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For macromolecular structures determined by cryogenic electron microscopy, no technique currently exists for mapping elements to defined locations, leading to errors in the assignment of metals and other ions, cofactors, substrates, inhibitors and lipids that play essential roles in activity and regulation. Elemental mapping in the electron microscope is well established for dose-tolerant samples but is challenging for biological samples, especially in a cryo-preserved state. Here we combine electron energy-loss spectroscopy with single-particle image processing to allow elemental mapping in cryo-preserved macromolecular complexes. Proof-of-principle data show that our method, reconstructed electron energy-loss (REEL) analysis, allows a three-dimensional reconstruction of electron energy-loss spectroscopy data, such that a high total electron dose is accumulated across many copies of a complex. Working with two test samples, we demonstrate that we can reliably localize abundant elements. We discuss the current limitations of the method and potential future developments. An approach combining electron energy-loss spectroscopy with image processing tools from single-particle cryo-electron microscopy enables elemental mapping in macromolecular complexes, paving the way for the accurate assignment of metals, ions, ligands and lipids.

In order to fully understand any complex biochemical system from a mechanistic point of view, it is necessary to have access to the three-dimensional structures of the molecular components involved. Septins and their oligomers, filaments and higher-order complexes are no exception. Indeed, the spontaneous recruitment of different septin monomers to specific positions along a filament represents a fascinating example of subtle molecular recognition. Over the last few years, the amount of structural information available about these important cytoskeletal proteins has increased dramatically. This has allowed for a more detailed description of their individual domains and the different interfaces formed between them, which are the basis for stabilizing higher-order structures such as hexamers, octamers and fully formed filaments. The flexibility of these structures and the plasticity of the individual interfaces have also begun to be understood. Furthermore, recently, light has been shed on how filaments may bundle into higher-order structures by the formation of antiparallel coiled coils involving the C-terminal domains. Nevertheless, even with these advances, there is still some way to go before we fully understand how the structure and dynamics of septin assemblies are related to their physiological roles, including their interactions with biological membranes and other cytoskeletal components. In this review, we aim to bring together the various strands of structural evidence currently available into a more coherent picture. Although it would be an exaggeration to say that this is complete, recent progress seems to suggest that headway is being made in that direction.

Cell survival under nutrient-deprived conditions relies on cells’ ability to adapt their organelles and rewire their metabolic pathways. In yeast, glucose depletion induces a stress response mediated by mitochondrial fragmentation and sequestration of cytosolic ribosomes on mitochondria. This cellular adaptation promotes survival under harsh environmental conditions; however, the underlying mechanism of this response remains unknown. Here, we demonstrate that upon glucose depletion protein synthesis is halted. Cryo-electron microscopy structure of the ribosomes show that they are devoid of both tRNA and mRNA, and a subset of the particles depicted a conformational change in rRNA H69 that could prevent tRNA binding. Our in situ structural analyses reveal that the hibernating ribosomes tether to fragmented mitochondria and establish eukaryotic-specific, higher-order storage structures by assembling into oligomeric arrays on the mitochondrial surface. Notably, we show that hibernating ribosomes exclusively bind to the outer mitochondrial membrane via the small ribosomal subunit during cellular stress. We identify the ribosomal protein Cpc2/RACK1 as the molecule mediating ribosomal tethering to mitochondria. This study unveils the molecular mechanism connecting mitochondrial stress with the shutdown of protein synthesis and broadens our understanding of cellular responses to nutrient scarcity and cell quiescence. Cells adapt to low glucose by halting protein synthesis and altering organelle shape. Here the authors showed that hibernating ribosomes tether to mitochondria and form arrays on the membrane, acting as a pro-survival mechanism in dormant yeast cells.

