Multi-omic cross-sectional cohort study of pre-malignant Barrett's esophagus reveals early structural variation and retrotransposon activity.

Barrett's esophagus is a pre-malignant lesion that can progress to esophageal adenocarcinoma. We perform a multi-omic analysis of pre-cancer samples from 146 patients with a range of outcomes, comprising 642 person years of follow-up. Whole genome sequencing reveals complex structural variants and LINE-1 retrotransposons, as well as known copy number changes, occurring even prior to dysplasia. The structural variant burden captures the most variance across the cohort and genomic profiles do not always match consensus clinical pathology dysplasia grades. Increasing structural variant burden is associated with: high levels of chromothripsis and breakage-fusion-bridge events; increased expression of genes related to cell cycle checkpoint, DNA repair and chromosomal instability; and epigenetic silencing of Wnt signalling and cell cycle genes. Timing analysis reveals molecular events triggering genomic instability with more clonal expansion in dysplastic samples. Overall genomic complexity occurs early in the Barrett's natural history and may inform the potential for cancer beyond the clinically discernible phenotype.

Longitudinal tracking of 97 esophageal adenocarcinomas using liquid biopsy sampling.

BACKGROUND: The incidence of esophageal adenocarcinoma (EAC) is rapidly rising and has a 5-year survival rate of <20%. Beyond TNM (tumor-node-metastasis) staging, no reliable risk stratification tools exist and no large-scale studies have profiled circulating tumor DNA (ctDNA) at relapse in EAC. Here we analyze the prognostic potential of ctDNA dynamics in EAC, taking into account clonal hematopoiesis with indeterminate potential (CHIP). PATIENTS AND METHODS: A total of 245 samples from 97 patients treated with neoadjuvant chemotherapy and surgery were identified from the prospective national UK Oesophageal Cancer Clinical and Molecular Stratification (OCCAMS) consortium data set. A pan-cancer ctDNA panel comprising 77 genes was used. Plasma and peripheral blood cell samples were sequenced to a mean depth of 7082× (range 2196-28 524) and ctDNA results correlated with survival. RESULTS: Characteristics of the 97 patients identified were as follows: 83/97 (86%) male, median age 68 years (SD 9.5 years), 100% cT3/T4, 75% cN+. EAC-specific drivers had higher variant allele fractions than passenger mutations. Using stringent quality criteria 16/79 (20%) were ctDNA positive following resection; recurrence was observed in 12/16 (75%) of these. As much as 78/97 (80%) had CHIP analyses that enabled filtering for CHIP variants, which were found in 18/78 (23%) of cases. When CHIP was excluded, 10/63 (16%) patients were ctDNA positive and 9/10 of these (90%) recurred. With correction for CHIP, median cancer-specific survival for ctDNA-positive patients was 10.0 months versus 29.9 months for ctDNA-negative patients (hazard ratio 5.55, 95% confidence interval 2.42-12.71; P = 0.0003). Similar outcomes were observed for disease-free survival. CONCLUSIONS: We demonstrate in a large, national, prospectively collected data set that ctDNA in plasma following surgery for EAC is prognostic for relapse. Inclusion of peripheral blood cell samples can reduce or eliminate false positives from CHIP. In future, post-operative ctDNA could be used to risk stratify patients into high- and low-risk groups for intensification or de-escalation of adjuvant chemotherapy.

Comparing subjective and objective evaluation of show jumping competition and warm-up arena surfaces.

The development of safety and quality standards for equestrian surfaces needs to be based on objective, repeatable measurements which allow comparisons between surfaces. These measurements should incorporate the assessment of surface performance by riders. This study provides data from objective and subjective assessment of functional properties of high-level show jumping competition and warm-up arenas. Twenty-five arenas in nine international show jumping events were evaluated by mechanical in-situ testing with a surface tester, rider assessments using visual analogue scales (198 riders provided 749 arena evaluations), descriptions of arena constructions and by laboratory tests of surface material. Mixed models were used to present subjective evaluation of rider perception of the functional properties for each arena while controlling for rider and event. The association between objective and subjective assessments were also explored creating mixed models, controlling for rider and event. Mechanical measurements of impact firmness, and to a lesser extent cushioning and grip, had a significant positive association with the riders' perception. Responsiveness as assessed by the Orono biomechanical surface tester (OBST) was negatively associated with the riders' perceptions, which suggests riders and the OBST had different concepts of this functional property and that further developments of the OBST might be necessary. Objectively measured uniformity showed no useful association with riders' perception. Even though arena assessments were made by top level riders, a substantial inter-rider variation was demonstrated.

