Helicobacter Hypothesis for Idiopathic Parkinsonism: Before and Beyond

We challenge the concept of idiopathic parkinsonism (IP) as inevitably progressive neurodegeneration, proposing a natural history of sequential microbial insults with predisposing host response. Proof-of-principle that infection can contribute to IP was provided by case studies and a placebo-controlled efficacy study of Helicobacter eradication. "Malignant" IP appears converted to "benign", but marked deterioration accompanies failure. Similar benefit on brady/hypokinesia from eradicating "low-density" infection favors autoimmunity. Although a minority of UK probands are urea breath test positive for Helicobacter , the predicted probability of having the parkinsonian label depends on the serum H. pylori antibody profile, with clinically relevant gradients between this "discriminant index" and disease burden and progression. In IP, H. pylori antibodies discriminate for persistently abnormal bowel function, and specific abnormal duodenal enterocyte mitochondrial morphology is described in relation to H. pylori infection. Slow intestinal transit manifests as constipation from the prodrome. Diarrhea may flag secondary small-intestinal bacterial overgrowth. This, coupled with genetically determined intense inflammatory response, might explain evolution from brady/hypokinetic to rigidity-predominant parkinsonism.     

Response to: “Response to Different measures of 'genome-wide' DNA methylation exhibit unique properties in placental and somatic tissues

We are responding to a Letter to the Editor addressing the Method section of our paper “Different measures of ‘genome-wide’ DNA methylation exhibit unique properties in placental and somatic tissues.” The letter raised concerns that the protocol for Epigentek’s MethylFlash kit was followed incorrectly based on the wording of an online publication of our article. We admittedly made an error in the language used to describe the MethylFlash protocol in our initial submission and thus this was corrected as soon as it was brought to our attention. However, the error was only in language and not procedure. We are confident that the protocol was followed as stated in the insert provided with the MethylFlash^TM Methylated DNA Quantification kit (Colorimetric).We are responding to a Letter to the Editor addressing the Method section of our paper “Different measures of ‘genome-wide’ DNA methylation exhibit unique properties in placental and somatic tissues” (Price ME, Cotton AM, PeÒaherrera MS, McFadden DE, Kobor MS, Robinson WP. Different measures of “genome-wide” DNA methylation exhibit unique properties in placental and somatic tissues. Epigenetics 2012; 7: 652–63; PMID: 22531475; 10.4161/epi.20221). The letter raised concerns that the protocol for Epigentek’s MethylFlash kit was followed incorrectly based on the wording of an online publication of our article. We admittedly made an error in the language used to describe the MethylFlash protocol in our initial submission and thus this was corrected as soon as it was brought to our attention. However, the error was only in language and not procedure. We are confident that the protocol was followed as stated in the insert provided with the MethylFlash^TM Methylated DNA Quantification kit (Colorimetric).     

Mold colonization during use of preservative-treated and untreated air filters, including HEPA filters from hospitals and commercial locations over an 8-year period (1996–2003)

High efficiency particulate arrestance (HEPA; 99.97% efficient at 0.3 m) filters, filters with ASHRAE particulate arrestance rating of 90–95% at 1 m (90–95% filters), and lower efficiency cellulosic-polyester filters from air conditioning systems in hospitals and commercial buildings were removed from the systems and examined microscopically for mold colonization. Cellulosic-type filters from systems with water entrainment problems typically were colonized, or became colonized upon incubation in moisture chambers. Species of Acremonium, Aspergillus, and Cladosporium were most common. With air filters of all types, treatment of filter media with an antimicrobial preservative tended to reduce or delay colonization. Mold colonization of HEPA and 90–95% filters was observed most often on the load surfaces, but two untreated HEPA filters were permeated with fungi, one with Aspergillus flavus, the other with Cladosporium sp. Air filters in heating, ventilating, and air conditioning (HVAC) systems, particularly those with chronic or periodic exposure to moisture, may serve as point sources for indoor molds.     

Induction and evolution of cytomegalovirus-specific CD4+ T cell clonotypes in rhesus macaques

CMV infection induces robust CD4+ T cell responses in immunocompetent hosts that orchestrate immune control of viral replication, dissemination, and disease. In this study, we characterized the clonotypic composition of CD4+ T cell populations specific for rhesus CMV (RhCMV) in chronically infected adult rhesus macaques (RM) and in juvenile RM undergoing primary RhCMV infection and subsequent secondary challenge with RhCMV. In adult RM with established chronic infection, RhCMV-specific CD4+ T cell populations exhibited stable, pauciclonal structures with skewed hierarchies dominated by two or three clonotypes. During primary infection, in contrast, the initial RhCMV-specific CD4+ T cell populations were highly polyclonal and progressive evolution to the chronic pattern manifest in adults occurred over the ensuing 2-3 years. Clear patterns of clonal succession were observed during this maturation process, such that clonotypes present in the acute phase were largely replaced over time. However, rechallenge with RhCMV expanded virus-specific CD4+ T cell clonotypes identified solely during acute infection. These findings indicate that, during persistent viral infection, substantial selection pressures and ongoing clonotype recruitment shape the specific CD4+ T cell repertoire and that rapidly exhausted or superseded clonotypes often remain within the memory T cell pool.     

