Application of low-rank approximation using truncated singular value decomposition for noise reduction in hyperpolarized 13 C NMR spectroscopy.

Dissolution dynamic nuclear polarization allows in vivo studies of metabolic flux using 13 C-hyperpolarized tracers by enhancing signal intensity by up to four orders of magnitude. The T1 for in vivo applications is typically in the range of 10-50 s for the different 13 C-enriched metabolic substrates; the exponential loss of polarization due to various relaxation mechanisms leads to a strong reduction of the signal-to-noise ratio (SNR). A common solution to the problem of low SNR is the accumulation/averaging of consecutive spectra. However, some limitations related to long delays between consecutive scans occur: in particular, following biochemical kinetics and estimate apparent enzymatic constants becomes time critical when measurement scans are repeated with the typical delay of about 3 T1 . Here we propose a method to dramatically reduce the noise, and therefore also the acquisition times, by computing, via truncated singular value decomposition, a low-rank approximation to the individual complex time-domain signals. Moreover, this approach has the additional advantage that the phase correction can be applied to the spectra already denoised, thus greatly reducing phase correction errors. We have tested the method on (1) simulated data; (2) performing dissolution of hyperpolarized 1-13 C-pyruvate in standard conditions and (3) in vivo data sets, using a porcine model injected with hyperpolarized Na-1-13 C-acetate. It was shown that the presented method reduces the noise level in all the experimental data sets, allowing the retrieval of signals from highly noisy data without any prior phase correction pre-processing. The effects of the proposed approach on the quantification of metabolic kinetics parameters have to be shown by full quantification studies.

No evidence of bovine papillomavirus type 1 or 2 infection in healthy equids.

BACKGROUND: There is a large body of evidence supporting bovine papillomavirus types 1 and 2 (BPV1; BPV2) as aetiological agents of equine sarcoids. However, there is conflicting data regarding BPV1/2 infection in sarcoid-free equids. OBJECTIVES: Data obtained between 2007 and 2017 by BPV1/2 screening of sarcoids and nonsarcoid tumours vs. samples from healthy equids are presented to help clarify this issue. STUDY DESIGN: Cross-sectional study. METHODS: Tumour material obtained from horses, donkeys and mules with confirmed sarcoids (n = 130), suspected sarcoids (n = 120), or nonsarcoid lesions (n = 70), skin biopsies from 102 tumour-free horses and dandruff/hair roots from 35 tumour-free donkeys and mules were screened for BPV1/2 infection. Sample DNA was extracted and validated by equine ß-actin PCR. BPV1/2 screening was performed by BPV1/2 E5-specific PCR allowing for the detection of less than 10 viral DNA molecules. Twenty-six amplicons were bidirectionally sequenced and compared to known E5 variants using BLAST program. RESULTS: BPV1/2 E5 PCR scored positive for 130/130 diagnosed sarcoids, 63/120 suspected sarcoids and 13/70 nonsarcoid lesions, whereas 137/137 DNA aliquots derived from tumour-free equids tested negative. On predicted E5 protein level, six different BPV1 E5 variants were identified. MAIN LIMITATIONS: The diagnosis of equine sarcoid was not confirmed in 120 lesions. CONCLUSIONS: Lack of BPV1/2 E5 DNA in tumour-free equids and the prevalence of sarcoid disease in young adult individuals suggest that the time span between initial infection and sarcoid development is short. This contrasts with the long phase of virus latency characterising infection of humans by carcinogenic papillomaviruses. Presence of BPV1/2 DNA in several cases of poor wound healing/hypergranulation and dermatitis points to these skin disorders being possibly co-induced by BPV1/2. PCR screening of tumour tissue/scrapings for BPV1/2 DNA represents a reliable tool for the rapid validation of a clinical diagnosis of equine sarcoid.

Equine papillomavirus type 2: An equine equivalent to human papillomavirus 16?

