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Mitochondrial fission is a critical cellular event to maintain organelle function. This multistep process is initiated by the enhanced recruitment and oligomerization of dynamin-related protein 1 (Drp1) at the surface of mitochondria. As such, Drp1 is essential for inducing mitochondrial division in mammalian cells, and homologous proteins are found in all eukaryotes. As a member of the dynamin superfamily of proteins (DSPs), controlled Drp1 self-assembly into large helical polymers stimulates its GTPase activity to promote membrane constriction. Still, little is known about the mechanisms that regulate correct spatial and temporal assembly of the fission machinery. Here we present a cryo-EM structure of a full-length Drp1 dimer in an auto-inhibited state. This dimer reveals two key conformational rearrangements that must be unlocked through intramolecular rearrangements to achieve the assembly-competent state observed in previous structures. This structural insight provides understanding into the mechanism for regulated self-assembly of the mitochondrial fission machinery., (© 2024. The Author(s).)

The mechanochemical GTPase dynamin-related protein 1 (Drp1) catalyzes mitochondrial and peroxisomal fission, but the regulatory mechanisms remain ambiguous. Here we find that a conserved, intrinsically disordered, six-residue Short Linear Motif at the extreme Drp1 C-terminus, named CT-SLiM, constitutes a critical allosteric site that controls Drp1 structure and function in vitro and in vivo. Extension of the CT-SLiM by non-native residues, or its interaction with the protein partner GIPC-1, constrains Drp1 subunit conformational dynamics, alters self-assembly properties, and limits cooperative GTP hydrolysis, surprisingly leading to the fission of model membranes in vitro. In vivo, the involvement of the native CT-SLiM is critical for productive mitochondrial and peroxisomal fission, as both deletion and non-native extension of the CT-SLiM severely impair their progression. Thus, contrary to prevailing models, Drp1-catalyzed membrane fission relies on allosteric communication mediated by the CT-SLiM, deceleration of GTPase activity, and coupled changes in subunit architecture and assembly-disassembly dynamics., (© 2024. The Author(s).)

Patient mutations have been identified throughout dynamin-related protein 1 (Drp1), the key protein mediator of mitochondrial fission. These changes generally impact young children and often result in severe neurological defects and, in some instances, death. Until now, the underlying functional defect leading to patient phenotypes has been largely speculative. We therefore analyzed six disease-associated mutations throughout the GTPase and middle domains (MD) of Drp1. The MD plays a role in Drp1 oligomerization, and three mutations in this region were predictably impaired in self-assembly. However, another mutant in this region (F370C) retained oligomerization capability on pre-curved membranes despite being assembly-limited in solution. Instead, this mutation impaired membrane remodeling of liposomes, which highlights the importance of Drp1 in generating local membrane curvature before fission. Two GTPase domain mutations were also observed in different patients. The G32A mutation was impaired in GTP hydrolysis both in solution and in the presence of lipid but remains capable of self-assembly on these lipid templates. The G223V mutation also exhibited decreased GTPase activity and was able to assemble on pre-curved lipid templates; however, this change impaired membrane remodeling of unilamellar liposomes similar to F370C. This demonstrates that the Drp1 GTPase domain also contributes to self-assembly interactions that drive membrane curvature. Overall, the functional defects caused by mutations in Drp1 are highly variable even for mutations that reside within the same functional domain. This study provides a framework for characterizing additional Drp1 mutations to provide a comprehensive understanding of functional sites within this essential protein., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Nocturnal hypoxaemia, which is common in chronic obstructive pulmonary disease (COPD) patients, is associated with skeletal muscle loss or sarcopenia, which contributes to adverse clinical outcomes. In COPD, we have defined this as prolonged intermittent hypoxia (PIH) because the duration of hypoxia in skeletal muscle occurs through the duration of sleep followed by normoxia during the day, in contrast to recurrent brief hypoxic episodes during obstructive sleep apnoea (OSA). Adaptive cellular responses to PIH are not known. Responses to PIH induced by three cycles of 8 h hypoxia followed by 16 h normoxia were compared to those during chronic hypoxia (CH) or normoxia for 72 h in murine C2C12 and human inducible pluripotent stem cell-derived differentiated myotubes. RNA sequencing followed by downstream analyses were complemented by experimental validation of responses that included both unique and shared perturbations in ribosomal and mitochondrial function during PIH and CH. A sarcopenic phenotype characterized by decreased myotube diameter and protein synthesis, and increased phosphorylation of eIF2α (Ser51) by eIF2α kinase, and of GCN-2 (general controlled non-derepressed-2), occurred during both PIH and CH. Mitochondrial oxidative dysfunction, disrupted supercomplex assembly, lower activity of Complexes I, III, IV and V, and reduced intermediary metabolite concentrations occurred during PIH and CH. Decreased mitochondrial fission occurred during CH. Physiological relevance was established in skeletal muscle of mice with COPD that had increased phosphorylation of eIF2α, lower protein synthesis and mitochondrial oxidative dysfunction. Molecular and metabolic responses with PIH suggest an adaptive exhaustion with failure to restore homeostasis during normoxia. KEY POINTS: Sarcopenia or skeletal muscle loss is one of the most frequent complications that contributes to mortality and morbidity in patients with chronic obstructive pulmonary disease (COPD). Unlike chronic hypoxia, prolonged intermittent hypoxia is a frequent, underappreciated and clinically relevant model of hypoxia in patients with COPD. We developed a novel, in vitro myotube model of prolonged intermittent hypoxia with molecular and metabolic perturbations, mitochondrial oxidative dysfunction, and consequent sarcopenic phenotype. In vivo studies in skeletal muscle from a mouse model of COPD shared responses with our myotube model, establishing the pathophysiological relevance of our studies. These data lay the foundation for translational studies in human COPD to target prolonged, nocturnal hypoxaemia to prevent sarcopenia in these patients., (© 2022 The Authors. The Journal of Physiology © 2022 The Physiological Society.)

