Multiplatform metabolomic interlaboratory study of a whole human stool candidate reference material from omnivore and vegan donors.

INTRODUCTION: Human metabolomics has made significant strides in understanding metabolic changes and their implications for human health, with promising applications in diagnostics and treatment, particularly regarding the gut microbiome. However, progress is hampered by issues with data comparability and reproducibility across studies, limiting the translation of these discoveries into practical applications. OBJECTIVES: This study aims to evaluate the fit-for-purpose of a suite of human stool samples as potential candidate reference materials (RMs) and assess the state of the field regarding harmonizing gut metabolomics measurements. METHODS: An interlaboratory study was conducted with 18 participating institutions. The study allowed for the use of preferred analytical techniques, including liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), and nuclear magnetic resonance (NMR). RESULTS: Different laboratories used various methods and analytical platforms to identify the metabolites present in human stool RM samples. The study found a 40% to 70% recurrence in the reported top 20 most abundant metabolites across the four materials. In the full annotation list, the percentage of metabolites reported multiple times after nomenclature standardization was 36% (LC-MS), 58% (GC-MS) and 76% (NMR). Out of 9,300 unique metabolites, only 37 were reported across all three measurement techniques. CONCLUSION: This collaborative exercise emphasized the broad chemical survey possible with multi-technique approaches. Community engagement is essential for the evaluation and characterization of common materials designed to facilitate comparability and ensure data quality underscoring the value of determining current practices, challenges, and progress of a field through interlaboratory studies.

Associations between SARS-CoV-2 Infection or COVID-19 Vaccination and Human Milk Composition: A Multi-Omics Approach.

BACKGROUND: The risk of contracting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via human milk-feeding is virtually nonexistent. Adverse effects of coronavirus disease 2019 (COVID-19) vaccination for lactating individuals are not different from the general population, and no evidence has been found that their infants exhibit adverse effects. Yet, there remains substantial hesitation among this population globally regarding the safety of these vaccines. OBJECTIVES: Herein, we aimed to determine if compositional changes in milk occur following infection or vaccination, including any evidence of vaccine components. METHODS: Using a subset of milk samples obtained as part of our broad studies examining the effects on milk of SARS-CoV-2 infection and COVID-19 vaccination, an extensive multiomics approach. RESULTS: We found that compared with unvaccinated individuals, SARS-CoV-2 infection was associated with significant compositional differences in 67 proteins, 385 lipids, and 13 metabolites. In contrast, COVID-19 vaccination was not associated with any changes in lipids or metabolites, although it was associated with changes in 13 or fewer proteins. Compositional changes in milk differed by vaccine. Changes following vaccination were greatest after 1-6 h for the mRNA-based Moderna vaccine (8 changed proteins), 3 d for the mRNA-based Pfizer (4 changed proteins), and adenovirus-based Johnson and Johnson (13 changed proteins) vaccines. Proteins that changed after both natural infection and Johnson and Johnson vaccine were associated mainly with systemic inflammatory responses. In addition, no vaccine components were detected in any milk sample. CONCLUSIONS: Together, our data provide evidence of only minimal changes in milk composition because of COVID-19 vaccination, with much greater changes after natural SARS-CoV-2 infection.

Metabolomic and transcriptomic remodeling of bone marrow myeloid cells in response to maternal obesity.

