Demonstrating the reliability of in vivo metabolomics based chemical grouping: towards best practice.

While grouping/read-across is widely used to fill data gaps, chemical registration dossiers are often rejected due to weak category justifications based on structural similarity only. Metabolomics provides a route to robust chemical categories via evidence of shared molecular effects across source and target substances. To gain international acceptance, this approach must demonstrate high reliability, and best-practice guidance is required. The MetAbolomics ring Trial for CHemical groupING (MATCHING), comprising six industrial, government and academic ring-trial partners, evaluated inter-laboratory reproducibility and worked towards best-practice. An independent team selected eight substances (WY-14643, 4-chloro-3-nitroaniline, 17α-methyl-testosterone, trenbolone, aniline, dichlorprop-p, 2-chloroaniline, fenofibrate); ring-trial partners were blinded to their identities and modes-of-action. Plasma samples were derived from 28-day rat tests (two doses per substance), aliquoted, and distributed to partners. Each partner applied their preferred liquid chromatography-mass spectrometry (LC-MS) metabolomics workflows to acquire, process, quality assess, statistically analyze and report their grouping results to the European Chemicals Agency, to ensure the blinding conditions of the ring trial. Five of six partners, whose metabolomics datasets passed quality control, correctly identified the grouping of eight test substances into three categories, for both male and female rats. Strikingly, this was achieved even though a range of metabolomics approaches were used. Through assessing intrastudy quality-control samples, the sixth partner observed high technical variation and was unable to group the substances. By comparing workflows, we conclude that some heterogeneity in metabolomics methods is not detrimental to consistent grouping, and that assessing data quality prior to grouping is essential. We recommend development of international guidance for quality-control acceptance criteria. This study demonstrates the reliability of metabolomics for chemical grouping and works towards best-practice.

Pathway-Based Approaches for Assessing Biological Hazards of Complex Mixtures of Contaminants: A Case Study in the Maumee River.

Assessment of ecological risks of chemicals in the field usually involves complex mixtures of known and unknown compounds. We describe the use of pathway-based chemical and biological approaches to assess the risk of chemical mixtures in the Maumee River (OH, USA), which receives a variety of agricultural and urban inputs. Fathead minnows (Pimephales promelas) were deployed in cages for 4 d at a gradient of sites along the river and adjoining tributaries in 2012 and during 2 periods (April and June) in 2016, in conjunction with an automated system to collect composite water samples. More than 100 industrial chemicals, pharmaceuticals, and pesticides were detected in water at some of the study sites, with the greatest number typically found near domestic wastewater treatment plants. In 2016, there was an increase in concentrations of several herbicides from April to June at upstream agricultural sites. A comparison of chemical concentrations in site water with single chemical data from vitro high-throughput screening (HTS) assays suggested the potential for perturbation of multiple biological pathways, including several associated with induction or inhibition of different cytochrome P450 (CYP) isozymes. This was consistent with direct effects of water extracts in an HTS assay and induction of hepatic CYPs in caged fish. Targeted in vitro assays and measurements in the caged fish suggested minimal effects on endocrine function (e.g., estrogenicity). A nontargeted mass spectroscopy-based analysis suggested that hepatic endogenous metabolite profiles in caged fish covaried strongly with the occurrence of pesticides and pesticide degradates. These studies demonstrate the application of an integrated suite of measurements to help understand the effects of complex chemical mixtures in the field. Environ Toxicol Chem 2021;40:1098-1122. © 2020 SETAC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Infrared spectroscopy and theory of the formaldehyde cation and its hydroxymethylene isomer.

Pulsed discharges in supersonic expansions containing the vapor of different precursors (formaldehyde, methanol) produce the m/z = 30 cations with formula [H2,C,O]+. The corresponding [H2,C,O]+ Ar complexes are produced under similar conditions with argon added to the expansion gas. These ions are mass selected in a time-of-flight spectrometer and studied with infrared laser photodissociation spectroscopy. Spectra in the 2300-3000 cm-1 region produce very different vibrational patterns for the ions made from different precursors. Computational studies with harmonic methods and various forms of anharmonic theory allow detailed assignment of these spectra to two isomeric species. Discharges containing formaldehyde produce primarily the corresponding formaldehyde radical cation, CH2O+, whereas those with methanol produce exclusively the cis- and trans-hydroxymethylene cations, HCOH+. The implications for the interstellar chemistry of these cations are discussed.

