Benchmarking digital PCR partition classification methods with empirical and simulated duplex data.

Digital PCR (dPCR) is a highly accurate technique for the quantification of target nucleic acid(s). It has shown great potential in clinical applications, like tumor liquid biopsy and validation of biomarkers. Accurate classification of partitions based on end-point fluorescence intensities is crucial to avoid biased estimators of the concentration of the target molecules. We have evaluated many clustering methods, from general-purpose methods to specific methods for dPCR and flowcytometry, on both simulated and real-life data. Clustering method performance was evaluated by simulating various scenarios. Based on our extensive comparison of clustering methods, we describe the limits of these methods, and formulate guidelines for choosing an appropriate method. In addition, we have developed a novel method for simulating realistic dPCR data. The method is based on a mixture distribution of a Poisson point process and a skew-$t$ distribution, which enables the generation of irregularities of cluster shapes and randomness of partitions between clusters ('rain') as commonly observed in dPCR data. Users can fine-tune the model parameters and generate labeled datasets, using their own data as a template. Besides, the database of experimental dPCR data augmented with the labeled simulated data can serve as training and testing data for new clustering methods. The simulation method is available as an R Shiny app.

Baselining physiological parameters in three muscles across three equine breeds. What can we learn from the horse?

Mapping-out baseline physiological muscle parameters with their metabolic blueprint across multiple archetype equine breeds, will contribute to better understanding their functionality, even across species. Aims: 1) to map out and compare the baseline fiber type composition, fiber type and mean fiber cross-sectional area (fCSA, mfCSA) and metabolic blueprint of three muscles in 3 different breeds 2) to study possible associations between differences in histomorphological parameters and baseline metabolism. Methods: Muscle biopsies [m. pectoralis (PM), m. vastus lateralis (VL) and m. semitendinosus (ST)] were harvested of 7 untrained Friesians, 12 Standardbred and 4 Warmblood mares. Untargeted metabolomics was performed on the VL and PM of Friesian and Warmblood horses and the VL of Standardbreds using UHPLC/MS/MS and GC/MS. Breed effect on fiber type percentage and fCSA and mfCSA was tested with Kruskal-Wallis. Breeds were compared with Wilcoxon rank-sum test, with Bonferroni correction. Spearman correlation explored the association between the metabolic blueprint and morphometric parameters. Results: The ST was least and the VL most discriminative across breeds. In Standardbreds, a significantly higher proportion of type IIA fibers was represented in PM and VL. Friesians showed a significantly higher representation of type IIX fibers in the PM. No significant differences in fCSA were present across breeds. A significantly larger mfCSA was seen in the VL of Standardbreds. Lipid and nucleotide super pathways were significantly more upregulated in Friesians, with increased activity of short and medium-chain acylcarnitines together with increased abundance of long chain and polyunsaturated fatty acids. Standardbreds showed highly active xenobiotic pathways and high activity of long and very long chain acylcarnitines. Amino acid metabolism was similar across breeds, with branched and aromatic amino acid sub-pathways being highly active in Friesians. Carbohydrate, amino acid and nucleotide super pathways and carnitine metabolism showed higher activity in Warmbloods compared to Standardbreds. Conclusion: Results show important metabolic differences between equine breeds for lipid, amino acid, nucleotide and carbohydrate metabolism and in that order. Mapping the metabolic profile together with morphometric parameters provides trainers, owners and researchers with crucial information to develop future strategies with respect to customized training and dietary regimens to reach full potential in optimal welfare.

From Corrosion Casting to Virtual Dissection: Contrast-Enhanced Vascular Imaging using Hafnium Oxide Nanocrystals.

Vascular corrosion casting is a method used to visualize the three dimensional (3D) anatomy and branching pattern of blood vessels. A polymer resin is injected in the vascular system and, after curing, the surrounding tissue is removed. The latter often deforms or even fractures the fragile cast. Here, a method is proposed that does not require corrosion, and is based on in situ micro computed tomography (micro-CT) scans. To overcome the lack of CT contrast between the polymer cast and the animals' surrounding soft tissue, hafnium oxide nanocrystals (HfO2 NCs) are introduced as CT contrast agents into the resin. The NCs dramatically improve the overall CT contrast of the cast and allow for straightforward segmentation in the CT scans. Careful design of the NC surface chemistry ensures the colloidal stability of the NCs in the casting resin. Using only 5 m% of HfO2 NCs, high-quality cardiovascular casts of both zebrafish and mice can be automatically segmented using CT imaging software. This allows to differentiate even µ $\umu$ m-scale details without having to alter the current resin injection methods. This new method of virtual dissection by visualizing casts in situ using contrast-enhanced CT imaging greatly expands the application potential of the technique.

