Hepatobiliary circulation and dominant urinary excretion of homogentisic acid in a mouse model of alkaptonuria.

Altered activity of specific enzymes in phenylalanine-tyrosine (phe-tyr) metabolism results in incomplete breakdown of various metabolite substrates in this pathway. Increased biofluid concentration and tissue accumulation of the phe-tyr pathway metabolite homogentisic acid (HGA) is central to pathophysiology in the inherited disorder alkaptonuria (AKU). Accumulation of metabolites upstream of HGA, including tyrosine, occurs in patients on nitisinone, a licenced drug for AKU and hereditary tyrosinaemia type 1, which inhibits the enzyme responsible for HGA production. The aim of this study was to investigate the phe-tyr metabolite content of key biofluids and tissues in AKU mice on and off nitisinone to gain new insights into the biodistribution of metabolites in these altered metabolic states. The data show for the first time that HGA is present in bile in AKU (mean [±SD] = 1003[±410] µmol/L; nitisinone-treated AKU mean [±SD] = 45[±23] µmol/L). Biliary tyrosine, 3(4-hydroxyphenyl)pyruvic acid (HPPA) and 3(4-hydroxyphenyl)lactic acid (HPLA) are also increased on nitisinone. Urine was confirmed as the dominant elimination route of HGA in untreated AKU, but with indication of biliary excretion. These data provide new insights into pathways of phe-tyr metabolite biodistribution and metabolism, showing for the first time that hepatobiliary excretion contributes to the total pool of metabolites in this pathway. Our data suggest that biliary elimination of organic acids and other metabolites may play an underappreciated role in disorders of metabolism. We propose that our finding of approximately 3.8 times greater urinary HGA excretion in AKU mice compared with patients is one reason for the lack of extensive tissue ochronosis in the AKU mouse model.

Determinants of tyrosinaemia during nitisinone therapy in alkaptonuria.

Nitisinone (NIT) produces inevitable but varying degree of tyrosinaemia. However, the understanding of the dynamic adaptive relationships within the tyrosine catabolic pathway has not been investigated fully. The objective of the study was to assess the contribution of protein intake, serum NIT (sNIT) and tyrosine pathway metabolites to nitisinone-induced tyrosinaemia in alkaptonuria (AKU). Samples of serum and 24-h urine collected during SONIA 2 (Suitability Of Nitisinone In Alkaptonuria 2) at months 3 (V2), 12 (V3), 24 (V4), 36 (V5) and 48 (V6) were included in these analyses. Homogentisic acid (HGA), tyrosine (TYR), phenylalanine (PHE), hydroxyphenylpyruvate (HPPA), hydroxyphenyllactate (HPLA) and sNIT were analysed at all time-points in serum and urine. Total body water (TBW) metabolites were derived using 60% body weight. 24-h urine and TBW metabolites were summed to obtain combined values. All statistical analyses were post-hoc. 307 serum and 24-h urine sampling points were analysed. Serum TYR from V2 to V6, ranging from 478 to 1983 µmol/L were stratified (number of sampling points in brackets) into groups < 701 (47), 701-900 (105), 901-1100 (96) and > 1100 (59) µmol/L. The majority of sampling points had values greater than 900 µmol/L. sPHE increased with increasing sTYR (p < 0.001). Tyrosine, HPPA and HPLA in serum and TBW all increased with rising sTYR (p < 0.001), while HPLA/TYR ratio decreased (p < 0.0001). During NIT therapy, adaptive response to minimise TYR formation was demonstrated. Decreased conversion of HPPA to HPLA, relative to TYR, seems to be most influential in determining the degree of tyrosinaemia.

Temporal adaptations in the phenylalanine/tyrosine pathway and related factors during nitisinone-induced tyrosinaemia in alkaptonuria.

