Gene transcripts expressed in equine white blood cells are potential biomarkers of extracorporeal shock wave therapy.

Extracorporeal shockwave therapy (ESWT) is a treatment applied to musculoskeletal injuries in equine athletes to alleviate pain and accelerate healing. ESWT also causes acute tissue damage. Therefore, its ability to act as an analgesic and cause tissue damage potentially increases the risk of a catastrophic event if used shortly before a strenuous competition such as horseracing. While ESWT is prohibited by many racing jurisdictions within 10 days prior to competition, a test to detect whether a horse has received ESWT is needed. ESWT changes the protein levels of inflammatory mediators in blood, and white blood cells (WBC) typically produce these proteins. Changes in gene expression precede changes in protein production; thus, it was hypothesized that WBC gene transcripts might serve as biomarkers of ESWT. To test this hypothesis, six thoroughbred horses received a single administration of ESWT to the distal limb, and WBC RNA was extracted from blood samples collected before (0 h) and after ESWT (2, 4, 6, 24, 48, and 72 h). Targeted and untargeted analyses evaluated the transcriptome using quantitative PCR (qPCR) and microarray. The expression of IL-1α, IL-1ß, TNF-α, IL-1Ra1, IL-1Ra2 and TGF-ß1, and BMPR1A in circulating WBCs was significantly up-regulated, while IFN-γ, ZNF483, TMEM80, CAH6, ENPP, and S8723 were significantly down-regulated at various time points following ESWT. These data support the hypothesis that changes in WBC gene transcripts could serve as biomarkers for ESWT.

Coxsackievirus Adenovirus Receptor Loss Impairs Adult Neurogenesis, Synapse Content, and Hippocampus Plasticity.

UNLABELLED: Although we are beginning to understand the late stage of neurodegenerative diseases, the molecular defects associated with the initiation of impaired cognition are poorly characterized. Here, we demonstrate that in the adult brain, the coxsackievirus and adenovirus receptor (CAR) is located on neuron projections, at the presynapse in mature neurons, and on the soma of immature neurons in the hippocampus. In a proinflammatory or diseased environment, CAR is lost from immature neurons in the hippocampus. Strikingly, in hippocampi of patients at early stages of late-onset Alzheimer's disease (AD), CAR levels are significantly reduced. Similarly, in triple-transgenic AD mice, CAR levels in hippocampi are low and further reduced after systemic inflammation. Genetic deletion of CAR from the mouse brain triggers deficits in adult neurogenesis and synapse homeostasis that lead to impaired hippocampal plasticity and cognitive deficits. We propose that post-translational CAR loss of function contributes to cognitive defects in healthy and diseased-primed brains. SIGNIFICANCE STATEMENT: This study addressed the role of the coxsackievirus and adenovirus receptor (CAR), a single-pass cell adhesion molecule, in the adult brain. Our results demonstrate that CAR is expressed by mature neurons throughout the brain. In addition, we propose divergent roles for CAR in immature neurons, during neurogenesis, and at the mature synapse. Notably, CAR loss of function also affects hippocampal plasticity.

Yinzhihuang attenuates ANIT-induced intrahepatic cholestasis in rats through upregulation of Mrp2 and Bsep expressions.

BACKGROUND: The treatment of intrahepatic cholestasis has been limited, and development of an effective drug is needed. Clinical studies have shown that Yinzhihuang (YZH), a traditional Chinese decoction, enhances bilirubin clearance. The goal of this study was to determine the protective effect of YZH on experimental intrahepatic cholestasis in young rats and to explore its underlying molecular mechanisms. METHODS: Intrahepatic cholestasis in rats was induced by α-naphthylisothiocyanate (ANIT) on days 1 and 8. The rats received YZH, ursodeoxycholic acid (UDCA), or vehicle for 9 d and were killed on either day 3 or day 10. Serum biomarkers, liver histology, and the distribution of protein and mRNA expression of Mrp2 and Bsep were analyzed. RESULTS: YZH treatment resulted in decreased levels of serum biomarkers except γ-glutamyl transpeptidase, attenuated liver histological injuries, increased protein expressions of Mrp2 and Bsep, and upregulated expressions of Mrp2 and Bsep mRNAs. The effects of YZH on serum biomarkers (aminotransferase, alanine aminotransferase, and direct bilirubin), liver histology, and Mrp2 mRNA expressions were significantly greater and earlier than those of UDCA. CONCLUSION: Our results suggest that YZH has protective effect against ANIT-induced intrahepatic cholestasis in rats, through upregulation of Mrp2 and Bsep expressions.

