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Posterior capsule opacification (PCO) is a post-surgery complication of cataract surgery, and lens epithelial cells (LECs) are involved in its development. A suppressive effect on LECs is exerted by the non specific chloride channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) exerts. Herein, the growth and migration inhibitory effects of NPPB on LECs were assessed, and the mechanism underlying the effects were investigated by focusing on Ca2+/CaMKII signaling. LECs were treated with different concentrations of NPPB, and the changes in cell viability, cell-cycle distribution, anchorage-dependent growth, migration, Ca2+ level, and CaMKII expression were evaluated. NPPB inhibited LECs' proliferation and induced G1 cell-cycle arrest in the cells. Regarding LECs' mobility, NPPB suppressed the cells' anchorage-dependent growth ability and inhibited their migration. Changes in cell phenotypes were associated with an increased intracellular Ca2+ level and down-regulation of CaMKII. Together these results confirmed the inhibitory effect of NPPB on the proliferation and migration of LECs, and the effect was shown to be associated with the induced level of Ca2+ and the inhibition of CaMKII signaling transduction.

While searching for novel small molecules for new organic pesticide agents against plant-parasitic nematodes, we found that the hexane extract from the roots of Senecio sinuatos and its main secondary metabolite, 3β-angeloyloxy-6β-hydroxyfuranoeremophil-1(10)-ene (1), possess nematicidal activity against the second stage juvenile (J2) of Meloidogyne incognita and Nacobbus aberrans. Both species reduce yield of various vegetable crops. These results encouraged us to synthesize esters 3-9 formed by diol 2, obtained by alkaline hydrolysis of 1 and acetic anhydride, benzoic acid, 2-nitrobenzoic acid, 2-bromobenzoic acid, 4-nitrobenzoic acid, 4-bromobenzoic acid, and 4-methoxybenzoic acid, respectively. The nematicidal activity of these esters was evaluated and compared with that of the free benzoic acids.Natural product 1 and derivatives 2-9 were obtained and characterized by their physical and spectroscopic properties, including one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) experiments; X-ray diffraction analysis established their absolute configuration. The nematicidal activity of compounds 1-9 was assessed in vitro against M. incognita and N. aberrans J2 and was compared to activity shown by benzoic acid, 2-nitrobenzoic acid, 2-bromobenzoic acid, 4-nitrobenzoic acid, 4-bromobenzoic acid, and 4-methoxybenzoic acid. The esters suppressed nematodes more than free benzoic acid. Nacobbus aberrans J2 were suppressed, with compounds 5, 6, and 8 being the most active.Esters formed by 3β,6β-dihydroxyfuranoeremophil-1(10)-ene and ortho- or para-substituted benzoic acids containing electron acceptor groups had nematicidal activity against N. aberrans. These compound can potentially serve as a model for the development of new organic nematicidal agents. © 2022 Society of Chemical Industry.

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 1100 (59) µmol/L. The majority of sampling points had values greater than 900 µmol/L. sPHE increased with increasing sTYR (p p p

Background: Tyrosinemia type 1 (HT1) is a rare metabolic disorder caused by a defect in the tyrosine catabolic pathway. Since HT1 patients are treated with NTBC, outcome improved and life expectancy greatly increased. However extensive neurocognitive and behavioural problems have been described, which might be related to treatment with NTBC, the biochemical changes induced by NTBC, or metabolites accumulating due to the enzymatic defect characterizing the disease.Objective: To study the possible pathophysiological mechanisms of brain dysfunction in HT1, we assessed blood and brain LNAA, and brain monoamine neurotransmitter metabolite levels in relation to behavioural and cognitive performance of HT1 mice.Design: C57BL/6 littermates were divided in three different experimental groups: HT1, heterozygous and wild-type mice (n = 10; 5 male). All groups were treated with NTBC and underwent cognitive and behavioural testing. One week after behavioural testing, blood and brain material were collected to measure amino acid profiles and brain monoaminergic neurotransmitter levels.Results: Irrespective of the genetic background, NTBC treatment resulted in a clear increase in brain tyrosine levels, whereas all other brain LNAA levels tended to be lower than their reference values. Despite these changes in blood and brain biochemistry, no significant differences in brain monoamine neurotransmitter (metabolites) were found and all mice showed normal behaviour and learning and memory.Conclusion: Despite the biochemical changes, NTBC and genotype of the mice were not associated with poorer behavioural and cognitive function of the mice. Further research involving dietary treatment of FAH−/− are warranted to investigate whether this reveals the cognitive impairments that have been seen in treated HT1 patients.

