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Abstract Background Up-regulation of S100A7 (Psoriasin), a small calcium-binding protein, is associated with the development of several types of carcinomas, but its function and possibility to serve as a diagnostic or prognostic marker have not been fully defined. In order to prepare antibodies to the protein for immunohistochemical studies we produced the recombinant S100A7 protein in E. coli. mRNA extracted from human tracheal tumor tissue which was amplified by RT-PCR to provide the region coding for the S100A7 gene. The amplified fragment was cloned in the vector pCR2.1-TOPO and sub-cloned in the expression vector pAE. The protein rS100A7 (His-tag) was expressed in E. coli BL21::DE3, purified by affinity chromatography on an Ni-NTA column, recovered in the 2.0 to 3.5 mg/mL range in culture medium, and used to produce a rabbit polyclonal antibody anti-rS100A7 protein. The profile of this polyclonal antibody was evaluated in a tissue microarray. Results The rS100A7 (His-tag) protein was homogeneous by SDS-PAGE and mass spectrometry and was used to produce an anti-recombinant S100A7 (His-tag) rabbit serum (polyclonal antibody anti-rS100A7). The molecular weight of rS100A7 (His-tag) protein determined by linear MALDI-TOF-MS was 12,655.91 Da. The theoretical mass calculated for the nonapeptide attached to the amino terminus is 12,653.26 Da (delta 2.65 Da). Immunostaining with the polyclonal anti-rS100A7 protein generated showed reactivity with little or no background staining in head and neck squamous cell carcinoma cells, detecting S100A7 both in nucleus and cytoplasm. Lower levels of S100A7 were detected in non-neoplastic tissue. Conclusions The polyclonal anti-rS100A7 antibody generated here yielded a good signal-to-noise contrast and should be useful for immunohistochemical detection of S100A7 protein. Its potential use for other epithelial lesions besides human larynx squamous cell carcinoma and non-neoplastic larynx should be explored in future.

Abstract Background Neutrophils are the most abundant leukocytes in peripheral blood and represent one of the most important elements of innate immunity. Recent subcellular proteomic studies have focused on the identification of human neutrophil proteins in various subcellular membrane and granular fractions. Although there are relatively few studies dealing with the analysis of the total extract of human neutrophils, many biological problems such as the role of chemokines, adhesion molecules, and other activating inputs involved in neutrophil responses and signaling can be approached on the basis of the identification of the total cellular proteins. Results Using gel-LC-MS/MS, 251 total cellular proteins were identified from resting human neutrophils. This is more than ten times the number of proteins identified by an initial proteome analysis of human neutrophils and almost five times the number of proteins identified by the first 2-DE map of extracts of rat polymorphonuclear leukocytes. Most of the proteins identified in the present study are well-known, but some of them, such as neutrophil-secreted proteins and centaurin beta-1, a cytoplasmic protein involved in the regulation of NF-κB activity, are described here for the first-time. Conclusion The present report provides new information about the protein content of human neutrophils. Importantly, our study resulted in the discovery of a series of proteins not previously reported to be associated with human neutrophils. These data are relevant to the investigation of comparative pathological states and models for novel classes of pharmaceutical drugs that could be useful in the treatment of inflammatory disorders in which neutrophils participate.

The Sup35 prion protein of budding yeast has been reported to undergo phase separation to form liquid droplets both at low pH in vitro and when energy depletion decreases the intracellular pH in vivo. It also has been shown using purified proteins that this phase separation is driven by the prion domain of Sup35 and does not re-quire its C-terminal domain. In contrast, we now find that a Sup35 fragment consisting of only the N-terminal prion domain and the M-domain does not phase separate in vivo; this phase separation of Sup35 requires the C-terminal domain, which binds Sup45 to form the translation termination complex. The phase-separated Sup35 not only colocalizes with Sup45 but also with Pub1, a stress granule marker protein. In addition, like stress granules, phase separation of Sup35 appears to require mRNA since cycloheximide treatment, which inhibits mRNA release from ribosomes, prevents phase separation of Sup35. Finally, unlike Sup35 in vitro, Sup35 condensates do not disassemble in vivo when the intracellular pH is increased. These results suggest that, in energy-depleted cells, Sup35 forms supramolecular assemblies that differ from the Sup35 liquid droplets that form in vitro.

The yeast Sup35 protein misfolds into the infectious [ PSI + ] prion, which is then propagated by the severing activity of the molecular chaperone, Hsp104. Unlike other yeast prions, this prion is unique in that it is efficiently cured by the overexpression as well as the inactivation of Hsp104. However, it is controversial whether curing by overexpression is due to the dissolution of the prion seeds by the trimming activity of Hsp104 or the asymmetric segregation of the prion seeds between mother and daughter cells which requires cell division. To answer this question, we conducted experiments and found no difference in the extent of curing between mother and daughter cells when half of the cells were cured by Hsp104 overexpression in one generation. Furthermore, curing was not affected by the lack of Sir2 expression, which was reported to be required for asymmetric segregation of the [ PSI + ] seeds. More importantly, when either hydroxyurea or ethanol were used to inhibit cell division, the extent of curing by Hsp104 overexpression was not significantly reduced. Therefore, the curing of [ PSI + ] by Hsp104 overexpression is not due to asymmetric segregation of the prion seeds, but rather their dissolution by Hsp104.

Auxilin participates in the uncoating of clathrin-coated vesicles (CCVs), thereby facilitating synaptic vesicle (SV) regeneration at presynaptic sites. Auxilin (DNAJC6/PARK19) loss-of-function mutations cause early-onset Parkinson's disease (PD). Here, we utilized auxilin knockout (KO) mice to elucidate the mechanisms through which auxilin deficiency and clathrin-uncoating deficits lead to PD. Auxilin KO mice display cardinal features of PD, including progressive motor deficits, α-synuclein pathology, nigral dopaminergic loss, and neuroinflammation. Significantly, treatment with L-DOPA ameliorated motor deficits. Unbiased proteomic and neurochemical analyses of auxilin KO brains indicated dopamine dyshomeostasis. We validated these findings by demonstrating slower dopamine reuptake kinetics in vivo, an effect associated with dopamine transporter misrouting into axonal membrane deformities in the dorsal striatum. Defective SV protein sorting and elevated synaptic autophagy also contribute to ineffective dopamine sequestration and compartmentalization, ultimately leading to neurodegeneration. This study provides insights into how presynaptic endocytosis deficits lead to dopaminergic vulnerability and pathogenesis of PD., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Parkinson's disease (PD) is a neurodegenerative disorder characterized by defective dopaminergic (DAergic) input to the striatum. Mutations in two genes encoding synaptically enriched clathrin-uncoating factors, synaptojanin 1 (SJ1) and auxilin, have been implicated in atypical Parkinsonism. SJ1 knock-in (SJ1-KI RQ ) mice carrying a disease-linked mutation display neurological manifestations reminiscent of Parkinsonism. Here we report that auxilin knockout (Aux-KO) mice display dystrophic changes of a subset of nigrostriatal DAergic terminals similar to those of SJ1-KI RQ mice. Furthermore, Aux-KO/SJ1-KI RQ double mutant mice have shorter lifespan and more severe synaptic defects than single mutant mice. These include increase in dystrophic striatal nerve terminals positive for DAergic markers and for the PD risk protein SV2C, as well as adaptive changes in striatal interneurons. The synergistic effect of the two mutations demonstrates a special lability of DAergic neurons to defects in clathrin uncoating, with implications for PD pathogenesis in at least some forms of this condition., (© 2023. The Author(s).)