Maternal obesity puts the offspring at high risk of developing obesity and cardio-metabolic diseases in adulthood. Here, using a mouse model of maternal high-fat diet (HFD)-induced obesity, we show that whole body fat content of the offspring of HFD-fed mothers (Off-HFD) increases significantly from very early age when compared to the offspring regular diet-fed mothers (Off-RD). We have previously shown significant metabolic and immune perturbations in the bone marrow of newly-weaned offspring of obese mothers. Therefore, we hypothesized that lipid metabolism is altered in the bone marrow Off-HFD in newly-weaned offspring of obese mothers when compared to the Off-RD. To test this hypothesis, we investigated the lipidomic profile of bone marrow cells collected from three-week-old offspring of regular and high fat diet-fed mothers. Diacylgycerols (DAGs), triacylglycerols (TAGs), sphingolipids and phospholipids, including plasmalogen, and lysophospholipids were remarkably different between the groups, independent of fetal sex. Levels of cholesteryl esters were significantly decreased in offspring of obese mothers, suggesting reduced delivery of cholesterol to bone marrow cells. This was accompanied by age-dependent progression of mitochondrial dysfunction in bone marrow cells. We subsequently isolated CD11b+ myeloid cells from three-week-old mice and conducted metabolomics, lipidomics, and transcriptomics analyses. The lipidomic profiles of these bone marrow myeloid cells were largely similar to that seen in bone marrow cells and included increases in DAGs and phospholipids alongside decreased TAGs, except for long-chain TAGs, which were significantly increased. Our data also revealed significant sex-dependent changes in amino acids and metabolites related to energy metabolism. Transcriptomic analysis revealed altered expression of genes related to major immune pathways including macrophage alternative activation, B-cell receptor signaling, TGFß signaling, and communication between the innate and adaptive immune systems. All told, this study revealed lipidomic, metabolomic, and gene expression abnormalities in bone marrow cells broadly, and in bone marrow myeloid cells particularly, in the newly-weaned offspring of obese mothers, which might at least partially explain the progression of metabolic and cardiovascular diseases in their adulthood.

PeakQC: A Software Tool for Omics-Agnostic Automated Quality Control of Mass Spectrometry Data.

Mass spectrometry is broadly employed to study complex molecular mechanisms in various biological and environmental fields, enabling 'omics' research such as proteomics, metabolomics, and lipidomics. As study cohorts grow larger and more complex with dozens to hundreds of samples, the need for robust quality control (QC) measures through automated software tools becomes paramount to ensure the integrity, high quality, and validity of scientific conclusions from downstream analyses and minimize the waste of resources. Since existing QC tools are mostly dedicated to proteomics, automated solutions supporting metabolomics are needed. To address this need, we developed the software PeakQC, a tool for automated QC of MS data that is independent of omics molecular types (i.e., omics-agnostic). It allows automated extraction and inspection of peak metrics of precursor ions (e.g., errors in mass, retention time, arrival time) and supports various instrumentations and acquisition types, from infusion experiments or using liquid chromatography and/or ion mobility spectrometry front-end separations and with/without fragmentation spectra from data-dependent or independent acquisition analyses. Diagnostic plots for fragmentation spectra are also generated. Here, we describe and illustrate PeakQC's functionalities using different representative data sets, demonstrating its utility as a valuable tool for enhancing the quality and reliability of omics mass spectrometry analyses.

Multi-scale signaling and tumor evolution in high-grade gliomas.

Although genomic anomalies in glioblastoma (GBM) have been well studied for over a decade, its 5-year survival rate remains lower than 5%. We seek to expand the molecular landscape of high-grade glioma, composed of IDH-wildtype GBM and IDH-mutant grade 4 astrocytoma, by integrating proteomic, metabolomic, lipidomic, and post-translational modifications (PTMs) with genomic and transcriptomic measurements to uncover multi-scale regulatory interactions governing tumor development and evolution. Applying 14 proteogenomic and metabolomic platforms to 228 tumors (212 GBM and 16 grade 4 IDH-mutant astrocytoma), including 28 at recurrence, plus 18 normal brain samples and 14 brain metastases as comparators, reveals heterogeneous upstream alterations converging on common downstream events at the proteomic and metabolomic levels and changes in protein-protein interactions and glycosylation site occupancy at recurrence. Recurrent genetic alterations and phosphorylation events on PTPN11 map to important regulatory domains in three dimensions, suggesting a central role for PTPN11 signaling across high-grade gliomas.

Exploring the ideas of young healthcare professionals from selected countries regarding rural proofing.