Lon is an ATP-dependent protease belonging to the "ATPase associated with diverse cellular activities" (AAA+) protein family. In humans, Lon is translated as a precursor and imported into the mitochondria matrix through deletion of the first 114 amino acid residues. In mice, embryonic knockout of lon is lethal. In humans, some dysfunctional lon mutations are tolerated but they cause a developmental disorder known as the CODAS syndrome. To gain a better understanding on the enzymology of human mitochondrial Lon, this study compares the structure-function relationship of the WT versus one of the CODAS mutants R721G to identify the mechanistic features in Lon catalysis that are affected. To this end, steady-state kinetics were used to quantify the difference in ATPase and ATP-dependent peptidase activities between WT and R721G. The K m values for the intrinsic as well as protein-stimulated ATPase were increased whereas the k cat value for ATP-dependent peptidase activity was decreased in the R721G mutant. The mutant protease also displayed substrate inhibition kinetics. In vitro studies revealed that R721G did not degrade the endogenous mitochondrial Lon substrate pyruvate dehydrogenase kinase isoform 4 (PDK4) effectively like WT hLon. Furthermore, the pyruvate dehydrogenase complex (PDH) protected PDK4 from hLon degradation. Using hydrogen deuterium exchange/mass spectrometry and negative stain electron microscopy, structural perturbations associated with the R721G mutation were identified. To validate the in vitro findings under a physiologically relevant condition, the intrinsic stability as well as proteolytic activity of WT versus R721G mutant towards PDK 4 were compared in cell lysates prepared from immortalized B lymphocytes expressing the respective protease. The lifetime of PDK4 is longer in the mutant cells, but the lifetime of Lon protein is longer in the WT cells, which corroborate the in vitro structure-functional relationship findings., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The geographic distribution of the Rocky Mountain wood tick, Dermacentor andersoni Stiles, was determined in Alberta, Canada, by drag sampling at 86 and 89 sites during 2011 and 2012, respectively. Tick density and prevalence varied between years, averaging (range) 1.0 (0-26.2) and 5.9 (0-110) ticks/1,000 m2 in 2011 and 2012, respectively. Ticks were detected at 24.4% and 42.7% of the sites sampled in each respective year. Tick density and presence declined in a northerly direction to 51.6°N and in a westerly direction to ca. 113°W, except for a small area of high density at the edge of the Rocky Mountains in the southeastern portion of the province. Ticks were most abundant in the Dry Mixedgrass and Montane natural subregions and in areas with Brown Chernozemic, Regosol, and Solodized Solonetzic great soil groups. A logistic regression model indicated that tick presence was increased in the Dry Mixedgrass natural subregion and in regions with greater temperatures during the previous summer and normal winter precipitation but was reduced in areas with Dark Brown Chernozemic soils. The model will be useful for predicting tick presence and the associated risk of tick-borne diseases in the province., (© Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada, 2021.)

