Succination is Increased on Select Proteins in the Brainstem of the NADH dehydrogenase (ubiquinone) Fe-S protein 4 (Ndufs4) Knockout Mouse,a Model of Leigh Syndrome

Elevated fumarate concentrations as a result of Krebs cycle inhibition lead to increases in protein succination, an irreversible post-translational modification that occurs when fumarate reacts with cysteine residues to generate S-(2-succino)cysteine (2SC). Metabolic events that reduce NADH re-oxidation can block Krebs cycle activity; therefore we hypothesized that oxidative phosphorylation deficiencies, such as those observed in some mitochondrial diseases, would also lead to increased protein succination. Using the Ndufs4 knockout (Ndufs4 KO) mouse, a model of Leigh syndrome, we demonstrate for the first time that protein succination is increased in the brainstem (BS), particularly in the vestibular nucleus. Importantly, the brainstem is the most affected region exhibiting neurodegeneration and astrocyte and microglial proliferation, and these mice typically die of respiratory failure attributed to vestibular nucleus pathology. In contrast, no increases in protein succination were observed in the skeletal muscle, corresponding with the lack of muscle pathology observed in this model. 2D SDS-PAGE followed by immunoblotting for succinated proteins and MS/MS analysis of BS proteins allowed us to identify the voltage-dependent anion channels 1 and 2 as specific targets of succination in the Ndufs4 knockout. Using targeted mass spectrometry, Cys⁷⁷ and Cys⁴⁸ were identified as endogenous sites of succination in voltage-dependent anion channels 2. Given the important role of voltage-dependent anion channels isoforms in the exchange of ADP/ATP between the cytosol and the mitochondria, and the already decreased capacity for ATP synthesis in the Ndufs4 KO mice, we propose that the increased protein succination observed in the BS of these animals would further decrease the already compromised mitochondrial function. These data suggest that fumarate is a novel biochemical link that may contribute to the progression of the neuropathology in this mitochondrial disease model.We previously identified the formation of S-(2-succino)cysteine (2SC)¹ (protein succination) as a result of the irreversible reaction of fumarate with reactive cysteine thiols (1, 2). Fumarate concentrations are increased during adipogenesis and adipocyte maturation (2, 3), and the excess of glucose and insulin leads to augmented protein succination in the adipose tissue of type 2 diabetic mice (4, 5). Protein succination is also specifically increased in fumarate hydratase deficient hereditary leiomyomatosis and renal cell carcinoma (HLRCC), because of the decreased conversion of fumarate to malate (6, 7). In both cases, intracellular fumarate concentrations are elevated; in fumarate hydratase deficient cells, the fumarate concentration is about 5 mm (8), whereas fumarate levels increase up to fivefold in adipocytes grown in the presence of high (30 mm) versus normal (5 mm) glucose concentrations (2). In the adipocyte the increase in fumarate and succinated proteins develops as a direct result of mitochondrial stress induced by nutrient excess. Mechanistically, excess glucose without increased ATP demand inhibits the electron transport chain resulting in an elevated NADH/NAD⁺ ratio. This inhibits NAD⁺-dependent Krebs cycle enzymes and leads to an increase in fumarate and protein succination (9). In support of this we have also shown that low concentrations of chemical uncouplers of oxidative phosphorylation (OXPHOS) can decrease fumarate concentrations and protein succination (9). The physiological consequences of protein succination include a decrease in the functionality of the target protein (8, 10–12), for example succination of adiponectin prevents the formation of multimeric complexes and reduces plasma adiponectin levels in diabetes (4). Considering the impact of glucotoxicity driven mitochondrial stress in the adipocyte, we predicted that deficiencies in OXPHOS associated with NADH accumulation would also result in increased protein succination.Mitochondrial respiratory chain disorders encompass a broad range of encephalopathies and myopathies associated with the defective assembly, activity or maintenance of the OXPHOS machinery (13), and are estimated to occur in about 1 in 5,000 live births (14). A common feature in most mitochondrial diseases (MD) is a failure to thrive because of reduced mitochondrial energy production; both the brain and muscle are usually affected because of their high dependence on oxidative metabolism (13). Leigh syndrome is one of the most common manifestations of MD and is characterized by progressive neurodegeneration with bilateral necrotizing lesions of the brainstem and basal ganglia, resulting in lactic acidosis, ataxia, seizures, dystonia, and respiratory failure (15, 16). Mutations in genes encoding the five complexes of the OXPHOS machinery can lead to Leigh syndrome; however, the majority of these mutations affect subunits of complexes I and IV (17), and both mitochondrial and nuclear encoded proteins may be affected (17–19). Complex I is a large (980 kDa) l-shaped protein assembly consisting of 45 peptides, with one flavin mononucleotide and eight iron–sulfur clusters (20). One of the first identified mutations of complex I encoded Ndufs4, a small (18 kDa) assembly protein (21–23). Ndufs4 assists in the final stages of complex I assembly, and its absence results in the formation of a smaller ∼830 kDa subcomplex that lacks the NADH dehydrogenase module and has significantly less electron shuttling activity than the intact holoenzyme (24, 25). Ndufs4 mutations are associated with brainstem deterioration in humans (26), and a recently described Ndufs4 knockout mouse (Ndufs4 KO) exhibits many of the clinical and neurological symptoms observed in human Leigh syndrome (27, 28).One of the most common clinical features of MD is lactic acidosis, derived from the accumulation of pyruvate and elevated NADH. Increased lactate or lactate:pyruvate ratios have been measured in the blood, urine, and cerebrospinal fluid of a large number of Leigh syndrome patients (15, 16). Increases in other organic acids in urine have also been reported (16), indicating that metabolic acidosis is a prominent clinical feature. Interestingly, a study designed to find new diagnostic metabolites in MD demonstrated that within certain age ranges the measurement of urinary fumarate and malate was a more useful discriminator of MD than lactate or other organic acids (29). Barshop''s findings support the hypothesis that MD derived from OXPHOS deficiencies may exhibit increased protein succination because of the accumulation of NADH and subsequently fumarate. In this study we report for the first time that protein succination is present in the brain in an animal model of Leigh syndrome, the Ndufs4 KO mouse, suggesting that this modification may be an important biochemical link between the genetic defect and the onset of neuropathology observed in Leigh syndrome.     

