Community assembly of the human piercing microbiome.

Predicting how biological communities respond to disturbance requires understanding the forces that govern their assembly. We propose using human skin piercings as a model system for studying community assembly after rapid environmental change. Local skin sterilization provides a 'clean slate' within the novel ecological niche created by the piercing. Stochastic assembly processes can dominate skin microbiomes due to the influence of environmental exposure on local dispersal, but deterministic processes might play a greater role within occluded skin piercings if piercing habitats impose strong selection pressures on colonizing species. Here we explore the human ear-piercing microbiome and demonstrate that community assembly is predominantly stochastic but becomes significantly more deterministic with time, producing increasingly diverse and ecologically complex communities. We also observed changes in two dominant and medically relevant antagonists (Cutibacterium acnes and Staphylococcus epidermidis), consistent with competitive exclusion induced by a transition from sebaceous to moist environments. By exploiting this common yet uniquely human practice, we show that skin piercings are not just culturally significant but also represent ecosystem engineering on the human body. The novel habitats and communities that skin piercings produce may provide general insights into biological responses to environmental disturbances with implications for both ecosystem and human health.

A new multiplex qPCR assay to detect and differentiate big cat species in the illegal wildlife trade.

All species of big cats, including tigers, cheetahs, leopards, lions, snow leopards, and jaguars, are protected under the Convention on the International Trade in Endangered Species (CITES). This is due in large part to population declines resulting from anthropogenic factors, especially poaching and the unregulated and illegal trade in pelts, bones, teeth and other products that are derived from these iconic species. To enhance and scale up monitoring for big cat products in this trade, we created a rapid multiplex qPCR test that can identify and differentiate DNA from tiger (Panthera tigris), cheetah (Acinonyx jubatus), leopard (Panthera pardus), lion (Panthera leo), snow leopard (Panthera uncia), and jaguar (Panthera onca) in wildlife products using melt curve analysis to identify each species by its unique melt peak temperature. Our results showed high PCR efficiency (> 90%), sensitivity (detection limit of 5 copies of DNA per PCR reaction) and specificity (no cross amplification between each of the 6 big cat species). When paired with a rapid (< 1 h) DNA extraction protocol that amplifies DNA from bone, teeth, and preserved skin, total test time is less than three hours. This test can be used as a screening method to improve our understanding of the scale and scope of the illegal trade in big cats and aid in the enforcement of international regulations that govern the trade in wildlife and wildlife products, both ultimately benefiting the conservation of these species worldwide.

Dynamic mapping of proteome trafficking within and between living cells by TransitID.

The ability to map trafficking for thousands of endogenous proteins at once in living cells would reveal biology currently invisible to both microscopy and mass spectrometry. Here, we report TransitID, a method for unbiased mapping of endogenous proteome trafficking with nanometer spatial resolution in living cells. Two proximity labeling (PL) enzymes, TurboID and APEX, are targeted to source and destination compartments, and PL with each enzyme is performed in tandem via sequential addition of their small-molecule substrates. Mass spectrometry identifies the proteins tagged by both enzymes. Using TransitID, we mapped proteome trafficking between cytosol and mitochondria, cytosol and nucleus, and nucleolus and stress granules (SGs), uncovering a role for SGs in protecting the transcription factor JUN from oxidative stress. TransitID also identifies proteins that signal intercellularly between macrophages and cancer cells. TransitID offers a powerful approach for distinguishing protein populations based on compartment or cell type of origin.

Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells.

The incorporation of light-responsive domains into engineered proteins has enabled control of protein localization, interactions and function with light. We integrated optogenetic control into proximity labeling, a cornerstone technique for high-resolution proteomic mapping of organelles and interactomes in living cells. Through structure-guided screening and directed evolution, we installed the light-sensitive LOV domain into the proximity labeling enzyme TurboID to rapidly and reversibly control its labeling activity with low-power blue light. 'LOV-Turbo' works in multiple contexts and dramatically reduces background in biotin-rich environments such as neurons. We used LOV-Turbo for pulse-chase labeling to discover proteins that traffic between endoplasmic reticulum, nuclear and mitochondrial compartments under cellular stress. We also showed that instead of external light, LOV-Turbo can be activated by bioluminescence resonance energy transfer from luciferase, enabling interaction-dependent proximity labeling. Overall, LOV-Turbo increases the spatial and temporal precision of proximity labeling, expanding the scope of experimental questions that can be addressed with proximity labeling.

