Changes in saliva biomarkers during a standardized increasing intensity field exercise test in endurance horses

Salivary biomarkers could be useful to evaluate stress, fitness level, and skeletal muscle damage associated to exercise in horses in an easy and non-painful way. Therefore, this study aims to evaluate if cortisol in saliva (sCor), salivary alpha-amylase (sAMY) and butyrylcholinesterase (sBChE) and lactate (sLA) and creatine kinase (sCK) in saliva of horses can show changes during a standardized exercise test, and if they are related to heart rate variability (HRV) parameters related to sympathetic and parasympathetic tone, fitness level or skeletal muscle damage. For this purpose, ten endurance horses were submitted to a standardized exercise test in field conditions. Saliva and blood were obtained at basal time (TB), after the seven bouts of velocity (T + 01 to T + 07), and 5, 15, 30, and 45 min later (T + 5, T + 15, T + 30, and T + 45). Five endurance horses in resting condition (control group) were also enrolled. HRV and fitness level parameters, and plasma CK as a marker of muscle damage were also evaluated. Salivaryalpha-amylase increased at T + 30 (P = 0.03), sBChE at T + 5 (P = 008), and sCK at T + 07 (P = 0.009) after the exercise test, with significant differences between the exercise and control groups’ results. The sCor did not show significant changes during the exercise test in the exercise group but higher concentration compared to the control horses (P < 0.001) were observed. sCor, sAMY, sBChE, and sCK showed a positive correlation (r values between 0.47 and 0.64) with the sympathetic tone and a negative correlation (r values between −0.37 and −0.56) with the parasympathetic tone. In conclusion, sAMY, sBChE, and sCK showed significant increases in ten endurance horses after an increasing intensity velocity exercise. Values of sCor, sAMY, sBChE, and sCK were associated with HRV, which is used to evaluate stress, and therefore, they could be potentially used to assess the exercise-related stress after a physical effort.     

Non-targeted metabolomics of saliva to explore potential biomarkers for gastric ulceration in pigs fed hemp

Gastric ulceration is a common disease in pig production worldwide and is associated with economic losses as well as animal health and welfare issues. The aim of this study was to explore potential salivary biomarkers for gastric ulceration in pigs. In addition, the aim was to study the effect of hemp on the incidence of gastric ulcers. Approximately 440 growing-finishing pigs in the period from 30 to 110 kg BW were allocated to four different diets: meal feed (Meal); pelleted feed (Pellets); pelleted feed added 4% hempseed cake (Hemp Cake); pelleted feed added 4% hempseed hulls (Hemp Hulls). The day before slaughter, saliva samples from each pig were collected. After slaughter, the stomachs were emptied to assess the consistency of the stomach content and examined for gastric ulceration using an index scale (0–10). Noticeable changes of the gastric mucosa (total index score ≥ 6) were observed in 291 pigs. The odds of having index scores 0–5 relative to index scores 6–8 and 9–10, respectively, were higher (P < 0.001) for pigs fed Meal compared to pigs fed Pellets. The odds of suffering from severe gastric ulcers tended (P = 0.08) to be lower in pigs fed Hemp Hulls compared to pigs fed Pellets. A non-targeted liquid chromatography mass spectrometry based metabolomics analysis was performed on saliva samples to determine any separation between pigs with healthy stomachs and those with gastric ulcers and to examine a possible correlation between gastric ulcer index and potential biomarkers. Partial least-squares discriminant analysis showed a separation between pigs with ulcers and those with healthy stomachs/hyperkeratosis (HK). Metabolites contributing to the separation between groups were identified. Levels of oxylipins deriving from linoleic acid were lower (P < 0.001) in pigs with ulcers compared to healthy/HK pigs. This may indicate a shift in the metabolic pathways towards more pro-inflammatory arachidonic acid-derived eicosanoids, which might reflect an increased inflammatory response. Thus, reduced levels of oxylipins derived from linoleic acid seemed to be associated with active gastric ulcers, and thereby they might function as biomarkers for gastric ulceration in pigs. In addition, supplementation of hempseed hulls had a beneficial effect on severe gastric ulcers, as hempseed hulls changed the consistency of the gastric content by conferring more solidness. However, it was not possible to observe any reliable separation between pigs fed pellets supplemented with hemp products and pigs fed non-supplemented pellets according to the identified salivary metabolites.     

