpiStem RNA-Amp™) applied to single cell equivalent levels of RNA (25-50 pg) using Affymetrix arrays. The EpiStem RNA-Amp™ kit exhibited the highest sensitivity and was therefore chosen for further testing. A comparison of Affymetrix array data from RNA-Amp™ cDNA generated from single MCF7 and MCF10A cells to reference controls of unamplified cDNA revealed a high degree of concordance. To assess the flexibility of the amplification system single cell RNA-Amp™ cDNA was also analysed using RNA-Seq and high-density qPCR, and showed strong cross-platform correlations. To exemplify the approach we used the system to analyse RNA profiles of small populations of rare cancer initiating cells (CICs) derived from a NSCLC patient-derived xenograft. RNA-Seq analysis was able to identify transcriptional differences in distinct subsets of CIC, with one group potentially enriched for metastasis formation. Pathway analysis revealed that the distinct transcriptional signatures demonstrated in the CIC subpopulations were significantly correlated with published stem-cell and epithelial-mesenchymal transition signatures.
The combined results confirm the sensitivity and flexibility of the RNA-Amp™ method and demonstrate the suitability of the approach for identifying clinically relevant signatures in rare, biologically important cell populations.
Many technologies are based on the hybridisation property of complementary nucleic acids; including microarrays, which use nucleic acid probes attached to a surface to estimate levels of ...complementary targets in labelled samples. Applications include expression studies, comparative genomic hybridisation and sequence capture. Nucleic acid hybridisation in solution is well studied. On microarrays it can be affected by the surface, reaction conditions, experimental noise and data-processing. There are established computational models of hybridisation and factors that affect it, based on thermodynamic principles. Additional research is required to build a model of microarray hybridisation, which could aid understanding, providing insights for experimental design. Microarray hybridisations often involve sequence mismatches between probe and target, due to variation between individuals, strains and breeds, or due to Cross-Species Hybridisation (CSH), where the array is used with a sample from a different species. CSH is common because array designs are only available for a limited number of species. Sequence mismatch effects on microarray hybridisations were investigated in experimental data, modelled and the model used to examine the impact of evolutionary distance on microarray CSH. Model parameterisation by simulated annealing suggested modifications. Mouse comparative genomic hybridisation data and strain-specific single nucleotide polymorphism allele information were combined to investigate sequence mismatch effects on microarray results. A strong, position-dependent effect was apparent, with mismatches near the centre of the probe being most destabilising. A new model of nucleic acid hybridisation was developed, and tested by replicating the qualitative aspects of the experimental data. Modules were added to model sequence evolution, signal intensity, noise and data processing. Simulations of CSH demonstrated good sensitivity and specificity at typical evolutionary distances when using long probes. Increasing evolutionary distance led to reduced power (sensitivity) and increased False Discovery Rate (FDR; lack of specificity), occurring sooner for shorter probes. If the hybridisation compared a sample from the array species with one from another species, there was an unexpected increase in power, due to an earlier increase in FDR, as many positives, both true and false, were declared. The nearest-neighbour thermodynamic parameters used in the model were compared with estimates from simulated annealing. They were greatly different, suggesting an adaptation to existing models may be necessary. These results suggest ensuring sequence mismatches are near probe ends if they can't be avoided in probe design. In CSH, for better results minimise evolutionary distance, use long probes and always compare two samples from the same species so that evolutionary distance has equal impact. This indicates how important modelling can be in understanding micromay hybridisations. Simulated annealing results suggest further study.
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•A murine model of cutaneous schistosome infection shows activation of the epidermis.•Epidermal keratinocyte precursor cells in the hair follicle expand after infection.•Infection ...leads to increased expression of pro-inflammatory genes in the epidermis.•Keratinocytes exposed to schistosome antigens in vitro secrete IL-1α and IL-1β.•Keratinocyte responses to schistosome infection and antigens mimic wound healing.
Keratinocytes constitute the majority of cells in the skin’s epidermis, the first line of defence against percutaneous pathogens. Schistosome larvae (cercariae) actively penetrate the epidermis to establish infection, however the response of keratinocytes to invading cercariae has not been investigated. Here we address the hypothesis that cercariae activate epidermal keratinocytes to promote the development of a pro-inflammatory immune response in the skin. C57BL/6 mice were exposed to Schistosoma mansoni cercariae via each pinna and non-haematopoietic cells isolated from epidermal tissue were characterised for the presence of different keratinocyte sub-sets at 6, 24 and 96h p.i. We identified an expansion of epidermal keratinocyte precursors (CD45−, CD326−, CD34+) within 24h of infection relative to naïve animals. Following infection, cells within the precursor population displayed a more differentiated phenotype (α6integrin−) than in uninfected skin. Parallel immunohistochemical analysis of pinnae cryosections showed that this expansion corresponded to an increase in the intensity of CD34 staining, specifically in the basal bulge region of hair follicles of infected mice, and a higher frequency of keratinocyte Ki67+ nuclei in both the hair follicle and interfollicular epidermis. Expression of pro-inflammatory cytokine and stress-associated keratin 6b genes was also transiently upregulated in the epidermal tissue of infected mice. In vitro exposure of keratinocyte precursors isolated from neonatal mouse skin to excretory/secretory antigens released by penetrating cercariae elicited IL-1α and IL-1β production, supporting a role for keratinocyte precursors in initiating cutaneous inflammatory immune responses. Together, these observations indicate that S.mansoni cercariae and their excretory/secretory products act directly upon epidermal keratinocytes, which respond by initiating barrier repair and pro-inflammatory mechanisms similar to those observed in epidermal wound healing.