Comparison of limb kinematics between collected and lengthened (medium/extended) trot in two groups of dressage horses on two different surfaces.

BACKGROUND: Dressage horses are often asked to work in lengthened paces during training and competition, but to date there is limited information about the biomechanics of dressage-specific paces. Preliminary work has shown increased fetlock extension in extended compared with collected paces, but further investigation of the kinematic differences between collected, medium and extended trot in dressage horses is warranted. OBJECTIVES: Investigation of the effect of collected vs. medium/extended trot on limb kinematics of dressage horses. STUDY DESIGN: Prospective kinematic evaluation. METHODS: Twenty clinically sound horses in active dressage training were used. Group 1: Ten young horses (≤6 years) were assessed at collected and medium trot and Group 2: Ten mature horses (≥9 years) were assessed at collected and extended trot. All horses were evaluated on two different surfaces. High speed motion capture (240 Hz) was used to determine kinematic variables. Fore- and hindlimb angles were measured at mid-stance. Descriptive statistics and mixed effect multilevel regression analyses were performed. RESULTS: Speed and stride length were reduced and stride duration increased at collected compared with medium/extended trot. Lengthened trot (medium/extended trot) was associated with increased fetlock extension in both the fore- and hindlimbs in both groups of horses. Changes were greater in mature horses compared with young horses. Shoulder and carpus angles were associated with forelimb fetlock angle. Hock angle was not significantly influenced by pace. Surface had no effect on fetlock or hock angles. MAIN LIMITATIONS: Only 2D motion analysis was carried out. Results may have differed in horses with more extreme gait characteristics. CONCLUSIONS: Medium/extended trot increases extension of the fore- and hindlimb fetlock joints compared with collected trot in both young and mature dressage horses, respectively.

Double blind investigation of the effects of oral supplementation of combined glucosamine hydrochloride (GHCL) and chondroitin sulphate (CS) on stride characteristics of veteran horses.

REASONS FOR PERFORMING STUDY: Oral chondroprotective supplements are commercially popular for veteran (and other athletic or arthritic) horses prone to joint degeneration, yet lack conclusive scientific support. OBJECTIVES: To quantify the effects of an oral joint supplement (combination glucosamine hydrochloride (GHCL), chondroitin sulphate (CS) and N-acetyl-D-glucosamine) in vivo on stride parameters of veteran horses. METHODS: Twenty veteran horses were randomly assigned to a treatment (n = 15) or placebo group (n = 5). Pre-treatment gait characteristics were recorded at trot using digital video footage (50 Hz). The range of joint motion, stride length, and swing and stance duration were assessed using 2-dimensional motion analysis. Treatment (or placebo) was administered daily for 12 weeks at the manufacturer's recommended dosage. Gait was reassessed every 4 weeks using the pre-treatment protocol. Double blind procedure was implemented throughout. Relationships between variables were analysed using General Linear Model. RESULTS: Differences occurred in the treated horses by week 8. Range of joint motion increased significantly in the elbow (P<0.05), stifle and hind fetlock (P<0.01). Stride length increased significantly (P<0.05) with treatment. Swing duration was significantly increased at week 12 (P<0.05), whilst stance duration remained constant. CONCLUSION: The oral chondroprotective offered symptomatic relief to veteran horses, evidenced by improved stride characteristics. POTENTIAL RELEVANCE: Oral GHCL and CS supplementation may improve welfare by alleviating symptoms of degenerative joint disease.

Brefeldin A and a synthetic peptide to ADP-ribosylation factor (ARF) inhibit regulated exocytosis in melanotrophs.

We investigated the role of ADP-ribosylation factor (ARF) in regulated exocytosis in patch-clamped rat melanotrophs. Addition of brefeldin A (BFA) to inhibit activation of endogenous ARF protein was found to attenuate regulated secretory activity monitored as changes in membrane capacitance (Cm). A synthetic peptide to amino acids 46-61 of ARF (P-14) was also found to inhibit Ca(2+)-induced secretory activity in these cells. This inhibition was not apparent with a scrambled amino acid sequence of ARF-P14 peptide. This paper provides the first patch-clamp study to suggest that the small GTP-binding protein ARF is required to trigger release of secretory granules from rat pituitary melanotrophs.

rab3-peptide stimulates exocytosis from mast cells via a pertussis toxin-sensitive mechanism.