Plasma interleukin-6 concentration in Standardbred racehorses determined by means of a novel validated ELISA.

OBJECTIVE: To evaluate plasma interleukin 6 (IL-6) concentration in Standardbred racehorses by means of a novel ELISA following validation of the assay for use with equine plasma samples. SAMPLE: Plasma samples obtained from 25 Thoroughbreds for use in assay validation and from 319 Standardbred racehorses at rest 2 to 2.5 hours prior to warm-up and racing. PROCEDURES: A sandwich ELISA was developed with equine anti-IL-6 polyclonal antibody and the biotin-streptavidin chemical interaction to enhance sensitivity. The assay was validated for specificity, sensitivity, precision, and accuracy by use of both recombinant and endogenous proteins. RESULTS: For the assay, cross-reactivity with other human and equine cytokines was very low or absent. Serial dilution of plasma samples resulted in proportional decreases in reactivity, indicating high specificity of the method. Partial replacement of detection antibody with capture antibody or pretreatment of samples with capture antibody caused assay signals to significantly decrease by 55%. The inter- and intra-assay precisions were ≤ 13.6% and ≤ 9.3%, respectively; inter- and intra-assay accuracies were within ranges of ± 14.1% and ± 8.6%, respectively, at concentrations from 78 to 5,000 pg/mL, and the sensitivity was 18 pg/mL. Plasma IL-6 concentration varied widely among the 319 Standardbreds at rest (range, 0 to 193,630 pg/mL; mean, 6,153 pg/mL; median, 376 pg/mL). CONCLUSIONS AND CLINICAL RELEVANCE: This ELISA method proved suitable for quantification of IL-6 concentration in equine plasma samples. Plasma IL-6 concentration was high (> 10,000 pg/mL) in 9.1% of the Standardbred racehorses, which warrants further investigation.

Incidence and related clinical factors of falls among older Chinese veterans in military communities: a prospective study.

[Purpose] The aim of this study was to determine fall incidence and explore clinical factors of falls among older Chinese veterans in military communities. [Subjects and Methods] We carried out a 12-month prospective study among 13 military communities in Beijing, China. Fall events were obtained by self-report to military community liaisons and monthly telephone interviews by researchers. [Results] Among the final sample of 447 older veterans, 86 fell once, 25 fell twice or more, and 152 falls occurred altogether. The incidence of falls and fallers were 342/1,000 person-years and 249/1,000 person-years. In Cox regression models, independent clinical factors associated with falls were visual acuity (RR=0.47), stroke (RR=2.43), lumbar diseases (RR=1.73), sedatives (RR=1.80), fall history in the past 6 months (RR=2.77), multiple chronic diseases (RR=1.53), multiple medications (RR=1.34), and five-repetition sit-to-stand test score (RR=1.41). Hearing acuity was close to being statistically significant. [Conclusion] The incidences of falls and fallers among older Chinese veterans were lower than those of Hong Kong and western countries. The clinical risk factors of falls were poor senses, stroke, lumbar diseases, taking sedatives, fall history in the past 6 months, having multiple chronic diseases, taking multiple medications, and poor physical function. The preventive strategies targeting the above risk factors are very significant for reducing falls.

Factors Associated with Performance-based Physical Function of Older Veterans of the PLAAF: A Pilot Study.