Hsp104 propagates the yeast prion [ PSI + ], the infectious form of Sup35, by severing the prion seeds, but when Hsp104 is overexpressed, it cures [ PSI + ] in a process that is not yet understood but may be caused by trimming, which removes monomers from the ends of the amyloid fibers. This curing was shown to depend on both the N-terminal domain of Hsp104 and the expression level of various members of the Hsp70 family, which raises the question as to whether these effects of Hsp70 are due to it binding to the Hsp70 binding site that was identified in the N-terminal domain of Hsp104, a site not involved in prion propagation. Investigating this question, we now find, first, that mutating this site prevents both the curing of [ PSI + ] by Hsp104 overexpression and the trimming activity of Hsp104. Second, we find that depending on the specific member of the Hsp70 family binding to the N-terminal domain of Hsp104, both trimming and the curing caused by Hsp104 overexpression are either increased or decreased in parallel. Therefore, the binding of Hsp70 to the N-terminal domain of Hsp104 regulates both the rate of [ PSI + ] trimming by Hsp104 and the rate of [ PSI + ] curing by Hsp104 overexpression.

The aggregation of huntingtin fragments with expanded polyglutamine repeat regions (HttpolyQ) that cause Huntington's disease depends on the presence of a prion with an amyloid conformation in yeast. As a result of this relationship, HttpolyQ aggregation indirectly depends on Hsp104 due to its essential role in prion propagation. We find that HttQ103 aggregation is directly affected by Hsp104 with and without the presence of [ RNQ + ] and [ PSI + ] prions. When we inactivate Hsp104 in the presence of prion, yeast cells have only one or a few large HttQ103 aggregates rather than numerous smaller aggregates. When we inactivate Hsp104 in the absence of prion, there is no significant aggregation of HttQ103, whereas with active Hsp104, HttQ103 aggregates accumulate slowly due to the severing of spontaneously nucleated aggregates by Hsp104. We do not observe either effect with HttQ103P, which has a polyproline-rich region downstream of the polyglutamine region, because HttQ103P does not spontaneously nucleate and Hsp104 does not efficiently sever the prion-nucleated HttQ103P aggregates. Therefore, the only role of Hsp104 in HttQ103P aggregation is to propagate yeast prion. In conclusion, because Hsp104 efficiently severs the HttQ103 aggregates but not HttQ103P aggregates, it has a marked effect on the aggregation of HttQ103 but not HttQ103P.

The yeast prion [URE3] propagates as a misfolded amyloid form of the Ure2 protein. Propagation of amyloid-based yeast prions requires protein quality control (PQC) factors, and altering PQC abundance or activity can cure cells of prions. Yeast antiprion systems composed of PQC factors act at normal abundance to restrict establishment of the majority of prion variants that arise de novo While these systems are well described, how they or other PQC factors interact with prion proteins remains unclear. To gain insight into such interactions, we identified mutations outside the Ure2 prion-determining region that destabilize [URE3]. Despite residing in the functional domain, 16 of 17 mutants retained Ure2 activity. Four characterized mutations caused rapid loss of [URE3] yet allowed [URE3] to propagate under prion-selecting conditions. Two sensitized [URE3] to Btn2, Cur1, and Hsp42, but in different ways. Two others reduced amyloid formation in vitro Of these, one impaired prion replication and the other apparently impaired transmission. Thus, widely dispersed sites outside a prion's amyloid-forming region can contribute to prion character, and altering such sites can disrupt prion propagation by altering interactions with PQC factors.

Prions are infectious proteins that self-propagate by changing from their normal folded conformation to a misfolded conformation. The misfolded conformation, which is typically rich in β-sheet, serves as a template to convert the prion protein into its misfolded conformation. In yeast, the misfolded prion proteins are assembled into amyloid fibers or seeds, which are constantly severed and transmitted to daughter cells. To cure prions in yeast, it is necessary to eliminate all the prion seeds. Multiple mechanisms of curing have been found including inhibiting severing of the prion seeds, gradual dissolution of the prion seeds, asymmetric segregation of the prion seeds between mother and daughter cells during cell division, and degradation of the prion seeds. These mechanisms, achieved by using different protein quality control machinery, are not mutually exclusive; depending on conditions, multiple mechanisms may work simultaneously to achieve curing. This review discusses the various methods that have been used to differentiate between these mechanisms of curing.

Fluorogenic analogues of α-tocopherol developed by our group have been instrumental in monitoring reactive oxygen species (ROS) within lipid membranes. Prepared as two-segment trap-reporter (chromanol-BODIPY) probes, photoinduced electron transfer (PeT) was utilized to provide these probes with an off/on switch mechanism warranting the necessary sensitivity. Herein, we rationalize within the context of Marcus theory of electron transfer how substituents on the BODIPY core and linker length joining the trap and reporter segments, tune PeT efficiency. DFT and electrochemical studies were used to estimate the thermodynamic driving force of PeT in our constructs. By tuning the redox potential over a 400 mV range, we observed over an order of magnitude increase in PeT efficiency. Increasing the linker length between the chromanol and BODIPY by 2.8 angstroms, in turn, decreased PeT efficiency 2.7-fold. Our results illustrate how substituent and linker choice enable "darkening" the off state of fluorogenic probes based on BODIPY fluorophores, by favoring PeT over radiative emission from the singlet excited state manifold. Ultimately, our work brings light to the sensitivity ceiling one may achieve in developing fluorogenic antioxidant analogues of α-tocopherol. The work provides general guidelines applicable to those developing fluorogenic probes based on PeT., (© 2018 The American Society of Photobiology.)

