Abstract Carboxylases are among the most important enzymes in nature as they catalyze the fixation of inorganic carbon (CO 2 ), a central step in the global carbon cycle. In addition to their well-known function in autotrophic CO 2 fixation, many carboxylases play a role in the heterotrophic assimilation of organic compounds. In this study, we provide genomic evidence for an assimilatory carboxylation pathway involved in acetone degradation in photoheterotrophic bacteria from metagenomes collected along a latitudinal transect of the Arctic Ocean. This curious metabolism was linked to a single population of Gammaproteobacteria ( Porticoccus arcticus ). P. arcticus has a streamlined genome compared to Porticoccus relatives but has maintained a complete acetone carboxylation pathway while acquiring multiple proteorhodopsin genes by lateral gene transfer. Arctic Ocean metatranscriptomes revealed the acetone carboxylase and rhodopsins genes were among the most highly expressed P. arcticus genes in oligotrophic Arctic surface waters. P. arcticus sequences were consistently detected, and often abundant (up to 9%), in a multiyear Arctic Ocean 16S rRNA time-series, supporting its ecological significance in Arctic marine systems. Overall, this work reports a metabolic module (acetone carboxylation) in the ocean that may allow photoheterotrophic bacteria to enhance their biosynthetic capacity via CO 2 assimilation.

The Arctic Ocean microbiomes experience extreme seasonal fluctuations in light, nutrient availability, and organic carbon supply. In this environment, neutral lipid storage may provide a key survival strategy. Here, we investigated the diversity, distribution, and ecological role of neutral lipid metabolism in Arctic microbiomes using metagenome-resolved analyses and global ocean comparisons. Arctic photic-zone microbiomes were strongly enriched in triacylglycerol (TAG) biosynthesis genes relative to other oceans, primarily due to picoeukaryotic phytoplankton, including the ecologically dominant Micromonas and Bathycoccus. In contrast, prokaryotic communities exhibited diverse TAG-degrading taxa and fatty acid transport systems, supporting a previously unrecognized lipotrophic bacterial guild exploiting phytoplankton-derived lipids as carbon and energy sources. Genome-resolved analyses further revealed distinct bacterial lipid-storage strategies: TAG-producing taxa preferentially encoded fatty acid uptake and carbohydrate utilization pathways, whereas polyhydroxyalkanoate-producing taxa were associated with aromatic compound degradation, linking terrestrial organic matter to lipid storage. Our results expand the known diversity of marine microbes capable of neutral lipid metabolism and identify microbial lipid cycling as a previously overlooked component of the Arctic carbon cycle. We propose that neutral lipid storage and turnover support microbial survival through the polar night while enhancing carbon transfer within Arctic microbial food webs under ongoing climate change.

Anthropogenic environmental changes impact freshwater biodiversity globally. While biodiversity assessments in freshwater environments have traditionally focused on individual groups of organisms or trophic levels, considering patterns of 'multitrophic biodiversity' across the food web provides a more comprehensive view of anthropogenic impacts and changes in communities along environmental gradients. Here we evaluate spatial biodiversity patterns in multitrophic communities of lake plankton (bacterioplankton-phytoplankton-zooplankton) across a lake-rich country: Canada. Capitalising on the first standardized survey at the country scale, we used random forest and structural equation models to identify natural and anthropogenic drivers of multitrophic alpha and beta diversity in 559 lakes from 11 ecozones. Our analyses revealed that multitrophic alpha diversity (assemblage richness) was positively related to water quality but negatively related to water clarity, while beta diversity (assemblage uniqueness) was negatively related to both water quality and water clarity. Agricultural land use in lake watersheds strongly affected water quality and clarity, thus indirectly shaping multitrophic alpha and beta diversity. Bacterio-, phyto- and zooplankton diversity were not well correlated and responded to distinct drivers, confirming the utility of multitrophic indices to provide an integrative view of diversity patterns. Finally, our analyses showed that facets of alpha and beta multitrophic diversity have contrasting responses to the indirect effect of a key human disturbance, watershed land use, thereby illustrating the value of considering multiple diversity facets in biodiversity assessments.