Inactivation of Prions and Amyloid Seeds with Hypochlorous Acid

Hypochlorous acid (HOCl) is produced naturally by neutrophils and other cells to kill conventional microbes in vivo. Synthetic preparations containing HOCl can also be effective as microbial disinfectants. Here we have tested whether HOCl can also inactivate prions and other self-propagating protein amyloid seeds. Prions are deadly pathogens that are notoriously difficult to inactivate, and standard microbial disinfection protocols are often inadequate. Recommended treatments for prion decontamination include strongly basic (pH ≥~12) sodium hypochlorite bleach, ≥1 N sodium hydroxide, and/or prolonged autoclaving. These treatments are damaging and/or unsuitable for many clinical, agricultural and environmental applications. We have tested the anti-prion activity of a weakly acidic aqueous formulation of HOCl (BrioHOCl) that poses no apparent hazard to either users or many surfaces. For example, BrioHOCl can be applied directly to skin and mucous membranes and has been aerosolized to treat entire rooms without apparent deleterious effects. Here, we demonstrate that immersion in BrioHOCl can inactivate not only a range of target microbes, including spores of Bacillus subtilis, but also prions in tissue suspensions and on stainless steel. Real-time quaking-induced conversion (RT-QuIC) assays showed that BrioHOCl treatments eliminated all detectable prion seeding activity of human Creutzfeldt-Jakob disease, bovine spongiform encephalopathy, cervine chronic wasting disease, sheep scrapie and hamster scrapie; these findings indicated reductions of ≥10³- to 10⁶-fold. Transgenic mouse bioassays showed that all detectable hamster-adapted scrapie infectivity in brain homogenates or on steel wires was eliminated, representing reductions of ≥~10^5.75-fold and >10⁴-fold, respectively. Inactivation of RT-QuIC seeding activity correlated with free chlorine concentration and higher order aggregation or destruction of proteins generally, including prion protein. BrioHOCl treatments had similar effects on amyloids composed of human α-synuclein and a fragment of human tau. These results indicate that HOCl can block the self-propagating activity of prions and other amyloids.     

