The TiHoCL panel for canine lymphoma: a feasibility study integrating functional genomics and network biology approaches for comparative oncology targeted NGS panel design.

Targeted next-generation sequencing (NGS) enables the identification of genomic variants in cancer patients with high sensitivity at relatively low costs, and has thus opened the era to personalized human oncology. Veterinary medicine tends to adopt new technologies at a slower pace compared to human medicine due to lower funding, nonetheless it embraces technological advancements over time. Hence, it is reasonable to assume that targeted NGS will be incorporated into routine veterinary practice in the foreseeable future. Many animal diseases have well-researched human counterparts and hence, insights gained from the latter might, in principle, be harnessed to elucidate the former. Here, we present the TiHoCL targeted NGS panel as a proof of concept, exemplifying how functional genomics and network approaches can be effectively used to leverage the wealth of information available for human diseases in the development of targeted sequencing panels for veterinary medicine. Specifically, the TiHoCL targeted NGS panel is a molecular tool for characterizing and stratifying canine lymphoma (CL) patients designed based on human non-Hodgkin lymphoma (NHL) research outputs. While various single nucleotide polymorphisms (SNPs) have been associated with high risk of developing NHL, poor prognosis and resistance to treatment in NHL patients, little is known about the genetics of CL. Thus, the ~100 SNPs featured in the TiHoCL targeted NGS panel were selected using functional genomics and network approaches following a literature and database search that shielded ~500 SNPs associated with, in nearly all cases, human hematologic malignancies. The TiHoCL targeted NGS panel underwent technical validation and preliminary functional assessment by sequencing DNA samples isolated from blood of 29 lymphoma dogs using an Ion Torrent™ PGM System achieving good sequencing run metrics. Our design framework holds new possibilities for the design of similar molecular tools applied to other diseases for which limited knowledge is available and will improve drug target discovery and patient care.

Human TH17 cells engage gasdermin E pores to release IL-1α on NLRP3 inflammasome activation.

It has been shown that innate immune responses can adopt adaptive properties such as memory. Whether T cells utilize innate immune signaling pathways to diversify their repertoire of effector functions is unknown. Gasdermin E (GSDME) is a membrane pore-forming molecule that has been shown to execute pyroptotic cell death and thus to serve as a potential cancer checkpoint. In the present study, we show that human T cells express GSDME and, surprisingly, that this expression is associated with durable viability and repurposed for the release of the alarmin interleukin (IL)-1α. This property was restricted to a subset of human helper type 17 T cells with specificity for Candida albicans and regulated by a T cell-intrinsic NLRP3 inflammasome, and its engagement of a proteolytic cascade of successive caspase-8, caspase-3 and GSDME cleavage after T cell receptor stimulation and calcium-licensed calpain maturation of the pro-IL-1α form. Our results indicate that GSDME pore formation in T cells is a mechanism of unconventional cytokine release. This finding diversifies our understanding of the functional repertoire and mechanistic equipment of T cells and has implications for antifungal immunity.

Convalescent COVID-19 Patients Without Comorbidities Display Similar Immunophenotypes Over Time Despite Divergent Disease Severities.

COVID-19, the disease caused by SARS-CoV-2 infection, can assume a highly variable disease course, ranging from asymptomatic infection, which constitutes the majority of cases, to severe respiratory failure. This implies a diverse host immune response to SARS-CoV-2. However, the immunological underpinnings underlying these divergent disease courses remain elusive. We therefore set out to longitudinally characterize immune signatures of convalescent COVID-19 patients stratified according to their disease severity. Our unique convalescent COVID-19 cohort consists of 74 patients not confounded by comorbidities. This is the first study of which we are aware that excludes immune abrogations associated with non-SARS-CoV-2 related risk factors of disease severity. Patients were followed up and analyzed longitudinally (2, 4 and 6 weeks after infection) by high-dimensional flow cytometric profiling of peripheral blood mononuclear cells (PBMCs), in-depth serum analytics, and transcriptomics. Immune phenotypes were correlated to disease severity. Convalescence was overall associated with uniform immune signatures, but distinct immune signatures for mildly versus severely affected patients were detectable within a 2-week time window after infection.

Exploring With Transcriptomic Approaches the Underlying Mechanisms of an Essential Oil-Based Phytogenic in the Small Intestine and Liver of Pigs.