INTRODUCTION: Globally, most countries struggle to meet the health needs of rural communities. This has resulted in rural areas performing poorly when compared to urban areas in terms of a range of health indicators. There have been few coherent or systematic strategies that target rural communities and address their needs within the rural context. Rural proofing, defined as the systematic application of a rural lens across policies and guidelines to ensure that they speak to these health needs, seeks to address this gap. The healthcare professionals (HCPs) who will be called upon to advocate for and lead the implementation of rural proofing efforts are those currently in training or early career stages. We thus sought to understand the perspectives of young HCPs regarding the concept of rural proofing. METHODS: The study adopted an interpretivist paradigm. Data were collected using semi-structured individual interviews and focus group discussions (FGDs). Selected HCPs who are in leadership in Rural Seeds, a movement for young HCPs, participated in the study. FGDs in the form of Rural Cafés were led by some Rural Seeds leaders who participated in the interviews and who showed interest in organising the discussions. Eleven exploratory interviews and six FGDs were conducted using Zoom. HCPs were from Australia, Europe, Africa, North America, South America, and Asia. Interviews and FGDs were conducted in English, recorded, and transcribed verbatim. Thematic analysis was then undertaken. RESULTS: Participants perceived the state of rural healthcare globally to be problematic. Access to care was seen as the most significant issue in rural health care, associated with the challenges of lack of equity in access, and limited funding and support for healthcare professionals and their career pathways. Despite varying understanding of the concept, rural proofing was seen to be of great value in improving rural health care. A number of ideas for applying rural proofing, with examples, were proposed from their perspectives as frontline healthcare providers. They particularly recognised the importance of addressing the local needs of rural communities and the needs of present and future HCPs. Implementation of rural proofing was seen to require the involvement of key stakeholders from a range of sectors at multiple levels. CONCLUSION: Given the state of rural health, young rural HCPs suggest that rural proofing strategies are needed as they have the potential to bring about equity in the delivery of health care in rural and remote communities. These strategies will assist in creating a more positive future for rural health care worldwide and motivate young HCPs to become involved in rural health care, as well as to increase their motivation to take an interest in health policy development. These strategies need to be applied at multiple levels, from national government to local contexts. It is also seen to be critically important to involve multiple levels of stakeholders, from politicians to healthcare providers and community members, in the process of rural proofing.

Influence of soil depth, irrigation, and plant genotype on the soil microbiome, metaphenome, and carbon chemistry.

IMPORTANCE: Carbon is cycled through the air, plants, and belowground environment. Understanding soil carbon cycling in deep soil profiles will be important to mitigate climate change. Soil carbon cycling is impacted by water, plants, and soil microorganisms, in addition to soil mineralogy. Measuring biotic and abiotic soil properties provides a perspective of how soil microorganisms interact with the surrounding chemical environment. This study emphasizes the importance of considering biotic interactions with inorganic and oxidizable soil carbon in addition to total organic carbon in carbonate-containing soils for better informing soil carbon management decisions.

Fungal organic acid uptake of mineral-derived K is dependent on distance from carbon hotspot.

IMPORTANCE: Fungal species are foundational members of soil ecosystems with vital contributions that support interspecies resource translocation. The minute details of these biogeochemical processes are poorly investigated. Here, we addressed this knowledge gap by probing fungal growth in a novel mineral-doped soil micromodel platform using spatially-resolved imaging methodologies. We found that fungi uptake K from K-rich minerals using organic acids exuded in a distance-dependent manner from a carbon-rich hotspot. While identification of specific mechanisms within soil remains challenging, our findings demonstrate the significance of reduced complexity platforms such as the mineral-doped micromodel in probing biogeochemical processes. These findings provide visualization into hyphal uptake and transport of mineral-derived nutrients in a resource-limited environment.

Vulvar Complex Apocrine Carcinoma in a Horse (Equus caballus): A Case Report and Review of Literature.

A 6-year-old Marwari mare presented with recurrent vulvar growth. The growth was surgically excised, fixed and processed routinely. Microscopically, neoplasm showed proliferation of epithelial and myoepithelial cells with tubulopapillary pattern. On immunohistochemistry, myoepithelial cells showed strong immunoreactivity with smooth muscle actin alpha and p63. On basis of histopathology and immunohistochemistry, tumour was diagnosed as complex apocrine carcinoma. This case report describes first confirm vulvar complex apocrine carcinoma in equines.

Graphene oxide exposure alters gut microbial community composition and metabolism in an in vitro human model.