[Purpose] This study investigated the factors associated with performance-based physical function of older veterans of the People's Liberation Army Air Force of China (PLAAF). [Subjects and Methods] A cross-sectional survey of 146 older veterans of the PLAAF was carried out. Their physical function was measured using the Chinese Mini-Physical Performance Testing (CM-PPT). The demographics and health status (including physical measures, blood chemical tests, chronic diseases, and number of morbidities) were collected from health examination reports and computer records of case history. Cognition was measured using the Mini-Mental Status Examination (MMSE). [Results] In multiple linear regressions, age, MMSE, Parkinsonism, and chronic obstructive pulmonary disease were independently associated with CM-PPT, while previous stroke and albumin level reached borderline statistical significance. The association between the number of morbidities and CM-PPT was significant after adjustment for MMSE and demographics. The CM-PPT of low (0 or 1), medium (2 to 4) and high count (5 or more) morbidities were 11.3±3.9, 10.2±4.1, 6.1±3.8 respectively, and the difference among these three groups was significant. [Conclusion] Some modified conditions and the number of chronic diseases might be associated with the physical function of older veterans of the PLAAF.

Identification of sample donor by 24-plex short tandem repeat in a post-race equine plasma containing dexamethasone.

BACKGROUND: Animal sport such as horseracing is tainted with drug abuse as are human sports. Treatment of racehorses on race day with therapeutic medications in most cases is banned, and thus, it is essential to monitor the illicit use of drugs in the racing horse to maintain integrity of racing, ensure fair competition and protect the health, safety and welfare of the horse, jockeys and drivers. In the event of a dispute over the identity of the sample donor, if the regulator can provide evidence that the DNA genotype profile of the post-race sample matched that of the alleged donor, then the potential drug violation case might be easily resolved without legal challenges. CASE DESCRIPTION: We present a case study of a racehorse sample that tested positive for dexamethasone in a post-race plasma sample in Pennsylvania (PA) but the result was challenged by the trainer of the horse. Dexamethasone is a synthetic glucocorticoid widely used in the management of musculoskeletal problems in horses but its presence in the horse during competition is banned by the PA Racing Commissions. The presence of dexamethasone in the post-competition plasma sample was confirmed using liquid chromatography-tandem mass spectrometry. However, this finding was challenged by the trainer of the horse alleging that the post-race sample was not collected from his/her horse and thus petitioned the Commission to be absolved of any wrong-doing. To resolve the dispute, a DNA test was ordered by the PA Racing Commission to identify the correct donor of the dexamethasone positive sample. For this purpose, a 24-plex short tandem repeat analysis to detect 21 equine markers and three human markers was employed. The results indicated that all the samples tested had identical DNA profiles and thus, it was concluded that the samples were collected from the same horse and that the probability of drawing a false conclusion was approximately zero (1.5 × 10(-15)). CONCLUSIONS: The plasma sample confirmed for the presence of dexamethasone was collected from the alleged horse.

Efficient use of retention time for the analysis of 302 drugs in equine plasma by liquid chromatography-MS/MS with scheduled multiple reaction monitoring and instant library searching for doping control.

Multiple drug target analysis (MDTA) used in doping control is more efficient than single drug target analysis (SDTA). The number of drugs with the potential for abuse is so extensive that full coverage is not possible with SDTA. To address this problem, a liquid chromatography tandem mass spectrometric method was developed for simultaneous analysis of 302 drugs using a scheduled multiple reaction monitoring (s-MRM) algorithm. With a known retention time of an analyte, the s-MRM algorithm monitors each MRM transition only around its expected retention time. Analytes were recovered from plasma by liquid-liquid extraction. Information-dependent acquisition (IDA) functionality was used to combine s-MRM with enhanced product ion (EPI) scans within the same chromatographic analysis. An EPI spectrum library was also generated for rapid identification of analytes. Analysis time for the 302 drugs was 7 min. Scheduled MRM improved the quality of the chromatograms, signal response, reproducibility, and enhanced signal-to-noise ratio (S/N), resulting in more data points. Reduction in total cycle time from 2.4 s in conventional MRM (c-MRM) to 1 s in s-MRM allowed completion of the EPI scan at the same time. The speed for screening and identification of multiple drugs in equine plasma for doping control analysis was greatly improved by this method.

Analysis of gabapentin in equine plasma with measurement uncertainty estimation by liquid chromatography-tandem mass spectrometry.