Oxidative stress, specifically lipid peroxidation, is a major driving force in neurodegenerative processes. However, the exact role of lipid peroxidation remains elusive as reliable real-time detection and quantification of lipid peroxyl radicals proves to be challenging in vitro and in vivo. Motivated by this methodological limitation, we have optimized conditions for real-time imaging and quantification of lipid peroxyl radical generation in primary neuron cultures using the lipophilic fluorogenic antioxidant H 4 BPMHC (8-((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)-methyl)-1,5-di(3-chloropropyl)-pyrromethene fluoroborate), an α-tocopherol analog probe. By subjecting neurons to different antioxidant conditions in the presence and absence of lipid peroxidation inducing stressors (Haber-Weiss reagents), we maximized H 4 BPMHC sensitivity and confirmed its potential to temporally resolve subtle and marked differences in lipid peroxidation levels in real-time. Herein we report imaging and quantification of homeostatic and induced lipid peroxidation in primary neuron cultures, supporting the use of this probe for investigating healthy and diseased states. Overall these results provide the necessary foundation and impetus towards using H 4 BPMHC for elucidating and mapping lipid peroxyl radical contributions to ROS-associated pathological processes in neurons., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Lipid peroxidation of polyunsaturated fatty acids in cells may occur via their catalytic autoxidation through peroxyl radicals under oxidative stress conditions. Lipid peroxidation is related to a number of pathologies, and may be invoked in new forms of regulated cell death, yet it may also have beneficial roles in cell signaling cascades. Antioxidants are a natural line of defense against lipid peroxidation, and may accordingly impact the biological outcome associated with the redox chemistry of lipid peroxidation. Critical to unraveling the physiological and pathological role of lipid peroxidation is the development of novel probes with the partition, chemical sensitivity and more importantly, molecular specificity, enabling the spatial and temporal imaging of peroxyl radicals in the lipid membranes of live cells, reporting on the redox status of the cell membrane. This review describes our recent progress to visualize lipid peroxidation in model membrane systems and in live cell studies. Our work portrays the mechanistic insight leading to the development of a highly sensitive probe to monitor lipid peroxyl radicals (LOO • ). It also describes technical aspects including reagents and fluorescence microscopy methodologies to consider in order to achieve the much sought after monitoring of rates of lipid peroxyl radical production in live cell studies, be it under oxidative stress but also under cell homeostasis. This review seeks to bring attention to the study of lipid redox reactions and to lay the groundwork for the adoption of fluorogenic antioxidant probeshancement and maximum intensity recorded in turn provide a benchmark to estimate, when compared to the control BODIPY dye lacking the intramolecular PeT based switch, the overall exte and related fluorescence microscopy methods toward gaining rich spatiotemporal information on lipid peroxidation in live cells., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The [URE3] yeast prion is the self-propagating amyloid form of the Ure2 protein. [URE3] is cured by overexpression of several yeast proteins, including Ydj1, Btn2, Cur1, Hsp42, and human DnaJB6. To better understand [URE3] curing, we used real-time imaging with a yeast strain expressing a GFP-labeled full-length Ure2 construct to monitor the curing of [URE3] over time. [URE3] yeast cells exhibited numerous fluorescent foci, and expression of the GFP-labeled Ure2 affected neither mitotic stability of [URE3] nor the rate of [URE3] curing by the curing proteins. Using guanidine to cure [URE3] via Hsp104 inactivation, we found that the fluorescent foci are progressively lost as the cells divide until they are cured; the fraction of cells that retained the foci was equivalent to the [URE3] cell fraction measured by a plating assay, indicating that the foci were the prion seeds. During the curing of [URE3] by Btn2, Cur1, Hsp42, or Ydj1 overexpression, the foci formed aggregates, many of which were 0.5 μm or greater in size, and [URE3] was cured by asymmetric segregation of the aggregated seeds. In contrast, DnaJB6 overexpression first caused a loss of detectable foci in cells that were still [URE3] before there was complete dissolution of the seeds, and the cells were cured. We conclude that GFP labeling of full-length Ure2 enables differentiation among the different [URE3]-curing mechanisms, including inhibition of severing followed by seed dilution, seed clumping followed by asymmetric segregation between mother and daughter cells, and seed dissolution.

The yeast [PSI + ] prion, which is the amyloid form of Sup35, has the unusual property of being cured not only by the inactivation of, but also by the overexpression of Hsp104. Even though this latter observation was made more than two decades ago, the mechanism of curing by Hsp104 overexpression has remained controversial. This question has been investigated in depth by our laboratory by combining live cell imaging of GFP-labeled Sup35 with standard plating assays of yeast overexpressing Hsp104. We will discuss why the curing of [PSI + ] by Hsp104 overexpression is not compatible with a mechanism of either inhibition of severing of the prion seeds or asymmetric segregation of the seeds. Instead, our recent data (J. Biol. Chem. 292:8630-8641) indicate that curing is due to dissolution of the prion seeds, which in turn is dependent on the trimming activity of Hsp104. This trimming activity decreases the size of the seeds by dissociating monomers from the fibers, but unlike Hsp104 severing activity, it does not increase the number of prion seeds. Finally, we will discuss the other factors that affect the curing of [PSI + ] by Hsp104 overexpression and how these factors may relate to the trimming activity of Hsp104.

Air pollution stimulates airway epithelial secretion through a cholinergic reflex that is unaffected in cystic fibrosis (CF), yet a strong correlation is observed between passive smoke exposure in the home and impaired lung function in CF children. Our aim was to study the effects of low smoke concentrations on cystic fibrosis transmembrane conductance regulator (CFTR) function in vitro. Cigarette smoke extract stimulated robust anion secretion that was transient, mediated by CFTR, and dependent on cAMP-dependent protein kinase activation. Secretion was initiated by reactive oxygen species (ROS) and mediated by at least two distinct pathways: autocrine activation of EP4 prostanoid receptors and stimulation of Ca 2+ store-operated cAMP signaling. The response was absent in cells expressing the most common disease-causing mutant F508del-CFTR. In addition to the initial secretion, prolonged exposure of non-CF bronchial epithelial cells to low levels of smoke also caused a gradual decline in CFTR functional expression. F508del-CFTR channels that had been rescued by the CF drug combination VX-809 (lumacaftor) + VX-770 (ivacaftor) were more sensitive to this downregulation than wild-type CFTR. The results suggest that CFTR-mediated secretion during acute cigarette smoke exposure initially protects the airway epithelium while prolonged exposure reduces CFTR functional expression and reduces the efficacy of CF drugs.

We describe herein a fluorescence-based assay to characterize and report on nucleophilic addition to carbonyl moieties and highlight the advantages a fluorescence-based assay and multiplex analysis can offer. The assay relies on the fluorogenic properties of meso -formyl boron-dipyrromethene (BODIPY) dyes that become emissive following nucleophilic addition. A reactivity palette is assembled based on the increasing electrophilic character of five meso -formyl BODIPY compounds tested. We show that increasing rates of emission enhancement correlate with the decreasing electrophilic character of BODIPY dyes in the presence of an acid catalyst and a nucleophile. These results are consistent with the rate-limiting step involving activation of the electrophile. Increasing product formation is shown to correlate with the increasing electrophilic character of the BODIPY dyes, as expected based on thermodynamics. In addition to providing rates of reaction, analysis of the fluorescence parameters for the reaction mixtures, including emission quantum yields and fluorescence lifetimes, enables us to determine the extent of reactant conversion at equilibrium (in our case the estimated yield of a transient species) and the presence of different products, without the need for isolation. We anticipate that our reactivity palette approach, combined with the in-depth fluorescence analysis discussed herein, will provide guidelines toward developing fluorogenic assays of reactivity offering multiplex information, beyond fluorescence intensity., Competing Interests: The authors declare no competing financial interest.