There is a great divide between the microbes active in natural environments and the organisms that may be grown in a laboratory setting. In this work we set out to cultivate representatives of the marine myxobacterial clade, a highly diverse, largely uncultured group of Gram-negative bacteria believed to have extensive biosynthetic potential. Sediment samples were collected from the St. Lawrence Estuary and Gulf and the presence of active marine myxobacteria was established through qPCR analysis of 16S rRNA gene and transcript abundances. In the expectation that the marine myxobacteria would exhibit predatory behaviour like their terrestrial counterparts, the sediment samples were then streaked on agar plates that contained common marine bacteria as the sole carbon source. Unexpectedly, in place of myxobacteria we isolated Pseudomonas, Bacillus, and Stenotropomonas spp., among others, revealing a generalized ability for these strains to break down living organic matter and suggesting that "bait" bacteria may be an effective approach for the cultivation of novel marine saprophytes.

Lake ecosystems are increasingly impacted by eutrophication and climate change. Whole-lake experiments have provided ecosystem-scale insights into the effects of freshwater stressors, yet these are constrained to the duration of monitoring programmes. Here, we leveraged multidecadal monitoring records and century-scale paleogenetic reconstructions for experimentally fertilised and unmanipulated lakes in the IISD Experimental Lakes Area of northwestern Ontario, Canada, to evaluate the responses of algal communities to nutrient and air temperature variation. We first validated the paleogenetic analysis of sediment DNA by demonstrating the synchrony of algal community changes with monitoring records. Algal communities underwent significant compositional shifts across experimental nutrient loading regimes and climate periods, with baseline assemblages informed by paleogenetics. Nonlinear regression modelling of algal community change in monitoring and paleogenetic time series showed the expected response that nutrients were strong drivers in fertilised lakes. Paleogenetic records reflected the century-scale impacts of climate warming and its combined effects with eutrophication, previously underestimated by monitoring. The synergy between eutrophication and warming points to eutrophic priming of the food web to respond to rising temperatures. Overall, the paleogenetic integration of algal diversity across habitats and seasons enables the detection of slow-acting climate change on lake ecosystems increasingly altered by nutrient pollution.

Aquatic fungi play key roles in organic matter decomposition and nutrient cycling, but the effects of lake conditions and food web interactions on fungal diversity are still largely unknown. Our study is the first to assess mycoplankton along a broad lake trophic gradient based on total phosphorus (TP) (2-2500 mu g/L) using DNA metabarcoding data from 369 Canadian lakes. Zoosporic fungi, Chytridiomycota in particular, dominated mycoplankton assemblages. Mycoplankton diversity declined -15 % along the trophic gradient. Community composition varied the most between oligotrophic and hypereutrophic lakes, with pH, TP and water temperature as main drivers. Notably, mycoplankton communities showed stronger correlations with eukaryotic phytoplankton than with physicochemical variables, underlining the importance of phytoplankton hosts and substrates. Chytrid taxa associated with Chrysophyta in acidic lakes differed from those associated with Chlorophyta and Cryptophyta in lakes within agricultural lands. Overall, our study highlights the essential role of phytoplankton in shaping mycoplankton diversity and communities.

Human activities such as agriculture and urban development are linked to water quality degradation. Canada represents a large and heterogeneous landscape of freshwater lakes, where variations in climate, geography and geology interact with land cover alteration to influence water quality differently across regions. In this study, we investigated the influence of water quality and land use on bacterial communities across 12 ecozones. At the pan-Canadian scale, total phosphorus (TP) was the most significant water quality variable influencing community structure, and the most pronounced shift was observed at 110 μg/L of TP, corresponding to the transition from eutrophic to hypereutrophic conditions. At the regional scale, water quality significantly explained bacterial community structure in all ecozones. In terms of land use effect, at the pan-Canadian scale, agriculture and, to a lesser extent, urbanisation were significant land use variables influencing community structure. Regionally, in ecozones characterised by extensive agriculture, this land cover variable was consistently significant in explaining community structure. Likewise, in extensively urbanised ecozones, urbanisation was consistently significant in explaining community structure. Overall, these results demonstrate that bacterial richness and community structure are influenced by water quality and shaped by agriculture and urban development in different ways.

