Stress-dependent condensate formation regulated by the ubiquitin-related modifier Urm1.

The ability of proteins and RNA to coalesce into phase-separated assemblies, such as the nucleolus and stress granules, is a basic principle in organizing membraneless cellular compartments. While the constituents of biomolecular condensates are generally well documented, the mechanisms underlying their formation under stress are only partially understood. Here, we show in yeast that covalent modification with the ubiquitin-like modifier Urm1 promotes the phase separation of a wide range of proteins. We find that the drop in cellular pH induced by stress triggers Urm1 self-association and its interaction with both target proteins and the Urm1-conjugating enzyme Uba4. Urmylation of stress-sensitive proteins promotes their deposition into stress granules and nuclear condensates. Yeast cells lacking Urm1 exhibit condensate defects that manifest in reduced stress resilience. We propose that Urm1 acts as a reversible molecular "adhesive" to drive protective phase separation of functionally critical proteins under cellular stress.

Mechanisms of readthrough mitigation reveal principles of GCN1-mediated translational quality control.

Readthrough into the 3' untranslated region (3' UTR) of the mRNA results in the production of aberrant proteins. Metazoans efficiently clear readthrough proteins, but the underlying mechanisms remain unknown. Here, we show in Caenorhabditis elegans and mammalian cells that readthrough proteins are targeted by a coupled, two-level quality control pathway involving the BAG6 chaperone complex and the ribosome-collision-sensing protein GCN1. Readthrough proteins with hydrophobic C-terminal extensions (CTEs) are recognized by SGTA-BAG6 and ubiquitylated by RNF126 for proteasomal degradation. Additionally, cotranslational mRNA decay initiated by GCN1 and CCR4/NOT limits the accumulation of readthrough products. Unexpectedly, selective ribosome profiling uncovered a general role of GCN1 in regulating translation dynamics when ribosomes collide at nonoptimal codons, enriched in 3' UTRs, transmembrane proteins, and collagens. GCN1 dysfunction increasingly perturbs these protein classes during aging, resulting in mRNA and proteome imbalance. Our results define GCN1 as a key factor acting during translation in maintaining protein homeostasis.

Comparative Studies in the A30P and A53T α-Synuclein C. elegans Strains to Investigate the Molecular Origins of Parkinson's Disease.

The aggregation of α-synuclein is a hallmark of Parkinson's disease (PD) and a variety of related neurological disorders. A number of mutations in this protein, including A30P and A53T, are associated with familial forms of the disease. Patients carrying the A30P mutation typically exhibit a similar age of onset and symptoms as sporadic PD, while those carrying the A53T mutation generally have an earlier age of onset and an accelerated progression. We report two C. elegans models of PD (PDA30P and PDA53T), which express these mutational variants in the muscle cells, and probed their behavior relative to animals expressing the wild-type protein (PDWT). PDA30P worms showed a reduced speed of movement and an increased paralysis rate, control worms, but no change in the frequency of body bends. By contrast, in PDA53T worms both speed and frequency of body bends were significantly decreased, and paralysis rate was increased. α-Synuclein was also observed to be less well localized into aggregates in PDA30P worms compared to PDA53T and PDWT worms, and amyloid-like features were evident later in the life of the animals, despite comparable levels of expression of α-synuclein. Furthermore, squalamine, a natural product currently in clinical trials for treating symptomatic aspects of PD, was found to reduce significantly the aggregation of α-synuclein and its associated toxicity in PDA53T and PDWT worms, but had less marked effects in PDA30P. In addition, using an antibody that targets the N-terminal region of α-synuclein, we observed a suppression of toxicity in PDA30P, PDA53T and PDWT worms. These results illustrate the use of these two C. elegans models in fundamental and applied PD research.

Mantodea of Panguana (Insecta: Dictyoptera).