BACKGROUND: Adaptations within the phenylalanine (PHE)/tyrosine (TYR) pathway during nitisinone (NIT) are not fully understood. OBJECTIVE: To characterise the temporal changes in metabolic features in NIT-treated patients with alkaptonuria. PATIENTS AND METHODS: Serum (s) and 24-urine (u) homogentisic acid (sHGA, uHGA24), TYR (sTYR, uTYR24), PHE (sPHE, uPHE24), hydroxyphenylpyruvate (sHPPA, uHPPA24), hydroxyphenyllactate (sHPLA, uHPLA24) and sNIT were measured at baseline (V1) and until month 48 (V6) in 69 NIT-treated patients, recommended to reduce protein intake. The 24-h urine urea (uUREA24), creatinine (uCREAT24) and body weight were also measured. Amounts of tyrosine metabolites in total body water (TBW) were derived by multiplying the serum concentrations by 60% body weight, and sum of TBW and urine metabolites resulted in combined values (c). RESULTS: uUREA24 and uCREAT24 decreased between V1 and V6 during NIT, whereas body weight and sNIT increased. Linear regression coefficient between uUREA24 and uCREAT24 was extremely strong (R = 0.84). sPHE, TBWPHE and cPHE24 increased gradually from V1 to V6. A decrease in cTYR24/cPHE24, sTYR/sPHE and TBWTYR/TBWPHE was seen from V2 to V6. Serum, 24-urine and combined TYR, HPPA and HPLA either remained stable or decreased from V2 to V6. DISCUSSION: The gradual increase in PHE suggests adaptation to increasing TYR during NIT therapy. The decrease in protein intake resulted in decreased muscle mass and increased weight gain. CONCLUSION: Progressive adaptation by decreasing PHE conversion to TYR occurs over time during NIT therapy. A low protein diet results in loss of muscle mass but also weight gain suggesting an increase in fat mass.

Characterization of changes in the tyrosine pathway by 24-h profiling during nitisinone treatment in alkaptonuria.

BACKGROUND: Although changes in the tyrosine pathway during nitisinone therapy are known, a complete characterization of the induced tyrosinaemia is lacking to improve disease management. PATIENTS AND METHODS: Our research aims were addressed by 24-h blood sampling. 40 patients with alkaptonuria (AKU), treated with 0, 1, 2, 4 and 8 mg nitisinone daily (n = 8), were studied over four weeks. Serum homogentisic acid (sHGA), tyrosine (sTYR), phenylalanine (sPHE), hydroxyphenylpyruvate (sHPPA), hydroxyphenyllactate (sHPLA) and nitisinone (sNIT) were measured at baseline and after four weeks. RESULTS: sNIT showed a clear dose-proportional response. sTYR increased markedly but with less clear-cut dose responses after nitisinone. Fasting and average 24-h (Cav) sTYR responses were similar. Individual patient sTYR 24-h profiles showed significant fluctuations during nitisinone therapy. At week 4, sTYR, sHPPA and sHPPL all showed dose-related increases compared to V0, with the greatest difference between 1 and 8 mg nitisinone seen for HPLA, while there was no change from V0 in sPHE. sHGA decreased to values around the lower limit of quantitation. DISCUSSION: There was sustained tyrosinaemia after four weeks of nitisinone therapy with significant fluctuations over the day in individual patients. Diet and degree of conversion of HPPA to HPLA may determine extent of nitisinone-induced tyrosinaemia. CONCLUSION: A fasting blood sample is recommended to monitor sTYR during nitisinone therapy Adaptations in HPPA metabolites as well as the inhibition of tyrosine aminotransferase could be contributing factors generating tyrosinaemia during nitisinone therapy.

A fully automatic system to assess foot collapse on lateral weight-bearing foot radiographs: A pilot study.

BACKGROUND: Foot collapse is primarily diagnosed and monitored using lateral weight-bearing foot x-ray images. There are several well-validated measurements which aid assessment. However, these are subject to inter- and intra-user variability. OBJECTIVE: To develop and validate a software system for the fully automatic assessment of radiographic changes associated with foot collapse; automatically generating measurements for calcaneal tilt, cuboid height and Meary's angle. METHODS: This retrospective study was approved by the Health Research Authority (IRAS 244852). The system was developed using lateral weight-bearing foot x-ray images, and evaluated against manual measurements from five clinical experts. The system has two main components: (i) a Random Forest-based point-finder to outline the bones of interest; and (ii) a geometry-calculator to generate the measurements based on the point positions from the point-finder. The performance of the point-finder was assessed using the point-to-point error (i.e. the mean absolute distance between each found point and the equivalent ground truth point, averaged over all points per image). For assessing the performance of the geometry-calculator, linear mixed models were fitted to estimate clinical inter-observer agreement and to compare the performance of the software system to that of the clinical experts. RESULTS: A total of 200 images were collected from 79 subjects (mean age: 56.4 years ±12.9 SD, 30/49 females/males). There was good agreement among all clinical experts with intraclass correlation estimates between 0.78 and 0.86. The point-finder achieved a median point-to-point error of 2.2 mm. There was no significant difference between the clinical and automatically generated measurements using the point-finder points, suggesting that the fully automatically obtained measurements are in agreement with the manually obtained measurements. CONCLUSIONS: The proposed system can be used to support and automate radiographic image assessment for diagnosing and managing foot collapse, saving clinician time, and improving patient outcomes.