Graphene oxide (GO) nanomaterials have unique physicochemical properties that make them highly promising for biomedical, environmental, and agricultural applications. There is growing interest in the use of GO and extensive in vitro and in vivo studies have been conducted to assess its nanotoxicity. Although it is known that GO can alter the composition of the gut microbiota in mice and zebrafish, studies on the potential impacts of GO on the human gut microbiome are largely lacking. This study addresses an important knowledge gap by investigating the impact of GO exposure- at low (25 mg/L) and high (250 mg/L) doses under both fed (nutrient rich) and fasted (nutrient deplete) conditions- on the gut microbial communitys' structure and function, using an in vitro model. This model includes simulated oral, gastric, small intestinal phase digestion of GO followed by incubation in a colon bioreactor. 16S rRNA amplicon sequencing revealed that GO exposure resulted in a restructuring of community composition. 25 mg/L GO induced a marked decrease in the Bacteroidota phylum and increased the ratio of Firmicutes to Bacteroidota (F/B). Untargeted metabolomics on the supernatants indicated that 25 mg/L GO impaired microbial utilization and metabolism of substrates (amino acids, carbohydrate metabolites) and reduced production of beneficial microbial metabolites such as 5-hydroxyindole-3-acetic acid and GABA. Exposure to 250 mg/L GO resulted in community composition and metabolome profiles that were very similar to the controls that lacked both GO and digestive enzymes. Differential abundance analyses revealed that 3 genera from the phylum Bacteroidota (Bacteroides, Dysgonomonas, and Parabacteroides) were more abundant after 250 mg/L GO exposure, irrespective of feed state. Integrative correlation network analysis indicated that the phylum Bacteroidota showed strong positive correlations to multiple microbial metabolites including GABA and 3-indoleacetic acid, are much larger number of correlations compared to other phyla. These results show that GO exposure has a significant impact on gut microbial community composition and metabolism at both low and high GO concentrations.

Rapid remodeling of the soil lipidome in response to a drying-rewetting event.

BACKGROUND: Microbiomes contribute to multiple ecosystem services by transforming organic matter in the soil. Extreme shifts in the environment, such as drying-rewetting cycles during drought, can impact the microbial metabolism of organic matter by altering microbial physiology and function. These physiological responses are mediated in part by lipids that are responsible for regulating interactions between cells and the environment. Despite this critical role in regulating the microbial response to stress, little is known about microbial lipids and metabolites in the soil or how they influence phenotypes that are expressed under drying-rewetting cycles. To address this knowledge gap, we conducted a soil incubation experiment to simulate soil drying during a summer drought of an arid grassland, then measured the response of the soil lipidome and metabolome during the first 3 h after wet-up. RESULTS: Reduced nutrient access during soil drying incurred a replacement of membrane phospholipids, resulting in a diminished abundance of multiple phosphorus-rich membrane lipids. The hot and dry conditions increased the prevalence of sphingolipids and lipids containing long-chain polyunsaturated fatty acids, both of which are associated with heat and osmotic stress-mitigating properties in fungi. This novel finding suggests that lipids commonly present in eukaryotes such as fungi may play a significant role in supporting community resilience displayed by arid land soil microbiomes during drought. As early as 10 min after rewetting dry soil, distinct changes were observed in several lipids that had bacterial signatures including a rapid increase in the abundance of glycerophospholipids with saturated and short fatty acid chains, prototypical of bacterial membrane lipids. Polar metabolites including disaccharides, nucleic acids, organic acids, inositols, and amino acids also increased in abundance upon rewetting. This rapid metabolic reactivation and growth after rewetting coincided with an increase in the relative abundance of firmicutes, suggesting that members of this phylum were positively impacted by rewetting. CONCLUSIONS: Our study revealed specific changes in lipids and metabolites that are indicative of stress adaptation, substrate use, and cellular recovery during soil drying and subsequent rewetting. The drought-induced nutrient limitation was reflected in the lipidome and polar metabolome, both of which rapidly shifted (within hours) upon rewet. Reduced nutrient access in dry soil caused the replacement of glycerophospholipids with phosphorus-free lipids and impeded resource-expensive osmolyte accumulation. Elevated levels of ceramides and lipids with long-chain polyunsaturated fatty acids in dry soil suggest that lipids likely play an important role in the drought tolerance of microbial taxa capable of synthesizing these lipids. An increasing abundance of bacterial glycerophospholipids and triacylglycerols with fatty acids typical of bacteria and polar metabolites suggest a metabolic recovery in representative bacteria once the environmental conditions are conducive for growth. These results underscore the importance of the soil lipidome as a robust indicator of microbial community responses, especially at the short time scales of cell-environment reactions. Video Abstract.