In horses, squamous cell carcinomas (SCC) commonly affect the external genitals. There is growing evidence that equine papillomavirus type 2 (EcPV2) infection promotes disease development. To assess the possible association of EcPV2 with equine SCCs of the head (HSCC), 15 HSCC DNA samples were screened by E6/E7, E2, and LCR PCR and amplicons were analysed for sequence variations. The physical form of EcPV2 in HSCC, genital lesions, and smegma from horses with SCC was then addressed using EcPV2 immunocapture PCR (IC/PCR) for detection of virion, and E6 vs. E2 qPCR to investigate possible integration events. Four of 15 HSCC tested positive for EcPV2 DNA and harboured known or novel genetic variants of E6, E7, E2 and the LCR. Eighteen of 35 sample extracts including 3/4 smegma samples scored positive by IC/PCR, suggesting that about 51% of tested extracts harboured virions. E6/E2 qPCR from tumour DNA revealed E2/E6 copies/cell ranging between <1 (E2; E6) and 797 (E2) or 1434 (E6). IC/PCR-positive smegma samples contained higher E2 and E6 copy numbers, ranging between 1490 and 4.95×105 (E2) or 2227 and 8.54×105 (E6) copies/cell. Together with IC/PCR results, this finding suggests that smegma can serve as a rich EcPV2 reservoir. HSCCs harboured significantly lower viral DNA amounts (<1-25 copies/cell) than most genital tumour and smegma DNA isolates. The majority of samples contained more E6 than E2 DNA, with E6:E2 ratios ranging between 0.88 and 4.12. Although not statistically significant (P>0.05), this finding suggests that EcPV2 can integrate into the equine host cell genome.

A simple surgical treatment for bovine digital dermatitis-associated white line lesions and sole ulcers.

Non-healing white line disease (nhWLD) and sole ulcers (nhSU) are seen increasingly in herds endemically affected with bovine digital dermatitis (BDD). In 35 cows with 42 nhWLD or nhSU lesions, the healing process was monitored for up to 28 or 38 days following extensive debridement of loose horn and infected corium under regional anaesthesia, and topical application of tetracycline spray with bandaging. By 28 days, 27/42 (64%) nhWLD and nhSU were completely covered by a new horn layer and this increased to 30/42 (71%) that had healed by 38 days. Lesion sizes on day 0 correlated with clinical healing within the study period. In view of this satisfying therapeutic result, the terms nhWLD and nhSU are proposed for BDD-associated white line disease (BDD-WLD) and BDD-associated sole ulcers (BDD-SU), respectively.

Occurrence of Treponema DNA in equine hoof canker and normal hoof tissue.

REASONS FOR PERFORMING STUDY: Equine hoof canker is a chronic pododermatitis of still unknown aetiology. Recent findings reported for 3 canker-bearing individuals are suggestive for Treponema spp. having a role in disease pathogenesis. OBJECTIVES: Based on this hypothesised association, we assessed a larger number of DNA samples from hooves with canker and normal hooves for the presence of treponemal DNA. STUDY DESIGN: Retrospective survey of archived material. METHODS: The study involved 71 archival, PCR-compatible DNA extractions purified from 59 canker samples obtained from 26 equine cases and from 12 hoof biopsies taken from 9 canker-free control horses. Presence of treponemal DNA was assessed by qualitative PCR using 4 different primer pairs recognising in sum a broad range of Treponema ssp. Obtained amplification products were identified by bidirectional sequencing and BLAST alignment. RESULTS: Treponemal DNA was detected in 37 of 59 canker DNA samples from 19 of 26 cases and in 9 of 12 hoof DNA samples from 7 of 9 healthy individuals. Canine oral Treponema sp. and Treponema medium ssp. bovis were the most frequently detected treponemal sequences in hoof canker, while control tissues were mainly shown to harbour Treponema refringens-like or canine oral Treponema-like DNA. All control samples tested negative for T. medium ssp. bovis DNA. CONCLUSIONS: Treponema DNA was detectable in the majority of hoof canker and control samples. The sample groups differed to some extent regarding identified Treponema phylotypes; however, this finding may be explained by the methodology used. Treponemes that are highly similar to bovine digital dermatitis treponemes are present in canker lesions. However, further work is needed to clarify the specific contribution of the identified Treponema phylotypes to the pathogenesis of disease.