Protein and DNA alkylation by endogenously produced electrophiles is associated with the pathogenesis of neurodegenerative diseases, to epigenetic alterations and to cell signaling and redox regulation. With the goal of visualizing, in real-time, the spatiotemporal response of the cell milieu to electrophiles, we have designed a fluorogenic BODIPY-acrolein probe, AcroB, that undergoes a >350-fold fluorescence intensity enhancement concomitant with protein adduct formation. AcroB enables a direct quantification of single post-translational modifications occurring on cellular proteins via recording fluorescence bursts in live-cell imaging studies. In combination with super-resolution imaging, protein alkylation events may be registered and individually counted, yielding a map of protein-electrophile reactions within the cell lipid milieu. Alkylation is predominantly observed within mitochondria, a source, yet not a sink, of AcroB adducts, illustrating that a mitochondrial constitutive excretion mechanism ensures rapid disposal of compromised proteins. Sorting within the Golgi apparatus and trafficking along microtubules and through the cell endo- and exocytic pathways can be next visualized via live-cell imaging. Our results offer a direct visualization of cellular response to a noncanonical acrolein warhead. We envision AcroB will enable new approaches for diagnosis of pathologies associated with defective cellular trafficking. AcroB may help elucidate key aspects of mitochondria electrophile adduct excretion and cell endocytic and exocytic pathways. Conceptually, AcroB provides a new paradigm on fluorescence microscopy studies where chemical perturbation is achieved and simultaneously reported by the probe.

Reactive oxygen species (ROS) and their associated byproducts have been traditionally associated with a range of pathologies. It is now believed, however, that at basal levels these molecules also have a beneficial cellular function in the form of cell signaling and redox regulation. Critical to elucidating their physiological role is the opportunity to visualize and quantify the production of ROS with spatiotemporal accuracy. Armed with a newly developed, extremely sensitive fluorogenic α-tocopherol analogue (H 4 BPMHC), we report herein the observation of steady concentrations of lipid peroxyl radicals produced in live cell imaging conditions. Imaging studies with H 4 BPMHC indicate that the rate of production of lipid peroxyl radicals in HeLa cells under basal conditions is 33 nM/h within the cell. Our work further provides indisputable evidence on the antioxidant role of Vitamin E, as lipid peroxidation was suppressed in HeLa cells both under basal conditions and in the presence of Haber-Weiss chemistry, generated by the presence of cumyl hydroperoxide and Cu 2+ in solution, when supplemented with the α-tocopherol surrogate, PMHC (2,2,5,7,8-pentamethyl-6-hydroxy-chromanol, an α-tocopherol analogue lacking the phytyl tail). H 4 BPMHC has the sensitivity needed to detect trace changes in oxidative status within the lipid membrane, underscoring the opportunity to illuminate the physiological relevance of lipid peroxyl radical production during cell homeostasis and disease.

Prions arise from proteins that have two possible conformations: properly folded and non-infectious or misfolded and infectious. The [ PSI + ] yeast prion, which is the misfolded and self-propagating form of the translation termination factor eRF3 (Sup35), can be cured of its infectious conformation by overexpression of Hsp104, which helps dissolve the prion seeds. This dissolution depends on the trimming activity of Hsp104, which reduces the size of the prion seeds without increasing their number. To further understand the relationship between trimming and curing, trimming was followed by measuring the loss of GFP-labeled Sup35 foci from both strong and weak [ PSI + ] variants; the former variant has more seeds and less soluble Sup35 than the latter. Overexpression of Saccharomyces cerevisiae Hsp104 (Sc-Hsp104) trimmed the weak [ PSI + ] variants much faster than the strong variants and cured the weak variants an order of magnitude faster than the strong variants. Overexpression of the fungal Hsp104 homologs from Schizosaccharomyces pombe (Sp-Hsp104) or Candida albicans (Ca-Hsp104) also trimmed and cured the weak variants, but interestingly, it neither trimmed nor cured the strong variants. These results show that, because Sc-Hsp104 has greater trimming activity than either Ca-Hsp104 or Sp-Hsp104, it cures both the weak and strong variants, whereas Ca-Hsp104 and Sp-Hsp104 only cure the weak variants. Therefore, curing by Hsp104 overexpression depends on both the trimming ability of the fungal Hsp104 homolog and the strength of the [ PSI + ] variant: the greater the trimming activity of the Hsp104 homolog and the weaker the variant, the greater the curing., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

We report two BODIPY based photosensitizers (Br2BOAc and I2BOAc) featuring an acetoxymethyl substituent at the meso-position. These photosensitizers show improved photostability against singlet oxygen, when compared to a BODIPY photosensitizer lacking the acetoxymethyl group. Both compounds were evaluated for photodynamic therapy against HeLa cells and photodynamic inactivation against E. coli bacteria. We show that the compounds readily embed in the lipid membranes of HeLa cervical cancer cells and efficiently induced light-dependent apoptosis at nanomolar concentration. Also, both compounds showed a substantial degree of photoinactivation of E. coli bacteria when used at low micromolar concentrations.

We report herein the synthesis and characterization of a fluorogenic analogue of ubiquinone designed to reversibly report on redox reactions in biological systems. The analogue, H2B-Q, consists of the redox-active quinone segment found in ubiquinone, 2,3-dimethoxy-1,4-benzoquinone, coupled to a boron-dipyrromethene (BODIPY) fluorophore segment that both imparts lipophilicity in lieu of the isoprenyl tail of ubiquinone, and reports on redox changes at the quinone/quinol segment. Redox sensing is mediated by a photoinduced electron transfer intramolecular switch. In its reduced dihydroquinone form, H2B-QH2 is highly emissive in nonpolar media (quantum yields 55-66%), while once oxidized, the resulting quinone H2B-Q emission is suppressed. Cyclic voltammetry of H2B-Q shows two reversible, 1-electron reduction peaks at -1.05 V and -1.37 V (vs ferrocene) on par with those of ubiquinone. Chemical reduction of H2B-Q by NaBH4 resulted in >200 fold emission enhancement. H2B-QH2 is shown to react with peroxyl radicals, a form of reactive oxygen species (ROS) as well as to cooperatively interact with chromanol (the active segment of α-tocopherol). Kinetic analysis further shows the antioxidant reactivity of the nonfluorescent intermediate semiquinone. We anticipate that the H2B-Q/H2B-QH2 off/on reversible couple may serve as a tool to monitor chemical redox processes in real-time and in a noninvasive manner.

