Roadmap for the next decade of plant programmed cell death research.

Programmed cell death (PCD) is fundamentally important for plant development, abiotic stress responses and immunity, but our understanding of its regulation remains fragmented. Building a stronger research community is required to accelerate progress in this area through knowledge exchange and constructive debate. In this Viewpoint, we aim to initiate a collective effort to integrate data across a diverse set of experimental models to facilitate characterisation of the fundamental mechanisms underlying plant PCD and ultimately aid the development of a new plant cell death classification system in the future. We also put forward our vision for the next decade of plant PCD research stemming from discussions held during the 31st New Phytologist workshop, 'The Life and Death Decisions of Plant Cells' that took place at University College Dublin in Ireland (14-15 June 2023). We convey the key areas of significant progress and possible future research directions identified, including resolving the spatiotemporal control of cell death, isolation of its molecular and genetic regulators, and harnessing technical advances for studying PCD events in plants. Further, we review the breadth of potential impacts of plant PCD research and highlight the promising new applications of findings from this dynamically evolving field.

Arsenic stress triggers active exudation of arsenic-phytochelatin complexes from Lupinus albus roots.

Arsenic contamination of soils threatens the health of millions globally through accumulation in crops. While plants detoxify arsenic via phytochelatin (PC) complexation and efflux of arsenite from roots, arsenite efflux mechanisms are not fully understood. Here, white lupin (Lupinus albus) was grown in semi-hydroponics and exudation of glutathione (GSH) derivatives and PCs in response to arsenic was scrutinised using LC-MS/MS. Inhibiting synthesis of PC precursor GSH with L-buthionine sulfoximine (BSO) or ABC transporters with vanadate drastically reduced (>22%) GSH-derivative and PC2 exudation, but not PC3 exudation. This was accompanied by arsenic hypersensitivity in plants treated with BSO and moderate sensitivity with vanadate treatment. Investigating arsenic-phytochelatin (As-PC) complexation revealed two distinct As-PC complexes, As bound to GSH and PC2 (GS-As-PC2) and As bound to PC3 (As-PC3), in exudates of As-treated lupin. Vanadate inhibited As-PC exudation, while BSO inhibited both the synthesis and exudation of As-PC complexes. These results demonstrate a role of GSH-derivatives and PC exudation in lupin arsenic tolerance and reveal As-PC exudation as a new potential mechanism contributing to active arsenic efflux in plants. Overall, this study uncovers insight into rhizosphere arsenic detoxification with potential to help mitigate pollution and reduce arsenic accumulation in crops.

Gut microbiota influence anastomotic healing in colorectal cancer surgery through modulation of mucosal proinflammatory cytokines.

OBJECTIVE: Colorectal cancer (CRC) is the third most diagnosed cancer, and requires surgical resection and reconnection, or anastomosis, of the remaining bowel to re-establish intestinal continuity. Anastomotic leak (AL) is a major complication that increases mortality and cancer recurrence. Our objective is to assess the causal role of gut microbiota in anastomotic healing. DESIGN: The causal role of gut microbiota was assessed in a murine AL model receiving faecal microbiota transplantation (FMT) from patients with CRC collected before surgery and who later developed or not, AL. Anastomotic healing and gut barrier integrity were assessed after surgery. Bacterial candidates implicated in anastomotic healing were identified using 16S rRNA gene sequencing and were isolated from faecal samples to be tested both in vitro and in vivo. RESULTS: Mice receiving FMT from patients that developed AL displayed poor anastomotic healing. Profiling of gut microbiota of patients and mice after FMT revealed correlations between healing parameters and the relative abundance of Alistipes onderdonkii and Parabacteroides goldsteinii. Oral supplementation with A. onderdonkii resulted in a higher rate of leaks in mice, while gavage with P. goldsteinii improved healing by exerting an anti-inflammatory effect. Patients with AL and mice receiving FMT from AL patients presented upregulation of mucosal MIP-1α, MIP-2, MCP-1 and IL-17A/F before surgery. Retrospective analysis revealed that patients with AL present higher circulating neutrophil and monocyte counts before surgery. CONCLUSION: Gut microbiota plays an important role in surgical colonic healing in patients with CRC. The impact of these findings may extend to a vast array of invasive gastrointestinal procedures.