Synthetic peptides of the putative effector domain of members of the rab3 gene family of small GTP-binding proteins have been shown to have potent actions on vesicular transport and exocytosis [1,2]. Here, we use similar rab3-effector domain peptides to study their role in intracellular signalling in mast cells. We find that rab3-like peptides stimulate exocytosis and decrease cyclic 3',5'-AMP levels in these cells when applied extracellularly. Cells pretreated with pertussis toxin (PtX) to selectively uncouple alpha i/alpha o type G proteins from their biological activators, however, did not respond to rab3 peptides. rab3-like peptides also induce a Ca2+ transient in mast cells. These observations provide evidence for functional coupling between an effector domain peptide sequence of rab3 protein and a PtX-sensitive G protein substrate.

Blot-sequencing of antibodies: application to analysis of V gene usage among anti-steroid monoclonal antibodies.

Automated gas-phase protein sequencing has been used to characterize variable regions of antibody heavy and light chains separated by SDS-polyacrylamide gel electrophoresis (PAGE) and electroblotted onto Immobilon polyvinylidene difluoride membranes ('blot-sequencing'). Starting from 100 micrograms of antibody, 20 or more residues of N-terminal VH and VL sequences can regularly be obtained, which is often sufficient to assign the V region to a known family or subgroup. We have applied the blot-sequencing method to analysis of VH and VL usage among a panel of monoclonal anti-steroid antibodies, namely anti-progesterone, anti-pregnanediol, anti-estrone and anti-testosterone. The results demonstrate restricted, repetitive usage of VL subgroups and VH families related to anti-steroid specificities. VL regions of the VK1 group were particularly associated with anti-progesterone, VK21 with anti-estrone, and VK8 and VK9 with anti-pregnanediol. VH regions of anti-progesterone antibodies were all derived from the VHVGAM3.8 family; anti-estrone and anti-pregnanediol antibodies were derived from the VH7183 and VH36-60 families. The latter two families appear to characterize antibodies raised against steroids conjugated to proteins via a sugar bridge. Differences in VH/VL combination were associated with diversity of antibody specificity. In order to extend the sequence data obtained by this technique and confirm family assignments, we have shown that internal V-region sequences can be obtained by limited chemical cleavage of whole antibody with cyanogen bromide, followed by separation of individual fragments by SDS-PAGE and blot-sequencing.

Experimental hemiparkinsonism in the rat following chronic unilateral infusion of MPP+ into the nigrostriatal dopamine pathway--III. Reversal by embryonic nigral dopamine grafts.

In a previous study conducted over six months, we demonstrated that 1-methyl-4-phenylpyridinium ion (MPP+) the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, chronically infused (10 micrograms/24 h for seven days) into one median forebrain bundle of the rat can cause long-lasting damage to the nigrostriatal dopamine system. The present study was carried out in animals 18-19 months after MPP+ infusion to determine firstly, if the lesion was indeed permanent and secondly, if embryonic nigral dopamine suspension grafts implanted into the dopamine-denervated neostriatum can reverse the neurochemical and behavioural deficits induced by MPP+. All the animals within the MPP(+)-lesioned group showed robust contralateral and ipsilateral turning in response to apomorphine (0.05 mg/kg) and methamphetamine (2.5 mg/kg), respectively, at each time point of testing. In the grafted animals there was a progressive significant reduction in the number of rotations in response to both apomorphine and methamphetamine over the three-month test period. Autoradiographic analysis of [125I]sulpiride binding to striatal sections showed a 27% increase in dopamine D2 receptor density in the ipsilateral striatum of MPP(+)-lesioned animals. This increase in D2 receptor density was completely abolished by the dopamine grafts so that the D2 receptor density in the grafted striatum was similar to the contralateral striatum of MPP(+)-lesioned animals. This increase in D2 receptor density was completely abolished by the dopamine grafts so that the D2 receptor density in the grafted striatum was similar to the contralateral striatum of the grafted animals or the ipsilateral striatum of control non-lesioned animals. In all the animals of the lesioned and grafted groups there was a complete loss of dopamine neurons in the ipsilateral substantia nigra as demonstrated by tyrosine hydroxylase-immunohistochemistry and in-situ hybridization histochemistry. In all the animals that received nigral dopamine grafts, numerous cells were localized within the grafts which contained tyrosine hydroxylase immunoreactivity and tyrosine hydroxylase mRNA. Moreover, immunohistochemical staining showed a dense network of tyrosine hydroxylase-positive fibres within the grafted striatum. The results of the present study are important in two respects. Firstly, they demonstrate that MPP+ infusions into the rat nigrostriatal dopamine pathway can produce a permanent degeneration of nigral dopamine neurons. Thus, in animals assessed 18-19 months after the initial MPP(+)-lesion there was no significant behavioural or neurochemical compensation with time. Secondly, the results clearly show that embryonic nigral dopamine grafts implanted into the dopamine-denervated striatum can reverse the behavioural and neurochemical deficits induced by MPP+.

