Oxidative bursts of single mitochondria mediate retrograde signaling toward the ER.

The emerging role of mitochondria as signaling organelles raises the question of whether individual mitochondria can initiate heterotypic communication with neighboring organelles. Using fluorescent probes targeted to the endoplasmic-reticulum-mitochondrial interface, we demonstrate that single mitochondria generate oxidative bursts, rapid redox oscillations, confined to the nanoscale environment of the interorganellar contact sites. Using probes fused to inositol 1,4,5-trisphosphate receptors (IP3Rs), we show that Ca2+ channels directly sense oxidative bursts and respond with Ca2+ transients adjacent to active mitochondria. Application of specific mitochondrial stressors or apoptotic stimuli dramatically increases the frequency and amplitude of the oxidative bursts by enhancing transient permeability transition pore openings. Conversely, blocking interface Ca2+ transport via elimination of IP3Rs or mitochondrial calcium uniporter channels suppresses ER-mitochondrial Ca2+ feedback and cell death. Thus, single mitochondria initiate local retrograde signaling by miniature oxidative bursts and, upon metabolic or apoptotic stress, may also amplify signals to the rest of the cell.

Reduced ER-mitochondria connectivity promotes neuroblastoma multidrug resistance.

Most cancer deaths result from progression of therapy resistant disease, yet our understanding of this phenotype is limited. Cancer therapies generate stress signals that act upon mitochondria to initiate apoptosis. Mitochondria isolated from neuroblastoma cells were exposed to tBid or Bim, death effectors activated by therapeutic stress. Multidrug-resistant tumor cells obtained from children at relapse had markedly attenuated Bak and Bax oligomerization and cytochrome c release (surrogates for apoptotic commitment) in comparison with patient-matched tumor cells obtained at diagnosis. Electron microscopy identified reduced ER-mitochondria-associated membranes (MAMs; ER-mitochondria contacts, ERMCs) in therapy-resistant cells, and genetically or biochemically reducing MAMs in therapy-sensitive tumors phenocopied resistance. MAMs serve as platforms to transfer Ca2+ and bioactive lipids to mitochondria. Reduced Ca2+ transfer was found in some but not all resistant cells, and inhibiting transfer did not attenuate apoptotic signaling. In contrast, reduced ceramide synthesis and transfer was common to resistant cells and its inhibition induced stress resistance. We identify ER-mitochondria-associated membranes as physiologic regulators of apoptosis via ceramide transfer and uncover a previously unrecognized mechanism for cancer multidrug resistance.

Bacterial catabolism of acetovanillone, a lignin-derived compound.

Bacterial catabolic pathways have considerable potential as industrial biocatalysts for the valorization of lignin, a major component of plant-derived biomass. Here, we describe a pathway responsible for the catabolism of acetovanillone, a major component of several industrial lignin streams. Rhodococcus rhodochrous GD02 was previously isolated for growth on acetovanillone. A high-quality genome sequence of GD02 was generated. Transcriptomic analyses revealed a cluster of eight genes up-regulated during growth on acetovanillone and 4-hydroxyacetophenone, as well as a two-gene cluster up-regulated during growth on acetophenone. Bioinformatic analyses predicted that the hydroxyphenylethanone (Hpe) pathway proceeds via phosphorylation and carboxylation, before ß-elimination yields vanillate from acetovanillone or 4-hydroxybenzoate from 4-hydroxyacetophenone. Consistent with this prediction, the kinase, HpeHI, phosphorylated acetovanillone and 4-hydroxyacetophenone. Furthermore, HpeCBA, a biotin-dependent enzyme, catalyzed the ATP-dependent carboxylation of 4-phospho-acetovanillone but not acetovanillone. The carboxylase's specificity for 4-phospho-acetophenone (kcat/KM = 34 ± 2 mM-1 s-1) was approximately an order of magnitude higher than for 4-phospho-acetovanillone. HpeD catalyzed the efficient dephosphorylation of the carboxylated products. GD02 grew on a preparation of pine lignin produced by oxidative catalytic fractionation, depleting all of the acetovanillone, vanillin, and vanillate. Genomic and metagenomic searches indicated that the Hpe pathway occurs in a relatively small number of bacteria. This study facilitates the design of bacterial strains for biocatalytic applications by identifying a pathway for the degradation of acetovanillone.