Since the EU approval of nitisinone in 2005, prognosis for patients with hereditary tyrosinaemia type 1 has changed dramatically, with patients living with the disease now reaching adulthood for the first time in history. This study aimed to assess the long-term safety and outcomes of nitisinone treatment in patients with hereditary tyrosinaemia type 1.We did a non-interventional, non-comparative, multicentre study in 77 sites across 17 countries in Europe and collected retrospective and prospective longitudinal data in patients with hereditary tyrosinaemia type 1 who were treated with oral nitisinone during the study period (Feb 21, 2005, to Sept 30, 2019). There were no specific exclusion criteria. Patients were followed-up with an investigator at least annually for as long as they were treated, or until the end of the study. The primary endpoints, occurrence of adverse events related to hepatic, renal, ophthalmic, haematological, or cognitive or developmental function, were assessed in the complete set (all patients already receiving treatment at the index date [Feb 21, 2005] or starting treatment thereafter) and the index set (the subset of patients who had their first dose on the index date or later only).315 patients were enrolled during the study period (complete set). Additionally, data from 24 patients who had liver transplantation or died during the post-marketing surveillance programme were retrieved (extended analysis set; 339 patients). Median treatment duration was 11·2 years (range 0·7-28·4); cumulative nitisinone exposure was 3172·7 patient-years. Patients who were diagnosed by neonatal screening started nitisinone treatment at median age 0·8 months versus 8·5 months in those who presented clinically. Incidences of hepatic, renal, ophthalmic, haematological, or cognitive or developmental adverse events were low. Occurrence of liver transplantation or death was more frequent the later that treatment was initiated (none of 70 patients who started treatment at age28 days vs 35 [13%] of 268 patients who started treatment at age ≥28 days). 279 (89%) of 315 patients were assessed as having either very good or good nitisinone treatment compliance. Treatment and diet compliance declined as patients aged. Suboptimal plasma phenylalanine and tyrosine levels were observed. The majority of patients were reported to have good overall clinical condition throughout treatment; 176 (87%) of 203 during the entire study, 98% following 1 year of treatment.Long-term nitisinone treatment was well tolerated and no new safety signals were revealed. Life-limiting hepatic disease appears to have been prevented by early treatment start. Neonatal screening was the most effective way of ensuring early treatment. Standardised monitoring of blood tyrosine, phenylalanine, and nitisinone levels has potential to guide individualised therapy.Swedish Orphan Biovitrum (Sobi).

A large alkaptonuria (AKU) cohort was studied to better characterise the poorly understood phenotype of aortic stenosis of rare disease AKU.Eighty-one patients attended the National Alkaptonuria Centre (NAC) between 2007 and 2020. Nine only attended once. Fifty-one attended more than once and received nitisinone 2 mg daily. Twenty-one attended at least twice without receiving nitisinone. Assessments included questionnaire analysis, standard transthoracic echocardiography, as well as photographs of ochronotic pigment in eyes and ears at baseline when 2 mg nitisinone was commenced, and yearly thereafter. Blood and urine samples were collected for chemical measurement. The prevalence of aortic stenosis and aortic valve replacement were 22.2 and 6.2% in the current group. Aortic maximum velocity (Vmax) was directly related to varying degrees to age (R = 0.58, p 0.001), systolic blood pressure (R = 0.32, p 0.05), serum homogentisic acid (sHGA) (R = 0.28, p 0.05), ochronosis scores (R = 0.72, p 0.001), and alkaptonuria severity score index (AKUSSI) (R = 0.58, p 0.001) on linear regression analysis. Age and ochronosis scores were significantly related to Vmax on multiple regression analysis (p 0.005). Nitisinone decreased sHGA, 24-h urine HGA (uHGAAortic valve disease is highly prevalent in this NAC cohort, and strongly associated with ochronosis and disease severity. Nitisinone decreases ochronosis and had a similar significant effect on Vmax.

Venturing onto the water for business or pleasure is not a risk-free activity. Despite the dangers facing crew and passengers there is little data on the characteristics of fatal accidents involving vessels in the water. The goal of this study was to review accident reports from the National Transportation Safety Board (NTSB) to determine characteristics of fatal marine accidents.Data was obtained from the Marine Accident Reports issued by the NTSB. Information regarding the number of people involved, fatalities and the accident itself was collected.Fifty-two accidents involving 5045 people from 1972 to 2019 were included in the study, with 468 fatalities reported. Of the fatalities, 155 (33.1%) were definitely on the vessel when they died, 49 (10.5%) were probably on the vessel, 65 (13.9%) were definitely or likely in the water, and the location of 199 (42.5%) was unknown. The most common cause of death was drowning (88, 18.8%), the most common accident cause was sinking (63.5%), and accidents most often started during nighttime hours (7pm-7am, 30, 57.7%).This study found that sinking was the most common accident cause for fatal marine accidents, drowning the most common cause of death, and where fatality location was known most were on the vessel when they died. This suggests that, particularly when a ship is in the process of sinking, it is of paramount importance to ensure passengers and crew are familiar with exit routes, are able to exit the vessel, and are instructed to do so in a timely manner.