For structures determined by single particle cryo-EM, no technique currently exists for mapping elements to defined locations, leading to errors in the assignment of metals and other ions, cofactors, substrates, inhibitors, and lipids that play essential roles in activity and regulation. Elemental mapping in the electron microscope is well established for dose-tolerant samples but is challenging for biological samples, especially in a cryo-preserved state. Here, we combine electron energy-loss spectroscopy (EELS) with single-particle image processing to allow elemental mapping in cryo-preserved macromolecular complexes. Proof-of-principle data show that our method, REEL-EM, allows 3D reconstruction of EELS data, such that a high total electron dose is accumulated across many copies of a complex. Working with two test samples, we demonstrate that we can reliably localise abundant elements. We discuss the current limitations of the method and potential future developments.Competing Interest StatementD.L. P.K., M.L., and H.M. are employed by CEOS GmbH, the manufacturer of the CEFID energy filter used in this work. The authors declare no further competing interests.

Cell survival under nutrient-deprived conditions relies on cells’ ability to adapt their organelles and to rewire their metabolic pathways. In the fission yeast Schizosaccharomyces pombe, nutrient depletion is an unfavorable condition for protein synthesis and triggers a response characterized by mitochondrial fragmentation and the sequestration of cytosolic ribosomes on mitochondria. The molecular mechanism underlying ribosomal sequestration remains elusive. In this study, we performed time-lapse in situ cryo-electron tomography and cryo-electron microscopy complemented by biochemical experiments to elucidate the molecular details of this adaptive response. Our analysis indicate that upon glucose depletion protein synthesis is halted, causing ribosomes to enter an inactive state characterized by a conformational change that obstructs the peptidyl transferase center. Our in situ experiments reveal the presence of oligomeric arrays of hibernating ribosomes tethered to the mitochondrial surface. Surprisingly, ribosomes bind to the outer mitochondrial membrane via the small ribosomal subunit, an interaction facilitated by the ribosomal protein RACK1-orthologue Cpc2. Our experiments show that ribosome tethering is important for cell survival under glucose depletion conditions. This study broadens our understanding of the cellular adaptations triggered by nutrient scarcity and the underlying molecular mechanisms that regulate cell quiescence.

The assembly of a septin filament requires that homologous monomers must distinguish between one another in establishing appropriate interfaces with their neighbours. To understand this phenomenon at the molecular level, we present the first four crystal structures of heterodimeric septin complexes. We describe in detail the two distinct types of G-interface present within the octameric particles which must polymerize to form filaments. These are formed between SEPT2 and SEPT6 and between SEPT7 and SEPT3, and their description permits an understanding of the structural basis for the selectivity necessary for correct filament assembly. By replacing SEPT6 by SEPT8 or SEPT11, it is possible to rationalize Kinoshita’s postulate which predicts the exchangeability of septins from within a subgroup. Switches I and II, which in classical small GTPases provide a mechanism for nucleotide-dependent conformational change, have been repurposed in septins to play a fundamental role in molecular recognition. Specifically, it is switch I which holds the key to discriminating between the two different G-interfaces. Moreover, residues which are characteristic for a given subgroup play subtle, but pivotal, roles in guaranteeing that the correct interfaces are formed. High resolution structures of septin heterodimeric complexes reveal new interactions Switches of small GTPases are repurposed in septins to play key roles in interface contacts The GTP present in catalytically inactive septins participates in molecular recognition Conservation of interface residues allows for subunit exchangeability from within septin subgroups Specific residues for each septin subgroup provide selectivity for proper filament assembly

The assembly of a septin filament requires that homologous monomers must distinguish between one another in establishing appropriate interfaces with their neighbours. To understand this phenomenon at the molecular level, we present the first four crystal structures of heterodimeric septin complexes. We describe in detail the two distinct types of G-interface present within the octameric particles which must polymerize to form filaments. These are formed between SEPT2 and SEPT6 and between SEPT7 and SEPT3, and their description permits an understanding of the structural basis for the selectivity necessary for correct filament assembly. By replacing SEPT6 by SEPT8 or SEPT11, it is possible to rationalize Kinoshita's postulate which predicts the exchangeability of septins from within a subgroup. Switches I and II, which in classical small GTPases provide a mechanism for nucleotide-dependent conformational change, have been repurposed in septins to play a fundamental role in molecular recognition. Specifically, it is switch I which holds the key to discriminating between the two different G-interfaces. Moreover, residues which are characteristic for a given subgroup play subtle, but pivotal, roles in guaranteeing that the correct interfaces are formed. Competing Interest Statement The authors have declared no competing interest.