Nano-bio-chips for high performance multiplexed protein detection: Determinations of cancer biomarkers in serum and saliva using quantum dot bioconjugate labels

The integration of semiconductor nanoparticle quantum dots (QDs) into a modular, microfluidic biosensor for the multiplexed quantitation of three important cancer markers, carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), and Her-2/Neu (C-erbB-2) was achieved. The functionality of the integrated sample processing, analyte capture and detection modalities was demonstrated using both serum and whole saliva specimens. Here, nano-bio-chips that employed a fluorescence transduction signal with QD-labeled detecting antibody were used in combination with antigen capture by a microporous agarose bead array supported within a microfluidics ensemble so as to complete the sandwich-type immunoassay. The utilization of QD probes in this miniaturized biosensor format resulted in signal amplification 30 times relative to that of standard molecular fluorophores as well as affording a reduction in observed limits of detection by nearly 2 orders of magnitude (0.02 ng/mL CEA; 0.11 pM CEA) relative to enzyme-linked immunosorbent assay (ELISA). Assay validation studies indicate that measurements by the nano-bio-chip system correlate to standard methods at R² = 0.94 and R² = 0.95 for saliva and serum, respectively. This integrated nano-bio-chip assay system, in tandem with next-generation fluorophores, promises to be a sensitive, multiplexed tool for important diagnostic and prognostic applications.     

Functional amino acid supplementation postweaning mitigates the response of normal birth weight more than for low birth weight pigs to a subsequent Salmonella challenge

Previous work has shown that dietary supplementation with key functional amino acids (FAA) improves growth performance and immune status of disease-challenged normal birth weight (NBW) pigs. It is not known whether FAA supplementation attenuates the effects of a subsequent disease challenge or whether this response is similar in low birth weight (LBW) pigs. The objective was to determine the effects of birth weight and FAA supplementation during the postweaning period in Salmonella-challenged pigs. Thirty-two LBW (1.08 ± 0.11 kg) and NBW (1.58 ± 0.11 kg) pigs were assigned to a nursery feeding program at weaning (25 d) for 31 days in a 2 × 2 factorial arrangement. Factors were birth weight category (LBW vs. NBW) and basal (FAA–) or supplemented FAA profile (FAA+; Thr, Met, and Trp at 120% of requirements). At d 31, pigs were placed onto a common grower diet and, after a 7-d adaptation period, were inoculated with Salmonella Typhimurium (ST; 2.2 × 10⁹ colony-forming units/mL) and monitored for 7-d postinoculation. Growth performance, rectal temperature, fecal score, indicators of gut health, ST shedding score in feces, intestinal ST colonization and translocation, and blood parameters of acute-phase response and antioxidant balance were measured pre- and postinoculation. Inoculation with ST increased temperature and fecal score, and the overall rectal temperature was higher in LBW compared to NBW pigs (P < 0.05). Postinoculation (d 7), reduced:oxidized glutathione was increased in NBW compared to LBW pigs (P < 0.05). Salmonella shedding and translocation to spleen were lower in NBW-FAA+ compared to NBW-FAA? pigs (P < 0.05). Postinoculation average daily gain was higher in NBW-FAA+ (P < 0.05) compared to the other groups. Postinoculation haptoglobin, superoxide dismutase, and colonic myeloperoxidase were increased in LBW-FAA? pigs (P < 0.05). Ileal alkaline phosphatase was decreased in LBW compared to NBW (P < 0.05). Overall, FAA supplementation represents a potential strategy to mitigate the effect of enteric disease challenge in NBW, but not LBW pigs.     