Ecological generalism and physiology mediate fish biogeographic ranges under ocean warming.

Climate-driven species redistributions are facilitated by niche modifications that increase a species's chances of establishment in novel communities. It is well understood how range-extending species adjust individual niche traits when entering novel environments, yet whether modification of ecological niche traits collectively alters the pace of range extensions or contractions remains unknown. We quantified habitat niche, abundance, physiological performance and cellular defence/damage of range-extending coral reef fishes and coexisting local temperate fishes along a 2000 km latitudinal gradient. We also assessed their dietary and behavioural niches, and establishment potential, to understand whether ecological generalism facilitates successful range extension of coral reef fishes. The coral reef fish that increased all ecological niches, showed stronger establishment, increased physiological performance and cellular damage, but decreased cellular defence at their cold-range edge, whereas tropical species that showed unmodified ecological niches showed lower establishment. One temperate species showed decreased abundance, habitat niche width and body condition, but increased cellular defence, cellular damage and energy reserves at their warm-trailing range, while other temperate species showed contrasting responses. Therefore, ecological generalists might be more successful than ecological specialists during the initial stages of climate change, with increasing future warming strengthening this pattern by physiologically benefitting tropical generalists but disadvantaging temperate specialists.

Body size mediates trophic interaction strength of novel fish assemblages under climate change.

Ecological similarity plays an important role in biotic interactions. Increased body size similarity of competing species, for example, increases the strength of their biotic interactions. Body sizes of many exothermic species are forecast to be altered under global warming, mediating shifts in existing trophic interactions among species, in particular for species with different thermal niches. Temperate rocky reefs along the southeast coast of Australia are located in a climate warming hotspot and now house a mixture of temperate native fish species and poleward range-extending tropical fishes (vagrants), creating novel species assemblages. Here, we studied the relationship between body size similarity and trophic overlap between individual temperate native and tropical vagrant fishes. Dietary niche overlap between vagrant and native fish species increased as their body sizes converged, based on both stomach content composition (short-term diet), stable isotope analyses (integrated long-term diet) and similarity in consumed prey sizes. We conclude that the warming-induced faster growth rates of tropical range-extending fish species at their cool water ranges will continue to converge their body size towards and strengthen their degree of trophic interactions and dietary overlap with co-occurring native temperate species under increasing ocean warming. The strengthening of these novel competitive interactions is likely to drive changes to temperate food web structures and reshuffle existing species community structures.

Redox Nanodomains Are Induced by and Control Calcium Signaling at the ER-Mitochondrial Interface.

The ER-mitochondrial interface is central to calcium signaling, organellar dynamics, and lipid biosynthesis. The ER and mitochondrial membranes also host sources and targets of reactive oxygen species (ROS), but their local dynamics and relevance remained elusive since measurement and perturbation of ROS at the organellar interface has proven difficult. Employing drug-inducible synthetic ER-mitochondrial linkers, we overcame this problem and demonstrate that the ER-mitochondrial interface hosts a nanodomain of H2O2, which is induced by cytoplasmic [Ca(2+)] spikes and exerts a positive feedback on calcium oscillations. H2O2 nanodomains originate from the mitochondrial cristae, which are compressed upon calcium signal propagation to the mitochondria, likely due to Ca(2+)-induced K(+) and concomitant water influx to the matrix. Thus, ER-mitochondrial H2O2 nanodomains represent a component of inter-organelle communication, regulating calcium signaling and mitochondrial activities.

Staining protocols affect use of otolith to estimate the demography of the damselfish sergeant major (Abudefduf vaigiensis).

