Acetyl-leucine slows disease progression in lysosomal storage disorders

Acetyl-DL-leucine is a derivative of the branched chain amino acid leucine. In observational clinical studies acetyl-DL-leucine improved symptoms of ataxia, in particular in patients with the lysosomal storage disorder, Niemann-Pick disease type C1. Here, we investigated acetyl-DL-leucine and its enantiomers acetyl-L-leucine and acetyl-D-leucine in symptomatic Npc1-/- mice and observed improvement in ataxia with both individual enantiomers and acetyl-DL-leucine. When acetyl-DL-leucine and acetyl-L-leucine were administered pre-symptomatically to Npc1-/- mice, both treatments delayed disease progression and extended life span, whereas acetyl-D-leucine did not. These data are consistent with acetyl-L-leucine being the neuroprotective enantiomer. Altered glucose and antioxidant metabolism were implicated as one of the potential mechanisms of action of the L enantiomer in Npc1-/- mice. When the standard of care drug miglustat and acetyl-DL-leucine were used in combination significant synergy resulted. In agreement with these pre-clinical data, when Niemann-Pick disease type C1 patients were evaluated after 12 months of acetyl-DL-leucine treatment, rates of disease progression were slowed, with stabilisation or improvement in multiple neurological domains. A beneficial effect of acetyl-DL-leucine on gait was also observed in this study in a mouse model of GM2 gangliosidosis (Sandhoff disease) and in Tay-Sachs and Sandhoff disease patients in individual cases of off-label-use. Taken together, we have identified an unanticipated neuroprotective effect of acetyl-L-leucine and underlying mechanisms of action in lysosomal storage diseases, supporting its further evaluation in clinical trials in lysosomal disorders

Cytokeratin-18 is a sensitive biomarker of alanine transaminase increase in a placebo-controlled, randomized, crossover trial of therapeutic paracetamol dosing (PATH-BP biomarker substudy)

Drug-induced liver injury (DILI) is a challenge in clinical medicine and drug development. Highly sensitive novel biomarkers have been identified for detecting DILI following a paracetamol overdose. The study objective was to evaluate biomarker performance in a 14-day trial of therapeutic dose paracetamol. The PATH-BP trial was a double-blind, placebo-controlled, crossover study. Individuals (n = 110) were randomized to receive 1 g paracetamol 4× daily or matched placebo for 2 weeks followed by a 2-week washout before crossing over to the alternate treatment. Blood was collected on days 0 (baseline), 4, 7, and 14 in both arms. Alanine transaminase (ALT) activity was measured in all patients. MicroRNA-122 (miR-122), cytokeratin-18 (K18), and glutamate dehydrogenase (GLDH) were measured in patients who had an elevated ALT on paracetamol treatment (≥50% from baseline). ALT increased in 49 individuals (45%). All 3 biomarkers were increased at the time of peak ALT (K18 paracetamol arm: 18.9 ± 9.7 ng/ml, placebo arm: 11.1 ± 5.4 ng/ml, ROC-AUC = 0.80, 95% CI 0.71–0.89; miR-122: 15.1 ± 12.9fM V 4.9 ± 4.7fM, ROC-AUC = 0.83, 0.75–0.91; and GLDH: 24.6 ± 31.1U/l V 12.0 ± 11.8U/l, ROC-AUC = 0.66, 0.49–0.83). All biomarkers were correlated with ALT (K18 r = 0.68, miR-122 r = 0.67, GLDH r = 0.60). To assess sensitivity, biomarker performance was analyzed on the visit preceding peak ALT (mean 3 days earlier). K18 identified the subsequent ALT increase (K18 ROC-AUC = 0.70, 0.59–0.80; miR-122 ROC-AUC = 0.60, 0.49–0.72, ALT ROC-AUC = 0.59, 0.48–0.70; GLDH ROC-AUC = 0.70, 0.50–0.90). Variability was lowest for ALT and K18. In conclusion, K18 was more sensitive than ALT, miR-122, or GLDH and has potential significant utility in the early identification of DILI in trials and clinical practice

Annual severity increment score as a tool for stratifying patients with Niemann-Pick disease type C and for recruitment to clinical trials