Microbial community origin and fate through a rural wastewater treatment plant.

Conventional wastewater treatment relies on a complex microbiota; however, much of this community is still to be characterized. To better understand the origin, dynamics and fate of bacteria within a wastewater treatment plant: untreated primary wastewater, activated sludge and post-treatment effluent were characterized. From 3163 exact sequence variants (ESVs), 860 were annotated to species-level. In primary wastewater, 28% of ESVs were putative bacterial species previously associated with humans, 14% with animals and 5% as common to the environment. Differential abundance analysis revealed significant relative reductions in ESVs from potentially human-associated species from primary wastewater to activated sludge, and significant increases in ESVs from species associated with nutrient cycling. Between primary wastewater and effluent, 51% of ESVs from human-associated species did not significantly differ, and species such as Bacteroides massiliensis and Bacteroides dorei increased. These findings illustrate that activated sludge increased extracellular protease and urease-producing species, ammonia and nitrite oxidizers, denitrifiers and specific phosphorus accumulators. Although many human-associated species declined, some persisted in effluent, including strains of potential health or environmental concern. Species-level microbial assessment may be useful for understanding variation in wastewater treatment efficiency as well as for monitoring the release of microbes into surface water and the wider ecosystem.

Accuracy, Sensitivity, and Specificity of the LLIS and ULL27 in Detecting Breast Cancer-Related Lymphedema.

INTRODUCTION: Breast cancer-related lymphedema occurs in up to 30% of women following axillary lymph node dissection (ALND) and less commonly following sentinel lymph node biopsy. To quantify disability in these patients, patient-reported outcome measures (PROMs) have proven useful; however, given the overlap of symptoms between ALND and lymphedema, examination of their accuracy, sensitivity, and specificity in detecting lymphedema in breast cancer patients undergoing ALND is needed. METHODS: The Lymphedema Life Impact Scale (LLIS) and the Upper Limb Lymphedema 27 scale (ULL27) were administered to patients who had undergone ALND at least 2 years prior and either did or did not develop lymphedema. Survey responses and the degree of disability were compared to generate receiver operator characteristic (ROC) curves, and the sensitivity and specificity of PROMs to diagnose lymphedema were analyzed. RESULTS: Both PROMs were highly accurate, sensitive, and specific for detecting lymphedema. The LLIS had an accuracy of 97%, sensitivity of 100%, and specificity of 84.8% at a cutoff of ≥ 5.88 overall percent impairment score (higher scores indicate worse disability). The ULL27 had an accuracy of 93%, sensitivity of 88.6%, and specificity of 90.9% at a cutoff of ≤ 83.3 global score (lower scores indicate worse disability). CONCLUSIONS: The LLIS and the ULL27 appear to be highly specific for lymphedema and capable of differentiating it from symptoms resulting from ALND alone. Our findings suggest that use of these questionnaires with a threshold may be effective for diagnosing lymphedema, potentially reducing the need for frequent clinic visits and time-consuming measurements.

Phytochelatin and coumarin enrichment in root exudates of arsenic-treated white lupin.

Soil contamination with toxic metalloids, such as arsenic, can represent a substantial human health and environmental risk. Some plants are thought to tolerate soil toxicity using root exudation, however, the nature of this response to arsenic remains largely unknown. Here, white lupin plants were exposed to arsenic in a semi-hydroponic system and their exudates were profiled using untargeted liquid chromatography-tandem mass spectrometry. Arsenic concentrations up to 1 ppm were tolerated and led to the accumulation of 12.9 µg As g-1 dry weight (DW) and 411 µg As g-1 DW in above-ground and belowground tissues, respectively. From 193 exuded metabolites, 34 were significantly differentially abundant due to 1 ppm arsenic, including depletion of glutathione disulphide and enrichment of phytochelatins and coumarins. Significant enrichment of phytochelatins in exudates of arsenic-treated plants was further confirmed using exudate sampling with strict root exclusion. The chemical tolerance toolkit in white lupin included nutrient acquisition metabolites as well as phytochelatins, the major intracellular metal-binding detoxification oligopeptides which have not been previously reported as having an extracellular role. These findings highlight the value of untargeted metabolite profiling approaches to reveal the unexpected and inform strategies to mitigate anthropogenic pollution in soils around the world.