Proper growth and patterning of blood vessels are critical for embryogenesis. Chemicals or environmental hormones may interfere with vascular growth and cause developmental defects. Nitrobenzoate-based compounds have been demonstrated to have a wide range of biological and pharmacological functions, leading to the development of numerous 4-nitrobenzoate derivatives for clinical application. In this study, we tested a novel nitrobenzoate-derived compound, X8, and investigated its effects on vascular development using zebrafish as a model organism. We first determined the survival rate of embryos after the addition of exogenous X8 (0.5, 1, 3, 5, and 10 μM) to the fish medium and determined a sublethal dose of 3 μM for use in further assays. We used transgenic fish to examine the effects of X8 treatment on vascular development. At 25–32 h postfertilization (hpf), X8 treatment impaired the growth of intersegmental vessels (ISVs) and caudal vein plexuses (CVPs). Moreover, X8-treated embryos exhibited pericardial edema and circulatory defects at 60–72 hpf, suggesting the effects of X8 in vasculature. Apoptosis tests showed that the vascular defects were likely caused by the inhibition of proliferation and migration. To investigate the molecular impacts underlying the defects in the vasculature of X8-treated fish, the expression levels of vascular markers, including ephrinb2, mrc1, and stabilin, were assessed, and the decreased expression of those genes was detected, indicating that X8 inhibited the expression of vascular genes. Finally, we showed that X8 treatment disrupted exogenous GS4012-induced angiogenesis in Tg(flk:egfp) zebrafish embryos. In addition, vascular defects were enhanced during cotreatment with X8 and the VEGFR2 inhibitor SU5416, suggesting that X8 treatment causes vascular defects mediated by disruption of VEGF/VEGFR2 signaling. Collectively, our findings indicate that X8 could be developed as a novel antiangiogenic agent.

BACKGROUND AND AIMS Apelin (APLN) is the endogenous ligand of its G protein-coupled receptor, apelin receptor (APJ). APLN serum levels are increased in human liver diseases. We evaluated whether the APLN-APJ axis regulates ductular reaction and liver fibrosis during cholestasis. APPROACH AND RESULTS We measured the expression of APLN and APJ and serum APLN levels in human primary sclerosing cholangitis (PSC) samples. Following bile duct ligation (BDL) or sham surgery, male wild-type (WT) mice were treated with ML221 (APJ antagonist) or saline for 1 week. WT and APLN-/- mice underwent BDL or sham surgery for 1 week. Multidrug resistance gene 2 knockout (Mdr2-/- ) mice were treated with ML221 for 1 week. APLN levels were measured in serum and cholangiocyte supernatants, and cholangiocyte proliferation/senescence and liver inflammation, fibrosis, and angiogenesis were measured in liver tissues. The regulatory mechanisms of APLN-APJ in (1) biliary damage and liver fibrosis were examined in human intrahepatic biliary epithelial cells (HIBEpiCs) treated with APLN and (2) hepatic stellate cell (HSC) activation in APLN-treated human HSC lines (HHSteCs). APLN serum levels and biliary expression of APLN and APJ increased in PSC samples. APLN levels were higher in serum and cholangiocyte supernatants from BDL and Mdr2-/- mice. ML221 treatment or APLN-/- reduced BDL-induced and Mdr2-/- -induced cholangiocyte proliferation/senescence, liver inflammation, fibrosis, and angiogenesis. In vitro, APLN induced HIBEpiC proliferation, increased nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) expression, reactive oxygen species (ROS) generation, and extracellular signal-regulated kinase (ERK) phosphorylation. Pretreatment of HIBEpiCs with ML221, diphenyleneiodonium chloride (Nox4 inhibitor), N-acetyl-cysteine (NAC, ROS inhibitor), or PD98059 (ERK inhibitor) reduced APLN-induced cholangiocyte proliferation. Activation of HHSteCs was induced by APLN but reduced by NAC. CONCLUSIONS The APLN-APJ axis induces cholangiocyte proliferation through Nox4/ROS/ERK-dependent signaling and HSC activation through intracellular ROS. Modulation of the APLN-APJ axis may be important for managing cholangiopathies.