Here we show the design, preparation, and characterization of a dormant singlet oxygen ((1)O2) photosensitizer that is activated upon its reaction with reactive oxygen species (ROS), including (1)O2 itself, in what constitutes an autocatalytic process. The compound is based on a two segment photosensitizer-trap molecule where the photosensitizer segment consists of a Br-substituted boron-dipyrromethene (BODIPY) dye. The trap segment consists of the chromanol ring of α-tocopherol, the most potent naturally occurring lipid soluble antioxidant. Time-resolved absorption, fluorescence, and (1)O2 phosphorescence studies together with fluorescence and (1)O2 phosphorescence emission quantum yields collected on Br2B-PMHC and related bromo and iodo-substituted BODIPY dyes show that the trap segment provides a total of three layers of intramolecular suppression of (1)O2 production. Oxidation of the trap segment with ROS restores the sensitizing properties of the photosensitizer segment resulting in ∼40-fold enhancement in (1)O2 production. The juxtaposed antioxidant (chromanol) and prooxidant (Br-BODIPY) antagonistic chemical activities of the two-segment compound enable the autocatalytic, and in general ROS-mediated, activation of (1)O2 sensitization providing a chemical cue for the spatiotemporal control of (1)O2.The usefulness of this approach to selectively photoactivate the production of singlet oxygen in ROS stressed vs regular cells was successfully tested via the photodynamic inactivation of a ROS stressed Gram negative Escherichia coli strain.

Cyclin-G-associated kinase (GAK), the ubiquitously expressed J-domain protein, is essential for the chaperoning and uncoating of clathrin that is mediated by Hsc70 (also known as HSPA8). Adjacent to the C-terminal J-domain that binds to Hsc70, GAK has a clathrin-binding domain that is linked to an N-terminal kinase domain through a PTEN-like domain. Knocking out GAK in fibroblasts caused inhibition of clathrin-dependent trafficking, which was rescued by expressing a 62-kDa fragment of GAK, comprising just the clathrin-binding and J-domains. Expressing this fragment as a transgene in mice rescued the lethality and the histological defects caused by knocking out GAK in the liver or in the brain. Furthermore, when both GAK and auxilin (also known as DNAJC6), the neuronal-specific homolog of GAK, were knocked out in the brain, mice expressing the 62-kDa GAK fragment were viable, lived a normal life-span and had no major behavior abnormalities. However, these mice were about half the size of wild-type mice. Therefore, the PTEN-like domains of GAK and auxilin are not essential for Hsc70-dependent chaperoning and uncoating of clathrin, but depending on the tissue, these domains appear to increase the efficiency of these co-chaperones., (© 2015. Published by The Company of Biologists Ltd.)

We report herein spectroscopy and computational results that illustrate an efficient intramolecular deactivation pathway for meso-unsaturated boron-dipyrromethene (BODIPY) dyes in their singlet excited state. Our results show that the mechanism hinges on the structural flexibility imparted by the boron atom and on the energetic stabilization conferred by extending the conjugation into the meso substituent, which is otherwise unconjugated in the ground state. Following photoexcitation, rotation along the dihedral angle of the meso-unsaturated group results in its conjugation at the expense of shifting one pyrrole moiety in dipyrrin out of the plane. Internal conversion to an energetically hot, ground-state species efficiently competes with emission. The mechanism applies to meso-vinyl, -formyl, and -iminyl moieties. The presence of methyl groups at positions C1 and C7 exacerbates the energetic penalty toward conjugation of the meso groups leading to a small energy gap between relaxed excited state and ground state and undetected emission quantum yields. Importantly, methyls at C1 and C7 prevent nonradiative deactivation in meso-aryl moieties, illustrating that when push comes to shove, the energetic (kinetic) barrier toward reaching conjugation is too large for aryl moieties but low enough for smaller groups to effectively compete with radiative transitions. Wisely chosen meso-unsaturated BODIPY dyes may serve as richly sensitive platforms for the preparation of novel fluorogenic substrates to monitor chemical reactions or to probe the rigidity of their surrounding environment.

The conversion of the properly folded prion protein, PrPc, to its misfolded amyloid form, PrPsc, occurs as the two proteins traffic along the endocytic pathway and PrPc is exposed to PrPsc. To determine the specific site of prion conversion, we knocked down various proteins in the endocytic pathway including Rab7a, Tsg101 and Hrs (also known as HGS). PrPsc was markedly reduced in two chronically infected cell lines by preventing the maturation of the multivesicular body, a process that begins in the early endosome and ends with the sorting of cargo to the lysosome. By contrast, knocking down proteins in the retromer complex, which diverts cargo away from the multivesicular body caused an increase in PrPsc levels. These results suggest that the multivesicular body is the major site for intracellular conversion of PrPc to PrPsc., (© 2015. Published by The Company of Biologists Ltd.)

Objective: Palatal lift appliances have a role in management of velopharyngeal dysfunction for immobile palates of adequate length where surgery is contraindicated. Conventional appliances involve acrylic/wire work adjustment over successive appointments until they can be tolerated without gagging. A novel appliance has been developed where the lifting plate is incrementally distalized by the patient and vertically adjusted to optimize soft palate positioning., Method: The design, construction, and utility of the appliance, which was developed in Dundee Dental Hospital, are described., Participants: The subject was a 12-year-old boy with a variant of Moebius syndrome and velopharyngeal dysfunction. Previous pharyngoplasty had been carried out and further surgery was contraindicated., Interventions: The appliance is constructed and fitted and the flexible spring arm is vertically adjusted to lift the soft palate. The screw is turned incrementally at home, extending the lifting plate posteriorly. Videofluoroscopy allows visualization of the appliance and soft palate positioning., Main Outcome Measures/results: The procedure improved soft palate positioning, as demonstrated by videofluoroscopy, and objective speech outcomes., Conclusions: The appliance was well tolerated and led to improved speech outcomes for the patient. Adjustments were quick and easy for both clinician and patient. Further studies are needed to definitively determine the efficacy of the appliance.

