Experimental design and reporting standards for metabolomics studies of mammalian cell lines.

Metabolomics is an analytical technique that investigates the small biochemical molecules present within a biological sample isolated from a plant, animal, or cultured cells. It can be an extremely powerful tool in elucidating the specific metabolic changes within a biological system in response to an environmental challenge such as disease, infection, drugs, or toxins. A historically difficult step in the metabolomics pipeline is in data interpretation to a meaningful biological context, for such high-variability biological samples and in untargeted metabolomics studies that are hypothesis-generating by design. One way to achieve stronger biological context of metabolomic data is via the use of cultured cell models, particularly for mammalian biological systems. The benefits of in vitro metabolomics include a much greater control of external variables and no ethical concerns. The current concerns are with inconsistencies in experimental procedures and level of reporting standards between different studies. This review discusses some of these discrepancies between recent studies, such as metabolite extraction and data normalisation. The aim of this review is to highlight the importance of a standardised experimental approach to any cultured cell metabolomics study and suggests an example procedure fully inclusive of information that should be disclosed in regard to the cell type/s used and their culture conditions. Metabolomics of cultured cells has the potential to uncover previously unknown information about cell biology, functions and response mechanisms, and so the accurate biological interpretation of the data produced and its ability to be compared to other studies should be considered vitally important.

Untargeted metabolomics of neuronal cell culture: A model system for the toxicity testing of insecticide chemical exposure.

Toxicity testing is essential for the protection of human health from exposure to toxic environmental chemicals. As traditional toxicity testing is carried out using animal models, mammalian cell culture models are becoming an increasingly attractive alternative to animal testing. Combining the use of mammalian cell culture models with screening-style molecular profiling technologies, such as metabolomics, can uncover previously unknown biochemical bases of toxicity. We have used a mass spectrometry-based untargeted metabolomics approach to characterize for the first time the changes in the metabolome of the B50 cell line, an immortalised rat neuronal cell line, following acute exposure to two known neurotoxic chemicals that are common environmental contaminants; the pyrethroid insecticide permethrin and the organophosphate insecticide malathion. B50 cells were exposed to either the dosing vehicle (methanol) or an acute dose of either permethrin or malathion for 6 and 24 hours. Intracellular metabolites were profiled by gas chromatography-mass spectrometry. Using principal components analysis, we selected the key metabolites whose abundance was altered by chemical exposure. By considering the major fold changes in abundance (>2.0 or <0.5 from control) across these metabolites, we were able to elucidate important cellular events associated with toxic exposure including disrupted energy metabolism and attempted protective mechanisms from excitotoxicity. Our findings illustrate the ability of mammalian cell culture metabolomics to detect finer metabolic effects of acute exposure to known toxic chemicals, and validate the need for further development of this process in the application of trace-level dose and chronic toxicity studies, and toxicity testing of unknown chemicals.

Metabolomic profiling and genomic analysis of wheat aneuploid lines to identify genes controlling biochemical pathways in mature grain.

Metabolomics is becoming an increasingly important tool in plant genomics to decipher the function of genes controlling biochemical pathways responsible for trait variation. Although theoretical models can integrate genes and metabolites for trait variation, biological networks require validation using appropriate experimental genetic systems. In this study, we applied an untargeted metabolite analysis to mature grain of wheat homoeologous group 3 ditelosomic lines, selected compounds that showed significant variation between wheat lines Chinese Spring and at least one ditelosomic line, tracked the genes encoding enzymes of their biochemical pathway using the wheat genome survey sequence and determined the genetic components underlying metabolite variation. A total of 412 analytes were resolved in the wheat grain metabolome, and principal component analysis indicated significant differences in metabolite profiles between Chinese Spring and each ditelosomic lines. The grain metabolome identified 55 compounds positively matched against a mass spectral library where the majority showed significant differences between Chinese Spring and at least one ditelosomic line. Trehalose and branched-chain amino acids were selected for detailed investigation, and it was expected that if genes encoding enzymes directly related to their biochemical pathways were located on homoeologous group 3 chromosomes, then corresponding ditelosomic lines would have a significant reduction in metabolites compared with Chinese Spring. Although a proportion showed a reduction, some lines showed significant increases in metabolites, indicating that genes directly and indirectly involved in biosynthetic pathways likely regulate the metabolome. Therefore, this study demonstrated that wheat aneuploid lines are suitable experimental genetic system to validate metabolomics-genomics networks.

Strong Born-A First of Its Kind National FASD Prevention Campaign in Australia Led by the National Aboriginal Community Controlled Health Organisation (NACCHO) in Collaboration with the Aboriginal Community Controlled Health Organisations (ACCHOs).