Gene expression in striatal grafts--I. Cellular localization of neurotransmitter mRNAs.

This study utilized the technique of in situ hybridization histochemistry to identify cells expressing neurotransmitter mRNAs in embryonic striatal tissue grafts implanted into the ibotenic acid-lesioned rat neostriatum. Synthetic 32P- or 35S-labelled oligodeoxyribonucleotide probes specific for prosomatostatin, proneuropeptide Y. proenkephalin, prodynorphin and preprotachykinin mRNAs and a 32P-labelled cRNA probe specific for glutamate decarboxylase mRNA were used to study the regional and cellular changes in these mRNA levels in the normal, lesioned and grafted neostriatum. The levels of neuropeptide Y mRNA and somatostatin mRNA were substantially increased in the striatal grafts compared with the intact control striata. The levels of glutamate decarboxylase mRNA in the grafts also appeared to be slightly elevated over those in the control striata. However, the levels of proenkephalin mRNA, prodynorphin mRNA and preprotachykinin mRNA were significantly lower in the grafts. The increased levels of neuropeptide Y mRNA and somatostatin mRNA in the grafts were due both to an increase in the number of labelled cells and to an increase in the cellular levels of each neuropeptide mRNA. In contrast, the cellular levels of proenkephalin mRNA, prodynorphin mRNA and preprotachykinin mRNA in the grafts were comparable, or elevated relative, to those in the intact striata but the density of cells expressing each of these mRNAs was reduced. Since neuropeptide Y and somatostatin are known to be present in medium to large aspiny striatal neurons (interneurons) and enkephalin, dynorphin and tachykinin peptides and GABA are localized in medium spiny striatal projection neurons, the above findings would indicate that there is a divergence in the levels of activity between these two neuronal populations in the striatal grafts. Our data suggest that the levels of gene expression and hence the functional neurotransmitter-synthesizing and releasing activity in the grafted neuron are different from those in the normal mature striatum.

Automated enzymatic assays for the determination of intestinal permeability probes in urine. 2. Mannitol.

A need for a simple method for the determination of mannitol in urine has arisen because of the use of this monosaccharide in intestinal permeability tests. A rapid spectrophotometric assay for mannitol is presented based on the use of the bacterial enzyme mannitol dehydrogenase. The technique has been automated for use on the Cobas-Bio (Roche) centrifugal analyser. The assay has been shown to be highly specific for mannitol and was not affected by high concentrations of glucose, lactose or lactulose in samples. Within assay coefficient of variation was in the range 0.3 to 1.1% and 0.6 to 2.4% between assays. The technique represents a significant improvement in terms of time, simplicity and precision on existing methods.

Sheep antiluteolytic interferon: cDNA sequence and analysis of mRNA levels.

A cloned cDNA has been isolated by probing a sheep blastocyst cDNA library using a synthetic oligonucleotide representing the N-terminal amino acid sequence of the antiluteolytic protein, ovine trophoblast protein-1. Sequence analysis of the cDNA confirms the 70% homology between the antiluteolysin and the interferon-alpha family of proteins; however, the sequence reported here differs at several points from previously reported amino acid and cDNA sequences for the antiluteolysin. In-vitro translation of day-16 poly(A)+ RNA indicated that antiluteolysin mRNA is a major constituent of total mRNA at this stage of blastocyst development, and Northern blotting confirmed that antiluteolysin mRNA production occurred between days 13 and 22 after oestrus. This is consistent with the stage at which embryonic extracts are antiluteolytic on administration in vivo. These and other data confirm that the ovine trophoblast antiluteolysin is an interferon, and suggest that at least five isoforms of this protein may exist.

Localization of vasopressin mRNA in the rat brain using in situ hybridization with short cRNA probes made from synthetic DNA templates.