Background/aims: Pyridoxamine dihydrochloride (Pyridorin™) blocks pathogenic oxidative pathways in the progression of diabetic nephropathy. The pyridoxamine pilot study was designed to test entry criteria and outcomes. Subjects had SCr 1.3-3.5 mg/dl, protein-to-creatinine ≥1,200 mg/g and used a surrogate outcome of ΔSCr over 52 weeks. Subjects had to be on a maximally tolerated dose of ACE/ARB for 3 months; stable other antihypertensive doses for 2 months; stable diuretic dose for 2 weeks, and BP ≤160/90 mm Hg; or enter a Pharmaco-Stabilization Phase (PSP). This pilot failed to detect an effect on ΔSCr in intent-to-treat analysis., Methods: We queried the locked clinical trial database for subgroups in which there was a treatment effect., Results: Subjects not requiring PSP and those with entry SCr <2.0 mg/dl had a treatment effect. Subjects entering PSP required more changes in antihypertensive medications and experienced larger ΔSCr over 52 weeks. PSP subjects with BP >140/90 mm Hg had no treatment effect, but those ≤140/90 mm Hg did., Conclusion: Time required for acute effects of ACE/ARB to stabilize is unknown, but these data suggest >3 months. Thus, subjects in the pivotal trial must be on ACE/ARB for 6 months. Frequent antihypertensive adjustment could engender SCr changes unrelated to CKD progression. Thus, we will require subjects to have BP ≤150/90 mm Hg and on stable antihypertensives for 26 weeks, or ≤140/90 mm Hg and on stable antihypertensives for 13 weeks. Since ΔSCr over 52 weeks is limited as a surrogate outcome, the pivotal trial uses a time-to-event analysis of baseline SCr to at least a 50% increase in SCr or ESRD as the primary outcome. This substantial ΔSCr is protected from noise and is clinically relevant. The pyridoxamine pilot provided critical information to inform the design of PIONEER-CSG-17, which we conducted under the SPA agreement with FDA.

We employed qualitative methods to explore how conflict over water collection and use impacts women, and the role that women play in water management and conflict resolution in Marsabit, Kenya. Conflicts between domestic and livestock water led to insufficient water for domestic use and intra-household conflict. Women's contributions to water management were valued, especially through informal initiatives, though involvement in statutory water management committees was not culturally appropriate. Promoting culturally appropriate ways to involve women in water management, rather than merely increasing the percentage of women on water committee, may reduce conflicts and increase women's access to domestic water supplies.

Here we formulate equations based solely on empirical Hammett substituent constants to predict the redox potentials for the electronic excited state of boron-dipyrromethene (BODIPY) dyes. We utilized computational, spectroscopic, and electrochemical techniques toward characterizing the effect of substitution at the positions C2, C6, and C8 of the 1,3,5,7-tetramethyl BODIPY core. Working with a library of 100 BODIPY dyes, we found that highest occupied molecular orbital (HOMO) energies calculated at the B3LYP 6-31g(d) level correlated linearly with the Hammett σm value for substituents at position C8 and with Hammett σp values for substituents at positions C2 and C6. In turn, we observed that LUMO energies correlated linearly with Hammett σp at position C8 and with Hammett σm at positions C2 and C6. Focusing on a subset of 26 dyes for which reduction potentials were either previously available or measured herein and ranged from -1.84 to -0.52 V (a full 1.3 V), we found a linear relationship between redox potentials in acetonitrile and HOMO and lowest unoccupied molecule orbital (LUMO) energies determined via density functional theory (DFT). A linear correlation was thus ultimately established between redox potentials in acetonitrile and Hammett substituent constants. Combining this with equations derived for the linear relationship existing between the zero vibrational energy of the excited BODIPY and Hammett substituent constants enabled us to provide the parameters toward predicting the oxidizing/reducing power of photoexcited 1,3,5,7,-tetramethyl BODIPY dyes in their singlet excited state.

The [PSI(+)] yeast prion is formed when Sup35 misfolds into amyloid aggregates. [PSI(+)], like other yeast prions, is dependent on the molecular chaperone Hsp104, which severs the prion seeds so that they pass on as the yeast cells divide. Surprisingly, however, overexpression of Hsp104 also cures [PSI(+)]. Several models have been proposed to explain this effect: inhibition of severing, asymmetric segregation of the seeds between mother and daughter cells, and dissolution of the prion seeds. First, we found that neither the kinetics of curing nor the heterogeneity in the distribution of the green fluorescent protein (GFP)-labeled Sup35 foci in partially cured yeast cells is compatible with Hsp104 overexpression curing [PSI(+)] by inhibiting severing. Second, we ruled out the asymmetric segregation model by showing that the extent of curing was essentially the same in mother and daughter cells and that the fluorescent foci did not distribute asymmetrically, but rather, there was marked loss of foci in both mother and daughter cells. These results suggest that Hsp104 overexpression cures [PSI(+)] by dissolution of the prion seeds in a two-step process. First, trimming of the prion seeds by Hsp104 reduces their size, and second, their amyloid core is eliminated, most likely by proteolysis.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson disease (PD), and common variants around LRRK2 are a risk factor for sporadic PD. Using protein-protein interaction arrays, we identified BCL2-associated athanogene 5, Rab7L1 (RAB7, member RAS oncogene family-like 1), and Cyclin-G-associated kinase as binding partners of LRRK2. The latter two genes are candidate genes for risk for sporadic PD identified by genome-wide association studies. These proteins form a complex that promotes clearance of Golgi-derived vesicles through the autophagy-lysosome system both in vitro and in vivo. We propose that three different genes for PD have a common biological function. More generally, data integration from multiple unbiased screens can provide insight into human disease mechanisms.

The impact of improved water, sanitation, and hygiene (WASH) access on mitigating illness is well documented, although impact of school-based WASH on school-aged children has not been rigorously explored. We conducted a cluster-randomized trial in Nyanza Province, Kenya to assess the impact of a school-based WASH intervention on diarrhoeal disease in primary-school pupils. Two study populations were used: schools with a nearby dry season water source and those without. Pupils attending 'water-available' schools that received hygiene promotion and water treatment (HP&WT) and sanitation improvements showed no difference in period prevalence or duration of illness compared to pupils attending control schools. Those pupils in schools that received only the HP&WT showed similar results. Pupils in 'water-scarce' schools that received a water-supply improvement, HP&WT and sanitation showed a reduction in diarrhoea incidence and days of illness. Our study revealed mixed results on the impact of improvements to school WASH improvements on pupil diarrhoea.

The Hsc70 cochaperone, G cyclin-associated kinase (GAK), has been shown to be essential for the chaperoning of clathrin by Hsc70 in the cell. In this study, we used conditional GAK knockout mouse embryonic fibroblasts (MEFs) to determine the effect of completely inhibiting clathrin-dependent trafficking on the cell cycle. After GAK was knocked out, the cells developed the unusual phenotype of having multiple centrosomes, but at the same time failed to divide and ultimately became senescent. To explain this phenotype, we examined the signaling profile and found that mitogenic stimulation of the GAK KO cells and the control cells were similar except for increased phosphorylation of Akt. In addition, the disruption of intracellular trafficking caused by knocking out GAK destabilized the lysosomal membranes, resulting in DNA damage due to iron leakage. Knocking down clathrin heavy chain or inhibiting dynamin largely reproduced the GAK KO phenotype, but inhibiting only clathrin-mediated endocytosis by knocking down adaptor protein (AP2) caused growth arrest and centrosome overduplication, but no DNA damage or senescence. We conclude that disruption of clathrin-dependent trafficking induces senescence accompanied by centrosome overduplication because of a combination of DNA damage and changes in mitogenic signaling that uncouples centrosomal duplication from DNA replication., (Published 2013. This article is a U.S. Government work and is in the public domain in the USA.)