ANCHOR: a 16S rRNA gene amplicon pipeline for microbial analysis of multiple environmental samples.

Analysis of 16S ribosomal RNA (rRNA) gene amplification data for microbial barcoding can be inaccurate across complex environmental samples. A method, ANCHOR, is presented and designed for improved species-level microbial identification using paired-end sequences directly, multiple high-complexity samples and multiple reference databases. A standard operating procedure (SOP) is reported alongside benchmarking against artificial, single sample and replicated mock data sets. The method is then directly tested using a real-world data set from surface swabs of the International Space Station (ISS). Simple mock community analysis identified 100% of the expected species and 99% of expected gene copy variants (100% identical). A replicated mock community revealed similar or better numbers of expected species than MetaAmp, DADA2, Mothur and QIIME1. Analysis of the ISS microbiome identified 714 putative unique species/strains and differential abundance analysis distinguished significant differences between the Destiny module (U.S. laboratory) and Harmony module (sleeping quarters). Harmony was remarkably dominated by human gastrointestinal tract bacteria, similar to enclosed environments on earth; however, Destiny module bacteria also derived from nonhuman microbiome carriers present on the ISS, the laboratory's research animals. ANCHOR can help substantially improve sequence resolution of 16S rRNA gene amplification data within biologically replicated environmental experiments and integrated multidatabase annotation enhances interpretation of complex, nonreference microbiomes.

Comparative Transcriptomic Approaches Exploring Contamination Stress Tolerance in Salix sp. Reveal the Importance for a Metaorganismal de Novo Assembly Approach for Nonmodel Plants.

Metatranscriptomic study of nonmodel organisms requires strategies that retain the highly resolved genetic information generated from model organisms while allowing for identification of the unexpected. A real-world biological application of phytoremediation, the field growth of 10 Salix cultivars on polluted soils, was used as an exemplar nonmodel and multifaceted crop response well-disposed to the study of gene expression. Sequence reads were assembled de novo to create 10 independent transcriptomes, a global transcriptome, and were mapped against the Salix purpurea 94006 reference genome. Annotation of assembled contigs was performed without a priori assumption of the originating organism. Global transcriptome construction from 3.03 billion paired-end reads revealed 606,880 unique contigs annotated from 1588 species, often common in all 10 cultivars. Comparisons between transcriptomic and metatranscriptomic methodologies provide clear evidence that nonnative RNA can mistakenly map to reference genomes, especially to conserved regions of common housekeeping genes, such as actin, α/ß-tubulin, and elongation factor 1-α. In Salix, Rubisco activase transcripts were down-regulated in contaminated trees across all 10 cultivars, whereas thiamine thizole synthase and CP12, a Calvin Cycle master regulator, were uniformly up-regulated. De novo assembly approaches, with unconstrained annotation, can improve data quality; care should be taken when exploring such plant genetics to reduce de facto data exclusion by mapping to a single reference genome alone. Salix gene expression patterns strongly suggest cultivar-wide alteration of specific photosynthetic apparatus and protection of the antenna complexes from oxidation damage in contaminated trees, providing an insight into common stress tolerance strategies in a real-world phytoremediation system.

Meta-transcriptomics indicates biotic cross-tolerance in willow trees cultivated on petroleum hydrocarbon contaminated soil.

BACKGROUND: High concentrations of petroleum hydrocarbon (PHC) pollution can be hazardous to human health and leave soils incapable of supporting agricultural crops. A cheap solution, which can help restore biodiversity and bring land back to productivity, is cultivation of high biomass yielding willow trees. However, the genetic mechanisms which allow these fast-growing trees to tolerate PHCs are as yet unclear. METHODS: Salix purpurea 'Fish Creek' trees were pot-grown in soil from a former petroleum refinery, either lacking or enriched with C10-C50 PHCs. De novo assembled transcriptomes were compared between tree organs and impartially annotated without a priori constraint to any organism. RESULTS: Over 45% of differentially expressed genes originated from foreign organisms, the majority from the two-spotted spidermite, Tetranychus urticae. Over 99% of T. urticae transcripts were differentially expressed with greater abundance in non-contaminated trees. Plant transcripts involved in the polypropanoid pathway, including phenylalanine ammonia-lyase (PAL), had greater expression in contaminated trees whereas most resistance genes showed higher expression in non-contaminated trees. CONCLUSIONS: The impartial approach to annotation of the de novo transcriptomes, allowing for the possibility for multiple species identification, was essential for interpretation of the crop's response treatment. The meta-transcriptomic pattern of expression suggests a cross-tolerance mechanism whereby abiotic stress resistance systems provide improved biotic resistance. These findings highlight a valuable but complex biotic and abiotic stress response to real-world, multidimensional contamination which could, in part, help explain why crops such as willow can produce uniquely high biomass yields on challenging marginal land.