Workflow enabling deepscale immunopeptidome, proteome, ubiquitylome, phosphoproteome, and acetylome analyses of sample-limited tissues.

Serial multi-omic analysis of proteome, phosphoproteome, and acetylome provides insights into changes in protein expression, cell signaling, cross-talk and epigenetic pathways involved in disease pathology and treatment. However, ubiquitylome and HLA peptidome data collection used to understand protein degradation and antigen presentation have not together been serialized, and instead require separate samples for parallel processing using distinct protocols. Here we present MONTE, a highly sensitive multi-omic native tissue enrichment workflow, that enables serial, deep-scale analysis of HLA-I and HLA-II immunopeptidome, ubiquitylome, proteome, phosphoproteome, and acetylome from the same tissue sample. We demonstrate that the depth of coverage and quantitative precision of each 'ome is not compromised by serialization, and the addition of HLA immunopeptidomics enables the identification of peptides derived from cancer/testis antigens and patient specific neoantigens. We evaluate the technical feasibility of the MONTE workflow using a small cohort of patient lung adenocarcinoma tumors.

Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells.

The incorporation of light-responsive domains into engineered proteins has enabled control of protein localization, interactions, and function with light. We integrated optogenetic control into proximity labeling (PL), a cornerstone technique for high-resolution proteomic mapping of organelles and interactomes in living cells. Through structure-guided screening and directed evolution, we installed the light-sensitive LOV domain into the PL enzyme TurboID to rapidly and reversibly control its labeling activity with low-power blue light. "LOV-Turbo" works in multiple contexts and dramatically reduces background in biotin-rich environments such as neurons. We used LOV-Turbo for pulse-chase labeling to discover proteins that traffick between endoplasmic reticulum, nuclear, and mitochondrial compartments under cellular stress. We also showed that instead of external light, LOV-Turbo can be activated by BRET from luciferase, enabling interaction-dependent PL. Overall, LOV-Turbo increases the spatial and temporal precision of PL, expanding the scope of experimental questions that can be addressed with PL.

Dynamic mapping of proteome trafficking within and between living cells by TransitID.

The ability to map trafficking for thousands of endogenous proteins at once in living cells would reveal biology currently invisible to both microscopy and mass spectrometry. Here we report TransitID, a method for unbiased mapping of endogenous proteome trafficking with nanometer spatial resolution in living cells. Two proximity labeling (PL) enzymes, TurboID and APEX, are targeted to source and destination compartments, and PL with each enzyme is performed in tandem via sequential addition of their small-molecule substrates. Mass spectrometry identifies the proteins tagged by both enzymes. Using TransitID, we mapped proteome trafficking between cytosol and mitochondria, cytosol and nucleus, and nucleolus and stress granules, uncovering a role for stress granules in protecting the transcription factor JUN from oxidative stress. TransitID also identifies proteins that signal intercellularly between macrophages and cancer cells. TransitID introduces a powerful approach for distinguishing protein populations based on compartment or cell type of origin.

Estimating biodiversity across the tree of life on Mount Everest's southern flank with environmental DNA.

Species composition in high-alpine ecosystems is a useful indicator for monitoring climatic and environmental changes at the upper limits of habitable environments. We used environmental DNA (eDNA) analysis to document the breadth of high-alpine biodiversity present on Earth's highest mountain, Mt. Everest (8,849 m a.s.l.) in Nepal's Khumbu region. In April-May 2019, we collected eDNA from ten ponds and streams between 4,500 m and 5,500 m. Using multiple sequencing and bioinformatic approaches, we identified taxa from 36 phyla and 187 potential orders across the Tree of Life in Mt. Everest's high-alpine and aeolian ecosystem. These organisms, all recorded above 4,500 m-an elevational belt comprising <3% of Earth's land surface-represents ∼16% of global taxonomic order estimates. Our eDNA inventory will aid future high-Himalayan biomonitoring and retrospective molecular studies to assess changes over time as climate-driven warming, glacial melt, and anthropogenic influences reshape this rapidly transforming world-renowned ecosystem.