Short antisense and sense RNA sequences were transcribed from annealed double stranded oligodeoxynucleotide templates containing the T7 RNA polymerase promotor sequence and its complementary sequence, together with 5' overhanging sequence corresponding to either the sense or antisense sequences of part of the rat vasopressin gene to produce after transcription 35S-labelled sense or antisense RNA for use in situ hybridization histochemistry. Labelled antisense RNA coding for a vasopressin sequence visualized sites of vasopressin mRNA expression (as did the 35S-labelled antisense oligodeoxynucleotide sequence), whereas sense RNA sequences revealed no specific sites of hybridization. This method represents an accessible, convenient and general method for the generation of cRNA probes suitable for use in situ hybridization.

Insulin-like growth factor II in the rat choroid plexus.

In situ hybridization histochemistry has been used to demonstrate the expression of the mRNA for insulin-like growth factor II (IGF-II) in the adult rat choroid plexus. IGF-II mRNA was found in the choroid plexus cells by using two different probes specific for different parts of IGF-II sequence. Parallel studies on consecutive sections showed that message for a choroid plexus marker transthyretin mRNA was also localized in the same choroid plexus cells.

Effect of insulin on hind-limb and whole-body leucine and protein metabolism in fed and fasted lambs.

1. A combination of isotope-dilution and arterio-venous difference techniques was used to determine rates of leucine metabolism and protein synthesis and degradation in a hind-limb preparation (predominantly muscle) and the whole body of eight lambs fed on milk to appetite and eight lambs fasted from 24 to 48 h. 2. Compared with fed lambs, fasted lambs showed decreased rates of protein synthesis in both whole body and hind-limb, and in hind-limb muscle, elevated rates of protein degradation. 3. The effects of two rates of insulin infusion on whole-body and hind-limb-muscle leucine metabolism, and in turn on protein metabolism, were determined. Insulin had no significant effect on leucine flux or oxidation (and hence protein synthesis and degradation) in whole-body or hind-limb muscle of fed lambs. In fasted lambs insulin progressively reduced arterial leucine concentration and whole-body leucine flux and oxidation, indicating a reduction in both protein synthesis and degradation. Insulin reduced the rate of leucine efflux from hind-limb muscle, which was followed by a reduction in leucine uptake. Insulin increased hind-limb-muscle glucose uptake in both fed and fasted lambs. 4. On the basis that hind-limb muscle was representative of skeletal muscle in general, we estimated that muscle accounted for the same percentage (about 27) of whole-body protein synthesis in both fed and fasted lambs. This percentage was unaffected by infusion of insulin, although the absolute rates differed in fed and fasted lambs.

The metabolism of circulating non-esterified fatty acids by the whole animal, hind-limb muscle and uterus of pregnant ewes.

1. The over-all and regional metabolism of non-esterified fatty acids (NEFA) was studied using a combination of isotopic and arteriovenous-difference techniques. 2. There was a common linear relationship, whether stearic, palmitic or oleic acids were used as tracer, between the arterial NEFA concentration and the rates of entry and oxidation. 3. Assuming that the tracer used reflected the metabolism of all the NEFA, the total entry rate in fed and fasted pregnant ewes was (mean +/- SE) 0.44 +/- 0.02 and 0.55 +/- 0.07 mmol/h per kg body-weight respectively. Oxidation of NEFA contributed (mean +/- SE) 34 +/- 5 and 58 +/- 7% to the respiratory carbon dioxide in fed and fasted animals, this accounting for (mean +/- SE) 46 +/- 6 and 59 +/- 3% of the respective entry rates. 4. Hind-limb muscle both utilized and produced NEFA. The mean gross fractional extraction (calculated from isotopic uptake) was (mean +/- SE) 9 +/- 1%. Gross utilization of any NEFA and appearance of 14CO2 across the muscle were linearly related to the arterial concentration of tracer fatty acid, irrespective of whether this was oleate or stearate. The amount of 14CO2 appearing was consistent with (mean +/- SE) 54 +/- 8% of the CO2 produced by the hind-limb being derived from NEFA oxidation. 5. Infused NEFA were partly converted to ketone bodies. Uptake and oxidation in the hind-limb of ketones formed in the liver could account for approximately 20% of the 14CO2 apparently produced in muscle from NEFA. Correction for this reduces the proportion of CO2 derived from NEFA to 43%. There was some indication that ketones were also produced from NEFA in the hind-limb. 6. NEFA were not a significant energy source for the gravid uterus. 7. An over-all view of energy sources for the whole animal and for hind-limb muscle in normal and fasted pregnant sheep was presented.