Phytogenics are plant-based feed additives utilized in animal nutrition to support animal growth and health. Worldwide restrictions and bans on the use of antibiotic growth promoters resulted in an increased demand for in-feed alternatives including phytogenics. However, several challenges remain for technology readiness in animal industry, especially regarding the standardization of the ingredients as well as our knowledge on the cellular mechanisms underlying their biological effects. In the present study, 32 weaned piglets were allocated for 28 days to four experimental diets, a control diet, a phytogenic feed additive (PFA) diet, or the same two diets but with the addition of oxidized oil (OO) at 10%. The last two diets aimed at evaluating the antioxidant properties of PFA. At the end of the trial, the ileum and the liver of the pigs were sampled, and RNA were isolated for profiling their transcriptome via RNA sequencing (RNA-Seq). In the ileum, the gene set enrichment analysis showed that the activity of several immune pathways (NF-kB, interferon α/ß, antimicrobial peptide, and collagen pathways) was reduced in piglets fed PFA compared to the control piglets. As expected, the addition of OO induced strong effects on the liver transcriptome and most likely accounted for the significant growth impairment. The likelihood ratio test across the four diets revealed a global response driven by the oxidative stress challenge with hundreds of genes associated with fatty acid ß-oxidation and peroxisome in the liver. The expression levels of those genes in the piglets fed OO+PFA were much less affected by the challenge. Collectively, the effects seen at day 28 suggest that substances in the PFA formulation provide anti-inflammatory and antioxidant properties. The use of RNA-Seq in animal nutrition allows exploring and deciphering novel mechanisms of natural growth promoters.

Research Note: Snapshot of the transcriptome via RNA sequencing in the ileum of broiler chickens fed subtherapeutic concentrations of avilamycin.

Antibiotics have played a critical role in sustaining and improving livestock production in the past decades, but the emergence of antimicrobial resistance has led several countries to ban or limit their use. Since then, in-feed alternatives have gained a lot of attention but the development of efficacious alternatives implies a better understanding of the mode of action of antibiotic growth promoters (AGP) when administered at subtherapeutic concentrations. In the present study, 120 broiler chickens per group (8 pens/group) were fed for 35 d with either basal feed (control group) or feed supplemented with avilamycin (AGP group; 10 g/1,000 kg of feed). At the end of the trial, the ileum from the small intestine of 5 birds per group was sampled, and RNA were isolated for profiling their transcriptome via RNA sequencing (RNA-Seq). As expected, the growth of chickens in the AGP group was significantly higher than in the control group. Overall, 66 differentially expressed genes (false discovery rate ≤ 0.05 and fold change ≥ 2 or ≤ -2) were found in the ileum of chickens fed avilamycin in comparison with the control group. The functional analysis showed reduced activity of genes related to signaling by interleukins, with IL-22, SOCS3, and certain antimicrobial peptides found multiple times in these pathways in the AGP group at day 35. In addition, higher activity was predicted in a module of genes related to lipid metabolism and transport in the avilamycin group. The use of RNA-Seq allowed a snapshot of the whole transcriptome at day 35 and aimed at delivering additional data on the host-centric hypothesis regarding the mode of action of AGP (i.e. immunomodulation, reduction of the immunological stress).

Optimisation of a droplet digital PCR for strain specific quantification of a probiotic Bifidobacterium animalis strain in poultry feed.

The use of probiotics in animal nutrition to provide health benefits is widely accepted. Bifidobacterium animalis (BAN) is an example of a commonly used beneficial strain. BAN is applied in a multi-strain feed additive for poultry. As part of the increased demand for tracking and tracing of feed additives within modern quality management, it is crucial to determine the quantity of the active strain after mixing the probiotic product into feed. A real-time PCR protocol, already developed some years ago, was replaced with a Droplet Digital PCR (ddPCR) assay, as this third generation PCR method is known for higher precision, sensitivity and does not require standard curves. Each sample is partitioned into thousands of small subsamples that are measured individually and an absolute result value for each sample is extrapolated via Poisson distribution. The following parameters were evaluated for the ddPCR assay: optimal annealing temperature (59 °C), concentration of primers (500 nM) and probe (400 nM), and PCR cycle number (50 cycles). The linearity of the optimised ddPCR assay was tested with BAN DNA extracted from pure culture. The obtained standard curve was linear (R2 = 0.9982) and the efficiency (E) of the method was 99.98%. To finalise the development, the Limit of Blank (LoB = 9.17 × 102 copies g-1), Limit of Detection (LoD = 1.15 × 103 copies g-1) and Limit of Quantification (LoQ = 1.57 × 103 copies g-1) for the assay were determined using poultry feed free of BAN and feed spiked with different concentrations of the strain. A BAN strain-specific, probe-based ddPCR assay for the quantification in poultry feed was developed.