In December 2019, a new type of SARS corona virus emerged from China and caused a globally pandemic corona virus disease (COVID-19). This highly infectious virus has been named as SARS-CoV-2 by the International Committee of the Taxonomy of Viruses. It has severely affected a large population and economy worldwide. Globally various scientific communities have been involved in studying this newly emerged virus and is lifecycle. Multiple diverse studies are in progress to design novel therapeutic agents, in which understanding of interactions between the target and drug ligand is a significant key for this challenge. Structures of proteins involved in the life cycle of the virus have been revealed in RCSB PDB by researchers. In this study, we employed molecular docking study of 4-Acetamido-3-nitrobenzoic acid (ANBA) with corona virus proteins (spike protein, spike binding domain with ACE2 receptor and Main protease, RNA-dependent RNA polymerase). Single crystal X-ray analysis and density functional theory calculations were carried out for ANBA to explore the structural and chemical-reactive parameters. Intermolecular interactions which are involved in the ligand-protein binding process are validated by Hirshfeld surface analysis. To study the behaviour of ANBA in a living organism and to calculate the physicochemical parameters, ADMET analysis was done using SwissADME and Osiris data warrior tools. Further, Toxicity of ANBA was predicted using pkCSM online software. Based on the molecular docking analysis, we introduce here a potent drug molecule that binds to the COVID-19 proteins.Communicated by Ramaswamy H. Sarma.

The brevijanazines are novel

Creatinine, a biologically important compound, is used to analyze kidney function and kidney diseases in the human body. The salt form of creatinine is used in the formation of drug materials like anti-HIV, antifungal, antiprotozoal, antiviral and antitumour compounds. Here we report the solid-state structures of three new crystalline salts, namely, creatininium (2-amino-1-methyl-4-oxo-4,5-dihydro-1H-imidazol-3-ium) bromide, C4H8N3O+·Br−, (I), creatininium 3-aminobenzoate, C4H8N3O+·C7H6NO2 −, (II), and creatininium 3,5-dinitrobenzoate, C4H8N3O+·C7H3N2O6 −, (III). These salts have been synthesized and characterized by single-crystal X-ray diffraction and Hirshfeld surface analysis. The structural chemistry of salts (I)–(III) and their crystal packing are discussed in detail. The primary interaction between the creatinine cation and the acid anion in the three salts is N—H...Br/O hydrogen bonds. In salt (I), the creatinine cation and bromide anion are connected through a pair of N—H...Br hydrogen bonds forming R 4 2(8) and R 4 2(12) ring motifs. In salts (II) and (III), the creatinine cation interacts with the corresponding anion via a pair of N—H...O hydrogen bonds. The crystal structure is further stabilized by C—H...O and O—H...O hydrogen bonds with the ring motifs R 2 2(8), R 2 1(7) and R 2 1(6). Furthermore, the crystal structures are stabilized by π–π, C—H...π, C—O...π and N—O...π stacking interactions. The contributions made by each hydrogen bond in maintaining the crystal structure stability has been quantified by Hirshfeld surface analysis.

Motor neuron degeneration and neuroinflammation are the most striking pathological features of amyotrophic lateral sclerosis (ALS). ALS currently has no cure and approved drugs have only a modest clinically therapeutic effect in patients. Drugs targeting different deleterious inflammatory pathways in ALS appear as promising therapeutic alternatives. Here, we have assessed the potential therapeutic effect of an electrophilic nitroalkene benzoic acid derivative, (E)-4-(2-nitrovinyl) benzoic acid (BANA), to slow down paralysis progression when administered after overt disease onset in SOD1(G93A) rats. BANA exerted a significant inhibition of NF-κB activation in NF-κB reporter transgenic mice and microglial cell cultures. Systemic daily oral administration of BANA to SOD1(G93A) rats after paralysis onset significantly decreased microgliosis and astrocytosis, and significantly reduced the number of NF-κB-p65-positive microglial nuclei surrounding spinal motor neurons. Numerous microglia bearing nuclear NF-κB-p65 were observed in the surrounding of motor neurons in autopsy spinal cords from ALS patients but not in controls, suggesting ALS-associated microglia could be targeted by BANA. In addition, BANA-treated SOD1(G93A) rats after paralysis onset showed significantly ameliorated spinal motor neuron pathology as well as conserved neuromuscular junction innervation in the skeletal muscle, as compared to controls. Notably, BANA prolonged post-paralysis survival by ~30%, compared to vehicle-treated littermates. These data provide a rationale to therapeutically slow paralysis progression in ALS using small electrophilic compounds such as BANA, through a mechanism involving microglial NF-κB inhibition. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13311-020-00953-z.