A Longitudinal HbA1c Model Elucidates Genes Linked to Disease Progression on Metformin.

One-third of type-2 diabetic patients respond poorly to metformin. Despite extensive research, the impact of genetic and nongenetic factors on long-term outcome is unknown. In this study we combine nonlinear mixed effect modeling with computational genetic methodologies to identify predictors of long-term response. In all, 1,056 patients contributed their genetic, demographic, and long-term HbA1c data. The top nine variants (of 12,000 variants in 267 candidate genes) accounted for approximately one-third of the variability in the disease progression parameter. Average serum creatinine level, age, and weight were determinants of symptomatic response; however, explaining negligible variability. Two single nucleotide polymorphisms (SNPs) in CSMD1 gene (rs2617102, rs2954625) and one SNP in a pharmacologically relevant SLC22A2 gene (rs316009) influenced disease progression, with minor alleles leading to less and more favorable outcomes, respectively. Overall, our study highlights the influence of genetic factors on long-term HbA1c response and provides a computational model, which when validated, may be used to individualize treatment.

A genome-wide association study identifies variants in KCNIP4 associated with ACE inhibitor-induced cough.

The most common side effect of angiotensin-converting enzyme inhibitor (ACEi) drugs is cough. We conducted a genome-wide association study (GWAS) of ACEi-induced cough among 7080 subjects of diverse ancestries in the Electronic Medical Records and Genomics (eMERGE) network. Cases were subjects diagnosed with ACEi-induced cough. Controls were subjects with at least 6 months of ACEi use and no cough. A GWAS (1595 cases and 5485 controls) identified associations on chromosome 4 in an intron of KCNIP4. The strongest association was at rs145489027 (minor allele frequency=0.33, odds ratio (OR)=1.3 (95% confidence interval (CI): 1.2-1.4), P=1.0 × 10(-8)). Replication for six single-nucleotide polymorphisms (SNPs) in KCNIP4 was tested in a second eMERGE population (n=926) and in the Genetics of Diabetes Audit and Research in Tayside, Scotland (GoDARTS) cohort (n=4309). Replication was observed at rs7675300 (OR=1.32 (1.01-1.70), P=0.04) in eMERGE and at rs16870989 and rs1495509 (OR=1.15 (1.01-1.30), P=0.03 for both) in GoDARTS. The combined association at rs1495509 was significant (OR=1.23 (1.15-1.32), P=1.9 × 10(-9)). These results indicate that SNPs in KCNIP4 may modulate ACEi-induced cough risk.

Infrared spectroscopy of the methanol cation and its methylene-oxonium isomer.

The carbenium ion with nominal formula [C,H4,O](+) is produced from methanol or ethylene glycol in a pulsed-discharge supersonic expansion source. The ion is mass selected, and its infrared spectrum is measured from 2000 to 4000 cm(-1) using laser photodissociation spectroscopy and the method of rare gas atom tagging. Computational chemistry predicts two isomers, the methanol and methylene-oxonium cations. Predicted vibrational spectra based on scaled harmonic and reduced dimensional treatments are compared to the experimental spectra. The methanol cation is the only isomer produced when methanol is used as a precursor. When ethylene glycol is used as the precursor, methylene-oxonium is produced in addition to the methanol cation. Theoretical results at the CCSD(T)/cc-pVTZ level show that methylene-oxonium is lower in energy than methanol cation by 6.4 kcal/mol, and is in fact the global minimum isomer on the [C,H4,O](+) potential surface. Methanol cation is trapped behind an isomerization barrier in our source, providing a convenient method to produce and characterize this transient species. Analysis of the spectrum of the methanol cation provides evidence for strong CH stretch vibration/torsion coupling in this molecular ion.