The Arctic Ocean (AO) is changing at an unprecedented rate, with ongoing sea ice loss, warming and freshening impacting the extent and duration of primary productivity over summer months. Surface microbial eukaryotes are vulnerable to such changes, but basic knowledge of the spatial variability of surface communities is limited. Here, we sampled microbial eukaryotes in surface waters of the Beaufort Sea from four contrasting environments: the Canada Basin (open ocean), the Mackenzie Trough (river-influenced), the Nuvuk region (coastal) and the under-ice system of the Canada Basin. Microbial community structure and composition varied significantly among the systems, with the most phylogenetically diverse communities being found in the more coastal systems. Further analysis of environmental factors showed potential vulnerability to change in the most specialised community, which was found in the samples taken in water immediately beneath the sea ice, and where the community was distinguished by rare species. In the context of ongoing sea ice loss, specialised ice-associated microbial assemblages may transition towards more generalist assemblages, with implications for the eventual loss of biodiversity and associated ecosystem function in the Arctic Ocean.

Climate change is profoundly impacting the Arctic, leading to a loss of multiyear sea ice and a warmer, fresher upper Arctic Ocean. The response of microbial communities to these climate-mediated changes is largely unknown. Here, we document the interannual variation in bacterial and archaeal communities across a 9-year time series of the Canada Basin that includes two historic sea ice minima (2007 and 2012). We report an overall loss of bacterial and archaeal community richness and significant shifts in community composition. The magnitude and period of most rapid change differed between the stratified water layers. The most pronounced changes in the upper water layers (surface mixed layer and upper Arctic water) occurred earlier in the time series, while changes in the lower layer (Pacific-origin water) occurred later. Shifts in taxonomic composition across time were subtle, but a decrease in Bacteroidota taxa and increase in Thaumarchaeota and Euryarchaeota taxa were the clearest signatures of change. This time series provides a rare glimpse into the potential influence of climate change on Arctic microbial communities; extension to the present day should contribute to deeper insights into the trajectory of Arctic marine ecosystems in response to warming and freshening.

Background: There is growing awareness of breast density in women attending breast cancer screening; however, it is unclear whether this awareness is associated with increased knowledge. This study aims to evaluate breast density knowledge among Australian women attending breast cancer screening. Method: This cross-sectional study was conducted on women undergoing breast cancer screening at The Queen Elizabeth Hospital Breast/Endocrine outpatient department. Participants were provided with a questionnaire to assess knowledge, awareness, and desire to know their own breast density. Result: Of the 350 women who participated, 61% were familiar with ‘breast density’ and 57% had ‘some knowledge’. Prior breast density notification (OR = 4.99, 95% CI = 2.76, 9.03; p = 0.004), awareness (OR = 4.05, 95% CI = 2.57, 6.39; p = 0.004), younger age (OR = 0.97, 95% CI = 0.96, 0.99; p = 0.02), and English as the language spoken at home (OR = 3.29, 95% CI = 1.23, 8.77; p = 0.02) were independent predictors of ‘some knowledge’ of breast density. A significant proportion of participants (82%) expressed desire to ascertain their individual breast density. Conclusions: While knowledge of breast density in this Australian cohort is generally quite low, we have identified factors associated with increased knowledge. Further research is required to determine optimal interventions to increase breast density knowledge.