Isolation of Treponema and Tannerella spp. from equine odontoclastic tooth resorption and hypercementosis related periodontal disease.

REASONS FOR PERFORMING STUDY: Red complex bacteria, i.e. Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia, are involved in the onset and progression of periodontal disease in man, yet seldom inhabit the oral cavity of healthy individuals. Periodontal disease is also encountered in horses, with equine odontoclastic tooth resorption and hypercementosis (EOTRH) constituting a particular form of disease. However, only little is known about the oral microbiome of healthy and periodontitis-affected equids. OBJECTIVE: We aimed to test the hypothesis that red complex bacteria are also associated with EOTRH-related periodontal disease. STUDY DESIGN: Controlled cross-sectional study. METHODS: We screened DNA purified from crevicular fluid derived from 23 EOTRH-affected and 21 disease-free horses for the presence of Treponema spp., Tannerella spp. and Porphyromonas gingivalis DNA by polymerase chain reaction. Subsequently, amplified DNA was bidirectionally sequenced and identified via BLAST analysis. RESULTS: Treponema and/or Tannerella DNA was detected in 100% of periodontitis-related samples and in 52.2% of DNA derived from healthy horses. Twenty-six amplicon sequences were 98-100% homologous to published bacterial sequences, which mostly corresponded to Treponema pectinovorum, oral Treponema clones JU025 and OMZ 840, and Tannerella forsythia. P. gingivalis DNA was only found in 3 EOTRH-related samples. Forty-three amplicon sequences revealed weaker homologies ranging between 80% and 97% to known Treponema or Tannerella strains, partly because of their heterogeneity, partly because they obviously represented so far unknown types. CONCLUSIONS: This is the first report in which known and novel Treponema and Tannerella spp. were isolated in association with EOTRH-related periodontal disease.

Automatic assignment of 1H-NMR spectra of small molecules.

A novel data-evaluation procedure for the automatic atom to peak or multiplet assignment of 1H-NMR spectra of small molecules has been developed using a fast and robust expert system. The applicability and reliability of the method are demonstrated by comparison of a manually assigned database of 1H-NMR spectra with the assignments produced by the automatic procedure. The results of this analysis show an excellent success ratio, indicating that this new algorithm can have a major impact as a time saving tool for the organic chemist. A new graphical feature used to illustrate both the stability and quality of the elementary assignments is also introduced.

Bovine papillomavirus type 1 and Equus caballus papillomavirus 2 in equine squamous cell carcinoma of the head and neck in a Connemara mare.

In January 2010, 18 months after excision of an ocular squamous cell carcinoma (SCC), a Connemara mare presented with anorexia and periorbital/parotideal lesions. Post mortem examination revealed these lesions as forming one entity, with 2 additional growths in the retropharyngeal region and the left jugular groove, respectively. The lesions were confirmed histopathologically as SCCs. Using PCR, peripheral blood mononuclear cells (PBMCs) from 2008 and 2010, tumour tissue, intact skin and vulval mucosa were screened for Equus caballus papillomavirus type 2 (EcPV-2) and bovine papillomavirus types 1 and 2 (BPV-1/2) DNA. Whereas PBMCs from 2008 scored negative, EcPV-2 DNA was present in PBMCs and SCCs from 2010. Furthermore, reverse transcription PCR revealed EcPV-2 E6 transcripts in these samples. BPV-1/2 DNA, but not RNA, was demonstrated in the periorbital/parotideal mass, the SCC of the jugular groove, vulval mucosa and intact skin, but not in the pharyngeal SCC and PBMCs. Sequencing revealed a 99% similarity of EcPV-2 amplicons with the published EcPV-2 sequence. BPV-1/2 amplicons corresponded to BPV type 1. This report is the first to describe co-presence of BPV-1 and EcPV-2 DNA in a pony affected by an uncommon form of nongenital SCC, and the detection of EcPV-2 transcripts in lesions and PBMCs.

Equus caballus papillomavirus-2 (EcPV-2): an infectious cause for equine genital cancer?

