Papillomavirus infection and squamous cell carcinoma in horses.

Squamous cell carcinoma (SCC) is a common disease that seriously impairs the health and welfare of affected horses and other equids. In humans, almost all cervical carcinomas, a high percentage of anogenital SCCs and a subset of SCCs of the head and neck are caused by high-risk human papillomavirus (hrHPV) infection. Since hrHPV-induced human cancers and equine SCC have similar cytological and histopathological features, it has been hypothesised that equine SCCs could also be induced by papillomaviruses. This review provides an overview of the current evidence for an aetiological association between papillomavirus infections and equine SCCs and SCC precursor lesions. SCC of apparently papillomavirus-unrelated aetiology are also discussed, as are recent advances in equine SCC prophylaxis.

Enzyme Armoring by an Organosilica Layer: Synthesis and Characterization of Hybrid Organic/Inorganic Nanobiocatalysts.

The availability of highly stable and reusable enzymes is one of the main challenges in bio-based industrial processes. Enzyme immobilization and encapsulation represent promising strategies to reach this goal. In this chapter, the synthetic strategy to produce hybrid organic/inorganic nanobiocatalysts (NBC) is reported. This strategy is based on the sequential immobilization of an enzyme on the surface of silica nanoparticles followed by the growth, at the surface of the nanoparticles, of a shielding layer which serves as an armor to protect the enzyme against denaturation/degradation. This armor is produced through a thickness-controlled organosilane poly-condensation onto the nanoparticle surface around the enzyme to form a protective organosilica layer. The armored nanobiocatalysts present enhanced catalytic activity and improved stability against heat, pH, chaotropic agents, proteases, and ultrasound. The method is versatile in that it can be successfully adapted to a number of different enzymes.

A Biocatalytic Nanomaterial for the Label-Free Detection of Virus-Like Particles.

The design of nanomaterials that are capable of specific and sensitive biomolecular recognition is an on-going challenge in the chemical and biochemical sciences. A number of sophisticated artificial systems have been designed to specifically recognize a variety of targets. However, methods based on natural biomolecular detection systems using antibodies are often superior. Besides greater affinity and selectivity, antibodies can be easily coupled to enzymatic systems that act as signal amplifiers, thus permitting impressively low detection limits. The possibility to translate this concept to artificial recognition systems remains limited due to design incompatibilities. Here we describe the synthesis of a synthetic nanomaterial capable of specific biomolecular detection by using an internal biocatalytic colorimetric detection and amplification system. The design of this nanomaterial relies on the ability to accurately grow hybrid protein-organosilica layers at the surface of silica nanoparticles. The method allows for label-free detection and quantification of targets at picomolar concentrations.

Enzyme Shielding in an Enzyme-thin and Soft Organosilica Layer.

The fragile nature of most enzymes is a major hindrance to their use in industrial processes. Herein, we describe a synthetic chemical strategy to produce hybrid organic/inorganic nanobiocatalysts; it exploits the self-assembly of silane building blocks at the surface of enzymes to grow an organosilica layer, of controlled thickness, that fully shields the enzyme. Remarkably, the enzyme triggers a rearrangement of this organosilica layer into a significantly soft structure. We demonstrate that this change in stiffness correlates with the biocatalytic turnover rate, and that the organosilica layer shields the enzyme in a soft environment with a markedly enhanced resistance to denaturing stresses.

Treponema DNA in bovine 'non-healing' versus common sole ulcers and white line disease.

Bovine digital dermatitis (BDD)-associated 'non-healing' white line disease (nhWLD) and 'non-healing' sole ulcers (nhSU) are increasingly encountered in cattle. Using established PCR protocols, 42 nhWLD/nhSU, 25 BDD and 15 common WLD DNA isolates were screened for the presence of Treponema DNA. Obtained amplicons were identified by gel electrophoresis and sequencing. Independent from their source, Treponema DNA was isolated from all lesions, but the lesion type varied with the detected Treponema phylotypes. Whilst Treponema pedis was omnipresent, T. medium was almost exclusively identified in BDD and associated nhWLD/nhSU lesions when compared to common WLD. This observation was confirmed by specific T. medium PCR scoring positive for all BDD and nhWLD/nhSU lesions, but for only 1/15 (6.7%) common WLD lesions. It is suggested that T. medium may have an active role in the pathogenesis of nhWLD/nhSU but further work is needed to verify this concept.