A genetic model for in vivo proximity labelling of the mammalian secretome.

Organ functions are highly specialized and interdependent. Secreted factors regulate organ development and mediate homeostasis through serum trafficking and inter-organ communication. Enzyme-catalysed proximity labelling enables the identification of proteins within a specific cellular compartment. Here, we report a BirA*G3 mouse strain that enables CRE-dependent promiscuous biotinylation of proteins trafficking through the endoplasmic reticulum. When broadly activated throughout the mouse, widespread labelling of proteins was observed within the secretory pathway. Streptavidin affinity purification and peptide mapping by quantitative mass spectrometry (MS) proteomics revealed organ-specific secretory profiles and serum trafficking. As expected, secretory proteomes were highly enriched for signal peptide-containing proteins, highlighting both conventional and non-conventional secretory processes, and ectodomain shedding. Lower-abundance proteins with hormone-like properties were recovered and validated using orthogonal approaches. Hepatocyte-specific activation of BirA*G3 highlighted liver-specific biotinylated secretome profiles. The BirA*G3 mouse model demonstrates enhanced labelling efficiency and tissue specificity over viral transduction approaches and will facilitate a deeper understanding of secretory protein interplay in development, and in healthy and diseased adult states.

Measuring protected-area effectiveness using vertebrate distributions from leech iDNA.

Protected areas are key to meeting biodiversity conservation goals, but direct measures of effectiveness have proven difficult to obtain. We address this challenge by using environmental DNA from leech-ingested bloodmeals to estimate spatially-resolved vertebrate occupancies across the 677 km2 Ailaoshan reserve in Yunnan, China. From 30,468 leeches collected by 163 park rangers across 172 patrol areas, we identify 86 vertebrate species, including amphibians, mammals, birds and squamates. Multi-species occupancy modelling shows that species richness increases with elevation and distance to reserve edge. Most large mammals (e.g. sambar, black bear, serow, tufted deer) follow this pattern; the exceptions are the three domestic mammal species (cows, sheep, goats) and muntjak deer, which are more common at lower elevations. Vertebrate occupancies are a direct measure of conservation outcomes that can help guide protected-area management and improve the contributions that protected areas make towards global biodiversity goals. Here, we show the feasibility of using invertebrate-derived DNA to estimate spatially-resolved vertebrate occupancies across entire protected areas.

Promoting equity and inclusion with student-driven initiatives.

Representation is crucial in building more inclusive communities in science, technology, engineering, mathematics, and medicine (STEMM) fields. STEMM Diversity is a student-driven initiative that was founded to promote equity, diversity, and inclusion (EDI) at McGill University. Here, we discuss the lessons learned while developing STEMM Diversity that can help guide others to develop their own student-driven initiatives.

Transgenes of genetically modified animals detected non-invasively via environmental DNA.

We demonstrate that simple, non-invasive environmental DNA (eDNA) methods can detect transgenes of genetically modified (GM) animals from terrestrial and aquatic sources in invertebrate and vertebrate systems. We detected transgenic fragments between 82-234 bp through targeted PCR amplification of environmental DNA extracted from food media of GM fruit flies (Drosophila melanogaster), feces, urine, and saliva of GM laboratory mice (Mus musculus), and aquarium water of GM tetra fish (Gymnocorymbus ternetzi). With rapidly growing accessibility of genome-editing technologies such as CRISPR, the prevalence and diversity of GM animals will increase dramatically. GM animals have already been released into the wild with more releases planned in the future. eDNA methods have the potential to address the critical need for sensitive, accurate, and cost-effective detection and monitoring of GM animals and their transgenes in nature.

Resistance, resilience, and functional redundancy of freshwater bacterioplankton communities facing a gradient of agricultural stressors in a mesocosm experiment.