Characterizing bacterial assemblages in sediments and aerosols at a dry lake bed in Australia using high-throughput sequencing

Dust storms are responsible for the transport of a large quantity of bacteria from arid regions. A severe drought in the first decade of the new millennium in Australia increased the incidence of dust transport further. The major aims of this study were to characterize the bacterial communities in aerosols and their associated source sediments using high-throughput sequencing (HTS) and to investigate the possibility of using HTS to link dust to its source, which has not been previously performed in this way. Four field campaigns were conducted at the recently evaporated saline playa Lake Gnarpurt in the Australian state of Victoria between 2008 and 2010 (3 in the austral summer, 1 in winter) to collect aerosol and sediment samples. Aerosol samples were collected on filters up to 150 m above the lake bed using a tethered helium-filled balloon. DNA was extracted from all samples using commercial kits, and the bacterial communities were examined using 454 HTS on the 16S rRNA gene. Over 200,000 sequences from 29 samples were analysed. In both sediment and aerosol samples, Salinimicrobium was the most abundant taxon; however, there was great variation and diversity across all samples. Analysis of similarities of the bacterial communities indicated that there was a significant overlap between the sediment samples and the aerosols collected above that location, showing that the bacteria in the air was derived from a subset of dust from a nearby source. The challenge remains to use bacterial profiling to link an aerosol sample to a distant source.     

In Vitro and In Vivo Characterization of the Alkaloid Nuciferine

Rationale

The sacred lotus (Nelumbo nucifera) contains many phytochemicals and has a history of human use. To determine which compounds may be responsible for reported psychotropic effects, we used in silico predictions of the identified phytochemicals. Nuciferine, an alkaloid component of Nelumbo nucifera and Nymphaea caerulea, had a predicted molecular profile similar to antipsychotic compounds. Our study characterizes nuciferine using in vitro and in vivo pharmacological assays.

Methods

Nuciferine was first characterized in silico using the similarity ensemble approach, and was followed by further characterization and validation using the Psychoactive Drug Screening Program of the National Institute of Mental Health. Nuciferine was then tested in vivo in the head-twitch response, pre-pulse inhibition, hyperlocomotor activity, and drug discrimination paradigms.

Results

Nuciferine shares a receptor profile similar to aripiprazole-like antipsychotic drugs. Nuciferine was an antagonist at 5-HT_2A, 5-HT_2C, and 5-HT_2B, an inverse agonist at 5-HT₇, a partial agonist at D_2, D₅ and 5-HT₆, an agonist at 5-HT_1A and D₄ receptors, and inhibited the dopamine transporter. In rodent models relevant to antipsychotic drug action, nuciferine blocked head-twitch responses and discriminative stimulus effects of a 5-HT_2A agonist, substituted for clozapine discriminative stimulus, enhanced amphetamine induced locomotor activity, inhibited phencyclidine (PCP)-induced locomotor activity, and rescued PCP-induced disruption of prepulse inhibition without induction of catalepsy.

Conclusions

The molecular profile of nuciferine was similar but not identical to that shared with several approved antipsychotic drugs suggesting that nuciferine has atypical antipsychotic-like actions.     