X-ray micro-computed tomography in willow reveals tissue patterning of reaction wood and delay in programmed cell death.

BACKGROUND: Variation in the reaction wood (RW) response has been shown to be a principle component driving differences in lignocellulosic sugar yield from the bioenergy crop willow. The phenotypic cause(s) behind these differences in sugar yield, beyond their common elicitor, however, remain unclear. Here we use X-ray micro-computed tomography (µCT) to investigate RW-associated alterations in secondary xylem tissue patterning in three dimensions (3D). RESULTS: Major architectural alterations were successfully quantified in 3D and attributed to RW induction. Whilst the frequency of vessels was reduced in tension wood tissue (TW), the total vessel volume was significantly increased. Interestingly, a delay in programmed-cell-death (PCD) associated with TW was also clearly observed and readily quantified by µCT. CONCLUSIONS: The surprising degree to which the volume of vessels was increased illustrates the substantial xylem tissue remodelling involved in reaction wood formation. The remodelling suggests an important physiological compromise between structural and hydraulic architecture necessary for extensive alteration of biomass and helps to demonstrate the power of improving our perspective of cell and tissue architecture. The precise observation of xylem tissue development and quantification of the extent of delay in PCD provides a valuable and exciting insight into this bioenergy crop trait.

Longitudinal multi-omics analysis of host microbiome architecture and immune responses during short-term spaceflight.

Maintenance of astronaut health during spaceflight will require monitoring and potentially modulating their microbiomes. However, documenting microbial shifts during spaceflight has been difficult due to mission constraints that lead to limited sampling and profiling. Here we executed a six-month longitudinal study to quantify the high-resolution human microbiome response to three days in orbit for four individuals. Using paired metagenomics and metatranscriptomics alongside single-nuclei immune cell profiling, we characterized time-dependent, multikingdom microbiome changes across 750 samples and 10 body sites before, during and after spaceflight at eight timepoints. We found that most alterations were transient across body sites; for example, viruses increased in skin sites mostly during flight. However, longer-term shifts were observed in the oral microbiome, including increased plaque-associated bacteria (for example, Fusobacteriota), which correlated with immune cell gene expression. Further, microbial genes associated with phage activity, toxin-antitoxin systems and stress response were enriched across multiple body sites. In total, this study reveals in-depth characterization of microbiome and immune response shifts experienced by astronauts during short-term spaceflight and the associated changes to the living environment, which can help guide future missions, spacecraft design and space habitat planning.

Dynamics of bacterial and archaeal communities during horse bedding and green waste composting.