Agricultural pollution with fertilizers and pesticides is a common disturbance to freshwater biodiversity. Bacterioplankton communities are at the base of aquatic food webs, but their responses to these potentially interacting stressors are rarely explored. To test the extent of resistance and resilience in bacterioplankton communities faced with agricultural stressors, we exposed freshwater mesocosms to single and combined gradients of two commonly used pesticides: the herbicide glyphosate (0-15 mg/L) and the neonicotinoid insecticide imidacloprid (0-60 µg/L), in high or low nutrient backgrounds. Over the 43-day experiment, we tracked variation in bacterial density with flow cytometry, carbon substrate use with Biolog EcoPlates, and taxonomic diversity and composition with environmental 16S rRNA gene amplicon sequencing. We show that only glyphosate (at the highest dose, 15 mg/L), but not imidacloprid, nutrients, or their interactions measurably changed community structure, favouring members of the Proteobacteria including the genus Agrobacterium. However, no change in carbon substrate use was detected throughout, suggesting functional redundancy despite taxonomic changes. We further show that communities are resilient at broad, but not fine taxonomic levels: 24 days after glyphosate application the precise amplicon sequence variants do not return, and tend to be replaced by phylogenetically close taxa. We conclude that high doses of glyphosate - but still within commonly acceptable regulatory guidelines - alter freshwater bacterioplankton by favouring a subset of higher taxonomic units (i.e., genus to phylum) that transiently thrive in the presence of glyphosate. Longer-term impacts of glyphosate at finer taxonomic resolution merit further investigation.

Irisin deletion induces a decrease in growth and fertility in mice.

BACKGROUND: Irisin, which is cleaved from fibronectin type III domain-containing protein 5 (Fndc5), plays an important role in energy homeostasis. The link between energy metabolism and reproduction is well known. However, the biological actions of irisin in reproduction remain largely unexplored. METHODS: In this study, we generated Fndc5 gene mutation to create irisin deficient mice. Female wild-type (WT) and Fndc5 mutant mice were fed with standard chow for 48 weeks. Firstly, the survival rate, body weight and fertility were described in mice. Secondly, the levels of steroid hormones in serum were measured by ELISA, and the estrus cycle and the appearance of follicles were determined by vaginal smears and ovarian continuous sections. Thirdly, mRNA-sequencing analysis was used to compare gene expression between the ovaries of Fndc5 mutant mice and those of WT mice. Finally, the effects of exogenous irisin on steroid hormone production was investigated in KGN cells. RESULTS: The mice lacking irisin presented increased mortality, reduced body weight and poor fertility. Analysis of sex hormones showed decreased levels of estradiol, follicle-stimulating hormone and luteinizing hormone, and elevated progesterone levels in Fndc5 mutant mice. Irisin deficiency in mice was associated with irregular estrus, reduced ratio of antral follicles. The expressions of Akr1c18, Mamld1, and Cyp19a1, which are involved in the synthesis of steroid hormones, were reduced in the ovaries of mutant mice. Exogenous irisin could promote the expression of Akr1c18, Mamld1, and Cyp19a1 in KGN cells, stimulating estradiol production and inhibiting progesterone secretion. CONCLUSIONS: Irisin deficiency was related to disordered endocrinology metabolism in mice. The irisin deficient mice showed poor growth and development, and decreased fertility. Irisin likely have effects on the expressions of Akr1c18, Mamld1 and Cyp19a1 in ovary, regulating the steroid hormone production. This study provides novel insights into the potential role of irisin in mammalian growth and reproduction.

Corrosion Protective Film Formation on Mg Alloys AZ 31 by Exposure to Dilute Selenite Solutions.

The study of protective film formation on Mg alloys by exposure to sodium selenite solutions was conducted. Anodic polarization studies, electrochemical impedance spectroscopy studies, morphological analysis, and Energy-dispersive X-ray spectroscopy were performed on AZ31 Mg alloy after coating treatment in different concentrations of sodium selenite. The corrosion resistance was improved by around 5 times compared with control. Improved resistance to localized corrosion was observed in the coatings treated by 5 mM or 10 mM sodium selenite. The protection mechanism was ascribed to the transformation of selenite to insoluble selenium, the formation of insoluble MgSeO3 hydrate, and polymerization of amorphous selenium.