Patellar ligament rupture in the dog: repair methods and patient outcomes in 43 cases.

The medical records of dogs receiving surgery for unilateral patellar ligament rupture between 1999 and 2012 at 12 multidisciplinary referral centres were reviewed. Forty-three cases were identified; 26 were traumatic in origin; almost one-third were iatrogenic, of which over three-quarters occurred as a complication following surgical stabilisation of patellar luxation. Treatment involved primary reapposition of the ligament (36 cases). The repair was protected by circumpatellar and/or transpatellar loop(s) of orthopaedic wire, nylon, polypropylene or polydioxanone suture (34 cases). Wire loops were more likely to require surgical removal compared with loops of other materials (P=0.0014). The stifle joint was immobilised postoperatively by the applications of a transarticular external skeletal fixator (taESF) in 17 cases and by external coaptation (EC) in 8 cases; in 18 cases, no postoperative joint immobilisation was provided. Complications specific to the method of immobilisation occurred in seven of the cases with taESF and six of the cases with EC. Revision surgery to address failure of repair was required in five cases. Outcome was classified as acceptable or good in over three-quarters of the cases (31/40) and poor in less than a quarter (9/40). These data highlight patellar ligament rupture as a complication of surgical stabilisation of patellar luxation.

Infrared spectroscopy of the acetyl cation and its protonated ketene isomer.

[C2,H3,O](+) ions are generated with a pulsed discharge in a supersonic expansion containing methyl acetate or acetone. These ions are mass selected and their infrared spectra are recorded via laser photodissociation and the method of argon tagging. Computational chemistry is employed to investigate structural isomers and their spectra. The acetyl cation (CH3CO(+)) is the global minimum and protonated ketene (CH2COH(+)) is the next lowest energy isomer (+176.2 kJ/mol). When methyl acetate is employed as the precursor, the infrared spectrum reveals that only the acetyl cation is formed. Partially resolved rotational structure reveals rotation about the C3 axis. When acetone is used as the precursor, acetyl is still the most abundant cation, but there is also a minor component of protonated ketene. Computations reveal a significant barrier to interconversion between the two isomers (+221 kJ/mol), indicating that protonated ketene must be obtained via kinetic trapping. Both isomers may be present in interstellar environments, and their implications for astrochemistry are discussed.

Genetic variants in transcription factors are associated with the pharmacokinetics and pharmacodynamics of metformin.

One-third of type 2 diabetes patients do not respond to metformin. Genetic variants in metformin transporters have been extensively studied as a likely contributor to this high failure rate. Here, we investigate, for the first time, the effect of genetic variants in transcription factors on metformin pharmacokinetics (PK) and response. Overall, 546 patients and healthy volunteers contributed their genome-wide, pharmacokinetic (235 subjects), and HbA1c data (440 patients) for this analysis. Five variants in specificity protein 1 (SP1), a transcription factor that modulates the expression of metformin transporters, were associated with changes in treatment HbA1c (P < 0.01) and metformin secretory clearance (P < 0.05). Population pharmacokinetic modeling further confirmed a 24% reduction in apparent clearance in homozygous carriers of one such variant, rs784888. Genetic variants in other transcription factors, peroxisome proliferator-activated receptor-α and hepatocyte nuclear factor 4-α, were significantly associated with HbA1c change only. Overall, our study highlights the importance of genetic variants in transcription factors as modulators of metformin PK and response.

IR spectroscopy of α- and ß-protonated pyrrole via argon complex photodissociation.

Protonated pyrrole cations are produced in a pulsed discharge/supersonic expansion source, mass-selected in a time-of-flight spectrometer, and studied with infrared photodissociation spectroscopy. Vibrational spectra in both the fingerprint and C-H/N-H stretching regions are obtained using the method of tagging with argon. Sharp vibrational structure is compared to IR spectra predicted by theory for the possible α-, ß-, and N-protonated structures. The spectral differences among these isomers are much larger than the frequency shifts due to argon attachment at alternative sites. Though α-protonation predominates thermodynamically, the kinetically favored ß-protonated species is also observed for the first time (in 3-4 times lower abundance under the conditions employed here). Theoretical investigations attribute the greater stability of α-protonated pyrrole to topological charge stabilization, rather than merely to the greater number of resonance contributors. The far-IR pattern of protonated pyrrole does not match the interstellar UIR bands.