Cathepsin L-like cysteine proteinase (DcCathL) from Delia coarctata (wheat bulb fly): Basis of insecticidal activity

A cDNA encoding a cathepsin L-like cysteine proteinase (DcCathL) was prepared from gut tissue of larvae of wheat bulb fly (Delia coarctata: Diptera). The predicted protein is a homologue of the product of Drosophila melanogaster gene Cp-1 (CG6692), and is similar to a sub-family of cysteine proteinases found in other insects which have roles in tissue remodelling during development, and moulting. Recombinant DcCathL was produced using the yeast Pichia pastoris as expression host, and showed hydrolytic activity in vitro towards the synthetic substrate Z-Phe-Arg-AMC with a pH optimum of 4.5. DcCathL was insecticidal to lepidopteran larvae when injected into haemolymph, causing mortality that was accompanied by systemic melanisation, suggesting that DcCathL was affecting the immune-related proteolytic activation cascade leading to production of active phenoloxidase. This process is normally negatively regulated by serpins in the haemolymph. Recombinant serpins from cabbage moth (Mamestra brassicae) did not inhibit DcCathL, and were susceptible to degradation by the enzyme in vitro in buffer and extracted haemolymph. When M. brassicae larvae were co-injected with a lethal dose of DcCathL and exogenous recombinant serpins, no mortality or systemic melanisation was observed, suggesting that the insecticidal effects of DcCathL in vivo result from degradation of endogenous serpins.     

Development of alpha-keto-based inhibitors of cruzain, a cysteine protease implicated in Chagas disease

Trypanosoma cruzi, a protozoan parasite, is the causative agent of Chagas disease, a major cause of cardiovascular disease in many Latin American countries. There is an urgent need to develop an improved therapy due to the toxicity of existing drugs and emerging drug resistance. Cruzain, the primary cysteine protease of T. cruzi, is essential for the survival of the parasite in host cells and therefore is an important target for the development of inhibitors as potential therapeutics. A novel series of alpha-ketoamide-, alpha-ketoacid-, alpha-ketoester-, and aldehyde-based inhibitors of cruzain has been developed. The inhibitors were identified by screening protease targeted small molecule libraries and systematically optimizing the P1, P2, P3, and P1' residues using specific structure-guided methods. A total of 20 compounds displayed picomolar potency in in vitro assays and three inhibitors representing different alpha-keto-based inhibitor scaffolds demonstrated anti-trypanosomal activity in cell culture. A 2.3A crystallographic structure of cruzain bound with one of the alpha-ketoester analogs is also reported. The structure and kinetic assay data illustrate the covalent binding, reversible inhibition mechanism of the inhibitor. Information on the compounds reported here will be useful in the development of new lead compounds as potential therapeutic agents for the treatment of Chagas disease and as biological probes to study the role that cruzain plays in the pathology. This study also demonstrates the validity of structure-guided approaches to focused library design and lead compound optimization.     

Species richness, environmental fluctuations, and temporal change in total community biomass

Theory and empirical results suggest that high biodiversity should often cause lower temporal variability in aggregate community properties such as total community biomass. We assembled microbial communities containing 2 to 8 species of competitors in aquatic microcosms and found that the temporal change in total community biomass was positively but insignificantly associated with diversity in a constant temperature environment. There was no evidence of any trend in variable temperature environments. Three non-exclusive mechanisms might explain the lack of a net stabilising effect of species richness on temporal change. (1) A direct destabilising effect of diversity on population level variances caused some populations to vary more when embedded in more diverse communities. (2) Similar responses of the different species to environmental variability might have limited any insurance effect of increased species richness. (3) Large differences in the population level variability of different species (i.e., unevenness) could weaken the relation between species richness and community level stability. These three mechanisms may outweigh the stabilising effects of increases in total community biomass with diversity, statistical averaging, and slightly more negative covariance in more diverse communities. Our experiment and analyses advocate for further experimental investigations of diversity-variability relations.     

Amine promiscuity and toxicology analysis

Drug discovery programs often face challenges to obtain sufficient duration of action of the drug (i.e. seek longer half-lives). If the pharmacodynamic response is driven by free plasma concentration of the drug then extending the plasma drug concentration is a valid approach. Half-life is dependent on the volume of distribution, which in turn can be dependent upon the ionization state of the molecule. Basic compounds tend to have a higher volume of distribution leading to longer half-lives. However, it has been shown that bases may also have higher promiscuity. In this work, we describe an analysis of in vitro pharmacological profiling and toxicology data investigating the role of primary, secondary, and tertiary amines in imparting promiscuity and thus off-target toxicity. Primary amines are found to be less promiscuous in in vitro assays and have improved profiles in in vivo toxicology studies compared to secondary and tertiary amines.