We present here the first illustrated checklist of the praying mantids (Mantodea) collected at the Panguana Field Station in Central Peru over the course of 50 years. The examination of over 430 specimens obtained mainly by light-trapping, but also other methods, revealed 44 species in 28 genera. Mantoida brunneriana, Mantoida cf. argentinae, Pseudomiopteryx cf. decipiens, Angela trifasciata, Liturgusa neblina, Cardioptera squalodon, Metriomantis cf. pilosella, Acontista festae, and Heterovates pardalina are new Peruvian records. Microphotina panguanensis n. sp. is new to science and the first species of the genus Microphotina described from the Western Amazon. The checklist of confirmed Peruvian Mantodea species is raised to 80. Thus, more than half of the currently known Peruvian Mantodea species is found at Panguana. We discuss the reasons for this diversity and comment on putative additional species which might be sampled if collection efforts are intensified. The results highlight the Conservation value of ACP Panguana for Western Amazonian Mantodea.

A natural product inhibits the initiation of α-synuclein aggregation and suppresses its toxicity.

The self-assembly of α-synuclein is closely associated with Parkinson's disease and related syndromes. We show that squalamine, a natural product with known anticancer and antiviral activity, dramatically affects α-synuclein aggregation in vitro and in vivo. We elucidate the mechanism of action of squalamine by investigating its interaction with lipid vesicles, which are known to stimulate nucleation, and find that this compound displaces α-synuclein from the surfaces of such vesicles, thereby blocking the first steps in its aggregation process. We also show that squalamine almost completely suppresses the toxicity of α-synuclein oligomers in human neuroblastoma cells by inhibiting their interactions with lipid membranes. We further examine the effects of squalamine in a Caenorhabditis elegans strain overexpressing α-synuclein, observing a dramatic reduction of α-synuclein aggregation and an almost complete elimination of muscle paralysis. These findings suggest that squalamine could be a means of therapeutic intervention in Parkinson's disease and related conditions.

Chirality of male genitalia in Otomantis casaica Giglio-Tos, 1915 (Mantodea: Hymenopodidae, Acromantinae).

Reversal of male genitalia are known in various insect orders, such as in Odonata, Orthoptera, Dermaptera, Hemiptera and Trichoptera (Schilthuizen 2007) and, within the Dictyoptera, in several species of Ectobiinae (Blattodea) (Bohn 1987), and Mantodea. Balderson (1978) first described reversal of the phallic complex in Stenomantis Saussure and Ciulfina Giglio-Tos, reporting this condition in eleven of 17 specimens representing two species of the latter-informally named as "Ciulfina sp.2" and "Ciulfina sp.7" (see Balderson 1978: 238). Subsequently, Anisyutkin & Gorochov (2004) reported the same condition at the time of describing Haania doroshenkoi from Cambodia. The male external genitalia within the Mantodea ("praying mantises") are markedly asymmetrical and generally develop in a single orientation (Klass 1997; Huber et al. 2007). Typically, the phallic complex consists of three phallic lobes surrounding the gonopore, all contained in a genital chamber between the ninth sternite and the paraprocts. Two of the three phallic lobes (phallomeres of La Greca 1955) are situated above the gonopore-one to the left and one to the right-while the third lies ventral to the genital opening. The right phallomere (RP) (Fig.1) ("right epiphallus" of Beier 1964) is usually dorsally positioned and its base extends almost completely across the wall of the genital chamber. The left phallomere (LP) (Fig.1) ("left epiphallus" of Beier 1964) is the most complex of the three lobes and it lies above the ventral phallomere (VP) (Fig.1) (hypophallus of Beier 1964).

An anticancer drug suppresses the primary nucleation reaction that initiates the production of the toxic Aß42 aggregates linked with Alzheimer's disease.