Measuring DNA quality by digital PCR using probability calculations.

BACKGROUND: Accurate methods to assess DNA integrity are needed for many biomolecular methods. A multiplex digital PCR (dPCR) method designed for interspaced target sequences can be used to assess sequence integrity of large DNA strands. The ratio of single positive partitions versus double positive partitions is then used to calculate the sheared DNA strands. However, this simple calculation is only valid with low DNA concentration. We here describe a method based on probability calculations which enables DNA quality analysis in a large dynamic range of DNA concentrations. RESULTS: Known DNA integrity percentages were mimicked using artificial double stranded DNA in low, intermediate and high DNA concentration scenarios, respectively 600, 12500 and 30000 copies of DNA per reaction. At low concentrations both methods were similar. However, at the intermediate concentration (12500 copies per reaction) the ratio based method started producing a larger error than the proposed probability calculation method with a mean relative error of 20.7 and 16.7 for the Bruner and the proposed method respectively. At the high concentration (30000 copies per reaction) only the proposed method provided accurate measurements with a mean relative error of 60.9 and 9.3 for the ratio based and the proposed method respectively. Furthermore, while both methods have a bias, it is constant for the proposed method, while it decreases with the integrity of the DNA for the ratio based method. The probability calculation equation was extended to 4 dimensions and a proof of concept experiment was performed, the data suggested that the 4 dimensional equation is valid. SIGNIFICANCE AND NOVELTY: We here validate a method of estimating DNA integrity with dPCR using multiple probe combinations, allowing fast and flexible DNA integrity analysis. Additionally, we extend the method from 2 to 4 plex for more accurate DNA integrity measurements.

Digital PCR: a tool in clostridial mutant selection and detection.

The ClosTron mutagenesis system has enabled researchers to efficiently edit the clostridial genome. Since site-specific insertion of the mobile ClosTron insert may cause errors, validation is key. In this paper we describe the use of digital PCR (dPCR) as an alternative tool in selecting clostridial mutant strains. Clostridium perfringens chitinase mutant strains were constructed in which the mobile ClosTron intron was inserted into one of the chitinase genes. On-target insertion of the mobile intron was validated through conventional PCR. In order to confirm the absence of off-target insertions, dPCR was used to determine the amount of the ClosTron intron as well as the amount of a reference gene, located in close proximity to the interrupted gene. Subsequently, mutant strains containing an equivalent amount of both genes were selected as these do not contain additional off-target mobile ClosTron inserts. The outcome of this selection procedure was confirmed through a validated PCR-based approach. In addition to its application in mutant selection, dPCR can be used in other aspects of clostridial research, such as the distinction and easy quantification of different types of strains (wildtype vs. mutant) in complex matrices, such as faecal samples, a process in which other techniques are hampered by bacterial overgrowth (plating) or inhibition by matrix contaminants (qPCR). This research demonstrates that dPCR is indeed a high-throughput method in the selection of clostridial insertion mutants as well as a robust and accurate tool in distinguishing between wildtype and mutant C. perfringens strains, even in a complex matrix such as faeces. KEY POINTS: • Digital PCR as an alternative in ClosTron mutant selection • Digital PCR is an accurate tool in bacterial quantification in a complex matrix • Digital PCR is an alternative tool with great potential to microbiological research.

Digital PCR Partition Classification.

BACKGROUND: Partition classification is a critical step in the digital PCR data analysis pipeline. A range of partition classification methods have been developed, many motivated by specific experimental setups. An overview of these partition classification methods is lacking and their comparative properties are often unclear, likely impacting the proper application of these methods. CONTENT: This review provides a summary of all available digital PCR partition classification approaches and the challenges they aim to overcome, serving as a guide for the digital PCR practitioner wishing to apply them. We additionally discuss strengths and weaknesses of these methods, which can further guide practitioners in vigilant application of these existing methods. This review provides method developers with ideas for improving methods or designing new ones. The latter is further stimulated by our identification and discussion of application gaps in the literature, for which there are currently no or few methods available. SUMMARY: This review provides an overview of digital PCR partition classification methods, their properties, and potential applications. Ideas for further advances are presented and may bolster method development.