Interrogating the role of the milk microbiome in mastitis in the multi-omics era.

There is growing interest in a functional understanding of milk-associated microbiota as there is ample evidence that host-associated microbial communities play an active role in host health and phenotype. Mastitis, characterized by painful inflammation of the mammary gland, is prevalent among lactating humans and agricultural animals and is associated with significant clinical and economic consequences. The etiology of mastitis is complex and polymicrobial and correlative studies have indicated alterations in milk microbial community composition. Recent evidence is beginning to suggest that a causal relationship may exist between the milk microbiota and host phenotype in mastitis. Multi-omic approaches can be leveraged to gain a mechanistic, molecular level understanding of how the milk microbiome might modulate host physiology, thereby informing strategies to prevent and ameliorate mastitis. In this paper, we review existing studies that have utilized omics approaches to investigate the role of the milk microbiome in mastitis. We also summarize the strengths and challenges associated with the different omics techniques including metagenomics, metatranscriptomics, metaproteomics, metabolomics and lipidomics and provide perspective on the integration of multiple omics technologies for a better functional understanding of the milk microbiome.

Standardized multi-omics of Earth's microbiomes reveals microbial and metabolite diversity.

Despite advances in sequencing, lack of standardization makes comparisons across studies challenging and hampers insights into the structure and function of microbial communities across multiple habitats on a planetary scale. Here we present a multi-omics analysis of a diverse set of 880 microbial community samples collected for the Earth Microbiome Project. We include amplicon (16S, 18S, ITS) and shotgun metagenomic sequence data, and untargeted metabolomics data (liquid chromatography-tandem mass spectrometry and gas chromatography mass spectrometry). We used standardized protocols and analytical methods to characterize microbial communities, focusing on relationships and co-occurrences of microbially related metabolites and microbial taxa across environments, thus allowing us to explore diversity at extraordinary scale. In addition to a reference database for metagenomic and metabolomic data, we provide a framework for incorporating additional studies, enabling the expansion of existing knowledge in the form of an evolving community resource. We demonstrate the utility of this database by testing the hypothesis that every microbe and metabolite is everywhere but the environment selects. Our results show that metabolite diversity exhibits turnover and nestedness related to both microbial communities and the environment, whereas the relative abundances of microbially related metabolites vary and co-occur with specific microbial consortia in a habitat-specific manner. We additionally show the power of certain chemistry, in particular terpenoids, in distinguishing Earth's environments (for example, terrestrial plant surfaces and soils, freshwater and marine animal stool), as well as that of certain microbes including Conexibacter woesei (terrestrial soils), Haloquadratum walsbyi (marine deposits) and Pantoea dispersa (terrestrial plant detritus). This Resource provides insight into the taxa and metabolites within microbial communities from diverse habitats across Earth, informing both microbial and chemical ecology, and provides a foundation and methods for multi-omics microbiome studies of hosts and the environment.

Primary hepatic lymphoma- A diagnostic dilemma.

Primary non-Hodgkin lymphoma of liver is a very rare malignancy. Here we report the case of a 50 year old female who presented with dull ache in the right hypochondrium and decreased appetite since 1 month. CT scan of abdomen and pelvis showed an enlarged liver with an ill- defined soft tissue lesion arising from left lobe measuring 13 × 9 cm suggestive of primary hepatic neoplasm. CT scan of chest, abdomen, and pelvis and whole body positron emission tomography showed no involvement of bone marrow, lymph nodes, spleen, or any other organ. Her liver function tests, alpha fetoprotein and carcinoembryonic antigen levels were normal. Serology was negative for viruses. Pathological examination favored diagnosis of Burkitt's lymphoma. Cytogenetic studies for MYC translocation t (8;14) is suggested for confirming the diagnosis since Ki 67 index is > 70% and not nearly 100% which is characteristic of Burkitt's lymphoma.

Comparative metabolomic analysis reveals shared and unique chemical interactions in sponge holobionts.