The conversion of the ß-amyloid (Aß) peptide into pathogenic aggregates is linked to the onset and progression of Alzheimer's disease. Although this observation has prompted an extensive search for therapeutic agents to modulate the concentration of Aß or inhibit its aggregation, all clinical trials with these objectives have so far failed, at least in part because of a lack of understanding of the molecular mechanisms underlying the process of aggregation and its inhibition. To address this problem, we describe a chemical kinetics approach for rational drug discovery, in which the effects of small molecules on the rates of specific microscopic steps in the self-assembly of Aß42, the most aggregation-prone variant of Aß, are analyzed quantitatively. By applying this approach, we report that bexarotene, an anticancer drug approved by the U.S. Food and Drug Administration, selectively targets the primary nucleation step in Aß42 aggregation, delays the formation of toxic species in neuroblastoma cells, and completely suppresses Aß42 deposition and its consequences in a Caenorhabditis elegans model of Aß42-mediated toxicity. These results suggest that the prevention of the primary nucleation of Aß42 by compounds such as bexarotene could potentially reduce the risk of onset of Alzheimer's disease and, more generally, that our strategy provides a general framework for the rational identification of a range of candidate drugs directed against neurodegenerative disorders.

Characterization of full-scale carbon contactors for siloxane removal from biogas using online Fourier transform infrared spectroscopy.

In this study, online Fourier transform infrared (FTIR) spectroscopy has been used to generate the first comprehensive characterization of full-scale carbon contactors for siloxane removal from biogas. Using FTIR, two clear operational regions within the exhaustion cycle were evidenced: an initial period of pseudo-steady state where the outlet siloxane concentration was consistently below the proposed siloxane limits; and a second period characterized by a progressive rise in outlet siloxane concentration during and after breakthrough. Due to the sharp breakthrough front identified, existing detection methods (which comprise field sampling coupled with laboratory-based chromatographic determination) are insufficiently responsive to define breakthrough, thus carbon contactors currently remain in service while providing limited protection to the combined heat and power engine. Integration of the exhaustion cycle to breakthrough identified average specific media capacities of 8.5-21.5 gsiloxane kg(-1)GAC, which are lower than that has been reported for vapour phase granular activated carbon (GAC). Further speciation of the biogas phase identified co-separation of organic compounds (alkanes and aromatics), which will inevitably reduce siloxane capacity. However, comparison of the five full-scale contactors identified that greater media capacity was accessible through operating contactors at velocities sufficient to diminish axial dispersion effects. In addition to enabling significant insight into gas phase GAC contactors, the use of FTIR for online control of GAC for siloxane removal is also presented.

A taxonomic revision of Otomantis Bolivar, 1890 (Mantodea: Hymenopodidae, Acromantinae) with description of five new species.

The African genus Otomantis Bolivar, 1890, is taxonomically treated via the re-description of its species on the basis of new morphological features (pronotum and male genitalia). Five new species, O. centralis sp. n. from D. R. of Congo and Angola, O. gracilis sp. n. from D. R. of Congo, O. trimacula sp. n. from Zambia and Malawi, O. bolivari sp. n. from Kenya and Tanzania and O. minima sp. n. from South Africa are described. The taxonomic position of the syntypes of O. capirica Giglio-Tos is revised. A lectotype is designated for the female of O. capirica. The female of O. rendalli (Kirby) and the male of O. aurita (Saussure & Zehntner) are described for the first time. Also provided are many new localities for all nominal species.  A key to the species of Otomantis is included for both male and female, each key fully illustrated. Finally, observations on species distributions and relationships are presented.

Increasing the enrolment of rural applicants to the faculty of medicine and addressing diversity by using a priority matrix approach to assign values to rural attributes.

In an external review of the admissions process for the Faculty of Medicine, University of Manitoba, Canada, it was suggested that admissions policies be modified to increase the enrolment of students more likely to practise in rural locations, by selecting a cohort of students with attributes reflecting potential for rural practice. A broad-based Working Group devised a framework for scoring personal attributes reflecting a potential for living and working in rural areas. This framework, based on established characteristics reported in the literature, valued applicants who had rural connections, a history of rural employment, a history of rural community service, or a combination of these attributes. Relative weights for the attributes were determined using a priority matrix approach. Historic admissions data, comprising applicants' rural origin (defined only by location of high school graduation), composite scores, and ranking, were reanalyzed to identify the magnitude of numerical constants that, when applied to composite scores, enhanced the relative ranking of eligible rural-origin applicants. This resulted in a hypothetical 29%-33% increase in the number of rural-origin students in incoming classes in those years. In the inaugural year of implementation of the policy and methodology, 60 admission offers (44.1%) were made to applicants with one or more rural attributes. Without adjustments, only 49 applicants with rural attributes (36%) would have been offered admission. This methodology resulted in a 22.4% increase in admission offers to applicants with rural attributes, and ushered in an incoming class that was more representative of the province's rural-urban demographics than in previous years. This methodology, although focused on rurality, could be equally applicable to any attribute, and to achieve greater diversity and equity among medical school applicants.