Background Dried blood spot monitoring of nitisinone and succinylacetone in hereditary tyrosinaemia type 1 patients is not widely available in the United Kingdom. Currently, biochemical monitoring utilizes urinary succinylacetone, blood spot tyrosine and phenylalanine monitoring, which can lack in convenience and accuracy, respectively. Methods We report the development of a dried blood spot assay for nitisinone and succinylacetone and analysed retrospective clinical and biochemical data for hereditary tyrosinaemia type 1 patients from a single UK centre. Results A total of 13 hereditary tyrosinaemia type 1 patients were evaluated. Eleven presented with liver dysfunction (two with associated renal tubulopathy) and two were detected by early sibling screening. All patients (age 0.03–22 months) were commenced on a tyrosine-/phenylalanine-restricted diet and nitisinone at diagnosis. Ten patients were on twice daily dosing and three were on single daily dosing at the start of monitoring. One patient from each dosing group swapped between dosing regimens at 20 years of age and 8 months of age, respectively. A total of 684 dried blood spot samples were analysed; 80% of nitisinone concentrations were between 9.2 and 27 µmol/L when succinylacetone was Conclusions Dried blood spot monitoring for nitisinone and succinylacetone concentrations in hereditary tyrosinaemia type 1 patients is a rapid and convenient method which allows physicians to individualize treatment plans and observe adherence to treatment.

Background The prognosis of patients with Hereditary Tyrosinemia Type 1 (HT-1) has greatly improved with early detection through newborn screening and the introduction of nitisinone (NTBC) therapy. A recent guideline calls for periodic monitoring of biochemical markers and NTBC levels to tailor treatment; however, this is currently only achieved through a combination of clinical laboratory tests. We developed a multiplexed assay measuring relevant amino acids, succinylacetone (SUAC), and NTBC in dried blood spots (DBS) to facilitate treatment monitoring. Methods Tyrosine, phenylalanine, methionine, NTBC and SUAC were eluted from DBS with methanol containing internal standards for each analyte and analyzed by liquid chromatography tandem mass spectrometry over 6.5 min in the multiple reaction monitoring positive mode. Results Pre-analytical and analytical factors were studied and demonstrated a reliable assay. Chromatography resolved an unknown substance that falsely elevates SUAC concentrations and was present in all samples. To establish control and disease ranges, the method was applied to DBS collected from controls (n = 284) and affected patients before (n = 2) and after initiation of treatment (n = 29). In the treated patients SUAC concentrations were within the normal range over a wide range of NTBC levels. Conclusions This assay enables combined, accurate measurement of revelevant metabolites and NTBC in order to simplify treatment monitoring of patients with HT-1. In addition, the use of DBS allows for specimen collection at home to facilitate more standardization in relation to drug and dietary treatment.

Skin pigmentation is a highly heterogeneous trait with diverse consequences worldwide. SLC24A5, encoding a potent K(+)-dependent Na(+)/Ca(2+) exchanger, is among the known color-coding genes that participate in melanogenesis by maintaining pH in melanosomes. Deficient SLC24A5 activity results in oculocutaneous albinism (OCA) type 6 in humans. In this study, by utilizing a exome sequencing (ES) approach, we identified two new variants [p. (Gly110Arg) and p. (IIe189Ilefs*1)] of SLC24A5 cosegregating with the OCA phenotype, including nystagmus, strabismus, foveal hypoplasia, albinotic fundus and vision impairment, in three large consanguineous Pakistani families. Both of these variants failed to rescue the pigmentation in zebrafish slc24a5 morphants, confirming the pathogenic effects of the variants. We also phenotypically characterized a commercially available zebrafish mutant line (slc24a5(ko)) that harbors a nonsense (p.Tyr208*) allele of slc24a5. Similar to morphants, homozygous slc24a5(ko) mutants had significantly reduced melanin content and pigmentation. Next, we used these slc24a5(ko) zebrafish mutants to test the efficacy of nitisinone, a compound known to increase ocular and fur pigmentation in OCA1 (TYR)-mutant mice. Treatment of slc24a5(ko) mutant zebrafish embryos with varying doses of nitisinone did not improve melanin production and pigmentation, suggesting that treatment with nitisinone is unlikely to be therapeutic in OCA6 patients.