Human cytomegalovirus multiple-strain infections and viral population diversity in haematopoietic stem cell transplant recipients analysed by high-throughput sequencing.

Human cytomegalovirus (HCMV) is an important opportunistic pathogen in allogeneic haematopoietic stem cell transplant (HSCT) recipients. High-throughput sequencing of target-enriched libraries was performed to characterise the diversity of HCMV strains present in this high-risk group. Forty-four HCMV-DNA-positive plasma specimens (median viral input load 321 IU per library) collected at defined time points from 23 HSCT recipients within 80 days of transplantation were sequenced. The genotype distribution for 12 hypervariable HCMV genes and the number of HCMV strains present (i.e. single- vs. multiple-strain infection) were determined for 29 samples from 16 recipients. Multiple-strain infection was observed in seven of these 16 recipients, and five of these seven recipients had the donor (D)/recipient (R) HCMV-serostatus combination D + R + . A very broad range of genotypes was detected, with an intrahost composition that was generally stable over time. Multiple-strain infection was not associated with particular virological or clinical features, such as altered levels or duration of antigenaemia, development of acute graft-versus-host disease or increased mortality. In conclusion, despite relatively low viral plasma loads, a high frequency of multiple-strain HCMV infection and a high strain complexity were demonstrated in systematically collected clinical samples from this cohort early after HSCT. However, robust evaluation of the pathogenic role of intrahost viral diversity and multiple-strain infection will require studies enrolling larger numbers of recipients.

Impact of severe acute respiratory syndrome coronavirus 2 on ectopic pregnancy management in the United Kingdom: a multicentre observational study.

OBJECTIVE: To describe the impact of coronavirus disease 2019 (COVID-19) on the management of women with ectopic pregnancy. DESIGN: A multicentre observational study comparing outcomes from a prospective cohort during the pandemic [COVID-19-ectopic pregnancy registry (CEPR)] compared with a historical pre-pandemic cohort [non-COVID-19-ectopic pregnancy registry (NCEPR)]. SETTING: Five London university hospitals. POPULATION AND METHODS: Consecutive patients diagnosed clinically and/or radiologically with ectopic pregnancy (March 2020-August 2020) were entered into the CEPR and results were compared with the NCEPR cohort (January 2019-June 2019). An adjusted analysis was performed for potentially confounding variables. MAIN OUTCOME MEASURES: Patient demographics, management (expectant, medical and surgical), length of treatment, number of hospital visits (non-surgical management), length of stay (surgical management) and 30-day complications. RESULTS: Three hundred and forty-one women met the inclusion criteria: 162 CEPR and 179 NCEPR. A significantly lower percentage of women underwent surgical management versus non-surgical management in the CEPR versus NCEPR (58.6%; 95/162 versus 72.6%; 130/179; P = 0.007). Among patients managed with expectant management, the CEPR had a significantly lower mean number of hospital visits compared with NCEPR (3.0, interquartile range [IQR] [3, 5] versus 9.0, [5, 14]; P = <0.001). Among patients managed with medical management, the CEPR had a significantly lower median number of hospital visits versus NCEPR (6.0, [5, 8] versus 9, [6, 10]; P = 0.003). There was no observed difference in complication rates between cohorts. CONCLUSION: Women were found to undergo significantly higher rates of non-surgical management during the COVID-19 first wave compared with a pre-pandemic cohort. Women managed non-surgically in the CPER cohort were also managed with fewer hospital attendances. This did not lead to an increase in observed complication rates. TWEETABLE ABSTRACT: A higher rate of non-surgical management of ectopic pregnancy during the COVID-19 pandemic did not increase complication rates.

Parameter estimation for discretely observed linear birth-and-death processes.