At‐line process analytical technology (PAT) for more efficient scale up of biopharmaceutical microfiltration unit operations

Tangential flow microfiltration (MF) is a cost‐effective and robust bioprocess separation technique, but successful full scale implementation is hindered by the empirical, trial‐and‐error nature of scale‐up. We present an integrated approach leveraging at‐line process analytical technology (PAT) and mass balance based modeling to de‐risk MF scale‐up. Chromatography‐based PAT was employed to improve the consistency of an MF step that had been a bottleneck in the process used to manufacture a therapeutic protein. A 10‐min reverse phase ultra high performance liquid chromatography (RP‐UPLC) assay was developed to provide at‐line monitoring of protein concentration. The method was successfully validated and method performance was comparable to previously validated methods. The PAT tool revealed areas of divergence from a mass balance‐based model, highlighting specific opportunities for process improvement. Adjustment of appropriate process controls led to improved operability and significantly increased yield, providing a successful example of PAT deployment in the downstream purification of a therapeutic protein. The general approach presented here should be broadly applicable to reduce risk during scale‐up of filtration processes and should be suitable for feed‐forward and feed‐back process control. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:108–115, 2016     

Burden of Liver Disease among Community-Based People Who Inject Drugs (PWID) in Chennai,India

Background and Objective

We characterize the burden of liver disease in a cohort of PWID in Chennai, India, with a high prevalence of HCV.

Materials and Methods

1,042 PWID were sampled through community outreach in Chennai. Participants underwent fasting blood draw, questionnaire and an examination that included liver stiffness assessment using transient elastography (Fibroscan) and assessment of steatosis via ultrasound.

Results

The median age was 39 years, all were male, 14.8% were HIV infected and 35.6% were HCV antibody positive, of whom 78.9% were chronically infected (HCV RNA positive). Median liver stiffness was 6.2 kPA; 72.9% had no evidence of or mild stiffness, 14.5% had moderate stiffness, and 12.6% had evidence of severe stiffness/cirrhosis. Prevalence of severe stiffness/cirrhosis was significantly higher among persons who were older, had a longer duration of injecting drugs, higher body mass index, higher prevalence of insulin resistance, higher prevalence of steatosis, higher HCV RNA levels and evidence of alcohol dependence. An estimated 42.1% of severe stiffness/cirrhosis in this sample was attributable to HCV. 529 (53.0%) had some evidence of steatosis. Prevalence of steatosis was higher among those who had larger waist circumference, insulin resistance, higher HDL cholesterol and a history of antiretroviral therapy.

Conclusions

We observed a high burden of liver disease in this relatively young cohort that was primarily driven by chronic HCV infection, metabolic factors (insulin resistance and steatosis) and heavy alcohol use. Interventions to improve access to HCV treatment and reduce alcohol use are needed to prevent further progression of liver disease.     

The influence of copper exposure on predator-prey interactions in aquatic insect communities

SUMMARY. 1. Vulnerability of two species of net-spinning caddisflies (Chimarra sp. and Hydropsyche morosa) to predation by the stonefly, Paragnetina media , was significantly greater in experimental streams dosed with copper (target concentration=6 μg 1^-1) than in control streams.
2. Results of stomach analyses showed that Hydropsychidae was the major component of stonefly diets in experimental streams and that the number of Hydropsychidae per stonefly gut was significantly greater in dosed streams than controls.
3. Since these results could not be predicted using single species bioassays, we suggest that such simple tests be supplemented with more environmentally realistic procedures that account for species interactions.     

Surface hydrophobicity and dispersal of Pseudomonas aeruginosa from biofilms

A novel method of cell culture was employed to control the growth-rate of bacterial biofilms [1]. Cell-surface hydrophobicity increased progressively with growth rate for planktonic, chemostatgrown Pseudomonas aeruginosa and also for cells, resuspended from the biofilms. Dependence of surface hydrophobicity upon growth rate was greater for the planktonic cells. Newly-formed daughter cells, shed from the biofilms, were in all cases more hydrophilic than their adherent counterparts and demonstrated only slight growth rate dependency for this property.