Methyl mercury, a toxic compound, is produced by anaerobic microbes and magnifies in aquatic food webs, affecting the health of animals and humans. The exploration of mercury methylators based on genomes is still limited, especially in the context of river ecosystems. To address this knowledge gap, we developed a genome catalogue of potential mercury-methylating microorganisms. This was based on the presence of hgcAB from the sediments of a river affected by two run-of-river hydroelectric dams, logging activities and a wildfire. Through the use of genome-resolved metagenomics, we discovered a unique and diverse group of mercury methylators. These were dominated by members of the metabolically versatile Bacteroidota and were particularly rich in microbes that ferment butyrate. By comparing the diversity and abundance of mercury methylators between sites subjected to different disturbances, we found that ongoing disturbances, such as the input of organic matter related to logging activities, were particularly conducive to the establishment of a mercury-methylating niche. Finally, to gain a deeper understanding of the environmental factors that shape the diversity of mercury methylators, we compared the mercury-methylating genome catalogue with the broader microbial community. The results suggest that mercury methylators respond to environmental conditions in a manner similar to the overall microbial community. Therefore, it is crucial to interpret the diversity and abundance of mercury methylators within their specific ecological context.

Histological grading of tumours is a well-established biomarker used to guide treatment in female breast cancer. However, its significance in male breast cancer remains unclear. This systematic review investigates the prognostic significance of tumour grade in relation to breast cancer-specific survival (BCSS) in male breast cancer patients undergoing surgery. MEDLINE, PUBMED Central and EMBASE databases were searched to identify randomised trials and observational studies related to male breast neoplasms, tumour grading, recurrence, and survival. A total of fifteen observational type studies were included in the review. A significant association between tumour grade and BCSS was reported in a majority of studies. This association was most evident with regard to high-grade (grade III) compared to low grade (grade I) tumours, with a significant relationship in 4 out of 4 studies. For intermediate-grade II tumours an association was demonstrated in a minority of studies. This study confirms an association between high-grade male breast cancers and poorer disease-specific survival, however, the significance of intermediate-grade tumours remains unclear. Further research is required to investigate the biology of male breast cancer in relation to histological grade and optimally define intermediate-grade disease.

The United Nations Environment Assembly (UNEA-3) have recognised the importance of the environment in the development, spread and transmission of antimicrobial resistance (AMR) to humans and animals. Such recognition calls for wider surveillance of antimicrobial resistance genes (ARG) in wastewater and other environmental reservoirs. For ARG surveillance to be valuable to regulators, it must enable source tracking and risk assessment. Adequate surveillance also requires the processing of a large number of samples at a relatively low cost, and a low detection limit to allow quantification of the riskiest ARGs. However, current methods for tracking ARGs have various limitations. The current study presents a multiplexed targeted amplicon sequencing approach for the detection of sequence variants of ARGs in environmental samples. To demonstrate the application of this technique, wastewater samples collected from the inlet to 16 treatment plants located along a 440-km transect of the St-Lawrence river in the province of Quebec (Canada) were analysed. Among the ARGs examined, between 3 and 45 nucleic acid sequence variants were detected demonstrating the high sequence diversity that occurs within genes originating from a single sample type and the information that is missed using traditional techniques. Using the PLSDB and Comprehensive Antibiotic Resistance Database (CARD), the risk of ARG sequence variants was inferred based upon their reported mobility and detection in pathogens. Results suggest that sequence variants within a single ARG class present different risks to public health. In the future, targeted amplicon sequencing could be a valuable tool in environmental studies for both risk assessment purposes and in AMR source tracking.