Mutation at positively selected positions in the binding site for HLA-C shows that KIR2DL1 is a more refined but less adaptable NK cell receptor than KIR2DL3

Through recognition of HLA class I, killer cell Ig-like receptors (KIR) modulate NK cell functions in human immunity and reproduction. Although a minority of HLA-A and -B allotypes are KIR ligands, HLA-C allotypes dominate this regulation, because they all carry either the C1 epitope recognized by KIR2DL2/3 or the C2 epitope recognized by KIR2DL1. The C1 epitope and C1-specific KIR evolved first, followed several million years later by the C2 epitope and C2-specific KIR. Strong, varying selection pressure on NK cell functions drove the diversification and divergence of hominid KIR, with six positions in the HLA class I binding site of KIR being targets for positive diversifying selection. Introducing each naturally occurring residue at these positions into KIR2DL1 and KIR2DL3 produced 38 point mutants that were tested for binding to 95 HLA- A, -B, and -C allotypes. Modulating specificity for HLA-C is position 44, whereas positions 71 and 131 control cross-reactivity with HLA-A*11:02. Dominating avidity modulation is position 70, with lesser contributions from positions 68 and 182. KIR2DL3 has lower avidity and broader specificity than KIR2DL1. Mutation could increase the avidity and change the specificity of KIR2DL3, whereas KIR2DL1 specificity was resistant to mutation, and its avidity could only be lowered. The contrasting inflexibility of KIR2DL1 and adaptability of KIR2DL3 fit with C2-specific KIR having evolved from C1-specific KIR, and not vice versa. Substitutions restricted to activating KIR all reduced the avidity of KIR2DL1 and KIR2DL3, further evidence that activating KIR function often becomes subject to selective attenuation.     

Genome-wide association study shows that microtia in Altay sheep is caused by a 76 bp duplication of HMX1

Microtia is a congenital malformation of the external ear that can be observed in many species including sheep. However, the genetic basis of microtia still remains unclear. Here, a GWAS was conducted to investigate the genetic basis underlying microtia. A total of 55 samples from 26 microtia and 29 normal animals were genotyped with Illumina OvineHD BeadChip. The strongest significant SNP was identified on OAR6, approximating the evolutionarily conserved region of the HMX1 gene, which is related to congenital malformations of the external ear in other species such as cattle and rats. Sequencing an evolutionarily conserved region surrounding HMX1 revealed a duplication of 76 bp, which is concordant with microtia, suggesting a dominant inheritance mode. Identification of this causal mutation in the HMX1 gene indicates the role of this particular gene in the development of the external ear and provides a genetic marker for selection against microtia.     

Prediction of climate warming impacts on plant species could be more complex than expected. Evidence from a case study in the Himalaya

Many studies have investigated the possible impact of climate change on the distributions of plant species. In the present study, we test whether the concept of potential distribution is able to effectively predict the impact of climate warming on plant species.Using spatial simulation models, we related the actual (current species distribution), potential (modelled distribution assuming unlimited dispersal) and predicted (modelled distribution accounting for wind-limited seed dispersal) distributions of two plant species under several warming scenarios in the Sagarmatha National Park (Nepal). We found that the two predicted distributions were, respectively, seven and nine times smaller than the potential ones. Under a +3 °C scenario, both species would likely lose their actual and predicted distributions, while their potential distributions would remain partially safe. Our results emphasize that the predicted distributions of plant species may diverge to a great extent from their potential distributions, particularly in mountain areas, and predictions of species preservation in the face of climate warming based on the potential distributions of plant species are at risk of producing overoptimistic projections.We conclude that the concept of potential distribution is likely to lead to limited or inefficacious conservation of plant species due to its excessively optimistic projections of species preservation. More robust strategies should utilize concepts such as “optimal reintroduction”, which maximizes the benefit–cost ratio of conservation activities by limiting reintroduction efforts to suitable areas that could not otherwise be reached by a species; moreover, such strategies maximize the probability of species establishment by excluding areas that will be endangered under future climate scenarios.     