This study assessed the otolith (sagittae, lapilli, and asterisci) increment deposition rate in the range-shifting damselfish, A. vaigiensis, using different concentrations of Alizain Red S and evaluated the impact of staining on increment width. Daily increment deposition was verified in all otolith types and presented clearer fluorescent markings in the lapilli and sagittae than the asterisci, with high stain concentration showing the best clarity. Higher stain concentrations were found to decrease increment width, suggesting care is needed when using stained otoliths as a proxy for growth for this species.

Metabolic adaptation to the chronic loss of Ca2+ signaling induced by KO of IP3 receptors or the mitochondrial Ca2+ uniporter.

Calcium signaling is essential for regulating many biological processes. Endoplasmic reticulum inositol trisphosphate receptors (IP3Rs) and the mitochondrial Ca2+ uniporter (MCU) are key proteins that regulate intracellular Ca2+ concentration. Mitochondrial Ca2+ accumulation activates Ca2+-sensitive dehydrogenases of the tricarboxylic acid (TCA) cycle that maintain the biosynthetic and bioenergetic needs of both normal and cancer cells. However, the interplay between calcium signaling and metabolism is not well understood. In this study, we used human cancer cell lines (HEK293 and HeLa) with stable KOs of all three IP3R isoforms (triple KO [TKO]) or MCU to examine metabolic and bioenergetic responses to the chronic loss of cytosolic and/or mitochondrial Ca2+ signaling. Our results show that TKO cells (exhibiting total loss of Ca2+ signaling) are viable, displaying a lower proliferation and oxygen consumption rate, with no significant changes in ATP levels, even when made to rely solely on the TCA cycle for energy production. MCU KO cells also maintained normal ATP levels but showed increased proliferation, oxygen consumption, and metabolism of both glucose and glutamine. However, MCU KO cells were unable to maintain ATP levels and died when relying solely on the TCA cycle for energy. We conclude that constitutive Ca2+ signaling is dispensable for the bioenergetic needs of both IP3R TKO and MCU KO human cancer cells, likely because of adequate basal glycolytic and TCA cycle flux. However, in MCU KO cells, the higher energy expenditure associated with increased proliferation and oxygen consumption makes these cells more prone to bioenergetic failure under conditions of metabolic stress.

Publisher Correction: Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.

Short-term resilience to climate-induced temperature increases for equatorial sea turtle populations.

Projection models are being increasingly used to manage threatened taxa by estimating their responses to climate change. Sea turtles are particularly susceptible to climate change as they have temperature-dependent sex determination and increased sand temperatures on nesting beaches could result in the 'feminisation' of hatchling sex ratios for some populations. This study modelled likely long-term trends in sand temperatures and hatchling sex ratios at an equatorial nesting site for endangered green turtles (Chelonia mydas) and critically endangered hawksbill turtles (Eretmochelys imbricata). A total of 1078 days of sand temperature data were collected from 28 logger deployments at nest depth between 2018 and 2022 in Papua New Guinea (PNG). Long-term trends in sand temperature were generated from a model using air temperature as an environmental proxy. The influence of rainfall and seasonal variation on sand temperature was also investigated. Between 1960 and 2019, we estimated that sand temperature increased by ~0.6°C and the average hatchling sex ratio was relatively balanced (46.2% female, SD = 10.7). No trends were observed in historical rainfall anomalies and projections indicated no further changes to rainfall until 2100. Therefore, the sex ratio models were unlikely to be influenced by changing rainfall patterns. A relatively balanced sex ratio such as this is starkly different to the extremely female-skewed hatchling sex ratio (>99% female) reported for another Coral Sea nesting site, Raine Island (~850 km West). This PNG nesting site is likely rare in the global context, as it is less threatened by climate-induced feminisation. Although there is no current need for 'cooling' interventions, the mean projected sex ratios for 2020-2100 were estimated 76%-87% female, so future interventions may be required to increase male production. Our use of long-term sand temperature and rainfall trends has advanced our understanding of climate change impacts on sea turtles.

Shape Expressions (ShEx) schemas for the FHIR R5 specification.