Infrared spectroscopy of the mass 31 cation: protonated formaldehyde vs methoxy.

Pulsed discharges containing methanol or ethanol produce ions having the nominal formula [C,H(3),O](+), i.e. m/z = 31. Similar ions resulting from electron impact ionization in mass spectrometers are long recognized to have either the CH(2)OH(+) protonated formaldehyde or CH(3)O(+) methoxy cation structures. The H(2)OCH(+) oxonio-methylene structure has also been suggested by computational chemistry. To investigate these structures, ions are expanded in a supersonic beam, mass-selected in a time-of-flight spectrometer, and studied with infrared laser photodissociation spectroscopy. Sharp bands in the O-H and C-H stretching and fingerprint regions are compared to computational predictions for the three isomeric structures and their vibrational spectra. Protonated formaldehyde is the most abundant isomer, but methoxy is also formed with significant abundance. The branching ratio of these two ion species varies with precursors and formation conditions.

The effect of external coaptation on plate deformation in an ex vivo model of canine pancarpal arthrodesis.

OBJECTIVES: Since external coaptation is applied clinically to prevent plate failure during healing in canine pancarpal arthrodesis (PCA), we tested the hypothesis that external coaptation does not significantly reduce plate strain in an experimental ex vivo model of canine PCA. METHODS: Ten thoracic limbs from healthy Greyhounds euthanatized for reasons un- related to the study were harvested and the carpus was stabilised with a dorsally applied 2.7/3.5 mm hybrid PCA plate. The strain in the plate adjacent to the most distal radial screw hole (R4) and the radial carpal bone (RCB) screw hole was measured as the limbs were loaded axially to a load that approximated that of controlled walking. Each limb was tested with and without external coaptation in place. RESULTS: Mean strain amplitude at the RCB was -177.2 µÎµ (± 20.78) without external coaptation. Following cast application, strain reduced significantly to -34.7 µÎµ (± 9.84) (p <0.002). Mean strain at R4 was -89.4 µÎµ (± 22.10) without external support and -66.9 µÎµ (± 10.74) following application of a cast. This reduction in recorded strain was not statistically significant. CLINICAL SIGNIFICANCE: The application of a cast to the distal portion of the limb significantly reduced strain in the 2.7/3.5 mm hybrid PCA plate, but the magnitude of the measured strain was low, suggesting that fatigue damage is unlikely to accumulate as a result of this type of loading and that external coaptation may not be necessary to prevent fatigue failure of the plate.

Clinical assessments of increased sensory sensitivity in dogs with cranial cruciate ligament rupture.

Dogs with chronic pain have a compromised quality of life. Repeatable and accurate sensory assessments form a means by which the hypersensitivity likely to reflect chronic pain may be quantified. These assessments can be applied to individuals to identify those that may benefit from improved analgesic relief. In this study four sensory assessments were evaluated in dogs presenting with a naturally occurring chronic painful condition (cranial cruciate ligament rupture, CCLR) and were compared with healthy control animals of similar age and weight. Inter-digital von Frey filament and thermal sensitivity tests revealed that the affected hind limb of dogs with CCLR was significantly more sensitive than the opposing limb. Static weight bearing and gait parameter scores were also reduced in the affected hind limb compared to the opposing hind limb of dogs with CCLR; no such differences were found between the hind limbs of healthy (control) dogs. The quantitative sensory tests permitted the differentiation of limbs affected by CCLR from healthy limbs. Dogs presenting with CCLR demonstrate objectively quantitative sensory sensitivities, which may require additional consideration in case management.