Birth-and-death processes are widely used to model the development of biological populations. Although they are relatively simple models, their parameters can be challenging to estimate, as the likelihood can become numerically unstable when data arise from the most common sampling schemes, such as annual population censuses. A further difficulty arises when the discrete observations are not equi-spaced, for example, when census data are unavailable for some years. We present two approaches to estimating the birth, death, and growth rates of a discretely observed linear birth-and-death process: via an embedded Galton-Watson process and by maximizing a saddlepoint approximation to the likelihood. We study asymptotic properties of the estimators, compare them on numerical examples, and apply the methodology to data on monitored populations.

Fatal acute haemolysis and methaemoglobinaemia in a man with renal failure and Alkaptonuria - Is nitisinone the solution?

Haemolysis and methaemoglobinaemia (MetHb) are rare metabolic complications that can occur in Alkaptonuria (AKU), for which there is no curative treatment. Presented is a case of a man who had AKU, and serves as a reminder of life-threatening complications that can occur with haemolysis and MetHb. This case presents an opportunity to revisit important considerations relating to the investigation and treatment of haemolysis and MetHb with a view to raising awareness, and in doing so hopefully reducing the uniformly fatal outcome. Additionally it is proposed that treatment of haemolysis and MetHb with nitisinone is considered as a potentially lifesaving treatment as it is believed that reducing the concentration of circulating homogentisic acid will reduce oxidative stress.

Quantification of the flux of tyrosine pathway metabolites during nitisinone treatment of Alkaptonuria.

Nitisinone decreases homogentisic acid (HGA) in Alkaptonuria (AKU) by inhibiting the tyrosine metabolic pathway in humans. The effect of different daily doses of nitisinone on circulating and 24 h urinary excretion of phenylalanine (PA), tyrosine (TYR), hydroxyphenylpyruvate (HPPA), hydroxyphenyllactate (HPLA) and HGA in patients with AKU was studied over a four week period. Forty AKU patients, randomised into five groups of eight patients, received doses of 1, 2, 4 or 8 mg of nitisinone daily, or no drug (control). Metabolites were analysed by tandem mass spectrometry in 24 h urine and serum samples collected before and after nitisinone. Serum metabolites were corrected for total body water and the sum of 24 hr urine plus total body water metabolites of PA, TYR, HPPA, HPLA and HGA were determined. Body weight and urine urea were used to check on stability of diet and metabolism over the 4 weeks of study. The sum of quantities of urine metabolites (PA, TYR, HPPA, HPLA and HGA) were similar pre- and post-nitisinone. The sum of total body water metabolites were significantly higher post-nitisinone (p < 0.0001) at all doses. Similarly, combined 24 hr urine:total body water ratios for all analytes were significantly higher post-nitisinone, compared with pre-nitisinone baseline for all doses (p = 0.0002 - p < 0.0001). Significantly higher concentrations of metabolites from the tyrosine metabolic pathway were observed in a dose dependant manner following treatment with nitisinone and we speculate that, for the first time, experimental evidence of the metabolite pool that would otherwise be directed towards pigment formation, has been unmasked.

Assessing the effect of nitisinone induced hypertyrosinaemia on monoamine neurotransmitters in brain tissue from a murine model of alkaptonuria using mass spectrometry imaging.

OBJECTIVE: Nitisinone induced hypertyrosinaemia is a concern in patients with Alkaptonuria (AKU). It has been suggested that this may alter neurotransmitter metabolism, specifically dopamine and serotonin. Herein mass spectrometry imaging (MSI) is used for the direct measurement of 2,4-diphenyl-pyranylium tetrafluoroborate (DPP-TFB) derivatives of monoamine neurotransmitters in brain tissue from a murine model of AKU following treatment with nitisinone. METHODS: Metabolite changes were assessed using MSI on DPP-TFB derivatised fresh frozen tissue sections directing analysis towards primary amine neurotransmitters. Matched tail bleed plasma samples were analysed using LC-MS/MS. Eighteen BALB/c mice were included in this study: HGD-/- (n = 6, treated with nitisinone-4 mg/L, in drinking water); HGD-/- (n = 6, no treatment) and HGD+/- (n = 6, no treatment). RESULTS: Ion intensity and distribution of DPP-TFB derivatives in brain tissue for dopamine, 3-methoxytyramine, noradrenaline, tryptophan, serotonin, and glutamate were not significantly different following treatment with nitisinone in HGD -/- mice, and no significant differences were observed between HGD-/- and HGD+/- mice that received no treatment. Tyrosine (10-fold in both comparisons, p = 0.003; [BALB/c HGD-/- (n = 6) and BALB/c HGD+/- (n = 6) (no treatment) vs. BALB/c HGD-/- (n = 6, treated)] and tyramine (25-fold, p = 0.02; 32-fold, p = 0.02) increased significantly following treatment with nitisinone. Plasma tyrosine and homogentisic acid increased (ninefold, p = < 0.0001) and decreased (ninefold, p = 0.004), respectively in HGD-/- mice treated with nitisinone. CONCLUSIONS: Monoamine neurotransmitters in brain tissue from a murine model of AKU did not change following treatment with nitisinone. These findings have significant implications for patients with AKU as they suggest monoamine neurotransmitters are not altered following treatment with nitisinone.