Lakes are heterogeneous ecosystems inhabited by a rich microbiome whose genomic diversity is poorly defined. We present a continental-scale study of metagenomes representing 6.5 million km2 of the most lake-rich landscape on Earth. Analysis of 308 Canadian lakes resulted in a metagenome-assembled genome (MAG) catalogue of 1,008 mostly novel bacterial genomospecies. Lake trophic state was a leading driver of taxonomic and functional diversity among MAG assemblages, reflecting the responses of communities profiled by 16S rRNA amplicons and gene-centric metagenomics. Coupling the MAG catalogue with watershed geomatics revealed terrestrial influences of soils and land use on assemblages. Agriculture and human population density were drivers of turnover, indicating detectable anthropogenic imprints on lake bacteria at the continental scale. The sensitivity of bacterial assemblages to human impact reinforces lakes as sentinels of environmental change. Overall, the LakePulse MAG catalogue greatly expands the freshwater genomic landscape, advancing an integrative view of diversity across Earth's microbiomes.

Biodiversity loss has accelerated over the past century and freshwater species overall are among those experiencing greatest declines. Genetic resources have the potential to help evaluate the full magnitude of this loss and represent a key tool to effectively allocate conservation resources and monitor the success of restoration efforts. The full power of genetic resources will be realized when the daunting task of referencing all DNA sequences of freshwater organisms is complete. Here, we quantified the availability and distribution of barcode and genome data for freshwater macroscopic organisms in Canada, a country rich in inland water resources and thus particularly vulnerable to aquatic species losses. Impressively, most inland water species (86 %) were represented by barcodes recorded in the BOLD Systems database, while very few had full genomes available (<4 %) in the NCBI database. We identified barcode data deficiencies in northern regions and for taxa assessed as most at risk or without sufficient information for conservation status classification. As expected, the speciose insect group had a lower-than-average number of records per species and a high proportion of data deficient species without adequate barcode coverage. This study highlights where future sequencing resources should be prioritized within initiatives such as the Canada BioGenome Project and BIOSCAN Canada and provides a workflow that could be applied internationally to inform conservation management plans and to mitigate biodiversity loss.

Lakes are sentinels of environmental changes within their watersheds including those induced by a changing climate and anthropogenic activities. In particular, contamination originating from point or non-point sources (NPS) within watersheds might be reflected in changes in the bacterial composition of lake water. We assessed the abundance of potentially pathogenic bacteria (PPB) sampled in 413 lakes within 8 southern Canadian ecozones that represent a wide diversity of lakes and watershed land use. The study objectives were (1) to explore the diversity of PPB; (2) to build a fecal multi-indicator from a cluster of co-occurring PPB; and (3) to predict the fecal multi-indicator over thousands of lakes. We identified bacterial taxa based on 16S rRNA amplicon sequencing and clustered 33 PPB matching taxa in the Canadian ePATHogen database using a Sørensen dissimilarity index on binary data across the sampled lakes. One cluster contained Erysipelothrix, Desulfovibrio, Bacteroides, Vibrio and Acholeplasma and was related to the NPS fraction of agriculture and pasture within the watershed as its main driver and thus it was determined as the fecal multi-indicator. We subsequently developed a fecal multi-indicator predictive model across 200 212 southern Canadian lakes which explained 55.1% of the deviance. Mapping the predictions showed higher fecal multi-indicator abundances in the Prairies and Boreal Plains compared to the other ecozones. These results represent the first attempt to map a potential fecal multi-indicator at the continental scale, which may be further improved in the future. Lastly, the study demonstrates the capacity of a multi-disciplinary approach leveraging both datasets derived from remote sensing and DNA sequencing to provide mapping information for public health governmental policies.

We study the microbiome of sea water collected from two locations of the Barbadian coral reefs. The two sites differ in several environmental and ecological variables including their endogenous benthic community and their proximity to urban development and runoffs from inland watersheds. The composition of the microbial communities was estimated using whole genome DNA shotgun sequencing with adjuvant measurements of chemical and environmental qualities. Although both sites exhibit a similar degree of richness, the less urbanized site (Maycocks reef at Hangman’s Bay) has a strong concentration of phototrophs whereas the more urbanized location (Bellairs reef at Folkstone) is enriched for copiotrophs, macroalgal symbionts and marine-related disease-bearing organisms from taxa scattered across the tree of life. Our results are concordant with previous profiles of warm ocean surface waters, suggesting our approach captures the state of each coral reef site, setting the stage for longitudinal studies of marine microbiome dynamics in Barbados.