Selection and validation of reference genes for RT-qPCR normalization of porcine alveolar macrophages (PAMs) for PRRSV studies.

Porcine alveolar macrophages (PAMs) are widely used for in vitro studies of porcine respiratory viruses. Gene expression in these cells is altered by viral infection and cellular immune response. Real-time reverse transcription polymerase chain reaction (RT-qPCR) is a powerful technique for analyzing these changes. In order to obtain reliable quantitative RT-qPCR data and come to sound conclusions, stable reference genes are needed for normalization of target gene expression. In the present study, we evaluated the expression stability of nine reference genes in PAMs during cultivation and upon porcine reproductive and respiratory syndrome virus (PRRSV) inoculation. Using geNorm and NormFinder algorithms, we identified PSAP and GAPDH as the most stable reference genes under all experimental conditions. The selected reference genes were used for the normalization of CD163 expression under different conditions. This study demonstrates that selection of appropriate reference genes is essential for normalization and validation of RT-qPCR data across all experimental conditions. This study provides a new set of stable reference genes for future studies with porcine respiratory viruses in PAMs.

Different local, innate and adaptive immune responses are induced by two commercial Mycoplasma hyopneumoniae bacterins and an adjuvant alone.

Introduction: Enzootic pneumonia still causes major economic losses to the intensive pig production. Vaccination against its primary pathogen, Mycoplasma hyopneumoniae, is carried out worldwide to control the disease and minimize clinical signs and performance losses. Nonetheless, the effects of both infection with, and vaccination against Mycoplasma hyopneumoniae on the innate and adaptive immune responses remain largely unknown. Therefore, we conducted a study in which piglets were injected once with a commercial bacterin V1 or V2, or the adjuvant of V1 (A) to investigate their effect on local, innate and adaptive immune responses. Methods: Three weeks after vaccination, piglets were challenge infected with M. hyopneumoniae and euthanized four weeks later to assess vaccine efficacy via macroscopic and microscopic evaluation of lung lesions. Blood and broncho-alveolar lavage fluid (BAL) samples were collected to measure antibody responses, cellular immunity, BAL cytokine levels and BAL M. hyopneumoniae DNA load as well as cytokine secretion by monocytes. Results: After vaccination, proliferation of antigen-specific CD3+ T cells and a higher percentage of TNF-α+ CD8+, and TNF-α+ and TNF-α+IFN-γ+ CD4+CD8+ T cells was seen in V1, while proliferation of or a significant increase in cytokine production by different T cell subsets could not be observed for animals from V2. Interestingly, LPS-stimulated blood monocytes from V1 and A secreted less IL-10 on D7. After challenge, higher levels of IgA, more IL-10 and less IL-1ß was detected in BAL from V1, which was not observed in V2. Animals from A had significantly more IL-17A in BAL. The macroscopic lung lesion score and the M. hyopneumoniae DNA load at euthanasia was lower in V1, but the microscopic lung lesion score was lower in both vaccinated groups. Discussion: In conclusion, these results indicate that the two commercial bacterins induced different local and adaptive immune responses, that the adjuvant alone can reduce anti-inflammatory innate immune responses, and that both vaccines had a different efficacy to reduce Mycoplasma-like lung lesions and M. hyopneumoniae DNA load in the lung.

Evaluation of Angiogenesis in an Acellular Porous Biomaterial Based on Polyhydroxybutyrate and Chitosan Using the Chicken Ex Ovo Chorioallantoic Membrane Model.