Liquid chromatography tandem mass spectrometry for plasma metadrenalines.

Evidence is accumulating which may result in plasma free metadrenalines (PMets) becoming the preferred test for diagnosing phaeochromocytoma and paraganglioma. Moreover, increased availability and benefits over other analytical methods like liquid chromatography with electrochemical detection and immunoassay are causing liquid chromatography tandem mass spectrometry (LC-MS/MS) to become the method of choice for PMet measurement. This review explores the evidence-base supporting this, and summarises published LC-MS/MS analytical methods for PMet analysis. Key aspects of methods (including SPE extraction, HILIC chromatography, MRM MS-detection and standardisation) are discussed. Common causes of analytical interference (e.g. ion suppression/enhancement, ionic cross talk, in source transformation and isobaric interferences) are outlined to illustrate the importance of sample purification and chromatographic resolution. The importance of supine, fasting sampling and Bayesian interpretation against supine, fasting reference intervals are explained, as well as the importance of age-specific reference intervals for normetadrenaline. Confounding factors like diet, drugs, renal function and acute illness are explored, along with potential strategies to address these (e.g. CKD-specific reference intervals).

Data on items of AKUSSI in Alkaptonuria collected over three years from the United Kingdom National Alkaptonuria Centre and the impact of nitisinone.

Alkaptonuria is a rare genetic disorder characterized by a high level of circulating (and urine) homogentisic acid (HGA), which contributes to ochronosis when it is deposited in connective tissue as a pigmented polymer. In an observational study carried out by National AKU Centre (NAC) in Liverpool, a total of thirty-nine AKU patients attended yearly visits in varying numbers. At each visit a mixture of clinical, joint and spinal assessments were carried out and the results calculated to yield an AKUSSI (Alkaptonuria Severity Score Index), see "Nitisinone arrests ochronosis and decreases rate of progression of Alkaptonuria: evaluation of the effect of nitisinone in the United Kingdom National Alkaptonuria Centre" (Ranganath at el., 2018). The aim of this data article is to produce visual representation of the change in the components of AKUSSI over 3 years, through radar charts. The metabolic effect of nitisinone is shown through box plots.

Nitisinone arrests ochronosis and decreases rate of progression of Alkaptonuria: Evaluation of the effect of nitisinone in the United Kingdom National Alkaptonuria Centre.

QUESTION: Does Nitisinone prevent the clinical progression of the Alkaptonuria? FINDINGS: In this observational study on 39 patients, 2 mg of daily nitisinone inhibited ochronosis and significantly slowed the progression of AKU over a three-year period. MEANING: Nitisinone is a beneficial therapy in Alkaptonuria. BACKGROUND: Nitisinone decreases homogentisic acid (HGA), but has not been shown to modify progression of Alkaptonuria (AKU). METHODS: Thirty-nine AKU patients attended the National AKU Centre (NAC) in Liverpool for assessments and treatment. Nitisinone was commenced at V1 or baseline. Thirty nine, 34 and 22 AKU patients completed 1, 2 and 3 years of monitoring respectively (V2, V3 and V4) in the VAR group. Seventeen patients also attended a pre-baseline visit (V0) in the VAR group. Within the 39 patients, a subgroup of the same ten patients attended V0, V1, V2, V3 and V4 visits constituting the SAME Group. Severity of AKU was assessed by calculation of the AKU Severity Score Index (AKUSSI) allowing comparison between the pre-nitisinone and the nitisinone treatment phases. RESULTS: The ALL (sum of clinical, joint and spine AKUSSI features) AKUSSI rate of change of scores/patient/month, in the SAME group, was significantly lower at two (0.32 ±â€¯0.19) and three (0.15 ±â€¯0.13) years post-nitisinone when compared to pre-nitisinone (0.65 ±â€¯0.15) (p < .01 for both comparisons). Similarly, the ALL AKUSSI rate of change of scores/patient/month, in the VAR group, was significantly lower at one (0.16 ±â€¯0.08) and three (0.19 ±â€¯0.06) years post-nitisinone when compared to pre-nitisinone (0.59 ±â€¯0.13) (p < .01 for both comparisons). Combined ear and ocular ochronosis rate of change of scores/patient/month was significantly lower at one, two and three year's post-nitisinone in both VAR and SAME groups compared with pre-nitisinone (p < .05). CONCLUSION: This is the first indication that a 2 mg dose of nitisinone slows down the clinical progression of AKU. Combined ocular and ear ochronosis progression was arrested by nitisinone.