Organic waste decomposition can make up substantial amounts of municipal greenhouse emissions during decomposition. Composting has the potential to reduce these emissions as well as generate sustainable fertilizer. However, our understanding of how complex microbial communities change to drive the chemical and biological processes of composting is still limited. To investigate the microbiota associated with organic waste decomposition, initial composting feedstock (Litter), three composting windrows of 1.5 months (Young phase), 3 months (Middle phase) and 12 months (Aged phase) old, and 24-month-old mature Compost were sampled to assess physicochemical properties, plant cell wall composition and the microbial community using 16S rRNA gene amplification. A total of 2,612 Exact Sequence Variants (ESVs) included 517 annotated as putative species and 694 as genera which together captured 57.7% of the 3,133,873 sequences, with the most abundant species being Thermobifida fusca, Thermomonospora chromogena and Thermobifida bifida. Compost properties changed rapidly over time alongside the diversity of the compost community, which increased as composting progressed, and multivariate analysis indicated significant variation in community composition between each time-point. The abundance of bacteria in the feedstock is strongly correlated with the presence of organic matter and the abundance of plant cell wall components. Temperature and pH are the most strongly correlated parameters with bacterial abundance in the thermophilic and cooling phases/mature compost respectively. Differential abundance analysis revealed 810 ESVs annotated as species significantly varied in relative abundance between Litter and Young phase, 653 between the Young and Middle phases, 1182 between Middle and Aged phases and 663 between Aged phase and mature Compost. These changes indicated that structural carbohydrates and lignin degrading species were abundant at the beginning of the thermophilic phase, especially members of the Firmicute and Actinobacteria phyla. A high diversity of species capable of putative ammonification and denitrification were consistently found throughout the composting phases, whereas a limited number of nitrifying bacteria were identified and were significantly enriched within the later mesophilic composting phases. High microbial community resolution also revealed unexpected species which could be beneficial for agricultural soils enriched with mature compost or for the deployment of environmental and plant biotechnologies. Understanding the dynamics of these microbial communities could lead to improved waste management strategies and the development of input-specific composting protocols to optimize carbon and nitrogen transformation and promote a diverse and functional microflora in mature compost.

Altered serum bile acid profile in fibromyalgia is associated with specific gut microbiome changes and symptom severity.

ABSTRACT: Alterations in the composition and function of the gut microbiome in women with fibromyalgia have recently been demonstrated, including changes in the relative abundance of certain bile acid-metabolizing bacteria. Bile acids can affect multiple physiological processes, including visceral pain, but have yet to be explored for association to the fibromyalgia gut microbiome. In this study, 16S rRNA sequencing and targeted metabolomic approaches were used to characterize the gut microbiome and circulating bile acids in a cohort of 42 women with fibromyalgia and 42 healthy controls. Alterations in the relative abundance of several bacterial species known to metabolize bile acids were observed in women with fibromyalgia, accompanied by significant alterations in the serum concentration of secondary bile acids, including a marked depletion of α-muricholic acid. Statistical learning algorithms could accurately detect individuals with fibromyalgia using the concentration of these serum bile acids. Serum α-muricholic acid was highly correlated with symptom severity, including pain intensity and fatigue. Taken together, these findings suggest serum bile acid alterations are implicated in nociplastic pain. The changes observed in the composition of the gut microbiota and the concentration of circulating secondary bile acids seem congruent with the phenotype of increased nociception and are quantitatively correlated with symptom severity. This is a first demonstration of circulating bile acid alteration in individuals with fibromyalgia, potentially secondary to upstream gut microbiome alterations. If corroborated in independent studies, these observations may allow for the development of molecular diagnostic aids for fibromyalgia as well as mechanistic insights into the syndrome.

Viral activation and ecological restructuring characterize a microbiome axis of spaceflight-associated immune activation.

Maintenance of astronaut health during spaceflight will require monitoring and potentially modulating their microbiomes, which play a role in some space-derived health disorders. However, documenting the response of microbiota to spaceflight has been difficult thus far due to mission constraints that lead to limited sampling. Here, we executed a six-month longitudinal study centered on a three-day flight to quantify the high-resolution microbiome response to spaceflight. Via paired metagenomics and metatranscriptomics alongside single immune profiling, we resolved a microbiome "architecture" of spaceflight characterized by time-dependent and taxonomically divergent microbiome alterations across 750 samples and ten body sites. We observed pan-phyletic viral activation and signs of persistent changes that, in the oral microbiome, yielded plaque-associated pathobionts with strong associations to immune cell gene expression. Further, we found enrichments of microbial genes associated with antibiotic production, toxin-antitoxin systems, and stress response enriched universally across the body sites. We also used strain-level tracking to measure the potential propagation of microbial species from the crew members to each other and the environment, identifying microbes that were prone to seed the capsule surface and move between the crew. Finally, we identified associations between microbiome and host immune cell shifts, proposing both a microbiome axis of immune changes during flight as well as the sources of some of those changes. In summary, these datasets and methods reveal connections between crew immunology, the microbiome, and their likely drivers and lay the groundwork for future microbiome studies of spaceflight.

Dietary Intake Is Unlikely to Explain Symptom Severity and Syndrome-Specific Microbiome Alterations in a Cohort of Women with Fibromyalgia.