ObjectivesThe aim of this study was to investigate the healing activity of andiroba (Carapa guianensis Aubl.) against oral mucositis (OM) induced by 5-fluorouracil in golden Syrian hamsters.Materials and methodsA total of 122 animals were randomized and divided into six groups: andiroba oil 100%, andiroba oil 10%, andiroba oil 10% refined, no treatment group, all n = 28; and negative control (NC) and cyclophosphamide (CPA) groups, both n = 5. OM was induced by intraperitoneal administration of 60 mg/kg 5-FU on days 0, 5 and 10 followed by mechanical trauma on the oral mucosa on days 1 and 2. From day 1 to day 15, the animals of the andiroba group were treated three times a day. On days 4, 8, 12 and 15, the mucosa was photographed and removed for clinical and histopathological analysis. The bone marrow of the femur was removed and the micronucleus test was performed to evaluate the cytotoxicity and genotoxicity. The data were subjected to analysis of variance, followed by the Tukey and Bonferroni test.ResultsTreatment with 100% andiroba oil reduced the degree of OM compared to that reported in the other groups (p < 0.05). Andiroba oil at both concentrations was not cytotoxic, but treatment with 100% andiroba oil showed a genotoxic potential (p < 0.001).ConclusionsFrequent administration of andiroba oil accelerated the healing process in an experimental model of 5-fluorouracil-induced OM. However, the genotoxicity of andiroba in other cell systems and under other conditions are being tested.Clinical relevanceThe use of andiroba in topical form may be associated with reduced intensity of OM. Seek therapeutic alternatives to minimize the pain and suffering that these side effects cause cancer patients is an important scientific step.

Cryptococcus spp. are human pathogens that cause 181,000 deaths per year. In this work, we systematically investigated the virulence attributes of Cryptococcus spp. clinical isolates and correlated them with patient data to better understand cryptococcosis. We collected 66 C. neoformans and 19 C. gattii clinical isolates and analyzed multiple virulence phenotypes and host–pathogen interaction outcomes. C. neoformans isolates tended to melanize faster and more intensely and produce thinner capsules in comparison with C. gattii. We also observed correlations that match previous studies, such as that between secreted laccase and disease outcome in patients. We measured Cryptococcus colony melanization kinetics, which followed a sigmoidal curve for most isolates, and showed that faster melanization correlated positively with LC3-associated phagocytosis evasion, virulence in Galleria mellonella and worse prognosis in humans. These results suggest that the speed of melanization, more than the total amount of melanin Cryptococcus spp. produces, is crucial for virulence.

Purpose To investigate the slope of age-related performance decrease of male master athletes competing the 100 m, 400 m, and 10,000 m running events. Methods Sample was composed by official data from World Masters Rankings for years 2013–2016. Age and performance data were collected from 2937 athletes between 30 and 105 years. Performance was plotted against age and calculated a trendline for polynomial regression for each event using three different data dispositions: Top-20—best 20 athletes of each age group, all years of analysis; Top-3—best three athletes of each age group, all years of analysis; and annual Top-3—best three athletes of each age group, each year analyzed separately. The age-related point of substantial performance decline was determined by two mathematical methods, D max, and log–log. Results The annual-Top-3 (age group Top-3 athletes in each year) disposition indicated an early performance decline in 10,000 m in comparison with the 100 and 400 m for both methods ( p  < 0.05). Top-3 (Top-3 athletes of each age group) analysis also indicated an earlier performance decline in 10,000 m ( D max : 61.2 years/log–log: 67.6 years), followed by 400 m (72.9 years/77.5 years) and 100 m (76.7 years/78.2 years). Conclusion In conclusion, the critical age after which the aging-related decline in masters athletes’ performance is accelerated, and occurs earlier in endurance runners than sprinters.