Clinical and biochemical assessment of depressive symptoms in patients with Alkaptonuria before and after two years of treatment with nitisinone.

OBJECTIVE: Concerns exist over hypertyrosinaemia that is observed following treatment with nitisinone. It has been suggested that tyrosine may compete with tryptophan for uptake into the central nervous system, and or inhibit tryptophan hydroxylase activity reducing serotonin production. At the National Alkaptonuria (AKU) Centre nitisinone is being used off-licence to treat AKU, and there is uncertainty over whether hypertyrosinaemia may alter mood. Herein results from clinical and biochemical assessments of depression in patients with AKU before and after treatment with nitisinone are presented. PATIENTS AND METHODS: 63 patients were included pre-nitisinone treatment, of these 39 and 32 patients were followed up 12 and 24 months after treatment. All patients had Becks Depression Inventory-II (BDI-II) assessments (scores can range from 0 to 63, the higher the score the more severe the category of depression), and where possible urinary monoamine neurotransmitter metabolites and serum aromatic amino acids were measured as biochemical markers of depression. RESULTS: Mean (±standard deviation) BDI-II scores pre-nitisinone, and after 12 and 24 months were 10.1(9.6); 9.8(10.0) and 10.5(9.9) (p ≥ 0.05, all visits). Paired scores (n = 32), showed a significant increase at 24 months compared to baseline 10.5(9.9) vs. 8.6 (7.8) (p = 0.03). Serum tyrosine increased at least 6-fold following nitisinone (p ≤ 0.0001, all visits), and urinary 3-methoxytyramine (3-MT) increased at 12 and 24 months (p ≤ 0.0001), and 5-hydroxyindole acetic acid (5-HIAA) decreased at 12 months (p = 0.03). CONCLUSIONS: BDI-II scores were significantly higher following 24 months of nitisinone therapy in patients that were followed up, however the majority of these patients remained in the minimal category of depression. Serum tyrosine and urinary 3-MT increased significantly following treatment with nitisinone. In contrast urinary 5-HIAA did not decrease consistently over the same period studied. Together these findings suggest nitisinone does not cause depression despite some observed effects on monoamine neurotransmitter metabolism.

Serum Amino Acid Profiling in Patients with Alkaptonuria Before and After Treatment with Nitisinone.

BACKGROUND: Alkaptonuria (AKU) is a rare inherited disorder of the tyrosine metabolic pathway. Our group is evaluating the use of the homogentisic acid-lowering agent nitisinone in patients with AKU. A major biochemical consequence of this treatment is hypertyrosinaemia. Herein we report the concentration of 20 serum amino acids over a 36-month period pre- and post-treatment with nitisinone. METHODS: Fasting serum samples were collected at baseline (pre-nitisinone), 3 (2 mg nitisinone every other day), 6, 12, 24 and 36 (2 mg nitisinone daily) months. Amino acids were measured using the Biochrom 30 high-performance liquid chromatography cation exchange system with ninhydrin detection. RESULTS: Fifty patients [21 female, mean age (±standard deviation) 54.1 (15.6) years (range 25-75); 29 male, mean age 49.3 (11.6) years (range 22-70 years)] were included. Following treatment mean tyrosine concentrations increased seven- to eight-fold (baseline, 69.8 µmol/L; 3 months, 670.7 µmol/L; 6 months, 666.4 µmol/L; 12 months, 692.9 µmol/L; 24 months, 649.4 µmol/L; 36 months, 724.8 µmol/L, p = <0.001 for all visits compared to baseline).At baseline mean phenylalanine, aspartic acid and arginine were outside the normal reference range. Following treatment the ratios of phenylalanine/tyrosine, phenylalanine/large neutral amino acids, arginine/branched chain amino acids and branched chain/aromatic amino acids decreased (p = <0.05), and the tyrosine/large neutral amino acid ratio increased (p = <0.0001). CONCLUSIONS: Marked hypertyrosinaemia was observed following treatment with nitisinone. Noteworthy changes were also observed in the ratio of several amino acids following treatment with nitisinone suggesting that the availability of amino acids for neurotransmitter biosynthesis and liver function may be altered following treatment with nitisinone.