BACKGROUND: Significant alterations were recently identified in the composition and putative function of the gut microbiome in women with fibromyalgia. As diet can influence the composition of the gut microbiome, differences in nutritional intake could, in theory, account for some of these specific fibromyalgia microbiome alterations. The current study aims to compare the diet of women with fibromyalgia to that of controls in order to explore possible associations between the intake of certain nutrients, symptom severity and gut microbiome composition. METHODS: The study population was comprised of 56 women with fibromyalgia and 68 controls. Dietary intake was assessed using the NIH Automated Self-Administered 24 h recall, following dietitian's instructions and the completion of a three-day dietary recall. The gut microbiome was assessed by 16S ribosomal RNA gene sequencing of stool samples. RESULTS: Most demographic and anthropometric characteristics were comparable between groups. The average energy and macronutrient intake (total and relative) and overall diet quality score were not different between patients and controls, nor were the main vitamins, minerals, fatty acids, alcohol, caffeine, sugar or fiber intakes. The daily intake of micronutrients and normalized macronutrients in women with fibromyalgia was largely not correlated with disease-specific measures, including pain intensity, fatigue, cognitive symptoms and quality of sleep, or with the relative quantity of almost any of the gut microbiome bacterial taxa differentially abundant in fibromyalgia. CONCLUSION: These data demonstrate that dietary intakes, as evaluated by self-reported questionnaires, probably cannot explain the syndrome-specific differences in gut microbiome or the clinical phenotype of fibromyalgia.

Different Temperature and UV Patterns Modulate Berry Maturation and Volatile Compounds Accumulation in Vitis sp.

Volatile compounds (VCs) in grapevine berries play an important role in wine quality; however, such compounds and vine development can be sensitive to environmental conditions. Due to this sensitivity, changes in temperature patterns due to global warming are likely to further impact grape production and berry composition. The aim of this study was to determine the possible effects of different growing-degree day accumulation patterns on berry ripening and composition at harvest. An experimental field was conducted using Vitis sp. L'Acadie blanc, in Nova Scotia, Canada. Using on-the-row mini-greenhouses, moderate temperature increase and reduced ultraviolet (UV) exposure were triggered in grapevines during pre-veraison (inflorescence to the beginning of berry softening), post-veraison (berry softening to full maturity), and whole season (inflorescence to full maturity), while controls were left without treatment. Free and bound VCs were extracted from berries sampled at three different phenological stages between veraison and maturity before analysis by gas chromatography-mass spectrometry (GC-MS). Berries from grapevines exposed to higher temperatures during early berry development (pre-veraison and whole) accumulated significantly higher concentrations of benzene derivatives 2-phenylethanol and benzyl alcohol at harvest, but lower concentrations of hydroxy-methoxy-substituted volatile phenols, terpenes, and C13-norisoprenoids than the control berries. These results illustrate the importance of different environmental interactions in berry composition and suggest that temperature could potentially modulate phenylpropanoid and mevalonate metabolism in developing berries. This study provides insights into the relationships between abiotic conditions and secondary metabolism in grapevine and highlights the significance of early developmental stages on berry quality at harvest.

Haven Health is About to Disrupt the U.S. Healthcare System.

Background: Healthcare costs are increasing in the U.S. Healthcare market is fragmented and opaque. The Amazon, JP Morgan Chase, and Berkshire Hathaway partnered to form an independent healthcare company 'Haven', designed to support cost-containment in health care and to forge a better patient experience. Limited information is available in the public domain about Haven's strategy to deliver cost-effective healthcare for their employees. Objective: To describe the impact of Haven Health of modern healthcare. Methods: We reviewed literature in Pubmed (MEDLINE database of references and abstracts on life sciences and biomedical topics from the USA National Library of Medicine at the National Institutes of Health) and the public domain. Results: Amazon's expertise and abilities in e-commerce such as logistics, supply, and big data management will support Haven's quest to resolve inefficiencies in health care. Haven may attempt to directly negotiate with providers, bypassing insurance companies, to establish themselves as an independent payor. Additionally, Haven to establish themselves as a provider by building new low-cost primary care clinics, focusing on chronic disease prevention. The new healthcare system may leverage machine learning, artificial intelligence and big data analysis to support its initiatives and other 'big data' analytics to drive it all. Conclusions: Heaven Health may use its expertise to disrupt everything from the pharmaceutical supply chain to primary care and telehealth. More research is needed to evaluate the impact of Haven Healthcare on disease outcomes and healthcare costs.