This work continues along a visionary path of using Semantic Web standards such as RDF and ShEx to make healthcare data easier to integrate for research and leading-edge patient care. The work extends the ability to use ShEx schemas to validate FHIR RDF data, thereby enhancing the semantic web ecosystem for working with FHIR and non-FHIR data using the same ShEx validation framework. It updates FHIR's ShEx schemas to fix outstanding issues and reflect changes in the definition of FHIR RDF. In addition, it experiments with expressing FHIRPath constraints (which are not captured in the XML or JSON schemas) in ShEx schemas. These extended ShEx schemas were incorporated into the FHIR R5 specification and used to successfully validate FHIR R5 examples that are included with the FHIR specification, revealing several errors in the examples.

Characterization of alkylguaiacol-degrading cytochromes P450 for the biocatalytic valorization of lignin.

Cytochrome P450 enzymes have tremendous potential as industrial biocatalysts, including in biological lignin valorization. Here, we describe P450s that catalyze the O-demethylation of lignin-derived guaiacols with different ring substitution patterns. Bacterial strains Rhodococcus rhodochrous EP4 and Rhodococcus jostii RHA1 both utilized alkylguaiacols as sole growth substrates. Transcriptomics of EP4 grown on 4-propylguaiacol (4PG) revealed the up-regulation of agcA, encoding a CYP255A1 family P450, and the aph genes, previously shown to encode a meta-cleavage pathway responsible for 4-alkylphenol catabolism. The function of the homologous pathway in RHA1 was confirmed: Deletion mutants of agcA and aphC, encoding the meta-cleavage alkylcatechol dioxygenase, grew on guaiacol but not 4PG. By contrast, deletion mutants of gcoA and pcaL, encoding a CYP255A2 family P450 and an ortho-cleavage pathway enzyme, respectively, grew on 4-propylguaiacol but not guaiacol. CYP255A1 from EP4 catalyzed the O-demethylation of 4-alkylguaiacols to 4-alkylcatechols with the following apparent specificities (kcat/KM): propyl > ethyl > methyl > guaiacol. This order largely reflected AgcA's binding affinities for the different guaiacols and was the inverse of GcoAEP4's specificities. The biocatalytic potential of AgcA was demonstrated by the ability of EP4 to grow on lignin-derived products obtained from the reductive catalytic fractionation of corn stover, depleting alkylguaiacols and alkylphenols. By identifying related P450s with complementary specificities for lignin-relevant guaiacols, this study facilitates the design of these enzymes for biocatalytic applications. We further demonstrated that the metabolic fate of the guaiacol depends on its substitution pattern, a finding that has significant implications for engineering biocatalysts to valorize lignin.

Deep Metric Learning for Scalable Gait-Based Person Re-Identification Using Force Platform Data.

Walking gait data acquired with force platforms may be used for person re-identification (re-ID) in various authentication, surveillance, and forensics applications. Current force platform-based re-ID systems classify a fixed set of identities (IDs), which presents a problem when IDs are added or removed from the database. We formulated force platform-based re-ID as a deep metric learning (DML) task, whereby a deep neural network learns a feature representation that can be compared between inputs using a distance metric. The force platform dataset used in this study is one of the largest and the most comprehensive of its kind, containing 193 IDs with significant variations in clothing, footwear, walking speed, and time between trials. Several DML model architectures were evaluated in a challenging setting where none of the IDs were seen during training (i.e., zero-shot re-ID) and there was only one prior sample per ID to compare with each query sample. The best architecture was 85% accurate in this setting, though an analysis of changes in walking speed and footwear between measurement instances revealed that accuracy was 28% higher on same-speed, same-footwear comparisons, compared to cross-speed, cross-footwear comparisons. These results demonstrate the potential of DML algorithms for zero-shot re-ID using force platform data, and highlight challenging cases.

Coral-reef fishes can become more risk-averse at their poleward range limits.