BACKGROUND: Sponges are ancient sessile metazoans, which form with their associated microbial symbionts a complex functional unit called a holobiont. Sponges are a rich source of chemical diversity; however, there is limited knowledge of which holobiont members produce certain metabolites and how they may contribute to chemical interactions. To address this issue, we applied non-targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) and gas chromatography mass spectrometry (GC-MS) to either whole sponge tissue or fractionated microbial cells from six different, co-occurring sponge species. RESULTS: Several metabolites were commonly found or enriched in whole sponge tissue, supporting the notion that sponge cells produce them. These include 2-methylbutyryl-carnitine, hexanoyl-carnitine and various carbohydrates, which may be potential food sources for microorganisms, as well as the antagonistic compounds hymenialdisine and eicosatrienoic acid methyl ester. Metabolites that were mostly observed or enriched in microbial cells include the antioxidant didodecyl 3,3'-thiodipropionate, the antagonistic compounds docosatetraenoic acid, and immune-suppressor phenylethylamide. This suggests that these compounds are mainly produced by the microbial members in the sponge holobiont, and are potentially either involved in inter-microbial competitions or in defenses against intruding organisms. CONCLUSIONS: This study shows how different chemical functionality is compartmentalized between sponge hosts and their microbial symbionts and provides new insights into how chemical interactions underpin the function of sponge holobionts. Video abstract.

Efficacy of Sagittal Split Fix Plates with Adjustable Slider for Intra-operative Identification and Correction of Condylar Sag in Sagittal Split Osteotomy-a Pilot Study.

Aim: To assess the efficacy of sagittal split plate with adjustable slider for intra-operative correction of condylar sag after bilateral sagittal split osteotomy. Subjects and Methods: Patients reporting for correction of mandibular skeletal deformities for correction with sagittal split osteotomy (SSRO) were enrolled in the study. Simple randomization method was followed for patient allocation. Patients in group A had undergone fixation sagittal split fix plates; in group B, miniplate fixation with monocortical screws was used. Occlusion was the key indicator of condylar sage that was checked at different time frames (intra-operatively T0, immediate T1, 6 months postoperatively T2). Preoperative, immediate and late postoperative (at 6 months and 1-year interval) and lateral cephalometric assessment was used to assess their stability. Results: Thirty-three patients were enrolled and 20 patients were included in the study. One patient of group A presented with central condylar sag that was identified intra-operatively and addressed immediately. All the patients in group B presented with type 2 peripheral condylar sag that was addressed by inter-maxillary elastics and orthodontics. Two patients in group A presented with mild degree of relapse at 6 months, which was comparable to the control group indicating good stability. Conclusion: Sagittal split plates appear to be efficacious for intra-operative identification and correction of condylar sag is associated with SSRO. Supplementary Information: The online version contains supplementary material available at 10.1007/s12663-022-01782-7.

A Histoplasma capsulatum Lipid Metabolic Map Identifies Antifungal Targets.

Lipids play a fundamental role in fungal cell biology, being essential cell membrane components and major targets of antifungal drugs. A deeper knowledge of lipid metabolism is key for developing new drugs and a better understanding of fungal pathogenesis. Here, we built a comprehensive map of the Histoplasma capsulatum lipid metabolic pathway by incorporating proteomic and lipidomic analyses. We performed genetic complementation and overexpression of H. capsulatum genes in Saccharomyces cerevisiae to validate reactions identified in the map and to determine enzymes responsible for catalyzing orphan reactions. The map led to the identification of both the fatty acid desaturation and the sphingolipid biosynthesis pathways as targets for drug development. We found that the sphingolipid biosynthesis inhibitor myriocin, the fatty acid desaturase inhibitor thiocarlide, and the fatty acid analog 10-thiastearic acid inhibit H. capsulatum growth in nanomolar to low-micromolar concentrations. These compounds also reduced the intracellular infection in an alveolar macrophage cell line. Overall, this lipid metabolic map revealed pathways that can be targeted for drug development. IMPORTANCE It is estimated that 150 people die per hour due to the insufficient therapeutic treatments to combat fungal infections. A major hurdle to developing antifungal therapies is the scarce knowledge on the fungal metabolic pathways and mechanisms of virulence. In this context, fungal lipid metabolism is an excellent candidate for developing drugs due to its essential roles in cellular scaffolds, energy storage, and signaling transductors. Here, we provide a detailed map of Histoplasma capsulatum lipid metabolism. The map revealed points of this fungus lipid metabolism that can be targeted for developing antifungal drugs.