Removal and recovery of phosphate from municipal wastewaters using a polymeric anion exchanger bound with hydrated ferric oxide nanoparticles.

A new type of ion exchange media which is highly selective for phosphate, and can be easily regenerated has been investigated. The media consists of hydrated ferric oxide nanoparticles dispersed within the pore structures of polymeric anion exchanger beads. The media combines the durability and mechanical strength of ion exchange resins with the high sorption capacity of ferric oxide for phosphate. The media was trialled in fixed bed mini column experiments with real final effluent from two UK sewage treatment works, one with treatment based on chemical precipitation with iron chloride salts into an activated sludge process (population >250,000), and one based on trickling filter treatment with no specific phosphorus removal process (population <10,000). Results show that the media has high capacity for removing phosphate, reaching capacity at 4000 and 1300 bed volumes for the chemical precipitation and trickling filter works respectively, with performance greatly exceeding that of a standard anion exchanger, Amberlite IRA-410. Also trialled was the media's ability to elute the phosphorus after breakthrough, with the aim of recovering and processing it into a useful product. A one step regenerative process using a single solution containing 4% NaOH and 2% NaCl was passed through the resin bed and the phosphorus concentration of each bed volume leaving the column analysed. 80% of the phosphorus was eluted in the first bed volume. Subsequent tests investigated the performance of the media after successive partial regenerations of one bed volume of the NaOH/NaCl solution. There was no loss of performance observed after ten regeneration cycles, and levels of eluted phosphate were consistently high. These results suggest that the media has high potential for the removal and recovery of phosphate from wastewater streams. Additionally, the small volume of regenerant required translates to a very small operational footprint.

Direct oxidation of guanine and 7,8-dihydro-8-oxoguanine in DNA by a high-valent chromium complex: a possible mechanism for chromate genotoxicity.

Intracellular reductive activation of the human carcinogen chromate, Cr(VI), is a necessary step in the formation of DNA lesions that lead to cancer. Reductive activation forms the transient metastable high-valent oxidation state of Cr(V) as a precursor to the final intracellularly stable oxidation state, Cr(III). In this study, we have used a model high-valent Cr(V) complex, N,N'-ethylenebis(salicylideneanimato)oxochromium(V), Cr(V)-Salen, to probe the mechanism of interaction between this oxidation state of chromium and DNA. This interaction was found to be specific toward the oxidation of the nucleic acid base guanine in unmodified single- and double-stranded oligonucleotides as measured by an increased level of DNA strand cleavage at these sites following piperidine treatment. Replacement of a single guanine residue in DNA with a more readily oxidized 7,8-dihydro-8-oxoguanine (8-oxo-G) base allowed for site-specific oxidation at this modified site within the DNA strand by the Cr(V)-Salen complex. HPLC and ESI-mass spectrometry were used to identify the modified guanine base lesions formed in the reaction of this high-valent chromium complex with the 8-oxo-G-containing DNA substrate. Two of these modified base lesions, identified as guanidinohydantoin and spiroiminodihydantoin, were found in the reaction of the Cr(V)-Salen complex with 8-oxo-G-modified DNA, while only one, spiroiminodihydantoin, was formed from oxidation of the 8-oxo-G nucleoside. A primer extension assay using the exo(-) Klenow fragment demonstrated polymerase arrest at the site of these base modifications as well as a high degree of misincorporation of adenine opposite the site of modification. These results suggest that mutations arising from G --> T transversions would predominate with these lesions. The mechanism of damage and base oxidation products for the interaction between high-valent chromium and DNA described herein may be relevant to the in vivo formation of DNA damage leading to cancer in chromate-exposed human populations. These results also suggest how high-valent chromium can act as a cocarcinogen with 8-oxo-G-forming xenobiotics.