Background: Lyme disease is an emerging vector-borne zoonotic disease of increasing public health importance in Canada. As part of its mandate, the Canadian Lyme Disease Research Network (CLyDRN) launched a pan-Canadian sentinel surveillance initiative, the Canadian Lyme Sentinel Network (CaLSeN), in 2019., Objectives: To create a standardized, national sentinel surveillance network providing a real-time portrait of the evolving environmental risk of Lyme disease in each province., Methods: A multicriteria decision analysis (MCDA) approach was used in the selection of sentinel regions. Within each sentinel region, a systematic drag sampling protocol was performed in selected sampling sites. Ticks collected during these active surveillance visits were identified to species, and Ixodes spp. ticks were tested for infection with Borrelia burgdorferi , Borrelia miyamotoi , Anaplasma phagocytophilum , Babesia microti and Powassan virus., Results: In 2019, a total of 567 Ixodes spp. ticks ( I. scapularis [n=550]; I. pacificus [n=10]; and I. angustus [n=7]) were collected in seven provinces: British Columbia, Manitoba, Ontario, Québec, New Brunswick, Nova Scotia and Prince Edward Island. The highest mean tick densities (nymphs/100 m 2 ) were found in sentinel regions of Lunenburg (0.45), Montréal (0.43) and Granby (0.38). Overall, the Borrelia burgdorferi prevalence in ticks was 25.2% (0%-45.0%). One I. angustus nymph from British Columbia was positive for Babesia microti , a first for the province. The deer tick lineage of Powassan virus was detected in one adult I. scapularis in Nova Scotia., Conclusion: CaLSeN provides the first coordinated national active surveillance initiative for tick-borne disease in Canada. Through multidisciplinary collaborations between experts in each province, the pilot year was successful in establishing a baseline for Lyme disease risk across the country, allowing future trends to be detected and studied., Competing Interests: Competing interests: None.

Passive and active surveillance for the blacklegged tick, Ixodes scapularis Say, in the Canadian province of Saskatchewan was conducted over a 9-yr period (2009-2017). More than 26,000 ixodid ticks, representing 10 species, were submitted through passive surveillance. Most (97%) of these were the American dog tick, Dermacentor variabilis (Say). Of the 65 I. scapularis adults submitted, 75% were collected from dogs. Infection rates of Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti in I. scapularis were 12%, 8%, and 0%, respectively. Although the I. scapularis submitted by passive surveillance were collected from five of seven ecoregions in central and southern Saskatchewan, they were most frequent in the Moist Mixed Grassland and Aspen Parklands. In contrast, no I. scapularis were collected from the extensive field sampling conducted at multiple sites in different ecoregions across the province. Hence, there is no evidence of I. scapularis having established a breeding population in Saskatchewan. Nonetheless, continued surveillance for blacklegged ticks is warranted given their important role as a vector of medically and veterinary important pathogens, and because they have recently become established across much of the southern portions of the neighboring province of Manitoba., (© Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada, 2019.)