As climate warms, tropical species are expanding their distribution to temperate ecosystems where they are confronted with novel predators and habitats. Predation strongly regulates ecological communities, and range-extending species that adopt an effective antipredator strategy have a higher likelihood to persist in non-native environments. Here, we test this hypothesis by comparing various proxies of antipredator and other fitness-related behaviours between range-extending tropical fishes and native-temperate fishes at multiple sites across a 730 km latitudinal range. Although some behavioural proxies of risk aversion remained unaltered for individual tropical fish species, in general they became more risk-averse (increased sheltering and/or flight initiation distance), and their activity level decreased poleward. Nevertheless, they did not experience a decline in body condition or feeding rate in their temperate ranges. Temperate fishes did not show a consistently altered pattern in their behaviours across range locations, even though one species increased its flight initiation distance at the warm-temperate location and another one had lowest activity levels at the coldest range location. The maintenance of feeding and bite rate combined with a decreased activity level and increased sheltering may be behavioural strategies adopted by range-extending tropical fishes, to preserve energy and maintain fitness in their novel temperate ecosystems.

Genetic and transcriptomic analyses support a switch to lytic phase in Epstein Barr virus infection as an important driver in developing Systemic Lupus Erythematosus.

To investigate the molecular mechanisms through which Epstein-Barr virus (EBV) may contribute to Systemic Lupus Erythematosus (SLE) pathogenesis, we interrogated SLE genetic risk loci for signatures of EBV infection. We first compared the gene expression profile of SLE risk genes across 459 different cell/tissue types. EBV-infected B cells (LCLs) had the strongest representation of highly expressed SLE risk genes. By determining an SLE risk allele effect on gene expression (expression quantitative trait loci, eQTL) in LCLs and 16 other immune cell types, we identified 79 SLE risk locus:gene pairs putatively interacting with EBV infection. A total of 10 SLE risk genes from this list (CD40, LYST, JAZF1, IRF5, BLK, IKZF2, IL12RB2, FAM167A, PTPRC and SLC15A) were targeted by the EBV transcription factor, EBNA2, differentially expressed between LCLs and B cells, and the majority were also associated with EBV DNA copy number, and expression level of EBV encoded genes. Our final gene network model based on these genes is suggestive of a nexus involving SLE risk loci and EBV latency III and B cell proliferation signalling pathways. Collectively, our findings provide further evidence to support the interaction between SLE risk loci and EBV infection that is in part mediated by EBNA2. This interplay may increase the tendency towards EBV lytic switching dependent on the presence of SLE risk alleles. These results support further investigation into targeting EBV as a therapeutic strategy for SLE.

Ocean warming and acidification degrade shoaling performance and lateralization of novel tropical-temperate fish shoals.

Gregarious behaviours are common in animals and provide various benefits such as food acquisition and protection against predators. Many gregarious tropical species are shifting poleward under current ocean warming, creating novel species and social interactions with local temperate taxa. However, how the dynamics of these novel shoals might be altered by future ocean warming and acidification remains untested. Here we evaluate how novel species interactions, ocean acidification and warming affect shoaling dynamics, motor lateralization and boldness of range-extending tropical and co-shoaling temperate fishes under controlled laboratory conditions. Fishes were exposed to 1 of 12 treatments (combinations of three temperature levels, two pCO2  levels and two shoal type levels: mixed species or temperate only) for 38 days. Lateralization (a measure of asymmetric expression of cognitive function in group coordination and predator escape) of tropical and temperate species was right-side biased under present-day conditions, but side bias significantly diminished in tropical and temperate fishes under ocean acidification. Ocean acidification also decreased shoal cohesion irrespective of shoaling type, with mixed-species shoals showing significantly lower cohesion than temperate-only shoals irrespective of climate stressors. Tropical fish became bolder under ocean acidification (after 4 weeks), and temperate fish became bolder with increasing temperature, while ocean acidification dampened temperate fish boldness. Our findings highlight the direct effect of climate stressors on fish behaviour and the interplay with the indirect effects of novel species interactions. Because strong shoal cohesion and lateralization are key determinants of species fitness, their degradation under ocean warming and acidification could adversely affect species performance in novel assemblages in a future ocean, and might slow down tropical species range extensions.

Behavioural generalism could facilitate coexistence of tropical and temperate fishes under climate change.