For over two decades, nitisinone (NTBC) has been successfully used to manipulate the tyrosine degradation pathway and save the lives of many children with hereditary tyrosinaemia type 1. More recently, NTBC has been used to halt homogentisic acid accumulation in alkaptonuria (AKU) with evidence suggesting its efficacy as a disease modifying agent. NTBC-induced hypertyrosinaemia has been associated with cognitive impairment and potentially sight-threatening keratopathy. In the context of a non-lethal condition (ie, AKU), these serious risks call for an evaluation of the wider impact of NTBC on the tyrosine pathway. We hypothesised that NTBC increases the tyrosine pool size and concentrations in tissues. In AKU mice tyrosine concentrations of tissue homogenates were measured before and after treatment with NTBC. In humans, pulse injection with l-[13 C9 ]tyrosine and l-[d8 ]phenylalanine was used along with compartmental modelling to estimate the size of tyrosine pools before and after treatment with NTBC. We found that NTBC increased tyrosine concentrations in murine tissues by five to nine folds. It also significantly increased the tyrosine pool size in humans (P

BACKGROUND: Alkaptonuria (AKU) is caused by homogentisate 1,2-dioxygenase deficiency that leads to homogentisic acid (HGA) accumulation, ochronosis and severe osteoarthropathy. Recently, nitisinone treatment, which blocks HGA formation, has been effective in AKU patients. However, a consequence of nitisinone is elevated tyrosine that can cause keratopathy. The effect of tyrosine and phenylalanine dietary restriction was investigated in nitisinone-treated AKU mice, and in an observational study of dietary intervention in AKU patients. METHODS: Nitisinone-treated AKU mice were fed tyrosine/phenylalanine-free and phenylalanine-free diets with phenylalanine supplementation in drinking water. Tyrosine metabolites were measured pre-nitisinone, post-nitisinone, and after dietary restriction. Subsequently an observational study was undertaken in 10 patients attending the National Alkaptonuria Centre (NAC), with tyrosine >700μmol/L who had been advised to restrict dietary protein intake and where necessary, to use tyrosine/phenylalanine-free amino acid supplements. RESULTS: Elevated tyrosine (813μmol/L) was significantly reduced in nitisinone-treated AKU mice fed a tyrosine/phenylalanine-free diet in a dose responsive manner. At 3 days of restriction, tyrosine was 389.3μmol/L, 274.8μmol/L and 144.3μmol/L with decreasing phenylalanine doses. In contrast, tyrosine was not effectively reduced in mice by a phenylalanine-free diet; at 3 days tyrosine was 757.3μmol/L, 530.2μmol/L and 656.2μmol/L, with no dose response to phenylalanine supplementation. In NAC patients, tyrosine was significantly reduced (p=0.002) when restricting dietary protein alone, and when combined with tyrosine/phenylalanine-free amino acid supplementation; 4 out of 10 patients achieved tyrosine

Several rare pediatric liver disorders are accompanied by ophthalmic signs whose awareness and early identification may be of value in confirming/accelerating their diagnosis. Many of these signs are asymptomatic and can only be detected with an ophthalmological examination. Corneal signs are described in patients with Wilson's disease, Alagille's syndrome and some liver storage diseases. Cataract plays an important role to diagnose galactosemia. Retinal involvement is seen in some peroxisomal disorders (e.g. Zellweger's syndrome), in mucopolysaccharidoses (pigmentary retinopathy), and in Niemann-Pick disease (macular cherry red spot). In mucopolysaccharidoses optic nerve can be involved as optic atrophy secondary to pigmentary retinopathy or to chronic papilledema. Children with neonatal cholestasis due to hypopituitarism may present septo-optic dysplasia. Several infectious agents have an ophthalmological/hepatic involvement in the fetal life and/or thereafter. Some mitochondrial liver diseases, such as Pearson's syndrome, present pigmentary retinopathy and a chronic progressive external ophthalmoplegia. Finally, some drugs while protecting the liver may damage the ocular system as seen with long-term glucocorticoids and Nitisinone administration. This review provides a synopsis of those conditions that hepatologists and ophthalmologists should share among themselves to better take care of patients. Synoptic tables are presented to facilitate the mutual understanding of the issues.

Two-stage hepatectomy for bilobar colorectal cancer liver metastases is potentially curative for selected patients. Histological growth patterns of colorectal liver metastases (desmoplastic, replacement, and pushing) have prognostic value. Our aim was to evaluate their association with pathologic response to preoperative treatment, second-stage hepatectomy completion, and survival in patients treated with a curative-intent 2-stage hepatectomy.In 67 patients planned for 2-stage hepatectomy, colorectal liver metastases resected from the first-stage hepatectomy were retrospectively evaluated for growth patterns and pathologic response according to Tumor Regression Grading, modified Tumor Regression Grading, and Blazer grading. Tumor Regression Grading 1 to 3, modified Tumor Regression Grading 1 to 3, and Blazer 0 and 1 defined good responders.Desmoplastic growth patterns (GP) were more frequent among good responders (Plt; .001). Second-stage hepatectomy completion was associated with desmoplastic growth patterns and pathologic response on univariate analysis and multivariable analyses (P = .017 and P = .041, respectively). Median follow-up was 84 months (95% confidence interval: 53.4 [not reached]). Nondesmoplastic GP patients and nonresponders had a poorer overall survival (hazard ratio = 3.86, 95% confidence interval: 2.11-7.07, Plt; .001 and hazard ratio = 2.14, 95% confidence interval: 1.19-3.83, P = .009, respectively) on univariate analysis. Nondesmoplastic growth pattern was the only factor associated with a poorer overall survival on multivariable analysis (hazard ratio = 4.17, 95% confidence interval: 1.79-9.74, Plt; .001). Nondesmoplastic GP was also associated with a poorer recurrence-free survival (hazard ratio = 2.05, 95% confidence interval: 1.13-3.70, P = .017).Desmoplastic GP could represent a useful morphological marker for early identification of patients who might benefit from 2-stage hepatectomy completion.