Nuclear translocation of cytosolic phospholipase A2 is induced by ATP depletion.

Phospholipase A(2) (PLA(2)) enzymes may play a role in cellular injury due to ATP depletion. Renal Madin-Darby canine kidney cells were subjected to ATP depletion to assess the effects of cellular energy metabolism on cytosolic PLA(2) (cPLA(2)) regulation. ATP depletion results in a decrease in soluble cPLA(2) activity and an increase in membrane-associated activity, which is reversed upon restoration of ATP levels by addition of dextrose. In ATP-depleted cells cPLA(2) mass shifts from cytosol to nuclear fractions. GFP-cPLA(2) is localized at the nuclear membrane of stably transfected ATP-depleted LLC-PK(1) cells under conditions where [Ca(2+)](i) is known to increase. cPLA(2) translocation does not occur if the increase in [Ca(2+)](i) increase is inhibited. If [Ca(2+)](i) is allowed to increase when ATP is depleted and the cells are then lysed, cPLA(2) remains associated with nuclear fractions even if the homogenate [Ca(2+)] is markedly reduced. In contrast, cPLA(2), which becomes associated with the nucleus when [Ca(2+)](i) is increased using ionophore, readily dissociates from the nuclear fractions of ATP-replete cells upon reduction of homogenate [Ca(2+)]. Okadaic acid inhibits the ATP depletion-induced association of cPLA(2) with nuclear fractions. Thus energy deprivation results in [Ca(2+)]-induced nuclear translocation, which is partially prevented by a phosphatase inhibitor.

Hypervalent chromium mimics reactive oxygen species as measured by the oxidant-sensitive dyes 2',7'-dichlorofluorescin and dihydrorhodamine.

Intracellular metabolism of the carcinogen chromate [Cr(VI)] produces the oxidative stress and oxidative DNA damage associated with its genotoxicity. Such oxidative stress has previously been measured by fluorescence using oxidant-sensitive dyes and attributed to the formation of reactive oxygen species (ROS). However, metabolism of Cr(VI) also produces Cr(IV) and Cr(V) which can directly damage biological macromolecules without forming ROS. We used the high-valence chromium species, bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) [Cr(V)-EHBA], to test whether high-valence chromium would also react with the oxidant-sensitive dyes 2',7'-dichlorofluorescin (DCFH) and dihydrorhodamine (DHR). Cr(V)-EHBA caused both dyes to fluoresce over a wide dynamic range and under conditions which indicated that Cr(V) had reacted directly with both dyes without first forming a diffusible radical species. Dimethylthiourea (DMTU) and ethanol did not affect Cr(V)-induced fluorescence in vitro or Cr(VI)-induced fluorescence in A549 cells. Under the same conditions, ethanol and DMTU increased the extent of hydrogen peroxide-induced fluorescence. As chromium-induced fluorescence was unaffected by radical scavengers and was qualitatively different from hydrogen peroxide-induced fluorescence, we conclude that DCF and R123 fluorescence in chromate-treated A549 cells is a qualitative and cumulative measure of intracellular Cr(V) formation and not ROS.

Highly swelling hydrogels from ordered galactose-based polyacrylates.