Oral valganciclovir versus ganciclovir as delayed pre-emptive therapy for patients after allogeneic hematopoietic stem cell transplant: a pilot trial (04-0274) and review of the literature.

BACKGROUND: Cytomegalovirus (CMV) infection is an important cause of morbidity and mortality after allogeneic hematopoietic stem cell transplant (HSCT). This pilot prospective randomized clinical trial compares valganciclovir (VGV) to ganciclovir (GCV) as pre-emptive therapy for CMV viremia in the post-allogeneic HSCT population. METHODS: Patients undergoing allogeneic HSCT who were at risk for CMV viremia were monitored post HSCT by weekly quantitative whole blood polymerase chain reaction. Pre-emptive therapy was delayed until the viral load (VL) was >10,000 copies/mL once, or >5000 copies/mL twice. Patients were randomized to either GCV 5 mg/kg twice a day (b.i.d.) for 7 days followed by daily GCV 5 mg/kg for up to 21 days, or VGV 900 mg b.i.d. for 7 days followed by 900 mg daily for up to 21 days. The primary endpoint was clearance of viremia (VL <5000 copies/mL) within 28 days of initiation of therapy. RESULT: In total, 37 patients were enrolled; 19 patients received treatment with VGV and 18 patients received treatment with GCV. The VGV was not inferior in efficacy to GCV as pre-emptive therapy, with rates of viral clearance at 28 days of 89.5% and 83%, respectively (P-value for non-inferiority = 0.030). Toxicities were similar between the 2 arms. No patients developed CMV disease. CONCLUSIONS: In this trial, the rates of clearance of viremia appear to be similar with VGV and GCV.

Electronic spectroscopy of CoNe+ via mass-selected photodissociation.

The CoNe(+) diatomic cation is produced by laser vaporization in a pulsed-nozzle source and studied with photodissociation spectroscopy at visible wavelengths. Vibronic structure is assigned to the (3)Π(2) ← (3)Δ(3) band system correlating to the Co(+)((3)P(2) ← (3)F(4)) + Ne asymptote. The origin band (13,529 cm(-1)) and a progression of 14 other vibrational bands are detected ending in the dissociation limit at 14,191 cm(-1). The excited state dissociation energy is therefore D(0)(') = 662 cm(-1), and an energetic cycle using this, the origin band energy, and the atomic transition produces a ground state dissociation energy of D(0)(") = 930 cm(-1). The excited state vibrational frequency is 116.1 cm(-1). A rotationally resolved study of the origin band confirms the electronic transition assignment and provides the bond distance of r(0)(") = 2.36 Å. The properties of CoNe(+) are compared to those of other CoRG(+) and MNe(+) complexes studied previously.

Pharmacokinetic differences in exposure to camphor after intraruminal dosing in selectively bred lines of goats.

A pharmacokinetic dosing study with camphor was used to determine whether selection lines of high-juniper-consuming goats (HJC, n = 12) and low-juniper-consuming goats (LJC, n = 12) differed in their respective disposition kinetics. Postdosing plasma camphor concentrations were used to examine whether a timed single blood sample collected after intraruminal administration of camphor would be a useful screening test to aid in the identification of HJC. Yearling female Boer x Spanish goats (n = 24) received a single intraruminal dose of monoterpene cocktail (0.270 g/kg of BW) containing 4 different monoterpenes that represented their composition previously reported for Ashe juniper (Juniperus ashei). Camphor, the predominant monoterpene in Ashe juniper, was 49.6% of the mix and was the monoterpene analyzed for this study. Blood samples were taken at 15 time points from 0 to 8 h after dosing. Concentrations of camphor were measured in plasma using solid phase extraction and gas chromatography/flame-ionization detection analysis. Maximal plasma concentration of camphor was greater for LJC than HJC (P = 0.01), and area under the curve extrapolated to infinity was greater for LJC than HJC (P < 0.01). Total systemic exposure (area under the curve) to camphor was 5 times less in HJC goats. We conclude that 1) HJC goats possess internal mechanisms to reduce the bioavailability of camphor, and 2) a blood sample taken at 45 min or at 60 min after intraruminal administration of camphor may be useful for identifying HJC individual animals from within large populations of goats.