Red and blond Mangalitza pigs display a signature of divergent directional selection in the SLC45A2 gene

The Mangalitza lard‐type pig breed is well known for its fat appearance and curly hair, and it is mainly distributed in Eastern Europe. Four main lines were created in the nineteenth century by artificial selection: Blond Mangalitza, Black Mangalitza, Swallow‐Belly Mangalitza and Red Mangalitza. The Swallow‐Belly line has a black coat combined with yellow‐blond throat and underbelly. In the current work, we aimed to investigate if the colourations of Mangalitza pigs are genetically determined by one or a few loci whose frequencies have been modified by artificial selection. The results of selection scans, with Hap FLK and BayeScan , and of a GWAS for coat colour highlighted the existence of one region on SSC16 (18–20 Mb) with potential effects on hair pigmentation (Red vs. Blond contrast). The analysis of the gene content of this region allowed us to detect the solute carrier family 45 member 2 (SLC45A2) locus as a candidate gene for this trait. The polymorphism of the SLC45A2 locus has been associated with reduced levels or the absence of melanin in several mammalian species. The genotyping of four missense polymorphisms evidenced that rs341599992:G > A and rs693695020:G > A SNPs are strongly but not fully associated with the red and blond coat colours of Mangalitza pigs, a result that was confirmed by performing a haplotype association test. The near fixation of alternative SLC45A2 genotypes in Red and Blond Mangalitza pigs provides a compelling example of the consequences of a divergent directional selection for coat colour in a domestic species.     

A comparative study of mutations in Escherichia coli and Salmonella typhimurium shows that codon conservation is strongly correlated with codon usage

Escherichia coli and Salmonella typhimurium are closely related species of enteric bacteria, having diverged from 120 to 160 million years ago, according to the estimate of Ochman & Wilson (1987. J. Mol. Evol.26, 74-86). In order to study base substitution mutations in the genomes of these bacteria, we have compared pairs of genes for the same product in the two species, and have selected a sample in which the protein length is the same in both E. coli and S. typhimurium. From the alignment of these gene pairs, we observe that frequently used codons are more conserved than infrequently used codons, i.e., the apparent mutation rate is higher for rare codons than for popular codons.     

Post-thaw boar sperm motility is affected by prolonged storage of sperm in liquid nitrogen. A retrospective study

Owing to the quick genetic turnover of the pig industry, most AI-boar sires live 2-3 yr, a period during which for 1-2 yr their semen is extended and used in liquid form for AI. Despite showing low cryosurvival, affecting fertility after AI, boar semen is frozen for easiness of transport overseas and reposition of valuable genetics. For the latter, semen is stored in liquid nitrogen (LN₂, cryostorage) for many years, a controversial practice. Here we studied how length of cryostorage could affect sperm quality. Straws (0.5 mL) frozen using the same cryopreservation protocol at one specific location from AI- sires of proven fertility were stored in LN₂ for up to 8 yr. Post-thaw sperm quality was evaluated after 2, 4 or 8 yr of cryostorage, always compared to early thawing (15 d after freezing). Sperm motility and kinematics were evaluated post-thaw using CASA and sperm viability was cytometrically evaluated using specific fluorophores. Sperm viability was not affected by length of cryostorage, but total and progressive sperm motility were lower (p < 0.01) in sperm samples cryostored for 4 or 8 yr compared to those thawed 15 d after freezing. Cryostorage time affected sperm kinetics, but with greater intensity in the samples cryostored for 4 yr (p < 0.001) than in those for 2 yr (p < 0.01). The fact that the major phenotypic characteristic of boar spermatozoa, motility, is constrained by time of cryostorage should be considered when building cryobanks of pig semen. Attention should be placed on the finding that >2 yr of cryostorage time can be particularly detrimental for the post-thaw motility of some sires, which might require increasing sperm numbers for AI.     