REASONS FOR PERFORMING STUDY: The aetiology of genital squamous cell carcinoma (SCC) in horses remains unknown, but the similarity to the disease in man, for which papillomavirus infection has been shown to be a causal factor, requires to be investigated in horses. HYPOTHESIS: One or more novel papillomaviruses cause equine genital SCC and its associated premalignant lesions. METHODS: DNA was extracted from samples of equine genital SCC and performed rolling circle amplification, in order to identify closed circular DNA viral genomes within the samples. The amplified DNA was subcloned and sequenced and the DNA sequence compared to that of other papillomavirus genomes. Using PCR primers developed from these genomic DNA sequences, studies were then carried out in order to identify the frequency at which the viral DNA could be identified in equine genital cancer samples from horses in both the UK, Australia and Austria. Finally, in situ hybridisation using specific probes developed from this DNA sequence were used to confirm the presence of the viral RNA sequences in the neoplastic cells in these lesions. RESULTS: The full length genome of a novel papillomavirus species was characterised from the equine genital SCC tissue and termed Equus caballus papillomavirus-2 (EcPV-2). Viral DNA and RNA was identified in the genital tumour samples, but not in the adjacent histologically normal tissue. EcPV-2 DNA could not be identified in equine ocular or nasal carcinomas or within the scrotal skin or in most smegma samples obtained from tumour-free horses. Sequencing of amplicons, generated from the archived equine genital tumours, identified variations within E1 and E6 on DNA and predicted protein level. CONCLUSIONS: A novel papillomavirus, EcPV-2, is likely to play a causal role in the pathogenesis of equine genital epithelial tumours. POTENTIAL RELEVANCE: Identification of a papillomavirus causal for genital carcinomas in horses may lead to development of a vaccine that could be used to prevent this serious disease in horses. This would be analogous to man, where vaccination against oncogenic papillomavirus species is currently being used to help prevent cervical cancer.

PERFIDI filters to suppress and/or quantify relaxation time components in multi-component systems: an example for fat-water systems.

Parametrically Enabled Relaxation FIlters with Double and multiple Inversion (PERFIDI) is an experimental NMR/MRI technique devised to analyze samples/voxels characterized by multi-exponential longitudinal relaxation. It is based on a linear combination of NMR sequences with suitable preambles composed of inversion pulses. Given any standard NMR/MRI sequence, it permits one to modify it in a way which will attenuate, in a predictable manner and before data acquisition, signals arising from components with different r rates (r=1/T1). Consequently, it is possible to define relatively simple protocols to suppress and/or to quantify signals of different components. This article describes a simple way to construct low-pass, high-pass and band-pass PERFIDI filters. Experimental data are presented in which the method has been used to separate fat and water proton signals. We also present a novel protocol for very fast determination of the ratio between the fat signal and the total signal which avoids any time-consuming magnetization recovery multi-array data acquisition. The method has been validated also for MRI, producing well T1-contrasted images.

A compact electron beam ion source with integrated Wien filter providing mass and charge state separated beams of highly charged ions.

A Wien filter was designed for and tested with a room temperature electron beam ion source (EBIS). Xenon charge state spectra up to the charge state Xe46+ were resolved as well as the isotopes of krypton using apertures of different sizes. The complete setup consisting of an EBIS and a Wien filter has a length of less than 1 m substituting a complete classical beamline setup. The Wien filter is equipped with removable permanent magnets. Hence total beam current measurements are possible via simple removal of the permanent magnets. In dependence on the needs of resolution a weak (0.2 T) or a strong (0.5 T) magnets setup can be used. In this paper the principle of operation and the design of the Wien filter meeting the requirements of an EBIS are briefly discussed. The first ion beam extraction and separation experiments with a Dresden EBIS are presented.

Differential plus polarographic determination of submicrogram quantities of carmustine and related compounds in biological samples.

A polarographic method was developed to determine the antineoplastic agent carmustine and other nitrosoureas, such as N-methyl-N-nitrosourea and N-cyclohexyl-N-nitrosourea, in biological fluids at levels well below 1 microgram/ml or g. The stability of carmustine in different media was investigated to prevent losses during administration or assay. Examples of nitrosourea determination in biological samples are given.