The chorioallantoic membrane (CAM) is a highly vascularized avian extraembryonic membrane widely used as an in vivo model to study angiogenesis and its inhibition in response to tissues, cells, or soluble factors. In recent years, the use of CAM has become an integral part of the biocompatibility testing process for developing biomaterials intended for regenerative strategies and tissue engineering applications. In this study, we used the chicken ex ovo CAM assay to investigate the angiogenic potential of innovative acellular biopolymer polyhydroxybutyrate/chitosan (PHB/CHIT) scaffold, which is intended for the treatment of hard tissue defects, depending on treatment with pro- and anti-angiogenic substances. On embryonic day (ED) 7, the experimental biomaterials were placed on the CAM alone or soaked in vascular endothelial growth factor (VEGF-A), saline solution (PHY), or tyrosine kinase inhibitor (SU5402). After 72 h, the formation of vessels was analyzed in the surrounding area of the scaffold and inside the pores of the implants, using markers of embryonic endothelium (WGA, SNA), myofibroblasts (α-SMA), and macrophages (KUL-01). The morphological and histochemical analysis showed strong angiogenic potential of untreated scaffolds without additional effect of the angiogenic factor, VEGF-A. The lowest angiogenic potential was observed in scaffolds soaked with SU5402. Gene expression of pro-angiogenic growth factors, i.e., VEGF-A, ANG-2, and VE-CAD, was upregulated in untreated scaffolds after 72 h, indicating a pro-angiogenic environment. We concluded that the PHB/CHIT has a strong endogenous angiogenic potential and could be promising biomaterial for the treatment of hard tissue defects.

Comparison of serum tryptase as a diagnostic oncological marker in canine versus human mast cell neoplasms.

Canine mast cell tumors (MCTs) are a promising translational model for human mast cell neoplasms with striking similarities such as the Darier's sign and mutations in the KIT gene. Whereas mast cell neoplasms are rare in humans, MCTs are the most frequent malignant neoplasms of the skin in dogs. In human systemic mastocytosis, serum tryptase is an important diagnostic criterion. Surprisingly, serum tryptase levels were not yet investigated in dogs with MCTs. Therefore, the aim of this study was to investigate whether serum tryptase levels in dogs with cutaneous MCTs were elevated compared to those of a non-MCT control group. As a secondary aim, it was investigated whether surgical manipulation caused an increase in serum tryptase in canine MCT patients. A total of 48 serum samples were collected from dogs with different grades of cutaneous MCTs (n = 24) and non-MCT controls (n = 24). In dogs with cutaneous MCTs, blood was collected prior to and within 1 h after surgery. Serum tryptase levels were measured using a commercially available canine-specific ELISA kit. No significant difference in serum tryptase levels was found between cutaneous MCT patients and non-MCT controls, nor in these levels before versus after surgery. Our findings in canine cutaneous MCTs are in accordance with human cutaneous mastocytosis, where serum tryptase levels tend to remain within the normal range. However, despite various similarities between aggressive mast cell tumors in dogs and humans, serum tryptase cannot be considered a diagnostic biomarker in dogs with cutaneous MCTs as part of a comparative oncologic strategy.

One cisplatin dose provides durable stimulation of anti-tumor immunity and alleviates anti-PD-1 resistance in an intraductal model for triple-negative breast cancer.

Aggressive triple-negative breast cancer (TNBC) is classically treated with chemotherapy. Besides direct tumor cell killing, some chemotherapeutics such as cisplatin provide additional disease reduction through stimulation of anti-tumor immunity. The cisplatin-induced immunomodulation in TNBC was here investigated in-depth using immunocompetent intraductal mouse models. Upon primary tumor transition to invasive carcinoma, cisplatin was injected systemically and significantly reduced tumor progression. Flow cytometric immunophenotyping was corroborated by immunohistochemical analyses and revealed both differential immune cell compositions and positivity for their programmed death (PD)-1 and PD-ligand (L)1 markers across body compartments, including the primary tumor, axillary lymph nodes and spleen. As key findings, a significant decrease in immunosuppressive and a concomitant increase in anti-tumor lymphocytic cell numbers were observed in the axillary lymph nodes and spleen, highlighting their importance in cisplatin-stimulated anti-tumor immunity. These immunomodulatory effects were already established following the first cisplatin dose, indicating that early cisplatin-mediated events may determine (immuno)therapeutic outcome. Furthermore, a single cisplatin dose sufficed to alleviate anti-PD-1 resistance in a 4T1-based model, providing add-on disease reduction without toxic side effects as seen upon multiple cisplatin dosing. Overall, these results highlight cisplatin as immunotherapeutic ally in TNBC, providing durable immunostimulation, even after a single dose.

Intestinal Exposure to Ceftiofur and Cefquinome after Intramuscular Treatment and the Impact of Ceftiofur on the Pig Fecal Microbiome and Resistome.