Gabapentin (GPT) is an antiepileptic drug that was approved in 1993 for use in the management of neurotrophic pain and as an adjunctive therapy for refractory partial seizure in humans. It is also being tested in veterinary medicine as an adjunctive medication in the treatment of pain due to laminitis, neuropathic, or chronic pain. Gabapentin is readily available by prescription and even on the internet; therefore, it has the potential of being used in racehorses to mask pain. It is for this reason that a sensitive liquid chromatography-tandem mass spectrometry method has now been developed for the analysis of GPT in equine plasma and for studying the pharmacokinetic and pharmacodynamic profiles of GPT in the horse. Sample preparation was by rapid protein precipitation with acetonitrile. Analyte separation was achieved on a reversed-phase ACE C(18) column and analyzed by a hybrid triple-quadrupole linear ion trap mass spectrometer in positive electrospray ionization mode. Limits of detection, quantification, and confirmation of GPT were 1, 10, and 20 ng/mL, respectively. Calibration curve showed excellent linearity within the 10-2500 ng/mL range (r(2) > 0.999). Intra- and interday precision defined by coefficient of variation was <10%. Intra- and interday accuracy (bias %) was within 90-110%. Measurement uncertainty estimation was 8.6%. The method has been successfully used in the analysis of GPT in equine plasma following its administration to research horses for pharmacokinetic studies and in routine forensic analysis for doping control in racehorses in the State of Pennsylvania.

Detection and confirmation of 60 anabolic and androgenic steroids in equine plasma by liquid chromatography-tandem mass spectrometry with instant library searching.

In 2008, Pennsylvania (PA) became the first State in the USA to ban and enforce the ban on the use of anabolic and androgenic steroids (AAS) in equine athletes by using plasma for analysis. To enforce the ban, a rapid and high-throughput method for analysis of 60 AAS in equine plasma was developed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Analytes were recovered from plasma by liquid-liquid extraction (LLE) using methyl tert-butyl ether, separated on a reversed-phase C18 column and analyzed by electrospray ionization mass spectrometry. Multiple-reaction monitoring (MRM) scan was employed for screening. When the MRM signal of an analyte exceeded 1000 counts per second (cps), information-dependent acquisition (IDA) triggered generation of an enhanced product ion (EPI) scan of the analyte. A library for the analytes was simultaneously established using the EPI spectrum. Unambiguous identification of any of the 60 AAS in a test sample was based on both the presence of MRM response within the correct retention time (t(R)) window and a qualitative match between EPI spectrum of the test sample and that of the reference drug standard stored in the library. Total analysis time was 7 min. The limit of detection (LOD) and limit of confirmation (LOC) for most of the analytes were 0.01-2 ng/mL and 0.1-10 ng/mL, respectively. Recovery of the analytes from plasma by LLE was 74-138%. The method was successfully verified and is routinely used in the screening of post-race equine plasma samples for the presence of these 60 AAS. The method is rapid, sensitive, reproducible, and reliable.

Determining the source of equine bloodstains by dinucleotide repeats.

A novel multiplex of independent dinucleotide tandem repeat (DTR) loci was previously described that is capable of not only discriminating human and equine DNA, but of identifying a single equine source. We report a case in which a bloodstained syringe and two needles were found during inspection of a barn by inspectors of the Pennsylvania Racing Commissions. Using the multiplex and single-locus detection, all 21 equine DTR markers were detected in a suspect horse and two evidence samples, indicating the evidence samples came from the suspect animal. Only six markers were detected in the third evidence sample because the volume of blood was limited. Following whole-genome amplification and single-locus PCR, the third evidence sample detected a total of 17 markers and the likelihood of identity (probability from suspect horse/probability from a random pacer) was 7.0 × 106. The DTR multiplex has some technical limitations, but it is already practical for casework.

[Identification of the active material of anti-hepatic fibrosis from Amydae Carapax].