Coral-reef fishes are shifting their distributions poleward in response to human-mediated ocean warming; yet, the consequences for recipient temperate fish communities remain poorly understood. Behavioural modification is often the first response of species to environmental change, but we know little about how this might shape the ongoing colonisation by tropical fishes of temperate-latitude ecosystems under climate change. In a global hotspot of ocean warming (southeast Australia), we quantified 14 behavioural traits of invading tropical and local co-occurring temperate fishes at 10 sites across a 730 km latitudinal gradient as a proxy of species behavioural niche space in different climate ranges (subtropical, warm-temperate and cold-temperate). We found that tropical fishes (four species) modified their behavioural niches as well as increased their overall behavioural niche breadth in their novel temperate ranges where temperate species predominate, but maintained a moderate to high niche segregation with native temperate species across latitudinal range position. Temperate species (three co-occurring species) also modified their niches, but in contrast to tropical species, experienced an increased niche breadth towards subtropical ranges. Alterations to feeding and shoaling behaviours contributed most to niche modifications in tropical and temperate species, while behaviours related to alertness and escape from potential threats contributed least. We here show that at warmer and colder range edges where community structures are being reshuffled due to climate change, behavioural generalism and niche modification are potential mechanisms adopted by tropical range extenders and native temperate fishes to adjust to novel species interactions under climate change.

Insights from an ancient gymnosperm lineage: ambient temperature and light and the timing of thermogenesis in cycad cones.

PREMISE: Although maintaining the appropriate mid-day timing of the diel thermogenic events of cones of the dioecious cycads Macrozamia lucida and M. macleayi is central to the survival of both plant and pollinator in this obligate pollination mutualism, the nature of the underlying mechanism remains obscure. We investigated whether it is under circadian control. Circadian mechanisms control the timing of many ecologically important processes in angiosperms, yet only a few gymnosperms have been studied in this regard. METHODS: We subjected cones to different ambient temperature and lighting regimens (constant temperature and darkness; stepwise cool/warm ambient temperatures in constant darkness; stepwise dark/light exposures at constant temperature) to determine whether the resulting timing of their thermogenic events was consistent with circadian control. RESULTS: Cones exposed to constant ambient temperature and darkness generated multiple temperature peaks endogenously, with an average interpeak-temperature period of 20.7 (±0.20) h that is temperature-compensated (Q10 = 1.02). Exposure to 24-h ambient temperature cycles (12 h cool/12 h warm, constant darkness) yielded an interpeak-temperature period of 24.0 (±0.05) h, accurately and precisely replicating the ambient temperature period. Exposure to 24-h photo-cycles (12 h light/12 h dark, constant ambient temperature) yielded a shorter, more variable interpeak-temperature period of 23 (±0.23) h. CONCLUSIONS: Our results indicate that cycad cone thermogenesis is under circadian clock control and differentially affected by ambient temperature and light cycles. Our data from cycads (an ancient gymnosperm lineage) adds to what little is known about circadian timing in gymnosperms, which have rarely been studied from the circadian perspective.

The ecological importance of the accuracy of environmental temperature measurements.

The implications of logger accuracy and precision are rarely considered prior to their application in many ecological studies. We assessed the accuracy and precision of three temperature data loggers widely used in ecological studies (Hobo®, iButton® and TinyTag®). Accuracy was highest in TinyTags (95% of readings were within 0.23°C of the true temperature) and lowest in HOBOs and iButtons (95% of were readings within 0.43°C and 0.49°C of the true temperature, respectively). The precision (standard deviation of the repeat measurements) was greatest in TinyTags (0.04°C), followed by iButtons (0.17°C) and then HOBOs (0.22°C). As a case study, we then considered how modelled estimates of sea turtle hatchling sex ratios (derived from temperature), could vary as a function of logger accuracy. For example, at 29°C when the mean sex ratio derived was 0.47 female, the sex ratio estimate from a single logger could vary between 0.40 and 0.50 for TinyTags and 0.29 and 0.56 for both HOBOs and iButtons. Our results suggest that these temperature loggers can provide reliable descriptions of sand temperature if they are not over-interpreted. Logger accuracy must be considered in future ecological studies in which temperature thresholds are important.