Optimization of antimicrobial treatment during a bacterial infection in livestock requires in-depth knowledge of the impact of antimicrobial therapy on the pathogen and commensal microbiota. Once administered antimicrobials and/or their metabolites are excreted either by the kidneys through urine and/or by the intestinal tract through feces, causing antimicrobial pressure and possibly the emergence of resistance in the gastro-intestinal tract. So far, the excretion of ceftiofur and cefquinome in the intestinal tract of pigs has not been described. The objective of this study was to investigate the excretion of ceftiofur and cefquinome in the different segments of the gut and feces after intramuscular administration. Therefore, 16 pigs were treated either with ceftiofur (n = 8) or cefquinome (n = 8), and feces were collected during the entire treatment period. The presence of ceftiofur and desfuroylceftiofuracetamide or cefquinome were quantified via liquid chromatography−tandem mass spectrometry. At the end of the treatment, pigs were euthanized, and samples from the duodenum, jejunum, ileum and cecum were analyzed. In feces, no active antimicrobial residues could be measured, except for one ceftiofur-treated pig. In the gut segments, the concentration of both antimicrobials increased from duodenum toward the ileum, with a maximum in the ileum (187.8 ± 101.7 ng·g−1 ceftiofur-related residues, 57.8 ± 37.5 ng·g−1 cefquinome) and sharply decreased in the cecum (below the limit of quantification for ceftiofur-related residues, 6.4 ± 4.2 ng·g−1 cefquinome). Additionally, long-read Nanopore sequencing and targeted quantitative polymerase chain reaction (qPCR) were performed in an attempt to clarify the discrepancy in fecal excretion of ceftiofur-related residues between pigs. In general, there was an increase in Prevotella, Bacteroides and Faecalibacterium and a decrease in Escherichia and Clostridium after ceftiofur administration (q-value < 0.05). The sequencing and qPCR could not provide an explanation for the unexpected excretion of ceftiofur-related residues in one pig out of eight. Overall, this study provides valuable information on the gut excretion of parenteral administered ceftiofur and cefquinome.

The granulation (t)issue: A narrative and scoping review of basic and clinical research of the equine distal limb exuberant wound healing disorder.

Exuberant granulation tissue (EGT) is often observed during second intention wound healing in horses. Despite its impact on wound care, the basic mechanisms leading to EGT are still unclear and effective strategies to prevent and/or treat EGT are lacking. The development of EGT is a poorly understood, multifactorial process involving hyperproliferating fibroblasts and malfunctional differentiation of keratinocytes, suboptimal wound contraction, dysfunctional vascularisation, and chronic inflammation. To consolidate and describe basic and clinical research literature on EGT and to identify knowledge gaps and opportunities for future research, a search was systematically conducted using predefined search terms. Subsequently, a scoping review was conducted using specific criteria to select the peer-reviewed literature that described methods to treat and/or prevent EGT. Proposed mechanisms of effects as well as results and main conclusions were extracted and tabulated. The systematic search resulted in 1062 publications in PubMed and 767 in Web of Science. Twenty additional studies were later included. Of these, 327 studies were reviewed for the narrative review on basic research and 35 controlled clinical trials were eligible for the scoping review. All 35 studies were conducted in university hospitals, and all but one involved surgically induced non-infected wounds. The study population was predominantly horses (n = 230) with a small number of ponies (n = 18) and donkeys (n = 14). In conclusion, there remains a strong need for evidence-based recommendations on EGT treatment, preferably using multi-centre studies that represent the general population of horses, include higher numbers of animals, and are performed in naturally occurring wounds. This narrative and scoping review also emphasises the importance of incorporating basic research knowledge in the study design of clinical trials.

Triplex digital PCR assays for the quantification of intact proviral HIV-1 DNA.

The development of an HIV-1 cure is hampered by the existence of a persistent (latent) reservoir that contains a small proportion of replication-competent intact proviruses which refuels viral replication upon treatment discontinuation. Therefore, an accurate evaluation and quantification of these (intact) proviruses is essential to determine the efficacy of HIV-1 cure strategies which aim to eliminate this reservoir. Here, we present two triplex digital PCR assays which resulted from a combination of two existing methods, the IPDA (a 2-colour digital PCR based method) and Q4PCR assays (4 colour qPCR method), and tested the functionality on a three-colour digital PCR platform. In the present paper, we provide a step-by-step experimental protocol for these triplex digital PCR assays and validate their performance on a latently infected Jurkat cell-line model and HIV-1 patient samples. Our data demonstrates the potential and flexibility of increasing the number of subgenomic regions of HIV-1 within the IPDA to acquire sensitive detection of the HIV-1 reservoir while benefitting from the advantages of a dPCR setup.