The Strong Born Campaign (2022-2025) was launched by the National Aboriginal Community Controlled Health Organisation (NACCHO) in 2023. Strong Born is the first of its kind national Aboriginal and Torres Strait Islander health promotion campaign to address Fetal Alcohol Spectrum Disorder (FASD) within Australia. Strong Born was developed to address a longstanding, significant gap in health promotion and sector knowledge on FASD, a lifelong disability that can result from alcohol use during pregnancy. Utilizing a strengths-based and culturally sound approach, NACCHO worked closely with the Aboriginal Community Controlled Health Organisations (ACCHOs) to develop the campaign through co-design, as described in this paper. Since its inception, the ACCHOs have continually invested in driving change towards improvements in Aboriginal health determinants and health promotion. The Strong Born Campaign developed culturally safe health promotion tool kits designed for the community and health sector staff and also offered communities the opportunity to apply for FASD Communications and Engagement Grants to engage in local campaign promotion. The tool kits have been disseminated to 92 ACCHOs across Australia. This paper describes the development of the Strong Born Campaign and activities following its launch in February 2023 from an Indigenous context within Australia, as described by NACCHO.

Nitric oxide synthase 3 deficiency limits adverse ventricular remodeling after pressure overload in insulin resistance.

Insulin resistance (IR) and systemic hypertension are independently associated with heart failure. We reported previously that nitric oxide synthase 3 (NOS3) has a beneficial effect on left ventricular (LV) remodeling and function after pressure-overload in mice. The aim of our study was to investigate the interaction of IR and NOS3 in pressure-overload-induced LV remodeling and dysfunction. Wild-type (WT) and NOS3-deficient (NOS3(-/-)) mice were fed either a standard diet (SD) or a high-fat diet (HFD) to induce IR. After 9 days of diet, mice underwent transverse aortic constriction (TAC). LV structure and function were assessed serially using echocardiography. Cardiomyocytes were isolated, and levels of oxidative stress were evaluated using 2',7'-dichlorodihydrofluorescein diacetate. Cardiac mitochondria were isolated, and mitochondrial respiration and ATP production were measured. TAC induced LV remodeling and dysfunction in all mice. The TAC-induced decrease in LV function was greater in SD-fed NOS3(-/-) mice than in SD-fed WT mice. In contrast, HFD-fed NOS3(-/-) developed less LV remodeling and dysfunction and had better survival than did HFD-fed WT mice. Seven days after TAC, oxidative stress levels were lower in cardiomyocytes from HFD-fed NOS3(-/-) than in those from HFD-fed WT. N(ω)-nitro-L-arginine methyl ester and mitochondrial inhibitors (rotenone and 2-thenoyltrifluoroacetone) decreased oxidative stress levels in cardiomyocytes from HFD-fed WT mice. Mitochondrial respiration was altered in NOS3(-/-) mice but did not worsen after HFD and TAC. In contrast with its protective role in SD, NOS3 increases LV adverse remodeling after pressure overload in HFD-fed, insulin resistant mice. Interactions between NOS3 and mitochondria may be responsible for increased oxidative stress levels in HFD-fed WT mice hearts.

Soluble guanylate cyclase-α1 is required for the cardioprotective effects of inhaled nitric oxide.

Reperfusion injury limits the benefits of revascularization in the treatment of myocardial infarction (MI). Breathing nitric oxide (NO) reduces cardiac ischemia-reperfusion injury in animal models; however, the signaling pathways by which inhaled NO confers cardioprotection remain uncertain. The objective of this study was to learn whether inhaled NO reduces cardiac ischemia-reperfusion injury by activating the cGMP-generating enzyme, soluble guanylate cyclase (sGC), and to investigate whether bone marrow (BM)-derived cells participate in the sGC-mediated cardioprotective effects of inhaled NO. Wild-type (WT) mice and mice deficient in the sGC α(1)-subunit (sGCα(1)(-/-) mice) were subjected to cardiac ischemia for 1 h, followed by 24 h of reperfusion. During ischemia and for the first 10 min of reperfusion, mice were ventilated with oxygen or with oxygen supplemented with NO (80 parts per million). The ratio of MI size to area at risk (MI/AAR) did not differ in WT and sGCα(1)(-/-) mice that did not breathe NO. Breathing NO decreased MI/AAR in WT mice (41%, P = 0.002) but not in sGCα(1)(-/-) mice (7%, P = not significant). BM transplantation was performed to restore WT BM-derived cells to sGCα(1)(-/-) mice. Breathing NO decreased MI/AAR in sGCα(1)(-/-) mice carrying WT BM (39%, P = 0.031). In conclusion, these results demonstrate that a global deficiency of sGCα(1) does not alter the degree of cardiac ischemia-reperfusion injury in mice. The cardioprotective effects of inhaled NO require the presence of sGCα(1). Moreover, our studies suggest that BM-derived cells are key mediators of the ability of NO to reduce cardiac ischemia-reperfusion injury.