OBJECTIVE: To identify the active material of anti-hepatic fibrosis from Amydae Carapax. METHODS: Membrane separation technology was adopted to screen active fraction in Amydae Carapax, and the active components were isolated from the active fraction using gel chromatography and high performance liquid chromatography. The purified active components in Amydae Carapax were further analyzed using 4700 series time-of-flight mass spectrometer. RESULTS: Proteins and peptides of Amydae Carapax with molecular weight less than 6000 were proved to have biological activity. 8 components (Bj1-Bj8) were isolated from the active fraction. Bj4, Bj6 and Bj7 were screened as active components. Bj7 was further purified, resulting in 7 components (Bj701-Bj707). Bj704 and Bj707 showed significant biological activity. Mass spectrometry showed three molecular ion peaks with highest abundance, i.e. m/e 526, 542 and 572, i.e. m/e 526, 542 and 572, in Bj707 -A The amino acid sequences of above three peptide compounds were NDDY (Asn-Asp-Asp-Tyr), NPNPT (Asn-Pro-Asn-Pro-Thr), and HGRFG (His-Gly-Arg-Phe-Gly), respectively. And M572 was the most abandunt components. CONCLUSION: Three active peptide compounds of anti-hepatic fibrosis of Amydae Carapax were identified.

Identification of racehorse and sample contamination by novel 24-plex STR system.

Proper identification of racehorses competing in an official race and maintenance of defensible chain of custody are important in doping control regulations. The purpose of this study was to develop a reliable multiplex PCR method for providing genetic evidence for matching donors to test samples by using short tandem repeat (STR) loci. Amplification of 21 STR loci from blood, urine or hair root was achieved in a single tube and STR length polymorphism was analyzed using fluorescent labeled capillary electrophoresis. This novel approach showed an allele confidence interval of 0.19-0.43 bp and size estimation error of 0-0.48 bp. In 90 thoroughbred (TB) and 171 standardbred (STB) horses, the method was highly discriminating and reproducible with probability of false identification of 1 in 10(11) (TB) and 1 in 10(13) (STB). All loci were highly polymorphic with an average probability of identity of 0.18 (TB) and 0.13 (STB), heterozygosity of 0.65 (TB) and 0.68 (STB), and polymorphism information content (PIC) of 0.62 (TB) and 0.69 (STB). The highest allele frequency also reflected the degree of polymorphism due to high correlation with PIC. To obtain evidence of sample tampering with human material, three human specific STR markers were included in the panel. This method is the first in the horseracing industry, specifically designed for racehorse identification and detection of equine sample contamination by human DNA.

Tissue-specific deletion of the coxsackievirus and adenovirus receptor protects mice from virus-induced pancreatitis and myocarditis.

In cultured cells, infection by group B coxsackievirus (CVB) is mediated by the coxsackievirus and adenovirus receptor (CAR), but the importance of this molecule in CVB-induced disease has not been determined. We generated mice with tissue-specific ablation of CAR within each of two major CVB target organs, the pancreas and heart. In the pancreas, deletion of CAR resulted in a significant reduction in both virus titers and virus-induced tissue damage. Similarly, cardiomyocyte-specific CAR deletion resulted in a marked reduction in virus titer, infection-associated cytokine production, and histopathology within the heart. Consistent with the in vivo phenotype, CAR-deficient cardiomyocytes resisted infection in vitro. These results demonstrate a critical function for CAR in the pathogenesis of CVB infection in vivo and in virus tropism for the heart and pancreas.

Microsatellite loci in urine supernatant and stored samples from racehorses.

OBJECTIVE: To evaluate whether urine supernatant contains amplifiable DNA and to determine factors that influence genotyping of samples from racehorses after storage and transportation. SAMPLE POPULATION: 580 urine, 279 whole blood, and 40 plasma samples obtained from 261 Thoroughbreds and Standardbreds. PROCEDURES: Genomic DNA was isolated from stored blood and urine samples collected from racehorses after competition. Quantified DNA was evaluated to determine whether 5 equine microsatellite loci (VHL20, HTG4, AHT4, HMS6, and HMS7) could be amplified by use of PCR techniques. Fragment size of each amplified locus was determined by use of capillary electrophoresis. RESULTS: High-molecular-weight and amplifiable DNA were recovered from refrigerated blood samples, but recovery from urine varied. Deoxyribonucleic acid was recovered from both urine supernatant and sediment. Freeze-thaw cycles of urine caused accumulation of amplifiable DNA in the supernatant and clearance of naked DNA. Repeated freeze-thaw cycles significantly decreased DNA yield and induced DNA degradation, which resulted in failure to detect microsatellite loci. Select drugs detected in test samples did not affect PCR amplification. Contaminants in DNA isolates inhibited PCR amplification and resulted in partial microsatellite profiles. CONCLUSIONS AND CLINICAL RELEVANCE: Properly stored urine and blood samples were successfully genotyped, but subjecting urine to freeze-thaw cycles was most detrimental to the integrity of DNA. Increasing the volume of urine used improved recovery of DNA.