Measuring Proviral HIV-1 DNA: Hurdles and Improvements to an Assay Monitoring Integration Events Utilising Human Alu Repeat Sequences.

Integrated HIV-1 DNA persists despite antiretroviral therapy and can fuel viral rebound following treatment interruption. Hence, methods to specifically measure the integrated HIV-1 DNA portion only are important to monitor the reservoir in eradication trials. Here, we provide an up-to-date overview of the literature on the different approaches used to measure integrated HIV-1 DNA. Further, we propose an implemented standard-curve free assay to quantify integrated HIV-1 DNA, so-called Alu-5LTR PCR, which utilises novel primer combinations. We tested the Alu-5LTR PCR in 20 individuals on suppressive ART for a median of nine years; the results were compared to those produced with the standard-free Alu-gag assay. The numbers of median integrated HIV-1 DNA copies were 5 (range: 1-12) and 14 (5-26) with the Alu-gag and Alu-5LTR, respectively. The ratios between Alu-gag vs Alu-5LTR results were distributed within the cohort as follows: most patients (12/20, 60%) provided ratios between 2-5, with 3/20 (15%) and 5/20 (25%) being below or above this range, respectively. Alu-5LTR assay sensitivity was also determined using an "integrated standard"; the data confirmed the increased sensitivity of the assay, i.e., equal to 0.25 proviruses in 10,000 genomes. This work represents an improvement in the field of measuring proviral HIV-1 DNA that could be employed in future HIV-1 persistence and eradication studies.

Comparison of Shifts in Skeletal Muscle Plasticity Parameters in Horses in Three Different Muscles, in Answer to 8 Weeks of Harness Training.

Training-induced follow-up of multiple muscle plasticity parameters in postural stability vs. locomotion muscles provides an integrative physiological view on shifts in the muscular metabolic machinery. It can be expected that not all muscle plasticity parameters show the same expression time profile across muscles. This knowledge is important to underpin results of metabolomic studies. Twelve non-competing Standardbred mares were subjected to standardized harness training. Muscle biopsies were taken on a non-training day before and after 8 weeks. Shifts in muscle fiber type composition and muscle fiber cross-sectional area (CSA) were compared in the m. pectoralis, the m. vastus lateralis, and the m. semitendinosus. In the m. vastus lateralis, which showed most pronounced training-induced plasticity, two additional muscle plasticity parameters (capillarization and mitochondrial density) were assessed. In the m. semitendinosus, additionally the mean minimum Feret's diameter was assessed. There was a significant difference in baseline profiles. The m. semitendinosus contained less type I and more type IIX fibers compatible with the most pronounced anaerobic profile. Though no baseline fiber type-specific and overall mean CSA differences could be detected, there was a clear post-training decrease in fiber type specific CSA, most pronounced for the m. vastus lateralis, and this was accompanied by a clear increase in capillary supply. No shifts in mitochondrial density were detected. The m. semitendinosus showed a decrease in fiber type specific CSA of type IIAX fibers and a decrease of type I fiber Feret's diameter as well as mean minimum Feret's diameter. The training-induced increased capillary supply in conjunction with a significant decrease in muscle fiber CSA suggests that the muscular machinery models itself toward an optimal smaller individual muscle fiber structure to receive and process fuels that can be swiftly delivered by the circulatory system. These results are interesting in view of the recently identified important fuel candidates such as branched-chain amino acids, aromatic amino acids, and gut microbiome-related xenobiotics, which need a rapid gut-muscle gateway to reach these fibers and are less challenging for the mitochondrial system. More research is needed with that respect. Results also show important differences between muscle groups with respect to baseline and training-specific modulation.

High Numbers of CD163-Positive Macrophages in the Fibrotic Region of Exuberant Granulation Tissue in Horses.