Is colorectal cancer a more aggressive disease in young patients? A population-based study from the Czech Republic

IntroductionThe incidence of colorectal cancer in young patients is increasing. The goal of this study was to investigate whether clinicopathological features and survival differed between young, middle-aged and elderly patients.MethodsThe Czech National Cancer Registry was searched to identify all cases of colorectal cancer between 1982 and 2014. Three subgroups of patients were created: young patients, defined as being between 18 and 40 years of age, middle-aged patients, defined as being between 41 and 74 years of age, and elderly patients, defined as being over the age of 75 years.ResultsA total of 192,241 patients diagnosed with colorectal cancer between the years 1982 and 2014 were included in the study. Out of these, 3,287 patients (1.7%) were between 18 and 40 years of age, 134,139 patients (69.8%) were between 41 and 74 years of age and 54,815 patients (28.5%) were 75 years of age or older. The young patients had a higher incidence of mucinous adenocarcinoma and signet ring cell carcinoma, more advanced disease and more rectal tumours than elderly patients. Nonetheless, young patients received treatment more frequently and had better cancer-specific survival than the older patients.ConclusionThe better prognosis in young patients is presumably due to their better physiological reserve and lower incidence of comorbidities. Efforts should be made in younger patients to diagnose early and treat aggressively.     

Drivers of understorey biomass: tree species identity is more important than richness in a young forest

林下生物量影响因素：幼龄林树种特性比丰富度更重要 生物多样性与生态系统功能的正相关关系已被广泛报道,其主要来源于对草原生态系统的研究。然而,该结论并不一定适用于更复杂的环境,例如具有不同垂直层次的森林。举例而言,已有研究表明上层乔木树种丰富度与林下生产力降低有关。树种丰富度是否会通过增加(由于生境异质性)或降低(通过增强竞争)资源的可利用性进而影响林下生产力,以及林下生产力是否受树种特性的影响更大,这些影响机制都可能会随着时间的推移而改变。此外,研究还表明,丰富度-生产力关系随着环境背景的变化而改变。本研究利用可以操控树种丰富度的实验林场研究了这些不同垂直层位里的时间和环境动态。在中国亚热带森林生物多样性与生态系统功能(BEF-China)研究计划的框架下,我们在3年时间里沿树种丰富度梯度反复采集林下生物量样本,研究了不同环境处理中树种丰富度、树种特性和时间对林下生物量的影响。尽管我们发现乔木层特性对林下生物量有显著和一致的影响,但是树种丰富度对后者却不具有这种影响。另外,在森林结构层之间,可能并不存在单一的、具有普遍性的上层乔木树种丰富度与林下生产力的相关关系,并且与上层乔木相关的环境因素(如透光率)对林下生产力的贡献程度会随着时间而变化。总体而言,我们的结果表明,在研究森林结构层之间的关系时应将时间动态变化考虑在内。     

A comparative analysis of total serum miRNA profiles identifies novel signature that is highly indicative of metastatic melanoma: a pilot study

Abstract

Context: Quantification of circulating microRNAs (miRNAs) has recently become feasible and reliable, with most efforts focusing on miRNAs overexpressed by cancer cells.

Objective: Identification of a characteristic circulating miRNAs profile in melanoma patients.

Methods: We conducted a pilot study comprised of unbiased qPCR comparison of serum miRNA profiles between metastatic melanoma patients and healthy donors.

Results: Loss of two normal serum-miRNAs, miR-29c and miR-324-3p, is highly indicative of metastatic melanoma. Hierarchical clustering analysis supported the results and clearly distinguished melanoma patients from healthy donors, metastatic colon and renal cancer patients.

Discussion and conclusions: This approach is independent of tumor heterogeneity and is expected to have superior biomarker performances.