Problem Statement: Gene expression profiling tests are becoming a critical part of the clinician's toolbox to guide treatment decisions for early-stage estrogen receptor positive (ER+) breast cancer patients. Women over the age of 50 with intermediate 21-gene Recurrence Scores can safely avoid chemotherapy, as endocrine therapy alone is not inferior to chemo-endocrine therapy in terms of disease-free and overall survival1. However, there are differences in chemotherapy benefit for women under the age of 50 which are not well understood. We have examined how age and the menstrual cycle affect the 21-gene signature using paired breast cancer samples2 and a mouse model of breast cancer3. Methods: To investigate whether age affects variability in the 21-gene signature, paired formalin-fixed paraffin-embedded invasive ER+ breast cancer samples were collected approximately 2 weeks apart (median age 48 years; age range 36-77 years; n=25). To determine whether fluctuations in ovarian hormones affect the 21-gene signature, ER+ mammary tumours were dissected from naturally cycling Mmtv-Pymt mice at either the estrus or diestrus phase of the ovarian cycle (estrus n=25; diestrus n=28). In both human and mouse studies, gene expression was assessed through quantitative real time-PCR, and a 21-gene experimental recurrence score based on the published Oncotype DX Recurrence Score was calculated. Results: There was a significant inverse association between patient age and discordance in recurrence score (Figure 1). For every one-year decrease in age, discordance in recurrence scores between paired samples increased by 0.08 units (95% CI: -0.14, -0.01; p=0.017). Discordance in recurrence scores for women under the age of 50 were driven primarily by proliferation- and HER2-associated genes. Tumours collected at diestrus in the mouse model show significant differences in expression of six 21-gene signature genes (Ki67, Ccnb1, Esr1, Erbb2, Grb7, Bag1; p≤0.05) and a significant increase in recurrence score compared to tumours dissected at estrus (Figure 2). Conclusion: Age and menstrual cycle stage may critically affect the 21-gene signature and treatment decision-making in premenopausal breast cancer patients. These findings emphasize the need for the consideration of patient age and menstrual cycle stage in development and application of gene expression profiling tests for breast cancer care. Disclosure of Interest: None to declare References 1. Sparano JA, Gray RJ, Makower DF, et al. Adjuvant Chemotherapy Guided by a 21-Gene Expression Assay in Breast Cancer. N Engl J Med 2018;379:111-21. 2. Bernhardt SM, Dasari P, Wrin J, et al. Discordance in 21-gene recurrence scores between paired breast cancer samples is inversely associated with patient age. Breast Cancer Res 2020;22:90. 3. Bernhardt SM, Dasari P, Glynn DJ, et al. Ovarian cycle stage critically affects 21-gene recurrence scores in Mmtv-Pymt mouse mammary tumours. BMC Cancer 2021;21:736.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Lakes are heterogenous ecosystems inhabited by a rich microbiome whose genomic diversity is poorly defined. We present a continental-scale study of metagenomes representing 6.5 million km 2 of the most lake-rich landscape on Earth. Analysis of 308 Canadian lakes resulted in a metagenome-assembled genome (MAG) catalogue of 1,008 mostly novel bacterial genomospecies. Lake trophic state was a leading driver of taxonomic and functional diversity among MAG assemblages, reflecting the responses of communities profiled by 16S rRNA amplicons and gene-centric metagenomics. Coupling the MAG catalogue with watershed geomatics revealed terrestrial influences of soils and land use on assemblages. Agriculture and human population density were drivers of turnover, indicating detectable anthropogenic imprints on lake bacteria at the continental scale. The sensitivity of bacterial assemblages to human impact reinforces lakes as sentinels of environmental change. Overall, the LakePulse MAG catalogue greatly expands the freshwater genomic landscape, advancing an integrative view of diversity across Earth’s microbiomes.