Exuberant granulation tissue (EGT) is a frequently encountered complication during second intention healing in equine distal limb wounds. Although it is still unknown what exactly triggers the formation of this tissue, previous research has revealed a persistent inflammatory response in these wounds. In this preliminary study we examined this inflammatory response in EGT-developing wounds as well as in experimental induced wounds. Immunohistological stainings were performed to detect primary inflammatory immune cells (MAC387 staining) as well as pro-resolution immune cells (CD163 staining). Our results show a significantly higher amount of MAC387+ and CD163+ cells in the fibrotic regions of EGT compared with the 19-day-old experimental wounds. This persistent high amount of fibrosis-promoting CD163+ cells in EGT suggests that the wound healing processes in EGT-developing wounds are arrested at the level of the proliferation phase.

Techniques used to assess intussusceptive angiogenesis: A systematic review.

Intussusceptive angiogenesis (IA) is an important physiological form of angiogenesis in which an existing vessel splits in two by the formation of an intraluminal tissue pillar. The presence of these intraluminal pillars form the hallmark of ongoing IA in growing vascular beds. However, their visualization is technically challenging. The goal of this systematic review was to investigate which techniques are being used to identify intraluminal pillars and to formulate important points to keep in mind when studying IA. A systematic literature search resulted in 154 evaluated articles of which the majority (65%) provided sufficient data to unambiguously demonstrate the presence of intraluminal pillars. Scanning electron microscopy imaging of vascular corrosion casts and serial sectioning of ultrathin sections are the most used techniques. New methods such as serial block face scanning electron microscopy and micro computed tomography (µCT) are gaining importance. Moreover, our results indicate that IA was studied in a variety of animals and tissues. IA is a biologically very relevant form of angiogenesis. Techniques to visualize intraluminal pillars need to have a minimal resolution of 1 µm and should provide information on the 3D-nature of the pillars. Optimally, several techniques are combined to demonstrate ongoing IA.

Development of a 3':5' digital PCR assay to determine horse mRNA integrity.

Accurate tools to measure RNA integrity are essential to obtain reliable gene expression data. The reverse transcription quantitative PCR (RT-qPCR) based 3':5' assay permits a direct determination of messenger RNA (mRNA) integrity. However, the use of standard curves and the possible effect of PCR inhibitors make this method cumbersome and prone to variation, especially in small samples. Here we developed a triplex digital PCR (dPCR) 3':5' assay for assessing RNA integrity in equine samples as rapid and simple alternative to RT-qPCR. This dPCR assay not only provides a straight forward analysis of the mRNA integrity, but also of its quantity.

Flexibility of equine bioenergetics and muscle plasticity in response to different types of training: An integrative approach, questioning existing paradigms.

Equine bioenergetics have predominantly been studied focusing on glycogen and fatty acids. Combining omics with conventional techniques allows for an integrative approach to broadly explore and identify important biomolecules. Friesian horses were aquatrained (n = 5) or dry treadmill trained (n = 7) (8 weeks) and monitored for: evolution of muscle diameter in response to aquatraining and dry treadmill training, fiber type composition and fiber cross-sectional area of the M. pectoralis, M. vastus lateralis and M. semitendinosus and untargeted metabolomics of the M. pectoralis and M. vastus lateralis in response to dry treadmill training. Aquatraining was superior to dry treadmill training to increase muscle diameter in the hindquarters, with maximum effect after 4 weeks. After dry treadmill training, the M. pectoralis showed increased muscle diameter, more type I fibers, decreased fiber mean cross sectional area, and an upregulated oxidative metabolic profile: increased ß-oxidation (key metabolites: decreased long chain fatty acids and increased long chain acylcarnitines), TCA activity (intermediates including succinyl-carnitine and 2-methylcitrate), amino acid metabolism (glutamine, aromatic amino acids, serine, urea cycle metabolites such as proline, arginine and ornithine) and xenobiotic metabolism (especially p-cresol glucuronide). The M. vastus lateralis expanded its fast twitch profile, with decreased muscle diameter, type I fibers and an upregulation of glycolytic and pentose phosphate pathway activity, and increased branched-chain and aromatic amino acid metabolism (cis-urocanate, carnosine, homocarnosine, tyrosine, tryptophan, p-cresol-glucuronide, serine, methionine, cysteine, proline and ornithine). Trained Friesians showed increased collagen and elastin turn-over. Results show that branched-chain amino acids, aromatic amino acids and microbiome-derived xenobiotics need further study in horses. They feed the TCA cycle at steps further downstream from acetyl CoA and most likely, they are oxidized in type IIA fibers, the predominant fiber type of the horse. These study results underline the importance of reviewing existing paradigms on equine bioenergetics.