Introduction: Nitisinone used in alkaptonuria (AKU) can result in keratopathy due to strongly increased tyrosine levels. Methods: This study aimed to investigate nutritional status and changes in plasma tyrosine and phenylalanine and urinary homogentisic acid (u-HGA) levels in 8 adult AKU patients (mean age, 56.3 ± 4.7 years) who were on tyrosine/phenylalanine-restricted diet together with 2 mg/day nitisinone. Results: The treatment period was 23.4 ± 6.9 months. Daily dietary protein intake was restricted to 0.8–1.0 g/kg/day. Daily tyrosine intake was restricted to 260–450 mg/day for females and 330–550 mg/day for males. Tyrosine/phenylalanine-free amino acid supplements accounted for an average of 56.1% of daily protein intake. The following assessments were performed: anthropometric and plasma tyrosine level measurements every 2 months; ophthalmological examination every 6 months, and nutritional laboratory analyses and measurements of plasma amino acids and u-HGA once in a year. It was targeted to keep the plasma tyrosine level 700 μmol/L was detected. The u-HGA level before and after the 1st year of treatment was 1,429.3 ± 1,073.4 mmol/mol creatinine and 33.6 ± 9.5 mmol/mol creatinine, respectively. None of the patients developed keratopathy or experienced weight loss and protein or micronutrient deficiency. Conclusion: AKU patients should receive tyrosine/phenylalanine-restricted diet for reducing plasma tyrosine level to the safe range. Tyrosine/phenylalanine-free amino acid supplements can be safely used to enhance dietary compliance. Keratopathy and nutrient deficiency should be frequently monitored.

Plasma concentrations of natriuretic peptides (NP) contribute to risk stratification and management of patients undergoing non-cardiac surgery. However, genetically determined variability in the levels of these biomarkers has been described previously. In the perioperative setting, genetic contribution to NP plasma level variability has not yet been determined. A cohort of 427 patients presenting for non-cardiac surgery was genotyped for single-nucleotide polymorphisms (SNPs) from the NPPA/NPPB locus. Haplotype population frequencies were estimated and adjusted haplotype trait associations for brain natriuretic peptide (BNP) and amino-terminal pro natriuretic peptide (NT-proBNP) were calculated. Five SNPs were included in the analysis. Compared to the reference haplotype TATAT (rs198358, rs5068, rs632793, rs198389, rs6676300), haplotype CACGC, with an estimated frequency of 4%, showed elevated BNP and NT-proBNP plasma concentrations by 44% and 94%, respectively. Haplotype CGCGC, with an estimated frequency of 9%, lowered NT-proBNP concentrations by 28%. ASA classification status III and IV, as well as coronary artery disease, were the strongest predictors of increased NP plasma levels. Inclusion of genetic information might improve perioperative risk stratification of patients based on adjusted thresholds of NP plasma levels.

Nitroaromatic compounds pose severe threats to public health and environmental safety. Nitro group removal via ammonia release is an important strategy for bacterial detoxification of nitroaromatic compounds, such as the conversion of 4-nitrobenzoate (4-NBA) to protocatechuate by the bacterial pnb operon. In contrast to the LysR-family transcriptional regulator PnbR in proteobacteria, the actinomycete-derived pnb locus (4-NBA degradation structural genes) formed an operon with the TetR-family transcriptional regulator gene pnbX, implying that it has a distinct regulatory mechanism. Here, pnbBA from the actinomycete Nocardioides sp. strain LMS-CY was biochemically confirmed to express 4-NBA degradation enzymes, and pnbX was essential for inducible degradation of 4-NBA. Purified PnbX-6His could bind the promoter probe of the pnb locus in vitro, and 4-NBA prevented this binding. 4-NBA could bind PnbX at a 1:1 molar ratio with KD = 26.7 ± 4.2 nM. Low-nanomolar levels of 4-NBA induced the transcription of the pnb operon in strain LMS-CY. PnbX bound a palindromic sequence motif (5'-TTACGTTACA-N8 -TGTAACGTAA-3') that encompasses the pnb promoter. This study identified a TetR-family repressor for the actinomycete-derived pnb operon that recognizes 10-8 M 4-NBA as its ligand, implying that nitro group removal of nitroaromatic compounds may be especially important for actinomycetes.