[Effect of Mailuoning injectable powder on experimental vascular occlusion angeitides in rats].

OBJECTIVE: To investigate the effect of Mailuoning injectable powder on experimental vascular occlusion angeitides in rats. METHODS: Rats were injected laurostearic acid into arteria cruralis to induce the model of experimental vascular occlusion angeitides, then we observed the changes of objective sign of rats, and analysed throm ranking through pathological section under electro-microscope. RESULTS: Mailuoning injectable powder could decrease the quantity of throm in blood vessel, and improve hemorrheoiogy. CONCLUSION: The results show that Mailuoning injectable powder has obvious therapeutical effect on experimental vascular occlusion angeitides in rats, and its mechanism may be related to the anti-throm in blood vessel and improving hemorrheology.

Cardiomyocyte-specific deletion of the coxsackievirus and adenovirus receptor results in hyperplasia of the embryonic left ventricle and abnormalities of sinuatrial valves.

The coxsackievirus and adenovirus receptor (CAR), which mediates infection by the viruses most commonly associated with myocarditis, is a transmembrane component of specialized intercellular junctions, including the myocardial intercalated disc; it is known to mediate cell-cell recognition, but its natural function is poorly understood. We used conditional gene targeting to investigate the possible functions of CAR during embryonic development, generating mice with both germline and tissue-specific defects in CAR expression. Homozygous germline deletion of CAR exon 2 or cardiomyocyte-specific gene deletion at embryonic day 10 (E10) mediated by Cre recombinase expressed under the control of the cardiac troponin T promoter resulted in death by E12.5; embryos showed marked cardiac abnormalities by E10.5, with hyperplasia of the left ventricular myocardium, distention of the cardinal veins, and abnormalities of sinuatrial valves. Within the hyperplastic left ventricle, increased numbers of proliferating cells were evident; persistent expression of N-myc in the hyperplastic myocardium and attenuated expression of the trabecular markers atrial natriuretic factor and bone morphogenic protein 10 indicated that proliferating cardiomyocytes had failed to differentiate and form normal trabeculae. In electron micrographs, individual CAR-deficient cardiomyocytes within the left ventricle appeared normal, but intercellular junctions were ill-formed or absent, consistent with the known function of CAR as a junctional molecule; myofibrils were also poorly organized. When cardiomyocyte-specific deletion occurred somewhat later (by E11, mediated by Cre under control of the alpha-myosin heavy chain promoter), animals survived to adulthood and did not have evident cardiac abnormalities. These results indicate that during a specific temporal window, CAR expression on cardiomyocytes is essential for normal cardiac development. In addition, the results suggest that CAR-mediated intercellular contacts may regulate proliferation and differentiation of cardiomyocytes within the embryonic left ventricular wall.

Structure and chromosomal localization of the murine coxsackievirus and adenovirus receptor gene.

We analyzed BAC genomic clones encoding the murine coxsackievirus and adenovirus receptor (mCAR). The mCAR gene is situated on the distal portion of murine chromosome 16, and is composed of at least eight exons, with intron-exon boundaries similar to those reported for the human CAR gene. We previously described two cDNAs encoding mCAR isoforms: the extracellular and transmembrane portions of both are encoded by exons 1-6; the cytoplasmic domain of mCAR 1 is encoded by exon 7, whereas mCAR 2 results from an RNA splice linking the proximal portion of exon 7 to an alternative exon 8. RT-PCR analysis of the mCAR RNA 5'-terminus suggests that transcription may begin 141-161 nucleotides upstream of the ATG translational start site.