Sample Preparation and Reporting Standards for Metabolomics of Adherent Mammalian Cells.

Metabolomics is an analytical technique that investigates the small molecules present within a biological system. Metabolomics of cultured cells allows profiling of the metabolic chemicals involved in a cell type-specific system and the response of that metabolome to external challenges, such as change in environment or exposure to drugs or toxins. The numerous benefits of in vitro metabolomics include a much greater control of external variables and reduced ethical concerns. There is potential for metabolomics of mammalian cells to uncover new information on mechanisms of action for drugs or toxins or to provide a more sensitive, human-specific early risk assessment in drug development or toxicology investigations. One way to achieve stronger biological outcomes from metabolomic data is via the use of these mammalian cultured cell models, particularly in a high-throughput context. With the sensitivity and quantity of data that metabolomics is able to provide, it is important to ensure that the sampling techniques have minimal interference when it comes to interpretation of any observed shifts in the metabolite profile. Here we describe a sampling procedure designed to ensure that the effects seen in metabolomic analyses are explained fully by the experimental factor and not other routine culture-specific activities.

Soluble guanylate cyclase α1-deficient mice: a novel murine model for primary open angle glaucoma.

Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide. The molecular signaling involved in the pathogenesis of POAG remains unknown. Here, we report that mice lacking the α1 subunit of the nitric oxide receptor soluble guanylate cyclase represent a novel and translatable animal model of POAG, characterized by thinning of the retinal nerve fiber layer and loss of optic nerve axons in the context of an open iridocorneal angle. The optic neuropathy associated with soluble guanylate cyclase α1-deficiency was accompanied by modestly increased intraocular pressure and retinal vascular dysfunction. Moreover, data from a candidate gene association study suggests that a variant in the locus containing the genes encoding for the α1 and ß1 subunits of soluble guanylate cyclase is associated with POAG in patients presenting with initial paracentral vision loss, a disease subtype thought to be associated with vascular dysregulation. These findings provide new insights into the pathogenesis and genetics of POAG and suggest new therapeutic strategies for POAG.

Midkine-A functions upstream of Id2a to regulate cell cycle kinetics in the developing vertebrate retina.

BACKGROUND: Midkine is a small heparin binding growth factor expressed in numerous tissues during development. The unique midkine gene in mammals has two paralogs in zebrafish: midkine-a (mdka) and midkine-b (mdkb). In the zebrafish retina, during both larval development and adult photoreceptor regeneration, mdka is expressed in retinal stem and progenitor cells and functions as a molecular component of the retina's stem cell niche. In this study, loss-of-function and conditional overexpression were used to investigate the function of Mdka in the retina of the embryonic zebrafish. RESULTS: The results show that during early retinal development Mdka functions to regulate cell cycle kinetics. Following targeted knockdown of Mdka synthesis, retinal progenitors cycle more slowly, and this results in microphthalmia, a diminished rate of cell cycle exit and a temporal delay of cell cycle exit and neuronal differentiation. In contrast, Mdka overexpression results in acceleration of the cell cycle and retinal overgrowth. Mdka gain-of-function, however, does not temporally advance cell cycle exit. Experiments to identify a potential Mdka signaling pathway show that Mdka functions upstream of the HLH regulatory protein, Id2a. Gene expression analysis shows Mdka regulates id2a expression, and co-injection of Mdka morpholinos and id2a mRNA rescues the Mdka loss-of-function phenotype. CONCLUSIONS: These data show that in zebrafish, Mdka resides in a shared Id2a pathway to regulate cell cycle kinetics in retinal progenitors. This is the first study to demonstrate the function of Midkine during retinal development and adds Midkine to the list of growth factors that transcriptionally regulate Id proteins.

Genetic modifiers of hypertension in soluble guanylate cyclase α1-deficient mice.

Nitric oxide (NO) plays an essential role in regulating hypertension and blood flow by inducing relaxation of vascular smooth muscle. Male mice deficient in a NO receptor component, the α1 subunit of soluble guanylate cyclase (sGCα1), are prone to hypertension in some, but not all, mouse strains, suggesting that additional genetic factors contribute to the onset of hypertension. Using linkage analyses, we discovered a quantitative trait locus (QTL) on chromosome 1 that was linked to mean arterial pressure (MAP) in the context of sGCα1 deficiency. This region is syntenic with previously identified blood pressure-related QTLs in the human and rat genome and contains the genes coding for renin. Hypertension was associated with increased activity of the renin-angiotensin-aldosterone system (RAAS). Further, we found that RAAS inhibition normalized MAP and improved endothelium-dependent vasorelaxation in sGCα1-deficient mice. These data identify the RAAS as a blood pressure-modifying mechanism in a setting of impaired NO/cGMP signaling.