Genetic diversity of equine herpesvirus 1 isolated from neurological, abortigenic and respiratory disease outbreaks.

Equine herpesvirus 1 (EHV-1) causes respiratory disease, abortion, neonatal death and neurological disease in equines and is endemic in most countries. The viral factors that influence EHV-1 disease severity are poorly understood, and this has hampered vaccine development. However, the N752D substitution in the viral DNA polymerase catalytic subunit has been shown statistically to be associated with neurological disease. This has given rise to the term "neuropathic strain," even though strains lacking the polymorphism have been recovered from cases of neurological disease. To broaden understanding of EHV-1 diversity in the field, 78 EHV-1 strains isolated over a period of 35 years were sequenced. The great majority of isolates originated from the United Kingdom and included in the collection were low passage isolates from respiratory, abortigenic and neurological outbreaks. Phylogenetic analysis of regions spanning 80% of the genome showed that up to 13 viral clades have been circulating in the United Kingdom and that most of these are continuing to circulate. Abortion isolates grouped into nine clades, and neurological isolates grouped into five. Most neurological isolates had the N752D substitution, whereas most abortion isolates did not, although three of the neurological isolates from linked outbreaks had a different polymorphism. Finally, bioinformatic analysis suggested that recombination has occurred between EHV-1 clades, between EHV-1 and equine herpesvirus 4, and between EHV-1 and equine herpesvirus 8.

Assessment of the Effect of Once Daily Nitisinone Therapy on 24-h Urinary Metadrenalines and 5-Hydroxyindole Acetic Acid Excretion in Patients with Alkaptonuria After 4 Weeks of Treatment.

BACKGROUND: One of the major metabolic consequences of using nitisinone to treat patients with alkaptonuria is that circulating tyrosine concentrations increase. As tyrosine is required for the biosynthesis of catecholamine neurotransmitters, it is possible that their metabolism is altered as a consequence. Herein we report the 24-h urinary excretion of normetadrenaline (NMA), metadrenaline (MA), 3-methoxytyramine (3-MT) (catecholamine metabolites) and 5-hydroxyindole acetic acid (5-HIAA, metabolite of serotonin) in a cohort of AKU patients before and after a 4-week treatment trial with nitisinone. MATERIALS AND METHODS: 24 h urinary excretions of NMA, MA, 3-MT and 5-HIAA were determined by liquid chromatography tandem mass spectrometry. Interassay coefficient of variation was <10% for all analytes measured, at all concentrations tested. RESULTS: Urine samples were assayed at baseline (pre-nitisinone, n = 36) and 4 weeks later; 7 received no nitisinone (4 male, mean age (±SD) 46.3 (16.4) years), and 29 received a daily dose of nitisinone [1 mg (n = 7, 6 male, mean age 45.9 (10.9) years), 2 mg (n = 8, 5 male, mean age 43.9 (13.7) years), 4 mg (n = 8, 5 male, mean age 47.3 (10.7) years) and 8 mg (n = 6, 4 male, mean age 53.8 (8.3) years)]. 3-MT concentrations increase significantly (p < 0.01, at all doses) following nitisinone therapy but not in a dose-dependent manner. NMA concentrations decreased (p < 0.05, at all doses) following nitisinone therapy at all doses. 5-HIAA concentrations decreased following nitisinone therapy and were significantly lower at a daily dose of 8 mg only (p < 0.05). CONCLUSIONS: This study shows that catecholamine and serotonin metabolism is altered by treatment with nitisinone.

Bioinformatics tools for analysing viral genomic data.