Nitroaromatic compounds are essential materials for chemical industry, but they are also potentially toxic environmental pollutants. Therefore, their sensitive detection and degradation are important concerns. The microbial degradation pathways of nitroaromatic compounds have been studied in detail, but their usefulness needs to be evaluated to understand their potential applications in bioremediation. Here, we developed a rapid and relatively sensitive assay system to evaluate the activities and substrate specificities of nitroaromatic dioxygenases involved in the oxidative biodegradation of nitroaromatic compounds. In this system, nitrous acid, which was released from the nitroaromatic compounds by the dioxygenases, was detected and quantified using the Saltzman reagent. Escherichia coli producing the 3-nitrobenzoic acid dioxygenase complex MnbAB from Comamonas sp. JS46 clearly showed the apparent substrate specificity of MnbAB as follows. MnbAB accepted not only 3-nitrobenzoic acid but also several other p- and m-nitrobenzoic acid derivatives as substrates, although it much preferred 3-nitrobenzoic acid to others. Furthermore, the presence of a hydroxy or an amino group at the ortho position of the nitro group decreased the activity of MnbAB. In addition, MnbAB accepted 2-(4-nitrophenyl)acetic acid as a substrate, which has one additional methylene group between the aromatic ring and the carboxy group of 3-nitrobenzoic acid. This is the first report about the detailed substrate specificity of MnbAB. Our system can be used for other nitroaromatic dioxygenases and contribute to their characterization.

Alkaptonuria (AKU, OMIM 203500) is a rare congenital disorder caused by a deficiency of the enzyme homogentisate-1,2,-dioxygenase. The long-term consequences of AKU are joint problems, cardiac valve abnormalities and renal problems. Landmark intervention studies with nitisinone 10 mg daily, suppressing an upstream enzyme activity, demonstrated its beneficial effects in AKU patients with established complications, which usually start to develop in the fourth decade. Lower dose of nitisinone in the range of 0.2–2 mg daily will already reduce urinary homogentisic acid (uHGA) excretion by > 90%, which may prevent AKU-related complications earlier in the course of the disease while limiting the possibility of side-effects related to the increase of plasma tyrosine levels caused by nitisinone. Future preventive studies should establish the lowest possible dose for an individual patient, the best age to start treatment and also collect evidence to which level uHGA excretion should be reduced to prevent complications.

Background While therapeutic advances have significantly improved the prognosis of patients with hereditary tyrosinemia type 1 (HT1), adherence to dietary and pharmacological treatments is essential for an optimal clinical outcome. Poor treatment adherence is well documented among patients with chronic diseases, but data from HT1 patients are scarce. This study evaluated pharmacological and dietary adherence in HT1 patients both directly, by quantifying blood levels nitisinone (NTBC) levels and metabolic biomarkers of HT1 [tyrosine (Tyr), phenylalanine (Phe), and succinylacetone]; and indirectly, by analyzing NTBC prescriptions from hospital pharmacies and via clinical interviews including the Haynes–Sackett (or self-compliance) test and the adapted Battle test of patient knowledge of the disease. Results This observational study analyzed data collected over 4 years from 69 HT1 patients (7 adults and 62 children; age range, 7 months–35 years) who were treated with NTBC and a low-Tyr, low-Phe diet. Adherence to both pharmacological and, in particular, dietary treatment was poor. Annual data showed that NTBC levels were lower than recommended in more than one third of patients, and that initial Tyr levels were high (> 400 µM) in 54.2–64.4% of patients and exceeded 750 µM in 25.8% of them. Remarkably, annual normalization of NTBC levels was observed in 29.4–57.9% of patients for whom serial NTBC determinations were performed. Poor adherence to dietary treatment was more refractory to positive reinforcement: 36.2% of patients in the group who underwent multiple analyses per year maintained high Tyr levels during the entire study period, and, when considering each of the years individually this percentage ranged from 75 to 100% of them. Indirect methods revealed percentages of non-adherent patients of 7.3 and 15.9% (adapted Battle and Haynes tests, respectively). Conclusions Despite initially poor adherence to pharmacological and especially dietary treatment among HT1 patients, positive reinforcement at medical consultations resulted in a marked improvement in NTBC levels, indicating the importance of systematic positive reinforcement at medical visits.