Background: Niemann-Pick disease type C (NPC) is a lysosomal storage disease with a heterogeneous neurodegenerative clinical course. Multiple therapies are in clinical trials and inclusion criteria are currently mainly based on age and neurological signs, not taking into consideration differential individual rates of disease progression. Results: In this study, we have evaluated a simple metric, denoted annual severity increment score (ASIS), that measures rate of disease progression and could easily be used in clinical practice. We show that ASIS is stable over several years and can be used to stratify patients for clinical trials. It achieves greater homogeneity of the study cohort relative to age-based inclusion and provides an evidence-based approach for establishing inclusion/exclusion criteria. In addition, we show that ASIS has prognostic value and demonstrate that treatment with an experimental therapy - acetyl-DL-leucine - is associated with a reduction in ASIS scores. Conclusion: ASIS has the potential to be a useful metric for clinical monitoring, trial recruitment, for prognosis and measuring response to therapy

Comparative in situ analyses of cell wall matrix polysaccharide dynamics in developing rice and wheat grain

Cell wall polysaccharides of wheat and rice endosperm are an important source of dietary fibre. Monoclonal antibodies specific to cell wall polysaccharides were used to determine polysaccharide dynamics during the development of both wheat and rice grain. Wheat and rice grain present near synchronous developmental processes and significantly different endosperm cell wall compositions, allowing the localisation of these polysaccharides to be related to developmental changes. Arabinoxylan (AX) and mixed-linkage glucan (MLG) have analogous cellular locations in both species, with deposition of AX and MLG coinciding with the start of grain filling. A glucuronoxylan (GUX) epitope was detected in rice, but not wheat endosperm cell walls. Callose has been reported to be associated with the formation of cell wall outgrowths during endosperm cellularisation and xyloglucan is here shown to be a component of these anticlinal extensions, occurring transiently in both species. Pectic homogalacturonan (HG) was abundant in cell walls of maternal tissues of wheat and rice grain, but only detected in endosperm cell walls of rice in an unesterified HG form. A rhamnogalacturonan-I (RG-I) backbone epitope was observed to be temporally regulated in both species, detected in endosperm cell walls from 12 DAA in rice and 20 DAA in wheat grain. Detection of the LM5 galactan epitope showed a clear distinction between wheat and rice, being detected at the earliest stages of development in rice endosperm cell walls, but not detected in wheat endosperm cell walls, only in maternal tissues. In contrast, the LM6 arabinan epitope was detected in both species around 8 DAA and was transient in wheat grain, but persisted in rice until maturity

Pathogenic mycobacteria achieve cellular persistence via lipid-mediated inhibition of the Niemann-Pick disease type C pathway

M.tuberculosis, the causative agent of human tuberculosis, is able to achieve long-term persistence within host organism macrophages. This persistence is achieved via the ability of the mycobacterium to prevent phagosomal-lysosomal fusion. The mechanisms by which fusion is inhibited remain incompletely understood. Here we provide evidence supporting a mechanistic link between infection with pathogenic mycobacteria and the cellular pathway defective in the rare lysosomal storage disorder Niemann-Pick disease type C (NPC). We observed that NPC phenotypes, including lipid storage and reduced lysosomal calcium release, can be induced in wild-type murine and human macrophages by infection with pathogenic mycobacteria. This phenotype induction did not occur following infection with the non-pathogenic M.smegmatis. Phenotype induction could be achieved in the absence of the mycobacteria using lipids from the mycobacterial cell walls. The importance of mycobacterial cell wall lipids to mycobacterial virulence has been well-documented. This lipid-mediated inhibition likely occurs through the NPC1 protein. Susceptibility to phenotype induction was inversely proportional to levels of functional NPC1, whilst a pre-existing dysfunction in the NPC pathway (either stemming from mutation or pharmacological inhibition) rendered cells less able to clear non-pathogenic mycobacteria. Finally, we demonstrate that therapies for NPC, particularly curcumin, are able to promote clearance of mycobacteria from infected macrophages. NPC therapies may hold promise for a new approach to the treatment of tuberculosis.</p

Novel configurations of Type I and II CRISPR/Cas systems in Corynebacterium diphtheriae