Objectives: We examined the impact of school water, sanitation, and hygiene (WASH) interventions on diarrhea-related outcomes among younger siblings of school-going children., Methods: We conducted a cluster-randomized trial among 185 schools in Kenya from 2007 to 2009. We assigned schools to 1 of 2 study groups according to water availability. Multilevel logistic regression models, adjusted for baseline measures, assessed differences between intervention and control arms in 1-week period prevalence of diarrhea and 2-week period prevalence of clinic visits among children younger than 5 years with at least 1 sibling attending a program school., Results: Among water-scarce schools, comprehensive WASH improvements were associated with decreased odds of diarrhea (odds ratio [OR] = 0.44; 95% confidence interval [CI] = 0.27, 0.73) and visiting a clinic (OR = 0.36; 95% CI = 0.19, 0.68), relative to control schools. In our separate study group of schools with greater water availability, school hygiene promotion and water treatment interventions and school sanitation improvements were not associated with differences in diarrhea prevalence between intervention and control schools., Conclusions: In water-scarce areas, school WASH interventions that include robust water supply improvements can reduce diarrheal diseases among young children.

We report here the preparation of a lipophilic fluorogenic antioxidant (Mito-Bodipy-TOH) that targets the inner mitochondrial lipid membrane (IMM) and is sensitive to the presence of lipid peroxyl radicals, effective chain carriers in the lipid chain autoxidation. Mito-Bodipy-TOH enables monitoring of the antioxidant status, i.e., the antioxidant load and ability to prevent lipid chain autoxidation, within the inner mitochondrial membrane of live cells. The new probe consists of 3 segments: a receptor, a reporter, and a mitochondria-targeting element, constructed, respectively, from an α-tocopherol-like chromanol moiety, a BODIPY fluorophore, and a triphenylphosphonium cation (TPP). The chromanol moiety ensures reactivity akin to that of α-tocopherol, the most potent naturally occurring lipid soluble antioxidant, while the BODIPY fluorophore and TPP ensure partitioning within the inner mitochondrial membrane. Mechanistic studies conducted either in homogeneous solution or in liposomes and in the presence of free radical initiators show that the antioxidant activity of Mito-Bodipy-TOH is on par with that of α-tocopherol. Studies conducted on live fibroblast cells further show the antioxidant depletion in the presence of methyl viologen (paraquat), a known agent of oxidative stress and source of superoxide radical anion (and indirectly, a causative of lipid peroxidation) within the mitochondria matrix. We recorded a ca. 8-fold emission enhancement with Mito-Bodipy-TOH in cells stressed with methyl viologen, whereas no enhancement was observed in control studies with untreated cells. Our findings underscore the potential of the new fluorogenic antioxidant Mito-Bodipy-TOH to study the chemical link between antioxidant load, lipid peroxidation and mitochondrial physiology.

We employed a cluster randomized trial design to measure the impact of a school based water, sanitation, and hygiene (WASH) improvement on pupil enrollment and on gender parity in enrollment, in primary schools in Nyanza Province, Kenya (2007-2009). Among schools with poor water access during the dry season, those that received a water supply, hygiene promotion and water treatment (HP&WT) and sanitation improvement, demonstrated increased enrollment (β=0.091 [0.009, 0.173] p=0.03), which translates to 26 additional pupils per school on average. The proportion of girls enrolled in school also increased by 4% (prevalence ratio (PR)=1.04 [1.00, 1.07] p=0.02). Among schools with better baseline water access during the dry season (schools that didn't receive a water source), we found no evidence of increased enrollment in schools that received a HP&WT intervention (β=0.016 [-0.039, 0.072] p=0.56) or the HP&WT and sanitation intervention (β=0.027 [-0.028, 0.082]p=0.34), and there was no evidence of improved gender parity (PR=0.99 [0.96, 1.02] p=0.59, PR=1.00 [0.97, 1.02] p=0.75, respectively). Our findings suggest that increased school enrollment and improved gender parity may be influenced by a comprehensive WASH program that includes an improved water source; schools with poor water access during the dry season may benefit most from these interventions.

Clathrin coats, which stabilize membrane curvature during endocytosis and vesicular trafficking, form highly polymorphic fullerene lattices. We used cryo-electron tomography to visualize coated particles in isolates from bovine brain. The particles range from ∼66 to ∼134nm in diameter, and only 20% of them (all ⩾80nm) contain vesicles. The remaining 80% are clathrin "baskets", presumably artifactual assembly products. Polyhedral models were built for 54 distinct coat geometries. In true coated vesicles (CVs), most vesicles are offset to one side, leaving a crescent of interstitial space between the coat and the membrane for adaptor proteins and other components. The latter densities are fewer on the membrane-proximal side, which may represent the last part of the vesicle to bud off. A small number of densities - presumably cargo proteins - are associated with the interior surface of the vesicles. The clathrin coat, adaptor proteins, and vesicle membrane contribute almost all of the mass of a CV, with most cargoes accounting for only a few percent. The assembly of a CV therefore represents a massive biosynthetic effort to internalize a relatively diminutive payload. Such a high investment may be needed to overcome the resistance of membranes to high curvature., (Published by Elsevier Inc.)

We assessed whether supplying soap to primary schools on a regular basis increased pupil hand washing and decreased Escherichia coli hand contamination. Multiple rounds of structured observations of hand washing events after latrine use were conducted in 60 Kenyan schools, and hand rinse samples were collected one time in a subset of schools. The proportion of pupils observed practicing hand washing with soap (HWWS) events was significantly higher in schools that received a soap provision intervention (32%) and schools that received soap and latrine cleaning materials (38%) compared with controls (3%). Girls and boys had similar hand washing rates. There were non-significant reductions in E. coli contamination among intervention school pupils compared with controls. Removing the barrier of soap procurement can significantly increase availability of soap and hand washing among pupils; however, we discuss limitations in the enabling policy and institutional environment that may have prevented reaching desired levels of HWWS.

Randomized clinical trials are considered the 'gold standard' in primary research for healthcare interventions. However, they can be expensive and time-consuming to set up and require many approvals to be in place before they can begin. This paper outlines how to determine what approvals are required for a trial, the background of each approval and the process for obtaining them.