High swelling galactose-based hydrogels have been prepared using a chemoenzymatic procedure. Regioselective acylation of beta-O-methyl-galactopyranoside in nearly anhydrous pyridine with lipase from Pseudomonas cepacia yields the 6-acryloyl derivative (Compound I). Further lipase-catalysed acylation of the monoacrylate derivative in nearly anhydrous acetone yielded 2,6-diacryloyl-beta-O-methyl galactopyranoside (Compound II) that can act as a cross-linker with a structure similar to that of the sugar-based monomer. The high selectivity of enzyme catalysis yielded apparently highly regular hydrogel networks with swelling ratios at equilibrium ranging from 170 to 1100. elastic moduli ranging from 0.005 to 0.088 MPa and calculated mesh sizes ranging from 1160 to 6600 A. These values are far higher than conventional uncharged or lightly charged hydrogels at similar elastic moduli. Gel swelling was fast, with 75% of the equilibrium swelling value reached in a fractional time of 0.17. Non-selective chemical acryloylation of beta-O-methyl galactopyranoside followed by polymerization yielded a far lower-swelling hydrogel than that obtained using selective enzyme catalysis. These results indicate that the highly regular polymer structure achieved by regioselective enzyme-catalysed acylation yields relatively strong and highly swellable materials. Sugar-based hydrogels, such as those described herein, may find particular use as biomaterials because of their high water content, homogeneity, stability and expected non-toxicity. A wide range of pore sizes can be attained, suggesting that they may also be especially useful as matrices for enzyme immobilization and controlled delivery of biological macromolecules.

The management of ear disease: guidelines for Aboriginal health care programs.

Middle ear disease represents a continuing burden of illness for children of circumpolar regions. Uncertainty in management has been shown to create a barrier to timely and adequate treatment. Consistent intervention strategies were sought for the management of middle ear disease in children of the central Canadian Arctic. The current literature was reviewed using MEDLINE. A consensus document was established in consultation with specialists in community medicine, infectious disease, otolaryngology, pediatrics, and community-based care providers. Definitive recommendations and supportive algorithms have been established for management of middle ear disease in Inuit children.

Prevalence of diabetes mellitus and obesity in the Keewatin District of the Canadian Arctic.

Through a medical chart review, the prevalence of diagnosed diabetes mellitus in Inuit of the Keewatin District of the Canadian Northwest Territories was determined to be 0.27%. All cases were in adults, and no cases of gestational diabetes were noted. The prevalence and pattern of obesity were determined from measurements of body mass index (BMI), skinfold thickness, and waist-hip ratio obtained during the 1990-91 Keewatin Health Assessment Study. Thirty-one percent of 414 randomly identified adults (29% of men, 37% of women) were overweight (BMI > 27). Central fat patterning was more prevalent in women and less prevalent in men from the Keewatin compared to the general Canadian population. Comparison of skinfold thickness values to published measurements obtained from earlier arctic surveys supports the hypothesis that changes in diet and activity levels associated with urbanization have resulted in increased obesity in the Inuit.

Follow-up of a decentralized colposcopy program for the investigation and management of cervical intraepithelial neoplasia in the central Canadian Arctic.

Cervical intraepithelial neoplasia (CIN) is the second leading cause of cancer in Canadian Inuit women, and the incidence ratio in this population is 3.1 times the Canadian average. In 1993 a program was developed in a regional northern health center (Churchill) to provide colposcopy and loop electrosurgery for women in the Keewatin District of the central Canadian Arctic. Data collected prospectively over the following 2.5 years are presented. One hundred and forty-six women were seen in 341 visits. Indication for referral included CIN I on Pap smear (54.1%), CIN II (34.9%), CIN III (9.6%), and carcinoma of the cervix (1.4%). Large loop excision of the transformation zone was performed at a rate of 9.7 procedures per 100 patient visits. Estimated travel cost-savings attributable to this northern program are $299,200 (Canadian). Use of portable colposcopy in patients' home communities is presently being considered in order to provide enhanced accessibility and further cost-effectiveness.

Enhancing diagnostic ultrasound programs utilizing wide-area image management technology.

Diagnostic ultrasound in remote sites is limited by attendant delay in interpretation and reporting. Technology advances were incorporated in an ultrasound program based in Churchill, Manitoba, and designed to meet the needs of the Central Canadian Arctic. Still-frame ultrasound images are digitized via a sonographer's picture archiving and communications system (PACS) workstation, and as they are collected they are transmitted over a dial-up Internet protocol network in Dicom 3.0 format to a tertiary care center. Received images are routed to a physician's PACS workstation, where they can be reviewed prior to the patient leaving the remote clinic. The impact of existing technology on a northern ultrasound program is discussed. The implications of technology enhancement are reviewed, with specific reference to remote low bandwidth sites.