Disordered metabolism in mice lacking irisin.

Irisin is a product of fibronectin type III domain-containing protein (Fndc5) and is involved in the regulation of adipokine secretion and the differentiation of osteoblasts and osteoclasts. In this study, we aimed to determine whether irisin lacking affects glucose/lipid and bone metabolism. We knocked out the Fndc5 gene to generate irisin-lacking mice. Remarkable, irisin lacking was related to poor 'browning response', with a bigger size of the intraperitoneal white adipose cell and decreased a number of brown adipose cells in brown adipose of interscapular tissue. The irisin lacking mice had hyperlipidemia and insulin resistance, reduced HDL-cholesterol level, increased LDL-cholesterol level, and decreased insulin sensitivity. The lacking of irisin was associated with reduced bone strength and bone mass in mice. The increased number of osteoclasts and higher expression of RANKL indicated increased bone resorption in irisin lacking mice. The level of IL-6 and TNF-α also increased in irisin lacking mice. The results showed that irisin lacking was related to decreased 'browning response', glucose/lipid metabolic derangement, and reduced bone mass with increased bone resorption. Further studies are needed to confirm these initial observations and explore the mechanisms underlying the effects of irisin on glucose/lipid and bone metabolism.

Community rescue in experimental phytoplankton communities facing severe herbicide pollution.

Community rescue occurs when ecological or evolutionary processes restore positive growth in a highly stressful environment that was lethal to the community in its ancestral form, thus averting biomass collapse in a deteriorating environment. Laboratory evidence suggests that community rescue is most likely in high-biomass communities that have previously experienced moderate doses of sublethal stress. We assessed this result under more natural conditions, in a mesocosm experiment with phytoplankton communities exposed to the ubiquitous herbicide glyphosate. We tested whether community biomass and prior herbicide exposure would facilitate community rescue after severe contamination. We found that prior exposure to glyphosate was a very strong predictor of the rescue outcome, while high community biomass was not. Furthermore, although glyphosate had negative effects on diversity, it did not influence community composition significantly, suggesting a modest role for genus sorting in this rescue process. Our results expand the scope of community rescue theory to complex ecosystems and confirm that prior stress exposure is a key predictor of rescue.

The correlation between UDP-glucuronosyltransferase polymorphisms and environmental endocrine disruptors levels in polycystic ovary syndrome patients.

BACKGROUND: In recent years, there has been an interest in whether environmental endocrine disruptors (EEDs) may contribute to the endocrine disorders in patients with polycystic ovary syndrome (PCOS). The clearance of EEDs from the human body is regulated by the glucuronidation of UDP-glucuronosyltransferases (UGT). This study aimed to analyze the relationship of UGT1A1, UGT2B7, and UGT2B15 polymorphisms with the metabolism of EEDs in patients with PCOS. METHODS: A total of 357 Chinese women (119 PCOS cases and 238 controls) were genotyped for polymorphisms of UGT1A1, UGT2B7, and UGT2B15. The plasma concentrations of EEDs were measured by the gas chromatography-mass spectrometry method. The association between UGT polymorphisms and the serum level of EEDs in patients with PCOS was analyzed. RESULTS: The UGT2B7 single nucleotide polymorphism was associated with an increased risk of PCOS. The homozygous polymorphism (TT) of UGT2B7 showed higher bisphenol A and PAEs concentrations in serum. However, a single nucleotide polymorphism on UGT2B15 expression was associated with a decreased risk of PCOS. Subjects homozygous for the T allele of UGT2B15 had a significant effect on phthalates in the blood. In addition, our results also showed that the homozygous polymorphism (TT) of UGT2B7 and UGT2B15 was associated with the capacity of the excretion of androgen in patients with PCOS. CONCLUSIONS: Our study reported the novel associations between the UGT polymorphisms and EEDs concentrations in patients with PCOS, supporting the relevance of genetic differences in EEDs metabolism, which might be considered as an etiology of PCOS.