The container-inhabiting Aedes mosquitoes are the major vectors transmitting dengue and several other arboviral diseases such as chikungunya and zika across the tropical world. Surveillance for immature Aedes, particularly pupae, is an effective tool for measuring dengue outbreak risk. While in Bangladesh, the greatest burden of dengue fever and dengue hemorrhagic fever cases has periodically been occurring since the first major outbreak in 2000, very limited research has yet been pursued to understand the dynamics of Aedes pupal production in this country. In this backdrop, this study was carried out to i) identify containers at household premises contributing to dengue vector productivity; ii) measure the extent of pupae productivity of household containers; and, iii) determine the effects of household ecological factors upon productivity of pupae in the city of Dhaka, Bangladesh. During the monsoon months of 2013, a total of 1,033 containers (674 wet and 363 dry) in 727 household premises in 12 wards of the city of Dhaka were inspected to measure container productivity and collect household ecological, and human behavioural data. The results reveal that the majority of immature mosquitoes (73.52% larvae and 84.91% pupae) developed in containers located outdoor that are used mostly for household chores. Plastic containers (57.55% of all immature mosquito-positive containers) used for household chores produce most of the immature mosquitos. The results of the zero-inflated negative binomial (ZINB) model reveal that pupae production significantly varies by container type (p-value = 0.0136) for the count regression group. However, when considering container size along with container type, container size is found significant for pupae production (p-value = 0.0041), showing that container size is confounded with the container type and the pupae production. Containers greater than 50 litres (L) are likely to produce 4.9 times more pupae than containers with <1L. Two household ecological factors are found to be significant (shade: p-value = 0.005 in the count regression group and type of water: p-value = 0.001 in the excess zero group) for pupae production. We found that containers with partial shade produce 4.6 times more pupae than without any shade, whereas in the excess zero group the expected number of observed zero pupae count is 86.5% lower in containers filled with rain water than those with tap water, tube-well water, ring well water and water from other sources. The most commonly used plastic-made containers (i.e., refrigerator trays, drums, buckets) and flower tubs/trays are the most abundant immature mosquito-positive containers. These findings would help the concerned authorities to formulate programs for changing human behaviour targeting the most productive containers for Aedes habitat management and vector control in the city of Dhaka, Bangladesh., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: 'Filth flies' feed and develop in excrement and decaying matter and can transmit enteric pathogens to humans and animals, leading to colonization and infection. Considering these characteristics, 'filth flies' are potential vectors for the spread of antimicrobial resistance (AMR). This review defines the role of flies in the spread of AMR and identifies knowledge gaps., Methods: The literature search (original articles, reviews indexed for PubMed) was restricted to the English language. References of identified studies were screened for additional sources., Results: 'Filth flies' are colonized with antimicrobial-resistant bacteria of clinical relevance. This includes extended spectrum beta-lactamase-, carbapenemase-producing and colistin-resistant (mcr-1 positive) bacteria. Resistant bacteria in flies often share the same genotypes with bacteria from humans and animals when their habitat overlap. The risk of transmission is most likely highest for enteric bacteria as they are shed in high concentration in excrements and are easily picked up by flies. 'Filth flies' can 'bio-enhance' the transmission of AMR as bacteria multiply in the digestive tract, mouthparts and regurgitation spots., Conclusion: To better understand the medical importance of AMR in flies, quantitative risk assessment models should be refined and fed with additional data (e.g. vectorial capacity, colonization dose). This requires targeted ecological, epidemiological and in vivo experimental studies., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Lyme disease is emerging in Canada due to geographic range expansion of the tick vector Ixodes scapularis Say. Recent areas of emergence include parts of the southeastern Canadian Prairie region. We developed a map of potential risk areas for future I. scapularis establishment in the Canadian Prairie Provinces. Six I. scapularis risk algorithms were developed using different formulations of three indices for environmental suitability: temperature using annual cumulative degree-days > 0 °C (DD > 0 °C; obtained from Moderate Resolution Imaging Spectroradiometer satellite data as an index of conditions that allow I. scapularis to complete its life cycle), habitat as a combined geolayer of forest cover and agricultural land use, and rainfall. The relative performance of these risk algorithms was assessed using receiver-operating characteristic (ROC) area under the curve (AUC) analysis with data on presence-absence of I. scapularis obtained from recent field surveillance in the Prairie Provinces accumulated from a number of sources. The ROC AUC values for the risk algorithms were significantly different (P < 0.01). The algorithm with six categories of DD > 0 °C, habitat as a simple dichotomous variable of presence or absence of forest, and normalized rainfall had the highest AUC of 0.74, representing "fair to good" performance of the risk algorithm. This algorithm had good (>80%) sensitivity in predicting positive I. scapularis surveillance sites, but low (50%) specificity as expected in this region where not all environmentally suitable habitats are expected to be occupied. Further prospective studies are needed to validate and perhaps improve the risk algorithm., (© Crown copyright 2017.)

A new Leu-enkephalin peptidomimetic designed to explore the hydrogen bond acceptor ability of the third peptide bond has been prepared and studied. This new analog is produced by replacing the third amide of Leu-enkephalin with a fluoroalkene. An efficient and innovative synthesis of the corresponding dipeptide surrogate Fmoc-Gly-ψ[(Z)CF═CH]-Phe-OH is described. The key step involves the alkylation of a tin dienolate from the less hindered face of its chiral sulfonamide auxiliary derived from camphor. Once its synthesis was complete, its incorporation into the peptidomimetic sequence was achieved on a solid support with chlorotrityl resin following the Fmoc strategy. The peptidomimetic was characterized using competition binding with [ 125 I]-deltorphin I on membrane extracts of HEK293 cells expressing the mouse delta opioid receptor (DOPr) and based on its abilities to inhibit the electrically induced contractions of the mouse vas deferens and to activate the ERK1/2 signaling pathway in DRGF11/DOPr-GFP cells. Together with our previous observations, our findings strongly suggest that the third amide bond of Leu-enkephalin primarily acts as a hydrogen bond acceptor in DOPr. Consequently, this amide bond can be successfully replaced by an ester, a thioamide, or a fluoroalkene without greatly impacting the binding or biological activity of the corresponding analogs. The lipophilicity (LogD 7.4 ) of the active analog was also measured. It appears that fluoroalkenes are almost as efficient at increasing the lipophilicity as normal alkenes.

There is an increasing risk of Lyme disease in Canada due to range expansion of the tick vector, Ixodes scapularis. The objectives of this article are to i) raise public awareness with the help of veterinarians on the emerging and expanding risk of Lyme disease across Canada, ii) review the key clinical features of Lyme disease in dogs, and iii) provide recommendations for veterinarians on the management of Lyme disease in dogs.