Clustered regularly interspaced short palindromic repeats (CRISPRs) are major barriers to recombination through recognition of invading nucleic acids, such as phage and plasmids, and promoting their degredation through the action of CRISPR associated (Cas) proteins. The genomic comparison of 17 Corynebacterium diphtheriae strains led to the identification of three novel CRISPR-Cas system variants, based on the Type II (Type II-C) or type I-E systems. The type II-C system was the most common (11/17 isolates) but it lacked the csn2 and cas4 genes that are involved in spacer acquisition. We also identified that this variant type II-C CRISPR-Cas system is present in other bacteria, and the first system was recently characterized in Neisseria meningitidis. In the remaining isolates, the type II-C system was replaced by a variant of type I-E (I-E-a), where the repeat arrays are inserted between the cas3 and cse1 genes. Three isolates with the type II-C system also possess an additional variant of type I-E (I-E-b), elsewhere in the genome, that exhibits a novel divergent gene organization within the cas operon. The nucleotide sequences of the palindromic repeats and the cas1 gene were phylogenetically incongruent to the core genome. The G+C content of the systems is lower (46.0-49.5 mol%) than the overall DNA G+C content (53 mol%), and they are flanked by mobile genetic elements, providing evidence that they were acquired in three independent horizontal gene transfer events. The majority of spacers lack identity with known phage or plasmid sequences, indicating that there is an unexplored reservoir of corynebacteriophages and plasmids. These novel CRISPR-Cas systems may represent a unique mechanism for spacer acquisitions and defence against invading DNA

Pathogenic mycobacteria achieve cellular persistence via lipid-mediated inhibition of the Niemann-Pick disease type C pathway

M.tuberculosis, the causative agent of human tuberculosis, is able to achieve long-term persistence within host organism macrophages. This persistence is achieved via the ability of the mycobacterium to prevent phagosomal-lysosomal fusion. The mechanisms by which fusion is inhibited remain incompletely understood. Here we provide evidence supporting a mechanistic link between infection with pathogenic mycobacteria and the cellular pathway defective in the rare lysosomal storage disorder Niemann-Pick disease type C (NPC). We observed that NPC phenotypes, including lipid storage and reduced lysosomal calcium release, can be induced in wild-type murine and human macrophages by infection with pathogenic mycobacteria. This phenotype induction did not occur following infection with the non-pathogenic M.smegmatis. Phenotype induction could be achieved in the absence of the mycobacteria using lipids from the mycobacterial cell walls. The importance of mycobacterial cell wall lipids to mycobacterial virulence has been well-documented. This lipid-mediated inhibition likely occurs through the NPC1 protein. Susceptibility to phenotype induction was inversely proportional to levels of functional NPC1, whilst a pre-existing dysfunction in the NPC pathway (either stemming from mutation or pharmacological inhibition) rendered cells less able to clear non-pathogenic mycobacteria. Finally, we demonstrate that therapies for NPC, particularly curcumin, are able to promote clearance of mycobacteria from infected macrophages. NPC therapies may hold promise for a new approach to the treatment of tuberculosis.This thesis is not currently available on ORA

Serratia‐based toxin cluster elements are associated with a type I fimbria

Abstract A soil bacterium in the Serratia genus, carrying a 153 kb conjugative amber disease‐associated plasmid (pADAP), is commercially exploited for population control of the New Zealand endemic pest beetle Costelytra giveni (Coleoptera: Scarabaeidae). The main insecticidal elements are an anti‐feeding prophage and the Sep ABC toxin complex (Tc). Homologs of pADAP, encoding variant Tcs, convey different beetle disease phenotypes. To investigate the correlation between variable bioactivity and the Tc variant, 76 Serratia plasmids were sequenced, resulting in the identification of four additional tc variants. All Serratia tc variants were found to be colocated with a conserved type 1 sef fimbrial‐like operon, indicating a conserved sef‐tc genetic island not observed outside of the Serratia genus. The conserved co‐location of the fimbrial and tc genes suggests the fimbriae somehow contribute to the lifestyle of Tc‐producing cells. Expression of the sef operon in a fim‐null Escherichia coli strain revealed fimbriae presence while a constructed sef‐deficient mutant showed no reduction of virulence or host colonization. Although no detectable contribution of Sef to amber disease in C. giveni was observed, the Sef adhesin sequences clustered similarly to the Serratia species encoding it, suggesting Sef has a species‐specific function