The field of viral genomics and bioinformatics is experiencing a strong resurgence due to high-throughput sequencing (HTS) technology, which enables the rapid and cost-effective sequencing and subsequent assembly of large numbers of viral genomes. In addition, the unprecedented power of HTS technologies has enabled the analysis of intra-host viral diversity and quasispecies dynamics in relation to important biological questions on viral transmission, vaccine resistance and host jumping. HTS also enables the rapid identification of both known and potentially new viruses from field and clinical samples, thus adding new tools to the fields of viral discovery and metagenomics. Bioinformatics has been central to the rise of HTS applications because new algorithms and software tools are continually needed to process and analyse the large, complex datasets generated in this rapidly evolving area. In this paper, the authors give a brief overview of the main bioinformatics tools available for viral genomic research, with a particular emphasis on HTS technologies and their main applications. They summarise the major steps in various HTS analyses, starting with quality control of raw reads and encompassing activities ranging from consensus and de novo genome assembly to variant calling and metagenomics, as well as RNA sequencing.

Le champ de la génomique virale et de la bio-informatique connaît actuellement un nouvel essor grâce à la technologie du séquençage à haut débit (SHD), qui permet de séquencer puis d'assembler rapidement un très grand nombre de génomes viraux, à un coût abordable. De surcroît, grâce à la puissance sans précédent des technologies du SHD, il est désormais possible d'analyser la diversité des virus au sein d'un hôte ainsi que la dynamique des quasi-espèces afin d'élucider d'importantes questions biologiques ayant trait à la transmission virale, à la résistance vis-à-vis des vaccins et au passage d'un hôte à l'autre. Le SHD permet également d'identifier rapidement des virus connus ou potentiellement nouveaux dans des échantillons de terrain ou cliniques, ce qui apporte de nouveaux outils pour la découverte des virus et la métagénomique. La bio-informatique joue un rôle central dans le développement des applications du SHD car ce domaine en constante évolution génère des séries de données aussi nombreuses que complexes dont le traitement et l'analyse requièrent en permanence de nouveaux algorithmes et logiciels. Les auteurs font rapidement le point sur les principaux outils de la bio-informatique utilisés dans la recherche sur les génomes viraux, en mettant particulièrement l'accent sur les technologies du SHD et sur leurs applications les plus importantes. Ils décrivent schématiquement les grandes étapes de différents types d'analyse recourant au SHD, depuis le contrôle qualité des lectures brutes jusqu'aux activités telles que l'assemblage de séquences consensus et de novo du génome, l'appel de variants et la métagénomique, et enfin le séquençage d'ARN.

El campo de la genómica vírica y la bioinformática conoce hoy un renovado dinamismo gracias a las técnicas de secuenciación de alto rendimiento, que permiten secuenciar con rapidez y rentabilidad, y a continuación ensamblar, un gran número de genomas víricos. Además, la potencia sin precedentes que ofrecen estas técnicas ha hecho posible analizar la diversidad vírica dentro de los anfitriones y la dinámica de cuasiespecies en relación con importantes interrogantes biológicos tocantes a la transmisión de virus, la resistencia a las vacunas o el salto de un anfitrión a otro. Con la secuenciación de alto rendimiento también es posible identificar con celeridad los virus tanto conocidos como eventualmente nuevos que estén presentes en muestras clínicas u obtenidas sobre el terreno, lo que aporta nuevas herramientas al arsenal disponible en los campos del descubrimiento de virus y la metagenómica. La bioinformática ha sido un factor capital en el auge de las aplicaciones de técnicas de secuenciación de alto rendimiento, pues continuamente se necesitan nuevos algoritmos y programas informáticos para procesar y analizar los vastos y complejos conjuntos de datos que se generan en un ámbito sujeto a tan rápida evolución. Los autores repasan brevemente las principales herramientas bioinformáticas que existen para la investigación en genómica vírica, prestando especial atención a las técnicas de secuenciación de alto rendimiento y sus principales aplicaciones. Asimismo, resumen las etapas básicas de diversos procedimientos de análisis por secuenciación de alto rendimiento, empezando por el control de calidad de las lecturas brutas y pasando por labores que van desde el ensamblaje del genoma con creación de secuencia consenso o ensamblaje de novo hasta la asignación de variantes (variant calling) o la metagenómica, sin olvidar la secuenciación de ARN.