Virus-like particles as virus substitutes to design artificial virus-recognition nanomaterials.

Functional recognition imprints of virus-like particles, at the surface of silica particles, were generated following a strategy based on protein-templated polycondensation of organosilanes.

Structure and mechanism of human UDP-xylose synthase: evidence for a promoting role of sugar ring distortion in a three-step catalytic conversion of UDP-glucuronic acid.

UDP-xylose synthase (UXS) catalyzes decarboxylation of UDP-D-glucuronic acid to UDP-xylose. In mammals, UDP-xylose serves to initiate glycosaminoglycan synthesis on the protein core of extracellular matrix proteoglycans. Lack of UXS activity leads to a defective extracellular matrix, resulting in strong interference with cell signaling pathways. We present comprehensive structural and mechanistic characterization of the human form of UXS. The 1.26-Å crystal structure of the enzyme bound with NAD(+) and UDP reveals a homodimeric short-chain dehydrogenase/reductase (SDR), belonging to the NDP-sugar epimerases/dehydratases subclass. We show that enzymatic reaction proceeds in three chemical steps via UDP-4-keto-D-glucuronic acid and UDP-4-keto-pentose intermediates. Molecular dynamics simulations reveal that the D-glucuronyl ring accommodated by UXS features a marked (4)C(1) chair to B(O,3) boat distortion that facilitates catalysis in two different ways. It promotes oxidation at C(4) (step 1) by aligning the enzymatic base Tyr(147) with the reactive substrate hydroxyl and it brings the carboxylate group at C(5) into an almost fully axial position, ideal for decarboxylation of UDP-4-keto-D-glucuronic acid in the second chemical step. The protonated side chain of Tyr(147) stabilizes the enolate of decarboxylated C(4) keto species ((2)H(1) half-chair) that is then protonated from the Si face at C(5), involving water coordinated by Glu(120). Arg(277), which is positioned by a salt-link interaction with Glu(120), closes up the catalytic site and prevents release of the UDP-4-keto-pentose and NADH intermediates. Hydrogenation of the C(4) keto group by NADH, assisted by Tyr(147) as catalytic proton donor, yields UDP-xylose adopting the relaxed (4)C(1) chair conformation (step 3).

EcPV-2 is transcriptionally active in equine SCC but only rarely detectable in swabs and semen from healthy horses.

Squamous cell carcinomas (SCC) are malignant tumours arising from keratinocytes. In horses, there is increasing evidence for Equus caballus papillomavirus type 2 (EcPV-2) being causally involved in SCC development. However, only little is known regarding intralesional transcription of the virus, and sparse information on the incidence of EcPV-2 infection in healthy equids is available so far. Using RT-PCR, total mRNA from 8 EcPV-2 DNA-positive and 1 EcPV-2 negative SCC/SCC precursor lesions was screened for the presence of EcPV-2 E6 and E1 transcripts. Using PCR, we tested 193 sample specimens (30 ocular swabs, 94 genital swabs, 54 semen and 15 milk samples) from a total of 161 apparently healthy horses for the presence of EcPV-2 genes E7 and E6 or E2. Positive results were confirmed by repeating the PCR reactions, and by amplicon sequencing. E6 mRNA was detectable in 8/8 EcPV-2 DNA-positive lesions, whereas only 3/8 scored positive for E1 mRNA. EcPV-2 PCR scored positive for DNA from 1/30 ocular swabs, 4/94 genital swabs, 0/54 semen and 0/15 milk samples, thus resulting in an overall detection rate of 5/193, i.e. 2.6%. The demonstrated presence of viral mRNA in all EcPV-2 DNA-positive lesions is suggestive for an active pathogenic role of the virus in SCC development. This finding and the low incidence of EcPV-2 DNA in healthy equids further strengthen the concept of an aetiologic association of EcPV-2 with equine SCC disease.