Under conditions of collision-induced dissociation (CID), anions of α-hydroxycarboxylic acids usually fragment to yield the distinctive hydroxycarbonyl anion (m/z 45) and/or the complementary product anion formed by neutral loss of formic acid (46 u). Further support for the known two-step mechanism, involving an ion-neutral complex for the formation of the hydroxycarbonyl anion from the carboxyl group, is herein provided by tandem mass spectrometric results and density functional theory computations on the glycolate, lactate and 3-phenyllactate ions. A fourth, structurally related α-hydroxycarboxylate ion, obtained by deprotonation of mandelic acid, showed only loss of carbon dioxide upon CID. Density functional theory computations on the mandelate ion indicated that similar energy inputs were required for a direct, phenyl-assisted decarboxylation and a postulated novel rearrangement to a carbonate ester, which yielded the benzyl oxide ion upon loss of CO2. Rearrangement of the glycolate ion led to expulsion of carbon monoxide, whereas the 3-phenyllactate ion showed the loss of water and formation of the benzyl anion and the benzyl radical as competing processes. The fragmentation pathways proposed for lactate and 3-phenyllactate are supported by isotopic labeling. The relative computed energies of saddle points and product ions for all proposed fragmentation pathways are consistent with the energies supplied during CID experiments and the observed relative intensities of product ions. The diverse reaction pathways characterized for this set of four α-hydroxycarboxylate ions demonstrate that it is crucial to understand the effects of structural variations when attempting to predict the gas-phase reactivity and CID spectra of carboxylate ions., (Copyright © 2013 John Wiley & Sons, Ltd.)

Handwashing with soap effectively reduces exposure to diarrhea-causing pathogens. Interventions to improve hygiene and sanitation conditions in schools within low-income countries have gained increased attention; however, their impact on schoolchildren's exposure to fecal pathogens has not been established. Our trial examined whether a school-based water, sanitation, and hygiene intervention reduced Escherichia coli contamination on pupils' hands in western Kenya. A hygiene promotion and water treatment intervention did not reduce risk of E. coli presence (relative risk [RR] = 0.92, 95% confidence interval [CI] = 0.54-1.56); the addition of new latrines to intervention schools significantly increased risk among girls (RR = 2.63, 95% CI = 1.29-5.34), with a non-significant increase among boys (RR = 1.36, 95% CI = 0.74-2.49). Efforts to increase usage of school latrines by constructing new facilities may pose a risk to children in the absence of sufficient hygiene behavior change, daily provision of soap and water, and anal cleansing materials.

Expression of huntingtin fragments with 103 glutamines (HttQ103) is toxic in yeast containing either the [PIN(+)] prion, which is the amyloid form of Rnq1, or [PSI(+)] prion, which is the amyloid form of Sup35. We find that HttQP103, which has a polyproline region at the C-terminal end of the polyQ repeat region, is significantly more toxic in [PSI(+)] yeast than in [PIN(+)], even though HttQP103 formed multiple aggregates in both [PSI(+)] and [PIN(+)] yeast. This toxicity was only observed in the strong [PSI(+)] variant, not the weak [PSI(+)] variant, which has more soluble Sup35 present than the strong variant. Furthermore, expression of the MC domains of Sup35, which retains the C-terminal domain of Sup35, but lacks the N-terminal prion domain, almost completely rescued HttQP103 toxicity, but was less effective in rescuing HttQ103 toxicity. Therefore, the toxicity of HttQP103 in yeast containing the [PSI(+)] prion is primarily due to sequestration of the essential protein, Sup35.

Water insecurity is a primary underlying determinant of global health disparities. While public health research on water insecurity has focused mainly on two dimensions, water access and adequacy, an anthropological perspective highlights the cultural or lifestyle dimension of water insecurity, and its implications for access/adequacy and for the phenomenology of water insecurity. Recent work in Bolivia has shown that scores on a water insecurity scale derived from ethnographic observations are associated with emotional distress. We extend this line of research by assessing the utility of a locally developed water insecurity scale, compared with standard measures of water access and adequacy, in predicting women's psychosocial distress in Ethiopia. In 2009-2010 we conducted two phases of research. Phase I was mainly qualitative and designed to identify locally relevant experiences of water insecurity, and Phase II used a quantitative survey to test the association between women's reported water insecurity and the Falk Self-Reporting Questionnaire (SRQ-F), a measure of psychosocial distress. In multiple regression models controlling for food insecurity and reported quantity of water used, women's water insecurity scores were significantly associated with psychosocial distress. Including controls for time required to collect water and whether water sources were protected did not further predict psychosocial distress. This approach highlights the social dimension of water insecurity, and may be useful for informing and evaluating interventions to improve water supplies., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Objectives: There has been increased attention to access to water, sanitation and hygiene (WASH) at schools in developing countries, but a dearth of empirical studies on the impact. We conducted a cluster-randomized trial of school-based WASH on pupil absence in Nyanza Province, Kenya, from 2007 to 2008., Methods: Public primary schools nested in three geographical strata were randomly assigned and allocated to one of three study arms [water treatment and hygiene promotion (WT & HP), additional sanitation improvement, or control] to assess the effects on pupil absence at 2-year follow-up., Results: We found no overall effect of the intervention on absence. However, among schools in two of the geographical areas not affected by post-election violence, those that received WT and HP showed a 58% reduction in the odds of absence for girls (OR 0.42, CI 0.21-0.85). In the same strata, sanitation improvement in combination with WT and HP resulted in a comparable drop in absence, although results were marginally significant (OR 0.47, 0.21-1.05). Boys were not impacted by the intervention., Conclusion: School WASH improvements can improve school attendance for girls, and mechanisms for gendered impacts should be explored. Incomplete intervention compliance highlights the challenges of achieving consistent results across all settings., (© 2011 Blackwell Publishing Ltd.)

Parkinson disease is caused by neuronal loss in the substantia nigra which manifests by abnormality of movement, muscle tone, and postural stability. Several genes have been implicated in the pathogenesis of Parkinson disease, but the underlying molecular basis is still unknown for ∼70% of the patients. Using homozygosity mapping and whole exome sequencing we identified a deleterious mutation in DNAJC6 in two patients with juvenile parkinsonism. The mutation was associated with abnormal transcripts and marked reduced DNAJC6 mRNA level. DNAJC6 encodes the HSP40 Auxilin, a protein which is selectively expressed in neurons and confers specificity to the ATPase activity of its partner Hcs70 in clathrin uncoating. In Auxilin null mice it was previously shown that the abnormally increased retention of assembled clathrin on vesicles and in empty cages leads to impaired synaptic vesicle recycling and perturbed clathrin mediated endocytosis. Endocytosis function, studied by transferring uptake, was normal in fibroblasts from our patients, likely because of the presence of another J-domain containing partner which co-chaperones Hsc70-mediated uncoating activity in non-neuronal cells. The present report underscores the importance of the endocytic/lysosomal pathway in the pathogenesis of Parkinson disease and other forms of parkinsonism.