Emerging frontiers in human milk microbiome research and suggested primers for 16S rRNA gene analysis.

Human milk is the ideal food for infants due to its unique nutritional and immune properties, and more recently human milk has also been recognized as an important source of bacteria for infants. However, a substantial amount of fundamental human milk microbiome information remains unclear, such as the origin, composition and function of the community and its members. There is emerging evidence to suggest that the diversity and composition of the milk microbiome might differ between lactation stages, due to maternal factors and diet, agrarian and urban lifestyles, and geographical location. The evolution of the methods used for studying milk microbiota, transitioning from culture dependent-approaches to include culture-independent approaches, has had an impact on findings and, in particular, primer selection within 16S rRNA gene barcoding studies have led to discrepancies in observed microbiota communities. Here, the current state-of-the-art is reviewed and emerging frontiers essential to improving our understanding of the human milk microbiome are considered.

Distinct Changes Occur in the Human Breast Milk Microbiome Between Early and Established Lactation in Breastfeeding Guatemalan Mothers.

Human breast milk contains a diverse community of bacteria, but as breast milk microbiome studies have largely focused on mothers from high income countries where few women breastfeed to 6 months, the temporal changes in the breast milk microbiome that occur during later lactation stages have not been explored. For this cross-sectional study, microbiota from breast milk samples of Mam-Mayan mothers living in eight remote rural communities in the Western Highlands of Guatemala were analyzed. All mothers delivered vaginally and breastfed their infants for 6 months. Breast milk from 76 unrelated mothers was used to compare two lactation stages, either "early" (6-46 days post-partum, n = 33) or "late" (109-184 days post-partum, n = 43). Breast milk microbial communities were assessed using 16S ribosomal RNA gene sequencing and lactation stages were compared using DESeq2 differential abundance analysis. A total of 1,505 OTUs were identified, including 287 which could be annotated as putative species. Among several maternal factors, lactation stage explained microbiome variance and inertia in ordination with the most significance (p < 0.001). Differential abundance analysis identified 137 OTUs as significantly higher in either early or late lactation. These included a general shift from Staphylococcus and Streptococcus species in early lactation to Sphingobium and Pseudomonas species in late lactation. Species enriched in early lactation included putative commensal bacteria known to colonize the infant oral and intestinal tracts whereas species enriched in late lactation had a uniform functional trait associated with aromatic compound degradation. Differentially abundant species also included several species which have not previously been reported within breast milk, such as Janthinobacterium agaricidamnosum, Novosphingobium clariflavum, Ottowia beijingensis, and Flavobacterium cucumis. These discoveries describe temporal changes to the breast milk microbiome of healthy Guatemalan mothers from early to late lactation. Collectively, these findings illustrate how studying under-represented human populations might advance our understanding of factors that modulate the human milk microbiome in low and middle income countries (LMIC).

Altered microbiome composition in individuals with fibromyalgia.

Fibromyalgia (FM) is a prevalent syndrome, characterised by chronic widespread pain, fatigue, and impaired sleep, that is challenging to diagnose and difficult to treat. The microbiomes of 77 women with FM and that of 79 control participants were compared using 16S rRNA gene amplification and whole-genome sequencing. When comparing FM patients with unrelated controls using differential abundance analysis, significant differences were revealed in several bacterial taxa. Variance in the composition of the microbiomes was explained by FM-related variables more than by any other innate or environmental variable and correlated with clinical indices of FM. In line with observed alteration in butyrate-metabolising species, targeted serum metabolite analysis verified differences in the serum levels of butyrate and propionate in FM patients. Using machine-learning algorithms, the microbiome composition alone allowed for the classification of patients and controls (receiver operating characteristic area under the curve 87.8%). To the best of our knowledge, this is the first demonstration of gut microbiome alteration in nonvisceral pain. This observation paves the way for further studies, elucidating the pathophysiology of FM, developing diagnostic aids and possibly allowing for new treatment modalities to be explored.