Afforestation and deforestation are key land-use changes across the world, and are considered to be dominant factors controlling ecosystem functioning and biodiversity. However, the responses of soil ...microbial communities to these land-use changes are not well understood. Because changes in soil microbial abundance and community structure have consequences for nutrient cycling, C-sequestration and long-term sustainability, we investigated impacts of land-use change, age of stand and soil physico-chemical properties on fungal and bacterial communities and their metabolic activities. This study was carried out at four sites in two geographical locations that were afforested on long-established pastures with
Pinus radiata D. Don (pine). Two of the sites were on volcanic soils and two on non-volcanic soils and stand age ranged from 5 to 20 y. Microbial communities were analysed by biochemical (phospho-lipid fatty acids; PLFA) and molecular (multiplex-terminal restriction fragment length polymorphism; M-TRFLP) approaches. Both site and stand age influenced microbial properties, with changes being least detectable in the 5-y-old stand. Land use was a key factor influencing soil metabolic activities as measured by physiological profiling using MicroResp. Pasture soils had higher microbial biomass (
P < 0.001), and metabolic activities (
P < 0.001), and basal respiration rates were up to 2.8-times higher than in the pine soils. Microbial abundance analysis by PLFA showed that the fungal to bacterial ratio was higher in the pine soils (
P < 0.01). Community analysis suggested that soil bacterial communities were more responsive to site (principal component 1;
P < 0.001) than to land use (principal component 5;
P < 0.001). In contrast, the fungal community was more affected by land-use change (principal component 1;
P < 0.001) than by site, although site still had some influence on fungal community structure (principal component 2;
P < 0.001). Redundancy analysis also suggested that bacterial and fungal communities responded differently to various soil abiotic properties, land-use change and location of sites. Overall, our results indicate that the change in land use from pasture to
P. radiata stands had a direct impact on soil fungal communities but an indirect effect, through its effects on soil abiotic properties, on bacterial communities. Most of the changes in bacterial communities could be explained by altered soil physico-chemical properties associated with afforestation of pastures.
We investigated the effect of afforestation and reforestation of pastures on methane oxidation and the methanotrophic communities in soils from three different New Zealand sites. Methane oxidation ...was measured in soils from two pine (Pinus radiata) forests and one shrubland (mainly Kunzea ericoides var. ericoides) and three adjacent permanent pastures. The methane oxidation rate was consistently higher in the pine forest or shrubland soils than in the adjacent pasture soils. A combination of phospholipid fatty acid (PLFA) and stable isotope probing (SIP) analyses of these soils revealed that different methanotrophic communities were active in soils under the different vegetations. The C₁₈ PLFAs (signature of type II methanotrophs) predominated under pine and shrublands, and C₁₆ PLFAs (type I methanotrophs) predominated under pastures. Analysis of the methanotrophs by molecular methods revealed further differences in methanotrophic community structure under the different vegetation types. Cloning and sequencing and terminal-restriction fragment length polymorphism analysis of the particulate methane oxygenase gene (pmoA) from different samples confirmed the PLFA-SIP results that methanotrophic bacteria related to type II methanotrophs were dominant in pine forest and shrubland, and type I methanotrophs (related to Methylococcus capsulatus) were dominant in all pasture soils. We report that afforestation and reforestation of pastures caused changes in methane oxidation by altering the community structure of methanotrophic bacteria in these soils.
The aim of this trial was to assess the effectiveness of quality improvement collaboratives to implement large-scale change in the National Health Service (NHS) in the UK, specifically for improving ...outcomes in patients undergoing primary, elective total hip or knee replacement.
We undertook a two-arm, cluster randomised controlled trial comparing the roll-out of two preoperative pathways: methicillin-sensitive Staphylococcus aureus (MSSA) decolonisation (infection arm) and anaemia screening and treatment (anaemia arm). NHS Trusts are public sector organisations that provide healthcare within a geographical area. NHS Trusts (n = 41) in England providing primary, elective total hip and knee replacements, but that did not have a preoperative anaemia screening or MSSA decolonisation pathway in place, were randomised to one of the two parallel collaboratives. Collaboratives took place from May 2018 to November 2019. Twenty-seven Trusts completed the trial (11 anaemia, 16 infection). Outcome data were collected for procedures performed between November 2018 and November 2019. Co-primary outcomes were perioperative blood transfusion (within 7 days of surgery) and deep surgical site infection (SSI) caused by MSSA (within 90 days post-surgery) for the anaemia and infection trial arms, respectively. Secondary outcomes were deep and superficial SSIs (any organism), length of hospital stay, critical care admissions and unplanned readmissions. Process measures included the proportion of eligible patients receiving each preoperative initiative.
There were 19,254 procedures from 27 NHS Trusts included in the results (6324 from 11 Trusts in the anaemia arm, 12,930 from 16 Trusts in the infection arm). There were no improvements observed for blood transfusion (anaemia arm 183 (2.9%); infection arm 302 (2.3%) transfusions; adjusted odds ratio 1.20, 95% CI 0.52-2.75, p = 0.67) or MSSA deep SSI (anaemia arm 8 (0.13%); infection arm 18 (0.14%); adjusted odds ratio 1.01, 95% CI 0.42-2.46, p = 0.98). There were no significant improvements in any secondary outcome. This is despite process measures showing the preoperative pathways were implemented for 73.7% and 61.1% of eligible procedures in the infection and anaemia arms, respectively.
Quality improvement collaboratives did not result in improved patient outcomes in this trial; however, there was some evidence they may support successful implementation of new preoperative pathways in the NHS.
Prospectively registered on 15 February 2018, ISRCTN11085475.
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