Management of laterally displaced proximal tibial physeal fractures in three dogs.

Fractures of the proximal tibial physis are uncommon in dogs, and are rarely associated with marked instability and lateral displacement of the proximal tibial epiphysis. Three dogs with proximal tibial physeal fractures demonstrating marked instability and lateral displacement were treated with two different principles of fixation. Healing of the physeal fracture was achieved with rigid internal fixation in one case, and with adaptational osteosynthesis supplemented with a temporary transarticular external skeletal fixator in two cases. Duration of surgery and technical difficulty was reduced using a modified adaptational osteosynthesis approach, suggesting that surgical treatment of these uncommon and challenging fractures may be more appropriately achieved by this technique.

Clinical and neurological characteristics of aortic thromboembolism in dogs.

OBJECTIVES: To characterise the clinical presentation and neurological abnormalities in dogs affected by aortic thromboembolism. METHODS: The medical records of 13 dogs diagnosed with aortic thromboembolism as the cause of the clinical signs, and where a complete neurological examination was performed, were reviewed retrospectively. RESULTS: The onset was acute in only four dogs, chronic in five dogs (with all of these presenting as exercise intolerance) or chronic with acute deterioration in four dogs. Dogs with an acute onset of clinical signs were more severely affected exhibiting neurological deficits, while dogs with a chronic onset of disease predominantly presented with the exercise intolerance and minimal deficits. The locomotor deficits included exercise intolerance with pelvic limb weakness (five of 13), pelvic limb ataxia (one of 13), monoparesis (two of 13), paraparesis (two of 13), non-ambulatory paraparesis (two of 13) and paraplegia (one of 13). There was an apparent male predisposition and the cavalier King charles spaniel was overrepresented. CLINICAL SIGNIFICANCE: The rate of onset of clinical signs appears to segregate dogs affected by aortic thromboembolism into two groups, with different clinical characteristics and outcomes. Dogs with an acute onset of the clinical signs tend to be more severely affected, while dogs with a chronic onset predominantly present with exercise intolerance. It is therefore important to consider aortic thromboembolism as a differential diagnosis in dogs with an acute onset of pelvic limb neurological deficits and in dogs with longer standing exercise intolerance.

The course of hepatitis C viraemia in transfusion recipients prior to availability of antiviral therapy.

Knowing the likely distribution of intervals from hepatitis C infection to first RNA-negativity is important in deciding about therapeutic intervention. Prospectively collected sera and data from the Transfusion-transmitted Viruses Study (1974-1980) provide specific dates of infection and pattern of alanine aminotransferase (ALT) elevations. We examined frequency, timing and correlates of spontaneous resolution for 94 acutely infected transfusion recipients followed for a median of 9.5 months. Later, follow-up sera (>10 years) were available for 27 of the 94 cases from a Veterans Administration (VA) Study (1989-1990). Twenty-five (27%) of the 94 cases were classified as probably resolved during the episode itself. First RNA negativity occurred at 6-50 weeks (median, 19.5 weeks) after infection, and 5-43 weeks (median, 11 weeks) after ALT elevation. Thirteen of the 25 cases remained RNA-negative subsequently; 12 others had 1-6 RNA-positive sera intercalated between first and last RNA-negative results. RNA negativity, therefore, began variably and was interrupted in 12 cases of 25 (48%) by transient RNA-positive sera. Five of these 25 patients who were RNA-negative in the last study specimen had late, Veterans Administration Study follow-up; none showed viraemia. Of the remaining 69 transfusion transmitted virus study recipients, whose last serum was RNA-positive, two cleared viraemia after the last study serum but before late follow-up. Eleven (16%) had 23 intercalated RNA-negative sera before last positivity. RNA status, therefore, needs monitoring for many months before judging the spontaneous outcome as transient negativity may occur. Resolution was significantly more common in women and symptomatic cases; it was not associated with viral load in the infectious donation, HCV genotype, or the recipient's age.